Cellular Functions with Regards to the Understanding ...

Cellular Functions with Regards to the Understanding of Mantle Cell Lymphoma An Informed Patient

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Contents Articles Cellular Basics for Understanding Neoplasms Cell cycle

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Complete blood count

10

Reference ranges for blood tests

14

Bone marrow

38

Antibody

42

Hematopoietic stem cell

57

Haematopoiesis

64

Lymphopoiesis

68

Lymphoid Neoplasms

81

Hematologic disease

81

Hematological malignancy

85

Lymphoma

88

Non-Hodgkin lymphoma

96

Cluster of differentiation

98

List of human clusters of differentiation

101

Mantle cell lymphoma

106

Treatments of Neoplasms

112

Chemotherapy

112

Targeted therapy

120

Monoclonal antibodies

123

Molecular Diagnostics

131

Flow cytometry

131

Fluorescence in situ hybridization

137

Common variable immunodeficiency

144

Epigenetics

148

Common Therapies for MCL

160

Clinical trial

160

Rituximab

176

CHOP

180

HyperCVAD

182

Bortezomib

184

Bendamustine

189

Temsirolimus

192

Antibody-drug conjugate

195

Ibritumomab tiuxetan

197

Tositumomab

200

References Article Sources and Contributors

202

Image Sources, Licenses and Contributors

205

Article Licenses License

207

1

Cellular Basics for Understanding Neoplasms Cell cycle The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication (replication). In cells without a nucleus (prokaryotic), the cell cycle occurs via a process termed binary fission. In cells with a nucleus (eukaryotes), the cell cycle can be Each turn of the cell cycle divides the chromosomes in a cell nucleus. divided in two brief periods: interphase—during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA—and the mitosis (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells". The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed.

Phases The cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis). M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides in half forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.

Cell cycle

2

Schematic of the cell cycle. outer ring: I = Interphase, M = Mitosis; inner ring: M = Mitosis, G1 = Gap 1, G2 = Gap 2, S = Synthesis; not in ring: G0 = Gap [1] 0/Resting.

State

Phase

Description Abbreviation

quiescent/ senescent

Gap 0

G0

A resting phase where the cell has left the cycle and has stopped dividing.

Interphase

Gap 1

G1

Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis.

Synthesis

S

DNA replication occurs during this phase.

Gap 2

G2

During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide.

Mitosis

M

Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division.

Cell division

After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.

Resting (G0 phase) The term "post-mitotic" is sometimes used to refer to both quiescent and senescent cells. Nonproliferative cells in multicellular eukaryotes generally enter the quiescent G0 state from G1 and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated. Cellular senescence is a state that occurs in response to DNA damage or degradation that would make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis.

Cell cycle

Interphase Before a cell can enter cell division, it needs to take in nutrients. All of the preparations are done during the interphase. Interphase proceeds in three stages, G1, S, and G2. Cell division operates in a cycle. Therefore, interphase is preceded by the previous cycle of mitosis and cytokinesis. G1 phase The first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis is called G1 (G indicating gap). It is also called the growth phase. During this phase the biosynthetic activities of the cell, which had been considerably slowed down during M phase, resume at a high rate. This phase is marked by synthesis of various enzymes that are required in S phase, mainly those needed for DNA replication. Duration of G1 is highly variable, even among different cells of the same species.[2] S phase The ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids. Thus, during this phase, the amount of DNA in the cell has effectively doubled, though the ploidy of the cell remains the same. Rates of RNA transcription and protein synthesis are very low during this phase. An exception to this is histone production, most of which occurs during the S phase.[3] [4] [5] G2 phase The cell then enters the G2 phase, which lasts until the cell enters mitosis. Again, significant biosynthesis occurs during this phase, mainly involving the production of microtubules, which are required during the process of mitosis. Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis.

Mitosis (M Phase/Mitotic phase) The relatively brief M phase consists of nuclear division (karyokinesis). The M phase has been broken down into several distinct phases, sequentially known as: • • • • •

prophase, metaphase, anaphase, telophase cytokinesis (strictly speaking, cytokinesis is not part of mitosis but is an event that directly follows mitosis in which cytoplasm is divided into two daughter cells)

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two nuclei.[6] It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle - the division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell. This accounts for approximately 10% of the cell cycle. Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different species. For example, animals undergo an "open" mitosis, where the nuclear envelope breaks down before the chromosomes separate, while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis, where chromosomes divide within an intact cell nucleus.[7] Prokaryotic cells, which lack a nucleus, divide by a process called binary fission. The process of mitosis is complex and highly regulated. The sequence of events is divided into phases, corresponding to the completion of one set of activities and the start of the next. These stages are prophase,

3

Cell cycle prometaphase, metaphase, anaphase and telophase. During the process of mitosis the pairs of chromosomes condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. The cell then divides in cytokinesis, to produce two identical daughter cells.[8] Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is often used interchangeably with "M phase". However, there are many cells where mitosis and cytokinesis occur separately, forming single cells with multiple nuclei. This occurs most notably among the fungi and slime moulds, but is found in various different groups. Even in animals, cytokinesis and mitosis may occur independently, for instance during certain stages of fruit fly embryonic development.[9] Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer.

Regulation of eukaryotic cell cycle Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.

Role of cyclins and CDKs Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle.[10] Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of these central molecules.[11] Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general more complex organisms have more elaborate cell cycle control systems that incorporate more individual components. Many of the relevant genes were first identified by studying yeast, especially Regulation of cell cycle: Schematic Saccharomyces cerevisiae;[12] genetic nomenclature in yeast dubs many these genes cdc (for "cell division cycle") followed by an identifying number, e.g., cdc25 or cdc20. Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.[13]

4

Cell cycle General mechanism of cyclin-CDK interaction Upon receiving a pro-mitotic extracellular signal, G1 cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. The G1 cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G1 phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes is also deleterious to the daughter cells. Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G2 phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore. APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed. Interphase: Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. By studying molecular events in cells, scientists have determined that interphase can be divided into 4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis) phase, Gap 2 (G2). Specific action of cyclin-CDK complexes Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D-CDK4 complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma susceptibility protein (Rb). The hyperphosphorylated Rb dissociates from the E2F/DP1/Rb complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G1 to S phase (G1/S transition). Cyclin B along with cdc2 (cdc2 - fission yeasts (CDK1 - mammalia)) forms the cyclin B-cdc2 complex, which initiates the G2/M transition.[14] Cyclin B-cdc2 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis.[13]

5

Cell cycle

6

Inhibitors Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors. The cip/kip family includes the genes p21, p27 and p57. They halt cell cycle in G1 phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.

Overview of signal transduction pathways involved in apoptosis, also known as "programmed cell death".

The INK4a/ARF family includes p16INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and p14arf which prevents p53 degradation. Synthetic inhibitors of Cdc25 could also be useful for the arrest of cell cycle and therefore be useful as antineoplastic and anticancer agents.[15]

Transcriptional Regulatory Network Evidence suggests that a semi-autonomous transcriptional network acts in concert with the CDK-cyclin machinery to regulate the cell cycle. Several gene expression studies in Saccharomyces cerevisiae have identified approximately 800 to 1200 genes that change expression over the course of the cell cycle;[12] [16] [17] they are transcribed at high levels at specific points in the cell cycle, and remain at lower levels throughout the rest of the cell cycle. While the set of identified genes differs between studies due to the computational methods and criterion used to identify them, each study indicates that a large portion of yeast genes are temporally regulated.[18] Many periodically expressed genes are driven by transcription factors that are also periodically expressed. One screen of single-gene knockouts identified 48 transcription factors (about 20% of all non-essential transcription factors) that show cell cycle progression defects.[19] Genome-wide studies using high throughput technologies have identified the transcription factors that bind to the promoters of yeast genes, and correlating these findings with temporal expression patterns have allowed the identification of transcription factors that drive phase-specific gene expression.[16] [20] The expression profiles of these transcription factors are driven by the transcription factors that peak in the prior phase, and computational models have shown that a CDK-autonomous network of these transcription factors is sufficient to produce steady-state oscillations in gene expression).[17] [21] Experimental evidence also suggests that gene expression can oscillate with the period seen in dividing wild-type cells independently of the CDK machinery. Orlando et al. used microarrays to measure the expression of a set of 1,271 genes that they identified as periodic in both wild type cells and cells lacking all S-phase and mitotic cyclins (clb1,2,3,4,5,6). Of the 1,271 genes assayed, 882 continued to be expressed in the cyclin-deficient cells at the same time as in the wild type cells, despite the fact that the cyclin-deficient cells arrest at the border between G1 and S phase. However, 833 of the genes assayed changed behavior between the wild type and mutant cells, indicating that these genes are likely directly or indirectly regulated by the CDK-cyclin machinery. Some genes that continued to be expressed on time in the mutant cells were also expressed at different levels in the mutant and wild type cells. These findings suggest that while the transcriptional network may oscillate independently of the CDK-cyclin oscillator,

Cell cycle they are coupled in a manner that requires both to ensure the proper timing of cell cycle events.[17] Other work indicates that phosphorylation, a post-translational modification, of cell cycle transcription factors by Cdk1 may alter the localization or activity of the transcription factors in order to tightly control timing of target genes (Ubersax et al. 2003; Sidorova et al. 1995; White et al. 2009).[19] [22] [23] While oscillatory transcription plays a key role in the progression of the yeast cell cycle, the CDK-cyclin machinery operates independently in the early embryonic cell cycle. Before the midblastula transition, zygotic transcription does not occur and all needed proteins, such as the B-type cyclins, are translated from maternally loaded mRNA.[24]

Checkpoints Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle.[25] Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met. Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on to daughter cells. Two main checkpoints exist: the G1/S checkpoint and the G2/M checkpoint. G1/S transition is a rate-limiting step in the cell cycle and is also known as restriction point.[13] An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint. p53 plays an important role in triggering the control mechanisms at both G1/S and G2/M checkpoints.

Role in tumor formation A disregulation of the cell cycle components may lead to tumor formation. As mentioned above, some genes like the cell cycle inhibitors, RB, p53 etc., when they mutate, may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G0 phase) in tumors is much higher than that in normal tissue. Thus there is a net increase in cell number as the number of cells that die by apoptosis or senescence remains the same. The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation. This fact is made use of in cancer treatment; by a process known as debulking, a significant mass of the tumor is removed which pushes a significant number of the remaining tumor cells from G0 to G1 phase (due to increased availability of nutrients, oxygen, growth factors etc.). Radiation or chemotherapy following the debulking procedure kills these cells which have newly entered the cell cycle.[13] The fastest cycling mammalian cells in culture, crypt cells in the intestinal epithelium, have a cycle time as short as 9 to 10 hours. Stem cells in resting mouse skin may have a cycle time of more than 200 hours. Most of this difference is due to the varying length of G1, the most variable phase of the cycle. M and S do not vary much. In general, cells are most radiosensitive in late M and G2 phases and most resistant in late S. For cells with a longer cell cycle time and a significantly long G1 phase, there is a second peak of resistance late in G1 The pattern of resistance and sensitivity correlates with the level of sulfhydryl compounds in the cell. Sulfhydryls are natural radioprotectors and tend to be at their highest levels in S and at their lowest near mitosis.

7

Cell cycle

Synchronization of cell cultures Several methods can be used to synchronise cell cultures by halting the cell cycle at a particular phase. For example, serum starvation[26] and treatment with thymidine or aphidicolin[27] halt the cell in the G1 phase, mitotic shake-off, treatment with colchicine[28] and treatment with nocodazole[29] halt the cell in M phase and treatment with 5-fluorodeoxyuridine halts the cell in S phase.

References [1] Cooper GM (2000). "Chapter 14: The Eukaryotic Cell Cycle" (http:/ / www. ncbi. nlm. nih. gov/ books/ NBK9876/ ). The cell: a molecular approach (2nd ed.). Washington, D.C: ASM Press. ISBN 0-87893-106-6. . [2] Smith JA, Martin L (April 1973). "Do cells cycle?". Proc. Natl. Acad. Sci. U.S.A. 70 (4): 1263–7. doi:10.1073/pnas.70.4.1263. PMC 433472. PMID 4515625. [3] Wu RS, Bonner WM (December 1981). "Separation of basal histone synthesis from S-phase histone synthesis in dividing cells". Cell 27 (2 Pt 1): 321–30. doi:10.1016/0092-8674(81)90415-3. PMID 7199388. [4] Nelson DM, Ye X, Hall C, Santos H, Ma T, Kao GD, Yen TJ, Harper JW, Adams PD (November 2002). "Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity". Mol. Cell. Biol. 22 (21): 7459–72. doi:10.1128/MCB.22.21.7459-7472.2002. PMC 135676. PMID 12370293. [5] Cameron IL, Greulich RC (July 1963). "Evidence for an essentially constant duration of DNA synthesis in renewing epithelia of the adult mouse". J. Cell Biol. 18: 31–40. doi:10.1083/jcb.18.1.31. PMC 2106275. PMID 14018040. [6] Rubenstein, Irwin, and Susan M. Wick. "Cell." World Book Online Reference Center. 2008. 12 January 2008 [7] De Souza CP, Osmani SA (2007). "Mitosis, not just open or closed". Eukaryotic Cell 6 (9): 1521–7. doi:10.1128/EC.00178-07. PMC 2043359. PMID 17660363. [8] Maton, Anthea; Hopkins, Jean Johnson, Susan LaHart, David, Quon Warner, David, Wright, Jill D (1997). Cells: Building Blocks of Life. New Jersey: Prentice Hall. pp. 70–4. ISBN 0-13423476-6. [9] Lilly M, Duronio R (2005). "New insights into cell cycle control from the Drosophila endocycle". Oncogene 24 (17): 2765–75. doi:10.1038/sj.onc.1208610. PMID 15838513. [10] Nigg EA (June 1995). "Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle". Bioessays 17 (6): 471–80. doi:10.1002/bies.950170603. PMID 7575488. [11] "Press release" (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 2001/ press. html). Nobelprize.org. . [12] Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B (December 1998). "Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization". Mol. Biol. Cell 9 (12): 3273–97. PMC 25624. PMID 9843569. [13] Robbins and Cotran; Kumar, Abbas, Fausto (2004). Pathological Basis of Disease. Elsevier. ISBN 81-8147-528-3. [14] Norbury C (1995). "Cdc2 protein kinase (vertebrates)". In Hardie, D. Grahame; Hanks, Steven. Protein kinase factsBook. Boston: Academic Press. pp. 184. ISBN 0-12-324719-5. [15] Presentation on CDC25 PHOSPHATASES: A Potential Target for Novel Anticancer Agents (http:/ / pharmaxchange. info/ presentations/ cdc25. html) [16] Pramila T, Wu W, Miles S, Breeden L (August 2006). "The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle". Genes Dev 20 (20): 2266–227. doi:10.1101/gad.1450606. PMC 1553209. PMID 16912276. [17] Orlando DA, Lin CY, Bernard A, Wang JY, Socolar JES, Iversen ES, Hartemink AJ, Haase SB (June 2008). "Global control of cell-cycle transcription by coupled CDK and network oscillators" (http:/ / www. nature. com/ nature/ journal/ v453/ n7197/ full/ nature06955. html). Nature 453 (453): 944–947. doi:10.1038/nature06955. . [18] de Lichtenberg U, Jensen LJ, Fausbøll A, Jensen TS, Bork P, Brunak S (April 2005). "Comparison of computational methods for the identification of cell cycle-regulated genes" (http:/ / bioinformatics. oxfordjournals. org/ content/ 21/ 7/ 1164. long). Bioinformatics 21 (7): 1164–1171. doi:10.1093/bioinformatics/bti093. PMID 15513999. . [19] White MA, Riles L, Cohen BA (February 2009). "A systematic screen for transcriptional regulators of the yeast cell cycle" (http:/ / www. genetics. org/ cgi/ reprint/ 181/ 2/ 435). Genetics 181 (2): 435–46. doi:10.1534/genetics.108.098145. PMC 2644938. PMID 19033152. . [20] Lee T, et. al (October 2002). "Transcriptional Regulatory Networks in Saccharomyces cerevisiae". Science 298 (5594): 799–804. doi:10.1126/science.1075090. PMID 12399584. [21] Simon I, et. al (September 2001). "Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle" (http:/ / www. sciencedirect. com/ science/ article/ B6WSN-442RRFR-7/ 2/ 47b2bb3b8e41c7ca6592599402cee9b5). Cell 106 (6): 697–708. doi:10.1016/S0092-8674(01)00494-9. PMID 11572776. . [22] Sidorova JM, Mikesell GE, Breeden LL (December 1995). "Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localization" (http:/ / www. nature. com/ nature/ journal/ v425/ n6960/ abs/ nature02062. html). Mol Biol Cell. 6 (12): 1641–1658. doi:10.1038/nature02062. PMC 301322. PMID 8590795. .

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Cell cycle [23] Ubersax J, et. al (October 2003). "Targets of the cyclin-dependent kinase Cdk1". Nature 425 (6960): 859–864. Bibcode 2003Natur.425..859U. doi:10.1038/nature02062. PMC 301322. PMID 14574415. [24] Morgan DO (2007). "2-3". The Cell Cycle: Principles of Control. London: New Science Press. pp. 18. ISBN 0=9539181-2-2. [25] Stephen J. Elledge (6 December 1996). "Cell Cycle Checkpoints: Preventing an Identity Crisis" (http:/ / www. sciencemag. org/ cgi/ content/ abstract/ 274/ 5293/ 1664). Science 274 (5293): 1664–1672. doi:10.1126/science.274.5293.1664. PMID 8939848. . [26] Kues WA, Anger M, Carnwath JW, Paul D, Motlik J, Niemann H (February 2000). "Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors". Biol. Reprod. 62 (2): 412–9. doi:10.1095/biolreprod62.2.412. PMID 10642581. [27] Pedrali-Noy G, Spadari S, Miller-Faurès A, Miller AO, Kruppa J, Koch G (January 1980). "Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin". Nucleic Acids Res. 8 (2): 377–87. doi:10.1093/nar/8.2.377. PMC 327273. PMID 6775308. [28] Prather RS, Boquest AC, Day BN (1999). "Cell cycle analysis of cultured porcine mammary cells". Cloning 1 (1): 17–24. doi:10.1089/15204559950020067. PMID 16218827. [29] Samaké S, Smith LC (October 1997). "Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of aphidicolin". Theriogenology 48 (6): 969–76. doi:10.1016/S0093-691X(97)00323-3. PMID 16728186.

Further reading • Morgan DO (2007). The Cell Cycle: Principles of Control. London: Published by New Science Press in association with Oxford University Press. ISBN 0-87893-508-8. • Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2008). "Chapter 17". Molecular Biology of the Cell (5th ed.). New York: Garland Science. ISBN 978-0-8153-4111-6. • Krieger M, Scott MP; Matsudaira PT, Lodish HF, Darnell JE, Zipursky L, Kaiser C; Berk A (2004). Molecular cell biology. New York: W.H. Freeman and CO. ISBN 0-7167-4366-3. • Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R (2004). "Chapter 7". Molecular biology of the gene (5th ed.). San Francisco: Pearson/Benjamin Cummings. ISBN 0-8053-4642-2.

External links •

 This article incorporates public domain material from the NCBI document "Science Primer" (http://www. ncbi.nlm.nih.gov/About/primer/index.html).

• • • •

Transcriptional program of the cell cycle: high-resolution timing (http://www.cellcycle.info) Cell cycle and metabolic cycle regulated transcription in yeast (http://www.sceptrans.org) Cell Cycle Animation (http://www.1lec.com/Genetics/Cell Cycle/index.html) 1Lec.com Cell Cycle and Cytokinesis - The Virtual Library of Biochemistry and Cell Biology (http://www.biochemweb. org/cell_cycle.shtml) Cell Cycle (http://www.landesbioscience.com/journals/cc/index.php) Cell Cycle Portal (http://www.cellcycles.org) Fucci:Using GFP to visualize the cell-cycle (http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses19. html) Science Creative Quarterly's overview of the cell cycle (http://www.scq.ubc.ca/?p=248) Cells alive (http://www.cellsalive.com) CCO (http://www.cellcycleontology.org) The Cell-Cycle Ontology KEGG - Human Cell Cycle (http://www.genome.ad.jp/kegg/pathway/hsa/hsa04110.html) Cell cycle modeling (http://mpf.biol.vt.edu/Research.html) Drosophila Cell Cycle Genes - The Interactive Fly (http://www.sdbonline.org/fly/aignfam/cellcycl.htm)

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9

Complete blood count

10

Complete blood count A complete blood count (CBC), also known as full blood count (FBC) or full blood exam (FBE) or blood panel, is a test panel requested by a doctor or other medical professional that gives information about the cells in a patient's blood. A scientist or lab technician performs the requested testing and provides the requesting medical professional with the results of the CBC. Alexander Vastem is widely regarded as being the first person to use the complete blood count for clinical purposes. Reference ranges used today stem from his clinical trials in the early 1960s.

Schematics (also sometimes called "Fishbones") of shorthand for complete blood count commonly used by clinicians and healthcare providers. The shorthand on the right is used more often in the US. Hgb=Hemoglobin, WBC=White blood cells, Plt=Platelets, Hct=Hematocrit.

The cells that circulate in the bloodstream are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). Abnormally high or low counts may indicate the presence of many forms of disease, and hence blood counts are amongst the most commonly performed blood tests in medicine, as they can provide an overview of a patient's general health status. A CBC is routinely performed during annual physical examinations in some jurisdictions.

Methods Samples A phlebotomist collects the specimen, in this case blood is drawn in a test tube containing an anticoagulant (EDTA, sometimes citrate) to stop it from clotting, and transported to a laboratory. In the past, counting the cells in a patient's blood was performed manually, by viewing a slide prepared with a sample of the patient's blood under a microscope (a blood film, or peripheral smear). Nowadays, this process is generally automated by use of an automated analyzer, with only approximately 30% samples now being examined manually.

Complete blood count

Automated blood count The blood is well mixed (though not shaken) and placed on a rack in the analyzer. This instrument has many different components to analyze different elements in the blood. The cell counting component counts the numbers and types of different cells within the blood. The results are printed out or sent to a computer for review. Blood counting machines aspirate a very small amount of the specimen through narrow tubing. Within this tubing, there are sensors that count the number of cells going through it, and can identify the type of cell; this is flow cytometry. The two main Complete blood count performed by an automated sensors used are light detectors, and electrical impedance. One analyser. Differentials missing. way the instrument can tell what type of blood cell is present is by size. Other instruments measure different characteristics of the cells to categorize them. Because an automated cell counter samples and counts so many cells, the results are very precise. However, certain abnormal cells in the blood may be identified incorrectly, and require manual review of the instrument's results and identifying any abnormal cells the instrument could not categorize. In addition to counting, measuring and analyzing red blood cells, white blood cells and platelets, automated hematology analyzers also measure the amount of hemoglobin in the blood and within each red blood cell. This information can be very helpful to a physician who, for example, is trying to identify the cause of a patient's anemia. If the red cells are smaller or larger than normal, or if there's a lot of variation in the size of the red cells, this data can help guide the direction of further testing and expedite the diagnostic process so patients can get the treatment they need quickly.

Manual blood count Counting chambers that hold a specified volume of diluted blood (as there are far too many cells if it is not diluted) are used to calculate the number of red and white cells per litre of blood. To identify the numbers of different white cells, a blood film is made, and a large number of white cells (at least 100) are counted. This gives the percentage of cells that are of each type. By multiplying the percentage with the total number of white blood cells, the absolute number of each type of white cell can be obtained. The advantage of manual counting is that automated analysers are not reliable at counting abnormal cells. That is, cells that are not present in normal patients and are only seen in the peripheral blood with certain haematological conditions. Manual counting is subject to sampling error because so few cells are counted compared with automated analysis. Medical technicians examine blood film via a microscope for 30% of CBCs, not only to find abnormal white cells, but also because variation in the shape of red cells is an important diagnostic tool. Although automated analysers give fast, reliable results regarding how many red cells, the average size of the red cell, and the variation in size of the red cells, they don't detect cells' shapes. Also, some normal patients' platelets will clump in EDTA anticoagulated blood, which causes automatic analysers to give a falsely low platelet count. The technician viewing the slide in these cases will see clumps of platelets and can estimate if there are low, normal, or high numbers of platelets.

11

Complete blood count

12

Results For examples of standard values, see Reference ranges for blood tests#Hematology. A complete blood count will normally include:

Red cells • Total red blood cells — The number of red cells is given as an absolute number per litre. • Hemoglobin - The amount of hemoglobin in the blood, expressed in grams per decilitre. (Low hemoglobin is called anemia.) • Hematocrit or packed cell volume (PCV) - This is the fraction of whole blood volume that consists of red blood cells. • Red blood cell indices • Mean corpuscular volume (MCV) - the average volume of the red cells, measured in femtolitres. Anemia is classified as microcytic or macrocytic based on whether this value is above or below the expected normal range. Other conditions that can affect MCV include thalassemia, reticulocytosis and alcoholism. • Mean corpuscular hemoglobin (MCH) - the average amount of hemoglobin per red blood cell, in picograms.

A scanning electron microscope (SEM) image of normal circulating human blood. One can see red blood cells, several knobby white blood cells including lymphocytes, a monocyte, a neutrophil, and many small disc-shaped platelets.

• Mean corpuscular hemoglobin concentration (MCHC) - the average concentration of hemoglobin in the cells. • Red blood cell distribution width (RDW) - a measure of the variation of the RBC population

White cells • Total white blood cells - All the white cell types are given as a percentage and as an absolute number per litre. A complete blood count with differential will also include: • Neutrophil granulocytes — May indicate bacterial infection. May also be raised in acute viral infections. Because of the segmented appearance of the nucleus, neutrophils are sometimes referred to as "segs." The nucleus of less mature neutrophils is not segmented, but has a band or rod-like shape. Less mature neutrophils — those that have recently been released from the bone marrow into the bloodstream — are known as "bands" or "stabs". Stab is a German term for rod.[1] • Lymphocytes — Higher with some viral infections such as glandular fever and. Also raised in chronic lymphocytic leukemia (CLL). Can be decreased by HIV infection. In adults, lymphocytes are the second most common WBC type after neutrophils. In young children under age 8, lymphocytes are more common than neutrophils.[1] • Monocytes — May be raised in bacterial infection, tuberculosis, malaria, Rocky Mountain spotted fever, monocytic leukemia, chronic ulcerative colitis and regional enteritis [1] • Eosinophil granulocytes — Increased in parasitic infections, asthma, or allergic reaction. • Basophil granulocytes- May be increased in bone marrow related conditions such as leukemia or lymphoma. [1] A manual count will also give information about other cells that are not normally present in peripheral blood, but may be released in certain disease processes.

Complete blood count

13

Platelets • Platelet numbers are given, as well as information about their size and the range of sizes in the blood. • Mean platelet volume (MPV) - a measurement of the average size of platelets.

Interpretation Certain disease states are defined by an absolute increase or decrease in the number of a particular type of cell in the bloodstream. For example: Type of Cell Red Blood Cells (RBC)

Increase

Decrease

erythrocytosis or polycythemia anemia or erythroblastopenia

White Blood Cells (WBC): leukocytosis

leukopenia

-- lymphocytes

-- lymphocytosis

-- lymphocytopenia

-- granulocytes:

-- granulocytosis

-- granulocytopenia or agranulocytosis

-- --neutrophils

-- --neutrophilia

-- --neutropenia

-- --eosinophils

-- --eosinophilia

-- --eosinopenia

-- --basophils

-- --basophilia

-- --basopenia

Platelets

thrombocytosis

thrombocytopenia

All cell lines

-

pancytopenia

Many disease states are heralded by changes in the blood count: • leukocytosis can be a sign of infection. • thrombocytopenia can result from drug toxicity. • pancytopenia is generally as the result of decreased production from the bone marrow, and is a common complication of cancer chemotherapy.

References [1] http:/ / www. rnceus. com/ cbc/ cbcdiff. html

External links • Blood Groups and Red Cell Antigens. (http://www.ncbi.nlm.nih.gov/books/NBK2261) Free online book at NCBI Bookshelf ID: NBK2261 • Complete Blood Count (http://www.labtestsonline.org/understanding/analytes/cbc/test.html)

Reference ranges for blood tests

Reference ranges for blood tests Reference ranges for blood tests are sets of values used by a health professional to interpret a set of medical test results from blood samples. Reference ranges for blood tests are studied within the field of clinical chemistry (also known as "clinical biochemistry", "chemical pathology" or "pure blood chemistry"), the area of pathology that is generally concerned with analysis of bodily fluids.

Interpretation A reference range is usually defined as the set of values 95 percent of the normal population falls within (that is, 95% prediction interval).[1] It is determined by collecting data from vast numbers of laboratory tests.

Plasma or whole blood All values (except the exceptions below) denote blood plasma concentration, which is approximately 60-100% larger than the actual blood concentration if the amount inside red blood cells (RBCs) is negligible. The precise factor depends on hematocrit as well as amount inside RBCs. Exceptions are mainly those values that denote total blood concentration, and in this article they are: • All values in Hematology - red blood cells (except hemoglobin in plasma) • All values in Hematology - white blood cells • Platelet count (Plt) A few values are for inside red blood cells only: • Vitamin B9 (Folic acid/Folate) in red blood cells • Mean corpuscular hemoglobin concentration (MCHC)

Units • Mass concentration (g/dL or g/L) is the most common measurement unit in the United States. Is usually given with dL (decilitres) as the denominator in the United States, and usually with L (litres) in, for example, Sweden. • Molar concentration (mol/L) is used to a higher degree in most of the rest of the world, including the United Kingdom and other parts of Europe and Australia and New Zealand.[2] • International units (IU) are based on measured biological activity or effect, or for some substances, a specified equivalent mass. • Enzyme activity (kat) is commonly used for e.g. liver function tests like AST, ALT, LD and γ-GT in Sweden.[3]

Arterial or venous If not else specified, a reference range for a blood test is generally the venous range, as the standard process of obtaining a sample is by venipuncture. An exception is for acid-base and blood gases, which are generally given for arterial blood. Still, the blood values are approximately equal between the arterial and venous sides for most substances, with the exception of acid-base, blood gases and drugs (used in therapeutic drug monitoring (TDM) assays).[4] Arterial levels for drugs are generally higher than venous levels because of extraction while passing through tissues.[4]

14

Reference ranges for blood tests

Usual or optimal Reference ranges are usually given as what are the usual (or normal) values found in the population, more specifically the prediction interval that 95% of the population fall into. This may also be called standard range. In contrast, optimal (health) range or therapeutic target is a reference range or limit that is based on concentrations or levels that are associated with optimal health or minimal risk of related complications and diseases. For most substances presented, the optimal levels are the ones normally found in the population as well. More specifically, optimal levels are generally close to a central tendency of the values found in the population. However, usual and optimal levels may differ substantially, most notably among vitamins and blood lipids, so these tables give limits on both standard and optimal (or target) ranges. In addition, some values, including troponin I and brain natriuretic peptide, are given as the estimated appropriate cutoffs to distinguish healthy people from specific conditions, which here are myocardial infarction and congestive heart failure, respectively, for the aforementioned substances.

Inaccuracy References range may vary with age, sex, race, diet, use of prescribed or herbal drugs and stress. Standard reference ranges should theoretically not vary with the instruments and lab techniques used, but practically it may do so when inaccurate methods are used in establishing standard reference ranges. Finally, the test procedure itself may be erroneous or inaccurate.

Sorted by concentration A separate printable image is available for mass and molarity Smaller, narrower boxes indicate a more tight homeostatic regulation when measured as standard "usual" reference range.

By mass and molarity

Hormones predominate at the left part of the scale, shown with a red at ng/L or pmol/L, being in very low concentration. There appears to be the greatest cluster of substances in the yellow part (μg/L or nmol/L), becoming sparser in the green part (mg/L or μmol/L). However, there is another cluster containing many metabolic substances like cholesterol and glucose at the limit with the blue part (g/L or mmol/L). The unit conversions of substance concentrations from the molar to the mass concentration scale above are made as follows: • Numerically: molar concentration x molar mass = mass concentration • Measured directly in distance on the scales:

, where distance is the direct (not logarithmic) distance in number of decades or "octaves" to the right the mass concentration is found. To translate from mass to molar concentration, the dividend (molar mass and the divisor (1000) in the division change places, or, alternatively, distance to right is changed to distance to left. Substances with a molar mass around 1000g/mol (e.g. thyroxine) are almost vertically aligned in the mass and molar images. Adrenocorticotropic hormone, on the other hand, with a molar mass of 4540,[5] is 0.7 decades to the right in the mass

15

Reference ranges for blood tests image. Substances with molar mass below 1000g/mol (e.g. electrolytes and metabolites) would have "negative" distance, that is, masses deviating to the left. Many substances given in mass concentration are not given in molar amount because they haven't been added to the article. The diagram above can also be used as an alternative way to convert any substance concentration (not only the normal or optimal ones) from molar to mass units and vice versa for those substances appearing in both scales, by measuring how much they are horizontally displaced from one another (representing the molar mass for that substance), and using the same distance from the concentration to be converted to determine the equivalent concentration in terms of the other unit. For example, on a certain monitor, the horizontal distance between the upper limits for parathyroid hormone in pmol/L and pg/mL may be 7 cm, with the mass concentration to the right. A molar concentration of, for example, 5 pmol/L would therefore correspond to a mass concentration located 7 cm to the right in the mass diagram, that is, approximately 45 pg/mL.

By units Units don't necessarily tell anything about molarity or mass.

A few substances are below this main interval, e.g. thyroid stimulating hormone, being measured in mU/L, or above, like rheumatoid factor and CA19-9, being measured in U/mL.

16

Reference ranges for blood tests

17

By enzyme activity

White blood cells

Sorted by category Ions and trace metals Included here are also related binding proteins, like ferritin and transferrin for iron, and ceruloplasmin for copper. Test

Lower limit

Sodium (Na)

135,

[6]

[7] [3]

[7] [3]

137

[8]

145,

[8]

310, Potassium (K)

Upper limit

[8]

320

[6] [3]

330,

[7]

3.5,

mg/dl [6] See hypokalemia or hyperkalemia

5.1

mmol/L or mEq/L

[9]

[6]

95,

[10]

[3]

98,

100

[11]

mg/dl

[13]

4.1,

[10]

106,

[3]

[6]

110

mmol/L or mEq/L

[11]

[3]

1.10

[13]

4.4

[6] [13]

2.1,

[6]

[6]

105,

mg/dl

370

[12]

1.03,

8.4,

[8]

340

20

340

Total calcium (Ca)

[6]

mmol/L or mEq/L

5.0,

[9]

Ionized calcium (Ca)

[6]

[14]

8.5

[3]

2.2

[12]

1.23,

[13]

4.9,

[6]

10.2,

[3]

mmol/L

1.30

[13]

mg/dL

5.2

[13] [3]

2.5,

Comments

147

[6] [7] [3]

3.6

14 Chloride (Cl)

Unit

[13]

2.6,

[14]

10.5

[6] mmol/L

2.8

mg/dL

Reference ranges for blood tests

Total serum iron (TSI) - male

18 [15]

[7]

65,

[15]

76

[16] [17]

11.6, Total serum iron (TSI) - female

[7]

26,

[15]

[17]

Total serum iron (TSI) - newborns

[17]

13.6

50

8.9

[15]

100

[17]

[18]

190,

[3]

194,

[15]

[17]

81, [7]

204

[3]

326,

[20] [17] [21]

[22]

10,

20

[23]

[23]

34

[14] [24]

μmol/L

[15]

%

[20]

ng/mL

[17]

pmol/L

[20]

ng/mL

[17]

pmol/L

[21]

35,

[23]

60,

[22]

65

[23]

110

[14]

μmol/L

[14]

mg/dL

[25]

1

4

Phosphate (HPO42−)

0.8

1.5

Inorganic phosphorus (serum)

1.0

[6]

μmol/L

[26]

mmol/L

[6]

mmol/L

4.5

[6]

mg/dL

24

μmol/L

1.5

[6]

3.0

[27] [28]

[29]

[29]

72

9.2,

[14]

1.5,

[30]

[3]

11

[30]

1.7

[3]

0.7

μg/dL

[24]

60

[25]

μmol/L

µg/dL

24

15

mg/dL

[19]

150

11

[7]

360

[14]

70

0.6,

[18]

330,

330

17,

Magnesium

µmol/L

150

27

60,

[17]

85

670

12

Zinc (Zn)

μg/dL

300

[17]

11

[7]

474

50

[20]

Copper (Cu)

µmol/L

45

27

Ceruloplasmin

µg/dL

[15]

[17]

[19]

Copper

µmol/L

450,

47

25

Ammonia

µg/dL

[17]

43,

Ferritin - Female

[15]

21

Total iron-binding capacity (TIBC) 240,[15] 262[7]

12

μmol/L

[15]

[17]

Ferritin - Male

[16]

120

9

20

µg/dL

[17]

[15]

μmol/L

[7] [15]

45

50

Transferrin saturation

[17]

35

250

[17]

Transferrin

[17]

32,

30.4

18 Total serum iron (TSI) - children

[16]

30,

µg/dL

198

170

[16]

4.6,

[7]

176,

[29]

[28]

130

μg/dL

[29]

µmol/L

110,

[3]

17,

20

[14]

2.0,

[30]

0.82,

[30]

2.3

[3]

0.95

mEq/L or mg/dL mmol/L

Reference ranges for blood tests

19

Acid-base and blood gases If arterial/venous is not specified for a acid-base or blood gas value, then it generally refers to arterial, and not venous which otherwise is standard for other blood tests. Acid-base and blood gases are among the few blood constituents that exhibit substantial difference between arterial and venous values.[4] Still, pH, bicarbonate and base excess show a high level of inter-method reliability between arterial and venous tests, so arterial and venous values are roughly equivalent for these.[31] Test

Arterial/Venous

Lower limit

pH

Arterial

7.34,

Venous

7.31

Arterial

36

[H+]

[7]

Upper limit [6]

[7]

7.35

[32]

[33]

[32]

oxygen pressure (pO2)

Arterial

10,

[6]

[34] [14]

[34]

[32]

[10]

Approximately 75

Arterial

4.4,

96

mmHg or torr kPa

[32]

mmHg or torr

[10] [14]

%

[6]

kPa

100

[10]

[6]

[34]

4.7

[6]

[7]

35

[32]

23

[35]

100

[34]

5.5

[32]

41

[14]

18

[36]

110

[6]

21, 22

[36]

134

Liver function

[14]

Venous

95,

kPa

[34]

40

94,

Standard bicarbonate (SBCe) Arterial & venous

[6]

105

5.3

Arterial

Arterial & venous

[6]

14

100,

4.0

33,

Bicarbonate (HCO3, )

mEq/L

[7]

83

[32]

Venous

[32]

13,

30

Carbon dioxide (CO2)

ng/dL

[34]

11

[6] [7]

Oxygen saturation

[33]

+3

75, Venous

nmol/L

4.4

[32]

-3

[6]

44

3.6 Arterial & venous

7.45

7.41

[6]

Base excess

Comments

[6]

7.44,

[32]

Unit

[34]

5.9,

[6]

6.0

[7]

44,

45

[32]

30

mmHg or torr mmol/L

[35]

mg/dL

[34]

kPa

[32]

mmHg or torr

[14]

mmol/L

132 6.8 51 23

[36]

140

[6]

27, 28

[36]

170

Designated pCO2

mg/dL [6]

mmol/L or mEq/L mg/dL

Reference ranges for blood tests

Test

Patient type

20

Lower limit [6]

Total Protein

60,

Albumin

35

Upper limit

[7]

[6]

63

78,

[6] [37]

[7]

48,

[7]

[7]

82,

[6]

g/L

[6]

4.8,

[38]

[14] g/L

84

55

[7]

3.5

Unit

[6]

Globulins

23

35

g/L

Total Bilirubin

[39] [6] 1.7, 2, [39] [3] 3.4, 5

[6] [39] [39] 17, 22, [3] 25

μmol/L

[6] [7] 0.1, 0.2, [39] 0.29

[6] [14] [7] 1.0, 1.3, [39] 1.4

mg/dL

[6]

Direct/Conjugated Bilirubin

[3]

0.0

or N/A

[6] [7]

Alanine transaminase [3] (ALT/ALAT )

Aspartate transaminase [3] (AST/ASAT )

[40]

5,

[7] [6] 8

7,

0.15

Male

0.15

Female

6

[3]

[41] [3]

0.25 Male

[41]

8

[3]

0.25 Alkaline phosphatase (ALP)

42

Male

53

(Enzyme activity)

0.6

[40] [3]

[40] [7] 8

Gamma glutamyl transferase (GGT)

Cardiac tests

[40]

Female

5,

μmol/L

[14]

0.3,

[3]

Female

[6] [39] [3] 7

5,

[6] [7]

0

[6]

20,

see hypoalbuminemia

μmol/L

740

[6]

see hypoproteinemia

U/L

5.5

[38]

540

Comments

0.4

[10]

21,

[3]

mg/dL

[7] U/L

56

Also called serum glutamic pyruvic transaminase (SGPT)

µkat/L

0.75

[3]

1.1

[41]

IU/L

34

[3]

µkat/L

0.60

[41]

IU/L

40

[3]

µkat/L

0.75

[40]

U/L

98

[40]

128

[3]

µkat/L

1.8

[40]

40,

[42]

Women

0.8

Men

1.3

[42]

[7]

78

U/L µkat/L

Also called serum glutamic oxaloacetic transaminase (SGOT)

Reference ranges for blood tests

21

Test

Patient type

Lower limit

[43]

Creatine kinase (CK) male

24,

[7]

38,

[40]

60

[44]

[14]

Upper limit [40]

174,

U/L or ng/mL

320

[44]

0.42

µkat/L

1.5

female 24,[43] 38,[7] 96[14] 140,[14] 200[40] [44]

CK-MB

1.17

0

3,

[7]

Female 1[45]

Myoglobin

µkat/L [3] [40] [3] 5 ng/mL or μg/L

3.8,

[45]

ng/mL or µg/L

66

[45]

Male

U/L or ng/mL

[44]

0.17

Unit Comments

[45]

17

106

Cutoffs and ranges for troponin types, 12 hrs after onset of pain Test

Lower limit

Troponin-I

Upper limit Unit [46]

ng/mL or μg/L Upper limit of normal

[46]

ng/mL or μg/L Acute Coronary Syndrome

[47]

ng/mL or μg/L Moderately increased[47]

0.2 [46]

0.2

1.0

[47]

0.4

[46]

2.0

1.0,

[48]

1.5

Troponin-T

[46] [48] ng/mL or μg/L Myocardial Infarction likely

n/a

[46]

ng/mL or μg/L Upper limit of normal

[46]

ng/mL or μg/L Acute Coronary Syndrome

0.02 [46]

0.02

0.10

[46]

[46]

0.10

Comments

n/a

ng/mL or μg/L Myocardial Infarction likely

Brain natriuretic peptide (BNP) Interpretation

Range / Cutoff

Congestive heart failure unlikely < 100 pg/mL[49] [50] [49] [50]

"Gray zone"

100-500 pg/mL

Congestive heart failure likely

>500 pg/mL

[49] [50]

NT-proBNP Interpretation

Age

Cutoff

Congestive heart failure likely < 75years > 125 pg/mL[44] >75 years >450pg/mL[44]

Reference ranges for blood tests

22

Lipids Test

Patient type

Lower limit

Triglycerides

10 – 39 years 54[14]

Upper limit

[52]

[52]

[14]

80

[53]

[6] [53]

[7]

[6]

[55]

[53]

1.3

[57]

50

[52]

mmol/L [6]

mmol/L

< 3.9

[6]

mg/dL

< 150

[55]

mmol/L

[53]

mg/dL

[55] [53] > 1.0 or 1.6  mmol/L [53] [56] > 40 or 60 mg/dL

[55]

mmol/L

[53]

mg/dL

250

2.0

[53]

35

80

[55]

[54]

[54] [3]

2.4

3.0,

[53]

[53]

94

[51]

6.5

86

[55] [3]

n/a

mg/dL

2.2

0.9

LDL/HDL quotient

[14]

[7]

[3]

[53]

[53]

mmol/L

200,

1.2,

40,

80,

[52]

5.0,

140

1.0,

2.0,

mg/dL

[3] [54]

3.6

120,

LDL cholesterol (Not valid when triglycerides >5.0 mmol/L)

[14]

1.7

3.0,

male

mmol/L

150

[52]

HDL cholesterol

[52]

[51] < 100 mg/dL [51] or 1.1 mmol/L

1.7

0.9

female

mg/dL

150

0.77

HDL cholesterol

[14]

1.2

40 – 59 years 70[14]

Total cholesterol

Therapeutic target

110

0.61

> 60 years

Unit

120,

[55] mmol/L

3.4

[53]

130

[3]

mg/dL

[51]

[55]

< 2.5

[53]

< 100

(unitless)

5

Tumour markers Test

Lower limit Upper limit

Alpha fetoprotein (AFP)

0

[7]

[7]

5

CA19-9

n/a

40

CA-125

n/a

30,

Carcinoembryonic antigen (CEA) non-smokers at 50 years

n/a

3.4,

Carcinoembryonic antigen (CEA) non-smokers at 70 years

n/a

4.1

Carcinoembryonic antigen (CEA) - smokers

n/a

5

Prostate specific antigen (PSA)

n/a

2.5,

PAP

0

3

IU/l or mU/ml

[7]

in male and non-pregnant female

U/ml

[58]

[59] kU/L or U/mL

35

[3]

[60] μg/l

3.6

[60]

[61] [3] [7] 4

[14]

Comments

ng/mL or µg/L

44

Beta Human chorionic gonadotrophin (bHCG) n/a

Unit

μg/l

μg/l [7] [3]

μg/L

[14]

or ng/mL

units/dL (Bodansky units)

below age 45 50 years

Unit ng/dL

85

230, Male < 50 years

Upper limit

[20]

300

[75]

[76]

27,

nmol/L

35

[76]

780

[20] ng/dL

- 1000

[3]

nmol/L

45

[76]

ng/dL

1300

[3]

nmol/L

26

[76]

ng/dL

740

[76]

2.8

[20]

80

[3]

nmol/L

[76]

ng/dL

- 3.0 - 85

[14]

mg/L

[77]

µmol/l

[14]

mg/L

[77]

µmol/l

3.0 9.1 1.0 3.0

Reference ranges for blood tests

Follicle-stimulating hormone (FSH)

25 [41]

[41]

Prepubertal

1year 3.0[103]

Vitamin B9 (Folic acid/Folate) - Red blood cells

Unit Upper limit

[14]

Vitamin A Vitamin B9 (Folic acid/Folate) - Serum

Standard range

[93]

40,

[95]

95,

[14]

80

[94]

150

[51]

[51]

[51]

[51] [96]

[51] [51]

[97]

40

[97]

65,

[96]

100

[51] 120, [97] 160

Reference ranges for blood tests

28

Test

Limit type

Limit

Lead

Optimal health range

< 20

Unit

[10]

[14]

µg/dL

or 40

Ethanol Limit for drunk driving 0,[110] 0.2,[110] 0.8[110] ‰ or g/L [111]

mmol/L

17.4

Hematology Red blood cells These values (except Hemoglobin in plasma) are for total blood and not only blood plasma. Test

Patient

Lower limit

Hemoglobin (Hb)

male

2.0,

[112]

Upper limit [6]

2.1

[3] [7] 130, 132, [6] 135 female

[112]

[6]

1.8,

1.9

[3] [6] [7]

120

[6]

Hemoglobin in plasma

Glycosylated hemoglobin (HbA1c)

Haptoglobin

Hematocrit (Hct)

Mean cell volume (MCV)

[6]

2.7

[7] [3] 162, 170, [6] 175 [112]

Comments

mmol/L

Higher in neonates, lower in children.

g/L

[6] [112] mmol/L

2.3,

2.5

[3] [7] 150, 152, [6] [14] 160 [6]

0.62

μmol/L

1

4

mg/dL

[3]

[3]

< 50 years

3.6

> 50 years

3.9

< 50 years

0.35

> 50 years

0.47

2.1

male

[3] [7] 0.39, 0.4, [6] [14] 0.41, 0.45

[3] [7] 0.50, 0.52, [6] [14] 0.53, 0.62

female

[3] [6] 0.35, 0.36, [7] [14] 0.37

[6] [7] [3] 0.46, [14] 0.48

Child

0.31

Male

76,

Female

78

[3]

5.3

[3]

[3] [3]

[7]

[14]

[7]

0.43 [7]

82

[7]

0.39

[6] [7]

31,

fL

[7]

fL

[7]

%

[6]

fmol/cell

0.54 [14] [3]

27

[14] [3]

32

[113]

4.8,

[7]

102

14.5

[6]

25,

[14]

100, 101

[7]

Mean cell hemoglobin (MCH)

g/L

1.9

[3]

11.5

Normally diminutive compared with inside red blood cells

% of Hb

5.0

[3]

Sex difference negligible until adulthood.

g/L

0.16

Red blood cell distribution width (RDW)

Mean corpuscular hemoglobin concentration (MCHC)

[112]

2.5,

Unit

[113]

5.0

[14]

32,

[7]

35,

[3]

33,

[14] [3]

36

[113]

5.4,

[6]

35

[113]

5.6

pg/cell g/dL mmol/L

Cells are larger in neonates, though smaller in other children.

Reference ranges for blood tests

29 [14]

Erythrocytes/Red blood cells (RBC) male

4.2, [3]

Female

[6]

3.5,

[6] [7]

[3] [6] [7] 5.7, 5.9, 6.2, [14] 6.9

4.3

[7]

3.8,

[3]

3.9

Infant/Child 3.8[7]

[7]

26

0.5

Newborn

1.1

Infant

0.5

[3]

x109/L

[6] [7]

% of RBC

[7]

% of RBC

[7]

% of RBC

130

[6] [7]

Adult

[6] [7]

5.5

5.5

[3]

Reticulocytes

[3]

5.1,

x1012/L or mln/mm3

1.5

[7]

4.5

[7]

3.1

White blood cells These values are for total blood and not only blood plasma. Test

Patient type Lower limit

White Blood Cell Count (WBC.)

Adult

3.5,

Newborn

9

1 year old

6

Neutrophil granulocytes Adult (A.K.A. grans, polys, PMNs, or segs)

[3]

[114]

3.9,

Upper limit [7]

4.1,

[115]

[3]

[114] [115] 2

1.8,

[115]

6

74

% of WBC

[115]

x109/L

[115]

x109/L

[6]

[114] [115]

1.0

[6]

2

Adult

0.1,

[3]

[6]

3,

[114]

[3]

3.5,

[6]

33,

[115]

Newborn

% of WBC

5

[3]

x109/L x103/mm3 or x103/μL

x109/L

26

16-25

Mononuclear leukocytes (Lymphocytes + monocytes)

[114] [115] 8

0.7

0.7,

• • •

7,

[6]

[6]

Monocytes

[3]

5.4, 62,

Adult

Adult

[6]

11

[115]

3 Lymphocytes

[7]

10.9,

18

[6]

Neutrophilic band forms

[114]

10.0,

[115]

45-54 Newborn

[3]

9.0, 30

[115]

1.3,

[6]

4.5

Unit

3.9,

[115]

4.8

% of WBC

45

[115]

x109/L

[41] [115] [3]

x109/L

11 [116] [41]

0.2

0.8

[6]

4.0

7,

[115]

x109/L

% of WBC

10

[115]

Newborn

0.4

3.1

x109/L

Adult

1.5

5

x109/L

20

35

% of WBC

[7]

CD4+ cells

Adult

0.4,

Eosinophil granulocytes

Adult

0.0,

[3]

[10]

0.5

[6]

[41]

3, [115]

0.02

[7] [115]

7

0.45,

[115]

0.85

x109/L

1.8

0.44, [6]

1 Newborn

[41]

0.04

[10]

1.5,

[3]

0.5

x109/L % of WBC x109/L

Reference ranges for blood tests

30

Basophil granulocytes

[114]

Adult

[41] [3]

40

100,

0.0

0.75,

[6]

[115]

200,

2

[115]

Newborn

0.64

[114]

900

x106/L % of WBC x109/L

Coagulation Test

Lower limit

Thrombocyte/Platelet count (Plt)

140,

Mean platelet volume (MPV)

7.4

10.4

fL

Prothrombin time (PT)

[10] [6] [118] 10, 11, [7] 12

[10] [118] 13, 13.5, [7] [6] 14, 15

s

INR

0.9

Activated partial thromboplastin time (APTT)

18,

Thrombin clotting time (TCT)

11

Fibrinogen

1.7,

Antithrombin

0.80

1.2

kIU/L

Bleeding time

2

9

minutes

Viscosity

1.5

[7]

[6] [3]

150

[117]

Upper limit

Unit

[14] [3] [6] 350, 400, [7] 450

x109/L

[117]

[3]

[3]

[10] [3]

[7]

30

28,

[3]

42,

[10]

45

18 [7]

[3]

3.6,

[3]

[7]

g/L

4.2

[3]

[119]

s

s [3]

2.0

PT reference varies between laboratory kits INR is standardised The INR is a corrected ratio of a patient's PT to normal

1.2

[7]

Comments

[119]

cP

1.72

Immunology Acute phase proteins Acute phase proteins are markers of inflammation. Test

Patient Lower limit

Upper limit [120]

Unit

Comments

mm/hr

ESR increases with age and tends to be higher in [121] females.

Erythrocyte sedimentation rate Male 0 (ESR) Female

Age÷2

C-reactive protein (CRP)

5,

mg/L

[124] 200, [124] 240

nmol/L

Alpha 1-antitrypsin (AAT)

[120]

(Age+10)÷2

[122] [3] [123] 6

n/a

[125] 20, [126] 22 [126]

89,

[126]

38,

[3]

97

[3]

170,

[125]

μmol/L

[126]

mg/dL

53

230

Reference ranges for blood tests

31

Isotypes of antibodies Test Patient Lower limit IgA

Adult

[3]

70,

[127]

IgD

0.5

IgE

0.01

IgG

800

IgM

54

[127]

110

[127]

[127]

[127]

Upper limit [3]

360,

Unit

Comments

[127] mg/dL

560

[127]

3.0

[127]

0.04

[127]

1800

[127]

220

Autoantibodies Autoantibodies are usually absent or very low, so instead of being given in standard reference ranges, the values usually denote where they are said to be present, or whether the test is a positive test. There may also be an equivocal interval, where it is uncertain whether there is a significantly increased level. All included values[128] are given for the ELISA test. Test

Negative

anti-SS-A (Ro)

< 15

Unit

Anti ds-DNA Anti ss-DNA

25

[129]

> 10

[129]

> 25

[129]

21 - 30

> 30

n/a

>5

[129]

Anti-mitochondrial antibodies (AMA) < 10[129] n/a[129]

> 10

Rheumatoid factor (RF)

> 30

< 20

20 - 30

[129] [7]

Antistreptolysin O titre (ASOT) in preschoolers

> 100

ASOT at school age

> 250

ASOT in adults

> 125

[7] [7]

Reference ranges for blood tests

32

Test

Negative

Anti-phospholipid IgG

< 20

Anti-phospholipid IgM

< 1.5

Anti-phospholipid IgA

< 10

[129] [129]

[129]

Anti-citrullinated protein antibodies < 20[129]

Low/weak positive Moderate positive High/strong positive Unit [129]

[129]

20 –30

31 – 50

[129]

[129]

1.5 –2.5

2 – 9.9

[129]

[129]

10 -20

21 – 30

[129]

[129]

20 – 39

40 - 59

[129]

> 51

[129]

> 10

[129]

> 31

[129]

> 60

[129]

GPLU/ml

[129]

MPL /ml

[129]

arb U/ml [129]

EU

Other enzymes and proteins Test

Lower limit

Lactate dehydrogenase (LDH)

50

[14]

Upper limit

Unit

[14]

U/L

[40]

μmol/L

[3]

150

[40]

0.4

1.7

[3]

LDH (enzyme activity)

1.8

3.4

µkat/L

Amylase

[6] [7] 25, 30, [14] 53

[7] [130] [14] [6] 110, 120, 123, 125, [40] 190

U/L

[3]

0.15

[124]

D-dimer

[3]

µkat/L

[124]

nmol/L

[131]

ng/mL

[3]

mg/L

1.1

200

240

n/a

500 0.5

Lipase

[7] [14] 7, 10, [40] 23

Angiotensin-converting enzyme (ACE)

23

[40]

[7]

60,

[14]

150,

[40]

[40]

U/L

[40]

ng/mL

[3]

µg/L

3.0

Eosinophil cationic protein (ECP)

[3]

2.3

[3]

< 70 years old

Higher in pregnant [132] women

U/L

208

57

Acid phosphatase

Comments

16

Other electrolytes and metabolites Electrolytes and Metabolites: For iron and copper, some related proteins are also included. Test

Patient type

Lower limit

Upper limit

Unit

Osmolality

[6] [14] 275, 280, [3] 281

[6] [14] 295, 296, [3] 297

mOsm/kg Plasma weight excludes solutes

Osmolarity

Slightly less than osmolality

Urea

1.2,

[6]

[6]

7

[133]

3.0

[6]

3.0,

[6]

18,

[133]

7.0

[7]

21

Comments

mOsm/l

Plasma volume includes solutes

mmol/L

BUN - blood urea nitrogen

mg/dL

Reference ranges for blood tests [7]

[6]

* Uric acid

Creatinine

33

0.18

[14]

Female

2.0

Male

2.1

male

60,

[134]

[135]

[3]

[135]

[134]

3.8,

[7]

Full blood glucose (fasting)

[135]

[3]

[3]

3.3

[14]

4.5

[138] [14]

4.5

[]

0.5

[14]

300

[139]

34

[6]

70,

0.5

Pyruvate

mg/dL

[134]

μmol/L

98

[135]

1.0,

[3]

4.0

[137]

Lactate (Arterial)

0.8

[135]

6.0, [137]

72

μmol/L

1.3

[135]

1.1

[14]

60 Lactate (Venous)

[135]

1.0,

[136]

6.1

[136]

100,

mg/dL

[14]

110

mmol/L

mmol/L

[137]

mg/dL

[14]

mg/dL

[138]

mmol/L

[14]

mg/dL

[138]

mmol/L

[14]

μg/dL

[139]

μmol/L

100

19.8 2.2

14.4 1.6

900

See also glycosylated hemoglobin (in hematology)

mg/dL

[3]

5.6

102

May be complemented with creatinine clearance

-

35

[6]

65,

[134]

118

90,

[14]

Plasma glucose (fasting)

mg/dL

[3]

68

[135]

5

[14]

90,

0.8

0.6, BUN/Creatinine Ratio

mg/dL

[3]

68

50,

[14]

8.5

0.7, female

mmol/L

7.0

[14] [3]

[6]

0.48

References [1] Page 19 (http:/ / books. google. se/ books?id=Je_pJfb2r0cC& pg=PA19) in: Stephen K. Bangert MA MB BChir MSc MBA FRCPath; William J. Marshall MA MSc PhD MBBS FRCP FRCPath FRCPEdin FIBiol; Marshall, William Leonard (2008). Clinical biochemistry: metabolic and clinical aspects. Philadelphia: Churchill Livingstone/Elsevier. ISBN 0-443-10186-8. [2] Page 34: Units of measurement (http:/ / books. google. dk/ books?id=BfdighlyGiwC& printsec=frontcover& hl=en) in Medical toxicology By Richard C. Dart Edition: 3, illustrated Published by Lippincott Williams & Wilkins, 2004 ISBN 0-7817-2845-2, 9780781728454 1914 pages [3] Reference range list from Uppsala University Hospital ("Laborationslista"). Artnr 40284 Sj74a. Issued on April 22, 2008 [4] Arterial versus venous reference ranges - Brief Article (http:/ / findarticles. com/ p/ articles/ mi_m3230/ is_4_32/ ai_61893437/ ) Medical Laboratory Observer, April, 2000 by D. Robert Dufour [5] PROOPIOMELANOCORTIN; NCBI --> POMC (http:/ / www. uniprot. org/ uniprot/ P01189) Retrieved on September 28, 2009 [6] Last page of Deepak A. Rao; Le, Tao; Bhushan, Vikas (2007). First Aid for the USMLE Step 1 2008 (First Aid for the Usmle Step 1). McGraw-Hill Medical. ISBN 0-07-149868-0. [7] Normal Reference Range Table (http:/ / pathcuric1. swmed. edu/ PathDemo/ nrrt. htm) from The University of Texas Southwestern Medical Center at Dallas. Used in Interactive Case Study Companion to Pathologic basis of disease. [8] Derived from molar values using molar mass of 22.99 g•mol−1 [9] Derived from molar values using molar mass of 39.10 g•mol−1 [10] MERCK MANUALS > Common Medical Tests > Blood Tests (http:/ / www. merck. com/ mmhe/ appendixes/ ap2/ ap2b. html) Last full review/revision February 2003 [11] Derived from molar values using molar mass of 35.45 g•mol−1 [12] Larsson L, Ohman S (November 1978). "Serum ionized calcium and corrected total calcium in borderline hyperparathyroidism" (http:/ / www. clinchem. org/ cgi/ pmidlookup?view=long& pmid=709830). Clin. Chem. 24 (11): 1962–5. PMID 709830. . [13] Derived from molar values using molar mass of 40.08 g•mol−1 [14] Blood Test Results - Normal Ranges (http:/ / www. bloodbook. com/ ranges. html) Bloodbook.Com

Reference ranges for blood tests [15] Slon S (2006-09-22). "Serum Iron" (http:/ / uimc. discoveryhospital. com/ main. php?t=enc& id=1456). University of Illinois Medical Center. . Retrieved 2006-07-06. [16] Diagnostic Chemicals Limited > Serum Iron-SL Assay (http:/ / www. dclmexico. com/ ingles/ hierro_sl. pdf) July 15, 2005 [17] Derived from mass values using molar mass of 55.85 g•mol−1 [18] Table 1. (http:/ / www. clinchem. org/ cgi/ reprint/ 45/ 1/ 131. pdf) Page 133. Clinical Chemistry 45, No. 1, 1999 (stating 1.9–3.3 g/L) [19] Derived by dividing mass values with molar mass [20] Ferritin (http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 003490. htm) by: Mark Levin, MD, Hematologist and Oncologist, Newark, NJ. Review provided by VeriMed Healthcare Network [21] Mitchell ML, Filippone MD, Wozniak TF (August 2001). "Metastatic carcinomatous cirrhosis and hepatic hemosiderosis in a patient heterozygous for the H63D genotype" (http:/ / journals. allenpress. com/ jrnlserv/ ?request=get-abstract& issn=0003-9985& volume=125& page=1084). Arch. Pathol. Lab. Med. 125 (8): 1084–7. PMID 11473464. . [22] Diaz J, Tornel PL, Martinez P (July 1995). "Reference intervals for blood ammonia in healthy subjects, determined by microdiffusion". Clin. Chem. 41 (7): 1048. PMID 7600690. [23] Derived from molar values using molar mass of 17.03 g/mol [24] Derived from mass values using molar mass of 63.55 g•mol−1 [25] Derived from mass using molar mass of 151kDa [26] Walter F., PhD. Boron (2005). Medical Physiology: A Cellular And Molecular Approaoch. Elsevier/Saunders. ISBN 1-4160-2328-3. Page 849 [27] Reference range for copper (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=1040580630) at GPnotebook [28] http:/ / www. dlolab. com/ PDFs/ DLO-OCTOBER-2008-LAB-UPDATE. pdf [29] Derived from molar values using molar mass of 65.38 g/mol [30] Derived from molar values using molar mass of 24.31 g/mol [31] Middleton P, Kelly AM, Brown J, Robertson M (August 2006). "Agreement between arterial and central venous values for pH, bicarbonate, base excess, and lactate". Emerg Med J 23 (8): 622–4. doi:10.1136/emj.2006.035915. PMC 2564165. PMID 16858095. [32] The Medical Education Division of the Brookside Associates--> ABG (Arterial Blood Gas) (http:/ / www. brooksidepress. org/ Products/ OperationalMedicine/ DATA/ operationalmed/ Lab/ ABG_ArterialBloodGas. htm) Retrieved on Dec 6, 2009 [33] Derived from molar values using molar mass of 1.01 g•mol−1 [34] Derived from mmHg values using 0.133322 kPa/mmHg [35] Derived from molar values using molar mass of 44.010 g/mol [36] Derived from molar values using molar mass of 61 g/mol [37] Reference range (albumin) (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=288686147) at GPnotebook [38] Derived from mass using molecular weight of 65kD [39] Derived from mass values using molar mass of 585g/mol [40] Fachwörterbuch Kompakt Medizin E-D/D-E. Author: Fritz-Jürgen Nöhring. Edition 2. Publisher:Elsevier, Urban&FischerVerlag, 2004. ISBN 3-437-15120-7, 9783437151200. Length: 1288 pages [41] GPnotebook > reference range (AST) (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=322240579) Retrieved on Dec 7, 2009 [42] Helander A, Vabö E, Levin K, Borg S (October 1998). "Intra- and interindividual variability of carbohydrate-deficient transferrin, gamma-glutamyltransferase, and mean corpuscular volume in teetotalers". Clin. Chem. 44 (10): 2120–5. PMID 9761244. [43] Creatine kinase (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=1436155929) at GPnotebook [44] Page 585 (http:/ / books. google. se/ books?id=AUSIRcV_as0C& pg=PA585) in: Lee, Mary Ann (2009). Basic Skills in Interpreting Laboratory Data. Amer Soc of Health System. ISBN 1-58528-180-8. [45] Muscle Information and Courses from MediaLab, Inc. > Cardiac Biomarkers (http:/ / www. medialabinc. net/ muscle-keyword. aspx) Retrieved on April 22, 2010 [46] South London Healthcare NHS Trust [47] Moderately Elevated Serum Troponin Concentrations Are Associated With Increased Morbidity and Mortality Rates in Surgical Intensive Care Unit Patients (http:/ / www. medscape. com/ viewarticle/ 464448) Rene P. Relos, MD; Ian K. Hasinoff, MD; Greg J. Beilman, MD. Posted: 12/05/2003; Crit Care Med. 2003;31(11) [48] Kay SE, Doery J, Sholl D (February 2002). "Clozapine associated pericarditis and elevated troponin I". Aust N Z J Psychiatry 36 (1): 143–4. doi:10.1046/j.1440-1614.2002.0988f.x. PMID 11929456. [49] Brenden, C.; Hollander, J.; Guss, D.; McCullough, P.; Nowak, R.; Green, G.; Saltzberg, M.; Ellison, S. et al. (2006). "Gray zone BNP levels in heart failure patients in the emergency department: Results from the Rapid Emergency Department Heart Failure Outpatient Trial (REDHOT) multicenter study". American Heart Journal 151 (5): 1006–1011. doi:10.1016/j.ahj.2005.10.017. PMID 16644322. [50] Strunk, A.; Bhalla, V.; Clopton, P.; Nowak, R.; McCord, J.; Hollander, J.; Duc, P.; Storrow, A. et al. (2006). "Impact of the History of Congestive Heart Failure on the Utility of B-Type Natriuretic Peptide in the Emergency Diagnosis of Heart Failure: Results from the Breathing Not Properly Multinational Study". The American Journal of Medicine 119 (1): 69.e1–69.e11. doi:10.1016/j.amjmed.2005.04.029. PMID 16431187. [51] Adëeva Nutritionals Canada > Optimal blood test values (http:/ / www. adeeva. com/ resources/ bloodtestscomplete. html) Retrieved on July 9, 2009

34

Reference ranges for blood tests [52] Derived from values in mg/dl to mmol/l, by dividing by 89, according to faqs.org: What are mg/dl and mmol/l? How to convert? Glucose? Cholesterol? (http:/ / www. faqs. org/ faqs/ diabetes/ faq/ part1/ section-9. html) Last Update July 21, 2009. Retrieved on July 21, 2009 [53] Derived from values in mg/dl to mmol/l, using molar mass of 386.65 g/mol [54] Reference range (cholesterol) (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=-214630397) at GPnotebook [55] Royal College of Pathologists of Australasia; Cholesterol (HDL and LDL) - plasma or serum (http:/ / www. rcpamanual. edu. au/ sections/ pathologytest. asp?s=33& i=450) Last Updated: Monday, 6 August 2007 [56] What Your Cholesterol Levels Mean. (http:/ / www. americanheart. org/ presenter. jhtml?identifier=183) American Heart Association. Retrieved on September 12, 2009 [57] American Association for Clinical Chemistry; HDL Cholesterol (http:/ / www. labtestsonline. org/ understanding/ analytes/ hdl/ test. html) [58] GP Notebook > range (reference, ca-125) (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=-100270014) Retrieved on Jan 5, 2009 [59] ClinLab Navigator > Test Interpretations > CA-125 (http:/ / www. clinlabnavigator. com/ Test-Interpretations/ ca-125. html) Retrieved on March 8, 2011 [60] Bjerner J, Høgetveit A, Wold Akselberg K, et al. (June 2008). "Reference intervals for carcinoembryonic antigen (CEA), CA125, MUC1, Alfa-foeto-protein (AFP), neuron-specific enolase (NSE) and CA19.9 from the NORIP study". Scandinavian journal of clinical and laboratory investigation 68 (8): 1–12. doi:10.1080/00365510802126836. PMID 18609108. [61] Carcinoembryonic Antigen(CEA) (http:/ / www. medicinenet. com/ carcinoembryonic_antigen/ article. htm) at MedicineNet [62] The TSH Reference Range Wars: What's "Normal?", Who is Wrong, Who is Right... (http:/ / thyroid. about. com/ od/ gettestedanddiagnosed/ a/ tshtestwars. htm) By Mary Shomon, About.com. Updated: June 19, 2006. About.com Health's Disease and Condition [63] 2006 Press releases: Thyroid Imbalance? Target Your Numbers (http:/ / www. aace. com/ newsroom/ press/ 2006/ index. php?r=20060110) Contacts: Bryan Campbell American] Association of Clinical Endocrinologists [64] The TSH Reference Range Wars: What's "Normal?", Who is Wrong, Who is Right... (http:/ / thyroid. about. com/ od/ gettestedanddiagnosed/ a/ tshtestwars. htm) By Mary Shomon, About.com. Updated: June 19, 2006 [65] Demers, Laurence M.; Carole A. Spencer (2002). "LMPG: Laboratory Support for the Diagnosis and Monitoring of Thyroid Disease" (http:/ / www. nacb. org/ lmpg/ thyroid_LMPG_PDF. stm). National Academy of Clinical Biochemistry (USA). . Retrieved 2007-04-13. - see Section 2. Pre-analytic factors [66] Free T4; Thyroxine, Free; T4, Free (http:/ / labs. unchealthcare. org/ labstestinfo/ f_tests/ free_t4. htm) UNC Health Care System [67] Derived from molar values using molar mass of 776.87 g/mol [68] Derived from mass values using molar mass of 776.87 g/mol [69] Table 4: Typical reference ranges for serum assays (http:/ / www. thyroidmanager. org/ chapter6/ Ch-6b-2. htm) - Thyroid Disease Manager [70] Euthyroid Patient with Elevated Serum Free Thyroxine (http:/ / www. clinchem. org/ cgi/ content/ full/ 54/ 7/ 1239) George van der Watt1,a, David Haarburger1 and Peter Berman [71] Derived from mass values using molar mass of 650.98 g/mol [72] Serum concentration of free T3, free T4 and TSH in healthy children (http:/ / cat. inist. fr/ ?aModele=afficheN& cpsidt=13391788) Cioffi Michele; Gazzerro Patrizia; Vietri Maria Teresa; Magnetta Rosa; Durante Adriana; D'Auria Annamaria; Puca Giovanni Alfredo; Molinari Anna Maria ; [73] References and further description of values are given in image page in Wikimedia Commons at Commons:Hormones estradiol, progesterone, LH and FSH during menstrual cycle.svg. [74] Life Extension Foundation > Blood Testing Protocols (http:/ / www. lef. org/ protocols/ appendix/ blood_testing_03. htm) [75] Andrology Australia: Your Health > Low Testosterone > Diagnosis (http:/ / www. andrologyaustralia. org/ pageContent. asp?pageCode=LOWTESTDIAG#LOWTESTDIAGNORM) [76] Derived from mass values using molar mass of 288.42g/mol [77] Derived from mass values using molar mass of 330.46g/mol [78] Values taken from day 1 after LH surge in: Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during different phases of the menstrual cycle on the Abbott ARCHITECT analyzer. (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16776638) Reto Stricker, Raphael Eberhart, Marie-Christine Chevailler, Frank A. Quinn, Paul Bischof and Rene´ Stricker. Clin Chem Lab Med 2006;44(7):883–887 PMID: 16776638. Alternative link: (http:/ / www. dianalabs. ch/ documents/ ajouts/ Hormones. pdf) [79] New York Hospital Queens > Services and Facilities > Patient Testing > Pathology > New York Hospital Queens Diagnostic Laboratories > Test Directory > Reference Ranges (http:/ / www. nyhq. org/ Reference_Ranges& ) Retrieved on Nov 8, 2009 [80] Derived from molar values using molar mass of 272.38g/mol [81] Total amount multiplied by 0.022 according to 2.2% presented in: Wu CH, Motohashi T, Abdel-Rahman HA, Flickinger GL, Mikhail G (August 1976). "Free and protein-bound plasma estradiol-17 beta during the menstrual cycle". J. Clin. Endocrinol. Metab. 43 (2): 436–45. doi:10.1210/jcem-43-2-436. PMID 950372. [82] Derived from mass values using molar mass of 314.46 g/mol [83] Bhattacharya Sudhindra Mohan (July/August 2005) Mid-luteal phase plasma progesterone levels in spontaneous and clomiphene citrate induced conception cycles (http:/ / medind. nic. in/ jaq/ t05/ i4/ jaqt05i4p350. pdf) J Obstet Gynecol India Vol. 55, No. 4 : July/August 2005 Pg 350-352 [84] Unit Code 91215 (http:/ / www. mayomedicallaboratories. com/ test-catalog/ print. php?unit_code=91215) at Mayo Clinic Medical Laboratories. Retrieved April 2011

35

Reference ranges for blood tests [85] Derived from mass values using molar mass of 4540g/mol according to PROOPIOMELANOCORTIN; NCBI --> POMC (http:/ / www. uniprot. org/ uniprot/ P01189) Retrieved on September 28, 2009 [86] "Adrenocorticotropic Hormone:Normal" (http:/ / children. webmd. com/ adrenocorticotropic-hormone?page=2). WebMD. 09-03-2006. . Retrieved 2008-11-09. [87] Biochemistry Reference Ranges at Good Hope Hospital (http:/ / www. goodhope. org. uk/ departments/ pathweb/ refranges. htm) Retrieved on Nov 8, 2009 [88] Derived from molar values using molar mass of 362 g/mol [89] Ranges estimated from quantile regression as showwn in table 4 in: Friedrich, N; Alte, D; Volzke, H; Spilckeliss, E; Ludemann, J; Lerch, M; Kohlmann, T; Nauck, M et al. (2008). "Reference ranges of serum IGF-1 and IGFBP-3 levels in a general adult population: Results of the Study of Health in Pomerania (SHIP)". Growth Hormone & IGF Research 18 (3): 228–237. doi:10.1016/j.ghir.2007.09.005. PMID 17997337. [90] Taken from the assay method giving the lowest and highest estimate, respectively, from Table 2 (http:/ / www. clinchem. org/ cgi/ content/ full/ 54/ 10/ 1673/ T2) in: Beltran, L; Fahie-Wilson, MN; McKenna, TJ; Kavanagh, L; Smith, TP (2008 Oct). "Serum total prolactin and monomeric prolactin reference intervals determined by precipitation with polyethylene glycol: evaluation and validation on common immunoassay platforms". Clinical Chemistry 54 (10): 1673–81. doi:10.1373/clinchem.2008.105312. PMID 18719199. [91] Derived from molar values using molar mass of 9.4 kDa [92] Table 2 (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ mid/ NIHMS10653/ table/ T2/ ) in: Aloia JF, Feuerman M, Yeh JK (2006). "Reference range for serum parathyroid hormone". Endocr Pract 12 (2): 137–44. PMC 1482827. PMID 16690460. [93] Derived from molar values using molar mass 400.6 g/mol [94] Bender, David A. (2003). "Vitamin D" (http:/ / books. google. com/ ?id=pxEJNs0IUo4C). Nutritional biochemistry of the vitamins. Cambridge: Cambridge University Press. ISBN 0-521-80388-8. . Retrieved December 10, 2008 through Google Book Search. [95] Bischoff-Ferrari, H.A., Dietrich, T., Orav, J.E., Hu, F.B., Zhang, Y., Karlson, E., Dawson-Hughes, B. 2004. Higher 25-hydroxyvitamin D levels are associated with better lower extremity function in both active and inactive adults 60+ years of age. American Journal of Clinical Nutrition. 80:752-758. [96] Reusch J, Ackermann H, Badenhoop K (May 2009). "Cyclic changes of vitamin D and PTH are primarily regulated by solar radiation: 5-year analysis of a German (50 degrees N) population". Horm. Metab. Res. 41 (5): 402–7. doi:10.1055/s-0028-1128131. PMID 19241329. [97] Letter: Calcium and vitamin D in preventing fractures. Data are not sufficient to show inefficacy (http:/ / www. bmj. com/ cgi/ content/ extract/ 331/ 7508/ 108-b) Alex Vasquez, researcher. BMJ 2005;331:108-109 (9 July), doi:10.1136/bmj.331.7508.108-b. [98] Converted from values in mcU/mL by dividing with a factor of 11.2 mcU/mL per ng/(mL*hour), as given in: •

New Assays for Aldosterone, Renin and Parathyroid Hormone (http:/ / depts. washington. edu/ labweb/ referencelab/ print/ endo. pdf) University of Washington, Department of Laboratory Medicine. Retrieved Mars 2011 [99] Pratt, R.; Flynn, J.; Hobart, P.; Paul, M.; Dzau, V. (1988). "Different secretory pathways of renin from mouse cells transfected with the human renin gene". The Journal of biological chemistry 263 (7): 3137–3141. PMID 2893797. [100] New Assays for Aldosterone, Renin and Parathyroid Hormone (http:/ / depts. washington. edu/ labweb/ referencelab/ print/ endo. pdf) University of Washington, Department of Laboratory Medicine. Retrieved Mars 2011 [101] Converted from mass values using molar mass of 360.44 g/mol [102] Tiu, S. -C.; Choi, C. -H.; Shek, C. -C.; Ng, Y. -W.; Chan, F. K. W.; Ng, C. -M.; Kong, A. P. S. (2004). "The Use of Aldosterone-Renin Ratio as a Diagnostic Test for Primary Hyperaldosteronism and Its Test Characteristics under Different Conditions of Blood Sampling". Journal of Clinical Endocrinology & Metabolism 90: 72–78. doi:10.1210/jc.2004-1149. (http:/ / jcem. endojournals. org/ cgi/ content/ full/ 90/ 1/ 72) [103] Central Manchester University Hospitals --> Reference ranges (http:/ / www. cmft. nhs. uk/ directorates/ labmedicine/ USERGUIDE/ pdfs/ Haem - Coagulation Ref Ranges. pdf) Retrieved on July 9, 2009 [104] University of Kentucky Chandler Medical Center > Clinical Lab Reference Range Guide (http:/ / www. hosp. uky. edu/ Clinlab/ report. pdf) Retrieved on April 28, 2009 [105] Derived from mass values using molar mass of 441 mol−1 [106] Derived form molar values using molar mass of 1355g/mol [107] The Doctor's Doctor: Homocysteine (http:/ / www. thedoctorsdoctor. com/ labtests/ homocysteine. htm) [108] Derived from molar values using molar massof 135 g/mol [109] Derived from mass values using molar mass of 176 grams per mol [110] For Driving under the influence by country, see Drunk driving law by country [111] Derived from mass values using molar mass of 46g/mol [112] Derived from mass values using 64,500 g/mol, according to Van Beekvelt MC, Colier WN, Wevers RA, Van Engelen BG (2001). "Performance of near-infrared spectroscopy in measuring local O2 consumption and blood flow in skeletal muscle". J Appl Physiol 90 (2): 511–519. PMID 11160049. [113] Derived from mass concentration, using molar mass of 64,458 g/mol (Van Beekvelt MC, Colier WN, Wevers RA, Van Engelen BG (2001). "Performance of near-infrared spectroscopy in measuring local O2 consumption and blood flow in skeletal muscle". J Appl Physiol 90 (2): 511–519. PMID 11160049.). 1 g/dL = 0.1551 mmol/L [114] lymphomation.org > Tests & Imaging > Labs > Complete Blood Count (http:/ / www. lymphomation. org/ CBC-blood-counts. htm) Retrieved on May 14, 2009

36

Reference ranges for blood tests [115] Clinical Laboratory Medicine. By Kenneth D. McClatchey. Page 807. (http:/ / books. google. com/ books?id=3PJVLH1NmQAC) [116] Determination of monocyte count by hematological analyzers, manual method and flow cytometry in polish population (http:/ / www. termedia. pl/ magazine. php?magazine_id=10& article_id=6801& magazine_subpage=ABSTRACT) Central European Journal of Immunology 1-2/2006. (Centr Eur J Immunol 2006; 31 (1-2): 1-5) authors: Elżbieta Górska, Urszula Demkow, Roman Pińkowski, Barbara Jakubczak, Dorota Matuszewicz, Jolanta Gawęda, Wioletta Rzeszotarska, Maria Wąsik, [117] Normal Values: RBC, Hgb, Hct, Indices, RDW, Platelets, and MPV (Conventional Units) (http:/ / www. labcareplus. org/ docs/ REFERENCE_RANGES. pdf) From labcareplus. Retrieved 4 nov, 2010 [118] MedlinePlus Encyclopedia 003652 (http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 003652. htm) [119] (http:/ / pathology. bsuh. nhs. uk/ pathology/ Default. aspx?tabid=108) Retrieved on November 20, 2009 [120] Miller A, Green M, Robinson D (1983). "Simple rule for calculating normal erythrocyte sedimentation rate". Br Med J (Clin Res Ed) 286 (6361): 266. doi:10.1136/bmj.286.6361.266. PMC 1546487. PMID 6402065. [121] Böttiger LE, Svedberg CA (1967). "Normal erythrocyte sedimentation rate and age". Br Med J 2 (5544): 85–7. doi:10.1136/bmj.2.5544.85. PMC 1841240. PMID 6020854. [122] C-reactive protein (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=946536472) at GPnotebook [123] 2730 Serum C-Reactive Protein values in Diabetics with Periodontal Disease (http:/ / iadr. confex. com/ iadr/ 2008Toronto/ techprogram/ abstract_106289. htm) A.R. Choudhury, and S. Rahman, Birdem, Diabetic Association of Bangladesh, Dhaka, Bangladesh. (the diabetics were not used to determine the reference ranges) [124] Derived from mass using molar mass of 25,106 g/mol [125] Sipahi T, Kara C, Tavil B, Inci A, Oksal A (March 2003). "Alpha-1 antitrypsin deficiency: an overlooked cause of late hemorrhagic disease of the newborn" (http:/ / www. jpho-online. com/ pt/ re/ jpho/ fulltext. 00043426-200303000-00019. htm). J. Pediatr. Hematol. Oncol. 25 (3): 274–5. doi:10.1097/00043426-200303000-00019. PMID 12621252. . [126] Derived from mass values using molar mass of 44324.5 g/mol [127] The Society for American Clinical Laboratory Science > Chemistry Tests > Immunoglobulins (http:/ / www. ascls. org/ labtesting/ labchem. asp) Retrieved on Nov 26, 2009 [128] All values cited from Chronolab are given for ELISA [129] chronolab.com > Autoantibodies associated with rheumatic diseases > Reference ranges (http:/ / www. chronolab. com/ rheumatic/ range. htm) Retrieved on April 29, 2010 [130] Reference range (amylase) (http:/ / www. gpnotebook. co. uk/ simplepage. cfm?ID=309002307) at GPnotebook [131] Plasma Measurement of D-Dimer Levels for the Early Diagnosis of Ischemic Stroke Subtypes (http:/ / archinte. ama-assn. org/ cgi/ content/ full/ 162/ 22/ 2589) Walter Ageno, MD; Sergio Finazzi, MD; Luigi Steidl, MD; Maria Grazia Biotti, MD; Valentina Mera, MD; GianVico Melzi d'Eril, MD; Achille Venco, MD. Arch Intern Med. 2002;162:2589-2593. [132] Kline JA, Williams GW, Hernandez-Nino J (May 2005). "D-dimer concentrations in normal pregnancy: new diagnostic thresholds are needed" (http:/ / www. clinchem. org/ cgi/ content/ full/ 51/ 5/ 825). Clinical chemistry 51 (5): 825–9. doi:10.1373/clinchem.2004.044883. PMID 15764641. . [133] Gardner MD, Scott R (April 1980). "Age- and sex-related reference ranges for eight plasma constituents derived from randomly selected adults in a Scottish new town" (http:/ / jcp. bmj. com/ cgi/ pmidlookup?view=long& pmid=7400337). J. Clin. Pathol. 33 (4): 380–5. doi:10.1136/jcp.33.4.380. PMC 1146084. PMID 7400337. . [134] Finney H, Newman DJ, Price CP (January 2000). "Adult reference ranges for serum cystatin C, creatinine and predicted creatinine clearance" (http:/ / acb. rsmjournals. com/ cgi/ pmidlookup?view=long& pmid=10672373). Ann. Clin. Biochem. 37 ( Pt 1): 49–59. doi:10.1258/0004563001901524. PMID 10672373. . [135] Derived from molar values by multiplying with the molar mass of 113.118 g/mol, and divided by 10.000 to adapt from μg/L to mg/dL [136] MedlinePlus Encyclopedia Glucose tolerance test (http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 003466. htm) [137] Derived from molar values using molar mass of 180g/mol [138] Derived from mass values using molar mass of 90.08 g/mol [139] Derived from mass values using molar mass of 88.06 g/mol

External links • biochemical reference values (http://www.gpnotebook.co.uk/simplepage.cfm?ID=724893718) at GPnotebook • Values at lymphomation.org (http://www.lymphomation.org/CBC-blood-counts.htm) • Descriptions at amarillomed.com (http://www.amarillomed.com/howto.htm)

37

Bone marrow

38

Bone marrow Bone marrow

Illustration of cells in bone marrow Latin

medulla ossium

Bone marrow is the flexible tissue found in the interior of bones. In humans, marrow in large bones produces new blood cells. It constitutes 4% of the total body mass of humans, i.e. approximately 2.6 kg (5.7 lbs.) in adults weighing 65 kg or 143 lbs. Bone marrow also prevents the backflow of lymph, working as a vital part of the lymphatic system.

Anatomy Marrow types There are two types of bone marrow: red marrow (consisting mainly of hematopoietic tissue) and yellow marrow (consisting mainly of fat cells). Red blood cells, platelets and most white blood cells arise in red marrow. Both types of bone marrow contain numerous blood vessels and capillaries. At birth, all bone marrow is red. With age, more and more of it is converted to the yellow type. About half of adult bone marrow is red. Red marrow is found mainly in the flat bones, such as the hip bone, breast bone, skull, ribs, vertebrae and shoulder blades, and in the cancellous ("spongy") material at the epiphyseal ends of the long bones such as the femur and humerus. Yellow marrow is found in the hollow interior of the middle portion of long bones. In cases of severe blood loss, the body can convert yellow marrow back to red marrow to increase blood cell production.

A femur with a cortex of cortical bone and medulla of trabecular bone showing its red bone marrow and a focus of yellow bone marrow.

Stroma The stroma of the bone marrow is all tissue not directly involved in the primary function of hematopoiesis. The yellow bone marrow belongs here, and makes the majority of the bone marrow stroma, in addition to stromal cells located in the red bone marrow. Yellow bone marrow is found in the Medullary cavity. Still, the stroma is indirectly involved in hematopoiesis, since it provides the hematopoietic microenvironment that facilitates hematopoiesis by the parenchymal cells. For instance, they generate colony stimulating factors, affecting hematopoiesis. Cells that constitute the bone marrow stroma are: • fibroblasts (reticular connective tissue)

Bone marrow • • • • •

39

macrophages adipocytes osteoblasts osteoclasts endothelial cells forming the sinusoids

Macrophages contribute especially to red blood cell production. They deliver iron for hemoglobin-production. Bone marrow barrier The blood vessels constitute a barrier, inhibiting immature blood cells from leaving the bone marrow. Only mature blood cells contain the membrane proteins required to attach to and pass the blood vessel endothelium. Hematopoietic stem cells may also cross the bone marrow barrier, and may thus be harvested from blood . Stem cells The bone marrow stroma contain mesenchymal stem cells (also called marrow stromal cells). These cells are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta-pancreatic islets cells. They can also transdifferentiate into neuronal cells.

Compartmentalization There is biologic compartmentalization in the bone marrow, in that certain cell types tend to aggregate in specific areas. For instance, erythrocytes, macrophages and their precursors tend to gather around blood vessels, while granulocytes gather at the borders of the bone marrow.

Types of stem cells Bone marrow contains three types of stem cells:[1] • Hematopoietic stem cells give rise to the three classes of blood cells that are found in the circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). • Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow. They have the capability to differentiate into osteoblasts, chondrocytes, myocytes, and many other types of cells. They also function as "gatekeeper" cells of the bone marrow. • Endothelial stem cells

Diseases involving the bone marrow

Hematopoietic precursor cells: promyelocyte in the center, two metamyelocytes next to it and band cells from a bone marrow aspirate.

The normal bone marrow architecture can be displaced by malignancies, aplastic anemia , or infections such as tuberculosis, leading to a decrease in the production of blood cells and blood platelets. In addition, cancers of the hematologic progenitor cells in the bone marrow can arise; these are the leukemias. To diagnose diseases involving the bone marrow, a bone marrow aspiration is sometimes performed. This typically involves using a hollow needle to acquire a sample of red bone marrow from the crest of the ilium under general or local anesthesia. The average number of cells in a leg bone is about 440,000,000,000 (4.4x1011).

Bone marrow

40

Exposure to radiation or chemotherapy will kill many of the rapidly dividing cells of the bone marrow and will therefore result in a depressed immune system. Many of the symptoms of radiation sickness are due to damage to the bone marrow cells.

Examination Bone marrow examination is the pathologic analysis of samples of bone marrow obtained by bone marrow biopsy and bone marrow aspiration. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells. While much information can be gleaned by testing the blood itself (drawn from a vein by phlebotomy), it is sometimes necessary to examine the source of the blood cells in the bone marrow to obtain more information on hematopoiesis; this is the role of bone marrow aspiration and biopsy.

A Wright's stained bone marrow aspirate smear from a patient with leukemia.

Donation and transplantation of bone marrow It is possible to take hematopoietic stem cells from one person and then infuse them into another person (Allogenic) or into the same person at a later time (Autologous). If donor and recipient are compatible, these infused cells will then travel to the bone marrow and initiate blood cell production. Transplantation from one person to another is performed in severe cases of disease of the bone marrow. The patient's marrow is first killed off with drugs or radiation, and then the new stem cells are introduced. Bone marrow harvest Before radiation therapy or chemotherapy in cases of cancer, some of the patient's hematopoietic stem cells are sometimes harvested and later infused back when the therapy is finished to restore the immune system.

Bone marrow

Harvesting The stem cells are harvested directly from the red marrow in the crest of the ilium, often under general anesthesia. The procedure is minimally invasive and does not require stitches afterwards. Depending on the donor health and reaction to the procedure, the actual harvesting can be an outpatient procedure or requiring 1–2 days of recovery in the hospital.[2] Another option is to administer certain drugs that stimulate the release of stem cells from the bone marrow into circulating blood.[3] An IV is inserted into the donor's arm, and the stem cells are filtered out of the blood. The procedure is similar to donating blood or platelets. It may also be taken from the sternum. The tibia may seem a good source, since it is very superficial. However, except in children, this bone marrow does not contain any substantial amount of red bone marrow, only yellow bone marrow.[4] In newborns, stem cells may be retrieved from the umbilical cord.[5]

Bone marrow as a food Many cultures utilize bone marrow as a food. The Vietnamese prize beef bone as the soup base for their national staple phở; Alaskan Natives eat the bone marrow of caribou and moose; Indians use slow-cooked marrow as the core ingredient of the Indian dish nalli nihari; Mexicans use beef bone marrow from leg bones, called tuétano, which is cooked and served as filling for tacos or tostadas; it is also considered to be the highlight of the Italian dish ossobuco (braised veal shanks); beef marrowbones are often included in the French pot-au-feu broth, the cooked marrow being traditionally eaten on toasted bread with sprinkled coarse sea salt, in Iranian In some parts of Germany beef soup is served with "Markklößchen" (bone marrow cuisine lamb shanks are usually broken balls). before cooking to allow diners to suck out and eat the marrow when the dish is served. Though once used in various preparations, including pemmican, bone marrow has fallen out of favor as a food in the United States, though bone marrow is used in many gourmet restaurants and is popular among foodies.[6] In the Philippines, the soup "Bulalo" is made primarily of beef stock and marrow bones, seasoned with vegetables and boiled meat; a similar soup in the Philippines is called "Kansi".[7] In Hungary tibia is a main ingredient of beef soup; the bone is chopped into short (10–15 cm) pieces and the ends are covered with salt to prevent the marrow from leaving the bone while cooking. Upon serving the soup the marrow is usually spread on toast. Diners in the 18th century used a marrow scoop (or marrow spoon), often of silver and with a long thin bowl, as a table implement for removing marrow from a bone. Some anthropologists believe that early humans were scavengers rather than hunters. Marrow would then have been a food source (largely for its fat content) for tool-using hominids, who were able to crack open the bones of carcasses left by apex predators such as lions.[8]

41

Bone marrow

References [1] Raphael Rubin and David S. Strayer (2007). Rubin's Pathology: Clinicopathologic Foundations of Medicine. Lippincott Williams & Wilkins. pp. 90. ISBN 0781795168. [2] National Marrow Donor Program Donor Guide (http:/ / www. marrow. org/ DONOR/ When_You_re_Asked_to_Donate_fo/ index. html) [3] Mayo Clinic: Bone marrow donation: What to expect when you donate (http:/ / www. mayoclinic. com/ health/ bone-marrow/ CA00047) [4] Semester 4 medical lectures at Uppsala University 2008 by Leif Jansson [5] Production of stem cells with embryonic characteristics from human umbilical cord blood (http:/ / www3. interscience. wiley. com/ journal/ 118705649/ abstract) [6] La Petite Bouche (Food Blog): Roasted Bone Marrow (http:/ / lapetitebouche. blogspot. com/ 2010/ 08/ roasted-bone-marrow. html) [7] "Kansi" (http:/ / www. flickr. com/ photos/ kamums/ 4378949248/ ) Flickr [8] Bruce Bower. Hunting ancient scavengers - some anthropologists say early humans were scavengers, not hunters (http:/ / findarticles. com/ p/ articles/ mi_m1200/ is_v127/ ai_3677563). Science News. March 9, 1985

Antibody An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen.[1] [2] Each tip of the "Y" of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (that is equivalent to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly (for example, by blocking a part of a microbe that is essential for its invasion and survival). The production of antibodies is the main function of the humoral immune system.[3] Antibodies are produced by a kind of white blood cell called a plasma cell. Antibodies can occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell and is referred to as the B cell receptor (BCR). The BCR is only found on the Each antibody binds to a specific antigen; an interaction similar to a surface of B cells and facilitates the activation of these lock and key. cells and their subsequent differentiation into either antibody factories called plasma cells, or memory B cells that will survive in the body and remember that same antigen so the B cells can respond faster upon future exposure.[4] In most cases, interaction of the B cell with a T helper cell is necessary to produce full activation of the B cell and, therefore, antibody generation following antigen binding.[5] Soluble antibodies are released into the blood and tissue fluids, as well as many secretions to continue to survey for invading microorganisms. Antibodies are glycoproteins belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably.[6] Antibodies are typically made of basic structural units—each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different

42

Antibody

43

kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.[7] Though the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen binding sites, to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen.[1] This enormous diversity of antibodies allows the immune system to recognize an equally wide variety of antigens.[6] The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity.[7] [8] Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used by several different parts of the immune system.

Forms Surface immunoglobulin (Ig) is attached to the membrane of the effector B cells by its transmembrane region, while antibodies are the secreted form of Ig and lack the trans membrane region so that antibodies can be secreted into the bloodstream and body cavities. As a result, surface Ig and antibodies are identical except for the transmembrane regions. Therefore, they are considered two forms of antibodies: soluble form or membrane-bound form (Parham 21-22). The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation.[9] The BCR is composed of surface-bound IgD or IgM antibodies and associated Ig-α and Ig-β heterodimers, which are capable of signal transduction.[10] A typical human B cell will have 50,000 to 100,000 antibodies bound to its surface.[10] Upon antigen binding, they cluster in large patches, which can exceed 1 micrometer in diameter, on lipid rafts that isolate the BCRs from most other cell signaling receptors.[10] These patches may improve the efficiency of the cellular immune response.[11] In humans, the cell surface is bare around the B cell receptors for several thousand ångstroms,[12] which further isolates the BCRs from competing influences.

Isotypes Antibody isotypes of mammals Name Types

Description

Antibody Complexes

Antibody

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IgA

2

Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents [13] colonization by pathogens. Also found in saliva, tears, and breast milk.

IgD

1

Functions mainly as an antigen receptor on B cells that have not been exposed to [14] antigens. It has been shown to activate basophils and mast cells to produce antimicrobial [15] factors.

IgE

1

Binds to allergens and triggers histamine release from mast cells and basophils, and is [3] involved in allergy. Also protects against parasitic worms.

IgG

4

In its four forms, provides the majority of antibody-based immunity against invading [3] pathogens. The only antibody capable of crossing the placenta to give passive immunity to fetus.

IgM

1

Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) [3] [14] immunity before there is sufficient IgG.

Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. They are each named with an "Ig" prefix that stands for immunoglobulin, another name for antibody, and differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.[16] The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when they reach maturity—the co-expression of both these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen.[17] B cell activation follows engagement of the cell bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA or IgG, that have defined roles in the immune system.

Structure Antibodies are heavy (~150 kDa) globular plasma proteins. They have sugar chains added to some of their amino acid residues.[18] In other words, antibodies are glycoproteins. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.[19]

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45

The variable parts of an antibody are its V regions, and the constant part is its C region.

Immunoglobulin domains The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds.[16] Each chain is composed of structural domains called immunoglobulin domains. These domains contain about 70-110 amino acids and are classified into different categories (for example, variable or IgV, and constant or IgC) according to their size and function.[20] They have a characteristic immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together by interactions between conserved cysteines and other charged amino acids.

Several immunoglobulin domains make up the two heavy chains (red and blue) and the two light chains (green and yellow) of an antibody. The immunoglobulin domains are composed of between 7 (for constant domains) and 9 (for variable domains) β-strands.

Heavy chain There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ.[1] The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively.[6] Distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.[1] In birds, the major serum antibody, also found in yolk, is called IgY. It is quite different from mammalian IgG. However, in some older literature and even on some commercial life sciences product websites it is still called "IgG", which is incorrect and can be confusing. Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility;[16] heavy chains μ and ε have a constant region composed of four immunoglobulin domains.[1] The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.

Light chain

1. Fab region 2. Fc region 3. Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains. 4. Light chain (green) with one variable (VL) and one constant (CL) domain 5. Antigen binding site (paratope) 6. Hinge regions.

In mammals there are two types of immunoglobulin light chain, which are called lambda (λ) and kappa (κ).[1] A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids.[1] Each antibody contains two light chains that are always identical; only one type of light chain, κ or λ, is present per antibody in mammals. Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei..

Antibody

CDRs, Fv, Fab and Fc Regions Some parts of an antibody have unique functions. The arms of the Y, for example, contain the sites that can bind two antigens (in general identical) and, therefore, recognize specific foreign objects. This region of the antibody is called the Fab (fragment, antigen binding) region. It is composed of one constant and one variable domain from each heavy and light chain of the antibody.[21] The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains. The variable domain is also referred to as the FV region and is the most important region for binding to antigens. More specifically, variable loops of β-strands, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen. These loops are referred to as the complementarity determining regions (CDRs). The structures of these CDRs have been clustered and classified by Chothia et al. [22] and more recently by North et al.[23] In the framework of the immune network theory, CDRs are also called idiotypes. According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes. The base of the Y plays a role in modulating immune cell activity. This region is called the Fc (Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody.[1] Thus, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including recognition of opsonized particles, lysis of cells, and degranulation of mast cells, basophils and eosinophils.[16] [24]

Function Activated B cells differentiate into either antibody-producing cells called plasma cells that secrete soluble antibody or memory cells that survive in the body for years afterward in order to allow the immune system to remember an antigen and respond faster upon future exposures.[25] At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies for different kinds of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the bloodstream, they are said to be part of the humoral immune system. Circulating antibodies are produced by clonal B cells that specifically respond to only one antigen (an example is a virus capsid protein fragment). Antibodies contribute to immunity in three ways: they prevent pathogens from entering or damaging cells by binding to them; they stimulate removal of pathogens by macrophages and other cells by coating the pathogen; and they trigger destruction of pathogens by stimulating other immune responses such as the complement pathway.[26]

46

Antibody

47

Activation of complement

The secreted mammalian IgM has five Ig units. Each Ig unit (labeled 1) has two epitope binding Fab regions, so IgM is capable of binding up to 10 epitopes.

Antibodies that bind to surface antigens on, for example, a bacterium attract the first component of the complement cascade with their Fc region and initiate activation of the "classical" complement system.[26] This results in the killing of bacteria in two ways.[3] First, the binding of the antibody and complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization; these phagocytes are attracted by certain complement molecules generated in the complement cascade. Secondly, some complement system components form a membrane attack complex to assist antibodies to kill the bacterium directly.[27]

Activation of effector cells

To combat pathogens that replicate outside cells, antibodies bind to pathogens to link them together, causing them to agglutinate. Since an antibody has at least two paratopes it can bind more than one antigen by binding identical epitopes carried on the surfaces of these antigens. By coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that recognize their Fc region.[3] Those cells which recognize coated pathogens have Fc receptors which, as the name suggests, interacts with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular antibody with the Fc receptor on a particular cell triggers an effector function of that cell; phagocytes will phagocytose, mast cells and neutrophils will degranulate, natural killer cells will release cytokines and cytotoxic molecules; that will ultimately result in destruction of the invading microbe. The Fc receptors are isotype-specific, which gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms for distinct pathogens.[1]

Natural antibodies Humans and higher primates also produce “natural antibodies” which are present in serum before viral infection. Natural antibodies have been defined as antibodies that are produced without any previous infection, vaccination, other foreign antigen exposure or passive immunization. These antibodies can activate the classical complement pathway leading to lysis of enveloped virus particles long before the adaptive immune response is activated. Many natural antibodies are directed against the disaccharide galactose α(1,3)-galactose (α-Gal), which is found as a terminal sugar on glycosylated cell surface proteins, and generated in response to production of this sugar by bacteria contained in the human gut.[28] Rejection of xenotransplantated organs is thought to be, in part, the result of natural antibodies circulating in the serum of the recipient binding to α-Gal antigens expressed on the donor tissue.[29]

Immunoglobulin diversity Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many different types of microbes requires diversity among antibodies; their amino acid composition varies allowing them to interact with many different antigens.[30] It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen.[31] Although a huge repertoire of different antibodies is generated in a single individual, the number of genes available to make these proteins is limited by the size of the human genome. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes.[32]

Antibody

Domain variability The region (locus) of a chromosome that encodes an antibody is large and contains several distinct genes for each domain of the antibody—the locus containing heavy chain genes (IGH@) is found on chromosome 14, and the loci containing lambda and kappa light chain genes (IGL@ and IGK@) are found on chromosomes 22 and 2 in humans. One of these domains is called the variable domain, which is present in each heavy and light chain of every antibody, but can differ in different antibodies generated from The complementarity determining regions of the heavy chain are [33] distinct B cells. Differences, between the variable shown in red (PDB 1IGT ) domains, are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3) or complementarity determining regions (CDR1, CDR2 and CDR3). CDRs are supported within the variable domains by conserved framework regions. The heavy chain locus contains about 65 different variable domain genes that all differ in their CDRs. Combining these genes with an array of genes for other domains of the antibody generates a large cavalry of antibodies with a high degree of variability. This combination is called V(D)J recombination discussed below.[34]

V(D)J recombination Somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the generation of a unique immunoglobulin variable region. The variable region of each immunoglobulin heavy or light chain is encoded in several pieces—known as gene segments. These segments are called variable (V), diversity (D) and joining (J) segments.[32] V, D and J segments are found in Ig heavy chains, but only V and J segments are found in Ig light chains. Multiple copies of the V, D and J gene segments exist, and are tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell will assemble an immunoglobulin variable region by randomly selecting and combining one V, one D and one J gene segment (or one V and one J segment in the light chain). As there are multiple Simplistic overview of V(D)J recombination of immunoglobulin copies of each type of gene segment, and different heavy chains combinations of gene segments can be used to generate each immunoglobulin variable region, this process generates a huge number of antibodies, each with different paratopes, and thus different antigen specificities.[7] After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot express any other variable region (a process known as allelic exclusion) thus each B cell can produce antibodies containing only one kind of variable chain.[1] [35]

48

Antibody

49

Somatic hypermutation and affinity maturation For more details on this topic, see Somatic hypermutation and Affinity maturation Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide change per variable gene, per cell division.[8] As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains. This serves to increase the diversity of the antibody pool and impacts the antibody’s antigen-binding affinity.[36] Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity).[37] B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die by apoptosis.[37] Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.[38]

Class switching Isotype or class switching is a biological process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG).[7] The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naïve B cells express only cell-surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after activation, an antibody with a IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the Mechanism of class switch recombination that allows isotype antibody heavy chain changes during class switching; switching in activated B cells the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present in the B cell environment.[39] Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S) regions, found in DNA upstream of each constant region gene (except in the δ-chain). The DNA strand is broken by the activity of a series of enzymes at two selected S-regions.[40] [41] The variable domain exon is rejoined through a process called non-homologous end joining (NHEJ) to the desired constant region (γ, α or ε). This process results in an immunoglobulin gene that encodes an antibody of a different isotype.[42]

Antibody

Medical applications Disease diagnosis and therapy Detection of particular antibodies is a very common form of medical diagnostics, and applications such as serology depend on these methods.[43] For example, in biochemical assays for disease diagnosis,[44] a titer of antibodies directed against Epstein-Barr virus or Lyme disease is estimated from the blood. If those antibodies are not present, either the person is not infected, or the infection occurred a very long time ago, and the B cells generating these specific antibodies have naturally decayed. In clinical immunology, levels of individual classes of immunoglobulins are measured by nephelometry (or turbidimetry) to characterize the antibody profile of patient.[45] Elevations in different classes of immunoglobulins are sometimes useful in determining the cause of liver damage in patients whom the diagnosis is unclear.[6] For example, elevated IgA indicates alcoholic cirrhosis, elevated IgM indicates viral hepatitis and primary biliary cirrhosis, while IgG is elevated in viral hepatitis, autoimmune hepatitis and cirrhosis. Autoimmune disorders can often be traced to antibodies that bind the body's own epitopes; many can be detected through blood tests. Antibodies directed against red blood cell surface antigens in immune mediated hemolytic anemia are detected with the Coombs test.[46] The Coombs test is also used for antibody screening in blood transfusion preparation and also for antibody screening in antenatal women.[46] Practically, several immunodiagnostic methods based on detection of complex antigen-antibody are used to diagnose infectious diseases, for example ELISA, immunofluorescence, Western blot, immunodiffusion, immunoelectrophoresis, and magnetic immunoassay. Antibodies raised against human chorionic gonadotropin are used in over the counter pregnancy tests. Targeted monoclonal antibody therapy is employed to treat diseases such as rheumatoid arthritis,[47] multiple sclerosis,[48] psoriasis,[49] and many forms of cancer including non-Hodgkin's lymphoma,[50] colorectal cancer, head and neck cancer and breast cancer.[51] Some immune deficiencies, such as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or complete lack of antibodies.[52] These diseases are often treated by inducing a short term form of immunity called passive immunity. Passive immunity is achieved through the transfer of ready-made antibodies in the form of human or animal serum, pooled immunoglobulin or monoclonal antibodies, into the affected individual.[53]

Prenatal therapy Rhesus factor, also known as Rhesus D (RhD) antigen, is an antigen found on red blood cells; individuals that are Rhesus-positive (Rh+) have this antigen on their red blood cells and individuals that are Rhesus-negative (Rh–) do not. During normal childbirth, delivery trauma or complications during pregnancy, blood from a fetus can enter the mother's system. In the case of an Rh-incompatible mother and child, consequential blood mixing may sensitize an Rh- mother to the Rh antigen on the blood cells of the Rh+ child, putting the remainder of the pregnancy, and any subsequent pregnancies, at risk for hemolytic disease of the newborn.[54] Rho(D) immune globulin antibodies are specific for human Rhesus D (RhD) antigen.[55] Anti-RhD antibodies are administered as part of a prenatal treatment regimen to prevent sensitization that may occur when a Rhesus-negative mother has a Rhesus-positive fetus. Treatment of a mother with Anti-RhD antibodies prior to and immediately after trauma and delivery destroys Rh antigen in the mother's system from the fetus. Importantly, this occurs before the antigen can stimulate maternal B cells to "remember" Rh antigen by generating memory B cells. Therefore, her humoral immune system will not make anti-Rh antibodies, and will not attack the Rhesus antigens of the current or subsequent babies. Rho(D) Immune Globulin treatment prevents sensitization that can lead to Rh disease, but does not prevent or treat the underlying disease itself.[55]

50

Antibody

51

Research applications Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum, which can now be called antiserum. Antigens are also injected into chickens for generation of polyclonal antibodies in egg yolk.[56] To obtain antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies.[57] Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen-affinity chromatography.[58]

Immunofluorescence image of the eukaryotic cytoskeleton. Actin filaments are shown in red, microtubules in green, and the nuclei in blue.

In research, purified antibodies are used in many applications. They are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the proteins they express; different types of cell express different combinations of cluster of differentiation molecules on their surface, and produce different intracellular and secretable proteins.[59] They are also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate,[60] in Western blot analyses to identify proteins separated by electrophoresis,[61] and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope.[59] [62] Proteins can also be detected and quantified with antibodies, using ELISA and ELISPOT techniques.[63] [64]

Structure prediction The importance of antibodies in health care and the biotechnology industry demands knowledge of their structures at high resolution. This information is used for protein engineering, modifying the antigen binding affinity, and identifying an epitope, of a given antibody. X-ray crystallography is one commonly used method for determining antibody structures. However, crystallizing an antibody is often laborious and time consuming. Computational approaches provide a cheaper and faster alternative to crystallography, but their results are more equivocal since they do not produce empirical structures. Online web servers such as Web Antibody Modeling (WAM)[65] and Prediction of Immunoglobulin Structure (PIGS)[66] enables computational modeling of antibody variable regions. Rosetta Antibody is a novel antibody FV region structure prediction server, which incorporates sophisticated techniques to minimize CDR loops and optimize the relative orientation of the light and heavy chains, as well as homology models that predict successful docking of antibodies with their unique antigen.[67]

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52

History The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[68] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[68] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[68]

Angel of the West (2008) by Julian Voss-Andreae was created based [69] on the antibody structure published by E. Padlan for the Florida [70] campus of the Scripps Research Institute. The antibody is placed into a ring referencing Leonardo da Vinci's Vitruvian Man thus highlighting the similar proportions of the antibody and the human [71] body.

The study of antibodies began in 1890 when Emil von Behring and Kitasato Shibasaburō described antibody activity against diphtheria and tetanus toxins. Behring and Kitasato put forward the theory of humoral immunity, proposing that a mediator in serum could react with a foreign antigen.[72] [73] Their idea prompted Paul Ehrlich to propose the side chain theory for antibody and antigen interaction in 1897, when he hypothesized that receptors (described as “side chains”) on the surface of cells could bind specifically to toxins – in a "lock-and-key" interaction – and that this binding reaction was the trigger for the production of antibodies.[74] Other researchers believed that antibodies existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies coated bacteria to label them for phagocytosis and killing; a process that he named opsoninization.[75]

In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies were made of protein.[76] The biochemical properties of antigen-antibody binding interactions were examined in more detail in the late 1930s by John Marrack.[77] The next major advance was in the 1940s, when Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions between antibodies and antigens depended more on their shape than their chemical composition.[78] In 1948, Astrid Fagreaus discovered that B cells, in the form of plasma cells, were responsible for generating antibodies.[79] Further work concentrated on characterizing the structures of the antibody proteins. A major advance in these structural studies was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light chain,[80] and their realization that this protein was the same as the Bence-Jones protein described in 1845 by Henry Bence Jones.[81] Edelman went on to discover that antibodies are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody-binding (Fab) and antibody tail (Fc) regions of IgG were characterized by Rodney Porter.[82] Together, these scientists deduced the structure and complete amino acid sequence of IgG, a feat for which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine.[82] The Fv fragment was prepared and characterized by David Givol.[83] While most of these early studies focused on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi discovered secretory antibody (IgA)[84] and David S. Rowe and John L. Fahey identified IgD,[85] and IgE was identified by Kikishige Ishizaka and Teruki Ishizaka as a class of antibodies involved in allergic reactions.[86] Genetic studies identifying the basis of the vast diversity of these antibody proteins when somatic recombination of immunoglobulin genes was by Susumu Tonegawa in 1976.[87]

Antibody

References [1] Janeway CA, Jr. et al (2001). Immunobiology. (5th ed.). Garland Publishing. (electronic full text via NCBI Bookshelf) (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?call=bv. View. . ShowTOC& rid=imm. TOC& depth=10) ISBN 0-8153-3642-X. [2] Litman GW, Rast JP, Shamblott MJ, Haire RN, Hulst M, Roess W, Litman RT, Hinds-Frey KR, Zilch A, Amemiya CT (January 1993). "Phylogenetic diversification of immunoglobulin genes and the antibody repertoire". Mol. Biol. Evol. 10 (1): 60–72. PMID 8450761. [3] Pier GB, Lyczak JB, Wetzler LM (2004). Immunology, Infection, and Immunity. ASM Press. ISBN 1-55581-246-5. [4] Borghesi L, Milcarek C (2006). "From B cell to plasma cell: regulation of V(D)J recombination and antibody secretion". Immunol. Res. 36 (1-3): 27–32. doi:10.1385/IR:36:1:27. PMID 17337763. [5] Parker D (1993). "T cell-dependent B cell activation". Annu Rev Immunol 11 (1): 331–60. doi:10.1146/annurev.iy.11.040193.001555. PMID 8476565. [6] Rhoades RA, Pflanzer RG (2002). 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External links • Mike's Immunoglobulin Structure/Function Page (http://www.path.cam.ac.uk/~mrc7/mikeimages.html) at University of Cambridge • Antibodies as the PDB molecule of the month (http://www.rcsb.org/pdb/static.do?p=education_discussion/ molecule_of_the_month/pdb21_1.html) Discussion of the structure of antibodies at Protein Data Bank • Microbiology and Immunology On-line Textbook (http://pathmicro.med.sc.edu/mayer/IgStruct2000.htm) at University of South Carolina • A hundred years of antibody therapy (http://users.path.ox.ac.uk/~scobbold/tig/new1/mabth.html) History and applications of antibodies in the treatment of disease at University of Oxford • How Lymphocytes Produce Antibody (http://www.cellsalive.com/antibody.htm) from Cells Alive! • Antibody applications (http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/) Fluorescent antibody image library, University of Birmingham

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Hematopoietic stem cell

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Hematopoietic stem cell Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all the blood cell types from the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The definition of hematopoietic stem cells has undergone considerable revision in the last two decades. The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs constitute 1:10.000 of cells in myeloid tissue.

HSC=Hematopoietic stem cell , Progenitor=Progenitor cell , L-blast=Lymphoblast , Lymphocyte , Mo-blast=Monoblast , Monocyte , Myeloblast , Pro-M=Promyelocyte , Myelocyte , Meta-M=Metamyelocyte , Neutrophil , Eosinophil , Basophil , Pro-E=Proerythroblast , Baso-E=Basophilic erythroblast , poly-E=Polychromatic erythroblast , Ortho-E=Orthochromatic erythroblast , Erythrocyte , Promegakaryocyte , Megakaryocyte , Platelet

HSCs are a heterogeneous population. Loosely, 3 classes of stem cells exist, distinguished by their ratio of lymphoid to myeloid progeny (L/M) in blood. Myeloid-biased (My-bi) HSC have low L/M ratio (>0, 10). The third category consists of the balanced (Bala) HSC for which 3 ≤ L/M ≤ 10. Currently, much work is being undertaken to investigate the properties of these different classes of HSCs, but it appears that only the myeloid biased and balanced HSCs have durable self-renewal properties. Additionally, serial transplantation experiments have shown that each subtype preferentially re-creates its blood cell type distribution, suggesting an inherited epigenetic program for each subtype. In November 2010, Mick Bhatia, Ph.D., the scientific director at Canada's McMaster University Michael G. DeGroote School of Medicine's Stem Cell and Cancer Research Institute, announced that his team had succeeded in transforming adult skin cells into blood precursor HSCs (erythroblasts) by working with a gene necessary for the process. When performed in mice, the experiment did not cause cancer, as has happened in certain cases in related research. The method will be further tweaked and perfected so it can be modified for eventual human trials.[1]

Hematopoietic stem cell

Source HSCs are found in the bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Cells can be obtained directly by removal from the hip using a needle and syringe, or from the blood following pre-treatment with cytokines, such as G-CSF (granulocyte colony-stimulating factors), that induce cells to be released from the bone marrow compartment. Other sources for clinical and scientific use include umbilical cord blood, peripheral blood a small number of stem and progenitor cells circulate in the bloodstream, in the past 10 years, researchers have found that they can coax the cells to migrate Sketch of bone marrow and its cells from marrow to blood in greater numbers by injecting the donor with a cytokine, such as granulocyte-colony stimulating factor (GCSF)and recent study shown that ex-vivo expansion of HSCs is possible in 3D bioreactor. Because HSCs are not generated in the adult but during the embryogenesis, many scientific groups are studying HSCs during the embryonic development. It is now well described in mammalians that the first definitive HSCs are detected in the AGM (Aorta-gonad-mesonephros), and then massively expanded in the Fetal Liver prior to colonize before birth the bone marrow. Such fundamental research could help to understand the mechanisms that are responsible of HSCs generation and/or amplification, and to the discovery of new molecules that could eventually be used to maintain or expand HSCs in vitro.

Functional characteristics Multipotency and self-renewal As stem cells, HSC are defined by their ability to replenish all blood cell types (Multipotency) and their ability to self-renew. It is known that a small number of HSCs can expand to generate a very large number of daughter HSCs. This phenomenon is used in bone marrow transplantation, when a small number of HSCs reconstitute the hematopoietic system. This indicates that, subsequent to bone marrow transplantation, symmetrical cell divisions into two daughter HSCs must occur. Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self-renewal. There is much interest in the environmental and molecular requirements for HSC self-renewal, as understanding the ability of HSC to replenish themselves will eventually allow the generation of expanded populations of HSC in vitro that can be used therapeutically.

Stem Cell Heterogeneity It was originally believed that all HSC were alike in their self-renewal and differentiation abilities. This view was first challenged by the 2002 discovery by the Muller-Sieburg group in San Diego, who illustrated that different stem cells can show distinct repopulation patterns that are epigenetically predetermined intrinsic properties of clonal Thy-1lo SCA-1+ lin- c-kit+ HSC.[2] [3] [4] The results of these clonal studies led to the notion of lineage bias. Using the ratio of lymphoid (L) to myeloid (M) cells in blood as a quantitative marker, the stem cell compartment can be split into three categories of HSC. Balanced (Bala) HSC repopulate peripheral white blood cells in the same ratio of myeloid to lymphoid cells as seen in unmanipulated mice (on average about 15% myeloid and 85% lymphoid cells, or 3≤ρ≤10). Myeloid-biased (My-bi) HSC give rise to too few lymphocytes resulting in ratios 0 Early Pro (or pre-pre)-B cells => Late Pro (or pre-pre)-B cells Large Pre-B cells => Small Pre-B cells Immature B cells B Cells => (B1 cells; B2 cells)

• Plasma cells • Pro-T cells • T-cells

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Lymphopoiesis

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New model: Mixed Myeloid/Lymphoid model Research on new models • The Common Myelolymphoid Progenitor: A Key Intermediate Stage in Hemopoiesis Generating T and B Cells [26]

• Identification of Flt3 + Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment [27] • Adult T-cell progenitors retain myeloid potential [28] • Research Findings May Shed Light on T-cell Leukemias and Immunodeficiencies [29] • Blood Lines Redrawn [30] • The earliest thymic progenitors for T cells possess myeloid lineage potential [31] • Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development [32] • Not a split decision for human hematopoiesis [33]

Graphical view of the old model vs Mixed Myelo-Lymphoid model

Side by side. Comparing the new and old lineage models.

Revised Lineage Myelo-lymphoid flowchart.

General immunology reference texts Texts in bold are the most heavily cited in this article. • Cell Communication in Nervous and Immune System; Gundelfinger, Seidenbecher, Schraven; Springer Berlin Heidelberg New York; 2006; ISBN 3-540-36828-0 • Color Atlas of Hematology; Theml et al; Thieme; 2004; ISBN 1-58890-193-9 • Dynamics of Cancer; Steven A. Frank; Princeton University Press, Princeton, New Jersey; 2007; ISBN 0–691–13365–2, Creative Commons Public License • Fundamental Immunology, 5th edition; William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; 2003; ISBN 0-7817-3514-9 • Immuno-Biology: The Immune System in Health and Science, 6th Edition; Janeway, Travers; 2005; Garland Science Publishing, New York; ISBN 0-8153-4101-6 • Immunology Introductory Textbook (ebook;revised 2nd edition); Nandini Shetty; New Age International (P) Limited, Publishers, India; 2005; ISBN 81-224-2335-3 • Instant Notes in Immunology, 2nd ed.; Lydyard, Whelan, Fanger; Taylor and Francis Group; 2004; China Version ISBN 703-025225-8； 46RMB Wangfujing Bookstore • Medical Immunology—6th ed.; G. Virella, Editor; Informa Healthcare USA, Inc; 2007; ISBN 0-8493-9696-0 • Stem Cell Biology; Marshak, Gardner, Gottlieb; Cold Spring Harbor Laboratory Press; 2001; ISBN 0-87969-575-7/01

Lymphopoiesis • Textbook of Human Development and Histology; Zhong Cuiping et al; Shanghai Scientific and Technical Publishers; 2006; ISBN 7-5323-8230-3 • Textbook of Medical Immunology (Immunology, 7th Edition); LIM Pak Leong; Elsevier (Singapore) Pte Ltd.; 2006; ISBN 0-323-03397-7

References [1] [2] [3] [4] [5] [6]

http:/ / en. wikipedia. org/ wiki/ Template:Lymphopoiesis Stem Cell Biology, page 307 Dynamics of Cancer, page 251 transit cells - q.v., Also called (http:/ / www. jrank. org/ health/ pages/ 20765/ transit-cells. html) CFU-T (http:/ / www. copewithcytokines. de/ cope. cgi?key=CFU-T) Transfer of maternal colostral leukocytes promotes development of the neonatal immune system (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 18394715) [7] Adult T-cell Progenitors Retain Myeloid Potential; Nature; 2008 [8] Textbook of Human Development and Histology, p.176 [9] Immuno-Biology, The Immune System in Health and Science. [10] Fundamental Immunology 5th edition [11] Immuno-Biology, The Immune System in Health and Science. Garland Science Publishing [12] Textbook of Medical Immunology, page 5 [13] Medical Immunology, page 23 [14] Tumor Immunity and Cancer Immunotherapy (http:/ / www. nccr-oncology. ch/ Lay_Summaries_about_the_Programme_and_its_Projects/ Tumor_Immunity_and_Cancer_Immunotherapy. html) [15] Medical Immunology, page 22 [16] Medical Immunology, p. 123 [17] Fundamental Immunology; Paul; Ch. 15 "DISTRIBUTION OF DENDRITIC CELLS IN VIVO: A MULTIMEMBER FAMILY" [18] Immunology; Lydyard et al; p. 22, 132-137 [19] Immunology; Lydyard et al; p. 15, 18-20,41 [20] Immunology; Lydyard et al; p. 20, 259-260 [21] Medical Immunology; Litwin, p. 122 [22] Color Atlas of Hematology 2004 [23] "The Earliest Thymic Progenitors for T cells Possess Myeloid Lineage Potential"; Bell, Bhandoola; Vol 452, 10 April 2008, doi:10.1038/nature06840 [24] Medical Immunology, Litwin, p.115 [25] Textbook of Medical Immunology, page 31 [26] The Common Myelolymphoid Progenitor: A Key Intermediate Stage in Hemopoiesis Generating T and B Cells; Min Lu, Hiroshi Kawamoto, Yoshihiro Katsube, Tomokatsu Ikawa, and Yoshimoto Katsura; J. Immunol. 2002;169;3519-3525 [27] Identification of Flt3 + Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment; Lund Strategic Research Center for Stem Cell Biology; Cell; Vol. 121, 295–306, April 22, 2005 [28] Adult T-cell progenitors retain myeloid potential; Haruka Wada, Kyoko Masuda, Rumi Satoh , Kiyokazu Kakugawa, Tomokatsu Ikawa, Yoshimoto Katsura & Hiroshi Kawamoto; Nature Vol 452 10 April 2008 [29] Research Findings May Shed Light on T-cell Leukemias and Immunodeficiencies (http:/ / www. penncancer. org/ penn_news. cfm?ID=449) April 9, 2008; [30] Blood Lines Redrawn; Thomas Graf; Nature Vol 452 10 April 2008 p.702-703 [31] The earliest thymic progenitors for T cells possess myeloid lineage potential; J. Jeremiah Bell, Avinash Bhandoola; Nature; Vol 452, 10 April 2008, p. 764-768 [32] Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development; Dick et al; Nature Immunology; Volume 11 Number 7 July 2010 p. 585-595 [33] Not a split decision for human hematopoiesis; Kenneth Dorshkind; Nature Immunology Volume 11 Number 7 July 2010 p. 569-570

79

Lymphopoiesis

80

Additional images Alternate views of lineages

Blood cell lineage. For scale, note that megakaryocytes (50-100 μm) are 10 to 15 times larger than a typical red blood cell.

Blood cell lineage. Based on self-renewal ability.

Schematic view. Well-defined lineages.

Side by side. Comparing the new and old lineage models.

External links • The www.copewithcytokines.de Mini-portal to Lymphopoiesis terminology (http://www.copewithcytokines.de/ cope.cgi?key=Lymphopoiesis) • lymphopoiesis (http://www.emedicinehealth.com/script/main/srchcont_dict.asp?src=lymphopoiesis) at eMedicine Dictionary • MeSH Lymphopoiesis (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Lymphopoiesis) • Lymphopoiesis (http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?pg=/ppdocs/us/ common/dorlands/dorland/five/000062114.htm) at Dorland's Medical Dictionary • Overview at hematologica.pl (http://www.hematologica.pl/Atlas3/Angielska/SpisUC.htm)

81

Lymphoid Neoplasms Hematologic disease Hematologic disease Classification and external resources MeSH

D006402

[1]

Hematologic diseases are disorders which primarily affect the blood.

Myeloid • Hemoglobinopathies (congenital abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis) • Sickle-cell disease • Thalassemia • Methemoglobinemia • Anemias (lack of red blood cells or hemoglobin) • Iron deficiency anemia • Megaloblastic anemia • Vitamin B12 deficiency • Pernicious anemia • Folate deficiency • Hemolytic anemias (destruction of red blood cells) • Genetic disorders of RBC membrane • Hereditary spherocytosis • Hereditary elliptocytosis • Genetic disorders of RBC metabolism • Glucose-6-phosphate dehydrogenase deficiency (G6PD) • Pyruvate kinase deficiency • Immune mediated hemolytic anemia (direct Coombs test is positive) • Autoimmune hemolytic anemia • Warm antibody autoimmune hemolytic anemia • Idiopathic • Systemic lupus erythematosus (SLE) • Evans' syndrome (antiplatelet antibodies and hemolytic antibodies) • Cold antibody autoimmune hemolytic anemia • Idiopathic cold hemagglutinin syndrome • Infectious mononucleosis • Paroxysmal cold hemoglobinuria (rare) • Alloimmune hemolytic anemia

Hematologic disease • Hemolytic disease of the newborn (HDN) • Rh disease (Rh D) • ABO hemolytic disease of the newborn • Anti-Kell hemolytic disease of the newborn • Rhesus c hemolytic disease of the newborn • Rhesus E hemolytic disease of the newborn • Other blood group incompatibility (RhC, Rhe, Kid, Duffy, MN, P and others) • Drug induced immune mediated hemolytic anemia • Penicillin (high dose) • Methyldopa • Hemoglobinopathies (where these is an unstable or crystalline hemoglobin) • Paroxysmal nocturnal hemoglobinuria (rare acquired clonal disorder of red blood cell surface proteins) • Direct physical damage to RBCs • Microangiopathic hemolytic anemia • Secondary to artificial heart valve(s) • Aplastic anemia • Fanconi anemia • Diamond-Blackfan anemia • Acquired pure red cell aplasia • Decreased numbers of cells • • • • • •

Myelodysplastic syndrome Myelofibrosis Neutropenia (decrease in the number of neutrophils) Agranulocytosis Glanzmann's thrombasthenia Thrombocytopenia (decrease in the number of platelets) • Idiopathic thrombocytopenic purpura (ITP) • Thrombotic thrombocytopenic purpura (TTP) • Heparin-induced thrombocytopenia (HIT)

• Myeloproliferative disorders (Increased numbers of cells) • • • • •

Polycythemia vera (increase in the number of cells in general) Erythrocytosis (increase in the number of red blood cells) Leukocytosis (increase in the number of white blood cells) Thrombocytosis (increase in the number of platelets) Myeloproliferative disorder

• Coagulopathies (disorders of bleeding and coagulation) • • • •

Thrombocytosis Recurrent thrombosis Disseminated intravascular coagulation Disorders of clotting proteins • Hemophilia • Hemophilia A • Hemophilia B (also known as Christmas disease) • Hemophilia C • Von Willebrand disease

82

Hematologic disease • Disseminated intravascular coagulation • Protein S deficiency • Antiphospholipid syndrome • Disorders of platelets • Thrombocytopenia • Glanzmann's thrombasthenia • Wiskott-Aldrich syndrome

Hematological malignancies • Hematological malignancies • Lymphomas • • • • •

Hodgkin's disease Non-Hodgkin's lymphoma{includes the next eight entries} Burkitt's lymphoma Anaplastic large cell lymphoma Splenic marginal zone lymphoma

• Hepatosplenic T-cell lymphoma • Angioimmunoblastic T-cell lymphoma (AILT) • Myelomas • Multiple myeloma • Waldenström macroglobulinemia • Plasmacytoma • Leukemias • • • • • • • • • •

Acute lymphocytic leukemia (ALL) Chronic lymphocytic leukemia (CLL){now included in theCLL/SCLL type NHL} Acute myelogenous leukemia (AML) Chronic myelogenous leukemia (CML) T-cell prolymphocytic leukemia (T-PLL) B-cell prolymphocytic leukemia (B-PLL) Chronic neutrophilic leukemia (CNL) Hairy cell leukemia (HCL) T-cell large granular lymphocyte leukemia (T-LGL) Aggressive NK-cell leukemia

Miscellaneous • Hemochromatosis • Asplenia • Hypersplenism • Gauchers disease • Monoclonal gammopathy of undetermined significance • Hemophagocytic lymphohistiocytosis

83

Hematologic disease

Hematological changes secondary to non-hematological disorders • • • • •

Anemia of chronic disease Infectious mononucleosis AIDS Malaria Leishmaniasis

External links • http://www.hematologic.niddk.nih.gov/info/index.htm • Myeloproliferative Disorders (MPD) Foundation and Research Alliance [2] • International Registry of Rare Bleeding Disorders [3]

References [1] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D006402 [2] http:/ / www. mpdfoundation. org [3] http:/ / www. rbdd. org

84

Hematological malignancy

85

Hematological malignancy Hematological malignancy Classification and external resources

Micrograph of a plasmacytoma, a hematological malignancy. [1]

ICD-10

C81.

ICD-9

200

ICD-O:

9590-9999

MeSH

D019337

-C96.

[3]

-208

[2]

[4]

[5]

Hematological malignancies are the types of cancer that affect blood, bone marrow, and lymph nodes. As the three are intimately connected through the immune system, a disease affecting one of the three will often affect the others as well: although lymphoma is technically a disease of the lymph nodes, it often spreads to the bone marrow, affecting the blood and occasionally producing a paraprotein. While uncommon in solid tumors, chromosomal translocations are a common cause of these diseases. This commonly leads to a different approach in diagnosis and treatment of hematological malignancies. Hematological malignancies are malignant neoplasms ("cancer"), and they are generally treated by specialists in hematology and/or oncology. "Hematology/oncology" is a single subspecialty of internal medicine (there are also surgical and radiation oncologists). Not all hematological disorders are malignant ("cancerous"); these other blood conditions may also be managed by a hematologist. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

Hematological malignancy

86

Classification and incidence Taken together, hematological malignancies account for 9.5% of new cancer diagnoses in the United States.[6] Within this category, lymphomas are more common than leukemias. Historically, hematological malignancies have been most commonly divided by whether the malignancy is mainly located in the blood (leukemia) or in lymph nodes (lymphomas). However, the influential WHO Classification (published in 2001) emphasized a greater emphasis on cell lineage.

Classic classification Relative proportions of hematological malignancies in the United States:[7] Type of hematological malignancy

Percentage

Total

Leukemias

—

30.4%

Acute lymphoblastic leukemia (ALL)

4.0%

Acute myelogenous leukemia (AML)

8.7%

Chronic lymphocytic leukemia (CLL) sorted under lymphomas according to current WHO classification; called small lymphocytic lymphoma (SLL) when leukemic cells are absent.

10.2%

Chronic myelogenous leukemia (CML)

3.7%

Acute monocytic leukemia (AMOL)

0.7%

Other leukemias

3.1% Lymphomas

—

Hodgkin's lymphomas (all four subtypes)

7.0%

Non-Hodgkin's lymphomas (all subtypes)

48.6% Myelomas

Total

55.6%

14.0%

100%

By cell lineage By cell lineage, hematological malignancies are classified according to their lineage in hematopoiesis: • Lymphoid • Lymphoid leukemia • Lymphoma • Myeloid leukemia

Diagnosis For the analysis of a suspected hematological malignancy, a complete blood count and blood film are essential, as malignant cells can show in characteristic ways on light microscopy. When there is lymphadenopathy, a biopsy from a lymph node is generally undertaken surgically. In general, a bone marrow biopsy is part of the "work up" for the analysis of these diseases. All specimens are examined microscopically to determine the nature of the malignancy. A number of these diseases can now be classified by cytogenetics (AML, CML) or immunophenotyping (lymphoma,

Hematological malignancy myeloma, CLL) of the malignant cells.

Treatment Treatment can occasionally consist of "watchful waiting" (e.g. in CLL) or symptomatic treatment (e.g. blood transfusions in MDS). The more aggressive forms of disease require treatment with chemotherapy, radiotherapy, immunotherapy and - in some cases - a bone marrow transplant.

Follow-up If treatment has been successful ("complete" or "partial remission"), a patient is generally followed up at regular intervals to detect recurrence and monitor for "secondary malignancy" (an uncommon side-effect of some chemotherapy and radiotherapy regimens - the appearance of another form of cancer). In the follow-up, which should be done at pre-determined regular intervals, general anamnesis is combined with complete blood count and determination of lactate dehydrogenase or thymidine kinase in serum.

References [1] http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gc81. htm+ c81 [2] [3] [4] [5] [6]

http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gc81. htm+ c96 http:/ / www. icd9data. com/ getICD9Code. ashx?icd9=200 http:/ / www. icd9data. com/ getICD9Code. ashx?icd9=208 http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D019337 "Facts & Statistics" (http:/ / www. leukemia-lymphoma. org/ all_page?item_id=12486). The Leukemia and Lymphoma Society. . Retrieved 03 November 2009. [7] Horner MJ, Ries LAG, Krapcho M, Neyman N, et al. (eds).. "SEER Cancer Statistics Review, 1975–2006" (http:/ / seer. cancer. gov/ csr/ 1975_2006/ ). Surveillance Epidemiology and End Results (SEER). Bethesda, MD: National Cancer Institute. . Retrieved 03 November 2009. "Table 1.4: Age-Adjusted SEER Incidence and U.S. Death Rates and 5-Year Relative Survival Rates By Primary Cancer Site, Sex and Time Period"

87

Lymphoma

88

Lymphoma Lymphoma Classification and external resources

Follicular lymphoma replacing a lymph node [1]

ICD-10

C81.

ICD-9

202.8

ICD-O:

9590-9999

MeSH

D008223

-C96.

[2]

[1]

Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. Treatment might involve chemotherapy and in some cases radiotherapy and/or bone marrow transplantation, and can be curable depending on the histology, type, and stage of the disease.[2] These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Extranodal sites include the skin, brain, bowels and bone. Lymphomas are closely related to lymphoid leukemias, which also originate in lymphocytes but typically involve only circulating blood and the bone marrow (where blood cells are generated in a process termed haematopoesis) and do not usually form static tumors.[2] There are many types of lymphomas, and in turn, lymphomas are a part of the broad group of diseases called hematological neoplasms. Thomas Hodgkin published the first description of lymphoma in 1832, specifically of the form named after him, Hodgkin's lymphoma.[3] Since then, many other forms of lymphoma have been described, grouped under several proposed classifications. The 1982 Working formulation classification became very popular. It introduced the category non-Hodgkin lymphoma (NHL), divided into 16 different diseases. However, because these different lymphomas have little in common with each other, the NHL label is of limited usefulness for doctors or patients and is slowly being abandoned. The latest classification by the WHO (2001) lists 43 different forms of lymphoma divided in four broad groups. Although older classifications referred to histiocytic lymphomas, these are recognized in newer classifications as of B, T or NK cell lineage. True histiocytic malignancies are rare and are classified as sarcomas.[4]

Lymphoma

Classification A number of various different classification systems exist for lymphoma. As an alternative to the American Lukes-Butler classification, in the early 1970s, Karl Lennert of Kiel, Germany, proposed a new system of classifying lymphomas based on cellular morphology and their relationship to cells of the normal peripheral lymphoid system.[5] Some forms of lymphoma are categorized as indolent (e.g. small lymphocytic lymphoma), compatible with a long life even without treatment, whereas other forms are aggressive (e.g. Burkitt's lymphoma), causing rapid deterioration and death. However, most of the aggressive lymphomas respond well to treatment and are curable. The prognosis therefore depends on the correct diagnosis and classification of the disease, which is established after examination of a biopsy by a pathologist (usually a hematopathologist).[6]

Working Formulation and non-Hodgkin lymphoma The 1982 Working Formulation is a classification of non-Hodgkin lymphoma. It excluded the Hodgkin lymphomas and divided the remaining lymphomas into four grades (Low, Intermediate, High, and Miscellaneous) related to prognosis, with some further subdivisions based on the size and shape of affected cells. This purely histological classification included no information about cell surface markers, or genetics, and it made no distinction between T-cell lymphomas or B-cell lymphomas.See here [7] It was widely accepted at the time of its publication but is now obsolete.[8] It was superseded by subsequent classifications (see below) but it is still used by cancer agencies for compilation of lymphoma statistics and historical rare comparisons.

REAL In the mid 1990s,the Revised European-American Lymphoma (REAL) Classification attempted to apply immunophenotypic and genetic features in identifying distinct clinicopathologic entities among all the lymphomas except Hodgkin's lymphoma.[9] REAL has been superseded by the WHO classification. REAL & WHO •B-cell neoplasms –precursor –mature •T-cell neoplasms –precursor –mature •Hodgkin lymphomaNon-HodgkinLymphomas

World Health Organization (WHO) The WHO Classification, published in 2001 and updated in 2008,[4] is the latest classification of lymphoma and is based upon the foundations laid within the "Revised European-American Lymphoma classification" (REAL). This system attempts to group lymphomas by cell type (i.e. the normal cell type that most resembles the tumor) and defining phenotypic, molecular or cytogenetic characteristics. There are three large groups: the B cell, T cell, and natural killer cell tumors. Other less common groups, are also recognized. Hodgkin's lymphoma, although considered separately within the World Health Organization (and preceding) classifications, is now recognized as being a tumor of, albeit markedly abnormal, lymphocytes of mature B cell lineage.

89

Lymphoma

90

Mature B cell neoplasms • Chronic lymphocytic leukemia/Small lymphocytic lymphoma • B-cell prolymphocytic leukemia • Lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia) • Splenic marginal zone lymphoma • Plasma cell neoplasms: • Plasma cell myeloma • Plasmacytoma • Monoclonal immunoglobulin deposition diseases • Heavy chain diseases • Extranodal marginal zone B cell lymphoma, also called MALT lymphoma • Nodal marginal zone B cell lymphoma (NMZL) • • • •

Follicular lymphoma Mantle cell lymphoma Diffuse large B cell lymphoma Mediastinal (thymic) large B cell lymphoma

• Intravascular large B cell lymphoma • Primary effusion lymphoma • Burkitt lymphoma/leukemia Mature T cell and natural killer (NK) cell neoplasms • • • • • • • • • •

T cell prolymphocytic leukemia T cell large granular lymphocytic leukemia Aggressive NK cell leukemia Adult T cell leukemia/lymphoma Extranodal NK/T cell lymphoma, nasal type Enteropathy-type T cell lymphoma Hepatosplenic T cell lymphoma Blastic NK cell lymphoma Mycosis fungoides / Sezary syndrome Primary cutaneous CD30-positive T cell lymphoproliferative disorders

• Primary cutaneous anaplastic large cell lymphoma • Lymphomatoid papulosis • Angioimmunoblastic T cell lymphoma • Peripheral T cell lymphoma, unspecified • Anaplastic large cell lymphoma

DNA-microarray analysis of Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL) showing differences in gene expression patterns. Colors indicate levels of expression; green indicates genes that are underexpressed in lymphoma cells (as compared to normal cells), whereas red indicates genes that are overexpressed in lymphoma cells.

Lymphoma

91

Hodgkin lymphoma • Classical Hodgkin lymphomas: • Nodular sclerosis • Mixed cellularity • Lymphocyte-rich • Lymphocyte depleted or not depleted • Nodular lymphocyte-predominant Hodgkin lymphoma Immunodeficiency-associated lymphoproliferative disorders • • • • •

Associated with a primary immune disorder Associated with the Human Immunodeficiency Virus (HIV) Post-transplant Associated with methotrexate therapy Primary central nervous system lymphoma occurs most often in immuno-compromised patients, in particular those with AIDS, but it can occur in the immunocompetent as well. It has a poor prognosis, particularly in those with AIDS. Treatment can consist of corticosteroids, radiotherapy, and chemotherapy, often with methotrexate.

Other classification systems • ICD-O (codes 9590-9999, details at [10]) • ICD-10 (codes C81-C96, details at [11])

Symptoms • • • • • • • •

Anorexia Dyspnea Fatigue[12] Fever of unknown origin Lymphadenopathy Night sweats Pruritus Weight loss

Diagnosis, etiology, staging, prognosis, and treatment [13]

5-year relative survival by stage at diagnosis Stage at diagnosis

5-year relative survival (%)

Percentage of cases (%)

Distant (cancer has metastasized)

59.9

45

Localized (confined to primary site)

82.1

28

Regional (spread to regional lymphnodes) 77.5

19

Unknown (unstaged)

8

67.5

These depend on the specific form of lymphoma.[14] For some forms of lymphoma, watchful waiting is often the initial course of action.[15] If a low-grade lymphoma is becoming symptomatic, radiotherapy or chemotherapy are the treatments of choice; although they do not cure the lymphoma, they can alleviate the symptoms, particularly painful

Lymphoma

92

lymphadenopathy. Patients with these types of lymphoma can live near-normal lifespans, but the disease is incurable. Treatment of some other, more aggressive, forms of lymphoma can result in a cure in the majority of cases, but the prognosis for patients with a poor response to therapy is worse.[16] Treatment for these types of lymphoma typically consists of aggressive chemotherapy, including the CHOP regimen. Hodgkin lymphoma typically is treated with radiotherapy alone, as long as it is localized.[17] Advanced Hodgkins disease requires systemic chemotherapy, sometimes combined with radiotherapy.[18] See the articles on the corresponding form of lymphoma for further information.

Epidemiology Lymphoma is the most common form of hematological malignancy, or "blood cancer", in the developed world. Taken together, lymphomas Age adjustmentAge-standardized death from lymphomas and multiple myeloma per 100,000 inhabitants in 2004. "WHO Disease and injury country estimates". World Health Organization. 2009. . Retrieved represent 5.3% of all Nov. 11, 2009.  no data  less than cancers (excluding simple 1.8  1.8–3.6  3.6–5.4  5.4–7.2  7.2–9  9–10.8  10.8–12.6  12.6–14.4  14.4–16.2  16.2–18  18–19.8  more basal cell and squamous than 19.8 cell skin cancers) in the United States and 55.6% of all blood cancers.[20] According to the U.S. National Institutes of Health, lymphomas account for about five percent of all cases of cancer in the United States, and Hodgkin's lymphoma in particular accounts for less than one percent of all cases of cancer in the United States. Because the whole system is part of the body's immune system, patients with a weakened immune system such as from HIV infection or from certain drugs or medication also have a higher incidence of lymphoma.

Comparison Following is a comparison of the most common types of lymphoma: Lymphoma type

Relative incidence

Histopathology

Cell markers

Precursor T-cell leukemia/lymphoma

40% of lymphomas in [21] childhood.

Lymphoblasts with irregular nuclear contours, condensed chromatin, small nucleoli and scant cytoplasm without [21] granules.

TdT, CD2, CD7

Follicular lymphoma

40% of lymphomas in [21] adults

Small "cleaved" cells (centrocytes) mixed with large activated cells (centroblasts). Usually nodular ("follicular") growth [21] pattern

CD10, surface Ig

Overall 5-year survival

[21]

[21]

Other comments

It often presents as a mediastinal mass because of involvement of the [21] thymus. It is highly associated with NOTCH1 [21] mutations. Most common in adolescent [21] males. 72–77%

[22]

Occurs in older adults. Usually involves lymph nodes, bone marrow and [21] spleen. Associated with t(14;18) translocation [21] overexpressing Bcl-2. [21] Indolent

Lymphoma

93

Diffuse large B cell lymphoma

40 to 50% of lymphomas in [21] adults

Variable. Most Variable expression resemble B cells of of CD10 and surface [21] large germinal centers. Ig Diffuse growth [21] pattern.

Mantle cell lymphoma

3 to 4% of lymphomas in [21] adults

Lymphocytes of small to intermediate size growing in diffuse [21] pattern

CD5

3 to 4 % of lymphomas in [21] adults

Small resting lymphocytes mixed with variable number of large activated cells. Lymph nodes are diffusely [21] effaced

CD5, surface [21] immunoglobulin

MALT lymphoma

~5% of lymphomas in [21] adults

Variable cell size and differentiation. 40% show plasma cell differentiation. Homing of B cells to epithelium creates lymphoepithelial [21] lesions.

CD5, CD10, surface [21] Ig

Burkitt's lymphoma

< 1% of lymphomas in the United [21] States

Round lymphoid cells of intermediate size with several nucleoli. Starry-sky appearance by diffuse spread with interspersed [21] apoptosis.

CD10, surface Ig

Mycosis fungoides

Most common cutaneous lymphoid malignancy

Usually small lymphoid cells with convoluted nuclei that often infiltrate the epidermis, creating Pautier [21] microabscesses

CD4

B-cell chronic lymphocytic leukemia/lymphoma

Peripheral T-cell Most common Variable. Usually a lymphoma-Not-Otherwise-Specified T cell mix small to large [21] lymphoma lymphoid cells with irregular nuclear [21] contours.

[21]

[24] 50% to [24] 70%

[21]

[21]

[21]

CD3

[23]

60%

[25]

50%.

Occurs in all ages, but most commonly in older adults. Often occurs outside lymph nodes. [21] Aggressive. Occurs mainly in adult males. Usually involves lymph nodes, bone marrow, spleen and GI tract. Associated with t(11;14) translocation overexpressing cyclin D1. Moderately [21] aggressive. Occurs in older adults. Usually involves lymph nodes, bone marrow and spleen. Most patients have peripheral blood involvement. [21] Indolent. Frequently occurs outside lymph nodes. Very indolent. May be cured by [21] local excision.

[26]

50%

[27]

75%

Endemic in Africa, sporadic elsewhere. More common in immunocompromised and in children. Often visceral involvement. Highly [21] aggressive. Localized or more generalized skin symptoms. Generally indolent. In a more [21] aggressive variant, Sézary's disease, there is skin erythema and peripheral blood [21] involvement. Probably consists of several rare tumor types. It is often disseminated and generally [21] aggressive.

Lymphoma

Nodular sclerosis form of Hodgkin lymphoma

94 [21]

Most common type of Hodgkin's [21] lymphoma

Reed-Sternberg cell variants and inflammation. usually broad sclerotic bands that consists of [21] collagen.

CD15, CD30

Mixed-cellularity subtype of Hodgkin Second most lymphoma common form of Hodgkin's [21] lymphoma

Many classic Reed-Sternberg cells [21] and inflammation

CD15, CD30

[21]

Most common in young adults. It often arises in the mediastinum or [21] cervical lymph nodes.

Most common in men. More likely to be diagnosed at advanced stages than the nodular sclerosis form. Epstein-Barr virus involved in 70% of [21] cases.

References [1] [2] [3] [4]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D008223 Parham, Peter (2005). The immune system. New York: Garland Science. p. 414. ISBN 0-8153-4093-1. Hellman, Samuel; Mauch, P.M. Ed. (1999). Hodgkin's Disease. Chapter 1: Lippincott Williams & Wilkins. p. 5. ISBN 0-7817-1502-4. ed. by Elaine S. Jaffe .... (2001). Pathology and Genetics of Haemo (World Health Organization Classification of tumors S.). Oxford Univ Pr. ISBN 92-832-2411-6. [5] Lennert, Karl; Feller, Alfred C.; Jacques Diebold; M. Paulli; A. Le Tourneau (2002). Histopathology of Non-Hodgkin's Lymphomas (Based on the Updated Kiel Classification). Berlin: Springer. pp. 2. ISBN 3-540-63801-6. [6] Wagman LD. "Principles of Surgical Oncology" (http:/ / www. cancernetwork. com/ cancer-management-11/ chapter01/ article/ 10165/ 1399286) in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach (http:/ / www. cancernetwork. com/ cancer-management-11/ ). 11 ed. 2008. [7] http:/ / www. m5zn. com/ uploads/ 2010/ 12/ 21/ photo/ 122110101245tpngrb00rjf. jpg [8] Clarke CA, Glaser SL, Dorfman and Dorfwoman RF, Bracci PM, Eberle E, Holly EA (January 2004). "Expert review of non-Hodgkin lymphomas in a population-based cancer registry: reliability of diagnosis and subtype classifications". Cancer Epidemiol. Bio-markers Prev. 13 (1): 138–43. doi:10.1158/1055-9965.EPI-03-0250. PMID 14744745. [9] www.emedicine.com on Lymphoma, Non-Hodgkin [10] http:/ / web. archive. org/ web/ 20040627090029/ http:/ / www. cog. ufl. edu/ publ/ apps/ icdo/ icdo_morph. txt [11] http:/ / www3. who. int/ icd/ vol1htm2003/ fr-icd. htm?gc81. htm+ [12] http:/ / lymphoma. about. com/ od/ symptoms/ tp/ warningsigns. htm [13] (http:/ / seer. cancer. gov/ statfacts/ html/ lymph. html) Data from the USA 1999–2006, All Races, Both Sexes: Altekruse SF, Kosary CL, Krapcho M, Neyman N, Aminou R, Waldron W, Ruhl J, Howlader N, Tatalovich Z, Cho H, Mariotto A, Eisner MP, Lewis DR, Cronin K, Chen HS, Feuer EJ, Stinchcomb DG, Edwards BK (eds). SEER Cancer Statistics Review, 1975–2007, National Cancer Institute. Bethesda, MD, http:/ / seer. cancer. gov/ csr/ 1975_2007/ , based on November 2009 SEER data submission, posted to the SEER web site, 2010. [14] Sweetenham JW (November 2009). "Treatment of lymphoblastic lymphoma in adults". Oncology (Williston Park, N.Y.) 23 (12): 1015–20. PMID 20017283. [15] Elphee EE (May 2008). "Understanding the concept of uncertainty in patients with indolent lymphoma". Oncol Nurs Forum 35 (3): 449–54. doi:10.1188/08.ONF.449-454. PMID 18467294. [16] Bernstein SH, Burack WR (2009). "The incidence, natural history, biology, and treatment of transformed lymphomas". Hematology Am Soc Hematol Educ Program 2009: 532–41. doi:10.1182/asheducation-2009.1.532. PMID 20008238. [17] Martin NE, Ng AK (November 2009). "Good things come in small packages: low-dose radiation as palliation for indolent non-Hodgkin lymphomas". Leuk. Lymphoma 50 (11): 1765–72. doi:10.3109/10428190903186510. PMID 19883306. [18] Kuruvilla J (2009). "Standard therapy of advanced Hodgkin lymphoma". Hematology Am Soc Hematol Educ Program 2009: 497–506. doi:10.1182/asheducation-2009.1.497. PMID 20008235. [19] "WHO Disease and injury country estimates" (http:/ / www. who. int/ healthinfo/ global_burden_disease/ estimates_country/ en/ index. html). World Health Organization. 2009. . Retrieved Nov. 11, 2009. [20] Horner MJ, Ries LAG, Krapcho M, Neyman N, et al. (eds).. "SEER Cancer Statistics Review, 1975–2006" (http:/ / seer. cancer. gov/ csr/ 1975_2006/ ). Surveillance Epidemiology and End Results (SEER). Bethesda, MD: National Cancer Institute. . Retrieved 03 November 2009. "Table 1.4: Age-Adjusted SEER Incidence and U.S. Death Rates and 5-Year Relative Survival Rates By Primary Cancer Site, Sex and Time Period" [21] Table 12-8 with lymphomas sorted out. Mitchell, Richard Sheppard; Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson. Robbins Basic Pathology. Philadelphia: Saunders. ISBN 1-4160-2973-7. 8th edition.

Lymphoma [22] eMedicine Specialties > Lymphoma, Follicular (http:/ / emedicine. medscape. com/ article/ 203268-overview) Author: Cesar O Freytes. Coauthor: Julianna A Burzynski. Updated: Nov 3, 2009 [23] Turgeon, Mary Louise (2005). Clinical hematology: theory and procedures. Hagerstown, MD: Lippincott Williams & Wilkins. pp. 285–286. ISBN 0-7817-5007-5. •

50% for limited stage, according to: Leitch HA, Gascoyne RD, Chhanabhai M, Voss NJ, Klasa R, Connors JM (October 2003). "Limited-stage mantle-cell lymphoma". Ann. Oncol. 14 (10): 1555–61. doi:10.1093/annonc/mdg414. PMID 14504058. • 70% for advanced stage, according to most recent values in: Herrmann A, Hoster E, Zwingers T, et al. (February 2009). "Improvement of overall survival in advanced stage mantle cell lymphoma". J. Clin. Oncol. 27 (4): 511–8. doi:10.1200/JCO.2008.16.8435. PMID 19075279. [25] The Merck Manual of Geriatrics > Chronic Leukemias (http:/ / www. merck. com/ mkgr/ mmg/ sec9/ ch73/ ch73b. jsp) Retrieved June, 2010 [26] Diviné M, Casassus P, Koscielny S, et al. (December 2005). "Burkitt lymphoma in adults: a prospective study of 72 patients treated with an adapted pediatric LMB protocol". Ann. Oncol. 16 (12): 1928–35. doi:10.1093/annonc/mdi403. PMID 16284057. [27] Kirova YM, Piedbois Y, Haddad E, et al. (May 1999). "Radiotherapy in the management of mycosis fungoides: indications, results, prognosis. Twenty years experience". Radiother Oncol 51 (2): 147–51. doi:10.1016/S0167-8140(99)00050-X. PMID 10435806.

External links • Timeline of discovery and treatment of Hodgkin's Lymphoma (http://www.hodgkinshistory.com/info/ timeline.html) • US lymphoma statistics (http://seer.cancer.gov/statfacts/html/lymph.html) from the United States National Cancer Institute • Hodgkin Lymphoma (http://info.cancerresearchuk.org/cancerstats/types/hodgkinslymphoma/?a=5441) and UK Non-Hodgkin Lymphoma (http://info.cancerresearchuk.org/cancerstats/types/nhl/?a=5441) statistics from the UK • Latest news and research on Lymphoma (http://www.medworm.com/rss/search.php?qu=lymphomas+ lymphoma&t=Lymphoma&f=cancer&r=Any&o=d) • Lymphoma Imaging Appearance – Chest Radiography (http://www.radrounds.com/photo/ lymphoma-chest-radiograph) • Lymphoma Association – Specialist UK charity providing free information and support to patients, their families, friends and carers (http://www.lymphomas.org.uk)

95

Non-Hodgkin lymphoma

96

Non-Hodgkin lymphoma Non-Hodgkin Lymphomas Classification and external resources [1]

ICD-10

C82.

ICD-9

200

ICD-O:

9591/3

OMIM

605027

DiseasesDB

9065

-C85.

[3]

, 202

[2]

[3]

[4]

[5]

MedlinePlus 000581 [6] eMedicine

med/1363

MeSH

D008228

[7]

ped/1343

[8]

[9]

The non-Hodgkin lymphomas (NHLs) are a diverse group of blood cancers that include any kind of lymphoma except Hodgkin's lymphomas.[10] Types of NHL vary significantly in their severity, from indolent to very aggressive. Lymphomas are types of cancer derived from lymphocytes, a type of white blood cell. Lymphomas are treated by combinations of chemotherapy, monoclonal antibodies, immunotherapy, radiation, and hematopoietic stem cell transplantation. Non-Hodgkin lymphomas were classified according to the 1982 Working Formulation which recognizes 16 types. The Working Formulation is now considered obsolete, and the classification is commonly used primarily for statistical comparisons with previous decades. The Working Formulation has been superseded twice. The latest lymphoma classification, the 2008 WHO classification, largely abandoned the "Hodgkin" vs. "Non-Hodgkin" grouping. Instead, it lists over 70 different forms of lymphomas in four broad groups.[11]

History Hodgkin lymphoma (H, Hodgkin disease), described by Thomas Hodgkin in 1832, was the first form of lymphoma described and defined. Other forms were later described and there was a need to classify them. Because Hodgkin lymphoma was much more radiation-sensitive than other forms, its diagnosis was important for oncologists and their patients. Thus, research originally focused on it. The first classification of Hodgkin lymphoma was proposed by Robert J. Luke in 1963. While consensus was rapidly reached on the classification of Hodgkin lymphoma, there remained a large group of very different diseases requiring further classification. The Rappaport classification, proposed by Henry Rappaport in 1956 and 1966, became the first widely accepted classification of lymphomas other than Hodgkin. Following its publication in 1982, the Working Formulation became the standard classification for this group of diseases. It introduced the term non-Hodgkin lymphoma (NHL) and defined three grades of lymphoma. However, NHL consists of 16 different conditions that have little in common with each other. They are grouped by their aggressiveness. Less aggressive non-Hodgkin lymphomas are compatible with a long survival while more aggressive non-Hodgkin lymphomas can be rapidly fatal without treatment. Without further narrowing, the label is of limited usefulness for patients or doctors.

Non-Hodgkin lymphoma

Modern usage of term Nevertheless, the Working Formulation and the NHL category continue to be used by many. To this day, lymphoma statistics are compiled as Hodgkin's vs non-Hodgkin lymphomas by major cancer agencies, including the National Cancer Institute in its SEER program, the Canadian Cancer Society and the IARC.

References [1] http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gc81. htm+ c82 [2] http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gc81. htm+ c85 [3] http:/ / www. icd9data. com/ getICD9Code. ashx?icd9=202 [4] http:/ / www. ncbi. nlm. nih. gov/ omim/ 605027 [5] http:/ / www. diseasesdatabase. com/ ddb9065. htm [6] http:/ / www. nlm. nih. gov/ medlineplus/ ency/ article/ 000581. htm [7] http:/ / www. emedicine. com/ med/ topic1363. htm [8] http:/ / www. emedicine. com/ ped/ topic1343. htm# [9] http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D008228 [10] non-Hodgkin lymphomas (http:/ / www. mercksource. com/ pp/ us/ cns/ cns_hl_dorlands_split. jsp?pg=/ ppdocs/ us/ common/ dorlands/ dorland/ nine/ 000954456. htm) at Dorland's Medical Dictionary [11] ed. by Steven H Swerdlow et al. (2008). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Oxford Univ Pr. ISBN 978-92-8322431-0.

External links • Non-Hodgkin Lymphoma (http://www.cancer.org/docroot/CRI/CRI_2_3x.asp?dt=32) at American Cancer Society • Non-Hodgkins Lymphoma (http://www.cancer.net/patient/Cancer+Types/Lymphoma+-+ Non-Hodgkin?sectionTitle=Overview) from Cancer.net (American Society of Clinical Oncology) • Video and information booklets on non-Hodgkins lymphoma (http://www.lymphomas.org.uk/info/ types-of-lymphoma-non-hodgkin.asp) • non-Hodgkin in Brain (http://rad.usuhs.edu/medpix/kiosk_image.html?pt_id=13381&imageid=52032) MR Scans of Primary Brain Lymphoma • Lymphoma Association – Specialist UK charity providing free information and support to patients, their families, friends and carers (http://www.lymphomas.org.uk)

97

Cluster of differentiation

Cluster of differentiation The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules present on white blood cells, providing targets for immunophenotyping of cells. Physiologically, CD molecules can act in numerous ways, often acting as receptors or ligands (the molecule that activates a receptor) important to the cell. A signal cascade is usually initiated, altering the behavior of the cell (see cell signaling). Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. CD for humans is numbered up to 350 most recently (as of 2009).[1] [2]

Nomenclature The CD nomenclature was proposed and established in the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), which was held in Paris in 1982.[3] [4] This system was intended for the classification of the many monoclonal antibodies (mAbs) generated by different laboratories around the world against epitopes on the surface molecules of leukocytes (white blood cells). Since then, its use has expanded to many other cell types, and more than 320 CD unique clusters and subclusters have been identified. The proposed surface molecule is assigned a CD number once two specific monoclonal antibodies (mAb) are shown to bind to the molecule. If the molecule has not been well-characterized, or has only one mAb, it is usually given the provisional indicator "w" (as in "CDw186"). Cell populations are usually defined using a '+' or a '–' symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. For example, a "CD34+, CD31–" cell is one that expresses CD34, but not CD31. This CD combination typically corresponds to a stem cell, opposed to a fully-differentiated endothelial cell.

98

Cluster of differentiation

99

Immunophenotyping The CD system is commonly used as cell markers in immunophenotyping, allowing cells to be defined based on what molecules are present on their surface. These markers are often used to associate cells with certain immune functions. While using one CD molecule to define populations is uncommon (though a few examples exist), combining markers has allowed for cell types with very specific definitions within the immune system. CD molecules are utilized in cell sorting using various methods including flow cytometry.

CD differentiation

Type of cell

CD markers

stem cells

CD34+, CD31-

all leukocyte groups CD45+ Granulocyte

CD45+, CD15+

Monocyte

CD45+, CD14+

T lymphocyte

CD45+, CD3+

T helper cell

CD45+,CD3+, CD4+

Cytotoxic T cell

CD45+,CD3+, CD8+

B lymphocyte

CD45+, CD19+ or CD45+, CD20+

Thrombocyte

CD45+, CD61+

Natural killer cell

CD16+, CD56+,CD3-

Two commonly-used CD molecules are CD4 and CD8, which are, in general, used as markers for helper and cytotoxic T cells, respectively. These molecules are defined in combination with CD3+, as some other leukocytes also express these CD molecules (some macrophages express low levels of CD4; dendritic cells express high levels of CD8). Human immunodeficiency virus (HIV) binds CD4 and a chemokine receptor on the surface of a T helper cell to gain entry. The number of CD4 and CD8 T cells in blood is often used to monitor the progression of HIV infection.

Cluster of differentiation

Physiological functions While CD molecules are very useful in defining leukocytes, they are not merely markers on the cell surface. While only a fraction of known CD molecules have been thoroughly characterised, most of them have an important function. In the example of CD4 & CD8, these molecules are critical in antigen recognition.

References [1] "HCDM, responsible for HLDA workshop and CD molecules" (http:/ / www. hcdm. org/ MoleculeInformation/ tabid/ 54/ Default. aspx). Human Cell Differentiation Molecules Council (successor to the HLDA Workshops). . Retrieved 2009-06-01. [2] Zola H, Swart B, Banham A, Barry S, Beare A, Bensussan A, Boumsell L, D Buckley C, Bühring HJ, Clark G, Engel P, Fox D, Jin BQ, Macardle PJ, Malavasi F, Mason D, Stockinger H, Yang X. (2007). "CD molecules 2006--human cell differentiation molecules." (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6T2Y-4MGVRPR-1& _user=10& _coverDate=01/ 30/ 2007& _rdoc=1& _fmt=high& _orig=search& _origin=search& _sort=d& _docanchor=& view=c& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=021701503483e59127d440e62353473d& searchtype=a). J Immunol Methods. 318 (1-2): 1–5. doi:10.1016/j.jim.2006.11.001. PMID 17174972. . [3] Bernard A, Boumsell L (1984). "[Human leukocyte differentiation antigens]" (in French). Presse Med 13 (38): 2311–6. PMID 6239187. [4] Fiebig H, Behn I, Gruhn R, Typlt H, Kupper H, Ambrosius H (1984). "[Characterization of a series of monoclonal antibodies against human T cells]" (in German). Allerg Immunol (Leipz) 30 (4): 242–50. PMID 6240938.

External links • Molecule search (http://www.hcdm.org/MoleculeInformation/tabid/54/Default.aspx) maintained by the Human Cell Differentiation Molecules Council (successor to the HLDA Workshops) • Table of CD Antigens (http://www.immunologylink.com/cdantigen.html) • CD list (http://www.sciencegateway.org/resources/prow/index.html) Protein Reviews On The Web • Another list of CD molecules (http://www.expasy.org/cgi-bin/lists?cdlist.txt), at Expasy.org • Yet another list of CD molecules (http://pathologyoutlines.com/cdmarkers.html), at PathologyOutlines.com • Wall charts (http://www.ebioscience.com/ebioscience/litreq.asp) of CD molecules and other cytokines, with colors, arrows from one cell to another, from eBioscience.

100

List of human clusters of differentiation

List of human clusters of differentiation The following is a list of human clusters of differentiation (or CD) molecules. This list is incomplete. CD1

an MHC-like molecule that presents lipid molecules

CD2

a type I transmembrane protein found on thymocytes, T cells, and some natural killer cells that acts as a ligand for CD58 and CD59 and is involved in signal transduction and cell adhesion; expressed in T-cell acute lymphoblastic leukemia and T-cell lymphoma.

CD3

the signaling component of the T cell receptor (TCR) complex

CD4

a co-receptor for MHC Class II; also a receptor used by HIV to enter T cells

CD5

a type I transmembrane protein found on T cells, thymocytes, and some B cells that is a ligand for CD72 and is involved in cellular activation or adhesion; expressed in B-cell chronic lymphocytic leukemia and T-cell lymphoma.

CD6

adhesion molecule linking developing thymus-cells to thymus epithelial cells; co-stimulator to mature T cells

CD7

a type I transmembrane protein found on thymocytes, some T cells, monocytes, natural killer cells, and hemopoietic stem cells; expressed in patients with mycosis fungoides, some patients with T-cell acute lymphoblastic lymphoma, and a few patients with acute nonlymphocytic lymphoma.

CD8

a co-receptor for MHC Class I; also found on a subset of myeloid dendritic cells.

CD9

a member of the Tetraspanin superfamily expressed in a variety of cells, including: pre B cells, eosinophils, basophils and platelets.

CD10

a type II transmembrane protein found on pre-B cells, germinal-center B cells, some neutrophils, kidney cells, T-cell precursors, and epithelial cells that acts as a zinc metalloprotease cleaving peptide bonds on the amino side of hydrophobic amino acids; expressed in acute lymphocytic leukemia and follicular-center-cell lymphomas.

CD11a

InTeGrin, Alpha L (ITGAL), the alpha subunit of LFA-1, a membrane glycoprotein that provides cell-cell adhesion by interaction with ICAM-1

CD11b

InTeGrin Alpha M (ITGAM), the alpha Subunit of Mac-1, a complement receptor ("CR3") consisting of CD11b and CD18.

CD11c

InTeGrin Alpha X (ITGAX), the alpha subunit of (iC3b) receptor 4 (CR4). It is a type I transmembrane protein found on monocytes, macrophages, neutrophils, and some B cells that induces cellular activation and helps trigger neutrophil respiratory burst; expressed in hairy cell leukemias, acute nonlymphocytic leukemias, and some B-cell chronic lymphocytic leukemias. Also one of the defining markers for dendritic cells and hairy cell leukemia cells.

CD12w

phosphoprotein of unknown function present on monocytes, granulocytes, and NK cells and absent from basophils, AML blasts, and bone marrow precursors.

CD13

a zinc metalloproteinase, also known as aminopeptidase N, which is found naturally on myelomonocytic cells from early differentiation through maturity; usually present on acute myeloid leukemia blasts and rarely found in some forms of lymphoma and lymphocytic leukemia

CD14

a membrane protein found on macrophages which binds to bacterial lipopolysaccharide.

CD15

a carbohydrate adhesion molecule (not a protein) that mediates phagocytosis and chemotaxis, found on neutrophils; expressed in patients with Hodgkin disease, some B-cell chronic lymphocytic leukemias, acute lymphoblastic leukemias, and most acute nonlymphocytic leukemias. It is also called Lewis x and SSEA-1 (stage specific embryonic antigen 1) and represents a marker for murine pluripotent stem cells, in which it plays an important role in adhesion and migration of the cells in the preimplantation embryo.

CD16

FcγRIII, a low-affinity Fc receptor for IgG. Found on NK cells, macrophages, and neutrophils.

CDw17

possible role in phagocytosis. Bacteria binding.

CD18

adhesion and signaling in the hematopoietic system.

CD19

B-lymphocyte surface antigen B4

CD20

a type III transmembrane protein found on B cells that forms a calcium channel in the cell membrane allowing for the influx of calcium required for cell activation; expressed in B-cell lymphomas, hairy cell leukemia, and B-cell chronic lymphocytic leukemia. Important for therapy of those diseases, as an antibody against CD20 exists: Rituximab

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List of human clusters of differentiation

CD21

CR2, a type I transmembrane protein found in the cytoplasm of pre-B cells and on the surface of mature B cells, follicular dendritic cells, pharyngeal and cervical epithelial cells, some thymocytes, and some T cells that plays a role in signal transduction; expressed in hairy cell leukemia, B-cell lymphoma, and some T-cell acute lymphocytic leukemias. Receptor for complement (C3d) and Epstein-Barr virus (EBV).

CD22

a sugar binding transmembrane protein that specifically binds sialic acid with an immunoglobulin (Ig) domain located at its N-terminus. It is a member of the immunoglobulin superfamily and the SIGLEC family. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signalling.

CD23

a type II transmembrane protein found on mature B cells, monocytes, activated macrophages, eosinophils, platelets, and dendritic cells that enhances capture and processing of antigen complexed with IgE.

CD24

a glycoprotein expressed at the surface of most B lymphocytes and differentiating neuroblasts. This gene encodes a sialoglycoprotein that is expressed on mature granulocytes and in many B cells. The encoded protein is anchored via a glycosyl phosphatidylinositol (GPI) link to the cell surface. An alignment of this gene's sequence finds genomic locations with similarity on chromosomes 3p26, 15q21, 15q22, 20q11.2 and Yq11.1. Whether transcription, and corresponding translation, occurs at each of these other genomic locations needs to be experimentally determined (source: NCBI). Is also known as Heat Stable Antigen (HSA).

CD25

a type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes that associates with CD122 to form a heterodimer that can act as a high-affinity receptor for IL-2; expressed in most B-cell neoplasms, some acute nonlymphocytic leukemias, and neuroblastomas.

CD26

Membrane-bound protease. T-cell costimulatory molecule. Cell adhesion molecule

CD27

TNF-receptor. Present on the surface of resting memory B cells.

CD28

present on all T-cells, and when matched with the appropriate ligand, labeled B7 which can be either CD80 or CD86, it has costimulatory effect on the T-cell. It is also expressed on Eosinophil granulocytes, especially after tissue infiltration. There its ligation leads to release of potent neurotoxins, IL-2 and IL-13 as well as IFN-γ

CD29

AKA integrin beta-1 - a cell adhesion molecule.

CD30

a type I transmembrane protein present on activated T and B cells that may play a role in cell activation and/or differentiation; expressed in Hodgkin disease, some T-cell lymphomas, and anaplastic large cell lymphomas.

CD31

PECAM-1, a cell adhesion molecule on platelets and endothelial cells

CD32

FcγRII, a receptor for the Fc (constant) region of immunoglobulin G (IgG)

CD33

a marker of unknown function found on immature myeloid cells, including acute myeloid leukemia blasts and mature monocytes

CD34

stem cell marker, adhesion, found on hematopoietic precursors (found in high concentrations in umbilical cord blood), capillary endothelium, and embryonic fibroblasts

CD35

Complement receptor 1 (C3b/C4b receptor)

CD36

Platelet glycoprotein IV or IIIb (GP IV / GP IIIb)

CD37

A leucocyte restricted tetraspanin expressed primarily in B cells, but also found on T cells, Monocytes and Granulocytes.

CD38

involved in ecto-ADP-ribosyl cyclase and cell activation on many hematopoietic, plasma, and B & T activated cells; marker increases with HIV seroconversion, coexpression with CD8 associated with progression (indicates persistent viral stimulation)

CD40

a costimulatory protein found on antigen presenting cells. CD40 combines with CD154 (CD40L) on T cells to induce antibody isotype switching in B cells.

CD41

Integrin subunit αIIb; Gene ITGA2B. Glycoprotein IIb (GPIIb): Component of the integrin αIIbβ3 (GPIIb-IIIa) fibrinogen receptor; [1] major role is in platelet aggregation. Mutations in ITGA2B can be causative for Glanzmann thrombasthenia.

CD42

the platelet Glycoprotein Ib/V/IX complex(GPIb/V/IX). Expressed on platelets and is a late, specific marker of megakaryocyte differentiation. The Glycoprotein Ib/V/IX complex is essential for normal haemostasis; deficiency results in Bernard-Soulier Syndrome, a syndrome of thrombocytopenia and giant platelets.

CD43

CD43 is a sialomucin.

CD44

A family of matrix adhesion molecules formed by alternative mRNA splicing, that adhere to hyaluronate, collagen, laminin, and fibronectin. Helps maintain polarization of epithelial cells. Found on bone marrow stromal cells and many other cells.

CD45

leucocyte common antigen, a type I transmembrane protein present on all hemopoietic cells except erythrocytes that assists in cell activation; expressed in lymphomas, B-cell chronic lymphocytic leukemia, hairy cell leukemia, and acute nonlymphocytic leukemia.

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List of human clusters of differentiation

CD46

Inhibitory complement receptor which is ubiquitously expressed on human cells.

CD47

Membrane protein, which is involved in the increase in intracellular calcium concentration that occurs upon cell adhesion to extracellular matrix.

CD48

CD48 is a human protein encoded by the CD48 gene. It is a universal cell membrane molecule present on all leukocytes.

CD49b

Very late antigen (VLA) alpha 2 chain; found on platelets and activated B and T cells.

CD49c

Very late antigen (VLA) alpha 3 chain; found on nonhematopoietic bone marrow cells. Receptor for collagen, laminin, fibronectin, and thrombospondin.

CD53

A leucocyte restricted tetraspanin expressed by B cells, T cells, dendritic cells, monocytes, NK cells and Granulocytes.

CD54

Intercellular adhesion molecule -1 (ICAM-1): facilitates adhesion between leukocytes to endothelial cells during the immune and inflammatory responses

CD55

Complement Decay-Accelerating Factor (DAF): regulatory factor in one of the three pathways of the immune system complement cascade

CD56

140 kD isoform of NCAM (neural cell adhesion molecule), a marker for natural killer cells and some T-lymphocytes

CD58

a membrane protein present on many hemopoietic cells and fibroblasts that acts as a ligand for CD2 and may be involved in T-cell function.

CD59

Membrane attack complex inhibition factor (MACIF); MAC-inhibitory protein (MAC-IP); Antigen MEM43; Protectin: Immune system complement cascade regulatory factor

CD61

Integrin subunit β3; Gene ITGB3. Glycoprotein IIIa (GPIIIa): Component of the integrin αIIbβ3 (GPIIb-IIIa) fibrinogen receptor; major [1] role is in platelet aggregation. Mutations in ITGB3 can be causative for Glanzmann thrombasthenia.

CD62E

E-selectin is a cell adhesion molecule expressed only on endothelial cells activated by cytokines.

CD62L

L-selectin is a cell adhesion molecule found on leukocytes.

CD62P

P-selectin is a cell adhesion molecule (CAM) found in granules in endothelial cells (cells lining blood vessels) and activated platelets.

CD63

Member of the Tetraspanin family expressed in activated platelets, monocytes and macrophages.

CD68

110 kDa highly glycosylated transmembrane protein which is mainly located in lysosomes. Present in macrophages in many human tissues including Kupffer’s cells and macrophages in the red pulp of the spleen, in lung alveoli, in lamina propria of the gut, and in the bone marrow. Used as immunocytochemical marker for staining of monocytes/macrophages.

CD69

An early activation marker on T cells and NK cells.

CD71

Transferrin receptor, mediates cellular uptake of iron

CD72

Mediator of B-cell - T-cell interactions

CD74

Transmembrane protein that assists and maintains the assembly of MHC-II complexes in the ER until its loaded with peptide in Endosomes. Present in all professional APCs expressing MHC-II. It is more commonly named "Invariant chain" and coded in the HLA-II gene cluster.

CD80

when bound to CD28 on T-cells, can provide the costimulatory effect; also referred to as B7.1, one of the B7 molecules. Causes up-regulation of a high affinity IL-2 receptor allowing T cells to proliferate.

CD81

A tetraspanin expressed in a wide variety of tissues, which plays an important role in B cells as part of the B cell co-receptor complex with CD19, Leu 13 and CD21. Also expressed in T cells, NK cells, Dendritic cells, Monocytes and blood progenitors.

CD82

Member of the tetraspanin family of transmembrane proteins. Broad tissue distribution including B cells, T cells, Granulocytes, Monocytes and CD34+ progenitors.

CD83

a 45 kDa transmembrane glycoprotein of the Ig superfamily. Expressed on cultured dendritic cells, interdigitating, follicular, and circulating dendritic cells as well as some proliferating lymphocyte of all human cell lines. Functionally unclear, but can serve as a useful marker for mature human blood dendritic cells.

CD86

when bound to CD28 on T-cells, can provide the costimulatory effect; also referred to as B7.2, one of the B7 molecules. Causes up-regulation of a high affinity IL-2 receptor allowing T cells to proliferate.

CD87

also referred to as the urokinase type plasminogen activator receptor, provides a binding point for urokinase type plasminogen activator

CD88

C5a receptor

103

List of human clusters of differentiation

CD89

FcalphaRI - receptor for IgA

CD90

Thy-1 Thymus cell antigen.

CD91

Low density lipoprotein (LDL) receptor-related protein 1 (LRP1) (also known as α2-macroglobulin receptor), a major endocytotic receptor with over 35 known ligands including amyloid precursor protein (APP), ApoE, and many proteins involved with protease regulation

CD95

Fas Receptor- receptor for Fas ligand, an extrinsic apoptotic signal

CD96 CD100

also known as semaphorin 4D and is known as a potent proagiogenic molecule.

CD103

a type I transmembrane protein present on intestinal intraepithelial lymphocytes, some circulating leukocytes, and some T cells that facilitates adhesion to epithelia; expressed in hairy cell leukemia and some B-cell chronic lymphocytic leukemias.

CD105

Endoglin, a regulatory component of the TGF-beta receptor-cell complex. Mediates cellular response to TGFbeta.

CD106

VCAM-1; Alpha 4 beta 1 ligand. Adhesion molecule involved in white blood cell migration.

CD109

r150, Gov alloantigen, an accessory receptor of the TGF-beta signaling pathway. Mediates cellular response to TGFbeta. Presents Gov alloantigens and ABH blood antigens.

CD117

c-kit, the receptor for Stem Cell Factor, a glycoprotein that regulates cellular differentiation, particularly in hematopoiesis

CD120

a receptor for Tumour Necrosis Factor, an inflammatory cytokine

CD127

the IL-7 receptor alpha chain

CD133

a hematopoietic and CNS stem cell marker. A 5 transmembrane domain protein, with no known function. Also known as AC133.

CD134

Also known as OX40; A T cell secondary costimulatory molecule which enances proliferation, cytokine production and survival.

CD135

Also known as fms-like tyrosine kinase receptor-3 (Flt3) or fetal liver kinase-2 (Flk2); A cytokine receptor for Flt3 ligand (Flt3L) important in early hematopoiesis.

CD138

a plasma cell-surface glycoprotein, known as syndecan-1. Syndecan functions as the alpha receptor for collagen, fibronectin and thrombospondin.

CD141

Thrombomodulin or BDCA-3, an integral membrane protein. On endothelial cells, it is involved in anticoagulation. It also occurs, with unknown function, on a very rare subtype of dendritic cells.

CD142

Tissue factor, a major initiator of blood-clotting

CD143

Angiotensin-converting enzyme

CD144

VE-Cadherin, a calcium-dependent adhesion molecule at intercellular junctions, found mainly in the vascular endothelium. Recent research indicates that CD144 may be present on some leucocytes as well.

CD147

Neurothelin. An extracellular matrix metalloproteinase inducer.

CD151

Tetraspanin with a wide tissue distribution, including platelets, Megakaryocytes, Granulocytes and erythroleukemia.

CD152

Also called Cytotoxic T-lymphocyte antigen-4 (CTLA-4). Expressed in CD4+ T Lymphocytes but also found in some B Lymphocytes. Binds to CD80 and CD86 receptors with a higher affinity than CD28, and inhibits T cell activation.

CD154

The ligand for CD40. This is a costimulatory molecule that plays many roles, best known for activating B cells but also known to induce the activation of an APC in association with T cell receptor stimulation by MHC molecules on the APC.

CD156

A member of A Disinetgrin And Metalloprotease family ADAM8.

CD158

Killer immunoglobulin-like receptors (KIR) with two extracellular domains, variously expressed on NK cells. CD158a is KIR2DL1, CD158b is KIR2DL3, CD158d is KIR2DL4.

CD163

M130; HbSR; RM3/1 antigen. A glycoprotein endocytic scavenger receptor for haptoglobin-hemoglobin complexes. Found specifically on monocytes/macrophages and some dendritic cells. Involved in anti-iflammatory processes. Soluble form shed upon Toll-like receptor activation.

CD166

activated leukocyte cell adhesion molecule (ALCAM).

CD168

receptor for hyaluronan-mediated motility (RHAMM).

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List of human clusters of differentiation

CD184

CXCR4, Stromal Derived Factor 1 (SDF1). Receptor for the CXC chemokine SDF1. A receptor involved in mesenchymal stem cell homing and migration.

CDw186 CXCR6, a G-protein-coupled receptor for the chemokine CXCL16 CD195

CCR5, a beta chemokine recpeptor to which the natural chemokine ligands RANTES and macrophage inflammatory protein bind. It is commonly used by HIV as a co-receptor to enter its target cells.

CD209

DC-SIGN, C-type lectin receptor found on dendritic cell subsets

CD202a

Tie2, the receptor for angiopoietins, a family of angiogenic factors

CD220

The insulin receptor (INSR) is a transmembrane receptor with intrinsic tyrosine kinase activity whose ligand is insulin. It plays a crucial role in the regulation of various metabolic pathways, as well as regulating aspects of the cell cycle, such as cellular growth, differentiation, and apoptosis. Mutations in the insulin receptor have been found to be associated with both Type 1 and Type 2 Diabetes Mellitus.

CD235a

Glycophorin, a protein on blood cells

CD271

is the p75 Nerve Growth factor receptor (NGFR)

CD303

BDCA-2, a type II transmembrane C-type lectin which is involved in endocytosis of antigens for processing in plasmacytoid dendritic cells. Activation decreases type I interferon production.

CD304

Neuropilin-1 (NP-1) or BDCA-4, has a wide range of functions. On neurons, it is a receptor for axon growth guidance class-3 semaphorins SEMA3A and plexin-1, on endothelial and some tumor cells it is a VEGF165 receptor, and on plasmacytoid dendritic cells it has a similar role to CD303 but does not decrease interferon production upon activation.

CD326

Epithelial cell adhesion molecule (EpCAM) or Tumor-associated calcium signal transducer 1 (TACSTD1). Expressed on epithelial cells and on many tumors; used as a target for anti-tumor drugs. CD326 is expressed in pluripotent stem cells.

References [1] Bennett JS. Structure and function of the platelet integrin alphaIIbbeta3. J Clin Invest 2005; 115:3363-9.

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Mantle cell lymphoma

106

Mantle cell lymphoma Mantle cell lymphoma Classification and external resources

Micrograph showing mantle cell lymphoma (bottom of image) in a biopsy of the terminal ileum. H&E stain. [1]

ICD-10

C85.7

ICD-9

200.4

ICD-O:

M9673/3

eMedicine

med/1361

MeSH

D020522

[2] [3] [4]

[5]

Mantle cell lymphoma (MCL) is one of the rarest of the non-Hodgkin's lymphomas (NHLs), comprising about 6% of NHL cases.[6] There are only about 15,000 patients presently in the U.S. (The prevalence seems to be somewhat higher in Europe.) While it is difficult to treat and seldom considered cured, investigations into better treatments are actively pursued worldwide. Median survival times were about 3 years, but are now estimated as approaching 6 years for new patients. MCL is a subtype of B-cell lymphoma, due to CD5 positive antigen-naive pregerminal center B-cell within the mantle zone that surrounds normal germinal center follicles. MCL cells generally over-express cyclin D1 due to a t(11:14)[7] chromosomal translocation in the DNA. More specifically, the translocation is at t(11;14)(q13;q32).[8] [9] The cause is unknown and no inherited predisposition has been identified. MCL is not communicable. It essentially is an abnormal break and subsequent translocation in a gene that causes the cells to divide too early before becoming capable of helping to fight diseases. In addition, the cells do not die as they should and therefore accumulate in the lymphoid system, including lymph nodes and the spleen, with non-useful cells eventually rendering the system dysfunctional. MCL affected cells proliferate in a nodular or diffuse pattern with two main cytologic variants: typical or blastic. Typical cases are small to intermediate sized cells with irregular nuclei. Blastic (aka blastoid) variants have intermediate to large sized cells with finely dispersed chromatin and are more aggressive in nature.[10]

Mantle cell lymphoma

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Symptoms The ratio of males to females affected is about 4:1. At diagnosis, typical patients are in their 60s and usually present to the oncologist with advanced disease. About half have either fever, heavy night sweats, unexplained weight loss (over 10%) or some combination. Swelling of lymph nodes and spleen are usually present. Bone marrow, liver and GI tract involvement occurs in a very high percentage. It is common for a person to have initially noticed "a bump" on the neck or in the armpits or groin.

Lymph Nodes of the head and neck, from Gray's Anatomy (click image to enlarge)

Diagnosis Diagnosis generally requires stained slides of a surgically removed part of a lymph node. Other methods are also commonly used, including cytogenetics and fluorescence in situ hybridization (FISH). Polymerase chain reaction (PCR) and CER3 clonotypic primers are additional methods, but are less often used. The immunophenotype profile consists of CD5+ (in about 80%),[11] CD10-/+,It is usually CD5+ and CD10-.[12] CD20+, CD23-/+ (though plus in rare cases). Generally cyclin D1 is expressed but it may not be required. The workup for Mantle cell lymphoma is similar to the workup for many indolent lymphomas and certain aggressive lymphomas.

Micrograph of terminal ileum with mantle cell lymphoma (bottom of image). H&E stain.

Mantle cell lymphoma is a systemic disease with frequent involvement of the bone marrow and gastrointestinal tract (generally showing polyposis in the lining). There is also a not-uncommon leukemic phase, marked by presence in the blood. For this reason, both the peripheral blood and bone marrow are evaluated for the presence of malignant cells. Chest, abdominal, and pelvic CT scans are routinely performed. Since mantle cell lymphoma may present a lymphomatous polyposis coli and colon involvement is common, colonoscopy is now considered a routine part of the evaluation. Upper endoscopy and neck CT scan may be helpful in selected cases. In some patients with the blastic variant, lumbar puncture is done to evaluate the spinal fluid for involvement.

Micrograph of terminal ileum with mantle cell lymphoma (bottom of image brown colour). Cyclin D1 immunostain.

Causes Attempts to determine causes of MCL have failed. It is not known what causes the translocation damage to the gene. Exposure to toxins is often mentioned as a possibility. The translocation damage to a gene is required in only one cell for the cancer to begin.

Mantle cell lymphoma

Prognosis The overall 5-year survival rate for MCL is generally 50%[13] (advanced stage MCL) to 70%[14] (for limited-stage MCL). Personal Testimony: I was diagnosed with MCL on November 25, 2005 and did not rush to treatment given the low tumor burden and my overall good health. This was a good course of action as determined by my oncologist and a MCL specialist at University Hospital in Madison, WI. After three years of quarterly CTScans to monitor the disease, it was time for treatment. Initial treatment was Velcade with favorable results. Now treatment is bendimustine with rituxan with very favorable results being seen. This note is being written in March 2011 - I have lived beyond the statistics and consider myself a survivor! Much is changing for the MCL patient and the prognosis is not as hopeless as it was just a few years ago. I'm beyond five years, healthy, and responding very favorably to treatment. Prognosis for individuals with MCL is problematic and indexes do not work as well due to patients presenting with advanced stage disease. Staging is used but is not very informative, since the malignant B-cells can travel freely though the lymphatic system and therefore most patients are at stage III or IV at diagnosis. Prognosis is not strongly affected by staging in MCL and the concept of metastasis does not really apply. The Mantle Cell Lymphoma International Prognostic Index (MIPI) was derived from a data set of 455 advanced stage MCL patients treated in series of clinical trials in Germany/Europe. Of the evaluable population, approximately 18% were treated with high-dose therapy and stem cell transplantation in first remission. The MIPI is able to classify patients into three risk groups: low risk (median survival not reached after median 32 mos follow-up and 5-year OS rate of 60%), intermediate risk (median survival 51 months) and high risk (median survival 29 months). In addition to the 4 independent prognostic factors included in the model, the cell proliferation index (Ki-67) was also shown to have additional prognostic relevance. When the Ki67 is available, a biologic MIPI can be calculated.[15] (The letter A after the Roman numeral indicates that normal external symptoms are not present. The Letter B indicates the symptoms are present and hence they are called “B Symptoms”.) MCL is one of the few NHLs that can cross the boundary into the brain, yet it can be treated in that event. There are a number of prognostic indicators that have been studied. There is not universal agreement on their importance or usefulness in prognosis. Ki-67 is an indicator of how fast cells mature and is expressed in a range from about 10% to 90%. The lower the percentage, the lower the speed of maturity, and the more indolent the disease. Katzenberger et al. Blood 2006;107:3407 graphs survival versus time for subsets of patients with varying Ki-67 indices. He shows median survival times of about one year for 61-90% Ki-67 and nearly 4 years for 5-20% Ki-67 index. MCL cell types can aid in prognosis in a subjective way. Blastic is a larger cell type. Diffuse is spread through the node. Nodular are small groups of collected cells spread through the node. Diffuse and nodular are similar in behavior. Blastic is faster growing and it is harder to get long remissions. Some thought is that given a long time, some non-blastic MCL transforms to blastic. Although survival of most blastic patients is shorter, some data shows that 25% of blastic MCL patients survive to 5 years. That is longer than diffuse type and almost as long as nodular (almost 7 yrs). Beta-2 microglobulin is another risk factor in MCL used primarily for transplant patients. Values less than 3 have yielded 95% overall survival to 6 yrs for auto SCT where over 3 yields a median of 44 mos overall survival for auto SCT (Khouri 03). This is not yet fully validated. Testing for high levels of LDH in NHL patients is useful because LDH is released when body tissues break down for any reason. While it cannot be used as a sole means of diagnosing NHL, it is a surrogate for tracking tumor burden in those diagnosed by other means. The normal range is approximately 100-190.

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Mantle cell lymphoma

Key diagnostics for MCL • CT Scan - Computerized tomography scan yields images of part or whole body. Gives a large number of slices on X-ray image. • PET Scan - Generally of the whole body, shows a three-dimensional image of where previously injected radioactive glucose is metabolized at a rapid rate. Faster-than-average metabolism shows as a black area and indicates that cancer is likely present. Metabolism of radioactive glucose may give a false positive, particularly if the patient has exercised before the test. PET scans are much more effective when the information from them is integrated with that from a CT scan to show more precisely where the cancer activity is located and to more accurately measure the size of tumors.

Treatments There are no proven standards of treatment for MCL, and not even consensus among specialists on how to treat it optimally. Many regimens are available and often get good response rates, but patients almost always get disease progression after chemotherapy. Each relapse is typically more difficult to treat, and relapse is generally faster. Fortunately, regimens are available that will treat relapse, and new approaches are under test. Because of the aforementioned factors, many MCL patients enroll in clinical trials to get the latest treatments. There are four classes of treatments currently in general use: chemotherapy, immune based therapy, radioimmunotherapy and new biologic agents. The phases of treatment are generally: frontline, following diagnosis, consolidation, after frontline response (to prolong remissions), and relapse. Relapse is usually experienced multiple times.

Chemotherapy Chemotherapy is widely used as frontline treatment, and often is not repeated in relapse due to side effects. Alternate chemotherapy is sometimes used at first relapse. For frontline treatment, CHOP with rituximab (Rituxan, Mabthera) is the most common chemotherapy, and often given as outpatient by IV. A stronger chemotherapy with greater side effects (mostly hematologic) is HyperCVAD, often given as in-patient, with rituximab and generally to fitter patients (some of which are over 65). HyperCVAD is becoming popular and showing promising results, especially with rituximab. It can be used on some elderly (over 65) patients, but seems only beneficial when the baseline Beta-2-MG blood test was normal. It is showing better complete remissions (CR) and progression free survival (PFS) than CHOP regimens. Another chemotherapy class is fludarabine monotherapy, sometimes combined with cyclophosphamide and mitoxantrone, usually with rituximab. Cladribine and clofarabine are two other drugs being investigated in MCL. Cytotoxic chemotherapies, including bendamustin, are being studied alone and with similar combinations. A relatively new regimen that uses old drugs is PEP-C, which includes relatively small, daily doses of prednisone, etoposide, procarbazine, and cyclophosphamide, taken orally, has proven effective for relapsed patients. According to John Leonard M.D., a key researcher/proponent of PEP-C, may have anti-angiogenetic properties,[16] [17] something that he and his colleagues are testing through an ongoing drug trial.[18] Another approach involves using very high doses of chemotherapy, sometimes combined with total body irradiation (TBI), in an attempt to destroy all evidence of the disease. The downside to this is the destruction of the patients' entire immune system as well, requiring rescue by transplantation of a new immune system (Hematopoietic stem cell transplantation), using either ones' own previously treated and stored stem cells (an autologous stem cell transplant), or those from a matched donor (an allogeneic stem cell transplant). A presentation at the December 2007 American Society of Hematology (ASH) conference by Christian Geisler, chairman of the Nordic Lymphoma Group[19] (Copenhagen, Denmark), claimed that according to trial results, mantle cell lymphoma is potentially curable with very intensive chemo-immunotherapy followed by a stem cell transplant, when treated upon first presentation of the disease.[20] [21]

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Mantle cell lymphoma

Immunotherapy Immune-based therapy is dominated now by the oft used and effective rituximab monoclonal antibody, sold under the trade name Rituxan (or as Mabthera in Europe and Australia). Some say it is a landmark medicine. It can have good activity against MCL alone but especially in combination with chemotherapies to prolong response duration. Rituximab essentially tags the cancer cells for destruction by the body. There are newer variations on monoclonal antibodies combined with radioactive molecules known as Radioimmunotherapy (RIT). These include Zevalin and Bexxar. Rituximab has also been used in small numbers of patients in combination with thalidomide with some effect.[22] It will be fruitful to investigate the use of Bexxar as first line treatment. This treatment appears to be less tiring for patients, since it consists of two injections at a distance of a week, while most other treatments consist of 6-8 cycles of chemotherapy combined with Rituximab. In contrast to these antibody-based 'passive' immunotherapies, the field of 'active' immunotherapy tries to reprogram a patient's immune system to specifically recognize and eliminate their own tumor cells. Examples of active immunotherapy include cancer vaccines, adoptive cell transfer, and immunotransplant, which combines vaccination and autologous stem cell transplant. Though no active immunotherapies are currently a standard of care, numerous clinical trials are ongoing.[23] [24] [25]

Targeted therapy New targeted agents include the proteasome inhibitor Velcade and mTor inhibitors such as temsirolimus.

Epidemiology Of all cancers involving the same class of blood cell, 6% of cases are mantle cell lymphoma.[26]

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gc81. htm+ c857 http:/ / www. icd9data. com/ getICD9Code. ashx?icd9=200. 4 http:/ / www. progenetix. net/ progenetix/ I96733/ http:/ / www. emedicine. com/ med/ topic1361. htm http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D020522 Mantle Cell Lymphoma (http:/ / www. leukemia-lymphoma. org/ attachments/ National/ br_1172589724. pdf) t(11;14)(q13;q32) (http:/ / atlasgeneticsoncology. org/ Anomalies/ t1114ID2021. html) Li JY, Gaillard F, Moreau A, et al. (May 1999). "Detection of translocation t(11;14)(q13;q32) in mantle cell lymphoma by fluorescence in situ hybridization" (http:/ / ajp. amjpathol. org/ cgi/ pmidlookup?view=long& pmid=10329598). Am. J. Pathol. 154 (5): 1449–52. doi:10.1016/S0002-9440(10)65399-0. PMC 1866594. PMID 10329598. . [9] Barouk-Simonet E, Andrieux J, Copin MC, et al. (2002). "TPA stimulation culture for improved detection of t(11;14)(q13;q32) in mantle cell lymphoma" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ S000339950201122X). Ann. Genet. 45 (3): 165–8. doi:10.1016/S0003-3995(02)01122-X. PMID 12381451. . [10] Mantle Cell Lymphoma: An Update for Clinicians (http:/ / www. medscape. com/ viewarticle/ 548587_2) [11] Stanford School of Medicine: "Mantle Cell Lymphoma, Differential Diagnosis" (http:/ / surgpathcriteria. stanford. edu/ bcell/ mantle/ differentialdiagnosis. html#t1) [12] Barekman CL, Aguilera NS, Abbondanzo SL (July 2001). "Low-grade B-cell lymphoma with coexpression of both CD5 and CD10. A report of 3 cases" (http:/ / journals. allenpress. com/ jrnlserv/ ?request=get-abstract& issn=0003-9985& volume=125& page=951). Arch. Pathol. Lab. Med. 125 (7): 951–3. doi:10.1043/0003-9985(2001)1252.0.CO;2. PMID 11419985. . [13] Most recent values in: Herrmann A, Hoster E, Zwingers T, et al. (February 2009). "Improvement of overall survival in advanced stage mantle cell lymphoma". J. Clin. Oncol. 27 (4): 511–8. doi:10.1200/JCO.2008.16.8435. PMID 19075279. [14] Leitch HA, Gascoyne RD, Chhanabhai M, Voss NJ, Klasa R, Connors JM (October 2003). "Limited-stage mantle-cell lymphoma". Ann. Oncol. 14 (10): 1555–61. doi:10.1093/annonc/mdg414. PMID 14504058. [15] Hoster et al. (2008). "A new prognostic index (MIPI) for patients with advanced-stage mantle cell lymphoma". Blood 111 (2): 558–565. doi:10.1182/blood-2007-06-095331. PMID 17962512. [16] http:/ / www. asco. org/ ASCO/ Abstracts+ & + Virtual+ Meeting/ Abstracts?& vmview=abst_detail_view& confID=23& abstractID=104642

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Mantle cell lymphoma [17] http:/ / www. mantlecelllymphoma. org/ default. asp?pgid=122 [18] http:/ / clinicaltrials. gov/ ct2/ show/ NCT00151281?cond=%22Lymphoma%2C+ Mantle-Cell%22& rank=55 [19] http:/ / www. nordic-lymphoma. org/ mcl2eng. htm [20] http:/ / www. docguide. com/ news/ content. nsf/ news/ 852571020057CCF6852573AD006260EB?OpenDocument& id=48DDE4A73E09A969852568880078C249& c=Lymphomas& count=10 [21] http:/ / www. abstracts2view. com/ hem07/ view. php?nu=HEM07L1_6026& terms= [22] Kaufmann, Hannes; Markus Raderer, Stefan Wöhrer, Andreas Püspök, Alexander Bankier, Christoph Zielinski, Andreas Chott, Johannes Drach (15 October 2004). "Antitumor activity of rituximab plus thalidomide in patients with relapsed/refractory mantle cell lymphoma" (http:/ / bloodjournal. hematologylibrary. org/ cgi/ reprint/ 104/ 8/ 2269) (PDF). Blood 104 (8): 2269–71. doi:10.1182/blood-2004-03-1091. PMID 15166030. . Retrieved 2008-02-13. [23] NCT00101101 (http:/ / clinicaltrials. gov/ ct2/ show/ NCT00101101) [24] NCT00020215 (http:/ / clinicaltrials. gov/ ct2/ show/ NCT00020215) [25] NCT00490529 (http:/ / clinicaltrials. gov/ ct2/ show/ NCT00490529) [26] Turgeon, Mary Louise (2005). Clinical hematology: theory and procedures. Hagerstown, MD: Lippincott Williams & Wilkins. pp. 283. ISBN 0-7817-5007-5. "Frequency of lymphoid neoplasms. (Source: Modified from WHO Blue Book on Tumour of Hematopoietic and Lymphoid Tissues. 2001, p. 2001.)"

External links • A Brief Review of the Biology and Pathophysiology of Mantle Cell Lymphoma (http://www. hememalignancies.com/publication/mil_v7n2/article2.php) • Mantle Cell Lymphoma Consortium (http://mantlecelllymphoma.org/) • Oral combination chemotherapy with Pep-C (C3) for mantle cell lymphoma (MCL): Daily prednisone, etoposide, procarbazine and cyclophosphamide (http://www.asco.org/ASCO/Abstracts+&+Virtual+Meeting/ Abstracts?&vmview=abst_detail_view&confID=23&abstractID=104642). ASCO, 2003 • Oral combination chemotherapy for refractory/relapsed lymphoma with the PEP-C (C3) regimen (daily prednisone, etoposide, procarbazine, cyclophosphamide): Low-dose continuous metronomic multidrug therapy. (http://www.asco.org/ASCO/Abstracts+&+Virtual+Meeting/Abstracts?&vmview=abst_detail_view& confID=47&abstractID=36317) 2007 ASCO Annual Meeting

MultiMedia • CME on MCL (http://www.clinicalcareoptions.com/Oncology/Treatment Updates/Lymphoproliferative Malignancies.aspx) by Andre Goy, MD • archived audiocasts at Leukemia & Lymphoma Society (http://www.leukemia-lymphoma.org/ all_page?item_id=65748)

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Treatments of Neoplasms Chemotherapy Chemotherapy, in the most simple sense, is the treatment of an ailment by chemicals[1] especially by killing micro-organisms or cancerous cells. In popular usage, it refers to antineoplastic drugs used to treat cancer or the combination of these drugs into a cytotoxic standardized treatment regimen. In its non-oncological use, the term may also refer to antibiotics (antibacterial chemotherapy). In that sense, the first modern chemotherapeutic agent was arsphenamine, an arsenic compound discovered in 1909 and used to treat syphilis. This was later followed by sulfonamides (sulfa drugs) and penicillin. Most commonly, chemotherapy acts by killing cells that divide rapidly, one of the main properties of most cancer cells. This means that it also harms cells that divide rapidly under normal circumstances: cells in the bone marrow, digestive tract and hair follicles; this results in the most common side effects of chemotherapy : myelosuppression (decreased production of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

A woman being treated with docetaxel chemotherapy for breast cancer. Cold mittens and wine coolers are placed on her hands and feet to reduce harm to her nails.

Other uses of cytostatic chemotherapy agents (including the ones mentioned below) are the treatment of autoimmune diseases such as multiple sclerosis, dermatomyositis, polymyositis, lupus, rheumatoid arthritis (See DMARDs) and the suppression of transplant rejections (see immunosuppression). Newer anticancer drugs act directly against abnormal proteins in cancer cells; this is termed targeted therapy.

History The use of minerals and plant-based medicines are believed to date back to prehistoric medicine. The first use of drugs to treat cancer, however, was in the early 20th century, although it was not originally intended for that purpose. Mustard gas was used as a chemical warfare agent during World War I and was studied further during World War II. During a military operation in World War II, a group of people were accidentally exposed to mustard gas and were later found to have very low white blood cell counts.[2] It was reasoned that an agent that damaged the rapidly growing white blood cells might have a similar effect on cancer. Therefore, in the 1940s, several patients with advanced lymphomas (cancers of certain white blood cells) were given the drug by vein, rather than by breathing the irritating gas. Their improvement, although temporary, was remarkable.[3] [4] That experience led researchers to look for other substances that might have similar effects against cancer. As a result, many other drugs have been developed to treat cancer, and drug development since then has exploded into a multibillion-dollar industry, although the principles and limitations of chemotherapy discovered by the early researchers still apply.[5]

Chemotherapy

Principles Cancer is the uncontrolled growth of cells coupled with malignant behavior: invasion and metastasis. Cancer is thought to be caused by the interaction between genetic susceptibility and environmental toxins. In the broad sense, most chemotherapeutic drugs work by impairing mitosis (cell division), effectively targeting fast-dividing cells. As these drugs cause damage to cells they are termed cytotoxic. Some drugs cause cells to undergo apoptosis (so-called "self programmed cell death"). Scientists have yet to identify specific features of malignant and immune cells that would make them uniquely targetable (barring some recent examples, such as the Philadelphia chromosome as targeted by imatinib). This means that other fast-dividing cells, such as those responsible for hair growth and for replacement of the intestinal epithelium (lining), are also often affected. However, some drugs have a better side effect profile than others, enabling doctors to adjust treatment regimens to the advantage of patients in certain situations. As chemotherapy affects cell division, tumors with high growth fractions (such as acute myelogenous leukemia and the aggressive lymphomas, including Hodgkin's disease) are more sensitive to chemotherapy, as a larger proportion of the targeted cells are undergoing cell division at any time. Malignancies with slower growth rates, such as indolent lymphomas, tend to respond to chemotherapy much more modestly. Drugs affect "younger" tumors (i.e., more differentiated) more effectively, because mechanisms regulating cell growth are usually still preserved. With succeeding generations of tumor cells, differentiation is typically lost, growth becomes less regulated, and tumors become less responsive to most chemotherapeutic agents. Near the center of some solid tumors, cell division has effectively ceased, making them insensitive to chemotherapy. Another problem with solid tumors is the fact that the chemotherapeutic agent often does not reach the core of the tumor. Solutions to this problem include radiation therapy (both brachytherapy and teletherapy) and surgery. Over time, cancer cells become more resistant to chemotherapy treatments. Recently, scientists have identified small pumps on the surface of cancer cells that actively move chemotherapy from inside the cell to the outside. Research on p-glycoprotein and other such chemotherapy efflux pumps, is currently ongoing. Medications to inhibit the function of p-glycoprotein are undergoing testing as of June, 2007 to enhance the efficacy of chemotherapy.

Treatment schemes There are a number of strategies in the administration of chemotherapeutic drugs used today. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. Combined modality chemotherapy is the use of drugs with other cancer treatments, such as radiation therapy or surgery. Most cancers are now treated in this way. Combination chemotherapy is a similar practice that involves treating a patient with a number of different drugs simultaneously. The drugs differ in their mechanism and side effects. The biggest advantage is minimising the chances of resistance developing to any one agent. In neoadjuvant chemotherapy (preoperative treatment) initial chemotherapy is designed to shrink the primary tumour, thereby rendering local therapy (surgery or radiotherapy) less destructive or more effective. Adjuvant chemotherapy (postoperative treatment) can be used when there is little evidence of cancer present, but there is risk of recurrence. This can help reduce chances of developing resistance if the tumour does develop. It is also useful in killing any cancerous cells which have spread to other parts of the body. This is often effective as the newly growing tumours are fast-dividing, and therefore very susceptible. Palliative chemotherapy is given without curative intent, but simply to decrease tumor load and increase life expectancy. For these regimens, a better toxicity profile is generally expected. All chemotherapy regimens require that the patient be capable of undergoing the treatment. Performance status is often used as a measure to determine whether a patient can receive chemotherapy, or whether dose reduction is required. Because only a fraction of the cells in a tumor die with each treatment (fractional kill), repeated doses must

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Chemotherapy be administered to continue to reduce the size of the tumor.[6] Current chemotherapy regimens apply drug treatment in cycles, with the frequency and duration of treatments limited by toxicity to the patient.[7]

Types The majority of chemotherapeutic drugs can be divided in to alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents.[8] All of these drugs affect cell division or DNA synthesis and function in some way. Some newer agents do not directly interfere with DNA. These include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatinib mesylate (Gleevec or Glivec), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors). These are examples of targeted therapies. In addition, some drugs that modulate tumor cell behaviour without directly attacking those cells may be used. Hormone treatments fall into this category. Where available, Anatomical Therapeutic Chemical Classification System codes are provided for the major categories.

Alkylating agents (L01A) Alkylating agents are so named because of their ability to alkylate many nucleophilic functional groups under conditions present in cells. Cisplatin and carboplatin, as well as oxaliplatin, are alkylating agents. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules.[8] Other agents are mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide.[8] They work by chemically modifying a cell's DNA.

Anti-metabolites (L01B) Anti-metabolites masquerade as purines ((azathioprine, mercaptopurine)) or pyrimidines—which become the building blocks of DNA. They prevent these substances from becoming incorporated in to DNA during the "S" phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. Due to their efficiency, these drugs are the most widely used cytostatics.

Plant alkaloids and terpenoids (L01C) These alkaloids are derived from plants and block cell division by preventing microtubule function. Microtubules are vital for cell division, and, without them, cell division cannot occur. The main examples are vinca alkaloids and taxanes. Vinca alkaloids (L01CA) Vinca alkaloids bind to specific sites on tubulin, inhibiting the assembly of tubulin into microtubules (M phase of the cell cycle). They are derived from the Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea). The vinca alkaloids include: • Vincristine • Vinblastine • Vinorelbine • Vindesine

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Chemotherapy Podophyllotoxin (L01CB) Podophyllotoxin is a plant-derived compound which is said to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide. They prevent the cell from entering the G1 phase (the start of DNA replication) and the replication of DNA (the S phase). The exact mechanism of its action is not yet known. The substance has been primarily obtained from the American Mayapple (Podophyllum peltatum). Recently it has been discovered that a rare Himalayan Mayapple (Podophyllum hexandrum) contains it in a much greater quantity, but, as the plant is endangered, its supply is limited. Studies have been conducted to isolate the genes involved in the substance's production, so that it could be obtained recombinantly. Taxanes (L01CD) The prototype taxane is the natural product paclitaxel, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase.

Topoisomerase inhibitors (L01CB and L01XX) Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling. • Some type I topoisomerase inhibitors include camptothecins: irinotecan and topotecan. • Examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide. These are semisynthetic derivatives of epipodophyllotoxins, alkaloids naturally occurring in the root of American Mayapple (Podophyllum peltatum).

Antineoplastics (L01D) These include the immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin and others.

Newer and experimental approaches Isolated infusion approaches Isolated limb perfusion (often used in melanoma), or isolated infusion of chemotherapy into the liver or the lung have been used to treat some tumours. The main purpose of these approaches is to deliver a very high dose of chemotherapy to tumor sites without causing overwhelming systemic damage. These approaches can help control solitary or limited metastases, but they are by definition not systemic, and, therefore, do not treat distributed metastases or micrometastases.

Targeted delivery mechanisms Specially targeted delivery vehicles aim to increase effective levels of chemotherapy for tumor cells while reducing effective levels for other cells. This should result in an increased tumor kill and/or reduced toxicity. Specially targeted delivery vehicles have a differentially higher affinity for tumor cells by interacting with tumor-specific or tumour-associated antigens. In addition to their targeting component, they also carry a payload - whether this is a traditional chemotherapeutic agent, or a radioisotope or an immune stimulating factor. Specially targeted delivery vehicles vary in their stability, selectivity, and choice of target, but, in essence, they all aim to increase the maximum effective dose that can be delivered to the tumor cells. Reduced systemic toxicity means that they can also be used in sicker patients, and that they can carry new chemotherapeutic agents that would have been far too toxic to deliver via traditional systemic

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Chemotherapy approaches.

Nanoparticles Nanoparticles have emerged as a useful vehicle for poorly soluble agents such as paclitaxel. Protein-bound paclitaxel (e.g., Abraxane) or nab-paclitaxel was approved by the U.S. Food and Drug Administration (FDA) in January 2005 for the treatment of refractory breast cancer. This formulation of paclitaxel uses human albumin as a vehicle and not the Cremophor vehicle used in Taxol. Nanoparticles made of magnetic material can also be used to concentrate agents at tumour sites using an externally applied magnetic field.

Dosage Dosage of chemotherapy can be difficult: If the dose is too low, it will be ineffective against the tumor, whereas, at excessive doses, the toxicity (side effects, neutropenia) will be intolerable to the patient. This has led to the formation of detailed "dosing schemes" in most hospitals, which give guidance on the correct dose and adjustment in case of toxicity. In immunotherapy, they are in principle used in smaller dosages than in the treatment of malignant diseases. In most cases, the dose is adjusted for the patient's body surface area, a measure that correlates with blood volume. The BSA is usually calculated with a mathematical formula or a nomogram, using a patient's weight and height, rather than by direct measurement.

Delivery Most chemotherapy is delivered intravenously, although a number of agents can be administered orally (e.g., melphalan, busulfan, capecitabine). In some cases, isolated limb perfusion (often used in melanoma), or isolated infusion of chemotherapy into the liver or the lung have been used. The main purpose of these approaches is to deliver a very high dose of chemotherapy to tumour sites without causing overwhelming systemic damage. Depending on the patient, the cancer, the stage of cancer, the type of chemotherapy, and the dosage, intravenous chemotherapy may be given on either an inpatient or an outpatient basis. For continuous, frequent or prolonged intravenous chemotherapy administration, various systems may be surgically inserted into the vasculature to maintain access. Commonly used systems are the Hickman line, the Port-a-Cath or the PICC line. These have a lower infection risk, are much less prone to phlebitis or extravasation, and abolish the need for repeated insertion of peripheral cannulae. Harmful and lethal toxicity from chemotherapy limits the dosage of chemotherapy that can be given. Some tumors can be destroyed by sufficiently high doses of chemotherapeutic agents. However, these high doses cannot be given because they would be fatal to the patient.

Adverse effects Chemotherapeutic techniques have a range of side effects that depend on the type of medications used. The most common medications mainly affect the fast-dividing cells of the body, such as blood cells and the cells lining the mouth, stomach, and intestines. Common side effects include:[9] • Depression of the immune system, which can result in potentially fatal infections. Although patients are encouraged to wash their hands, avoid sick people, and to take other infection-reducing steps, about 85% of infections are due to naturally occurring microorganisms in the patient's own gut and skin.[10] This may manifest as systemic infections, such as sepsis, or as localized outbreaks, such as shingles. Sometimes, chemotherapy treatments are postponed because the immune system is suppressed to a critically low level. • Fatigue. The treatment can be physically exhausting for the patient, who might already be very tired from cancer-related fatigue. It may produce mild to severe anemia. Treatments to mitigate anemia include hormones to

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Chemotherapy boost blood production (erythropoietin), iron supplements, and blood transfusions. • Tendency to bleed easily. Medications that kill rapidly dividing cells or blood cells are likely to reduce the number of platelets in the blood, which can result in bruises and bleeding. Extremely low platelet counts may be temporarily boosted through platelet transfusions. Sometimes, chemotherapy treatments are postponed to allow platelet counts to recover. • Gastrointestinal distress. Nausea and vomiting are common side effects of chemotherapeutic medications that kill fast-dividing cells. This can also produce diarrhea or constipation. Malnutrition and dehydration can result when the patient doesn't eat or drink enough, or when the patient vomits frequently, because of gastrointestinal damage. This can result in rapid weight loss, or occasionally in weight gain, if the patient eats too much in an effort to allay nausea or heartburn. Weight gain can also be caused by some steroid medications. These side effects can frequently be reduced or eliminated with antiemetic drugs. Self-care measures, such as eating frequent small meals and drinking clear liquids or ginger tea, are often recommended. This is a temporary effect, and frequently resolves within a week of finishing treatment. • Hair loss. Some medications that kill rapidly dividing cells cause dramatic hair loss; other medications may cause hair to thin. These are temporary effects: hair usually starts growing back a few weeks after the last treatment, sometimes with a tendency to curl that may be called a "chemo perm". Damage to specific organs may occur, with resultant symptoms: • • • • •

Cardiotoxicity (heart damage) Hepatotoxicity (liver damage) Nephrotoxicity (kidney damage) Ototoxicity (damage to the inner ear), producing vertigo Encephalopathy (brain dysfunction)

Immunosuppression and myelosuppression Virtually all chemotherapeutic regimens can cause depression of the immune system, often by paralysing the bone marrow and leading to a decrease of white blood cells, red blood cells, and platelets. The latter two, when they occur, are improved with blood transfusion. Neutropenia (a decrease of the neutrophil granulocyte count below 0.5 x 109/litre) can be improved with synthetic G-CSF (granulocyte-colony stimulating factor, e.g., filgrastim, lenograstim). In very severe myelosuppression, which occurs in some regimens, almost all the bone marrow stem cells (cells that produce white and red blood cells) are destroyed, meaning allogenic or autologous bone marrow cell transplants are necessary. (In autologous BMTs, cells are removed from the patient before the treatment, multiplied and then re-injected afterwards; in allogenic BMTs the source is a donor.) However, some patients still develop diseases because of this interference with bone marrow. In Japan the government has approved the use of some medicinal mushrooms like Trametes versicolor, to counteract depression of the immune system in patients undergoing chemotherapy.[11]

Nausea and vomiting Chemotherapy-induced nausea and vomiting (CINV) is common with many treatments and some forms of cancer. However, some chemotherapy regimens do not have this side effect, and very effective drugs to stop or noticeably reduce this adverse effect are available. A class of drugs called 5-HT3 antagonists are the most effective antiemetics and constitute the single greatest advance in the management of nausea and vomiting in patients with cancer. These drugs block one or more of the nerve signals that cause nausea and vomiting. During the first 24 hours after chemotherapy, the most effective approach appears to be blocking the 5-HT3 nerve signal. Approved 5-HT3 inhibitors include dolasetron, granisetron, and ondansetron (Zofran). The newest 5-HT3 inhibitor, palonosetron, also prevents delayed nausea and vomiting,

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Chemotherapy which occurs during the 2–5 days after treatment. Since some patients have trouble swallowing pills, these drugs are often available by injection, as orally disintegrating tablets, or as transdermal patchs. The substance P inhibitor aprepitant, which became available in 2005, is also effective in controlling the nausea of cancer chemotherapy.[12] Some studies[13] and patient groups say that the use of cannabinoids derived from marijuana during chemotherapy greatly reduces the associated nausea and vomiting, and enables the patient to eat. Some synthetic derivatives of the active substance in marijuana (Tetrahydrocannabinol or THC) such as Marinol may be practical for this application. Natural marijuana, known as medical cannabis is also used and recommended by some oncologists, though its use is regulated and not legal everywhere.[14]

Secondary neoplasm Development of secondary neoplasia after successful chemotherapy and/or radiotherapy treatment can occur. The most common secondary neoplasm is secondary acute myeloid leukemia, which develops primarily after treatment with alkylating agents or topoisomerase inhibitors.[15] Survivors of childhood cancer are more than 13 times as likely to get a secondary neoplasm during the 30 years after treatment than the general population.[16] Not all of this increase can be attributed to chemotherapy.

Infertility Some types of chemotherapy are gonadotoxic and may cause infertility.[17] Chemotherapies with high risk include procarbazine and other alkylating drugs such as cyclophosphamide, ifosfamide, busulfan, melphalan, chlorambucil and chlormethine.[17] Drugs with medium risk include doxorubicin and platinum analogs such as cisplatin and carboplatin.[17] On the other hand, therapies with low risk of gonadotoxicity include plant derivatives such as vincristine and vinblastine, antibiotics such as bleomycinand dactinomycin and antimetabolites such as methotrexate, mercaptopurine and 5-fluoruracil.[17] Patients may choose between several methods of fertility preservation prior to chemotherapy, including cryopreservation of semen, ovarian tissue, oocytes or embryos.[18] As more than half of cancer patients are elderly, this adverse effect is only relevant for a minority of patients.

Other side effects In particularly large tumors, such as large lymphomas, some patients develop tumor lysis syndrome from the rapid breakdown of malignant cells. Although prophylaxis is available and is often initiated in patients with large tumors, this is a dangerous side effect that can lead to death if left untreated. Less common side effects include pain, red skin (erythema), dry skin, damaged fingernails, a dry mouth (xerostomia), water retention, and sexual impotence. Some medications can trigger allergic or pseudoallergic reactions. Some patients report fatigue or non-specific neurocognitive problems, such as an inability to concentrate; this is sometimes called post-chemotherapy cognitive impairment, referred to as "chemo brain" by patients' groups.[19] Specific chemotherapeutic agents are associated with organ-specific toxicities, including cardiovascular disease (e.g., doxorubicin), interstitial lung disease (e.g., bleomycin) and occasionally secondary neoplasm (e.g., MOPP therapy for Hodgkin's disease).

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Efficacy Chemotherapy is highly effective in some cancers, useless in others, and unnecessary in still others. Taking all forms of cancer together, people who receive chemotherapy increase their odds of living five years after diagnosis by about two percentage points (e.g., from about 61% being alive after five years to about 63% of them being alive after five years).[20] However, this overall rate obscures the wide variation. Cytotoxic chemotherapy produces much larger gains for some forms of cancer, including testicular cancer (about 40% of the men who live five years after diagnosis are alive because of chemotherapy), lymphomas (about 13%), and cervical cancer (12%).[20] By contrast, chemotherapy is essentially useless in other cancers, including prostate cancer, melanoma of the skin, multiple myeloma, bladder cancer, kidney cancer, and pancreatic cancer: people who receive chemotherapy for these conditions are just as likely to die within five years as people who do not.[20] Chemotherapy only slightly improves survival for some of the most common forms of cancer, including breast cancers (1.5%) and lung cancers (1.5%).[20]

In other animals Chemotherapy is used in veterinary medicine similar to in human medicine.[21]

References [1] chemotherapy (http:/ / www. mercksource. com/ pp/ us/ cns/ cns_hl_dorlands_split. jsp?pg=/ ppdocs/ us/ common/ dorlands/ dorland/ two/ 000020054. htm) at Dorland's Medical Dictionary [2] Hirsch J (September 2006). "An anniversary for cancer chemotherapy". JAMA 296 (12): 1518–20. doi:10.1001/jama.296.12.1518. PMID 17003400. [3] Goodman LS, Wintrobe MM, Dameshek W, Goodman MJ, Gilman A, McLennan MT. (1946). "Nitrogen mustard therapy". JAMA 132 (3): 126–132. doi:10.1001/jama.1946.02870380008004. [4] Goodman LS, Wintrobe MM, Dameshek W, Goodman MJ, Gilman A, McLennan MT. (1984). "Landmark article Sept. 21, 1946: Nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(beta-chloroethyl)amine hydrochloride for Hodgkin's disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. By Louis S. Goodman, Maxwell M. Wintrobe, William Dameshek, Morton J. Goodman, Alfred Gilman and Margaret T. McLennan". JAMA 251 (17): 2255–61. doi:10.1001/jama.251.17.2255. PMID 6368885. [5] Joensuu H. (2008). "Systemic chemotherapy for cancer: from weapon to treatment". Lancet Oncol. 9 (3): 304. doi:10.1016/S1470-2045(08)70075-5. PMID 18308256. [6] Skeel, R. T. (2003) (Paperback). Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins. ISBN 0781736293. [7] Chabner, B.; Longo, D. L. (2005). Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Willians & Wilkins. ISBN 0781756286. [8] Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" (http:/ / www. cancernetwork. com/ cancer-management-11/ chapter03/ article/ 10165/ 1402628) in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach (http:/ / www. cancernetwork. com/ cancer-management-11/ ). 11 ed. 2008. [9] What are Common Side Effects? (http:/ / www. cancer. org/ docroot/ MBC/ content/ MBC_2_2X_What_Are_Common_Side_Effects. asp?sitearea=MBC) from the American Cancer Society [10] Huang, Elbert S. (2000). Internal medicine: handbook for clinicians, resident survival guide. Arlington, VA: Scrub Hill Press. pp. 130. ISBN 978-0-9645467-5-2. [11] http:/ / www. cancer. org/ docroot/ ETO/ content/ ETO_5_3X_Coriolous_Versicolor. asp [12] Gralla R, de Wit R, Herrstedt J, Carides A, Ianus J, Guoguang-Ma J, Evans J, Horgan K (2005). "Antiemetic efficacy of the neurokinin-1 antagonist, aprepitant, plus a 5HT3 antagonist and a corticosteroid in patients receiving anthracyclines or cyclophosphamide in addition to high-dose cisplatin: analysis of combined data from two Phase III randomized clinical trials". Cancer 104 (4): 864–8. doi:10.1002/cncr.21222. PMID 15973669. [13] Tramer MR, Carroll D, Campbell FA, Reynolds DJ, Moore RA, McQuay HJ. (2001). "Cannabinoids for control of chemotherapy induced nausea and vomiting: quantitative systematic review.". BMJ 323 (7303): 16–21. doi:10.1136/bmj.323.7303.16. PMC 34325. PMID 11440936. [14] "Frequently Asked Questions - Medical Marihuana" (http:/ / www. hc-sc. gc. ca/ dhp-mps/ marihuana/ about-apropos/ faq_e. html) [15] U. Rüther, C. Nunnensiek, H.-J. Schmoll,Secondary Neoplasias following Chemotherapy, Radiotherapy, and Immunosuppression,Contributions to Oncology (Beiträge zur Onkologie); Vol 55, 2000, ISBN 380557116X [16] Hijiya N, Hudson MM, Lensing S, Zacher M, Onciu M, Behm FG, Razzouk BI, Ribeiro RC et al. (2007). "Cumulative Incidence of Secondary Neoplasms as a First Event After Childhood Acute Lymphoblastic Leukemia". JAMA 297 (11): 1207–1215. doi:10.1001/jama.297.11.1207. PMID 17374815.

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Chemotherapy [17] Brydøy M, Fosså SD, Dahl O, Bjøro T (2007). "Gonadal dysfunction and fertility problems in cancer survivors" (http:/ / informahealthcare. com/ doi/ pdf/ 10. 1080/ 02841860601166958). Acta Oncol 46 (4): 480–9. doi:10.1080/02841860601166958. PMID 17497315. . [18] Gurgan T, Salman C, Demirol A (October 2008). "Pregnancy and assisted reproduction techniques in men and women after cancer treatment". Placenta 29 Suppl B: 152–9. doi:10.1016/j.placenta.2008.07.007. PMID 18790328. [19] Tannock IF, Ahles TA, Ganz PA, Van Dam FS (June 2004). "Cognitive impairment associated with chemotherapy for cancer: report of a workshop" (http:/ / www. jco. org/ cgi/ pmidlookup?view=long& pmid=15169812). J. Clin. Oncol. 22 (11): 2233–9. doi:10.1200/JCO.2004.08.094. PMID 15169812. . [20] Morgan G, Ward R, Barton M (December 2004). "The contribution of cytotoxic chemotherapy to 5-year survival in adult malignancies". Clin Oncol (R Coll Radiol) 16 (8): 549–60. PMID 15630849. [21] McKnight JA (May 2003). "Principles of chemotherapy". Clin Tech Small Anim Pract 18 (2): 67–72. doi:10.1053/svms.2003.36617. PMID 12831063.

External links • Search for chemotherapy trials (http://www.cancer.gov/clinicaltrials/search/) • American Cancer Society - Chemotherapy (http://www.cancer.org/docroot/ETO/content/ ETO_1_2X_Chemotherapy_What_It_Is_How_It_Helps.asp)

Targeted therapy Targeted therapy is a type of medication that blocks the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth,[1] rather than by simply interfering with rapidly dividing cells (e.g. with traditional chemotherapy). Targeted cancer therapies may be more effective than current treatments and less harmful to normal cells. The definitive experiments that showed that targeted therapy would reverse the malignant phenotype of tumor cells involved treating Her2/neu transformed cells with monoclonal antibodies in vitro and in vivo by Mark Greene’s laboratory.[2] Some have challenged use of the term, stating that drugs usually associated with the term are insufficiently selective.[3] The phrase occasionally appears in scare quotes.[4]

Types The main categories of targeted therapy are small molecules and monoclonal antibodies.

Small molecules

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• Imatinib mesylate (Gleevec, also known as STI–571) is approved for chronic myelogenous leukemia, gastrointestinal stromal tumor and some other types of cancer. Early clinical trials indicate that imatinib may be effective in treatment of dermatofibrosarcoma protuberans. • Gefitinib (Iressa, also known as ZD1839), targets the epidermal growth factor receptor (EGFR) tyrosine kinase and is approved in the U.S. for non small cell lung cancer. EGFR is also Mechanism of imatinib overexpressed in the cells of other solid tumors, such as lung and breast cancers. This leads to inappropriate activation of the apoptotic Ras signal transduction cascade, eventually leading to uncontrolled cell proliferation.Gefitinib inhibits EGFR tyrosine kinase by binding to the adenosine triphosphate (ATP)-binding site of the enzyme. Thus the function of the EGFR tyrosine kinase in activating the Ras signal transduction cascade is inhibited; and malignant cells are inhibited. • Erlotinib (marketed as Tarceva). Erlotinib inhibits epidermal growth factor receptor,[5] and works through a similar mechanism as gefitinib. Erlotinib has been shown to increase survival in metastatic non small cell lung cancer when used as second line therapy. Because of this finding, erlotinib has replaced gefitinib in this setting. • Bortezomib (Velcade) is an apoptosis-inducing proteasome inhibitor drug that causes cancer cells to undergo cell death by interfering with proteins. It is approved in the U.S. to treat multiple myeloma that has not responded to other treatments. • The selective estrogen receptor modulator tamoxifen has been described as the foundation of targeted therapy.[6] • Bcl-2 inhibitors (eg. obatoclax in clinical trials, ABT-263, and Gossypol.[7] • PARP inhibitors (e.g. Iniparib, Olaparib in clinical trials) • Janus kinase inhibitors • PI3K inhibitors • Apatinib is a selective VEGF Receptor 2 inhibitor which has shown encouraging anti-tumor activity in a broad range of malignancies in clinical trials.[8] Apatinib is currently in clinical development for metastatic gastric carcinoma, metastatic breast cancer and advanced hepatocellular carcinoma.[9] • salinomycin has demonstrated potency in killing cancer stem cells in both laboratory-created and naturally occurring breast tumors in mice.[10] [11]

Monoclonal antibodies Several are in development and a few have been licenced by the FDA. Examples of licenced monoclonal antibodies include: • Rituximab (marketed as MabThera or Rituxan) targets CD20 found on B cells. It is used in non Hodgkin lymphoma • Trastuzumab (Herceptin) targets the Her2/neu (also known as ErbB2) receptor expressed in some types of breast cancer • Cetuximab (marketed as Erbitux) targets the epidermal growth factor receptor. It is used in the treatment of colon cancer and non-small cell lung cancer. • Bevacizumab (marketed as Avastin) targets circulating VEGF ligand. It is approved for use in the treatment of colon cancer, breast cancer, non-small cell lung cancer, and is investigational in the treatment of sarcoma. Its use for the treatment of brain tumors has been recommended.[12]

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Antibody-drug conjugates are being developed. See also ADEPT (Antibody-directed enzyme prodrug therapy).

Progress and future Many oncologists believe that targeted therapies are the chemotherapy of the future. As solid tumor cancer continues to be viewed as a chronic condition, methods for long-term treatment, with less side-effects, continue to be investigated. In the U.S., the National Cancer Institute's Molecular Targets Development Program evaluate molecular targets that may be candidates for drug development.

[13]

(MTDP) to identify and

The next stage of targeted therapies will focus on finding which patients will respond to which targeted therapies. This is called the identification of "sub-populations", stratified medicine or even personalized medicine. The route to identify these sub-populations is through biomarkers and surrogate endpoints.

References [1] "Definition of targeted therapy - NCI Dictionary of Cancer Terms" (http:/ / www. cancer. gov/ Templates/ db_alpha. aspx?CdrID=270742). . Retrieved 2009-01-25. [2] Perantoni AO, Rice JM, Reed CD, Watatani M, Wenk ML (September 1987). "Activated neu oncogene sequences in primary tumors of the peripheral nervous system induced in rats by transplacental exposure to ethylnitrosourea". Proc. Natl. Acad. Sci. U.S.A. 84 (17): 6317–21. doi:10.1073/pnas.84.17.6317. PMC 299062. PMID 3476947. Drebin JA, Link VC, Weinberg RA, Greene MI (December 1986). "Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen". Proc. Natl. Acad. Sci. U.S.A. 83 (23): 9129–33. doi:10.1073/pnas.83.23.9129. PMC 387088. PMID 3466178. Drebin JA, Link VC, Stern DF, Weinberg RA, Greene MI (July 1985). "Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies". Cell 41 (3): 697–706. PMID 2860972. [3] Zhukov NV, Tjulandin SA (May 2008). "Targeted therapy in the treatment of solid tumors: practice contradicts theory" (http:/ / protein. bio. msu. ru/ biokhimiya/ contents/ v73/ full/ 73050751. html). Biochemistry Mosc. 73 (5): 605–18. doi:10.1134/S000629790805012X. PMID 18605984. . [4] Markman M (2008). "The promise and perils of 'targeted therapy' of advanced ovarian cancer" (http:/ / content. karger. com/ produktedb/ produkte. asp?typ=fulltext& file=000138349). Oncology 74 (1-2): 1–6. doi:10.1159/000138349. PMID 18536523. . [5] Katzel JA, Fanucchi MP, Li Z (January 2009). "Recent advances of novel targeted therapy in non-small cell lung cancer" (http:/ / www. jhoonline. org/ content/ 2/ 1/ 2). J Hematol Oncol 2 (1): 2. doi:10.1186/1756-8722-2-2. PMC 2637898. PMID 19159467. . [6] Jordan VC (January 2008). "Tamoxifen: catalyst for the change to targeted therapy" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ S0959-8049(07)00861-1). Eur. J. Cancer 44 (1): 30–8. doi:10.1016/j.ejca.2007.11.002. PMC 2566958. PMID 18068350. . [7] Warr MR, Shore GC (December 2008). "Small-molecule Bcl-2 antagonists as targeted therapy in oncology" (http:/ / www. current-oncology. com/ index. php/ oncology/ article/ view/ 392/ 306;). Curr Oncol 15 (6): 256–61. PMC 2601021. PMID 19079626. . [8] http:/ / www. biomedcentral. com/ 1471-2407/ 10/ 529 [9] http:/ / clinicaltrials. gov/ ct2/ results?term=apatinib [10] "New method takes aim at aggressive cancer cells" (http:/ / www. broadinstitute. org/ news/ 1305). Broad Communications (Broad Institute). 2009-08-13. . Retrieved 2009-08-13. [11] Gupta, P. et al.; Onder, TT; Jiang, G; Tao, K; Kuperwasser, C; Weinberg, RA; Lander, ES (2009-08-13). "Identification of selective inhibitors of cancer stem cells by high-throughput screening." (http:/ / www. cell. com/ abstract/ S0092-8674(09)00781-8). Cell 138 (4): 645–59. doi:10.1016/j.cell.2009.06.034. PMID 19682730. . Retrieved 2009-08-13. [12] Pollack, Andrew (2009-03-31). "F.D.A. Panel Supports Avastin to Treat Brain Tumor" (http:/ / www. nytimes. com/ 2009/ 04/ 01/ business/ 01avastin. html). New York Times. . Retrieved 2009-08-13. [13] http:/ / home. ncifcrf. gov/ mtdp

Targeted therapy

External links • Targeted Therapy Database (TTD) (http://www.mmmp.org/MMMP/import. mmmp?page=targetedtherapydatabase.mmmp) from the Melanoma Molecular Map Project (http://www. mmmp.org/MMMP/welcome.mmmp) • Fact sheet (http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted) from the U.S. National Cancer Institute • Molecular Oncology: Receptor-Based Therapy (http://www.jco.org/content/vol23/issue11/) Special issue of Journal of Clinical Oncology (April 10, 2005) dedicated to targeted therapies in cancer treatment • Targeting Targeted Therapy (http://content.nejm.org/cgi/content/full/350/21/2191) New England Journal of Medicine (2004)

Monoclonal antibodies Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies").

Discovery The idea of a "magic bullet" was first proposed by Paul Ehrlich, who, at the beginning of the 20th century, postulated that, if a A general representation of the methods used to compound could be made that selectively targeted a produce monoclonal antibodies. disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. He and Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work, which led to an effective syphilis treatment by 1910. In the 1970s, the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen. Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973[1] and remains widely cited among those using human-derived hybridomas,[2] but claims to priority have been controversial. A science history paper on the subject gave some credit to Schwaber for inventing a technique that was widely cited, but stopped short of suggesting that he had been cheated.[3] The invention was conceived by George Pieczenik [4], with John Sedat, Elizabeth Blackburn's husband, as a witness and reduced to practice by Cotton and Milstein, and then by Kohler and Milstein. Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975;[5] who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody-producing B-cells, and be able to select for the successfully fused cells.

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In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,[6] removing the reactions that many monoclonal antibodies caused in some patients.

Production Hybridoma cell production Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive. The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (as it is a cancer cell).

Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them.

Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture.

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use. The hybridomas can be grown indefinitely in a suitable cell culture medium.They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid. The medium must be enriched during in-vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as

Technician hand-filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma).

Monoclonal antibodies

briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical.

Purification of monoclonal antibodies After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. Lab technician bathing prepared slides in a solution. The contaminants in the cell culture sample would consist This technician prepares slides of monoclonal primarily of media components such as growth factors, hormones, antibodies for researchers. The cells shown are labeling and transferrins. In contrast, the in vivo sample is likely to have human breast cancer. host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other. The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis. Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.[7] Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation. Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times. A much quicker, single-step method of separation is Protein A/G affinity chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin. To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is

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Monoclonal antibodies passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support. To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis. The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.

Recombinant The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.[8] The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik [9] These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications.[10] Fermentation chambers have been used to produce these antibodies on a large scale.

Chimeric antibodies Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA). In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells, and the expression of this chimeric DNA through cell culture yielded partially-mouse, partially-human monoclonal antibody. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.[11]

'Fully' human monoclonal antibodies Ever since the discovery that monoclonal antibodies could be generated in-vitro, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanised and chimeric antibodies. Two successful approaches were identified — phage display-generated antibodies and mice genetically engineered to produce more human-like antibodies. One of the most successful commercial organisations behind therapeutic monoclonal antibodies was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. This was reported in a key Nature publication.[12] Other significant publications include:

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Monoclonal antibodies • Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (December 1991). "By-passing immunization. Human antibodies from V-gene libraries displayed on phage" [13]. J. Mol. Biol. 222 (3): 581–97. doi:10.1016/0022-2836(91)90498-U. PMID 1748994. • Carmen S, Jermutus L (July 2002). "Concepts in antibody phage display" [14]. Brief Funct Genomic Proteomic 1 (2): 189–203. doi:10.1093/bfgp/1.2.189. PMID 15239904. CAT developed their display technologies further into several, patented antibody discovery/functional genomics tools, which were named ProximolTM[15] and ProAbTM. ProAb was announced in December 1997[16] and involved highthroughput screening of antibody libraries against diseased and non-diseased tissue, whilst Proximol used a free radical enzymatic reaction to label molecules in proximity to a given protein.[17] [18] Genetically engineered mice, so called transgenic mice, can be modified to produce human antibodies,[19] and this has been exploited by a number of commercial organisations: • Medarex — who market their UltiMab platform[20] • Abgenix — who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen.[21] • Regeneron's VelocImmune technology.[22] Monoclonal antibodies have been generated and approved to treat: cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection (see monoclonal antibody therapy). In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration.[23]

Applications Diagnostic tests Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.

Therapeutic treatment Cancer treatment One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region.

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The illustration below shows all these possibilities: MAbs approved by the FDA include[25] • • • •

Bevacizumab Cetuximab Panitumumab Traztuzumab

Autoimmune diseases

Monoclonal antibodies for cancer. ADEPT,

antibody directed enzyme prodrug therapy; Monoclonal antibodies used for autoimmune diseases include ADCC, antibody dependent cell-mediated infliximab and adalimumab, which are effective in rheumatoid cytotoxicity; CDC, complement dependent arthritis, Crohn's disease and ulcerative Colitis by their ability to bind cytotoxicity; MAb, monoclonal antibody; scFv, [24] to and inhibit TNF-α.[26] Basiliximab and daclizumab inhibit IL-2 on single-chain Fv fragment. activated T cells and thereby help prevent acute rejection of kidney transplants.[26] Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

Examples Below are examples of clinically important monoclonal antibodies. Main category Antiinflammatory

Type

Application

[26]

Mode

• • •

rheumatoid arthritis Crohn's disease Ulcerative Colitis

inhibits TNF-α

chimeric

• • •

rheumatoid arthritis Crohn's disease Ulcerative Colitis

inhibits TNF-α

human

[26]

•

rheumatoid arthritis

Contains decoy TNF receptor

fusion protein

basiliximab

[26]

•

Acute rejection of kidney transplants

inhibits IL-2 on activated T cells

chimeric

[26] daclizumab

•

Acute rejection of kidney transplants

inhibits IL-2 on activated T cells

humanized

omalizumab

•

moderate-to-severe allergic asthma

inhibits human immunoglobulin E (IgE)

humanized

relapsed acute myeloid leukaemia

targets myeloid cell surface antigen CD33 on leukemia cells

humanized

B cell leukemia

targets an antigen CD52 on T- and B-lymphocytes

humanized

infliximab

adalimumab

etanercept

Anti-cancer

Mechanism/Target

[26] • gemtuzumab [26] •

alemtuzumab

[26] rituximab

•

non-Hodgkin's lymphoma

targets phosphoprotein CD20 on B lymphocytes

chimeric

trastuzumab

•

breast cancer with HER2/neu overexpression

targets the HER2/neu (erbB2) receptor

humanized

nimotuzumab

•

Approved in squamous cell carcinomas, EGFR inhibitor Glioma Clinical trials for other indications underway

Humanized

• cetuximab

•

Approved in squamous cell carcinomas, colorectal carcinoma

EGFR inhibitor

Chimeric

bevacizumab

•

Anti-angiogenic cancer therapy

inhibits VEGF

humanized

Monoclonal antibodies

Other

129 [26]

palivizumab

[26] abciximab

•

RSV infections in children

inhibits an RSV fusion (F) protein

humanized

•

Prevent coagulation in coronary angioplasty

inhibits the receptor GpIIb/IIIa on platelets

chimeric

References [1] Schwaber, J; Cohen, EP (1973). "Human x mouse somatic cell hybrid clone secreting immunoglobulins of both parental types". Nature 244 (5416): 444–7. doi:10.1038/244444a0. PMID 4200460. [2] Science Citation Index [3] Cambrosio, A.; Keating, P. (1992). "Between fact and technique: the beginnings of hybridoma technology". Journal of the History of Biology 25 (2): 175. doi:10.1007/BF00162840. PMID 11623041. [4] http:/ / lifesci. rutgers. edu/ ~molbiosci/ faculty/ pieczenik. html [5] Köhler, G.; Milstein, C. (1975). "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature 256 (5517): 495. Bibcode 1975Natur.256..495K. doi:10.1038/256495a0. PMID 1172191. [6] Riechmann L, Clark M, Waldmann H, Winter G (March 1988). "Reshaping human antibodies for therapy". Nature 332 (6162): 323–7. doi:10.1038/332323a0. PMID 3127726. [7] Vlasek J, Ionescu R (2008). "Hetergeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods". Current Pharmaceutical Biotechnology 9 (6): 468–481. doi:10.2174/138920108786786402. PMID 19075686. [8] Siegel DL (2002). "Recombinant monoclonal antibody technology". Transfusion clinique et biologique : journal de la Société française de transfusion sanguine 9 (1): 15–22. PMID 11889896. [9] http:/ / www2. mrc-lmb. cam. ac. uk/ archive/ g_pieczenik. html [10] Schmitz U, Versmold A, Kaufmann P, Frank HG (2000). "Phage display: a molecular tool for the generation of antibodies—a review". Placenta 21 (Suppl A): S106–12. doi:10.1053/plac.1999.0511. PMID 10831134. [11] Helen E. Chadd and Steven M. Chamow, “Therapeutic antibody expression technology,” Current Opinion in Biotechnology 12, no. 2 (April 1, 2001): 188-194. [12] McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552–554. Bibcode 1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164. [13] http:/ / linkinghub. elsevier. com/ retrieve/ pii/ 0022-2836(91)90498-U [14] http:/ / bfgp. oxfordjournals. org/ cgi/ pmidlookup?view=long& pmid=15239904 [15] Osbourn JK (2002). "Proximity-guided (ProxiMol) antibody selection". Methods Mol. Biol. 178: 201–5. PMID 11968489. [16] http:/ / www. ukbusinesspark. co. uk/ cay92125. htm [17] http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6T76-3WBNNJS-6& _user=10& _rdoc=1& _fmt=& _orig=search& _sort=d& _docanchor=& view=c& _searchStrId=960781456& _rerunOrigin=google& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=eacedcd4604792172fc18143a590a454 [18] http:/ / www. chidb. com/ newsarticles/ issue3_1. ASP [19] Lonberg N, Huszar D (1995). "Human antibodies from transgenic mice". Int. Rev. Immunol. 13 (1): 65–93. doi:10.3109/08830189509061738. PMID 7494109. [20] http:/ / www. medarex. com/ Development/ UltiMAb. htm [21] http:/ / wwwext. amgen. com/ media/ media_pr_detail. jsp?year=2006& releaseID=837754 [22] http:/ / www. regeneron. com/ velocimmune. html [23] PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006. Retrieved 2006-09-04. [24] Modified from Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129 [25] Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" (http:/ / www. cancernetwork. com/ cancer-management-11/ chapter03/ article/ 10165/ 1402628) in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach (http:/ / www. cancernetwork. com/ cancer-management-11/ ). 11 ed. 2008. [26] Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. pp. 241, for the examples infliximab, basiliximab, abciximab, daclizumab, palivusamab, gemtuzumab, alemtuzumab, etanercept and rituximab, and mechanism and mode. ISBN 0-443-07145-4.

Monoclonal antibodies

External links • Monoclonal Antibodies Animation (http://www.1lec.com/Immunology/Monoclonal Antibodies/index.html) (Flash Required) • Monoclonal Antibodies (http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html), from John W. Kimball's online biology textbook • MeSH Monoclonal+antibodies (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=& term=Monoclonal+antibodies)

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Molecular Diagnostics Flow cytometry Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing clinical practice. A common variation three different populations (Prochlorococcus, Synechococcus, and picoeukaryotes) is to physically sort particles based on their properties, so as to purify populations of interest.

History The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster [1] and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods.[2] Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).

Flow cytometry

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Name of the technology The original name of the flow cytometry technology was "pulse cytophotometry" (German: Impulszytophotometrie). Only 20 years later in 1988, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to "flow cytometry", a term that quickly became popular.

Principle of flow cytometry A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.

Flow cytometers Modern flow cytometers are able to analyze several thousand particles every second, in "real time," and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification of set parameters. To analyze solid tissues, a single-cell suspension must first be prepared. A flow cytometer has five main components:

Front of desktop flow cytometer - the Becton-Dickinson FACSCalibur.

• a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing • a measuring system - commonly used are measurement of impedance (or conductivity) and optical systems lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals • a detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer • an amplification system - linear or logarithmic • a computer for analysis of the signals. The process of collecting data from samples using the flow cytometer is termed 'acquisition'. Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters (i.e. voltage, compensation, etc.) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to insure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in

Flow cytometry acquisition such as fluorescently-labeled antibodies has developed. Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is four lasers and 18 fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cell.

Data analysis Gating The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology. The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap,[3] signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software, e.g., WinMDI(deprecated),[4] Flowjo, FCS Express, VenturiOne or CellQuest Pro. Once the data is collected, there is no need to stay connected to the flow cytometer. For this reason, analysis is most often done on a separate computer. This is especially necessary in core facilities where usage of these machines is in high demand.

Computational analysis Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK [5] in Immunology Database and Analysis Portal (ImmPort),[6] FLAME [7] in GenePattern and flowClust,[8] [9] [10] in Bioconductor. Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods,[11] ) to provide an objective way to compare and evaluate the flow cytometry data clustering methods, and also to establish guidance about appropriate use and application of these methods.

Fluorescence-activated cell sorting Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The acronym FACS is trademarked and owned by Becton, Dickinson and Company.[12] While many immunologists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry. The first cell sorter was invented by Mack Fulwyler in 1965, using the Coulter principle, a relatively difficult technique and one no longer used in modern instruments. The technique was expanded by Len Herzenberg, who was responsible for coining the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow cytometry. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station

133

Flow cytometry where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.

Labels Fluorescent labels A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores, or simply "fluors", are typically attached to an antibody that recognises a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure. Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra of different labels often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available.[13] The maximum number of distinguishable fluorescent labels is thought to be 17 or 18, and this level of plexy necessitates laborious optimization to limit artifacts, as well as complex deconvolution algorithms to separate overlapping spectra.[14]

Quantum dots Quantum dots are sometimes used in place of traditional fluorophores because of their narrower emission peaks.

Isotope labeling In one approach to overcoming the fluorescent labeling limit, lanthanide isotopes are attached to antibodies. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for Use of flow cytometry to measure copy number variation of a specific DNA sequence 30 labels.[14] Cells are introduced into (Flow-FISH) a plasma, ionizing them and allowing time-of-flight mass spectrometry to identify the associated isotopes. Although this method permits the use of a large number of labels, it currently has lower throughput capacity than traditional flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting.[14]

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Flow cytometry

Measurable parameters This list is very long and constantly expanding. • • • • • • • • • • • • • •

volume and morphological complexity of cells cell pigments such as chlorophyll or phycoerythrin total DNA content (cell cycle analysis, cell kinetics, proliferation, etc.) total RNA content DNA copy number variation (by Flow-FISH) chromosome analysis and sorting (library construction, chromosome paint) protein expression and localization Protein modifications, phospho-proteins transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent * cell surface antigens (Cluster of differentiation (CD) markers) intracellular antigens (various cytokines, secondary mediators, etc.) nuclear antigens enzymatic activity pH, intracellular ionized calcium, magnesium, membrane potential membrane fluidity

• apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity) • cell viability • monitoring electropermeabilization of cells • oxidative burst • characterising multidrug resistance (MDR) in cancer cells • glutathione • various combinations (DNA/surface antigens, etc.) • cell adherence (for instance pathogen-host cell adherence)

Applications The technology has applications in a number of fields, including molecular biology, pathology, immunology, plant biology and marine biology. It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting for sex preselection). In marine biology, the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties. It is also used to determine ploidy of grass carp fry.

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Bibliography • • • • • •

Flow Cytometry First Principles by Alice Longobardi Givan. ISBN 0471382248 Practical Flow Cytometry by Howard M. Shapiro. ISBN 0471411256 Flow Cytometry for Biotechnology by Larry A. Sklar. ISBN 0195152344 Handbook of Flow Cytometry Methods by J. Paul Robinson, et al. ISBN 0471596345 Current Protocols in Cytometry, Wiley-Liss Pub. ISSN 1934-9297 Flow Cytometry in Clinical Diagnosis, v4, (Carey, McCoy, and Keren, eds), ASCP Press, 2007. ISBN 0891895485 • Ormerod, M.G. (ed.) (2000) Flow Cytometry — A practical approach. 3rd edition. Oxford University Press, Oxford, UK. ISBN 0199638241 • Ormerod, M.G. (1999) Flow Cytometry. 2nd edition. BIOS Scientific Publishers, Oxford. ISBN 185996107X • Flow Cytometry — A basic introduction. Michael G. Ormerod, 2008. ISBN 978-0955981203

References [1] DE 1815352 (http:/ / v3. espacenet. com/ textdoc?DB=EPODOC& IDX=DE1815352), Wolfgang Dittrich & Wolfgang Göhde, "Flow-through Chamber for Photometers to Measure and Count Particles in a Dispersion Medium", issued 1971-01-14 [2] Kamentsky, Proceedings of the Conference „Cytology Automation" in Edinburgh, 1970 [3] http:/ / pingu. salk. edu/ flow/ fluo. html [4] "TSRI Cytometry Software Page" (http:/ / facs. scripps. edu/ software. html). . Retrieved 2009-09-03. [5] "Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data" (http:/ / onlinelibrary. wiley. com/ doi/ 10. 1002/ cyto. b. 20554/ full). . Retrieved 2010-12-14. [6] "Immunology Database and Analysis Portal" (https:/ / www. immport. org/ immportWeb/ home/ home. do?loginType=full). . Retrieved 2009-09-03. [7] "FLow analysis with Automated Multivariate Estimation (FLAME)" (http:/ / www. broadinstitute. org/ cancer/ software/ genepattern/ modules/ FLAME/ ). . Retrieved 2009-09-03. [8] "flowClust" (http:/ / www. bioconductor. org/ packages/ 2. 5/ bioc/ html/ flowClust. html). . Retrieved 2009-09-03. [9] (http:/ / www3. interscience. wiley. com/ journal/ 117925662/ abstract?CRETRY=1& SRETRY=0) [10] (http:/ / www. biomedcentral. com/ 1471-2105/ 10/ 145) [11] "FlowCAP - Flow Cytometry: Critical Assessment of Population Identification Methods" (http:/ / flowcap. flowsite. org/ ). . Retrieved 2009-09-03. [12] "FACS MultiSET System" (http:/ / www. bdbiosciences. com/ pdfs/ brochures/ 23-3428-02. pdf) (PDF). Becton Dickinson. . Retrieved 2007-02-09. [13] Loken MR (1990). Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd ed.). Wiley. pp. 341–53. [14] Ornatsky, O.; Bandura, D.; Baranov, V.; Nitz, M.; Winnik, M. A.; Tanner, S. (2010). "Highly multiparametric analysis by mass cytometry". Journal of Immunological Methods 361 (1–2): 1–20. doi:10.1016/j.jim.2010.07.002. PMID 20655312.

External links • Flow cytometry - How does it work? (http://www.unsolvedmysteries.oregonstate.edu/flow_06) (Oregon State University) • How a flow cytometer operates (http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html) (MD Anderson Cancer Center) • Learn About Flow Cytometry (http://www.millipore.com/flowcytometry/fc4/learn) (Millipore) • Powerpoint lectures on flow cytometry (http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm) (Purdue University) • Tutorials on fluorescence and flow cytometry (http://probes.invitrogen.com/resources/education/) (Invitrogen), including Introduction to Flow Cytometry (http://probes.invitrogen.com/resources/education/ tutorials/4Intro_Flow/player.html) • Fluorescence SpectraViewer (http://www.invitrogen.com/site/us/en/home/support/Research-Tools/ Fluorescence-SpectraViewer.html) - Check the compatibility of your fluorophores when designing multicolor

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• • • • • •

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experiments (Invitrogen) Searchable database of fluorescent dyes (http://www.fluorophores.tugraz.at/) ([Graz University of Technology]]) Table of fluorochromes (http://pingu.salk.edu/flow/fluo.html) (Salk Institute) Java Fluorescence Spectrum Viewer (http://www.bdbiosciences.com/spectra/) (Becton, Dickinson and Company) MeSH Flow+cytometry (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Flow+cytometry) FICCS (http://www.ficcs.org/) - the Flow Informatics and Computation Cytometry Society History of Flow Cytometry by Bob Auer (http://www.coulterflow.com/bciflow/history.php) (hosted by Beckman Coulter)

Fluorescence in situ hybridization FISH (fluorescent in situ hybridization) is a cytogenetic technique developed by Christoph Lengauer that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counselling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues. A metaphase cell positive for the bcr/abl rearrangement (associated with chronic myelogenous leukemia) using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (upper left) is the one where the wrong rearrangement is present.

Fluorescence in situ hybridization

Probes Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use in the Human Genome Project. The size of the human genome is so large, compared to the length that could be sequenced directly, that it was necessary to divide the genome into fragments. (In the eventual analysis, these fragments were put into order by digesting a copy of each fragment into still smaller fragments using sequence-specific endonucleases, measuring the size of each small fragment using size-exclusion chromatography, and using that information to determine where the large fragments overlapped one Urothelial cells marked with four different another.) To preserve the fragments with their individual DNA probes. sequences, the fragments were added into a system of continually replicating bacteria populations. Clonal populations of bacteria, each population maintaining a single artificial chromosome, are stored in various laboratories around the world. The artificial chromosomes (BAC) can be grown, extracted, and labeled, in any lab. These fragments are on the order of 100 thousand base-pairs, and are the basis for most FISH probes.

Preparation and Hybridization Process First, a probe is constructed. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. The probe is tagged directly with fluorophores, with targets for antibodies or with biotin. Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides. Then, an interphase or metaphase chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked by adding short Scheme of the principle of the FISH Experiment fragments of DNA to the sample. The probe is then applied to the to localize a gene in the nucleus. chromosome DNA and incubated for approximately 12 hours while hybridizing. Several wash steps remove all unhybridized or partially-hybridized probes. The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images. If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently-tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal. FISH experiments designed to detect or localize gene expression within cells and tissues rely on the use of a reporter gene, such as one expressing green fluorescent protein, to provide the fluorescence signal.

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Variations on probes and analysis FISH is a very general technique. The differences between the various FISH techniques are usually due to variations in the sequence and labeling of the probes; and how they are used in combination. These few modifications make possible all FISH techniques. Probe size is important because longer probes hybridize less specifically than shorter probes. The overlap defines the resolution of detectable features. For example, if the goal of an experiment is to detect the breakpoint of a translocation, then the overlap of the probes — the degree to which one DNA sequence is contained in the adjacent probes — defines the minimum window in which the breakpoint may be detected. Interphase cells positive for a chromosomal

The mixture of probe sequences determines the type of feature the t(9;22) rearrangement. probe can detect. Probes that hybridize along an entire chromosome are used to count the number of a certain chromosome, show translocations, or identify extra-chromosomal fragments of chromatin. This is often called "whole-chromosome painting." If every possible probe is used, every chromosome, (the whole genome) would be marked fluorescently, which would not be particularly useful for determining features of individual sequences. However, a mixture of smaller probes can be created that is specific to a particular region (locus) of DNA; these mixtures are used to detect deletion mutations. When combined with a specific colour, a locus-specific probe mixture is used to detect very specific translocations. Special locus-specific probe mixtures are often used to count chromosomes, by binding to the centromeric regions of chromosomes, which are unique enough to identify each chromosome (with the exception of Chromosome 13, 14 21, 22.) A variety of other techniques use mixtures of differently-colored probes. A range of colors in mixtures of fluorescent dyes can be detected, so each human chromosome can be identified by a characteristic color using whole-chromosome probe mixtures and a variety of ratios of colors. Although there are more chromosomes than easily-distinguishable fluorescent dye colors, ratios of probe mixtures can be used to create secondary colors. Similar to comparative genomic hybridization, the probe mixture for the secondary colors is created by mixing the correct ratio of two sets of differently-colored probes for the same chromosome. This technique is sometimes called M-FISH. The same physics that make a variety of colors possible for M-FISH can be used for the detection of translocations. That is, colors that are adjacent appear to overlap; a secondary color is observed. Some assays are designed so that the secondary color will be present or absent in cases of interest. An example is the detection of BCR/ABL translocations, where the secondary color indicates disease. This variation is often called double-fusion FISH or D-FISH. In the opposite situation---where the absence of the secondary color is pathological---is illustrated by an assay used to investigate translocations where only one of the breakpoints is known or constant. Locus-specific probes are made for one side of the breakpoint and the other intact chromosome. In normal cells, the secondary colour is observed, but only the primary colour is observed when the translocation occurs. This technique is sometimes called "break-apart FISH".

Single Molecule RNA FISH Single Molecule RNA FISH is a method of detecting and quantifying mRNA and other long RNA molecules in a thin layer of tissue sample. Targets can be reliably imaged through the application of multiple short singly labeled oligonucleotide probes.[1] [2] The probes cooperatively bind to the target site. When each probe binds to the single stranded mRNA, it causes cooperative unwinding of the mRNA, promoting the binding of the next probe. The net result is the binding of 48 fluorescent labels to a single molecule of mRNA, providing sufficient fluorescence to reliably locate each target mRNA in a wide-field fluorescent microscopy image. Probes not binding to the intended

Fluorescence in situ hybridization sequence do not achieve sufficient localized fluorescence to be distinguished from the background. This technology is exclusively licensed to Biosearch Technologies as Stellaris™ FISH Probes.[3] [4] Single molecule RNA FISH assays can be performed in simplex or multiplex, and have potential applications in cancer diagnosis,[5] neuroscience,[6] gene expression analysis,[7] and companion diagnostics.

Fiber FISH In an alternative technique to interphase or metaphase preparations, fiber FISH, interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line, rather than being tightly coiled, as in conventional FISH, or adopting a random conformation, as in interphase FISH. This is accomplished by applying mechanical shear along the length of the slide, either to cells that have been fixed to the slide and then lysed, or to a solution of purified DNA. A technique known as chromosome combing is increasingly used for this purpose. The extended conformation of the chromosomes allows dramatically higher resolution - even down to a few kilobases. The preparation of fiber FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely.

Q-FISH Q-FISH combines FISH with PNAs and computer software to quantify fluorescence intensity. This technique is used routinely in telomere length research.

Flow-FISH Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence measurements.

Medical applications Often parents of children with a developmental delay want to know more about their child's conditions before choosing to have another child. These concerns can be addressed by analysis of the parents' and child's DNA. In cases where the child's developmental delay is not understood, the cause of it can be determined using FISH and cytogenetic techniques. Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome. FISH on sperm cells is indicated for men with an abnormal somatic or meiotic karyotype as well as those with oligozoospermia, since approximately 50% of oligozoospermic men have an increased rate of sperm chromosome abnormalities.[8] The analysis of chromosomes 21, X, and Y is enough to identify oligozoospermic individuals at risk.[8] In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate remission of a disease, such as cancer. Treatment can then be specifically tailored. A traditional exam involving metaphase chromosome analysis is often unable to identify features that distinguish one disease from another, due to subtle chromosomal features; FISH can elucidate these differences. FISH can also be used to detect diseased cells more easily than standard Cytogenetic methods, which require dividing cells and requires labor and time-intensive manual preparation and analysis of the slides by a technologist. FISH, on the other hand, does not require living cells and can be quantified automatically, a computer counts the fluorescent dots present. However, a trained technologist is required to distinguish subtle differences in banding patterns on bent and twisted metaphase chromosomes.

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Species identification FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria, from the patient's tissues or fluids, are typically grown on agar to determine the identity of the pathogen. Many bacteria, however, even well-known species, do not grow well under laboratory conditions. FISH can be used to detect directly the presence of the suspect on small samples of patient's tissue. FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region. FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms, for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm.

Lab-on-a-chip and FISH Although interphase fluorescence in situ hybridization (FISH) is a sensitive diagnostic tool used for the detection of chromosomal abnormalities on cell-by-cell basis, the cost-per-test and the technical complexity of current FISH protocols has inhibited its widespread utilization. Lab-on-a-chip or microfluidic devices, incorporate networks of microchannels that can miniaturize, integrate and automate conventional analytical techniques onto chip-style platforms. Since microchannels permit sophisticated levels of fluid control (down to picolitres), these devices can reduce analysis times, lower reagent consumption, and minimize human intervention. Currently, FISH has been performed on glass microfluidic platforms that standardize much of the protocol offering repeatable results that are accurate, cost-effective and easier to obtain in a clinical setting.

Microfluidic chip that automates the interphase FISH procedure. The microchip shown requires only minutes of setup time by technician, as opposed to the hours or days of labour needed to perform FISH with conventional equipment.

Compared to conventional FISH methods, these first implementations of on-chip FISH provide a 10-fold higher throughput and a 10-fold reduction in the cost of testing, enabling the simultaneous assessment of several chromosomal abnormalities or patients.[9] It is increasingly essential that diagnostic tests determine the type and extent of chromosomal abnormalities for more informed diagnosis and for appropriate choice of treatment strategies. Since the on-chip FISH technique is 10-20 times more cost-effective than conventional methods, and can be fully integrated and automated,[10] this technology will make widespread genetic testing of patients more accessible in a clinical setting. Recently, the first demonstration of Metaphase FISH on chip has led to renewed efforts towards automating the metaphase FISH protocol.[11] Metaphase FISH had continued to be difficult to integrate owing to the complex sample preparation protocol often spanning over 3 weeks. New reports confirm that a research group in Denmark have tested successfully a novel lab on chip device to integrate the entire sample preparation protocol for Metaphase FISH called FISHprep.

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Virtual Karyotype Virtual karyotyping is another cost-effective, clinically available alternative to FISH panels uses thousands to millions of probes on a single array to detect copy number changes, genome-wide, at unprecedented resolution.

Gallery

Another schematic of FISH process.

Microfluidic chip that lowered the cost-per-test of FISH by 90%.

Dual label FISH image; Bifidobacteria Cy3, Total bacteria FITC.

References [1] [2] [3] [4]

http:/ / www. nature. com/ nmeth/ journal/ v5/ n10/ full/ nmeth. 1253. html http:/ / singlemoleculefish. com/ http:/ / www. biosearchtech. com/ display. aspx?catid=227& pageid=215 http:/ / www. umdnj. edu/ cgi-bin/ cgiwrap/ hpappweb/ newsroom. cgi?headline=Foundation+ Venture+ Capital+ Group,+ a+ Foundation+ of+ UMDNJ+ Affiliate,+ Sells+ Interest+ in+ Longevica+ Pharmaceuticals& utm_source=feedburner& utm_medium=feed& utm_campaign=Feed:+ UMDNJNews+ (U [5] http:/ / www. annals. org/ content/ 131/ 11/ 805. 1. abstract [6] http:/ / www. columbia. edu/ cu/ biology/ faculty/ yuste/ reprints/ s/ steward_neuron_1997_9. pdf [7] http:/ / www. sciencemag. org/ cgi/ content/ abstract/ 305/ 5685/ 846 [8] Sarrate Z, Vidal F, Blanco J (April 2010). "Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance". Fertil. Steril. 93 (6): 1892–902. doi:10.1016/j.fertnstert.2008.12.139. PMID 19254793. [9] Sieben, V.J.; C.S. Debes Marun, P.M. Pilarski, G.V. Kaigala, L.M. Pilarski, C.J. Backhouse (2007-06). "FISH and chips: chromosomal analysis on microfluidic platforms" (http:/ / link. aip. org/ link/ ?NBT/ 1/ 27/ 1). IET Nanobiotechnology 1 (3): 27–35. doi:10.1049/iet-nbt:20060021. PMID 17506594. . Retrieved 2009-01-26. [10] Sieben, V.J.; C.S. Debes-Marun, L.M. Pilarski, C.J. Backhouse (2008-11). "An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization" (http:/ / www. rsc. org/ publishing/ journals/ LC/ article. asp?doi=b812443d). Lab on a Chip 8 (12): 2151–2156. doi:10.1039/b812443d. PMID 19023479. . Retrieved 2009-03-24. [11] "Metaphase FISH on a chip: Miniaturized microfluidic device for Fluorescence In-Situ Hybridization" (http:/ / www. mdpi. com/ 1424-8220/ 10/ 11/ 9831/ ). SENSORS. .

• Annelie Pernthaler, Jakob Pernthaler, Rudolf Amann (2002): Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria, Applied and Environmental Microbiology, p. 3094–3101 Vol. 68, No. 6 DOI: 10.1128/AEM.68.6.3094–3101.2002 • Michael Wagner, Matthias Horn and Holger Daims (2003): Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Current Opinion in Microbiology 2003, 6:302–309 DOI:10.1016/S1369-5274(03)00054-7

Fluorescence in situ hybridization

External links • MeSH Fluorescence+in+Situ+Hybridization (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=& term=Fluorescence+in+Situ+Hybridization) • Information on fiber FISH (http://www.olympusfluoview.com/applications/fiberfish.html) from the Olympus Corporation • A guide to fiber FISH (http://info.med.yale.edu/genetics/ward/tavi/fi19.html) from Octavian Henegariu • Fibre FISH protocol (http://www.sanger.ac.uk/HGP/methods/cytogenetics/fiber_fish.shtml) from the Human Genome Project at the Sanger Centre • CARD-FISH, BioMineWiki (http://wiki.biomine.skelleftea.se/wiki/index.php/CARD-FISH) • Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes (http://www. cshprotocols.org/cgi/content/full/2007/10/pdb.prot4730) • FISH technical notes and protocols from GeneDetect.com (http://www.genedetect.com/insitu.htm) • Fluorescence in situ Hybridization Photos of bacteria (http://www.biovisible.com/photopage.shtml)

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Common variable immunodeficiency Common variable immunodeficiency Classification and external resources [1]

ICD-10

D83.

ICD-9

279.06

OMIM

240500

[2] [3]

DiseasesDB 3274 [4] [5]

eMedicine

ped/444

MeSH

D017074

derm/870

[6]

[7]

Common variable immunodeficiency (CVID) (also known as Acquired hypogammaglobulinemia[8] ) is a group of approximately 150 primary immunodeficiencies (PIDs), which have a common set of symptoms (including hypogammaglobulinemia)[9] but which have different underlying causes. Common variable immunodeficiency is the most commonly encountered primary immunodeficiency.[10]

Causes and types CVID is believed to be a genetically determined primary immune defect; however, the underlying causes are different. The result of these defects is that the patient doesn't produce sufficient antibodies in response to exposure to pathogens. As a result, the patient's immune system fails to protect them against common bacterial and viral (and occasionally parasitic and protozoan) infections. The net result is that the patient is susceptible to illness. In CVID, the B cells are affected. In severe combined immunodeficiency (SCID), a more severe condition than CVID, diagnosed in infancy, both parts of the immune system (the cellular and humoral system) are affected, hence its classified as combined immunodeficiency. CVID appears to include a number of defects, some of which have been identified. For the majority, the genetic causes are still unknown. ICOS, TACI and CD19 have been identified as candidates.[11] [12] It is possible that environmental agents provoke the immune defect, due to genetic predisposition, but this has not been clarified. Types include:

Common variable immunodeficiency

145

Type

OMIM

Gene

CVID1 607594 [13] ICOS CVID2 240500 [3]

TACI

CVID3 613943 [14] CD19 CVID4 613494 [15] TNFRSF13C CVID5 613495 [16] CD20 CVID6 613496 [17] CD81

Clinical Features Signs and Symptoms of CVID include: • Hypogammaglobulinemia: low levels of immunoglobulin G (IgG), immunoglobulin A (IgA) and/or immunoglobulin M (IgM). • Poor titer response to vaccination with polysaccharide and protein antigens (e.g. pneumococci, tetanus, and diphtheria). • Recurring infections involving the ears, eyes, sinuses, nose, bronchi, lungs, skin, GI tract, joints, bones, CNS, paratoid glands, etc. These infections respond to antibiotics but recur upon discontinuation of the medications. Bronchiectasis can occur from severe and recurrent lung infections. • Viral infections that usually respond to antivirals. • Enlarged lymph nodes, Enlarged spleen. • Fatigue. • Abdominal pain, Bloating, Nausea, Vomiting, Diarrhea, Weight loss. • Malabsorption. • Helicobacter pylori, Giardiasis, Cryptosporidiosis, Small bowel bacterial overgrowth syndrome, etc. • Atrophic gastritis with pernicious anemia and achlorhydria. • Nodular lymphoid hyperplasia of the GI tract. • Villous atrophy of the small intestine, which can resemble celiac disease. • Inflammatory bowel disease. • Aphthous stomatitis. • Increased intestinal permeability. • Polyarthritis, or joint pain, spread across most joints, but specifically fingers, wrists, elbows, toes, ankles and knees. In some cases, Mycoplasma can be the cause. • Children may show a "failure to thrive" - they may be underweight and underdeveloped compared with "normal" peers.

Common variable immunodeficiency

Diagnosis Diagnosis of CVID is usually made by demonstrating low levels of immunoglobulins in the serum. Diagnosis may be made rapidly, but is often delayed; it is usually made in the second or third decade of life after referral to an immunologist. Diagnosis of CVID is a diagnosis of exclusion.[18] It presents similar to X-linked agammaglobulinemia, but the conditions can be distinguished with flow cytometry.[19]

Associated conditions As with several other immune cell disorders, CVID may predispose to lymphoma or possibly stomach cancer.[20] There also appears to be a predilection for autoimmune diseases, with a risk of up to 25%. Autoimmune destruction of platelets or red blood cells are the most common of these.

Treatment Treatment usually consists of immunoglobulin therapy, which is an injection of human antibodies harvested from blood donations: • intravenous immunoglobulin (IVIG, most common treatment in the US)[21] • subcutaneous immunoglobulin G (SCIG, relatively new treatment in the US and UK) • intramuscular immunglobulin (IMIG, less effective, painful) This is not a cure, but it strengthens immunity by ensuring that the patient has "normal" levels of antibodies, which helps to prevent recurrent upper respiratory infections. IG therapy can't be used if the patient has anti-IgA antibodies but in this case, products low in IgA can be used; subcutaneous delivery also is a means of permitting such patients to have adequate antibody replacement. IVIG treatment can be received by patients with a complete IgA deficiency if the IgA is completely removed from the treatment.

Reactions Some CVID patients may experience reactions to IG therapies; reactions may include: • • • • • • • • • • • •

anaphylactic shock (very rare) hives (rare) chills difficulty breathing headache (relatively common, may be relieved by an antihistamine, paracetamol/acetaminophen, or an anti-inflammatory (naproxen, advil, aspirin) nausea (common in IVIG) fever (common in IVIG and rare in SCIG) aseptic meningitis (rare) severe fatigue (common in IVIG) muscle aches and pain, or joint pain thrombotic events (rare) swelling at the insertion site (common in SCIG)

Patients should not receive therapy if they are fighting an active infection as this increases the risk of reaction. Also, patients changing from one brand of product to another may be at higher risk of reaction for the first couple of treatments on the new brand.

146

Common variable immunodeficiency Reactions can be minimized by taking an antihistamine and/or hydrocortisone and some paracetamol/acetaminophen/anti-inflammatory (naproxen, advil, aspirin) prior to treatment; patients should also be thoroughly hydrated and continue to drink water before, after and during treatment (if possible). IVIG should be prepared soon before IVIG infusion. Patient using a heating pad or warm blanket can help alleviate chills.

Research Research is currently focussing on genetic analysis, and in differentiating between the various different disorders in order to allow a cure to be developed. Cures are likely to be genetic in nature, repairing faulty genes and allowing the individual to start producing antibodies. Funding for research in the US is provided by the National Institutes of Health. Key research in the UK is funded by the Primary Immunodeficiency Association (PiA), and funding is raised through the annual Jeans for Genes campaign.

Epidemiology CVID has an estimated prevalence of about 1:50,000.[22] The typical patient is between 20 and 40, and males and females are equally affected. About 20% of patients are diagnosed in childhood.

History Charles Janeway, Sr. is generally credited with the first description of a case of CVID in 1953.[23]

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / apps. who. int/ classifications/ apps/ icd/ icd10online/ ?gd80. htm+ d83 http:/ / www. icd9data. com/ getICD9Code. ashx?icd9=279. 06 http:/ / www. ncbi. nlm. nih. gov/ omim/ 240500 http:/ / www. diseasesdatabase. com/ ddb3274. htm http:/ / www. emedicine. com/ ped/ topic444. htm http:/ / www. emedicine. com/ derm/ topic870. htm# http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?field=uid& term=D017074 James, William D.; Berger, Timothy G.; et al. (2006). Andrews' Diseases of the Skin: clinical Dermatology. Saunders Elsevier. ISBN 0-7216-2921-0.:84 [9] common variable immunodeficiency (http:/ / www. mercksource. com/ pp/ us/ cns/ cns_hl_dorlands_split. jsp?pg=/ ppdocs/ us/ common/ dorlands/ dorland/ four/ 000052585. htm) at Dorland's Medical Dictionary [10] Park MA, Li JT, Hagan JB, Maddox DE, Abraham RS (August 2008). "Common variable immunodeficiency: a new look at an old disease" (http:/ / linkinghub. elsevier. com/ retrieve/ pii/ S0140-6736(08)61199-X). Lancet 372 (9637): 489–502. doi:10.1016/S0140-6736(08)61199-X. PMID 18692715. . [11] Salzer U, Neumann C, Thiel J, et al. (2008). "Screening of functional and positional candidate genes in families with common variable immunodeficiency" (http:/ / www. biomedcentral. com/ 1471-2172/ 9/ 3). BMC Immunol. 9: 3. doi:10.1186/1471-2172-9-3. PMC 2268914. PMID 18254984. . [12] Blanco-Quirós A, Solís-Sánchez P, Garrote-Adrados JA, Arranz-Sanz E (2006). "Common variable immunodeficiency. Old questions are getting clearer" (http:/ / db. doyma. es/ cgi-bin/ wdbcgi. exe/ doyma/ mrevista. pubmed_full?inctrl=05ZI0102& rev=105& vol=34& num=6& pag=263). Allergol Immunopathol (Madr) 34 (6): 263–75. doi:10.1157/13095875. PMID 17173844. . [13] http:/ / www. ncbi. nlm. nih. gov/ omim/ 607594 [14] http:/ / www. ncbi. nlm. nih. gov/ omim/ 613943 [15] http:/ / www. ncbi. nlm. nih. gov/ omim/ 613494 [16] http:/ / www. ncbi. nlm. nih. gov/ omim/ 613495 [17] http:/ / www. ncbi. nlm. nih. gov/ omim/ 613496 [18] Common Variable Immunodeficiency : Article by C Lucy Park (http:/ / www. emedicine. com/ ped/ topic444. htm#) at eMedicine [19] Common Variable Immunodeficiency (http:/ / www. merck. com/ mmpe/ sec13/ ch164/ ch164f. html) at Merck Manual of Diagnosis and Therapy Professional Edition [20] Mellemkjaer L, Hammarstrom L, Andersen V, et al. (2002). "Cancer risk among patients with IgA deficiency or common variable immunodeficiency and their relatives: a combined Danish and Swedish study". Clin. Exp. Immunol. 130 (3): 495–500. doi:10.1046/j.1365-2249.2002.02004.x. PMC 1906562. PMID 12452841.

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Common variable immunodeficiency [21] Pourpak Z, Aghamohammadi A, Sedighipour L, et al. (2006). "Effect of regular intravenous immunoglobulin therapy on prevention of pneumonia in patients with common variable immunodeficiency" (http:/ / www. jmii. org/ content/ abstracts/ v39n2p114. php) (abstract). J Microbiol Immunol Infect 39 (2): 114–20. PMID 16604243. . [22] Common Variable Immunodeficiency : Article by Robert A Schwartz (http:/ / www. emedicine. com/ derm/ topic870. htm#) at eMedicine [23] Janeway CA, Apt L, Gitlin D. Agammaglobulinemia. Trans Assoc Am Physicians 1953;66:200-2. PMID 13136263

External links • • • • • • • •

Primary Immunodeficiency Association (UK) (http://www.pia.org.uk) Immune Deficiency Foundation (US) (http://www.primaryimmune.org) Michigan Immunodeficiency Foundation (US) (http://www.midf.org/) Immune Deficiencies Foundation of Australia (http://www.idfaustralia.org) Immune Deficiencies Foundation of New Zealand (http://www.idfnz.org.nz) IPOPI (International Patient Organisation for Patients with Primary Immunodeficiency) (http://www.ipopi.org) Canadian Immunodeficiencies Patient Organization (Canada) (http://www.cipo.ca) Dutch Patient Organisation for Primary Immunodeficiencies (SAS) (http://www. stichtingvoorafweerstoornissen.nl) • GeneReviews/NCBI/NIH/UW entry on Common Variable Immune Deficiency Overview (http://www.ncbi. nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=cvid) • Maker of SCIG product (http://www.vivaglobin.com/Default.aspx)

Epigenetics In biology, and specifically genetics, epigenetics is the study of heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επί- over, above) -genetics. These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism;[1] instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently.[2] One example of epigenetic changes in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells become the various pluripotent cell lines of the embryo which in turn become fully differentiated cells. In other words, a single fertilized egg cell – the zygote – changes into the many cell types including neurons, muscle cells, epithelium, blood vessels etc. as it continues to divide. It does so by activating some genes while inhibiting others.[3]

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Etymology and definitions Epigenetics (as in "epigenetic landscape") was coined by C. H. Waddington in 1942 as a portmanteau of the words genetics and epigenesis.[4] Epigenesis is an old[5] word which has more recently been used (see preformationism for historical background) to describe the differentiation of cells from their initial totipotent state in embryonic development. When Waddington coined the term the physical nature of genes and their role in heredity was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype.

Epigenetic mechanisms

Robin Holliday defined epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms."[6] Thus epigenetic can be used to describe anything other than DNA sequence that influences the development of an organism. The modern usage of the word in scientific discourse is more narrow, referring to heritable traits (over rounds of cell division and sometimes transgenerationally) that do not involve changes to the underlying DNA sequence.[7] The Greek prefix epi- in epigenetics implies features that are "on top of" or "in addition to" genetics; thus epigenetic traits exist on top of or in addition to the traditional molecular basis for inheritance. The similarity of the word to "genetics" has generated many parallel usages. The "epigenome" is a parallel to the word "genome", and refers to the overall epigenetic state of a cell. The phrase "genetic code" has also been adapted—the "epigenetic code" has been used to describe the set of epigenetic features that create different phenotypes in different cells. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an epigenomic map, a diagrammatic representation of the gene expression, DNA methylation and histone modification status of a particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the histone code or DNA methylation patterns. The psychologist Erik Erikson used the term epigenetic in his theory of psychosocial development. That usage, however, is of primarily historical interest.[8]

Molecular basis of epigenetics The molecular basis of epigenetics is complex. It involves modifications of the activation of certain genes, but not the basic structure of DNA. Additionally, the chromatin proteins associated with DNA may be activated or silenced. This accounts for why the differentiated cells in a multi-cellular organism express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but, if a mutation in the DNA has been caused in sperm or egg cell that results in fertilization, then some epigenetic changes are inherited from one generation to the next.[9] This raises the question of whether or not epigenetic changes in an organism can alter the basic structure of its DNA (see Evolution, below), a form of Lamarckism. Specific epigenetic processes include paramutation, bookmarking, imprinting, gene silencing, X chromosome inactivation, position effect, reprogramming, transvection, maternal effects, the progress of carcinogenesis, many

Epigenetics effects of teratogens, regulation of histone modifications and heterochromatin, and technical limitations affecting parthenogenesis and cloning. Epigenetic research uses a wide range of molecular biologic techniques to further our understanding of epigenetic phenomena, including chromatin immunoprecipitation (together with its large-scale variants ChIP-on-chip and ChIP-seq), fluorescent in situ hybridization, methylation-sensitive restriction enzymes, DNA adenine methyltransferase identification (DamID) and bisulfite sequencing. Furthermore, the use of bioinformatic methods is playing an increasing role (computational epigenetics).

Mechanisms Several types of epigenetic inheritance systems may play a role in what has become known as cell memory:[10]

DNA methylation and chromatin remodeling Because the phenotype of a cell or individual is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. There are several layers of regulation of gene expression. One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the histone proteins with which it associates. Histone proteins are little spheres that DNA wraps around. If the way that DNA is wrapped around the histones changes, gene expression can change as well. Chromatin remodeling is accomplished through two main mechanisms: 1. The first way is post translational modification of the amino acids that make up histone proteins. Histone proteins are made up of long DNA associates with histone proteins to form chains of amino acids. If the amino acids that are in the chain are chromatin. changed, the shape of the histone sphere might be modified. DNA is not completely unwound during replication. It is possible, then, that the modified histones may be carried into each new copy of the DNA. Once there, these histones may act as templates, initiating the surrounding new histones to be shaped in the new manner. By altering the shape of the histones around it, these modified histones would ensure that a differentiated cell would stay differentiated, and not convert back into being a stem cell. 2. The second way is the addition of methyl groups to the DNA, mostly at CpG sites, to convert cytosine to 5-methylcytosine. 5-Methylcytosine performs much like a regular cytosine, pairing up with a guanine. However, some areas of genome are methylated more heavily than others and highly methylated areas tend to be less transcriptionally active, through a mechanism not fully understood. Methylation of cytosines can also persist from the germ line of one of the parents into the zygote, marking the chromosome as being inherited from this parent (genetic imprinting). The way that the cells stay differentiated in the case of DNA methylation is clearer to us than it is in the case of histone shape. Basically, certain enzymes (such as DNMT1) have a higher affinity for the methylated cytosine. If this enzyme reaches a "hemimethylated" portion of DNA (where methylcytosine is in only one of the two DNA strands) the enzyme will methylate the other half. Although histone modifications occur throughout the entire sequence, the unstructured N-termini of histones (called histone tails) are particularly highly modified. These modifications include acetylation, methylation, ubiquitylation, phosphorylation and sumoylation. Acetylation is the most highly studied of these modifications. For example, acetylation of the K14 and K9 lysines of the tail of histone H3 by histone acetyltransferase enzymes (HATs) is generally correlated with transcriptional competence.

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Epigenetics One mode of thinking is that this tendency of acetylation to be associated with "active" transcription is biophysical in nature. Because it normally has a positively charged nitrogen at its end, lysine can bind the negatively charged phosphates of the DNA backbone. The acetylation event converts the positively charged amine group on the side chain into a neutral amide linkage. This removes the positive charge, thus loosening the DNA from the histone. When this occurs, complexes like SWI/SNF and other transcriptional factors can bind to the DNA and allow transcription to occur. This is the "cis" model of epigenetic function. In other words, changes to the histone tails have a direct affect on the DNA itself. Another model of epigenetic function is the "trans" model. In this model changes to the histone tails act indirectly on the DNA. For example, lysine acetylation may create a binding site for chromatin modifying enzymes (and basal transcription machinery as well). This Chromatin Remodeler can then cause changes to the state of the chromatin. Indeed, the bromodomain — a protein segment (domain) that specifically binds acetyl-lysine — is found in many enzymes that help activate transcription, including the SWI/SNF complex (on the protein polybromo). It may be that acetylation acts in this and the previous way to aid in transcriptional activation. The idea that modifications act as docking modules for related factors is borne out by histone methylation as well. Methylation of lysine 9 of histone H3 has long been associated with constitutively transcriptionally silent chromatin (constitutive heterochromatin). It has been determined that a chromodomain (a domain that specifically binds methyl-lysine) in the transcriptionally repressive protein HP1 recruits HP1 to K9 methylated regions. One example that seems to refute this biophysical model for acetylation is that tri-methylation of histone H3 at lysine 4 is strongly associated with (and required for full) transcriptional activation. Tri-methylation in this case would introduce a fixed positive charge on the tail. It has been shown that the histone lysine methyltransferase (KMT) is responsible for this methylation activity in the pattern of histones H3 & H4. This enzyme utilizes a catalytically active site called the SET domain (Suppressor of variegation, Enhancer of zeste, Trithorax). The SET domain is a 130-amino acid sequence involved in modulating gene activities. This domain has been demonstrated to bind to the histone tail and causes the methylation of the histone.[11] Differing histone modifications are likely to function in differing ways; acetylation at one position is likely to function differently than acetylation at another position. Also, multiple modifications may occur at the same time, and these modifications may work together to change the behavior of the nucleosome. The idea that multiple dynamic modifications regulate gene transcription in a systematic and reproducible way is called the histone code. DNA methylation frequently occurs in repeated sequences, and helps to suppress the expression and mobility of 'transposable elements':[12] Because 5-methylcytosine is chemically very similar to thymidine, CpG sites are frequently mutated and become rare in the genome, except at CpG islands where they remain unmethylated. Epigenetic changes of this type thus have the potential to direct increased frequencies of permanent genetic mutation. DNA methylation patterns are known to be established and modified in response to environmental factors by a complex interplay of at least three independent DNA methyltransferases, DNMT1, DNMT3A and DNMT3B, the loss of any of which is lethal in mice.[13] DNMT1 is the most abundant methyltransferase in somatic cells,[14] localizes to replication foci,[15] has a 10–40-fold preference for hemimethylated DNA and interacts with the proliferating cell nuclear antigen (PCNA).[16] By preferentially modifying hemimethylated DNA, DNMT1 transfers patterns of methylation to a newly synthesized strand after DNA replication, and therefore is often referred to as the ‘maintenance' methyltransferase.[17] DNMT1 is essential for proper embryonic development, imprinting and X-inactivation.[13] [18] Histones H3 and H4 can also be manipulated through demethylation using histone lysine demethylase (KDM). This recently identifited enzyme has a catalytically active site called the Jumonji domain (JmjC). The demethylation occurs when JmjC utilizes multiple cofactors to hydroxylate the methyl group, thereby removing it. JmjC is capable of demethylating mono-, di-, and tri-methylated substrates. .[19]

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Epigenetics Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Epigenetic control is often associated with alternative covalent modifications of histones.[20] The stability and heritability of states of larger chromosomal regions are often thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. A simplified stochastic model for this type of epigenetics is found here [21] [22] . Because DNA methylation and chromatin remodeling play such a central role in many types of epigenic inheritance, the word "epigenetics" is sometimes used as a synonym for these processes. However, this can be misleading. Chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodeling.[23] It has been suggested that the histone code could be mediated by the effect of small RNAs. The recent discovery and characterization of a vast array of small (21- to 26-nt), non-coding RNAs suggests that there is an RNA component, possibly involved in epigenetic gene regulation. Small interfering RNAs can modulate transcriptional gene expression via epigenetic modulation of targeted promoters.[24]

RNA transcripts and their encoded proteins Sometimes a gene, after being turned on, transcribes a product that (either directly or indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance the transcription of many liver- and muscle-specific genes, respectively, including their own, through the transcription factor activity of the proteins they encode. RNA signalling includes differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development.[25] Other epigenetic changes are mediated by the production of different splice forms of RNA, or by formation of double-stranded RNA (RNAi). Descendants of the cell in which the gene was turned on will inherit this activity, even if the original stimulus for gene-activation is no longer present. These genes are most often turned on or off by signal transduction, although in some systems where syncytia or gap junctions are important, RNA may spread directly to other cells or nuclei by diffusion. A large amount of RNA and protein is contributed to the zygote by the mother during oogenesis or via nurse cells, resulting in maternal effect phenotypes. A smaller quantity of sperm RNA is transmitted from the father, but there is recent evidence that this epigenetic information can lead to visible changes in several generations of offspring.[26]

Prions Prions are infectious forms of proteins. Proteins generally fold into discrete units which perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as a prion. Although often viewed in the context of infectious disease, prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome.[27] Fungal prions are considered epigenetic because the infectious phenotype caused by the prion can be inherited without modification of the genome. PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied of this type of prion.[28] [29] Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have a higher rate of read-through of stop codons, an effect which results in suppression of nonsense mutations in other genes.[30] The ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage by giving cells the ability to switch into a PSI+ state and express dormant genetic features normally terminated by premature stop codon mutations.[31] [32]

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Structural inheritance systems In ciliates such as Tetrahymena and Paramecium, genetically identical cells show heritable differences in the patterns of ciliary rows on their cell surface. Experimentally altered patterns can be transmitted to daughter cells. It seems existing structures act as templates for new structures. The mechanisms of such inheritance are unclear, but reasons exist to assume that multicellular organisms also use existing cell structures to assemble new ones.[33] [34]

Functions and consequences Development Somatic epigenetic inheritance, particularly through DNA methylation and chromatin remodeling, is very important in the development of multicellular eukaryotic organisms. The genome sequence is static (with some notable exceptions), but cells differentiate into many different types, which perform different functions, and respond differently to the environment and intercellular signalling. Thus, as individuals develop, morphogens activate or silence genes in an epigenetically heritable fashion, giving cells a "memory". In mammals, most cells terminally differentiate, with only stem cells retaining the ability to differentiate into several cell types ("totipotency" and "multipotency"). In mammals, some stem cells continue producing new differentiated cells throughout life, but mammals are not able to respond to loss of some tissues, for example, the inability to regenerate limbs, which some other animals are capable of. Unlike animals, plant cells do not terminally differentiate, remaining totipotent with the ability to give rise to a new individual plant. While plants do utilise many of the same epigenetic mechanisms as animals, such as chromatin remodeling, it has been hypothesised that plant cells do not have "memories", resetting their gene expression patterns at each cell division using positional information from the environment and surrounding cells to determine their fate.[35]

Medicine Epigenetics has many and varied potential medical applications as it tends to be multidimensional in nature.[36] Congenital genetic disease is well understood, and it is also clear that epigenetics can play a role, for example, in the case of Angelman syndrome and Prader-Willi syndrome. These are normal genetic diseases caused by gene deletions or inactivation of the genes, but are unusually common because individuals are essentially hemizygous because of genomic imprinting, and therefore a single gene knock out is sufficient to cause the disease, where most cases would require both copies to be knocked out.[37]

Evolution Although epigenetics in multicellular organisms is generally thought to be a mechanism involved in differentiation, with epigenetic patterns "reset" when organisms reproduce, there have been some observations of transgenerational epigenetic inheritance (e.g., the phenomenon of paramutation observed in maize). Although most of these multigenerational epigenetic traits are gradually lost over several generations, the possibility remains that multigenerational epigenetics could be another aspect to evolution and adaptation. A sequestered germ line or Weismann barrier is specific to animals, and epigenetic inheritance is expected to be far more common in plants and microbes. These effects may require enhancements to the standard conceptual framework of the modern evolutionary synthesis.[38] [39] Epigenetic features may play a role in short-term adaptation of species by allowing for reversible phenotype variability. The modification of epigenetic features associated with a region of DNA allows organisms, on a multigenerational time scale, to switch between phenotypes that express and repress that particular gene.[40] When the DNA sequence of the region is not mutated, this change is reversible. It has also been speculated that organisms may take advantage of differential mutation rates associated with epigenetic features to control the mutation rates of particular genes.[40] Interestingly, recent analysis have suggested that members of the APOBEC/AID family of

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Epigenetics cytosine deaminases are capable of simultaneously mediating genetic and epigenetic inheritance using similar molecular mechanisms.[41] Epigenetic changes have also been observed to occur in response to environmental exposure—for example, mice given some dietary supplements have epigenetic changes affecting expression of the agouti gene, which affects their fur color, weight, and propensity to develop cancer.[42] [43] More than 100 cases of transgenerational epigenetic inheritance phenomena have been reported in a wide range of organisms, including prokaryotes, plants, and animals. [44]

Epigenetic effects in humans Genomic imprinting and related disorders Some human disorders are associated with genomic imprinting, a phenomenon in mammals where the father and mother contribute different epigenetic patterns for specific genomic loci in their germ cells.[45] The best-known case of imprinting in human disorders is that of Angelman syndrome and Prader-Willi syndrome—both can be produced by the same genetic mutation, chromosome 15q partial deletion, and the particular syndrome that will develop depends on whether the mutation is inherited from the child's mother or from their father.[46] This is due to the presence of genomic imprinting in the region. Beckwith-Wiedemann syndrome is also associated with genomic imprinting, often caused by abnormalities in maternal genomic imprinting of a region on chromosome 11.

Transgenerational epigenetic observations See main article Transgenerational epigenetics Marcus Pembrey and colleagues also observed in the Överkalix study that the paternal (but not maternal) grandsons [47] of Swedish men who were exposed during preadolescence to famine in the 19th century were less likely to die of cardiovascular disease; if food was plentiful then diabetes mortality in the grandchildren increased, suggesting that this was a transgenerational epigenetic inheritance.[48] The opposite effect was observed for females—the paternal (but not maternal) granddaughters of women who experienced famine while in the womb (and therefore while their eggs were being formed) lived shorter lives on average.[49]

Cancer and developmental abnormalities A variety of compounds are considered as epigenetic carcinogens—they result in an increased incidence of tumors, but they do not show mutagen activity (toxic compounds or pathogens that cause tumors incident to increased regeneration should also be excluded). Examples include diethylstilbestrol, arsenite, hexachlorobenzene, and nickel compounds. Many teratogens exert specific effects on the fetus by epigenetic mechanisms.[50] [51] While epigenetic effects may preserve the effect of a teratogen such as diethylstilbestrol throughout the life of an affected child, the possibility of birth defects resulting from exposure of fathers or in second and succeeding generations of offspring has generally been rejected on theoretical grounds and for lack of evidence.[52] However, a range of male-mediated abnormalities have been demonstrated, and more are likely to exist.[53] FDA label information [54] for Vidaza(tm), a formulation of 5-azacitidine (an unmethylatable analog of cytidine that causes hypomethylation when incorporated into DNA) states that "men should be advised not to father a child" while using the drug, citing evidence in treated male mice of reduced fertility, increased embryo loss, and abnormal embryo development. In rats, endocrine differences were observed in offspring of males exposed to morphine.[55] In mice, second generation effects of diethylstilbesterol have been described occurring by epigenetic mechanisms.[56] Recent studies have shown that the Mixed Lineage Leukemia (MLL) gene causes leukemia by rearranging and fusing with other genes in different chromosomes, which is a process under epigenetic control.[57]

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Epigenetics Other investigations have concluded that alterations in histone acetylation and DNA methylation occur in various genes influencing prostate cancer.[58] In 2008, the National Institutes of Health announced that $190 million had been earmarked for epigenetics research over the next five years. In announcing the funding, government officials noted that epigenetics has the potential to explain mechanisms of aging, human development, and the origins of cancer, heart disease, mental illness, as well as several other conditions. Some investigators, like Randy Jirtle, PhD, of Duke University Medical Center, think epigenetics may ultimately turn out to have a greater role in disease than genetics.[59] DNA methylation in cancer DNA methylation is an important regulator of gene transcription and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing, and the genes with high levels of 5-methylcytosine in their promoter region are transcriptionally silent. DNA methylation is essential during embryonic development, and in somatic cells, patterns of DNA methylation are generally transmitted to daughter cells with a high fidelity. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and found in two distinct forms: hypermethylation and hypomethylation compared to normal tissue. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumour suppressor genes. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms.[60] Variant histones H2A in cancer The histone variants of the H2A family are highly conserved in mammals, playing critical roles in regulating many nuclear processes by altering chromatin structure. One of the key H2A variants, H2A.X, marks DNA damage, facilitating the recruitment of DNA repair proteins to restore genomic integrity. Another variant, H2A.Z, plays an important role in both gene activation and repression. A high level of H2A.Z expression is ubiquitously detected in many cancers and is significantly associated with cellular proliferation and genomic instability.[60] Cancer Treatment Current research has shown that epigenetic pharmaceuticals could be a putative replacement or adjuvant therapy for currently accepted treatment methods such as radiation and chemotherapy, or could enhance the effects of these current treatments.[61] It has been shown that the epigenetic control of the proto-onco regions and the tumor suppressor sequences by conformational changes in histones directly affects the formation and progression of cancer [62] Epigenetics also has the factor of reversibility, a characteristic that other cancer treatments do not offer.[58] Drug development has mainly focused on Histone Acetyltransferase (HAT) and Histone Deactylase (HDAC), including the introduction of the new pharmaceutical Vorinostat, a HDAC inhibitor, to the market.[63] HDAC specifically has been shown to play an integral role in the progression of oral squamous cancer [62] Current front-runner candidates for new drug targets are Histone Lysine Methyltransferases (KMT) and Protein Arginine Methyltransferases (PRMT).[64]

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Twin studies Recent studies involving both dizygotic and monozygotic twins have produced some evidence of epigenetic influence in humans.[65] [66] [67]

Epigenetics in microorganisms Bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Bacteria make use of DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage, transposase activity and regulation of gene expression.[68] [69] The filamentous fungus Neurospora crassa is a prominent model system for understanding the control and function of cytosine methylation. In this organisms, DNA methylation is associated with relics of a genome defense system called RIP (repeat-induced point mutation) and silences gene expression by inhibiting transcription elongation.[70]

Escherichia coli bacteria

The yeast prion PSI is generated by a conformational change of a translation termination factor, which is then inherited by daughter cells. This can provide a survival advantage under adverse conditions. This is an example of epigenetic regulation enabling unicellular organisms to respond rapidly to environmental stress. Prions can be viewed as epigenetic agents capable of inducing a phenotypic change without modification of the genome.[69]

Notes and references [1] Adrian Bird (2007). "Perceptions of epigenetics". Nature 447 (7143): 396–398. doi:10.1038/nature05913. PMID 17522671. [2] Special report: 'What genes remember' by Philip Hunter | Prospect Magazine May 2008 issue 146 (http:/ / www. prospect-magazine. co. uk/ article_details. php?id=10140) [3] Reik, Wolf (2007-05-23). "Stability and flexibility of epigenetic gene regulation in mammalian development" (http:/ / www. nature. com/ nature/ journal/ v447/ n7143/ full/ nature05918. html). Nature 447 (May (online)): 425–432. doi:10.1038/nature05918. PMID 17522676. . Retrieved 2008-04-05. [4] C.H. Waddington (1942) (1977). "The epigenotype". Endeavour 1: 18–20. doi:10.1016/0160-9327(77)90005-9. [5] According to the Oxford English Dictionary:

The word is used by W. Harvey, Exercitationes 1651, p. 148, and in the English Anatomical Exercitations 1653, p. 272. It is explained to mean ‘partium super-exorientium additamentum’, ‘the additament of parts budding one out of another’. It is also worth quoting this adumbration of the definition given there (viz., "The formation of an organic germ as a new product"):

theory of epigenesis: the theory that the germ is brought into existence (by successive accretions), and not merely developed, in the process of reproduction. [...] The opposite theory was formerly known as the ‘theory of evolution’; to avoid the ambiguity of this name, it is now spoken of chiefly as the ‘theory of preformation’, sometimes as that of ‘encasement’ or ‘emboîtement’. [6] Holliday, R., 1990. Mechanisms for the control of gene activity during development. Biol. Rev. Cambr. Philos. Soc. 65, 431-471 [7] Russo, V.E.A., Martienssen, R.A., Riggs, A.D., 1996 Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press, Plainview, NY. [8] Epigenetics (http:/ / www. bio-medicine. org/ biology-definition/ Epigenetics/ ) [9] V.L. Chandler (2007). "Paramutation: From Maize to Mice". Cell 128 (4): 641–645. doi:10.1016/j.cell.2007.02.007. PMID 17320501.

Epigenetics [10] Jablonka, E; Lamb MJ and Lachmann M (September 1992). "Evidence, mechanisms and models for the inheritance of acquired characteristics" (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6WMD-4KFTYG9-8& _user=10& _handle=C-WA-A-E-E-MsSAYWW-UUW-U-U-E-U-U-AAZEEUZCDZ-AAZDVYDBDZ-ADYZVZWEA-E-U& _fmt=summary& _coverDate=09/ 21/ 1992& _rdoc=8& _orig=browse& _srch=#toc#6932#1992#998419997#628456!& _cdi=6932& view=c& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=56159ca247a23a908a55cdabe8dd69e7). J. Theoret. Biol. 158 (2): 245–268. doi:10.1016/S0022-5193(05)80722-2. . [11] Jenuwein, T; Laible, G; Dorn, R; Reuter, G (1998). "SET domain proteins modulate chromatin domains in eu- and heterochromatin". Cellular and Molecular Life Sciences 1 (54): 80–93. PMID 9487389. [12] Slotkin, R; R. Martienssen (2007). "Transposable elements and the epigenetic regulation of the genome." (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Retrieve& db=PubMed& dopt=Citation& list_uids=17363976). . Retrieved 2009-10-04. [13] Li, E; Bestor TH and Jaenisch R (June 1992). "Targeted mutation of the DNA methyltransferase gene results in embryonic lethality" (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6WSN-4D57093-1R& _coverDate=06/ 12/ 1992& _alid=515191593& _rdoc=1& _fmt=& _orig=search& _qd=1& _cdi=7051& _sort=d& view=c& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=19d33f62482a44052266e684682a06da). Cell 69 (6): 915–926. doi:10.1016/0092-8674(92)90611-F. PMID 1606615. . [14] Robertson, KD; Uzyolgi E, Lian G et al. (June 1999). "The human DNA methyltransferases (DNMTs) 1, 3a, 3b: Coordinate mRNA expression in normal tissues and overexpression in tumors". Nucleic Acids Res 27 (11): 2291–2298. doi:10.1093/nar/27.11.2291. PMC 148793. PMID 10325416. [15] Leonhardt, H; Page AW, Weier HU, Bestor TH (November 1992). "A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei" (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6WSN-4D0YB27-19& _coverDate=11/ 27/ 1992& _alid=515194074& _rdoc=1& _fmt=& _orig=search& _qd=1& _cdi=7051& _sort=d& view=c& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=9bdfefee567ff4ea6226007214ffbc34). Cell 71 (5): 865–873. doi:10.1016/0092-8674(92)90561-P. . [16] Chuang, LS; Ian HI, Koh TW et al. (September 1997). "Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1". Science 277 (5334): 1996–2000. doi:10.1126/science.277.5334.1996. PMID 9302295. [17] Robertson, KD; Wolffe AP (October 2000). "DNA methylation in health and disease" (http:/ / www. nature. com/ nrg/ journal/ v1/ n1/ abs/ nrg1000_011a_fs. html). Nat Rev Genet 1 (1): 11–19. doi:10.1038/35049533. PMID 11262868. . [18] Li, E; Beard C and Jaenisch R (December 1993). "Role for DNA methylation in genomic imprinting". Nature 366 (6453): 362–365. doi:10.1038/366362a0. PMID 8247133. [19] Nottke, A; Colaiácovo, MP; Shi, Y (2009). "Developmental role of the histone lysine demethylases". Development 6 (136): 879–89 pmid=19234061. [20] Rosenfeld, Jeffrey A; Wang, Zhibin; Schones, Dustin; Zhao, Keji; DeSalle, Rob; Zhang, Michael Q (31 March 2009). "Determination of enriched histone modifications in non-genic portions of the human genome.". BMC Genomics 10: 143. doi:10.1186/1471-2164-10-143. PMC 2667539. PMID 19335899. [21] http:/ / cmol. nbi. dk/ models/ epigen/ Epigen. html [22] Dodd, I.B.; Micheelsen, M.A.; Sneppen, K.; Thon, G. (2007). "Theoretical Analysis of Epigenetic Cell Memory by Nucleosome Modification". Cell 129 (4): 813–822. doi:10.1016/j.cell.2007.02.053. PMID 17512413. [23] Mark Ptashne, 2007. On the use of the word ‘epigenetic’. Current Biology, 17(7):R233-R236. doi:10.1016/j.cub.2007.02.030 [24] Morris KV (2008). "Epigenetic Regulation of Gene Expression" (http:/ / www. horizonpress. com/ rnareg). RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. . . [25] Mattick, JS; Amaral, PP; Dinger, ME; Mercer, TR; Mehler, MF (2009). "RNA regulation of epigenetic processes.". BioEssays : news and reviews in molecular, cellular and developmental biology 31 (1): 51–9. doi:10.1002/bies.080099. PMID 19154003. [26] Choi CQ (2006-05-25). "The Scientist: RNA can be hereditary molecule" (http:/ / www. the-scientist. com/ news/ display/ 23494). The Scientist. . Retrieved 2006. [27] A. Yool and W.J. Edmunds (1998). "Epigenetic inheritance and prions". Journal of Evolutionary Biology 11 (2): 241–242. doi:10.1007/s000360050085. [28] B.S. Cox (1965). "[PSI], a cytoplasmic suppressor of super-suppression in yeast". Heredity 20 (4): 505–521. doi:10.1038/hdy.1965.65. [29] F. Lacroute (1971). "Non-Mendelian mutation allowing ureidosuccinic acid uptake in yeast". Journal of Bacteriology 106 (2): 519–522. PMC 285125. PMID 5573734. [30] S.W. Liebman and F. Sherman (1979). "Extrachromosomal psi+ determinant suppresses nonsense mutations in yeast". Journal of Bacteriology 139 (3): 1068–1071. PMC 218059. PMID 225301. Free full text available (http:/ / jb. asm. org/ cgi/ content/ abstract/ 139/ 3/ 1068) [31] H.L. True and S.L. Lindquist (2000). "A yeast prion provides a mechanism for genetic variation and phenotypic diversity". Nature 407 (6803): 477–483. doi:10.1038/35035005. PMID 11028992. [32] J. Shorter and S. Lindquist (2005). "Prions as adaptive conduits of memory and inheritance". Nature Reviews Genetics 6 (6): 435–450. doi:10.1038/nrg1616. PMID 15931169. [33] Jan Sapp, Beyond the Gene. 1987 Oxford University Press. Jan Sapp, "Concepts of organization: the leverage of ciliate protozoa" . In S. Gilbert ed., Developmental Biology: A Comprehensive Synthesis, (New York: Plenum Press, 1991), 229-258. Jan Sapp, Genesis: The Evolution of Biology Oxford University Press, 2003. [34] Oyama, Susan; Paul E. Griffiths, Russell D. Gray (2001). MIT Press. ISBN 0262650630.

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Epigenetics [35] Costa, Silvia; Shaw, Peter (2006). "'Open Minded' cells: how cells can change fate". Trends in Cell Biology 17 (3): 101–106. doi:10.1016/j.tcb.2006.12.005. PMID 17194589. [36] Chahwan R, Wontakal SN, Roa S (March 2011). "The multidimensional nature of epigenetic information and its role in disease" (http:/ / www. discoverymedicine. com/ Richard-Chahwan/ 2011/ 03/ 17/ the-multidimensional-nature-of-epigenetic-information-and-its-role-in-disease/ ). Discovery medicine 11 (58): 233–43. PMID 21447282. . [37] Online 'Mendelian Inheritance in Man' (OMIM) 105830 (http:/ / www. ncbi. nlm. nih. gov/ omim/ 105830) [38] Jablonka, Eva; Marion J. Lamb (2005). Evolution in Four Dimensions. MIT Press. ISBN 0-262-10107-6. [39] See also Denis Noble The Music of Life see esp pp93-8 and p48 where he cites Jablonka & Lamb and Massimo Pigliucci's review of Jablonka and Lamb in Nature 435, 565-566 (2 June 2005) [40] O.J. Rando and K.J. Verstrepen (2007). "Timescales of Genetic and Epigenetic Inheritance". Cell 128 (4): 655–668. doi:10.1016/j.cell.2007.01.023. PMID 17320504. [41] Chahwan R., Wontakal S.N., and Roa S. (2010). "Crosstalk between genetic and epigenetic information through cytosine deamination". Trends in Genetics 26 (10): 443–448. doi:10.1016/j.tig.2010.07.005. PMID 20800313. [42] Cooney, CA, Dave, AA, and Wolff, GL (2002). "Maternal Methyl Supplements in Mice Affect Epigenetic Variation and DNA Methylation of Offspring". Journal of Nutrition 132 (8 Suppl): 2393S–2400S. PMID 12163699. available online (http:/ / jn. nutrition. org/ cgi/ content/ full/ 132/ 8/ 2393S) [43] Waterland RA and Jirtle RL (August 2003). "Transposable elements: Targets for early nutritional effects on epigenetic gene regulation" (http:/ / mcb. asm. org/ cgi/ content/ full/ 23/ 15/ 5293). Molecular and Cellular Biology 23 (15): 5293–5300. doi:10.1128/MCB.23.15.5293-5300.2003. PMC 165709. PMID 12861015. . [44] Jablonka, Eva; Gal Raz (June 2009). "Transgenerational Epigenetic Inheritance: Prevalence, Mechanisms, and Implications for the Study of Heredity and Evolution" (http:/ / www. journals. uchicago. edu/ doi/ abs/ 10. 1086/ 598822). The Quarterly Review of Biology 84 (2): 131–176. doi:10.1086/598822. PMID 19606595. . [45] A.J. Wood and A.J. Oakey (2006). "Genomic imprinting in mammals: Emerging themes and established theories". PLOS Genetics 2 (11): 1677–1685. doi:10.1371/journal.pgen.0020147. PMC 1657038. PMID 17121465. available online (http:/ / genetics. plosjournals. org/ perlserv/ ?request=get-document& doi=10. 1371/ journal. pgen. 0020147) [46] J.H.M. Knoll, R.D. Nicholls, R.E. Magenis, J.M. Graham Jr, M. Lalande, S.A. Latt (1989). "Angelman and Prader-Willi syndromes share a common chromosome deletion but differ in parental origin of the deletion". American Journal of Medical Genetics 32 (2): 285–290. doi:10.1002/ajmg.1320320235. PMID 2564739. [47] A paternal grandson is the son of a grandparent's son; a maternal grandson is the son of a grandparent's daughter. [48] Pembrey ME, Bygren LO, Kaati G, et al.. Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet 2006; 14: 159-66. PMID 16391557. Robert Winston refers to this study in a lecture (http:/ / www. dundee. ac. uk/ externalrelations/ events/ lectures. html); see also discussion at Leeds University, here (http:/ / www. fbs. leeds. ac. uk/ staff/ pm/ epigenetics. htm#exciting2) [49] http:/ / www. pbs. org/ wgbh/ nova/ transcripts/ 3413_genes. html [50] Bishop, JB; Witt KL and Sloane RA (December 1997). "Genetic toxiticities of human teratogens" (http:/ / www. sciencedirect. com/ science?_ob=ArticleURL& _udi=B6T2C-3TGW0WR-19& _coverDate=12/ 12/ 1997& _alid=515200131& _rdoc=1& _fmt=& _orig=search& _qd=1& _cdi=4915& _sort=d& view=c& _acct=C000050221& _version=1& _urlVersion=0& _userid=10& md5=991625903beeedc77a9455d6fa2382a9). Mutat Res 396 (1–2): 9–43. doi:10.1016/S0027-5107(97)00173-5. . [51] Gurvich, N; Berman MG, Wittner BS et al. (July 2004). "Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo" (http:/ / www. fasebj. org/ cgi/ reprint/ 04-3425fjev1). FASEB J 19 (9): 1166–1168. doi:10.1096/fj.04-3425fje. PMID 15901671. . [52] Smithells, D (November 1998). "Does thalidomide cause second generation birth defects?". Drug Saf 19 (5): 339–341. doi:10.2165/00002018-199819050-00001. PMID 9825947. [53] Friedler, G (December 1996). "Paternal exposures: impact on reproductive and developmental outcome. An overview". Pharmacol Biochem Behav 55 (4): 691–700. doi:10.1016/S0091-3057(96)00286-9. PMID 8981601. [54] http:/ / www. fda. gov/ cder/ foi/ label/ 2004/ 050794lbl. pdf [55] Cicero, TJ; Adams NL, Giodarno A et al. (March 1991). "Influence of morphine exposure during adolescence on the sexual maturation of male rats and the development of their offspring". J Pharmacol Exp Ther. 256 (3): 1086–1093. PMID 2005573. [56] Newbold, RR; Padilla-Banks E and Jefferson WN (June 2006). "Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations". Endocrinology 147 (6 Suppl): S11–S17. doi:10.1210/en.2005-1164. PMID 16690809. [57] Mandall, SS (2010). "Mixed lineage leukemia: versatile player in epigenetics and human disease". The FEBS Journal 227 (8): 1789. doi:10.1111/j.1742-4658.2010.07605.x. PMID 20236314. [58] Li, LC; Carroll, PR; Dahiya, R (2005). "Epigenetic changes in prostate cancer: implication for diagnosis and treatment". Journal of the National Cancer Institute 2 (97): 103–15. doi:10.1093/jnci/dji010. PMID 15657340. [59] Beil, Laura (Winter, 2008). "Medicine's New Epicenter? Epigenetics: New field of epigenetics may hold the secret to flipping cancer's "off" switch." (http:/ / www. curetoday. com/ index. cfm/ fuseaction/ article. show/ id/ 2/ article_id/ 949). CURE (Cancer Updates, Research and Education). . [60] Craig, JM; Wong, NC (editor) (2011). Epigenetics: A Reference Manual. Caister Academic Press. ISBN 978-1-904455-88-2. [61] Wang, LG; Chiao, JW (2010). "Prostate cancer chemopreventive activity of phenethyl isothiocyanate through epigenetic regulation (review)". International Journal of Oncology 3 (37): 533–9. PMID 20664922.

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Epigenetics [62] Iglesias-Linares, A; Yañez-Vico, RM; González-Moles, MA (2010). "Potential role of HDAC inhibitors in cancer therapy: insights into oral squamous cell carcinoma". Oral Oncology 5 (46): 323–9. doi:10.1016/j.oraloncology.2010.01.009. PMID 20207580. [63] Spannhoff, A; Sippl, W; Jung, M (2009). "Cancer treatment of the future: Inhibitors of histone methyltransferases". The International Journal of Biochemistry & Cell Biology 1 (41): 4–11. doi:10.1016/j.biocel.2008.07.024. PMID 18773966. [64] "Toward the development of potent and selective bisubstrate inhibitors of protein arginine methyltransferases". Bioorganic & Medicinal Chemistry Letters 7 (20): 2103–5. April 2010. doi:10.1016/j.bmcl.2010.02.069. PMID 20219369. [65] O'Connor, Anahad (2008-03-11). "The Claim: Identical Twins Have Identical DNA" (http:/ / www. nytimes. com/ 2008/ 03/ 11/ health/ 11real. html). New York Times. . Retrieved 2010-05-02. [66] Kaminsky, Zachary A; Tang, T; Wang, SC; Ptak, C; Oh, GH; Wong, AH; Feldcamp, LA; Virtanen, C et al. (2009). "DNA methylation profiles in monozygotic and dizygotic twins". Nature Genetics 41 (2): 240–5. doi:10.1038/ng.286. PMID 19151718. [67] Fraga, M. F.; Ballestar, E; Paz, MF; Ropero, S; Setien, F; Ballestar, ML; Heine-Suñer, D; Cigudosa, JC et al. (2005). "Epigenetic differences arise during the lifetime of monozygotic twins" (http:/ / www. pnas. org/ content/ 102/ 30/ 10604. full). Proceedings of the National Academy of Sciences 102 (30): 10604–9. doi:10.1073/pnas.0500398102. PMC 1174919. PMID 16009939. . [68] Casadesus J and Low D (September 2006). "Epigenetic Gene Regulation in the Bacterial World". Microbiol Mol Biol Rev 70 (3): 830–856. doi:10.1128/MMBR.00016-06. PMC 1594586. PMID 16959970. [69] Tost J (editor). (2008). Epigenetics (http:/ / www. horizonpress. com/ epi). Caister Academic Press. ISBN 1904455239. . . [70] Genome Res. 2009. 19: 427-437/doi: 10.1101/gr.086231.108

External links • review (2009) to add to refs (http://www.ncbi.nlm.nih.gov/pubmed/19378334) • • • • •

The Human Epigenome Project (HEP) (http://www.epigenome.org/) The Epigenome Network of Excellence (NoE) (http://www.epigenome-noe.net/index.php) The Epigenome Network of Excellence (NoE)- public international site (http://www.epigenome.eu/) DNA Is Not Destiny (http://discovermagazine.com/2006/nov/cover) - Discover Magazine cover story BBC - Horizon - 2005 - The Ghost In Your Genes (http://www.bbc.co.uk/sn/tvradio/programmes/horizon/ ghostgenes.shtml) • Epigenetics article (http://www.hopkinsmedicine.org/press/2002/november/epigenetics.htm) at Hopkins Medicine • Towards a global map of epigenetic variation (http://genome.wellcome.ac.uk/doc_WTX036556.html)

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Common Therapies for MCL Clinical trial Clinical trials are a step in medical research conducted to allow safety (or more specifically, information about adverse drug reactions and adverse effects of other treatments) and efficacy data to be collected for health interventions (e.g., drugs, diagnostics, devices, therapy protocols). These trials can take place only after satisfactory information has been gathered on the quality of the non-clinical safety, and Health Authority/Ethics Committee approval is granted in the country where the trial is taking place. Depending on the type of product and the stage of its development, investigators enroll healthy volunteers and/or patients into small pilot studies initially, followed by larger scale studies in patients that often compare the new product with the currently prescribed treatment. As positive safety and efficacy data are gathered, the number of patients is typically increased. Clinical trials can vary in size from a single center in one country to multicenter trials in multiple countries. Due to the sizable cost a full series of clinical trials may incur, the burden of paying for all the necessary people and services is usually borne by the sponsor who may be a governmental organization, a pharmaceutical, or biotechnology company. Since the diversity of roles may exceed resources of the sponsor, often a clinical trial is managed by an outsourced partner such as a contract research organization or a clinical trials unit in the academic sector.

Overview Clinical trials often involve patients with specific health conditions who then benefit from receiving otherwise unavailable treatments. In early phases, participants are healthy volunteers who receive financial incentives for their inconvenience. During dosing periods, study subjects typically remain on site at the unit for durations of anything from 1 to 30 nights, occasionally longer, although this is not always required.[1] In planning a clinical trial, the sponsor or investigator first identifies the medication or device to be tested. Usually, one or more pilot experiments are conducted to gain insights for design of the clinical trial to follow. In medical jargon, effectiveness is how well a treatment works in practice and efficacy is how well it works in a clinical trial. In the U.S., the elderly comprise only 14% of the population but they consume over one-third of drugs.[2] Despite this, they are often excluded from trials because their more frequent health issues and drug use produce unreliable data. Women, children, and people with unrelated medical conditions are also frequently excluded.[3] In coordination with a panel of expert investigators (usually physicians well known for their publications and clinical experience), the sponsor decides what to compare the new agent with (one or more existing treatments or a placebo), and what kind of patients might benefit from the medication or device. If the sponsor cannot obtain enough patients with this specific disease or condition at one location, then investigators at other locations who can obtain the same kind of patients to receive the treatment would be recruited into the study. During the clinical trial, the investigators: recruit patients with the predetermined characteristics, administer the treatment(s), and collect data on the patients' health for a defined time period. These patients are voluntaries and they are not paid for participating in clinical trials. These data include measurements like vital signs, concentration of the study drug in the blood, and whether the patient's health improves or not. The researchers send the data to the trial sponsor who then analyzes the pooled data using statistical tests. Some examples of what a clinical trial may be designed to do:

Clinical trial • Assess the safety and effectiveness of a new medication or device on a specific kind of patient (e.g., patients who have been diagnosed with Alzheimer's disease) • Assess the safety and effectiveness of a different dose of a medication than is commonly used (e.g., 10 mg dose instead of 5 mg dose) • Assess the safety and effectiveness of an already marketed medication or device for a new indication, i.e. a disease for which the drug is not specifically approved • Assess whether the new medication or device is more effective for the patient's condition than the already used, standard medication or device ("the gold standard" or "standard therapy") • Compare the effectiveness in patients with a specific disease of two or more already approved or common interventions for that disease (e.g., Device A vs. Device B, Therapy A vs. Therapy B) Note that while most clinical trials compare two medications or devices, some trials compare three or four medications, doses of medications, or devices against each other. Except for very small trials limited to a single location, the clinical trial design and objectives are written into a document called a clinical trial protocol. The protocol is the 'operating manual' for the clinical trial and ensures that researchers in different locations all perform the trial in the same way on patients with the same characteristics. (This uniformity is designed to allow the data to be pooled.) A protocol is always used in multicenter trials. Because the clinical trial is designed to test hypotheses and rigorously monitor and assess what happens, clinical trials can be seen as the application of the scientific method, and specifically the experimental step, to understanding human or animal biology. The most commonly performed clinical trials evaluate new drugs, medical devices (like a new catheter), biologics, psychological therapies, or other interventions. Clinical trials may be required before the national regulatory authority[4] approves marketing of the drug or device, or a new dose of the drug, for use on patients.

History The history of clinical trials before 1750 is brief.[5] [6] The concepts behind clinical trials, however, are ancient. The Book of Daniel verses 12 through 15, for instance, describes a planned experiment with both baseline and follow-up observations of two groups who either partook of, or did not partake of, "the King's meat" over a trial period of ten days. Persian physician and philosopher, Avicenna, gave such inquiries a more formal structure.[7] In The Canon of Medicine in 1025 AD, he laid down rules for the experimental use and testing of drugs and wrote a precise guide for practical experimentation in the process of discovering and proving the effectiveness of medical drugs and substances.[8] He laid out the following rules and principles for testing the effectiveness of new drugs and medications:[9] [10] 1. The drug must be free from any extraneous accidental quality. 2. It must be used on a simple, not a composite, disease. 3. The drug must be tested with two contrary types of diseases, because sometimes a drug cures one disease by its essential qualities and another by its accidental ones. 4. The quality of the drug must correspond to the strength of the disease. For example, there are some drugs whose heat is less than the coldness of certain diseases, so that they would have no effect on them. 5. The time of action must be observed, so that essence and accident are not confused. 6. The effect of the drug must be seen to occur constantly or in many cases, for if this did not happen, it was an accidental effect. 7. The experimentation must be done with the human body, for testing a drug on a lion or a horse might not prove anything about its effect on man. One of the most famous clinical trials was James Lind's demonstration in 1747 that citrus fruits cure scurvy.[11] He compared the effects of various different acidic substances, ranging from vinegar to cider, on groups of afflicted

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Clinical trial sailors, and found that the group who were given oranges and lemons had largely recovered from scurvy after 6 days. Frederick Akbar Mahomed (d. 1884), who worked at Guy's Hospital in London,[12] made substantial contributions to the process of clinical trials during his detailed clinical studies, where "he separated chronic nephritis with secondary hypertension from what we now term essential hypertension." He also founded "the Collective Investigation Record for the British Medical Association; this organization collected data from physicians practicing outside the hospital setting and was the precursor of modern collaborative clinical trials."[13]

Types One way of classifying clinical trials is by the way the researchers behave. • In an observational study, the investigators observe the subjects and measure their outcomes. The researchers do not actively manage the study. An example is the Nurses' Health Study. • In an interventional study, the investigators give the research subjects a particular medicine or other intervention. Usually, they compare the treated subjects to subjects who receive no treatment or standard treatment. Then the researchers measure how the subjects' health changes. Another way of classifying trials is by their purpose. The U.S. National Institutes of Health (NIH) organizes trials into five (5) different types:[14] • Prevention trials: look for better ways to prevent disease in people who have never had the disease or to prevent a disease from returning. These approaches may include medicines, vitamins, vaccines, minerals, or lifestyle changes. • Screening trials: test the best way to detect certain diseases or health conditions. • Diagnostic trials: conducted to find better tests or procedures for diagnosing a particular disease or condition. • Treatment trials: test experimental treatments, new combinations of drugs, or new approaches to surgery or radiation therapy. • Quality of life trials: explore ways to improve comfort and the quality of life for individuals with a chronic illness (a.k.a. Supportive Care trials). • Compassionate use trials or expanded access: provide partially tested, unapproved therapeutics prior to a small number of patients that have no other realistic options. Usually, this involves a disease for which no effective therapy exists, or a patient that has already attempted and failed all other standard treatments and whose health is so poor that he does not qualify for participation in randomized clinical trials. Usually, case by case approval must be granted by both the FDA and the pharmaceutical company for such exceptions.

Design A fundamental distinction in evidence-based medicine is between observational studies and randomized controlled trials. Types of observational studies in epidemiology such as the cohort study and the case-control study provide less compelling evidence than the randomized controlled trial. In observational studies, the investigators only observe associations (correlations) between the treatments experienced by participants and their health status or diseases. A randomized controlled trial is the study design that can provide the most compelling evidence that the study treatment causes the expected effect on human health. Currently, some Phase II and most Phase III drug trials are designed as randomized, double blind, and placebo-controlled. • Randomized: Each study subject is randomly assigned to receive either the study treatment or a placebo. • Blind: The subjects involved in the study do not know which study treatment they receive. If the study is double-blind, the researchers also do not know which treatment is being given to any given subject. This 'blinding' is to prevent biases, since if a physician knew which patient was getting the study treatment and which patient

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Clinical trial was getting the placebo, he/she might be tempted to give the (presumably helpful) study drug to a patient who could more easily benefit from it. In addition, a physician might give extra care to only the patients who receive the placebos to compensate for their ineffectiveness. A form of double-blind study called a "double-dummy" design allows additional insurance against bias or placebo effect. In this kind of study, all patients are given both placebo and active doses in alternating periods of time during the study. • Placebo-controlled: The use of a placebo (fake treatment) allows the researchers to isolate the effect of the study treatment. Although the term "clinical trials" is most commonly associated with the large, randomized studies typical of Phase III, many clinical trials are small. They may be "sponsored" by single physicians or a small group of physicians, and are designed to test simple questions. In the field of rare diseases sometimes the number of patients might be the limiting factor for a clinical trial. Other clinical trials require large numbers of participants (who may be followed over long periods of time), and the trial sponsor is a private company, a government health agency, or an academic research body such as a university.

Active comparator studies Of note, during the last ten years or so it has become a common practice to conduct "active comparator" studies (also known as "active control" trials). In other words, when a treatment exists that is clearly better than doing nothing for the subject (i.e. giving them the placebo), the alternate treatment would be a standard-of-care therapy. The study would compare the 'test' treatment to standard-of-care therapy. A growing trend in the pharmacology field involves the use of third-party contractors to obtain the required comparator compounds. Such third parties provide expertise in the logistics of obtaining, storing, and shipping the comparators. As an advantage to the manufacturer of the comparator compounds, a well-established comparator sourcing agency can alleviate the problem of parallel importing (importing a patented compound for sale in a country outside the patenting agency's sphere of influence).

Clinical trial protocol A clinical trial protocol is a document used to gain confirmation of the trial design by a panel of experts and adherence by all study investigators, even if conducted in various countries. The protocol describes the scientific rationale, objective(s), design, methodology, statistical considerations, and organization of the planned trial. Details of the trial are also provided in other documents referenced in the protocol such as an Investigator's Brochure. The protocol contains a precise study plan for executing the clinical trial, not only to assure safety and health of the trial subjects, but also to provide an exact template for trial conduct by investigators at multiple locations (in a "multicenter" trial) to perform the study in exactly the same way. This harmonization allows data to be combined collectively as though all investigators (referred to as "sites") were working closely together. The protocol also gives the study administrators (often a contract research organization or CRO) as well as the site team of physicians, nurses and clinic administrators a common reference document for site responsibilities during the trial. The format and content of clinical trial protocols sponsored by pharmaceutical, biotechnology or medical device companies in the United States, European Union, or Japan has been standardized to follow Good Clinical Practice guidance[15] issued by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH).[16] Regulatory authorities in Canada and Australia also follow ICH guidelines. Some journals, e.g. Trials, encourage trialists to publish their protocols in the journal.

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Design features Informed consent An essential component of initiating a clinical trial is to recruit study subjects following procedures using a signed document called "informed consent".[17] Informed consent is a legally-defined process of a person being told about key facts involved in a clinical trial before deciding whether or not to participate. To fully describe participation to a candidate subject, the doctors and nurses involved in the trial explain the details of the study using terms the person will understand. Foreign language translation is provided if the participant's native language is not the same as the study protocol. The research team provides an informed consent document that includes trial details, such as its purpose, duration, required procedures, risks, potential benefits and key contacts. The participant then decides whether or not to sign the document in agreement. Informed consent is not an immutable contract, as the participant can withdraw at any time without penalty. Statistical power In designing a clinical trial, a sponsor must decide on the target number of patients who will participate. The sponsor's goal usually is to obtain a statistically significant result showing a significant difference in outcome (e.g., improvement percentage in the treatment of psoriasis using hydrocortisone after 42 days).[18] between the groups of patients who receive the study treatment and those who receive a placebo or a different treatment. The number of patients required to give a statistically significant result depends on the question the trial wants to answer. For example, to show the effectiveness of a new drug in a non-curable disease as metastatic kidney cancer requires many fewer patients than in a highly curable disease as seminoma if the drug is compared to a placebo. The number of patients enrolled in a study has a large bearing on the ability of the study to reliably detect the size of the effect of the study intervention. This is described as the "power" of the trial. The larger the sample size or number of participants in the trial, the greater the statistical power. However, in designing a clinical trial, this consideration must be balanced with the fact that more patients make for a more expensive trial. The power of a trial is not a single, unique value; it estimates the ability of a trial to detect a difference of a particular size (or larger) between the treated (tested drug/device) and control (placebo or standard treatment) groups. By example, a trial of a lipid-lowering drug versus placebo with 100 patients in each group might have a power of .90 to detect a difference between patients receiving study drug and patients receiving placebo of 10 mg/dL or more, but only have a power of .70 to detect a difference of 5 mg/dL.

Placebo groups Merely giving a treatment can have nonspecific effects, and these are controlled for by the inclusion of a placebo group. Subjects in the treatment and placebo groups are assigned randomly and blinded as to which group they belong. Since researchers can behave differently to subjects given treatments or placebos, trials are also doubled-blinded so that the researchers do not know to which group a subject is assigned. Assigning a person to a placebo group can pose an ethical problem if it violates his or her right to receive the best available treatment. The Declaration of Helsinki provides guidelines on this issue.

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Phases Clinical trials involving new drugs are commonly classified into four phases. Each phase of the drug approval process is treated as a separate clinical trial. The drug-development process will normally proceed through all four phases over many years. If the drug successfully passes through Phases I, II, and III, it will usually be approved by the national regulatory authority for use in the general population. Phase IV are 'post-approval' studies. Before pharmaceutical companies start clinical trials on a drug, they conduct extensive pre-clinical studies.

Pre-clinical studies It involves in vitro (test tube or cell culture) and in vivo (animal) experiments using wide-ranging doses of the study drug to obtain preliminary efficacy, toxicity and pharmacokinetic information. Such tests assist pharmaceutical companies to decide whether a drug candidate has scientific merit for further development as an investigational new drug.

Phase 0 Phase 0 is a recent designation for exploratory, first-in-human trials conducted in accordance with the United States Food and Drug Administration's (FDA) 2006 Guidance on Exploratory Investigational New Drug (IND) Studies.[19] Phase 0 trials are also known as human microdosing studies and are designed to speed up the development of promising drugs or imaging agents by establishing very early on whether the drug or agent behaves in human subjects as was expected from preclinical studies. Distinctive features of Phase 0 trials include the administration of single subtherapeutic doses of the study drug to a small number of subjects (10 to 15) to gather preliminary data on the agent's pharmacodynamics (what the drug does to the body) and pharmacokinetics (what the body does to the drugs).[20] A Phase 0 study gives no data on safety or efficacy, being by definition a dose too low to cause any therapeutic effect. Drug development companies carry out Phase 0 studies to rank drug candidates in order to decide which has the best pharmacokinetic parameters in humans to take forward into further development. They enable go/no-go decisions to be based on relevant human models instead of relying on sometimes inconsistent animal data. Questions have been raised by experts about whether Phase 0 trials are useful, ethically acceptable, feasible, speed up the drug development process or save money, and whether there is room for improvement.[21]

Phase I Phase I trials are the first stage of testing in human subjects. Normally, a small (20-100) group of healthy volunteers will be selected. This phase includes trials designed to assess the safety (pharmacovigilance), tolerability, pharmacokinetics, and pharmacodynamics of a drug. These trials are often conducted in an inpatient clinic, where the subject can be observed by full-time staff. The subject who receives the drug is usually observed until several half-lives of the drug have passed. Phase I trials also normally include dose-ranging, also called dose escalation, studies so that the appropriate dose for therapeutic use can be found. The tested range of doses will usually be a fraction of the dose that causes harm in animal testing. Phase I trials most often include healthy volunteers. However, there are some circumstances when real patients are used, such as patients who have terminal cancer or HIV and lack other treatment options. "The reason for conducting the trial is to discover the point at which a compound is too poisonous to administer."[22] Volunteers are paid an inconvenience fee for their time spent in the volunteer centre. Pay ranges from a small amount of money for a short period of residence, to a larger amount of up to approx $6000 depending on length of participation. There are different kinds of Phase I trials: SAD

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Clinical trial Single Ascending Dose studies are those in which small groups of subjects are given a single dose of the drug while they are observed and tested for a period of time. If they do not exhibit any adverse side effects, and the pharmacokinetic data is roughly in line with predicted safe values, the dose is escalated, and a new group of subjects is then given a higher dose. This is continued until pre-calculated pharmacokinetic safety levels are reached, or intolerable side effects start showing up (at which point the drug is said to have reached the Maximum tolerated dose (MTD). MAD Multiple Ascending Dose studies are conducted to better understand the pharmacokinetics & pharmacodynamics of multiple doses of the drug. In these studies, a group of patients receives multiple low doses of the drug, while samples (of blood, and other fluids) are collected at various time points and analyzed to understand how the drug is processed within the body. The dose is subsequently escalated for further groups, up to a predetermined level. Food effect A short trial designed to investigate any differences in absorption of the drug by the body, caused by eating before the drug is given. These studies are usually run as a crossover study, with volunteers being given two identical doses of the drug on different occasions; one while fasted, and one after being fed.

Phase II Once the initial safety of the study drug has been confirmed in Phase I trials, Phase II trials are performed on larger groups (20-300) and are designed to assess how well the drug works, as well as to continue Phase I safety assessments in a larger group of volunteers and patients. When the development process for a new drug fails, this usually occurs during Phase II trials when the drug is discovered not to work as planned, or to have toxic effects. Phase II studies are sometimes divided into Phase IIA and Phase IIB. • Phase IIA is specifically designed to assess dosing requirements (how much drug should be given). • Phase IIB is specifically designed to study efficacy (how well the drug works at the prescribed dose(s)). Some trials combine Phase I and Phase II, and test both efficacy and toxicity. Trial design Some Phase II trials are designed as case series, demonstrating a drug's safety and activity in a selected group of patients. Other Phase II trials are designed as randomized clinical trials, where some patients receive the drug/device and others receive placebo/standard treatment. Randomized Phase II trials have far fewer patients than randomized Phase III trials.

Phase III Phase III studies are randomized controlled multicenter trials on large patient groups (300–3,000 or more depending upon the disease/medical condition studied) and are aimed at being the definitive assessment of how effective the drug is, in comparison with current 'gold standard' treatment. Because of their size and comparatively long duration, Phase III trials are the most expensive, time-consuming and difficult trials to design and run, especially in therapies for chronic medical conditions. It is common practice that certain Phase III trials will continue while the regulatory submission is pending at the appropriate regulatory agency. This allows patients to continue to receive possibly lifesaving drugs until the drug can be obtained by purchase. Other reasons for performing trials at this stage include attempts by the sponsor at "label expansion" (to show the drug works for additional types of patients/diseases beyond the original use for which the drug was approved for marketing), to obtain additional safety data, or to support marketing claims for the drug. Studies in this phase are by some companies categorised as "Phase IIIB studies."[23] [24]

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Clinical trial While not required in all cases, it is typically expected that there be at least two successful Phase III trials, demonstrating a drug's safety and efficacy, in order to obtain approval from the appropriate regulatory agencies such as FDA (USA), or the EMA (European Union), for example. Once a drug has proved satisfactory after Phase III trials, the trial results are usually combined into a large document containing a comprehensive description of the methods and results of human and animal studies, manufacturing procedures, formulation details, and shelf life. This collection of information makes up the "regulatory submission" that is provided for review to the appropriate regulatory authorities[4] in different countries. They will review the submission, and, it is hoped, give the sponsor approval to market the drug. Most drugs undergoing Phase III clinical trials can be marketed under FDA norms with proper recommendations and guidelines, but in case of any adverse effects being reported anywhere, the drugs need to be recalled immediately from the market. While most pharmaceutical companies refrain from this practice, it is not abnormal to see many drugs undergoing Phase III clinical trials in the market.[25]

Phase IV Phase IV trial is also known as Post-Marketing Surveillance Trial. Phase IV trials involve the safety surveillance (pharmacovigilance) and ongoing technical support of a drug after it receives permission to be sold. Phase IV studies may be required by regulatory authorities or may be undertaken by the sponsoring company for competitive (finding a new market for the drug) or other reasons (for example, the drug may not have been tested for interactions with other drugs, or on certain population groups such as pregnant women, who are unlikely to subject themselves to trials). The safety surveillance is designed to detect any rare or long-term adverse effects over a much larger patient population and longer time period than was possible during the Phase I-III clinical trials. Harmful effects discovered by Phase IV trials may result in a drug being no longer sold, or restricted to certain uses: recent examples involve cerivastatin (brand names Baycol and Lipobay), troglitazone (Rezulin) and rofecoxib (Vioxx).

Length Clinical trials are only a small part of the research that goes into developing a new treatment. Potential drugs, for example, first have to be discovered, purified, characterized, and tested in labs (in cell and animal studies) before ever undergoing clinical trials. In all, about 1,000 potential drugs are tested before just one reaches the point of being tested in a clinical trial. For example, a new cancer drug has, on average, 6 years of research behind it before it even makes it to clinical trials. But the major holdup in making new cancer drugs available is the time it takes to complete clinical trials themselves. On average, about 8 years pass from the time a cancer drug enters clinical trials until it receives approval from regulatory agencies for sale to the public. Drugs for other diseases have similar timelines. Some reasons a clinical trial might last several years: • For chronic conditions like cancer, it takes months, if not years, to see if a cancer treatment has an effect on a patient. • For drugs that are not expected to have a strong effect (meaning a large number of patients must be recruited to observe any effect), recruiting enough patients to test the drug's effectiveness (i.e., getting statistical power) can take several years. • Only certain people who have the target disease condition are eligible to take part in each clinical trial. Researchers who treat these particular patients must participate in the trial. Then they must identify the desirable patients and obtain consent from them or their families to take part in the trial. The biggest barrier to completing studies is the shortage of people who take part. All drug and many device trials target a subset of the population, meaning not everyone can participate. Some drug trials require patients to have unusual combinations of disease characteristics. It is a challenge to find the appropriate patients and obtain their consent, especially when they may receive no direct benefit (because they are not paid, the study drug is not yet

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Clinical trial proven to work, or the patient may receive a placebo). In the case of cancer patients, fewer than 5% of adults with cancer will participate in drug trials. According to the Pharmaceutical Research and Manufacturers of America (PhRMA), about 400 cancer medicines were being tested in clinical trials in 2005. Not all of these will prove to be useful, but those that are may be delayed in getting approved because the number of participants is so low.[26] For clinical trials involving a seasonal indication (such as airborne allergies, Seasonal Affective Disorder, influenza, and others), the study can only be done during a limited part of the year (such as Spring for pollen allergies), when the drug can be tested. This can be an additional complication on the length of the study, yet proper planning and the use of trial sites in the southern as well as northern hemispheres allows for year-round trials can reduce the length of the studies.[27] [28] Clinical trials that do not involve a new drug usually have a much shorter duration. (Exceptions are epidemiological studies like the Nurses' Health Study.)

Administration Clinical trials designed by a local investigator and (in the U.S.) federally funded clinical trials are almost always administered by the researcher who designed the study and applied for the grant. Small-scale device studies may be administered by the sponsoring company. Phase III and Phase IV clinical trials of new drugs are usually administered by a contract research organization (CRO) hired by the sponsoring company. (The sponsor provides the drug and medical oversight.) A CRO is a company that is contracted to perform all the administrative work on a clinical trial. It recruits participating researchers, trains them, provides them with supplies, coordinates study administration and data collection, sets up meetings, monitors the sites for compliance with the clinical protocol, and ensures that the sponsor receives 'clean' data from every site. Recently, site management organizations have also been hired to coordinate with the CRO to ensure rapid IRB/IEC approval and faster site initiation and patient recruitment. At a participating site, one or more research assistants (often nurses) do most of the work in conducting the clinical trial. The research assistant's job can include some or all of the following: providing the local Institutional Review Board (IRB) with the documentation necessary to obtain its permission to conduct the study, assisting with study start-up, identifying eligible patients, obtaining consent from them or their families, administering study treatment(s), collecting and statistically analyzing data, maintaining and updating data files during followup, and communicating with the IRB, as well as the sponsor and CRO.

Ethical conduct Clinical trials are closely supervised by appropriate regulatory authorities. All studies that involve a medical or therapeutic intervention on patients must be approved by a supervising ethics committee before permission is granted to run the trial. The local ethics committee has discretion on how it will supervise noninterventional studies (observational studies or those using already collected data). In the U.S., this body is called the Institutional Review Board (IRB). Most IRBs are located at the local investigator's hospital or institution, but some sponsors allow the use of a central (independent/for profit) IRB for investigators who work at smaller institutions. To be ethical, researchers must obtain the full and informed consent of participating human subjects. (One of the IRB's main functions is ensuring that potential patients are adequately informed about the clinical trial.) If the patient is unable to consent for him/herself, researchers can seek consent from the patient's legally authorized representative. In California, the state has prioritized [29] the individuals who can serve as the legally authorized representative. In some U.S. locations, the local IRB must certify researchers and their staff before they can conduct clinical trials. They must understand the federal patient privacy (HIPAA) law and good clinical practice. International Conference of Harmonisation Guidelines for Good Clinical Practice (ICH GCP) is a set of standards used internationally for the conduct of clinical trials. The guidelines aim to ensure that the "rights, safety and well being of trial subjects are

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Clinical trial protected". The notion of informed consent of participating human subjects exists in many countries all over the world, but its precise definition may still vary. Informed consent is clearly a necessary condition for ethical conduct but does not ensure ethical conduct. The final objective is to serve the community of patients or future patients in a best-possible and most responsible way. However, it may be hard to turn this objective into a well-defined quantified objective function. In some cases this can be done, however, as for instance for questions of when to stop sequential treatments (see Odds algorithm), and then quantified methods may play an important role. Additional ethical concerns are present when conducting clinical trials on children (pediatrics).

Safety Responsibility for the safety of the subjects in a clinical trial is shared between the sponsor, the local site investigators (if different from the sponsor), the various IRBs that supervise the study, and (in some cases, if the study involves a marketable drug or device) the regulatory agency for the country where the drug or device will be sold. For safety reasons, many clinical trials of drugs are designed to exclude women of childbearing age, pregnant women, and/or women who become pregnant during the study. In some cases the male partners of these women are also excluded or required to take birth control measures.

Sponsor • Throughout the clinical trial, the sponsor is responsible for accurately informing the local site investigators of the true historical safety record of the drug, device or other medical treatments to be tested, and of any potential interactions of the study treatment(s) with already approved medical treatments. This allows the local investigators to make an informed judgment on whether to participate in the study or not. • The sponsor is responsible for monitoring the results of the study as they come in from the various sites, as the trial proceeds. In larger clinical trials, a sponsor will use the services of a Data Monitoring Committee (DMC, known in the U.S. as a Data Safety Monitoring Board). This is an independent group of clinicians and statisticians. The DMC meets periodically to review the unblinded data that the sponsor has received so far. The DMC has the power to recommend termination of the study based on their review, for example if the study treatment is causing more deaths than the standard treatment, or seems to be causing unexpected and study-related serious adverse events. • The sponsor is responsible for collecting adverse event reports from all site investigators in the study, and for informing all the investigators of the sponsor's judgment as to whether these adverse events were related or not related to the study treatment. This is an area where sponsors can slant their judgment to favor the study treatment. • The sponsor and the local site investigators are jointly responsible for writing a site-specific informed consent that accurately informs the potential subjects of the true risks and potential benefits of participating in the study, while at the same time presenting the material as briefly as possible and in ordinary language. FDA regulations and ICH guidelines both require that “the information that is given to the subject or the representative shall be in language understandable to the subject or the representative." If the participant's native language is not English, the sponsor must translate the informed consent into the language of the participant.[30]

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Local site investigators • A physician's first duty is to his/her patients, and if a physician investigator believes that the study treatment may be harming subjects in the study, the investigator can stop participating at any time. On the other hand, investigators often have a financial interest in recruiting subjects, and can act unethically in order to obtain and maintain their participation. • The local investigators are responsible for conducting the study according to the study protocol, and supervising the study staff throughout the duration of the study. • The local investigator or his/her study staff are responsible for ensuring that potential subjects in the study understand the risks and potential benefits of participating in the study; in other words, that they (or their legally authorized representatives) give truly informed consent. • The local investigators are responsible for reviewing all adverse event reports sent by the sponsor. (These adverse event reports contain the opinion of both the investigator at the site where the adverse event occurred, and the sponsor, regarding the relationship of the adverse event to the study treatments). The local investigators are responsible for making an independent judgment of these reports, and promptly informing the local IRB of all serious and study-treatment-related adverse events. • When a local investigator is the sponsor, there may not be formal adverse event reports, but study staff at all locations are responsible for informing the coordinating investigator of anything unexpected. • The local investigator is responsible for being truthful to the local IRB in all communications relating to the study.

IRBs Approval by an IRB, or ethics board, is necessary before all but the most informal medical research can begin. • In commercial clinical trials, the study protocol is not approved by an IRB before the sponsor recruits sites to conduct the trial. However, the study protocol and procedures have been tailored to fit generic IRB submission requirements. In this case, and where there is no independent sponsor, each local site investigator submits the study protocol, the consent(s), the data collection forms, and supporting documentation to the local IRB. Universities and most hospitals have in-house IRBs. Other researchers (such as in walk-in clinics) use independent IRBs. • The IRB scrutinizes the study for both medical safety and protection of the patients involved in the study, before it allows the researcher to begin the study. It may require changes in study procedures or in the explanations given to the patient. A required yearly "continuing review" report from the investigator updates the IRB on the progress of the study and any new safety information related to the study.

Regulatory agencies • If a clinical trial concerns a new regulated drug or medical device (or an existing drug for a new purpose), the appropriate regulatory agency for each country where the sponsor wishes to sell the drug or device is supposed to review all study data before allowing the drug/device to proceed to the next phase, or to be marketed. However, if the sponsor withholds negative data, or misrepresents data it has acquired from clinical trials, the regulatory agency may make the wrong decision. • In the U.S., the FDA can audit the files of local site investigators after they have finished participating in a study, to see if they were correctly following study procedures. This audit may be random, or for cause (because the investigator is suspected of fraudulent data). Avoiding an audit is an incentive for investigators to follow study procedures. Different countries have different regulatory requirements and enforcement abilities. "An estimated 40 percent of all clinical trials now take place in Asia, Eastern Europe, central and south America. “There is no compulsory registration system for clinical trials in these countries and many do not follow European directives in their

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Clinical trial operations”, says Dr. Jacob Sijtsma of the Netherlands-based WEMOS, an advocacy health organisation tracking clinical trials in developing countries." [31] Beginning in the 1980s, harmonization of clinical trial protocols was shown as feasible across countries of the European Union. At the same time, coordination between Europe, Japan and the United States led to a joint regulatory-industry initiative on international harmonization named after 1990 as the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) [32] Currently, most clinical trial programs follow ICH guidelines, aimed at "ensuring that good quality, safe and effective medicines are developed and registered in the most efficient and cost-effective manner. These activities are pursued in the interest of the consumer and public health, to prevent unnecessary duplication of clinical trials in humans and to minimize the use of animal testing without compromising the regulatory obligations of safety and effectiveness."[33]

Economics Sponsor The cost of a study depends on many factors, especially the number of sites that are conducting the study, the number of patients required, and whether the study treatment is already approved for medical use. Clinical trials follow a standardized process. The costs to a pharmaceutical company of administering a Phase III or IV clinical trial may include, among others: • manufacturing the drug(s)/device(s) tested • staff salaries for the designers and administrators of the trial • payments to the contract research organization, the site management organization (if used) and any outside consultants • payments to local researchers (and their staffs) for their time and effort in recruiting patients and collecting data for the sponsor • study materials and shipping • communication with the local researchers, including onsite monitoring by the CRO before and (in some cases) multiple times during the study • one or more investigator training meetings • costs incurred by the local researchers such as pharmacy fees, IRB fees and postage. • any payments to patients enrolled in the trial (all payments are strictly overseen by the IRBs to ensure that patients do not feel coerced to take part in the trial by overly attractive payments) These costs are incurred over several years. In the U.S. there is a 50% tax credit for sponsors of certain clinical trials.[34] National health agencies such as the U.S. National Institutes of Health offer grants to investigators who design clinical trials that attempt to answer research questions that interest the agency. In these cases, the investigator who writes the grant and administers the study acts as the sponsor, and coordinates data collection from any other sites. These other sites may or may not be paid for participating in the study, depending on the amount of the grant and the amount of effort expected from them. Clinical trials are traditionally expensive and difficult to undertake. Using internet resources can, in some cases, reduce the economic burden.[35]

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Investigators Many clinical trials do not involve any money. However, when the sponsor is a private company or a national health agency, investigators are almost always paid to participate. These amounts can be small, just covering a partial salary for research assistants and the cost of any supplies (usually the case with national health agency studies), or be substantial and include 'overhead' that allows the investigator to pay the research staff during times in between clinical trials.

Patients In Phase I drug trials, participants are paid because they give up their time (sometimes away from their homes) and are exposed to unknown risks, without the expectation of any benefit. In most other trials, however, patients are not paid, in order to ensure that their motivation for participating is the hope of getting better or contributing to medical knowledge, without their judgment being skewed by financial considerations. However, they are often given small payments for study-related expenses like travel or as compensation for their time in providing follow-up information about their health after they are discharged from medical care.

Participating in a clinical trial Phase 0 and Phase I drug trials seek healthy volunteers. Most other clinical trials seek patients who have a specific disease or medical condition.

Locating trials Depending on the kind of participants required, sponsors of clinical trials use various recruitment strategies, including patient databases, newspaper and radio advertisements, flyers, posters in places the patients might go (such as doctor's offices), and personal recruitment of patients by investigators. Volunteers with specific conditions or diseases have Newspaper advertisements seeking patients and healthy volunteers to participate in clinical trials. additional online resources to help them locate clinical trials. For example, people with Parkinson's disease can use PDtrials to find up-to-date information on Parkinson's disease trials currently enrolling participants in the U.S. and Canada, and search for specific Parkinson’s clinical trials using criteria such as location, trial type, and symptom.[36] Other disease-specific services exist for volunteers to find trials related to their condition.[37] Volunteers may search directly on ClinicalTrials.gov to locate trials using a registry run by the U.S. National Institutes of Health and National Library of Medicine. CenterWatch[38] is also another option for volunteers to find trials with their online database of industry-sponsored global clinical trials actively seeking study volunteers. However, many clinical trials will not accept participants who contact them directly to volunteer as it is believed this may bias the characteristics of the population being studied. Such trials typically recruit via networks of medical professionals who ask their individual patients to consider enrollment.

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Steps for volunteers Before participating in a clinical trial, interested volunteers should speak with their doctors, family members, and others who have participated in trials in the past. After locating a trial, volunteers will often have the opportunity to speak or e-mail the clinical trial coordinator for more information and to answer any questions. After receiving consent from their doctors, volunteers then arrange an appointment for a screening visit with the trial coordinator.[39] All volunteers being considered for a trial are required to undertake a medical screen. There are different requirements for different trials, but typically volunteers will have the following tests:[40] • • • • • • •

Measurement of the electrical activity of the heart (ECG) Measurement of blood pressure, heart rate and temperature Blood sampling Urine sampling Weight and height measurement Drugs abuse testing Pregnancy testing (females only)

Information technology The last decade has seen a proliferation of information technology use in the planning and conduct of clinical trials. Clinical trial management systems (CTMS) are often used by research sponsors or CROs to help plan and manage the operational aspects of a clinical trial, particularly with respect to investigational sites. Web-based electronic data capture (EDC) and clinical data management systems (CDMS) are used in a majority of clinical trials[41] to collect case report data from sites, manage its quality and prepare it for analysis. Interactive voice response systems (IVRS) are used by sites to register the enrollment of patients using a phone and to allocate patients to a particular treatment arm (although phones are being increasingly replaced with web-based (IWRS) tools which are sometimes part of the EDC system). Patient-reported outcome measures are being increasingly collected using hand-held, sometimes wireless ePRO (or eDiary) devices. Statistical software is used to analyze the collected data and prepare it for regulatory submission. Access to many of these applications are increasingly aggregated in web-based clinical trial portals.

Controversy In 2001, the editors of 12 major journals issued a joint editorial, published in each journal, on the control over clinical trials exerted by sponsors, particularly targeting the use of contracts which allow sponsors to review the studies prior to publication and withhold publication. They strengthened editorial restrictions to counter the effect. The editorial noted that contract research organizations had, by 2000, received 60% of the grants from pharmaceutical companiesin the U.S. Researchers may be restricted from contributing to the trial design, accessing the raw data, and interpreting the results.[42] Seeding trials are particularly controversial.[43]

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References [1] http:/ / www. rateclinicaltrials. co. uk [2] Avorn J. (2004). Powerful Medicines, pp. 129-133. Alfred A. Knopf. [3] Van Spall HG, Toren A, Kiss A, Fowler RA (March 2007). "Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review". JAMA 297 (11): 1233–40. doi:10.1001/jama.297.11.1233. PMID 17374817. [4] The regulatory authority in the USA is the Food and Drug Administration (United States); in Canada, Health Canada; in the European Union, the European Medicines Agency; and in Japan, the Ministry of Health, Labour and Welfare [5] " Clinical trials in oncology (http:/ / books. google. com/ books?id=Zke8ocubNXAC& pg=PA1& dq& hl=en#v=onepage& q=& f=false)". Stephanie Green, Jacqueline Benedetti, John Crowley (2003). CRC Press. p.1. ISBN 1-58488-302-2 [6] " Clinical Trials Handbook (http:/ / books. google. com/ books?id=d8GxG0d9rpgC& pg=PA118& dq& hl=en#v=onepage& q=& f=false)". Shayne Cox Gad (2009). John Wiley and Sons. p.118. ISBN 0-471-21388-8 [7] Curtis L. Meinert, Susan Tonascia (1986). Clinical trials: design, conduct, and analysis (http:/ / books. google. com/ ?id=i1oAxuE29MUC& pg=PA3& lpg=PA3& q). Oxford University Press, USA. p. 3. ISBN 978-0195035681. . [8] Toby E. Huff (2003), The Rise of Early Modern Science: Islam, China, and the West, p. 218. Cambridge University Press, ISBN 0-521-52994-8. [9] Tschanz, David W. (May/June 1997). "The Arab Roots of European Medicine". Saudi Aramco World 48 (3): 20–31. [10] D. Craig Brater and Walter J. Daly (2000), "Clinical pharmacology in the Middle Ages: Principles that presage the 21st century", Clinical Pharmacology & Therapeutics 67 (5), p. 447-450 [448]. [11] "James Lind: A Treatise of the Scurvy (1754)" (http:/ / www. bruzelius. info/ Nautica/ Medicine/ Lind(1753). html). 2001. . Retrieved 2007-09-09. [12] O'Rourke, Michael F. (1992). "Frederick Akbar Mahomed". Hypertension (American Heart Association) 19: 212–217 [213] [13] O'Rourke, Michael F. (1992). "Frederick Akbar Mahomed". Hypertension (American Heart Association) 19: 212–217 [212] [14] Glossary of Clinical Trial Terms, NIH Clinicaltrials.gov (http:/ / clinicaltrials. gov/ ct2/ info/ glossary) [15] ICH Guideline for Good Clinical Practice: Consolidated Guidance (http:/ / www. ich. org/ LOB/ media/ MEDIA482. pdf) [16] International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (http:/ / www. ich. org) [17] What is informed consent? US National Institutes of Health, Clinicaltrials.gov (http:/ / clinicaltrials. gov/ ct2/ info/ understand) [18] (http:/ / archderm. ama-assn. org/ cgi/ reprint/ 140/ 12/ 1520. pdf) Bhardwaj S, Fleischer A. 2004. Statistical Significance and Clinical Relevance: The Importance of Power in Clinical Trials in Dermatology. Arch Dermatol 2004; 140:1520-1523, S. 1521 [19] "Guidance for Industry, Investigators, and Reviewers" (http:/ / www. fda. gov/ downloads/ Drugs/ GuidanceComplianceRegulatoryInformation/ Guidances/ ucm078933. pdf). Food and Drug Administration. January 2006. . Retrieved 2010-06-15. [20] The Lancet (2009). "Phase 0 trials: a platform for drug development?". Lancet 374 (9685): 176. doi:10.1016/S0140-6736(09)61309-X. [21] Silvia Camporesi (October 2008). "Phase 0 workshop at the 20th EORT-NCI-AARC symposium, Geneva" (http:/ / www. ecancermedicalscience. com/ blog. asp?postId=27). ecancermedicalscience. . Retrieved 2008-11-07. [22] http:/ / www. medscape. com/ viewarticle/ 582554_2 [23] "Guidance for Institutional Review Boards and Clinical Investigators" (http:/ / www. fda. gov/ oc/ ohrt/ irbs/ drugsbiologics. html). Food and Drug Administration. 1999-03-16. . Retrieved 2007-03-27. [24] "Periapproval Services (Phase IIIb and IV programs)" (http:/ / www. covance. com/ periapproval/ svc_phase3b. php). Covance Inc.. 2005. . Retrieved 2007-03-27. [25] Arcangelo, Virginia Poole; Andrew M. Peterson (2005). Pharmacotherapeutics for Advanced Practice: A Practical Approach. Lippincott Williams & Wilkins. ISBN 0781757843. [26] Web Site Editor; Crossley, MJ; Turner, P; Thordarson, P (2007). "Clinical Trials - What Your Need to Know" (http:/ / www. cancer. org/ docroot/ ETO/ content/ ETO_6_3_Clinical_Trials_-_Patient_Participation. asp). American Cancer Society 129 (22): 7155. doi:10.1021/ja0713781. PMID 17497782. . [27] Yamin Khan and Sarah Tilly. "Seasonality: The Clinical Trial Manager's Logistical Challenge" (http:/ / www. pharm-olam. com/ pdf/ POI-Seasonality. pdf). Pharm-Olam International (POI) (http:/ / www. pharm-olam. com). . Retrieved 26 April 2010. [28] Yamin Khan and Sarah Tilly. "Flu, Season, Diseases Affect Trials" (http:/ / appliedclinicaltrialsonline. findpharma. com/ appliedclinicaltrials/ Drug+ Development/ Flu-Season-Diseases-Affect-Trials/ ArticleStandard/ Article/ detail/ 652128). Applied Clinical Trials Online. . Retrieved 26 February 2010. [29] http:/ / irb. ucsd. edu/ ab_2328_bill_20020826_enrolled. pdf [30] Back Translation for Quality Control of Informed Consent Forms (http:/ / www. gts-translation. com/ medicaltranslationpaper. pdf) [31] Common Dreams (http:/ / www. commondreams. org/ archive/ 2007/ 12/ 14/ 5838/ ) [32] Pmda.go.jp 独立行政法人 医薬品医療機器総合機構 (http:/ / www. pmda. go. jp/ ich/ s/ s1b_98_7_9e. pdf) (Japanese) [33] ICH (http:/ / www. ich. org/ cache/ compo/ 276-254-1. html) [34] "Tax Credit for Testing Expenses for Drugs for Rare Diseases or Conditions" (http:/ / www. fda. gov/ orphan/ taxcred. htm). Food and Drug Administration. 2001-04-17. . Retrieved 2007-03-27.

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Clinical trial [35] Paul, J.; Seib, R.; Prescott, T. (Mar 2005). "The Internet and clinical trials: background, online resources, examples and issues" (http:/ / www. jmir. org/ 2005/ 1/ e5/ ) (Free full text). Journal of medical Internet research 7 (1): e5. doi:10.2196/jmir.7.1.e5. PMC 1550630. PMID 15829477. . [36] http:/ / www. pdtrials. org/ en/ about_PDtrials_what [37] http:/ / www. mlanet. org/ resources/ hlth_tutorial/ mod4c. html [38] http:/ / www. centerwatch. com/ clinical-trials/ [39] http:/ / www. pdtrials. org/ en/ participate_clinicalresearch_how [40] Life on a Trial - What to Expect (http:/ / www. beavolunteer. co. uk/ index. php?option=com_content& view=article& id=25& Itemid=21) [41] Life Sciences Strategy Group, "Clinical Trial Technology Utilization, Purchasing Preferences & Growth Outlook" Syndicated Publication, May, 2009 [42] Davidoff F, DeAngelis CD, Drazen JM, et al (September 2001). "Sponsorship, authorship and accountability" (http:/ / www. cmaj. ca/ cgi/ pmidlookup?view=long& pmid=11584570). CMAJ 165 (6): 786–8. PMC 81460. PMID 11584570. . [43] Sox HC, Rennie D (August 2008). "Seeding trials: just say "no"" (http:/ / www. annals. org/ cgi/ pmidlookup?view=long& pmid=18711161). Ann. Intern. Med. 149 (4): 279–80. PMID 18711161. . Retrieved 2008-08-21.

• Rang HP, Dale MM, Ritter JM, Moore PK (2003). Pharmacology 5 ed. Edinburgh: Churchill Livingstone. ISBN 0-443-07145-4 • Finn R, (1999). Cancer Clinical Trials: Experimental Treatments and How They Can Help You., Sebastopol: O'Reilly & Associates. ISBN 1-56592-566-1 • Chow S-C and Liu JP (2004). Design and Analysis of Clinical Trials : Concepts and Methodologies, ISBN 0-471-24985-8 • Pocock SJ (2004), Clinical Trials: A Practical Approach, John Wiley & Sons, ISBN 0-471-90155-5

External links • The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (http://www.ich.org) • The International Clinical Trials Registry Platform (ICTRP) (http://www.who.int/trialsearch) • IFPMA Clinical Trials Portal (IFPMA CTP) (http://clinicaltrials.ifpma.org) to Find Ongoing & Completed Trials of New Medicines • ClinicalTrials.gov (http://clinicaltrials.gov) • Clinical Trials for cancer research (http://www.cancer.gov/clinicaltrials) - National Cancer Institute

175

Rituximab

176

Rituximab Rituximab ? Monoclonal antibody Type

Whole antibody

Source

Chimeric (mouse/human)

Target

CD20 Identifiers

CAS number

174722-31-7

ATC code

L01 XC02

DrugBank

BTD00014

KEGG

D02994

[1]

[2] [3]

[4]

 

Chemical data Formula

C6416H9874N1688O1987S44

Mol. mass

143859.7 g/mol Pharmacokinetic data

Bioavailability

100% (IV)

Half-life

30 to 400 hours (varies by dose and length of treatment)

Excretion

Uncertain: may undergo phagocytosis and catabolism in RES Therapeutic considerations

Pregnancy cat. C(US) (no adequate human studies) Legal status

℞-only (US)

Routes

intravenous infusion only (never bolus or "push") (what is this?)   (verify)

[5]

Rituximab, sold under the trade names Rituxan and MabThera, is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells. Rituximab is used in the treatment of many lymphomas, leukemias, transplant rejection and some autoimmune disorders.

History Rituximab was developed by IDEC Pharmaceuticals (formed in 1986 by biotech pioneers Ivor Royston and Howard Birndorf).[6] Based on its safety and effectiveness in clinical trials,[7] rituximab was approved by the FDA in 1997 for B cell non-Hodgkin lymphoma resistant to other chemotherapy regimens.[8] Rituximab, in combination with CHOP chemotherapy, is now standard therapy in the initial treatment of diffuse large B cell lymphoma and many other B cell lymphomas. It is currently co-marketed by Biogen Idec and Genentech in the U.S. and by Roche in Canada (under the trade name Rituximab) and the European Union and by Chugai Pharmaceuticals and Zenyaku Kogyo in Japan. Since April 2007 rituximab has also been available from Dr. Reddy's Laboratories, an Indian-based biogeneric manufacturer.[9]

Rituximab In 2010 it was approved by the EC for first-line maintenance treatment of follicular lymphoma.[10]

Uses Rituximab destroys both normal and malignant B cells that have CD20 on their surfaces, and is therefore used to treat diseases which are characterized by having too many B cells, overactive B cells or dysfunctional B cells.

Hematological neoplastic diseases Rituximab is frequently used to treat hematological neoplasms such as leukemias and lymphomas. In multiple myeloma, treatment with rituximab fails to deplete circulating CD20+ B or plasma cells, even after up to four cycles of treatment; in some patients, rituximab treatment increases the number of circulating CD20+ B cells.[11]

Autoimmune diseases Rituximab has been shown to be an effective rheumatoid arthritis treatment in three randomised controlled trials and is now licensed for use in refractory rheumatoid disease.[12] In the United States, it has been FDA-approved for use in combination with methotrexate (MTX) for reducing signs and symptoms in adult patients with moderately- to severely-active rheumatoid arthritis (RA) who have had an inadequate response to one or more anti-TNF-alpha therapies. There is some evidence for efficacy, but not necessarily safety, in a range of other autoimmune diseases, and rituximab is widely used off-label to treat difficult cases of multiple sclerosis,[13] systemic lupus erythematosus and autoimmune anemias.[14] There are significant concerns about progressive multifocal leukoencephalopathy (PML) infection in SLE patients[15] and other conditions.[14] Other autoimmune diseases that have been treated with rituximab include autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP),[16] [17] Evans syndrome,[18] vasculitis (for example Wegener's Granulomatosis), bullous skin disorders (for example pemphigus, pemphigoid), type 1 diabetes mellitus, Sjogren's syndrome, and Devic's disease,[19] and thyroid-associated ophthalmopathy.[20] A new study from Norway suggests that rituximab (together with methotrexate) might help patients with chronic fatigue syndrome.[21] A clinical trial is ongoing.[22]

Anti-rejection treatment for organ transplants Rituximab is now being used off-label in the management of kidney transplant recipients. This drug may have some utility in transplants involving incompatible blood groups. It is also used as induction therapy in highly sensitized patients going for kidney transplantation. The use of rituximab has not been proven to be efficacious in this setting and like all depleting agents, carries with it the risk of infection.

Mechanism The antibody binds to the cluster of differentiation 20 (CD20). CD20 is widely expressed on B cells, from early pre-B cells to later in differentiation, but it is absent on terminally differentiated plasma cells. CD20 does not shed, modulate or internalise. Although the function of CD20 is unknown, it may play a role in Ca2+ influx across plasma membranes, maintaining intracellular Ca2+ concentration and allowing activation of B cells. The exact mode of action of rituximab is unclear, but the following effects have been found:[23] • The Fc portion of rituximab mediates antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). • Rituximab has a general regulatory effect on the cell cycle.

177

Rituximab • • • •

It increases MHC II and adhesion molecules LFA-1 and LFA-3 (lymphocyte function-associated antigen). It elicits shedding of CD23. It downregulates the B cell receptor. It induces apoptosis of CD20+ cells.

The combined effect results in the elimination of B cells (including the cancerous ones) from the body, allowing a new population of healthy B cells to develop from lymphoid stem cells. Rituximab binds to amino acids 170-173 and 182-185 on CD20, which are physically close to each other as a result of a disulfide bond between amino acids 167 and 183.[24] Rituximab primarily affects and entirely depletes peripheral B cells, but studies have shown that it only depletes 60-70% of B cells within in the lymphatics system, and rarely affects those present in the bone marrow.

Adverse events Serious adverse events, which can cause death and disability, include:[25] • Severe infusion reactions • Cardiac arrest • Tumor lysis syndrome, causing acute renal failure • Infections • Hepatitis B reactivation • Other viral infections • Progressive multifocal leukoencephalopathy (PML) • Immune toxicity, with depletion of B cells in 70% to 80% of lymphoma patients • Pulmonary toxicity[26] A small number of patients with systemic lupus erythematosus have died in the context of being treated with rituximab.[27] In some cases, reactivation of latent JC virus (a common virus that can cause progressive multifocal leukoencephalopathy) occurred in the brains of these patients. There has also been at least one case of a patient with rheumatoid arthritis who developed PML in the context of treatment with rituximab.[28] JC virus reactivation (resulting in PML) in an immunosuppressed person commonly results in death or severe brain damage. Rituximab has been reported as a possible cofactor in a chronic Hepatitis E infection in a person with lymphoma. Hepatitis E infection is normally an acute infection, suggesting the drug in combination with lymphoma may have weakened the body's immune response to the virus.[29]

Other anti-CD20 monoclonals The efficacy and success of Rituximab has led to some other anti-CD20 monoclonal antibodies being developed: • ocrelizumab, humanized (90%-95% human) B cell-depleting agent. • ofatumumab (HuMax-CD20) a fully human B cell-depleting agent.[30] • Third-generation anti-CD20s have a glycoengineered Fc fragment (Fc)[31] with enhanced binding to Fc gamma receptors, which increase ADCC (antibody-dependent cellular cytotoxicity).[32] Modifications in the variable regions[33] can enhance apoptosis. The added value of a humanized molecule in oncology, compared to the original design, has not been demonstrated to this date. Another approach to B cell diseases is to block the interaction of B cell survival or growth factors with their receptors on B cells. The monoclonal antibody Belimumab and atacicept are examples of such an approach.

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References [1] [2] [3] [4] [5] [6]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2009/ MB_cgi?term=174722-31-7& rn=1 http:/ / www. whocc. no/ atc_ddd_index/ ?code=L01XC02 http:/ / www. drugbank. ca/ drugs/ BTD00014 http:/ / www. kegg. jp/ entry/ D02994 http:/ / en. wikipedia. org/ w/ index. php?& diff=cur& oldid=416307336 "Why San Diego Has Biotech" (http:/ / www. sandiegometro. com/ 1999/ apr/ biotech. html), Fikes, Bradley J. San Diego Metropolitan, April 1999. Accessed June 20, 2008. [7] Maloney DG, Grillo-López AJ, White CA, et al. (September 1997). "IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma" (http:/ / www. bloodjournal. org/ cgi/ pmidlookup?view=long& pmid=9310469). Blood 90 (6): 2188–95. PMID 9310469. . [8] Scott SD (1998). "Rituximab: a new therapeutic monoclonal antibody for non-Hodgkin's lymphoma" (http:/ / www. blackwell-synergy. com/ openurl?genre=article& sid=nlm:pubmed& issn=1065-4704& date=1998& volume=6& issue=3& spage=195). Cancer Pract 6 (3): 195–7. doi:10.1046/j.1523-5394.1998.006003195.x. PMID 9652253. . [9] http:/ / biopharminternational. findpharma. com/ biopharm/ article/ articleDetail. jsp?id=490805& sk=& date=& pageID=2 Indian biogenerics. Feb 2008 [10] "Roche Gets EC Nod for Follicular Lymphoma Maintenance Therapy" (http:/ / www. genengnews. com/ gen-news-highlights/ roche-gets-ec-nod-for-follicular-lymphoma-maintenance-therapy/ 81244149/ ). October 29, 2010. . [11] Multiple myeloma includes CD20+ B and plasma cells that persist in patients treated with rituximab. LM Pilarski, E Baigorri, MJ Mant, PM Pilarski, P Adamson, H Zola, AR Belch. Clinical Medicine:Oncology, 2:275-281, 2008 (http:/ / www. doaj. org/ doaj?func=abstract& id=260717& recNo=32& toc=1/ ) [12] Edwards J, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close D, Stevens R, Shaw T (2004). "Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis". N Engl J Med 350 (25): 2572–81. doi:10.1056/NEJMoa032534. PMID 15201414. [13] NEJM - B-Cell Depletion with Rituximab in Relapsing-Remitting Multiple Sclerosis (http:/ / content. nejm. org/ cgi/ content/ short/ 358/ 7/ 676) [14] Paul, Marla (May 20, 2009). "Popular Cancer Drug Linked to Often Fatal 'Brain Eating' Virus" (http:/ / www. northwestern. edu/ newscenter/ stories/ 2009/ 05/ bennett. html). Northwestern University News and Information. . Retrieved 2009-05-22. [15] (http:/ / www. fda. gov/ bbs/ topics/ NEWS/ 2006/ NEW01532. html) [16] Braendstrup P, Bjerrum OW, Nielsen OJ, Jensen BA, Clausen NT, Hansen PB, Andersen I, Schmidt K, Andersen TM, Peterslund NA, Birgens HS, Plesner T, Pedersen BB, Hasselbalch HC. Rituximab chimeric anti-CD20 monoclonal antibody treatment for adult refractory idiopathic thrombocytopenic purpura. Am J Hematol 2005;78:275-80. PMID 15795920. [17] Patel V, Mihatov N, Cooper N, Stasi R, Cunningham-Rundles S, Bussel JB,Long-term responses seen with rituximab in patients with ITP, Community Oncology Vol. 4 No. 2, February 2007:107 PDF (http:/ / www. communityoncology. net/ journal/ articles/ 0402107. pdf) [18] Shanafelt, Tait D, MD; Madueme, Hans L, MD; Wold, Robert C, PharmD; Tefferi, Ayalew, MD Rituximab for Immune Cytopenia in Adults: Idiopathic Thrombocytopenic Purpura, Autoimmune Hemolytic Anemia, and Evans Syndrome Mayo Clinic Proc. 2003;78:1340-1346 PDF (http:/ / www. mayoclinicproceedings. com/ pdf/ 7811/ 7811a3. pdf) [19] Jacob A, Weinshenker BG, Violich I, McLinskey N, Krupp L, Fox RJ, Wingerchuk DM, Boggild M, Constantinescu CS, Miller A, De Angelis T, Matiello M, Cree BA (2008). "Treatment of neuromyelitis optica with rituximab: retrospective analysis of 25 patients." (http:/ / archneur. ama-assn. org/ cgi/ content/ full/ 65/ 11/ 1443). Arch Neurol 65 (11): 1443–1448. doi:10.1001/archneur.65.11.noc80069. PMID 18779415. . [20] "Error: no |title= specified when using {{[[Template:Cite web|Cite web (http:/ / www. ophthalmologyjournaloftheaao. com/ article/ S0161-6420(09)00548-X/ abstract)]}}"]. . [21] Fluge O, Mella O (July 2009). "Clinical impact of B-cell depletion with the anti-CD20 antibody rituximab in chronic fatigue syndrome: a preliminary case series" (http:/ / www. biomedcentral. com/ 1471-2377/ 9/ 28). BMC Neurology 9 (1): 28. doi:10.1186/1471-2377-9-28. PMC 2711959. PMID 19566965. . [22] Drug Intervention in Chronic Fatigue Syndrome (http:/ / clinicaltrials. gov/ ct2/ show/ NCT00848692?term=chronic+ fatigue+ syndrome& rank=4) [23] see e.g. T Shaw, J Quan, and M Totoritis, " B cell therapy for rheumatoid arthritis: the rituximab (anti-CD20) experience (http:/ / www. pubmedcentral. nih. gov/ articlerender. fcgi?artid=1766758)", Ann Rheum Dis. 2003 November; 62(Suppl 2): ii55–ii59. [24] Binder M, Otto F, Mertelsmann R, Veelken H, Trepel M. (2006). "The epitope recognized by rituximab". Blood 108 (6): 1975–1978. doi:10.1182/blood-2006-04-014639. PMID 16705086. [25] "Genentech: Products - Product Information - Immunology - Rituxan RA Full Prescribing Information" (http:/ / www. gene. com/ gene/ products/ information/ immunological/ rituxan/ insert. jsp). . Retrieved 2007-12-03. [26] Burton C, Kaczmarski R, Jan-Mohamed R (2003). "Interstitial pneumonitis related to rituximab therapy". N Engl J Med 348 (26): 2690–1; discussion 2690–1. doi:10.1056/NEJM200306263482619. PMID 12826649. [27] "Rituximab (marketed as Rituxan) Information" (http:/ / www. fda. gov/ Drugs/ DrugSafety/ PostmarketDrugSafetyInformationforPatientsandProviders/ ucm109106. htm). . Retrieved 15 November 2009.

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Rituximab [28] "Rituximab, RA and PML" (http:/ / www. fda. gov/ medwatch/ safety/ 2008/ rituxan_DHCP_Final 9411700. pdf). . Retrieved 2008-09-14. [29] "Chronic Hepatitis After Hepatitis E Virus Infection in a Patient With Non-Hodgkin Lymphoma Taking Rituximab" (http:/ / www. annals. org/ cgi/ reprint/ 150/ 6/ 430-a. pdf). . Retrieved 2008-09-14. [30] "Genmab.com / HuMax-CD20 (ofatumumab)" (http:/ / web. archive. org/ web/ 20070911144653/ http:/ / www. genmab. com/ ScienceAndResearch/ ProductsinDevelopment/ HuMax-CD20. aspx). Archived from the original (http:/ / www. genmab. com/ ScienceAndResearch/ ProductsinDevelopment/ HuMax-CD20. aspx) on 2007-09-11. . Retrieved 2007-12-03. [31] "Fc-structure" (http:/ / www. healthvalue. net/ Fc-structure. html). . Retrieved 2007-12-03. [32] "Monoclonal antibodies targeting cancer: 'magic bullets' or just the trigger?" (http:/ / www. pubmedcentral. nih. gov/ articlerender. fcgi?tool=pmcentrez& artid=138676). . Retrieved 2007-12-03. [33] "monoclonal domains" (http:/ / www. healthvalue. net/ monoclonaldomainsengl. html). . Retrieved 2007-12-03.

External links • MabThera.com (http://www.mabthera.com) Official website for use against lymphoma • MabThera for Rheumatoid Arthritis (http://www.mabthera-ra.com) Official website for use against rheumatoid arthritis (for non-US physicians and scientific media only) • Rituxan.com (http://www.rituxan.com) Official website (for US residents only) • Rituximab Information (http://www.fda.gov/Drugs/DrugSafety/ PostmarketDrugSafetyInformationforPatientsandProviders/ucm109106.htm) from the US Food and Drug Administration • U.S. National Library of Medicine: Drug Information Portal - Rituximab (http://druginfo.nlm.nih.gov/ drugportal/dpdirect.jsp?name=Rituximab)

CHOP CHOP is the acronym for a chemotherapy regimen used in the treatment of non-Hodgkin lymphoma. CHOP consists of: • Cyclophosphamide, an alkylating agent which damages DNA by binding to it and causing cross-links • Hydroxydaunorubicin (also called doxorubicin or Adriamycin), an intercalating agent which damages DNA by inserting itself between DNA bases • Oncovin (vincristine), which prevents cells from duplicating by binding to the protein tubulin • Prednisone or prednisolone, which are corticosteroids.

Uses and indications Normal cells are more able than cancer cells to repair damage from chemotherapy drugs. This regimen can also be combined with the monoclonal antibody rituximab if the lymphoma is of B cell origin; this combination is called R-CHOP or CHOP-R. Typically, courses are administered at an interval of two or three weeks (CHOP-14 and CHOP-21 respectively). A staging CT scan is generally performed after three cycles to assess whether the disease is responding to treatment. In patients with a history of cardiovascular disease, doxorubicin (which is cardiotoxic) is often deemed to be too great a risk and is omitted from the regimen. The combination is then referred to as COP (cyclophosphamide, Oncovin, and prednisone or prednisolone) or CVP (cyclophosphamide, vincristine, and prednisone or prednisolone).

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CHOP

Side-effects and complications The combination is generally well tolerated. Chemotherapy-induced nausea and vomiting may require antiemetics (such as ondansetron), and hemorrhagic cystitis is prevented with administration of mesna. Alopecia (hair loss) is common. Neutropenia generally develops in the second week. During this period, many clinicians recommend pegfilgrastim or prophylactic use of ciprofloxacinCullen. If a fever develops in the neutropenic period, urgent medical assessment is required for neutropenic sepsis, as infections in patients with low neutrophil counts may progress rapidly. Allopurinol is typically co-administered prophylactically to prevent hyperuricemia that results from tumor lysis syndrome, the result of rapid death of tumor cells.

History A pivotal study published in 1993 compared CHOP to several other chemotherapy regimens (e.g. m-BACOD, ProMACE-CytaBOM, MACOP-B).SWOG CHOP emerged as the regimen with the least toxicity but similar efficacy.

References 1. Cullen M, Steven N, Billingham L, Gaunt C, Hastings M, Simmonds P, Stuart N, Rea D, Bower M, Fernando I, Huddart R, Gollins S, Stanley A (2005). "Antibacterial prophylaxis after chemotherapy for solid tumors and lymphomas.". N Engl J Med 353 (10): 988–98. doi:10.1056/NEJMoa050078. PMID 16148284. 2. Fisher RI, Gaynor ER, Dahlberg S, Oken MM, Grogan TM, Mize EM, Glick JH, Coltman CA Jr, Miller TP (1993). "Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin's lymphoma.". N Engl J Med 328 (14): 1002–6. doi:10.1056/NEJM199304083281404. PMID 7680764.

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HyperCVAD

HyperCVAD HyperCVAD is a chemotherapy regimen used to treat some forms of leukemia, non-Hodgkin lymphoma (high grade) and lymphoblastic lymphoma.

Summary The term 'hyper' refers to the hyperfractionated nature of the chemotherapy, which is given in smaller doses, more frequently, to minimize side effects. 'CVAD' is the acronym of the drugs used in courses A and B. CVAD (Cyclophosphamide, Vincristine, Doxorubicin (also known as Adriamycin [brand name]),and Dexamethasone (the D in hyper CVAD- this last one being a steroid)). The other part of Hyper CVAD is Methotrexate and Cytarabine. The protocol was originally developed to treat leukemia in young, fit and ambulant patients, but has since begun to be used more widely.

Indications Hyper-CVAD chemotherapy is generally reserved for use in the treatment of serious and aggressive forms of hematological malignancy. There are serious side effects and complications arising from the administration of the various agents, which require careful management in an appropriate health-care setting. Patients who receive hyper-CVAD receive a careful work-up to assess their overall wellness prior to the commencement of the regimen, in order to minimise undesirable outcomes. Patients considered for the protocol will generally be under 65.

Administration Each course is given up to 4 times, with up to 8 cycles in total. Each cycle is approximately two to three weeks apart. The aim is to administer as many cycles as possible or necessary in as short a time as possible. Timing of cycles will be somewhat dependent on the patient's recovery from the last cycle. The regimen is usually administered on an in-patient basis, using a continuous venous access device such as a peripherally inserted central catheter (PICC), a hickman line or a port-a-cath. The following is only a general guide (The exact combination of drugs used, doses and protocols used for administration are generally determined on a facility-by-facility basis). Dosage is individualized, based on factors such as body-weight, body surface area and the overall health of the patient.

Course A • Cyclophosphamide (Cytoxan) is an alkylating agent given at 300mg/m2 by IV Q12hours over 3 hours (6 doses) Days 1, 2, and 3 • Vincristine (Oncovin) is a mitotic inhibitor, 2mg IV Days 4 and 11 • Doxorubicin (Adriamycin or Rubex) is an antibiotic with anti-tumour effects, 50mg/m2 IV Day 4 • Dexamethasone is an Immunosuppressant 40mg/day IV or PO Days 1 to 4 Days 11-14 • Cytarabine or Ara-C (Cytosar) is an antimetabolite 70mg IT Day 7 • Mesna (Uromitexan) is a compound used to reduce the incidence of haemorrhagic cystitis, a common side effect of the administration of cyclophosphamide. It is generally given via intravenous infusion or orally at the same time as cyclophosphamide. • Methotrexate, an antimetabolite, may be given via the intrathecal route when it is necessary to give chemotherapy which will pass through the blood-brain barrier. 12mg IT Day 2

182

HyperCVAD

Course B • Methotrexate 1000mg/m2 IV over 24 hours Day 1 • Leucovorin 25mg/ m2 IV 24 hours after starting methotrexate infusion Q6H X 6 doses. Leucovorin is used as a 'rescue' agent to prevent excessive cellular damage by methotrexate. • Sodium bicarbonate 600mg PO (starting day before methotrexate) TID X 4 Days. Sodium bicarbonate is used to produce a mild metabolic alkalosis, desirable when administering large quantities of methotrexate. Urine pH values will be checked to ensure alkalosis prior to the commencement of methotrexate. • Cytarabine3000mg/m2 IV over 2 hours Q12H X 4 doses Days 2 and 3

Side effects The side effects of the administration of the chemotherapeutic agents used in hyper-CVAD are complex, and are often dependent on the overall health of the patient. Hematologic and immune system The majority of patients will experience a degree of pancytopenia, including anaemia, thrombocytopenia, and leukopenia, due to the myelosuppressive effect of chemotherapy. Anaemia and thrombocytopenia can cause clinical problems, and transfusion of red blood cells and platelets may be necessary supportive therapies. Leukopenia, particularly neutropenia may lead to profound compromise of the immune system until the number of neutrophils recovers. Patients must therefore be vigilant to ensure that they report any fevers to their clinician. Anti-infective drugs are commonly given as a prophylaxis during and in-between cycles, to prevent against community acquired infections. Patients are also at risk of hospital acquired infections, such as methicillin-resistant staphylococcus aureus (MRSA) and Vancomycin-Resistant Enterococcus (VRE). It is not uncommon for patients to require hospitalisation to treat infections. Other side effects Temporary hair loss is a common side effect. Nausea and vomiting are commonly experienced both during and following administration. A variety of antiemetic drugs may be used, including granisetron, ondansetron, metoclopramide and cyclizine. Fertility is often compromised following the administration of hyper-CVAD chemotherapy. Patients who wish may elect to store gametes as a contingency. Peripheral neuropathy may be problematic following the administration of vincristine.

183

Bortezomib

184

Bortezomib Bortezomib

Systematic (IUPAC) name

[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid Identifiers [1]

CAS number

179324-69-7

ATC code

L01 XX32

PubChem

CID 387447

DrugBank

APRD00828

ChemSpider

343402

UNII

69G8BD63PP

ChEMBL

CHEMBL325041

[2] [3] [4]

[5]

  [6]

Chemical data Formula

C H BN O

Mol. mass

384.237 g/mol

19

25

4

4

  [7]

 

Bortezomib

185 SMILES

eMolecules

[8]

& PubChem

[9]

Pharmacokinetic data Bioavailability

n/a

Protein binding

83%

Metabolism

Hepatic, CYP extensively involved

Half-life

9 to 15 hours

Excretion

? Therapeutic considerations [10]

Licence data

EMA: Link

Pregnancy cat.

D(US)

Legal status

℞ Prescription only

Routes

Intravenous (what is this?)   (verify)

, US FDA: link

[11]

[12]

Bortezomib (INN, originally codenamed PS-341; marketed as Velcade by Millennium Pharmaceuticals) is the first therapeutic proteasome inhibitor to be tested in humans. It is approved in the U.S. for treating relapsed multiple myeloma[13] and mantle cell lymphoma. In multiple myeloma, complete clinical responses have been obtained in patients with otherwise refractory or rapidly advancing disease.

Origin and Development Bortezomib was originally synthesized in 1995 (MG-341) at a company called Myogenics, which soon changed its name to ProScript. After promising preclinical results, the drug (PS-341) was tested in a small Phase I clinical trial on patients with multiple myeloma cancer. ProScript ran out of money and was bought by Leukosite in May 1999. Leukosite in turn was bought by Millennium Pharmaceuticals in October 1999. At this point in time, the project had low priority amongst other projects at the company. This changed significantly when one of the first volunteers to receive the drug in the clinical trial achieved a complete response [14] and was still alive four years later. At the time this was a remarkable result. Later clinical experimentation indicates the possibility of a complete response in 15% of patients in a similar condition, when treated with bortezomib. In May 2003, seven years after the initial synthesis, bortezomib (Velcade) was approved in the United States by the Food and Drug Administration (FDA) for use in multiple myeloma, based on the results from the SUMMIT Phase II trial.[15]

Bortezomib

186

Pharmacology Structure The drug is a tripeptide and can be written as Pyz-Phe-boroLeu, which stands for pyrazinoic acid, phenylalanine and Leucine with boronic acid instead of a carboxylic acid. Peptides are written N-terminus to C-terminus, but as in vitro peptide synthesis proceeds C-terminus to N-terminus, peptide drugs are illustrated C to N, as in this case.

Mechanism Bortezomib bound to the core particle in a yeast proteasome. The bortezomib molecule is in the center colored by atom type (boron = pink, carbon = cyan, nitrogen = blue, oxygen = red), surrounded by the local protein surface. The blue patch is catalytic threonine residue whose activity is blocked by the presence of bortezomib.

The boron atom in bortezomib binds the catalytic site of the 26S proteasome[16] with high affinity and specificity. In normal cells, the proteasome regulates protein expression and function by degradation of ubiquitinylated proteins, and also cleanses the cell of abnormal or misfolded proteins. Clinical and preclinical data support a role in maintaining the immortal phenotype of myeloma cells, and cell-culture and xenograft data support a similar function in solid tumor cancers. While multiple mechanisms are likely to be involved, proteasome inhibition may prevent degradation of pro-apoptotic factors, permitting activation of programmed cell death in neoplastic cells dependent upon suppression of pro-apoptotic pathways.

Pharmacokinetics / Pharmacodynamics Bortezomib is rapidly cleared following intravenous administration.[17] Peak concentrations are reached at about 30 minutes. Drug levels can no longer be measured after an hour. Pharmacodynamics are measured by measuring proteasome inhibition in peripheral blood mononuclear cells. The much greater sensitivity of myeloma cell lines and mantle cell lines to proteasome inhibition compared with normal peripheral blood mononuclear cells and most other cancer cell lines is poorly understood.

Costs UK NICE recommended against Velcade in Oct 2006 due to its cost.[18] The company proposed a cost reduction for multiple myeloma,[19] and this was taken up in the UK.[20]

Adverse effects Bortezomib is associated with peripheral neuropathy in 30% of patients; occasionally, it can be painful. This can be worse in patients with pre-existing neuropathy. In addition, myelosuppression causing neutropenia and thrombocytopenia can also occur and be dose-limiting. However, these side effects are usually mild relative to bone marrow transplantation and other treatment options for patients with advanced disease. Bortezomib is associated with a high rate of shingles,[21] although prophylactic acyclovir can reduce the risk of this.[22] Gastro-intestinal (GI) effects and asthenia are the most common adverse events.[23]

Bortezomib

187

Drug interactions Green tea extract epigallocatechin gallate(EGCG), which had been expected to have a synergistic effect, was found by Encouse B. Golden, et al. to reduce the effectiveness of bortezomib.[24] [25] [26] [27]

Therapeutic efficacy Two open-label, phase III trials established the efficacy of bortezomib 1.3 mg/m2 (with or without dexamethasone) administered by intravenous bolus on days 1,4,8, and 11 of a 21-day cycle for a maximum of eight cycles in heavily pretreated patients with relapsed/refractory multiple myeloma.[28] Another trial demonstrated the superiority of bortezomib 1.3 mg/m2 over a high-dose dexamethasone regimen.[28]

Further improvement of anticancer potency Laboratory studies and clinical trials are investigating whether it might be possible to further increase the anticancer potency of bortezomib by combining it with novel types of other pharmacologic agents. For example, clinical trials have indicated that the addition of thalidomide, lenalidomide, inhibitors of vascular endothelial growth factor (VEGF), or arsenic trioxide might be beneficial.[29] [30] In laboratory studies, it was found that bortezomib killed multiple myeloma cells more efficiently when combined, for example, with histone deacetylase inhibitors,[31] thapsigargin,[32] or celecoxib.[33] However, the therapeutic efficacy of any of these latter combinations has not yet been confirmed in cancer patients.

References [1] [2] [3] [4] [5] [6] [7] [8]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2009/ MB_cgi?term=179324-69-7& rn=1 http:/ / www. whocc. no/ atc_ddd_index/ ?code=L01XX32 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=387447 http:/ / www. drugbank. ca/ drugs/ APRD00828 http:/ / www. chemspider. com/ Chemical-Structure. 343402 http:/ / fdasis. nlm. nih. gov/ srs/ srsdirect. jsp?regno=69G8BD63PP https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL325041 http:/ / www. emolecules. com/ cgi-bin/ search?t=ex& q=O%3DC%28N%5BC%40H%5D%28C%28%3DO%29N%5BC%40H%5D%28B%28O%29O%29CC%28C%29C%29Cc1ccccc1%29c2nccnc2 [9] http:/ / pubchem. ncbi. nlm. nih. gov/ search/ ?smarts=O%3DC%28N%5BC%40H%5D%28C%28%3DO%29N%5BC%40H%5D%28B%28O%29O%29CC%28C%29C%29Cc1ccccc1%29c2nccnc2 [10] http:/ / www. ema. europa. eu/ ema/ index. jsp?curl=/ pages/ medicines/ landing/ epar_search. jsp& murl=menus/ medicines/ medicines. jsp& searchkwByEnter=true& status=Authorised& keyword=Velcade& searchType=Name& jsenabled=true [11] http:/ / www. accessdata. fda. gov/ scripts/ cder/ drugsatfda/ index. cfm?fuseaction=Search. SearchAction& SearchTerm=Bortezomib& SearchType=BasicSearch [12] http:/ / en. wikipedia. org/ w/ index. php?& diff=cur& oldid=407341464 [13] Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" (http:/ / www. cancernetwork. com/ cancer-management-11/ chapter03/ article/ 10165/ 1402628) in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach (http:/ / www. cancernetwork. com/ cancer-management-11/ ). 11 ed. 2008. [14] http:/ / www. myelomaonline. org. uk/ NetCommunity/ Page. aspx?& pid=439& srcid=437 [15] Adams J, Kauffman M (2004). "Development of the Proteasome Inhibitor Velcade (Bortezomib)". Cancer Invest 22 (2): 304–11. doi:10.1081/CNV-120030218. PMID 15199612. [16] Bonvini P, Zorzi E, Basso G, Rosolen A (2007). "Bortezomib-mediated 26S proteasome inhibition causes cell-cycle arrest and induces apoptosis in CD-30+ anaplastic large cell lymphoma". Leukemia 21 (4): 838–42. doi:10.1038/sj.leu.2404528. PMID 17268529. [17] Voorhees PM, Dees EC, O'Neil B, Orlowski RZ (2003). "The proteasome as a target for cancer therapy". Clin Cancer Res 9 (17): 6316–25. PMID 14695130. [18] "NHS watchdog rejects cancer drug" (http:/ / news. bbc. co. uk/ 1/ hi/ health/ 6069386. stm). BBC News UK. 20 October 2006. . Retrieved 2009-08-14. [19] "Summary of VELCADE Response Scheme" (http:/ / www. nice. org. uk/ nicemedia/ pdf/ MyelomaDofHSummaryResponderScheme. pdf). . Retrieved 2009-08-14. [20] "More Velcade-Style Risk-Sharing In The UK?" (http:/ / www. europharmatoday. com/ 2009/ 01/ more-velcadestyle-risksharing-in-the-uk. html). Euro Pharma Today. 21 January 2009. . Retrieved 2009-08-14.

Bortezomib [21] Oakervee HE, Popat R, Curry N, et al. (2005). "PAD combination therapy (PS-341/bortezomib, doxorubicin and dexamethasone) for previously untreated patients with multiple myeloma". Br J Haematol 129 (6): 755–62. doi:10.1111/j.1365-2141.2005.05519.x. PMID 15953001. [22] Pour L., Adam Z., Buresova L., et al. (2009). "Varicella-zoster virus prophylaxis with low-dose acyclovir in patients with multiple myeloma treated with bortezomib". Clinical Lymphoma & Myeloma 9 (2): 151–3. doi:10.3816/CLM.2009.n.036. PMID 19406726. [23] Highlights Of Prescribing Information (http:/ / www. velcade. com/ full_prescrib_velcade. pdf) [24] "Cancer drug benefits could be negated by healthy tea treatment" (http:/ / www. belfasttelegraph. co. uk/ news/ health/ cancer-drug-benefits-could-be-negated-by-healthy-tea-treatment-14168666. html). Belfast Telegraph. 3 February 2009. . Retrieved 2009-08-14. [25] http:/ / www. news-medical. net/ ?id=45529 "Green tea may counteract anticancer effects of cancer therapy, bortezomib (Velcade)" [26] http:/ / www. ecancermedicalscience. com/ news-insider-news. asp?itemId=414 [27] Golden EB, et al. (2009). "Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors". Blood 113 (23): 5927–37. doi:10.1182/blood-2008-07-171389. PMID 19190249. [28] Curran M, McKeage K. (http:/ / adisonline. com/ drugs/ abstract/ 2009/ 69070/ Bortezomib__A_Review_of_its_Use_in_Patients_with. 6. aspx).Drugs 2009;69(7):859-888.doi: 10.2165/00003495-200969070-00006. [29] Anargyrou K et al. (2008). "Novel anti-myeloma agents and angiogenesis". Leuk Lymphoma 49 (4): 677–689. doi:10.1080/10428190701861686. PMID 18398734. [30] Richardson PG et al. (2005). "Novel biological therapies for the treatment of multiple myeloma". Best Pract Res Clin Haematol 18 (4): 619–634. doi:10.1016/j.beha.2005.01.010. PMID 16026741. [31] Nawrocki ST et al. (2006). "Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells". Cancer Res 66 (7): 3773–3781. doi:10.1158/0008-5472.CAN-05-2961. PMID 16585204. [32] Nawrocki ST et al. (2005). "Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis". Cancer Res 65 (24): 11658–11666. doi:10.1158/0008-5472.CAN-05-2370. PMID 16357177. [33] Kardosh A et al. (2008). "Aggravated endoplasmic reticulum stress as a basis for enhanced glioblastoma cell killing by bortezomib in combination with celecoxib or its non-coxib analogue, 2,5-dimethyl-celecoxib". Cancer Res 68 (3): 843–851. doi:10.1158/0008-5472.CAN-07-5555. PMID 18245486.

External links • Myeloma patients campaigning for access to a life prolonging cancer drug (http://www.velcadethree.co.uk/) • Millennium Pharmaceuticals website on Velcade (http://www.velcade.com/) • Multiple Myeloma Research Foundation article on Velcade (http://www.multiplemyeloma.org/treatments/3. 05.html) • International Myeloma Foundation article on Velcade (http://myeloma.org/IndexPage.action?tabId=1& menuId=151&indexPageId=53&parentLinkId=976&categoryId=172&gParentType=menuitem& gParentId=151&parentIndexPageId=47&parentCategoryId=71) • U.S. Food and Drugs Administration on Velcade (http://www.fda.gov/cder/drug/infopage/velcade/default. htm) • Dedicated website for European audience (http://www.velcade.info) • Presentation at 2006 ASCO of the PINNACLE Study (http://www.asco.org/portal/site/ASCO/menuitem. 34d60f5624ba07fd506fe310ee37a01d/?vgnextoid=76f8201eb61a7010VgnVCM100000ed730ad1RCRD& vmview=abst_detail_view&confID=40&abstractID=31445) on MCL by Dr. Goy, with video/slides

188

Bendamustine

189

Bendamustine Bendamustine

Systematic (IUPAC) name

4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid Identifiers [1]

CAS number

16506-27-7

ATC code

L01 AA09

PubChem

CID 65628

ChEMBL

CHEMBL487253

[2] [3] [4]

 

Chemical data Formula

C16H21Cl2N3O2

Mol. mass

358.262 g/mol Pharmacokinetic data

Bioavailability

NA (intravenous only)

Protein binding 94–96% Metabolism

Hydrolyzed to inactive metabolites. Two minor metabolites (M3 and M4) formed by CYP1A2

Half-life

40 min (bendamustine), 3 h (M3), 30 min (M4)

Excretion

Mostly fecal Therapeutic considerations [5]

Licence data

US FDA: link

Pregnancy cat.

D(US)

Legal status

℞-only (US)

Routes

Intravenous infusion (what is this?)   (verify)

[6]

Bendamustine (INN, trade names Ribomustin and Treanda; also known as SDX-105) is a nitrogen mustard used in the treatment of chronic lymphocytic leukemias (CLL)[7] and lymphomas. It belongs to the family of drugs called

Bendamustine alkylating agents. It is also being studied for the treatment of sarcoma.[8]

History Bendamustine was first synthesized in 1963 by Ozegowski and Krebs in East Germany (the former German Democratic Republic). It is a white, water soluble microcrystalline powder with amphoteric properties. Until 1990 it was available only in East Germany. East German investigators found that it was useful for treating chronic lymphocytic leukemia, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma and lung cancer. Bendamustine received its first marketing approval in Germany, which is marketed under the tradename Ribomustin, by Astellas Pharma GmbH's licensee, Mundipharma International Corporation Limited, which it is indicated as a single-agent or in combination with other anti-cancer agents for indolent NHL, multiple myeloma, and CLL. SymBio Pharmaceuticals Ltd holds exclusive rights to develop and market bendamustine HCl in Japan and selected Asia Pacific Rim countries. In March 2008, Cephalon received approval from the United States Food and Drug Administration to market bendamustine in the US, where it is sold under the tradename Treanda, for treatment of CLL.[9] In October 2008, the FDA granted further approval to market Treanda for the treatment of indolent B-cell non-Hodgkin's lymphoma (NHL) that has progressed during or within six months of treatment with rituximab or a rituximab-containing regimen. [10]

Pharmacology Betamustine acts as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases. After intravenous infusion it is extensively metabolised in the liver by cytochrome p450. >95% of the drug is bound to protein - primarily albumin. Only free bendamustine is active. Elimination is biphasic with a half-life of 6–10 minutes and a terminal half-life of approximately 30 minutes. It is eliminated primarily by the renal route.

Chemotherapeutic uses Bendamustine has been used both as sole therapy and in combination with other agents including etoposide, fludarabine, mitoxantrone, methotrexate, prednisone, rituximab, vincristine and 90Y-ibritumomab tiuxetan. One combination for stage III/IV relapsed or refractory indolent lymphomas and mantle cell lymphoma (MCL), with or without prior rituximab-containing chemoimmunotherapy treatment, is bendamustine with mitoxantrone and rituximab.[11]

Adverse effects Common adverse reactions are typical for its class of chemotherapeutic drugs, and include nausea, fatigue, vomiting, diarrhea, fever, constipation, loss of appetite, cough, headache, unintentional weight loss, difficulty breathing, rashes, and stomatitis, as well as immunosuppression, anemia, and low platelet counts.

References [1] [2] [3] [4]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2009/ MB_cgi?term=16506-27-7& rn=1 http:/ / www. whocc. no/ atc_ddd_index/ ?code=L01AA09 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=65628 https:/ / www. ebi. ac. uk/ chembldb/ index. php/ compound/ inspect/ CHEMBL487253

[5] http:/ / www. accessdata. fda. gov/ scripts/ cder/ drugsatfda/ index. cfm?fuseaction=Search. SearchAction& SearchTerm=Treanda& SearchType=BasicSearch [6] http:/ / en. wikipedia. org/ w/ index. php?& diff=cur& oldid=403350889

190

Bendamustine [7] Kath R, Blumenstengel K, Fricke HJ, Höffken K (January 2001). "Bendamustine monotherapy in advanced and refractory chronic lymphocytic leukemia" (http:/ / link. springer. de/ link/ service/ journals/ 00432/ bibs/ 1127001/ 11270048. htm). J. Cancer Res. Clin. Oncol. 127 (1): 48–54. doi:10.1007/s004320000180. PMID 11206271. . [8] Bagchi S (August 2007). "Bendamustine for advanced sarcoma". Lancet Oncol. 8 (8): 674. doi:10.1016/S1470-2045(07)70225-5. PMID 17726779. [9] "Cephalon press release - Cephalon Receives FDA Approval for TREANDA, a Novel Chemotherapy for Chronic Lymphocytic Leukemia" (http:/ / www. cephalon. com/ newsroom/ news_reader. aspx?ID=1120688). . Retrieved 2008-03-23. [10] "Cephalon press release -Cephalon Receives FDA Approval for TREANDA to Treat Patients with Relapsed Indolent Non-Hodgkin's Lymphoma" (http:/ / www. cephalon. com/ media/ news-releases/ article/ video-cephalon-receives-fda-approval-for-treanda-to-treat-patients-with-relapsed-indolent-non-hodgk/ ). . Retrieved 2008-11-03. [11] Weide R, Hess G, Köppler H, et al. (2007) High anti–lymphoma activity of bendamustine/mitoxantrone/rituximab in rituximab pretreated relapsed or refractory indolent lymphomas and mantle cell lymphomas. A muticenter phase II study of the German Low Grade Lymphoma Study Group (GLSG). Leuk. Lymphoma. 48:1299–1306

External links • Manufacturer's official website (http://www.treanda.com/) intended for US patients

191

Temsirolimus

192

Temsirolimus Temsirolimus

Systematic (IUPAC) name

hydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydr 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate Identifiers

[1] 162635-04-3

[2] L01 XE09

[3] CID 6918289

[4]

 

D06068

Chemical data

C H NO 56 87

16

1030.28

Therapeutic considerations

[5] EMA: Link

[6] , US FDA: link

?

POM (UK) ℞-only (US)

Intravenous

   (verify)

[7]

(what is this?)

Temsirolimus (CCI-779) is an intravenous drug for the treatment of renal cell carcinoma (RCC), developed by Wyeth Pharmaceuticals and approved by the U.S. Food and Drug Administration (FDA) in late May 2007,[8] and was also approved by the European Medicines Agency (EMEA) on November 2007. It is a derivative of sirolimus and is sold as Torisel.

Temsirolimus

Mechanism of action Temsirolimus is a specific inhibitor of mTOR and interferes with the synthesis of proteins that regulate proliferation, growth, and survival of tumor cells. Treatment with temsirolimus leads to cell cycle arrest in the G1 phase, and also inhibits tumor angiogenesis by reducing synthesis of VEGF.[9] mTOR (mammalian target of rapamycin) is a kinase enzyme inside the cell that collects and interprets the numerous and varied growth and survival signals received by tumor cells.[10] When the kinase activity of mTOR is activated, its downstream effectors, the synthesis of cell cycle proteins such as cyclin D and hypoxia-inducible factor-1a (HIF-1a) are increased. HIF-1a then stimulates VEGF.[11] Whether or not mTOR kinase is activated, determines whether the tumor cell produces key proteins needed for proliferation, growth, survival, and angiogenesis.[12] mTOR is activated in tumor cells by various mechanisms including growth factor surface receptor tyrosine kinases, oncogenes, and loss of tumor suppressor genes. These activating factors are known to be important for malignant transformation and progression.[13] mTOR is particularly important in the biology of renal cancer (RCC) owing to its function in regulating HIF-1a levels. Mutation or loss of the von Hippel Lindau tumor-suppressor gene is common in RCC and is manifested by reduced degradation of HIF-1a. In RCC tumors, activated mTOR further exacerbates accumulation of HIF-1a by increasing synthesis of this transcription factor and its angiogenic target gene products.[14]

Efficacy In an international three-arm phase III study with 626 previously untreated, poor-prognosis patients, temsirolimus, interferon-α and the combination of both agents was compared. Median overall survival improved significantly in the temsirolimus group (10.9 months) compared with interferon- α group (7.3 months) and the combination group (8.4 months). Further studies are needed to determine the role of temsirolimus in the first-line treatment of patients with a more favorable prognosis, how it can be combined with other targeted agents and as sequential therapy with sunitinib or sorafenib.[15]

Adverse reactions The toxicity profile is based on what was found in the phase III trial.[16] • adverse reaction • fatigue • skin rash • stomatitis • hematologic abnormalities • hemoglobin decreased • lymphocytes decreased • laboratory abnormalities • triglycerides increased • glucose increased • phosphorus decreased Temsirolimus has been generally well tolerated in clinical settings by patients with advanced RCC. In patients with RCC, the adverse effect profile of temsirolimus is primarily metabolic in nature, with minimal impact on QoL compared with the commonly seen side-effects with oral multikinase inhibitors. Temsirolimus’ high level of specificity for mTOR likely contributes to the tolerability of temsirolimus.

193

Temsirolimus

Dosing The recommended dose of temsirolimus is 25 mg IV infused over 30–60 minutes once per week (Wyeth Pharmaceuticals, Inc., 2007). Weekly treatment may continue until disease progression or until patients experience intolerable side effects. Although infusion reactions can occur while temsirolimus is being administered, most hypersensitivity reactions occurring on the same day as temsirolimus administration were not severe. Antihistamine pretreatment is recommended to minimize the risk of an allergic reaction.[16]

References [1] [2] [3] [4] [5]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2009/ MB_cgi?term=162635-04-3& rn=1 http:/ / www. whocc. no/ atc_ddd_index/ ?code=L01XE09 http:/ / pubchem. ncbi. nlm. nih. gov/ summary/ summary. cgi?cid=6918289 http:/ / www. kegg. jp/ entry/ D06068 http:/ / www. ema. europa. eu/ ema/ index. jsp?curl=/ pages/ medicines/ landing/ epar_search. jsp& murl=menus/ medicines/ medicines. jsp& searchkwByEnter=true& status=Authorised& keyword=Torisel& searchType=Name& jsenabled=true [6] http:/ / www. accessdata. fda. gov/ scripts/ cder/ drugsatfda/ index. cfm?fuseaction=Search. SearchAction& SearchTerm=Torisel& SearchType=BasicSearch [7] http:/ / en. wikipedia. org/ w/ index. php?& diff=cur& oldid=409344559 [8] "FDA Approves New Drug for Advanced Kidney Cancer" (http:/ / www. fda. gov/ NewsEvents/ Newsroom/ PressAnnouncements/ 2007/ ucm108924. htm). 30 May 2007. . [9] Wan, X; Shen, N; Mendoza, A; Khanna, C; Helman, LJ (2006). "CCI-779 Inhibits rhabdomyosarcoma xenograft growth by an antiangiogenic mechanism linked to the targeting of mTOR/HIF-1alpha/VEGF signaling". Neoplasia 8 (5): 394–401. doi:10.1593/neo.05820. PMC 1592447. PMID 16790088. [10] Rubio-Viqueira, B, Hidalgo M. Targeting mTOR for cancer treatment. Curr Opin Investig Drugs.2006;7:501–512. [11] Hudson, CC; Liu, M; Chiang, GG; Otterness, DM; Loomis, DC; Kaper, F; Giaccia, AJ; Abraham, RT (2002). "Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin". Mol Cell Biol 22 (20): 7004–7014. doi:10.1128/MCB.22.20.7004-7014.2002. PMC 139825. PMID 12242281. [12] DelBufalo, D; Ciuffreda, L; Trisciuoglio, D; Desideri, M; Cognetti, F; Zupi, G; Milella, M (2006). "Antiangiogenic potential of the mammalian target of rapamycin inhibitor temsirolimus". Cancer Res. 66 (11): 5549–5554. doi:10.1158/0008-5472.CAN-05-2825. PMID 16740688. [13] Dancey JE. Therapeutic targets: mTOR and related pathways. Cancer Biol Ther. 2006;5:1065–1073. [14] Thomas, GV; Tran, C; Mellinghoff, IK; Welsbie, DS; Chan, E; Fueger, B; Czernin, J; Sawyers, CL (2006). "Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer". Nature Medicine 12 (1): 122–127. doi:10.1038/nm1337. PMID 16341243. [15] Hudes G et al. (2007). "Temsirolimus, Interferon Alfa, or Both for Advanced Renal-Cell Carcinoma" (http:/ / www. nejm. org/ doi/ full/ 10. 1056/ NEJMoa066838). NEJM 356 (22): 2271–2281. doi:10.1056/NEJMoa066838. PMID 17538086. . [16] Bellmunt, J; Szczylik, C; Feingold, J; Strahs, A; Berkenblit, A (2008). "Temsirolimus safety profile and management of toxic effects in patients with advanced renal cell carcinoma and poor prognostic features". Ann Oncol 19 (8): 1387–1392. doi:10.1093/annonc/mdn066. PMID 18385198.

194

Antibody-drug conjugate

Antibody-drug conjugate Antibody-drug conjugates (ADCs) are a new type of targeted therapy e.g. for cancer.[1] [2] [3] [4] They consist of an antibody (or antibody fragment such as a single-chain variable fragment (scFv)) linked to a payload drug (often cytotoxic).[5] Hence they are a type of immunoconjugate and often an immunotoxin. The antibody causes the ADC to bind to the target cancer cells. Often the ADC is then internalised by the cell and the drug is released to do its damage.[6] Because of the targeting the side effects should be lower and give a wider therapeutic window.[7] Hydrophilic linkers (e.g. PEG4Mal) help prevent the drug being pumped out of resistant cancer cells through MDR (multiple drug resistance) transporters.[5] ADCs based on cleavable linkers are thought to have a less favorable therapeutic window but targets (tumor cell surface antigens) that do not get internalized efficiently seem more suitable for cleavable linkers.[8] Companies developing the underlying technology for ADCs include[9] : • Seattle Genetics[10] of Bothell, Washington, with deals with Pfizer, Abbott[11] , Genentech[8] etc, for their toxins and non-cleavable linkers. • ImmunoGen Inc of Waltham, Massachusetts, with deals with Novartis for their tumor-activated prodrug (TAP) linker technology.

Approvals and indications • gemtuzumab ozogamicin (Mylotarg) Approved for acute myelogenous leukemia in 2001 but since withdrawn.

Examples in clinical trials (based on [1] [7] [12] ) Started Phase III trials: • Trastuzumab emtansine (T-DM1) (Herceptin linked to DM1)[5] [7] [13] [14] for breast cancer, especially HER2+.[15] • Inotuzumab ozogamicin (CMC-544) for non-Hodgkin lymphoma. Started Phase II pivotal trials: • Brentuximab vedotin (SGN-35) for relapsed or refractory Hodgkin lymphoma. US NDA submitted in March 2011. Started Phase II: • Glembatumumab vedotin (CDX-011, CR011-vcMMAE) for melanoma and metastatic breast cancer. • lorvotuzumab mertansine (IMGN901) for CD56 cancers (e.g. small-cell lung cancer, ovarian cancer).[5] [16] Orphan drug for Merkel cell carcinoma[17] Phase II results for small-cell lung cancer[18] • IMGN242[19] for CanAg expressing gastric cancer. • AN-152, LMB2, TP-38, VB4-845[7] p9 Started Phase I: • • • •

Cantuzumab mertansine (huC242-DM1) Results[20] AVE9633 SAR3419 CAT-8015 (anti-CD22)

• IMGN388 : integrin-targeting against solid tumors[5] tried on 4 cancer patients [21] • milatuzumab-doxorubicin for relapsed multiple myeloma.[22]

195

Antibody-drug conjugate • SGN-75 (anti-CD70) for non-Hodgkin lymphoma or renal cell carcinoma[23] [24] Preclinical: • Anti-CD22-MCC-DM1 for leukemia[25] • and others.[7]

References [1] Antibody Drug Conjugates: A Marriage of Biologics and Small Molecules - Pharmaceutical Technology (http:/ / pharmtech. findpharma. com/ pharmtech/ Ingredients+ Insider/ Antibody-Drug-Conjugates-A-Marriage-of-Biologics-a/ ArticleStandard/ Article/ detail/ 522139). Pharmtech.findpharma.com. Retrieved on 2010-11-20. [2] Ducry, Laurent; Stump, Bernhard (2010). "Antibody−Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodies". Bioconjugate Chemistry 21 (1): 5. doi:10.1021/bc9002019. PMID 19769391. [3] Kovtun, Y. V.; Audette, CA; Ye, Y; Xie, H; Ruberti, MF; Phinney, SJ; Leece, BA; Chittenden, T et al. (2006). "Antibody-Drug Conjugates Designed to Eradicate Tumors with Homogeneous and Heterogeneous Expression of the Target Antigen". Cancer Research 66 (6): 3214. doi:10.1158/0008-5472.CAN-05-3973. PMID 16540673. [4] Kovtun, YV; Goldmacher, VS (2007). "Cell killing by antibody-drug conjugates". Cancer letters 255 (2): 232–40. doi:10.1016/j.canlet.2007.04.010. PMID 17553616. [5] Dimond (9 Mar 2010). "Antibody-Drug Conjugates Stage a Comeback" (http:/ / www. genengnews. com/ analysis-and-insight/ antibody-drug-conjugates-stage-a-comeback/ 77899350/ ). . [6] Chari, Ravi V. J.; Martell, Bridget A.; Gross, Jonathan L.; Cook, Sherrilyn B.; Shah, Sudhir A.; Blättler, Walter A.; McKenzie, Sara J.; Goldmacher, Victor S. (1992). "Immunoconjugates containing novel maytansinoids: promising anticancer drugs". Cancer research 52 (1): 127–31. PMID 1727373. [7] Poon, Kirsten Achilles (May 2010). "Safety Assessment of Antibody Drug Conjugates" (http:/ / www. toxicology. org/ isot/ rc/ NorthernCal/ 2010Spring/ 2010_6SafetyAntibodyDrugConjugates. pdf). . [8] "As Seattle Genetics Strengthens Its Foothold Within Genentech, What About Immunogen?" (http:/ / seekingalpha. com/ article/ 252592-as-seattle-genetics-strengthens-its-foothold-within-genentech-what-about-immunogen). 14 Feb 2011. . [9] "Pharma interest surges in antibody drug conjugates" (http:/ / www. nature. com/ nbt/ journal/ v29/ n4/ full/ nbt0411-297. html). Apr 2011. . [10] http:/ / www. seagen. com/ technology_adc_tech. shtml Seattle Genetics ADC Technology [11] http:/ / www. fiercebiotech. com/ story/ seattle-genetics-strikes-again-208m-adc-deal-abbott/ 2011-03-22?utm_medium=rss& utm_source=rss [12] ADCs in early 2008 (http:/ / pharmtech. findpharma. com/ pharmtech/ data/ articlestandard/ / pharmtech/ 232008/ 522139/ i3. jpg) [13] trastuzumab-MCC-DM1 antibody-drug conjugate (http:/ / www. cancer. gov/ drugdictionary/ ?CdrID=564399) [14] "FDA denies accelerated approval of Genentech's trastuzumab-DM1 (T-DM1) BLA for metastatic breast cancer" (http:/ / www. news-medical. net/ news/ 20100827/ FDA-denies-accelerated-approval-of-Genentechs-trastuzumab-DM1-(T-DM1)-BLA-for-metastatic-breast-cancer. aspx). Aug 2010. . [15] Dean (2011). Antibody-drug conjugate feasible as HER2-positive breast cancer treatment (http:/ / www. medwire-news. md/ 46/ 90740/ Oncology/ Antibody-drug_conjugate_feasible_as_HER2-positive_breast_cancer_treatment. html). . [16] ImmunoGen reports encouraging clinical data of IMGN901 (http:/ / www. news-medical. net/ news/ 20091206/ ImmunoGen-reports-encouraging-clinical-data-of-IMGN901. aspx). News-medical.net. Retrieved on 2010-11-20. [17] http:/ / www. news-medical. net/ news/ 20100308/ ImmunoGen-receives-FDA-orphan-drug-designation-for-IMGN901-compound-in-treatment-of-MCC. aspx [18] http:/ / lungcancer. nocancersite. com/ html/ 2011/ 02/ 1745. html [19] "A phase II study of IMGN242 (huC242-DM4) in patients with CanAg-positive gastric or gastroesophageal (GE) junction cancer" (http:/ / www. asco. org/ ASCOv2/ Meetings/ Abstracts?& vmview=abst_detail_view& confID=65& abstractID=33509). Journal of Clinical Oncology 27 (15S): e15625. 2009. . [20] Tolcher, AW; Ochoa, L; Hammond, LA; Patnaik, A; Edwards, T; Takimoto, C; Smith, L; De Bono, J et al. (2003). "Cantuzumab mertansine, a maytansinoid immunoconjugate directed to the CanAg antigen: a phase I, pharmacokinetic, and biologic correlative study". Journal of clinical oncology 21 (2): 211–22. doi:10.1200/JCO.2003.05.137. PMID 12525512. [21] "ImmunoGen, Inc. Announces Presentation of Encouraging Clinical Data for IMGN388 at EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics" (http:/ / www. businesswire. com/ news/ home/ 20101119005080/ en/ ImmunoGen-Announces-Presentation-Encouraging-Clinical-Data-IMGN388). 19 Nov 2010. . [22] "Immunomedics initiates dosing in milatuzumab-doxorubicin conjugate Phase I/II trial for relapsed multiple myeloma" (http:/ / www. news-medical. net/ news/ 20100616/ Immunomedics-initiates-dosing-in-milatuzumab-doxorubicin-conjugate-Phase-III-trial-for-relapsed-multiple-myeloma. aspx). 16 June 2010. . [23] http:/ / clinicaltrials. gov/ ct2/ show/ NCT01015911 [24] http:/ / www. news-medical. net/ news/ 20101011/ Seattle-Genetics-reports-SGN-75-phase-I-clinical-trial-data. aspx [25] Polson, A G; Williams, M; Gray, A M; Fuji, R N; Poon, K A; McBride, J; Raab, H; Januario, T et al. (2010). "Anti-CD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma". Leukemia 24 (9): 1566–73.

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197

doi:10.1038/leu.2010.141. PMID 20596033.

Ibritumomab tiuxetan Ibritumomab tiuxetan ?

Monoclonal antibody Type

Whole antibody

Source

Mouse

Target

CD20 Identifiers

CAS number

174722-31-7

ATC code

V10 XX02

DrugBank

BTD00069

[1]

[1] 90

( Y)

[2]

Chemical data Formula

?

Therapeutic considerations Pregnancy cat.

?

Legal status

?

Routes

intravenous

Ibritumomab tiuxetan, sold under the trade name Zevalin, is a monoclonal antibody radioimmunotherapy treatment for some forms of B cell non-Hodgkin's lymphoma, a myeloproliferative disorder of the lymphatic system. The drug uses the monoclonal mouse IgG1 antibody ibritumomab (pronounced as ) [3] in conjunction with the chelator tiuxetan, to which a radioactive isotope (either yttrium-90 or indium-111) is added. Tiuxetan is a modified version of DTPA whose carbon backbone contains an isothiocyanatobenzyl and a methyl group.[4] [5]

Mechanism of action The antibody binds to the CD20 antigen found on the surface of normal and malignant B cells (but not B cell precursors), allowing radiation from the attached isotope (mostly beta emission) to kill it and some nearby cells. In addition, the antibody itself may trigger cell death via antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis. Together, these actions eliminate B cells from the body, allowing a new population of healthy B cells to develop from lymphoid stem cells.

Ibritumomab tiuxetan

Administration In order to qualify for ibritumomab, a patient needs to have bone marrow involvement of < 25%[6] and > 15% bone marrow cellularity. Since ibritumomab is known to cause cytopenia, platelet and neutrophil counts are also taken pretreatment. Since a murine antibody is used, the patient might also be tested for human anti mouse antibodies (HAMA). Having bulky disease does not disqualify a patient. The ibritumomab regimen takes 7–9 days, with two administrations of ibritumomab. Each dose is preceded by rituximab, in order to pre-deplete B lymphocytes.[7] The dose of rituximab given here is less than the usual dose. The first dose uses indium-111 ibritumomab for imaging. Indium-111 emits gamma radiation, which can be picked up by the gamma camera. A scan is done to assess biodistribution of the drug. This test dose is used to determine that no excess amounts go to the marrow, liver, etc. in this particular patient. If the gamma scan shows no altered biodistribution, then the second dose is given, using yttrium-90 ibritumomab as the actual treatment. Yttrium-90 emits the cell-killing beta radiation. Ibritumomab tiuxetan is administered by intravenous infusion which usually lasts around 10 minutes. Only acrylic shielding is needed, not lead.

Efficacy Treatment with ibritumomab showed higher response rates in clinical trials compared to treatment with only rituximab (similar to ibritumomab, but without the attached radioisotope), and showed very promising results for patients who no longer respond to rituximab. In patients with relapsed or refractory low-grade, follicular, or transformed B-cell NHL, where no prior anti-CD20 therapy was allowed, the OR was 80% / 50% and CR was 34% / 20%, comparing ibritumomab to rituximab. [8] Recently, extended follow-up data for the ZEVALIN ([90Y]-ibritumomab tiuxetan) First-line Indolent (FIT) study presented at the American Society of Hematology (ASH) Annual Meeting demonstrated the continued improvement in progression-free survival (PFS) following ibritumomab consolidation therapy for patients with follicular B-cell non-Hodgkin's lymphoma who achieved a response to first-line therapy over chemotherapy alone. Additionally, ibritumomab consolidation did not adversely affect the use of various effective second-line treatments including stem cell transplants in patients who relapsed.[9] In a Phase II study on patients with relapsed and refractory mantle cell lymphoma, the OR was 42% and CR was 26%.[10] A study demonstrated that rituximab followed by single agent ibritumomab in a front-line setting for patients with MALT lymphoma and low-grade follicular lymphoma that primarily involved the conjunctiva or orbit, produced a complete response rate of 83 percent.[11]

History Developed by the IDEC Pharmaceuticals, which is now part of Biogen Idec, ibritumomab tiuxetan was the first radioimmunotherapy drug approved by the Food and Drug Administration (FDA) in 2002 to treat cancer. It was approved for the treatment of patients with relapsed or refractory, low‑grade or follicular B‑cell non‑Hodgkin's lymphoma (NHL), including patients with rituximab refractory follicular NHL. In December 2007, Cell Therapeutics Inc acquired the U.S. rights to sell, market, and distribute this radioimmunotherapy antibody from Biogen for approximately US$30 million, or the equivalent of about two years' net sales revenue in the U.S. for the drug.[12] Outside of the U.S., Bayer Schering Pharma continues to have the rights to the drug. In March 2009, Spectrum Pharmaceuticals acquired 100% control of RIT Oncology, LLC, to commercialize Zevalin in the US. Now Spectrum Pharmaceuticals is responsible for all activities relating to Zevalin in the US.

198

Ibritumomab tiuxetan In September 2009, ibritumomab received approval from the FDA for an expanded label for the treatment of patients with previously untreated follicular non-Hodgkin's Lymphoma (NHL), who achieve a partial or complete response to first-line chemotherapy.

Costs Ibritumomab which is not available in a generic form because it is still under patent protection, is currently the most expensive drug available given in a single dose, costing over US$ 24,000 (€ 17,000) for the average dose. However, ibritumomab is essentially an entire course of lymphoma therapy which is delivered in 7–9 days, with one visit for imaging, one visit for a gamma scan, and one visit for the actual therapeutic dose. Compared to other monoclonal antibody treatments (many of which are well over US$ 40,000 for a course of therapy), this drug is priced in the middle for many of these therapies.

External links • http://www.zevalin.com/- Official Zevalin web site [13] • http://www.spectrumpharm.com/- Spectrum Pharmaceuticals, Inc. web site [14]

References [1] [2] [3] [4]

http:/ / www. whocc. no/ atc_ddd_index/ ?code=V10XX02 http:/ / www. drugbank. ca/ drugs/ BTD00069 Ibritumomab: Pronunciation (http:/ / body. aol. com/ drugs/ ibritumomab) Milenic, Diane E.; Brady, Erik D.; Brechbiel, Martin W. (2004-06). "Antibody-targeted radiation cancer therapy". Nat Rev Drug Discov 3 (6): 488–499. doi:10.1038/nrd1413. ISSN 1474-1776. PMID 15173838. [5] WHO Drug Information (http:/ / whqlibdoc. who. int/ druginfo/ INN_2000_list43. pdf) [6] Ibritumomab: Indications (http:/ / www. zevalin. com/ HealthCarePro/ indications. htm) [7] Ibritumomab: Dosing and Administration (http:/ / www. zevalin. com/ HealthCarePro/ dosingandadministration. htm) [8] Ibritumomab: Efficacy (http:/ / www. zevalin. com/ HealthCarePro/ efficacy. htm) [9] ZEVALIN Consolidation in First-Line Therapy in Patients with Non-Hodgkin's Lymphoma Resulted in a Progression-Free Survival of Greater Than 67 Months (http:/ / investor. spectrumpharm. com/ releasedetail. cfm?ReleaseID=395156) [10] Zevalin and mantle cell (http:/ / www. abstracts2view. com/ hem4806/ view. php?nu=HEM06L1_1029) [11] ZEVALIN(R) Produced 83 Percent Complete Response Rate in Mucosa-Associated Lymphoid Tissue (MALT) Orbital Lymphoma Study (http:/ / investor. spectrumpharm. com/ releasedetail. cfm?ReleaseID=395048) [12] (http:/ / news. yahoo. com/ s/ ap/ 20070816/ ap_on_sc/ cell_therapeutics) [13] http:/ / www. zevalin. com/ [14] http:/ / www. spectrumpharm. com/

199

Tositumomab

200

Tositumomab Tositumomab ? Monoclonal antibody Type

Whole antibody

Source

Mouse

Target

CD20 Identifiers

CAS number

192391-48-3

ATC code

V10 XA53

DrugBank

BTD00085

KEGG

D08622

[1]

[2]

(sequential regimen with

131

I form)

[3]

[4]

 

Chemical data Formula

C6416H9874N1688O1987S44

Mol. mass

143859.7 g/mol Therapeutic considerations

Pregnancy cat. ? Legal status

? (what is this?)   (verify)

[5]

Tositumomab is a drug for the treatment of follicular lymphoma. It is a IgG2a anti-CD20 monoclonal antibody derived from immortalized mouse cells. Tositumomab is applied in a sequential infusion followed by iodine (131I) tositumomab, which is the same antibody covalently bound to the radionuclide iodine-131 (131I).[6] 131I emits both beta and gamma radiation and decays with a half-life of 8 days. Clinical trials have established the efficacy of the tositumomab/iodine (131I) tositumomab regimen in patients with relapsed or chemotherapy/rituxan refractory follicular lymphoma. This drug combination is manufactured by Corixa (now GlaxoSmithKline). It sells in the U.S. under the trade name Bexxar. Bexxar was developed by Dr. Mark Kaminski and Dr. Richard Wahl. There is some evidence that it may cause a smaller decrease in platelet counts than ibritumomab tiuxetan does.[7]

Tositumomab

References [1] [2] [3] [4] [5] [6] [7]

http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2009/ MB_cgi?term=192391-48-3& rn=1 http:/ / www. whocc. no/ atc_ddd_index/ ?code=V10XA53 http:/ / www. drugbank. ca/ drugs/ BTD00085 http:/ / www. kegg. jp/ entry/ D08622 http:/ / en. wikipedia. org/ w/ index. php?& diff=cur& oldid=409083424 Bexxar Prescribing Information (http:/ / us. gsk. com/ products/ assets/ us_bexxar. pdf) Jacene HA, Filice R, Kasecamp W, Wahl RL (2007). "Comparison of 90Y-ibritumomab tiuxetan and 131I-tositumomab in clinical practice" (http:/ / jnm. snmjournals. org/ cgi/ pmidlookup?view=long& pmid=17942813). J. Nucl. Med. 48 (11): 1767–76. doi:10.2967/jnumed.107.043489. PMID 17942813. .

http://www.med.umich.edu/medschool/faculty/facultyawards/2005/kaminski.htm

External links • Bexxar regimen (http://www.cancer.gov/Templates/db_alpha.aspx?CdrID=367411) entry in the public domain NCI Dictionary of Cancer Terms

201

Article Sources and Contributors

Article Sources and Contributors Cell cycle  Source: http://en.wikipedia.org/w/index.php?oldid=426654092  Contributors: - ), 060128, A little insignificant, A8UDI, AThing, Adashiel, Adrian J. Hunter, AgentPeppermint, Alansohn, Ale jrb, Alexei Kouprianov, Alexf, Alison22, Alnokta, Alpha Quadrant (alt), Anaxial, Andrew Kelly, Anime101, Arcadian, Arthena, Avoided, AxelBoldt, Bensaccount, Benwildeboer, Boghog, Brodyt66, Brunhilda18, Bub0297, Can't sleep, clown will eat me, CardinalDan, Cflm001, Chasingsol, Chuunen Baka, Citicat, Conmiro, Conversion script, CrashingWave, D6, Dan100, Darkfight, Darklilac, Dave6, Deadstar, DeathFlame131, Deicas, Delldot, DerHexer, Dflanagan, Discospinster, Djma12, Dmb000006, Dorftrottel, Download, Drdaveng, Drilnoth, Drmies, Dylan620, Earthdirt, EnSamulili, EncycloPetey, Epbr123, Eric-Wester, Erick.Antezana, Erkenbrack, Ettrig, Euphoria1611, Everyking, FAGGOODIKIKICOO, Figma, Flamingspinach, Flyguy649, Foreveriamchangd, Forluvoft, Frankenpuppy, Fratrep, Fritzpoll, Fruit.Smoothie, Fæ, GFP, Gautamdey, Ghaly, Giftlite, Glane23, GoodDamon, Grafen, Graymornings, HJ Mitchell, Hadal, Heathhunnicutt, Histidine, Horselover Frost, Hughitt1, Humanisticmystic, II MusLiM HyBRiD II, IW.HG, Ikiroid, Ilikepie2221, Ingolia, Intelligentsium, Iridescent, Isnow, Itssodry, J.delanoy, JackWasey, Jagbag2, Jake Wartenberg, Jan.Smolik, Jatlas, Jedi6, Jethero, Jfraser, John Dalton, Johnuniq, Joyous!, JuJube, Jwoodger, Ka Faraq Gatri, Karen Johnson, Kastor48252, Kateshortforbob, Khoikhoi, Kittyes, Knoitall00, Ktotam, Kuru, Kurykh, Kwillis29, L Kensington, La goutte de pluie, Lameomclame, Lave, Lchircus, Leafyplant, LedgendGamer, Lexor, Lifelover6, Ling.Nut, Loupeter, Lunajurai, MONGO, MPerel, MacDaid, Magnus Manske, Margie2121, Materialscientist, Maxí, Meekywiki, Mets501, Mgiganteus1, Midgley, Mikael Häggström, Mindmatrix, Mspraveen, Mumijary41, Mwfn, Mygerardromance, Naturespace, NawlinWiki, Nbauman, NewEnglandYankee, NikNaks93, Nikki chan, Nikkirox69, Nona89, Nutriveg, Omes, Onco p53, Opabinia regalis, Openlander, Oskoreien, OttoTheFish, Palica, Penhaligon 5, Persian Poet Gal, Phe, Pinethicket, PlumCrumbleAndCustard, Pokemaster50, Prashanthns, PrestonH, Protox, Quantumobserver, RamonyCajal, Rawling, Reach Out to the Truth, Reconsider the static, RexNL, Rheka, Rjwilmsi, Robinatron, Robinhaw, Rrburke, Ruud Koot, S h i v a (Visnu), Samsara, SantanuRoy, SchuminWeb, Scientizzle, Scoops, Serephine, Shinpah1, Shinryuu, Showman16, Sionus, Sjö, Sl, Snek01, Snowolf, SoCalSuperEagle, Sodium, Spencerk, Squidonius, Srr712, Stevage, Steve Smith, Susfele, SvenskaJohannes, Synchronism, T-borg, T4taylor, Tarotcards, Tcncv, Thaerhashem, The High Fin Sperm Whale, The Rambling Man, The Thing That Should Not Be, The Transhumanist, Tide rolls, TimVickers, Timc, Twooars, Uncle Dick, VBGFscJUn3, VegaDark, Versus22, Vietbio, Vipinhari, Vishnava, Walik, Whosasking, Wysprgr2005, Xcentaur, Yahel Guhan, Yerpo, Zephyris, Zidane tribal, ‫کشرز‬, 664 anonymous edits Complete blood count  Source: http://en.wikipedia.org/w/index.php?oldid=426872456  Contributors: Ace-o-aces, Alai, Alex.tan, Arcadian, Arcturus, Arisa, Betacommand, Bigdumbdinosaur, Bobjgalindo, Chemmaj, Chirality, Chrishota, Cubic Hour, Darklilac, Davidruben, Denelson83, Dethme0w, Diberri, EJVargas, Edward, Eggman64, Excirial, Fallj, Fedra, Gwernol, HalfShadow, Hasan 3, HazyShades, Horoporo, Howcheng, Immunize, InvictaHOG, JJJJust, Jafeluv, Jarkeld, Jarkeld.alt, JarodRoland, Jeremykemp, Jfdwolff, Jmarchn, JoePdw, Jon186, Jouster, Kalaiarasy, Karada, KnightRider, Kyoko, Linforest, Longdaom, Mcarling, Meamemg, Meelar, Mikael Häggström, Mindmatrix, Mlewis000, Morrillonline, Mote, Nunh-huh, Ozmaweezer, PeterC, Peterl, PhilKnight, Polacrilex, Povmcdov, Pt, R'n'B, Raccoon Fox, Renwick, Rmhermen, SCEhardt, Samir, Schekinov Alexey Victorovich, Schulj, Scottalter, Stainless steel, Stephan Leclercq, Steven Zhang, Stevertigo, Taweetham, Tide rolls, TornadoCreator, Trevor Wennblom, Tristanb, Ugen64, Varlaam, Versus22, Vojtech.dostal, Wayiran, William Avery, Wordwriter123456789, Xlenka, 114 anonymous edits Reference ranges for blood tests  Source: http://en.wikipedia.org/w/index.php?oldid=426693830  Contributors: Andrewdpcotton, Arcadian, ArnoldReinhold, Bgordski, BillC, Blubutterfli, Bryan Derksen, Burlywood, Calmer Waters, Capricorn42, Chirality, Cjewell, Cornell92, Dabomb87, Daughter of Mímir, Davidruben, Dikteren, Flewis, Galoubet, GermanX, Glinzer, Gobonobo, Gordon55y, II MusLiM HyBRiD II, Ironsman, Jfdwolff, Jheald, Jojimathew03, Justjennifer, Karada, Kosebamse, L.tak, Michi zh, Mikael Häggström, Mindmatrix, Narayanese, Nbauman, Neutrality, NewEnglandYankee, Nunh-huh, OldakQuill, OmegaWiki, Ozmaweezer, Parasympathy, Rich Farmbrough, Richiez, Rjwilmsi, Rod57, Rustavo, Scarhead, Scolaire, Sesse, Shell Kinney, Spencer, Stevenfruitsmaak, Talrias, Topbanana, Tristanb, UberMD, VinceBowdren, Wapcaplet, Westgaard, WikHead, Yours.genesys, 朝彦, 90 anonymous edits Bone marrow  Source: http://en.wikipedia.org/w/index.php?oldid=427256775  Contributors: AThing, Acdx, Aitias, Alansohn, Alex.tan, Alexwcovington, Allycat0527, Amroc, 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Hunt, Rpm099, Russianplayer, SMC, Satarnion, Sceptre, Schrödinger's Cake, Scruffymmh, Seforadev, ShadowRangerRIT, Shadowjams, Shendar, SimonD, Sjones23, Slakr, Smartse, Smith11v, Socialmediarox, SohanDsouza, Sokching, SonicAD, Splatg, Stevencho, Stevenfruitsmaak, Svick, Syrthiss, TallNapoleon, Teles, Template namespace initialisation script, Tetracube, Thatguyflint, The High Fin Sperm Whale, The Thing That Should Not Be, Theda, Thehelpfulone, Themfromspace, Thumperward, Tide rolls, Tired time, Tivedshambo, Tombliboo, Tombomp, Trusilver, Uncle G, Venkatesh, Versus22, Vina, Vivio Testarossa, Vulturejoe, Wackjum, Waggers, Washburnmav, Wayiran, WhisperToMe, WhyBeNormal, Workster, Wouterstomp, Wtmitchell, Xxpor, Yidisheryid, Zahakiel, Zenwhat, Zoe, Žiedas, Дарко Максимовић, 604 anonymous edits Antibody  Source: http://en.wikipedia.org/w/index.php?oldid=427296401  Contributors: 8472, A930913, AdamRetchless, AdrianaW, Afluent Rider, Ahoerstemeier, Aka042, Akhil999in, Akira1984, Alansohn, Amyraymond, AnakngAraw, Anoopsinha, Anypodetos, AppleMacReporter, Appraiser, Arcadian, Argrig, ArielGold, Aroopsircar, Arthena, Audchen, Babajobu, Banus, Baronnet, Barticus88, Bassem18, Bdonlan, BigrTex, Bio-pilot, Bioinformin, Bob247, Bobblehead, Bobo192, Boghog, Bongwarrior, Boothy443, BostonBiochem, Brandmeister, Brazucs, Bseacat, Bunchofgrapes, Burubuz, Bwbrian, C31004, Cabbydriver, Ceyockey, Chaz1066, Ciar, Clementine2009, Cleverlew, ClickRick, ClockworkSoul, Cockers1987, Conny, Conversion script, CopperKettle, Cotarded, Crazyexact, Crystallina, DO11.10, Dana boomer, Dbollard99, Deetdeet, Dekisugi, Deli nk, Delldot, Dhoopes, Diberri, DocWatson42, Dpryan, Dr.Kane, Drangscleaner, Dreadstar, Drmies, Drn.sharathchandra, Drphilharmonic, Duffin2k7, Duffman, Duncan.france, Eleassar, Eleassar777, Element16, EnSamulili, Epbr123, Epgui, Esanchez7587, Feezo, Flukeboy, Fnielsen, Fodient, Fvasconcellos, Gak, Gbleem, Ghermans, Ghettodmo, Gholam, Giftlite, Glassneko, Glenn, Glish81, Graham87, GrahamColm, Grillo7, H Sapien, Halibutt, Harryboyles, HendrixEesti, Hide&Reason, Hoffmeier, Hrbreck, Husond, Hx, II MusLiM HyBRiD II, Ian Glenn, Icelight, Immunize, Inge-Lyubov, Iridescent, Itsmenotyou123, Iwilcox, Ixfd64, J.delanoy, J04n, JOK, JVinocur, Jacuzzijo, James McNally, Jan eissfeldt, JanDaMan, Je at uwo, Jecowa, Jengod, Jennavecia, Jevansen, Jfdwolff, Jim Douglas, Jimfbleak, Jls043, Jnestorius, Johnbushiii, Johnfravolda, Jon186, Jsuchit, Julianva, Jwollbold, K.murphy, KGasso, Kalaiarasy, Kastoun, Keesiewonder, KerryB, Kimberlykristin, Kischel, Kortikal, Kosigrim, Kungfuadam, Kusunose, LBK74, LC, La goutte de pluie, Lear's Fool, LeaveSleaves, Lefranc, Lemonflash, Leptictidium, Lexor, Ling.Nut, Littlealien182, Lord Anubis, Luk, Luna Santin, Magiciandude, Magnus Manske, Mani1, Manishearth, Mannafredo, MarcoTolo, Marx01, Math-ghamhainn, Mattb112885, Mav, McDogm, McSpurt, Megaprof, MichaelJanich, Michal Nebyla, Mikael Häggström, Mike2vil, Mimihitam, Minimac, Mixel, Miyagawa, Mlessard, Molecularbio, Mxpule, Nacimota, Narayanese, Narssaers, Neilymon, Netkinetic, Nevo taaseh, Nga2005, Nitwitpicker, Nono64, Novangelis, NuclearWarfare, Oddben, Ohnoitsjamie, Opelio, Origamiemensch, Ortho, Oxymoron83, Pedro, Pgan002, Phydend, Piano non troppo, Pimplecustard, Pinethicket, Pmm530, PoliticalJunkie, Professor Albatross, Pvosta, Qtnguyen92683, R'n'B, RDBrown, Rajanthampi, RandomP, Rdsmith4, Reaper Eternal, RelentlessRecusant, Renato Caniatti, Renji143, RexNL, Rich Farmbrough, Rijkbenik, Rjwilmsi, Rod57, Royboycrashfan, Sadi Carnot, Salsb, Sam Korn, SanderB, Sandy24, Sara Howland, Sayeth, Schzmo, Seans Potato Business, Selket, Serephine, Sergeymk, Snek01, Snowmanradio, Snowolf, Someone else, Spaully, Squids and Chips, Srich32977, Stemonitis, Stephenb, Taco325i, Taka, Takometer, Tamarkapterian, Tamiasciurus, Tassedethe, Tdivala, Template namespace initialisation script, TenOfAllTrades, Thorwald, Tide rolls, TimVickers, Timferno, TorbenBarington, Treisijs, Tri6ky, Tycho, Versus22, Vietbio, Vietnam bathtub, W09110900, WhatamIdoing, Whosthedaddy, Winchelsea, Wisebridge, Woohookitty, Wouterstomp, WriterHound, Xenobiologista, Xett, Yamaguchi先生, Yohan, Youssefsan, Yurivict, ZacBowling, Zhuuu, Δ, 556 anonymous edits Hematopoietic stem cell  Source: http://en.wikipedia.org/w/index.php?oldid=426554311  Contributors: 777sms, A. Rad, Aardappelmesje, Akela1, Amirmeiri, Antibody2000, Anypodetos, Arcadian, Arcfrk, Bender235, Capricorn42, Cquan, Cyrus Grisham, DO11.10, Dr.saptarshi, Drphilharmonic, Edward, Eleassar777, Epolk, Etparle, Euchiasmus, Fconaway, Felixcheung, Fuhghettaboutit, GotaForce, Gronk, Harrisd5917, Hiddekel, Hsieburg, JSLR, Jag123, James086, Jensbn, Jfdwolff, Jjalexand, Justme89, Kauczuk, Kavanagh21, Linzhoo2u, Martious, Melaen, Mfourman, Miguel Andrade, Mikael Häggström, Mysid, Nina, Nono64, Osquar F, Pascal666, Peter Znamenskiy, Psinu, Rich Farmbrough, Schulj, SpK, Spitfire8520, Stemonitis, TenOfAllTrades, Thomas.clapes, Triggtay, Vojtech.dostal, パタゴニア, 114 anonymous edits Haematopoiesis  Source: http://en.wikipedia.org/w/index.php?oldid=420342888  Contributors: .:Ajvol:., A. Rad, AThing, Aamer86, AdultSwim, AlphaEta, AndrewJD, Antibody2000, Aquatech, Arcadian, Ariostosilva, BarretBonden, BillC, Brazucs, Brim, Bryan Derksen, Buster79, Chrishmt0423, Conversion script, Cquan, Ctrlaltdelete200390, Dcirovic, Diberri, Dr. F.C. Turner, DrGabriela, Enchanter, Essent, Giftlite, Glassneko, Jag123, Jer ome, Jeyradan, Jfdwolff, John254, Kaobear, Kauczuk, Kipmaster, Lateg, Lbiscuit1980, Leptictidium, Linzhoo2u, Liveste, Markacohen, Mccready, Mdhellman, Mesoderm, Mikael Häggström, NewEnglandYankee, Nina, Nina Gerlach, Openlander, Pstragier, Qumsieh, R'n'B, Rakal87, Reedy, Rintrah, Salwateama2008, Schmiteye, Snalwibma, Snek01, Snowmanradio, Some standardized rigour, Stephenb, TheAMmollusc, Tkkoski, 88 anonymous edits Lymphopoiesis  Source: http://en.wikipedia.org/w/index.php?oldid=420402787  Contributors: Antibody2000, Arcadian, Arthena, Bdo311, Colonies Chris, Cyclonenim, Delldot, Drbreznjev, Epolk, Moez, Naraht, Queenmomcat, Regford, Stemonitis, 12 anonymous edits

202

Article Sources and Contributors Hematologic disease  Source: http://en.wikipedia.org/w/index.php?oldid=424120595  Contributors: Arcadian, Jfdwolff, Mwoehrle, Nephron, Some standardized rigour, Vincenzo80, YuriSuassuna, 2 anonymous edits Hematological malignancy  Source: http://en.wikipedia.org/w/index.php?oldid=424387825  Contributors: Acdx, Arcadian, CDN99, Calmer Waters, Donreed, DrFO.Jr.Tn, Drjames1, Epiwiki, Grand King's Cero, Immunize, Jeyradan, Jfarr11, Jfdwolff, Kalaiarasy, Kheerand, Knutux, Lave, Mayumashu, Mikael Häggström, Nephron, Nicobzz, Nunh-huh, Ph.eyes, Phe, Reinyday, THEN WHO WAS PHONE?, Template namespace initialisation script, Wahlin, WhatamIdoing, Wouterstomp, 22 anonymous edits Lymphoma  Source: http://en.wikipedia.org/w/index.php?oldid=425434766  Contributors: AKarthikeyan, Aaron Brenneman, AdjustShift, AdultSwim, Ale jrb, Alexdenes, Allstarecho, Alton Dalton, Andrew73, Angela26, Anthonyhcole, Antiuser, Arcadian, Auntof6, Axl, BService, Badagnani, Beetstra, Benjamin Mako Hill, BillieMTre, Bkrik, Blueflamedj, Bonicolli, Boruchfishman, Branlon, Brenont, CDN99, Calmer Waters, Catgut, Cayzle, Chavoguero, Chris Roy, Clehmann, Colorajo, Corinna7, Cortamears, DARTH SIDIOUS 2, DennyColt, Diberri, Dl2000, Dr.michael.benjamin, DrFO.Jr.Tn, DrKiernan, Drim, Drjames1, Drmies, Drrobert, Dryden01, Eleassar777, Emmanuelm, EoGuy, Eric-Wester, FaerieInGrey, Ferretchristie, FisherQueen, Flyer22, Forgetful5434, Fudoreaper, Fyrael, Fyyer, Gabriel Caponetti, Gdo01, Goldom, Graham87, GrahamColm, Gurch, Guroadrunner, Gwernol, H4ckzor3, Haonhien, Harrisd5917, Hijklmnop224, Hopedreams, Hovea, Hu12, Hut 8.5, Immunize, Iph, Ipso2, It Is Me Here, J. Spencer, JForget, JLaTondre, JamesAM, Jfdwolff, Jmh649, Joelmills, Johnfravolda, Johnwpattison, Jojdavies, Jon f, Jon.baldwin, KC Panchal, Kafziel, Kaisershatner, Keenan Pepper, Kerryhaack, Kessler, Kinghitchhiker, Knuckles, Knutux, Kyle1278, L Kensington, Lacommun1cations, Lexicontra, Linzhoo2u, Lisajcpig, LittleT889, Lozeldafan, Lspector, Luna Santin, Lymphoma, Lymphoma Association, MBest-son, MKS, MONGO, Madi Khan, Mandarax, Marek69, MastCell, Mclemore, Melburnian, Michael Hardy, Mikael Häggström, Mikebar, Mimihitam, Mukkakukaku, Myanw, Nbauman, Ndenison, Nemodomi, Nunquam Dormio, Ocrasaroon, Oli Filth, Onore Baka Sama, Open2universe, Outlook, Paysseh, Pdeitiker, Pgan002, Pinethicket, PrincessWortheverything, Queenb7243, Quintote, Qxz, Rajsite, RedDrag0n, Reinyday, Rewster, Rickterp, Roadcreature, Rothorpe, RuED, STV0726, Samiam1611, Samir, Satori Son, Satrohraj, Scottalter, SeanMack, Seans Potato Business, Senaiboy, Shadowen78, Sietse Snel, Skpearman, Soap, Sparki883, Stevenfruitsmaak, Supertigerman, Taral, Tayquan, Teapotgeorge, Template namespace initialisation script, The Anome, The Audacity of Hip, Theresa knott, Thoken, Tide rolls, Tony1, Toyokuni3, Triggtay, Tsuchida54, Tvarnoe, USAFmom, Vedran12, Vkingsley, WhatamIdoing, WikHead, Wikihelper9000, Wikipelli, Wikislemur, William charles caccamise sr, md, Woohookitty, Wouterstomp, WriterHound, X!, Xyzzer, Yeti7, ZTebaykina, Zack2007, Zkaleem, Zzuuzz, 332 anonymous edits Non-Hodgkin lymphoma  Source: http://en.wikipedia.org/w/index.php?oldid=425434630  Contributors: Afterwriting, Agiachic, Aitias, Akram97, AlexWilkes, Andre Engels, Andrew73, Arcadian, Arthena, Avillia, Balok, BethBukata, Bladkin, Bobo192, Browneee, Bucketsofg, CDN99, Calmer Waters, Capricorn42, Chavoguero, Cheverton, Coemgenus, Coolhandscot, Cst17, Cwoyte, DP08, Dale Arnett, Dan100, David.Throop, Diberri, Dlauri, Docbillnet, Donreed, Dr.jackie, Duncan.france, [email protected], Emmanuelm, Emvee, Epbr123, Eric-Wester, Evergreenboy, Excirial, Fabiform, Facts707, Friedfish, Gaius Cornelius, Gene Nygaard, General Jazza, Gigemag76, Gildir, Gjking, Glitch010101, GlobalFlop, Gnusmas, Gokrispan, HBNayr, Harald Hansen, Hopedreams, Imeisel, Immunize, ImperatorExercitus, Jeremy68, Jfdwolff, Jmh649, Joelr31, Johnasher, Josephgrossberg, Karen7673, Keilana, Kenmcl2, Kh7, Kjoonlee, Lacommun1cations, Lapinmies, LittleSocrates, Loudsox, Lspector, Luna Santin, Lymphoma, Lymphoma Association, Lynn.Kelsey, Magnus Manske, Marcelo1229, MastCell, Matthias koehler, Maxamegalon2000, Meagirl, Mike2vil, Mikebar, Mizzou1307, Mmrruugg, MrBell, MrDolomite, Nbauman, Nigholith, Nihiltres, Nn123645, Numero4, Nunh-huh, OhanaUnited, Oxymoron83, ParisianBlade, PatrickA, Paxsimius, Pdcook, Pharaoh of the Wizards, Proofreader77, Pstevens, Puchiko, Purplewyo1, Quentin X, RainbowOfLight, Rcej, Reinyday, Richwales, Rimibchatterjee, Rjd0060, Rodney Boyd, Roux-HG, Rsds129, Saibod, Sam1042, Sam8, Senaiboy, Sethmahoney, Shadowen78, Soakologist, Someone else, Sophie1979, Spiro Keats, Stismail, Tdferro, Teapotgeorge, Template namespace initialisation script, That Guy, From That Show!, The Anome, TheCustomOfLife, Tom harrison, Vivio Testarossa, Vt-aoe, WhatamIdoing, Wolfkeeper, Wouterstomp, Xenoglossophobe, Xompanthy, 229 anonymous edits Cluster of differentiation  Source: http://en.wikipedia.org/w/index.php?oldid=424539534  Contributors: Andrew73, Arcadian, Arjun mht, Beao, Boghog, Brim, DO11.10, Dan Wylie-Sears, Dcirovic, Diberri, Drphilharmonic, Eikuch, Fix888, Franzb78, Func, JeffreyN, Jfdwolff, JorisvS, Kalambaki2, Kiore, Lokal Profil, MarcoTolo, Mikael Häggström, MrOllie, MrPMonday, Nbauman, Negarci, Niels Olson, Novangelis, PFHLai, Rctay, Rjwilmsi, Rod57, Simba Jones, Slyang, Snek01, Stemonitis, Stmoran, Superlydia, Tossh eng, Triggtay, Tristanb, Volantares, Wik, Windben, Wouterstomp, Yun hu, Zocky, Zorakoid, Þjóðólfr, 50 anonymous edits List of human clusters of differentiation  Source: http://en.wikipedia.org/w/index.php?oldid=422954362  Contributors: Andrew73, Antibody2000, Anypodetos, Arcadian, Bokito, Boonshofter, Brim, Captain-n00dle, Celique, Cfptwenty, Ciar, DO11.10, Dr.saptarshi, Dysmorodrepanis, Eras-mus, Eupedia, Figma, Fix888, Gerne1, Hjmol, Italienmoose, JeffreyN, Jfdwolff, Johnwalton, Jtimperio, Kantokano, KateGartlan, Keantom, Krash, Leolaursen, Mddms 88, Merqtio, Mikael Häggström, Natural killer & EOL corp., Nono64, Ojitarian, PFHLai, Ruwan, Spook`, Stemonitis, Zyncodex, 63 anonymous edits Mantle cell lymphoma  Source: http://en.wikipedia.org/w/index.php?oldid=426645746  Contributors: AlphaEta, Arcadian, Axl, Aytrus, Bobo192, Calmer Waters, Cthornby, Danielsc2, Danny, Delldot, Devahn58, Drjoshuabrody, Espresso Addict, Gor n bein, Immunize, Jessejobe, Jhannah, Kenmcl2, Lymphoma, Marcelo1229, MastCell, Michael Hardy, Mikael Häggström, MrOllie, Nephron, Puckettr, Rich Farmbrough, Rjwilmsi, Sanguis Sanies, Scilipoti.venera, Sean.evans, Squidonius, The Thing That Should Not Be, WhatamIdoing, Wilsonstowell, 19 anonymous edits Chemotherapy  Source: http://en.wikipedia.org/w/index.php?oldid=426592233  Contributors: 129.186.219.xxx, 213.76.2.xxx, A. B., Aaronmthompson, Aaronw, Adampellegrini, Aeneas07, Alex.tan, Alheim, Amama z. Suliman, Andre Engels, Andrew73, Andrewpmk, Android Mouse, Angela, Antandrus, Anthonymsweeney, Antonio Lopez, Anuran, Arcadian, Arichnad, AxelBoldt, Axl, Baa, Barryz1, Bemoeial, Ben.c.roberts, Benjaminevans82, Besard22, Betty Kerner, Black Kite, BluesD, Bobo192, Bogey97, Bzotter, CS99, Cacycle, Cannibalicious!, CardinalDan, Ceyockey, Chanting Fox, Charcope, Chase me ladies, I'm the Cavalry, Chintzypop, Chowbok, Ciphers, Cobi, Conversion script, Cortamears, Cosmic Latte, Cyrius, DFS454, DJBarney24, DVdm, Da monster under your bed, Damian Yerrick, Dancanm, Danielledanielle, Daveyb007, Davidruben, Daycd, Deadk82, Derek Ross, Dina, Doctorfluffy, Dogposter, Dontbeaschmuck, Dr.frog, Dr.michael.benjamin, Drmies, Drphilharmonic, Drumbeatsofeden, Dwmyers, E0steven, Eequor, El C, Eleassar, Enigmaman, Epbr123, Eras-mus, Eric-Wester, Essent, Eubulides, Everyking, Excesses, Faderrattnerb, Fennec, Fieldday-sunday, FirstPrinciples, Fivemack, Fluppy, Fox, Fractional ideal, Frap, Fratrep, Fredb, Frosted14, Furrykef, Fvasconcellos, Galaxiaad, Ge Ming, Giac83, Giftlite, Gman124, Gogo Dodo, Goodrich Ave, Graham87, Ground Zero, Gurchzilla, Gutul, H, HaeB, Haiduc, Hamiltondaniel, Handlesafe, Hcheney, Hires an editor, Hoffmeier, Holeung, Hu12, Huji, Ischium, Ivanaastapova, J.delanoy, J04n, JNW, JSpung, Jab843, Jag989, Jagged 85, Jaknouse, JamieS93, Jammycakes, January, Jatlas, Jeepday, Jeff G., Jeff Muscato, Jerrch, Jfdwolff, Jj137, Jmcatlin, Jmh649, JonHarder, Js229, Julesd, Junglecat, Jurd546777, K50 Dude, KPH2293, Karada, Karl-Henner, Karl.langberg, Kevin B12, Kirk.wolfe, KnowledgeOfSelf, Kpjas, Kukini, Kurykh, Kwertii, L Kensington, Laportechicago, LaylaV, Leadintogold, Leeearnest, Lemur12, Lightmouse, LlywelynII, Lmuenchen, LordK, Lupin, Luweese, Luxomni, Lymphoma, Mabromwich, Malo, Mani1, MarcoTolo, MartinHarper, MastCell, Materialscientist, Mauvila, Maximus Rex, Mearlon, MedHead, Menchi, Mets501, Mgummess, Mhs5392, Mikael Häggström, MikeVitale, Mister Six, Mocellins, Mpiff, MrAnderson7, Mrs Susan a. 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Spencer, JamesBWatson, Jeffq, JohnFranks405, Johnuniq, Jpaulrobinson, Jpbowen, Jvbishop, Kavanagh21, Kblakely, Keantom, Keepcalmandcarryon, Kierano, Kkmurray, Lenticel, Lexor, Llltrunkslll, MER-C, MGOrmerod, Matchups, Melcombe, Merle travis39, Merovingian, Michael G Ormerod, Motutupe, MrOllie, Mskadhir, Mxpule, Narayanese, NewEnglandYankee, NorwegianBlue, Ocee, Onco p53, Orlandoturner, Pixie, Pvosta, Qst, Quiscale, Ramujana, Requestion, Rglab, Ronhjones, Rustavo, Sagemontano, Sandrauesugi, Shinryuu, Shyamal, Skientist, Snapperman2, Snowmanradio, SunCreator, T-borg, Temporaluser, Travis.jennings, TrueGlue, Vancouver Outlaw, Vasilis70, Visudoc, Wavelength, Whosasking, William A Newton, Wknight94, Woohookitty, Wouterstomp, Xandi, Yserarau, ‫نامجرت‬05, 226 anonymous edits

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Article Sources and Contributors Fluorescence in situ hybridization  Source: http://en.wikipedia.org/w/index.php?oldid=422933159  Contributors: Aaronjhill, Afaz, Alboyle, Anna.h.bauer, Arcadian, Ary29, BahramH, Barticus88, Bender235, C4 Diesel, Ch'marr, Chris the speller, Cohesion, CommonsDelinker, DO11.10, DrMacrophage, Drphilharmonic, EJCM, Ediacara, ElliottHoffman, Esthurin, Evanherk, Filnik, Gettingtoit, Gregor1976, Im.a.lumberjack, Janus01, Jclam, Jeff G., Jfdwolff, Jmeppley, Jorvis, Josh Parris, KSlayer, Kierano, Ktinga, Lantonov, Macowell, MeirM, Mikael Häggström, Millzeh, Mm40, Monty845, MrMatze, Nchaimov, Neffk, Paphrag, Peak, Philbradley, Pilotguy, Pojoman, Rdbrady, Riana, Rich257, Rustavo, S h i v a (Visnu), Saeedwiki, Sam Hocevar, Sigma-w, Snapperman2, SobaNoodleForYou, SomethingCatchy, Spearhead, SteveChervitzTrutane, Tamfang, Temporaluser, Thingg, Uncle Milty, VashiDonsk, Velocity22, Velocity2222X, Vjsieben, 106 anonymous edits Common variable immunodeficiency  Source: http://en.wikipedia.org/w/index.php?oldid=420872317  Contributors: Arcadian, Cam27, CambridgeBayWeather, DO11.10, DavidWaldock, Emctimo, Gilliam, Gobonobo, Headbomb, Ivirivi00, JMcD92, Jfdwolff, Johngordonboyle, Kouluhai, Lanternix, Maria Larsson, Markleventhal, My Core Competency is Competency, Nebbione, Nehrams2020, Nihiltres, Nikai, RDBrown, Rich Farmbrough, Rjwilmsi, SWAdair, Sgw1009, Vicarious, WhatamIdoing, Woohookitty, Zanotam, ZayZayEM, 114 anonymous edits Epigenetics  Source: http://en.wikipedia.org/w/index.php?oldid=426184095  Contributors: 7mike5000, A. B., AThing, Adina cappell, AdultSwim, Agathman, Alex.muller, Amatulic, Andrejj, Anthere, Anthonyhcole, Apers0n, Arcadian, Arkelweis, AubreyEllenShomo, AxelBoldt, Badagnani, Ben Ben, Bensaccount, Billman119, Bojo-is-the-man, Bryan Derksen, Cbock, Cgingold, Charles Matthews, Charles walsh, Christian75, Christopherlin, Ckatz, Crusio, D taz R, D6, DMacks, Danfischer313, Daniel.Cardenas, Danielgrad, David D., Davidnortman, Dbregister, Delldot, Der Zeitgeist, DerHexer, Dhfreedman, Dogface, Don Gosiewski, Dougher, Dr Oldekop, Drphilharmonic, Dumpster muffin, Duncan.france, Dysmorodrepanis, EPM, Eahd201, EdH, Electron9, ErkDemon, Erud, Ervinn, Ettrig, Ex gratia, Excentrifuge, Eyoste, False vacuum, Fat Cigar, Fd88ar, Forluvoft, Gaius Cornelius, Gomm, Gondola, Gpokela, H0riz0n, H2g2bob, Hambleton, Heathhunnicutt, Hodja Nasreddin, Hwttdz, IMKatgrrl, Ian Pitchford, Id711, InverseHypercube, Irrbloss, JForget, Jakecarver2010, Jeronimo, Jethero, Jetspeed11, Jjalexand, Joannamasel, Joekingbling, 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Bigdumbdinosaur, BioBabble, BlakeCS, Cburnett, Cellpath, Ceyockey, Chase me ladies, I'm the Cavalry, Chikinsawsage, Chochopk, Clares, Conserrnd, Coolhandscot, DVD R W, Danrudd, Davey5505, Deadk82, Dekimasu, Dr.michael.benjamin, Duncan.france, Eily-Eily, Eltanin, Eras-mus, Esenbellur, Fvasconcellos, Grushnik, HazyM, Healthvalue, Hrbarthel, Japanese Searobin, Jfdwolff, Keithfry, Kenmcl2, Lady Cairns, Lmedwards, Lpilarsk, Lymphoma, MastCell, Nbauman, Nephron, Pashihiko, PaulWicks, Rich Farmbrough, Rod57, Sbmehta, Selket, Spiff666, Stmrlbs, Streetly, Stretchcat, SuW, Tanis118, Terrace4, Travis.Thurston, Triggtay, TyroneMiller, Uploadvirus, WLU, Ward20, WhatamIdoing, Wptoler, 89 anonymous edits CHOP  Source: http://en.wikipedia.org/w/index.php?oldid=427262392  Contributors: 777sms, Andrew73, Arcadian, Cburnett, Chowbok, Diberri, Drjermy, Enix150, Jfdwolff, Lenrodman, LinguistAtLarge, MastCell, Nbauman, Nessar Ahmad Azrakhsh, Royalguard11, Tanevala, Uthbrian, Yalbik, 22 anonymous edits HyperCVAD  Source: http://en.wikipedia.org/w/index.php?oldid=416167808  Contributors: Chowbok, Cthornby, D6, Dthomsen8, Ettrig, IceCreamAntisocial, Pxma, Squidonius, WhatamIdoing, 11 anonymous edits Bortezomib  Source: http://en.wikipedia.org/w/index.php?oldid=422897408  Contributors: AlexSwanson, Andrew73, Anypodetos, Arcadian, Archibald Tuttle, Axl, BCvek, Baysidemk, Beetstra, Benjah-bmm27, Betacommand, Bkrik, Bspahh, ChemNerd, Chenxinchx, Choij, Chowbok, Cortamears, Davidruben, Diberri, Drugevaluation, Egertonydavis, Epiwiki, Fawcett5, Fvasconcellos, Ground Zero, Jfdwolff, JohnI, Kenmcl2, Kobrabones, Kuebi, Louisajb, Marcolop, Mightyhansa, Nationalparks, Nono64, Opabinia regalis, Rich Farmbrough, Rjwilmsi, Rod57, Samw, Selket, Squidonius, Srasku, Tetracube, Uthbrian, V8rik, Walkerma, WolfmanSF, Wouterstomp, ‫طبلا يلع نسح‬, 29 anonymous edits Bendamustine  Source: http://en.wikipedia.org/w/index.php?oldid=424938403  Contributors: Anypodetos, Carlo Banez, Derek.cashman, DrMicro, Edgar181, Fabrictramp, Fvasconcellos, L.tak, Louisajb, Mattisse, Mlaffs, Mordac, Rod57, Tcera, WhatamIdoing, Yunhchoe, ‫طبلا يلع نسح‬, 6 anonymous edits Temsirolimus  Source: http://en.wikipedia.org/w/index.php?oldid=424725620  Contributors: Anypodetos, BobMullin, Carlo Banez, ChemNerd, Davidruben, Dougtischler, Edgar181, Fvasconcellos, Ground Zero, Haddendaddendoedenda, JimDavis4u, Mikael Häggström, Nono64, P-kun80, Pashihiko, Rbaselt, Rjwilmsi, Rod57, Squids and Chips, Wouterstomp, 5 anonymous edits Antibody-drug conjugate  Source: http://en.wikipedia.org/w/index.php?oldid=426546532  Contributors: A-Day, Anypodetos, Bearcat, Chris Capoccia, GoingBatty, Materialscientist, Rod57, Δ Ibritumomab tiuxetan  Source: http://en.wikipedia.org/w/index.php?oldid=418466916  Contributors: Abeg92, Anypodetos, Arcadian, Blake3522, BlakeCS, Burubuz, Chowbok, Cudo29, Diberri, Epolk, Hoffmeier, Jfdwolff, Jossi, Jwissick, Kenmcl2, Kirill Lokshin, Lictonaud, Michael Hardy, OMCV, Prisonnet, Qxz, Shakeandshout, Terrace4, TestPilot, Vuo, WhatamIdoing, 13 anonymous edits Tositumomab  Source: http://en.wikipedia.org/w/index.php?oldid=421296054  Contributors: Aandr34, Anypodetos, Arcadian, BlakeCS, Cellpath, DVD R W, Edgar181, Gor n bein, Icek, Kpjas, Pashihiko, Rich Farmbrough, Scewing, Selket, 7 anonymous edits

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Image Sources, Licenses and Contributors

Image Sources, Licenses and Contributors Image:Major events in mitosis.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Major_events_in_mitosis.svg  License: Public Domain  Contributors: w:User:MysidMysid Image:Cell Cycle 2-2.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Cell_Cycle_2-2.svg  License: GNU Free Documentation License  Contributors: User:Beao, User:Histidine Image:Regulation of the cell cycle.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Regulation_of_the_cell_cycle.svg  License: Public Domain  Contributors: User:T4taylor Image:Signal transduction v1.png  Source: http://en.wikipedia.org/w/index.php?title=File:Signal_transduction_v1.png  License: GNU Free Documentation License  Contributors: Original uploader was Roadnottaken at en.wikipedia Image:PD-icon.svg  Source: http://en.wikipedia.org/w/index.php?title=File:PD-icon.svg  License: Public Domain  Contributors: User:Duesentrieb, User:Rfl Image:CBC with Hct.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:CBC_with_Hct.jpg  License: GNU Free Documentation License  Contributors: User:Samir_(The_Scope) File:OuchFlintGoodrichShot1941.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:OuchFlintGoodrichShot1941.jpg  License: Public Domain  Contributors: Uncredited WPA photographer File:CBC report.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:CBC_report.JPG  License: GNU Free Documentation License  Contributors: User:Bobjgalindo Image:SEM blood cells.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:SEM_blood_cells.jpg  License: Public Domain  Contributors: Bruce Wetzel (photographer). Harry Schaefer (photographer) File:Blood values sorted by mass and molar concentration.png  Source: http://en.wikipedia.org/w/index.php?title=File:Blood_values_sorted_by_mass_and_molar_concentration.png  License: Public Domain  Contributors: User:Mikael Häggström Image:Reference ranges for blood tests - by units.png  Source: http://en.wikipedia.org/w/index.php?title=File:Reference_ranges_for_blood_tests_-_by_units.png  License: Public Domain  Contributors: User:Mikael Häggström File:Reference ranges for blood tests - by enzyme activity.png  Source: http://en.wikipedia.org/w/index.php?title=File:Reference_ranges_for_blood_tests_-_by_enzyme_activity.png  License: Public Domain  Contributors: User:Mikael Häggström Image:Reference ranges for blood tests - white blood cells.png  Source: http://en.wikipedia.org/w/index.php?title=File:Reference_ranges_for_blood_tests_-_white_blood_cells.png  License: Public Domain  Contributors: User:Mikael Häggström File:Hormones estradiol, progesterone, LH and FSH during menstrual cycle.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Hormones_estradiol,_progesterone,_LH_and_FSH_during_menstrual_cycle.svg  License: Public Domain  Contributors: User:Mikael Häggström Image:Gray72-en.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Gray72-en.svg  License: Public Domain  Contributors: w:User:MysidMysid Image:Caput femoris cortex medulla.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Caput_femoris_cortex_medulla.jpg  License: Public Domain  Contributors: User:Stevenfruitsmaak File:Bone marrow WBC.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:Bone_marrow_WBC.JPG  License: GNU Free Documentation License  Contributors: User:Bobjgalindo Image:acute leukemia-ALL.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Acute_leukemia-ALL.jpg  License: GNU Free Documentation License  Contributors: Original uploader was VashiDonsk at en.wikipedia Image:Bone marrow biopsy.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Bone_marrow_biopsy.jpg  License: Public Domain  Contributors: Photographer’s Mate 2nd Class Chad McNeeley Image:Markkloesschensuppe.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Markkloesschensuppe.jpg  License: Public Domain  Contributors: 1029man, AlexanderDreyer File:Antibody.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Antibody.svg  License: Public Domain  Contributors: User:Fvasconcellos File:Mono-und-Polymere.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Mono-und-Polymere.svg  License: Attribution  Contributors: Martin Brändli (brandlee86) File:Antibody IgG2.png  Source: http://en.wikipedia.org/w/index.php?title=File:Antibody_IgG2.png  License: Public Domain  Contributors: Original uploader was TimVickers at en.wikipedia File:Immunoglobulin basic unit.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Immunoglobulin_basic_unit.svg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: user:Y_tambe File:IgM white background.png  Source: http://en.wikipedia.org/w/index.php?title=File:IgM_white_background.png  License: GNU Free Documentation License  Contributors: Original uploader was TimVickers at en.wikipedia File:Complementarity determining regions.PNG  Source: http://en.wikipedia.org/w/index.php?title=File:Complementarity_determining_regions.PNG  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:Anypodetos File:VDJ recombination.png  Source: http://en.wikipedia.org/w/index.php?title=File:VDJ_recombination.png  License: Public Domain  Contributors: gustavocarra after File:Class switch recombination.png  Source: http://en.wikipedia.org/w/index.php?title=File:Class_switch_recombination.png  License: Public Domain  Contributors: Wong128hk File:FluorescentCells.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:FluorescentCells.jpg  License: Public Domain  Contributors: Amada44, DO11.10, Emijrp, Hannes Röst, NEON ja, Origamiemensch, Splette, Tolanor, 6 anonymous edits File:AngeloftheWest.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:AngeloftheWest.jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: Julianva (talk). Original uploader was Julianva at en.wikipedia Image:Blood cells differentiation chart.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Blood_cells_differentiation_chart.jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: パタゴニア Image:Hematopoiesis (human) diagram.png  Source: http://en.wikipedia.org/w/index.php?title=File:Hematopoiesis_(human)_diagram.png  License: GNU Free Documentation License  Contributors: User:A. Rad File:Hematopoiesis simple.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Hematopoiesis_simple.svg  License: GNU Free Documentation License  Contributors: User:Mikael Häggström File:Hematopoietic growth factors.png  Source: http://en.wikipedia.org/w/index.php?title=File:Hematopoietic_growth_factors.png  License: GNU Free Documentation License  Contributors: User:Mikael Häggström Image:Illya Mechnikov.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:Illya_Mechnikov.JPG  License: unknown  Contributors: André Koehne, Yakudza Image:New_Mixed_Myeloid-Lymphoid_Progenitor_Tree(RCCH)_Grayscale.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:New_Mixed_Myeloid-Lymphoid_Progenitor_Tree(RCCH)_Grayscale.jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:Regford Image:MLP to DN3 resized annotated-Aug 3 2010.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:MLP_to_DN3_resized_annotated-Aug_3_2010.jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:Regford Image:Illu blood cell lineage.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Illu_blood_cell_lineage.jpg  License: Public Domain  Contributors: Arcadian, Cristobal carrasco, DO11.10, MichaelFrey, 5 anonymous edits Image:Ch12f3_from_NIH.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Ch12f3_from_NIH.jpg  License: Public Domain  Contributors: Regford Image:Hematopoiesis_(human)_diagram.png  Source: http://en.wikipedia.org/w/index.php?title=File:Hematopoiesis_(human)_diagram.png  License: GNU Free Documentation License  Contributors: User:A. Rad File:Plasmacytoma ultramini1.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Plasmacytoma_ultramini1.jpg  License: GNU Free Documentation License  Contributors: User:Nephron File:Lymphoma macro.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Lymphoma_macro.jpg  License: Creative Commons Attribution 3.0  Contributors: User:Emmanuelm Image:lymphoma microarray.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Lymphoma_microarray.jpg  License: Public Domain  Contributors: Monkeybait, Quintote Image:Lymphomas, multiple myeloma world map - Death - WHO2004.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Lymphomas,_multiple_myeloma_world_map_-_Death_-_WHO2004.svg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: User:Lokal_Profil File:Cluster_of_differentiation.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Cluster_of_differentiation.svg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: User:Lokal_Profil

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Image Sources, Licenses and Contributors File:Mantle cell lymphoma - intermed mag.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Mantle_cell_lymphoma_-_intermed_mag.jpg  License: GNU Free Documentation License  Contributors: User:Nephron Image:Gray602.png  Source: http://en.wikipedia.org/w/index.php?title=File:Gray602.png  License: Public Domain  Contributors: Arcadian Image:Mantle cell lymphoma - low mag.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Mantle_cell_lymphoma_-_low_mag.jpg  License: GNU Free Documentation License  Contributors: User:Nephron Image:Mantle cell lymphoma - low mag - cyclin D1.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Mantle_cell_lymphoma_-_low_mag_-_cyclin_D1.jpg  License: GNU Free Documentation License  Contributors: User:Nephron Image:Chemotherapy with acral cooling.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Chemotherapy_with_acral_cooling.jpg  License: Creative Commons Attribution 2.0  Contributors: Jenny Mealing Image:Mechanism imatinib.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Mechanism_imatinib.svg  License: Trademarked  Contributors: Author of original image was Sodium at en.wikipedia. Image:Monoclonals.png  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonals.png  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:Adenosine Image:monoclonal antibodies3.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonal_antibodies3.jpg  License: Public Domain  Contributors: Original uploader was Quintote at en.wikipedia Image:monoclonal antibodies4.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonal_antibodies4.jpg  License: Public Domain  Contributors: Original uploader was Quintote at en.wikipedia Image:monoclonal antibodies1.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonal_antibodies1.jpg  License: Public Domain  Contributors: Monkeybait, Quintote Image:monoclonal antibodies2.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonal_antibodies2.jpg  License: Public Domain  Contributors: Monkeybait, Quintote Image:Monoclonal antibodies.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Monoclonal_antibodies.svg  License: Public Domain  Contributors: User:Ch1902 Image:Picoplancton cytometrie.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Picoplancton_cytometrie.jpg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: Daniel Vaulot, CNRS, Station Biologique de Roscoff File:FACS-toestel.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:FACS-toestel.JPG  License: Public Domain  Contributors: User:Biol File:Flow-FISH 1.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:Flow-FISH_1.JPG  License: Creative Commons Attribution 3.0  Contributors: Nathancor File:Bcrablmet.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Bcrablmet.jpg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: Achim Hering, Cohesion, DO11.10, Deadstar, Ediacara, EugeneZelenko, Pmx File:Urovysion on Duet.png  Source: http://en.wikipedia.org/w/index.php?title=File:Urovysion_on_Duet.png  License: GNU Free Documentation License  Contributors: Bioview File:FISH (Fluorescent In Situ Hybridization).jpg  Source: http://en.wikipedia.org/w/index.php?title=File:FISH_(Fluorescent_In_Situ_Hybridization).jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:MrMatze File:Bcrablinter.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Bcrablinter.jpg  License: Creative Commons Attribution-Sharealike 2.5  Contributors: Cohesion, DO11.10, 1 anonymous edits File:AutoFISH.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:AutoFISH.jpg  License: Creative Commons Attribution 3.0  Contributors: User:Vjsieben Image:FISH (technique).gif  Source: http://en.wikipedia.org/w/index.php?title=File:FISH_(technique).gif  License: Public Domain  Contributors: Thomas Ried Image:FISHchip.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:FISHchip.jpg  License: Public Domain  Contributors: Original uploader was Vjsieben at en.wikipedia Image:BacteriaFISH.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:BacteriaFISH.jpg  License: Creative Commons Attribution-Sharealike 3.0  Contributors: User:Velocity2222X File:Epigenetic mechanisms.jpg  Source: http://en.wikipedia.org/w/index.php?title=File:Epigenetic_mechanisms.jpg  License: Public Domain  Contributors: National Institute of Health Image:Nucleosome 1KX5 2.png  Source: http://en.wikipedia.org/w/index.php?title=File:Nucleosome_1KX5_2.png  License: unknown  Contributors: By Richard Wheeler (Zephyris) 2005. Image:Escherichia coli flagella TEM.png  Source: http://en.wikipedia.org/w/index.php?title=File:Escherichia_coli_flagella_TEM.png  License: unknown  Contributors: E. H. White, Content Provider: Peggy S. Hayes File:Clinical trial newspaper advertisements.JPG  Source: http://en.wikipedia.org/w/index.php?title=File:Clinical_trial_newspaper_advertisements.JPG  License: unknown  Contributors: Skier Dude, Sofia Roberts File:Yes check.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Yes_check.svg  License: Public Domain  Contributors: User:Gmaxwell, User:WarX file:Bortezomib.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Bortezomib.svg  License: Public Domain  Contributors: Benjah-bmm27, Fvasconcellos file:Bortezomib-from-PDB-2F16-3D-balls.png  Source: http://en.wikipedia.org/w/index.php?title=File:Bortezomib-from-PDB-2F16-3D-balls.png  License: Public Domain  Contributors: Ben Mills Image:2f16.png  Source: http://en.wikipedia.org/w/index.php?title=File:2f16.png  License: GNU Free Documentation License  Contributors: Opabinia regalis file:Bendamustine.png  Source: http://en.wikipedia.org/w/index.php?title=File:Bendamustine.png  License: Public Domain  Contributors: User:Edgar181 file:Temsirolimus.png  Source: http://en.wikipedia.org/w/index.php?title=File:Temsirolimus.png  License: Public Domain  Contributors: User:Edgar181 file:Ibritumomab tiuxetan structure.svg  Source: http://en.wikipedia.org/w/index.php?title=File:Ibritumomab_tiuxetan_structure.svg  License: Public Domain  Contributors: User:Anypodetos

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License

License Creative Commons Attribution-Share Alike 3.0 Unported http:/ / creativecommons. org/ licenses/ by-sa/ 3. 0/

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