Multiplex PCR for gastroenteric pathogens - CLI

Weekly news updates on www.cli-online.com | September 2013 | Volume 37

Multiplex PCR for gastroenteric pathogens

Pg.12 Compact hematology analyser

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POCT analyser

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Also in this issue : Automation system

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Interference in thyroid Gene testing for mass market Pg. 16 function tests Pg. 20

POC diagnosis of diabetes Pg. 26

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EDITOR’S LETTER

3

A breakthrough in timely ovarian cancer diagnosis? While globally ovarian cancer is the eighth most common cancer in women, in the developed countries (with the exception of Japan) the disease is much more prevalent. In Europe it is the fifth most frequently diagnosed cancer in women, with an average lifetime risk of 1 in 70, and in both Europe and North America the disease accounts for over 5% of all female cancer deaths. In addition, unlike with most other cancers, the five year survival rate of only 45% has barely improved in the last 30 years. This poor prognosis is largely due to the non-specific symptoms, resulting in diagnosis at Stage III or IV when the tumour has already metastasized. But if ovarian cancer is diagnosed early, the five year survival rate exceeds 90%. Much work in recent decades has concentrated on finding a simple screening method that would allow more timely diagnosis; so far none has had a significant effect on mortality. An assay for the most frequently used biomarker, CA125, was developed around 30 years ago. Normally elevated in the serum of patients diagnosed with symptomatic ovarian cancer, CA125 is ideal in disease management, but its use to enable early disease detection has remained controversial. Specificity is very limited as the serum level is raised in several benign conditions (such as endometriosis) as well as in other cancers. In addition sensitivity is only about 50% in patients with Stage I or II disease. More recently human epididymis protein 4 (HE4) has been advocated as a useful marker for ovarian cancer detection. Its level is not elevated as a result of benign pelvic disease so its specificity is higher than CA125, but levels of HE4 are also raised in some other cancers. Recent work on ovarian cancer screening has suggested that screening utilizing a combination of these two biomarkers

may be the best approach for early disease detection. Now exciting preliminary data from the Anderson Cancer Center have just been published. Over 4,000 women, healthy at the start of the study, were classified into three risk groups based

– September 2013

Frances Bushrod, Ph.D.

on a mathematical model- the ROCA- incorporating their age and CA125 serum level. Followup over eleven years was dependent on the evolving perceived risk. The US researchers were ‘cautiously optimistic’ about this approach, but await results from

a similar trial in the UK, involving more than 200,000 women, which will be available within two years. Hopefully, though, screening using the ROCA will lead to more timely diagnosis and thus a better survival rate for ovarian cancer patients.

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Contents FRONT COVER

Weekly news updates on www.cli-online.com | September 2013 | Volume 37

Multiplex PCR for gastroenteric pathogens

Pg.12 Compact hematology analyser

Pg.30

POCT analyser

Testing directed at specific organisms misses important gastroenteric infections. Systematically testing all fecal samples using multiplex PCR for common viral, bacterial and parasitic pathogens allows laboratories to increase diagnostic yield, improve workflow, reduce waste and turn-around times.

Pg.29

Also in this issue : Automation system

Pg.32

Gene testing for mass market Pg. 16

Interference in thyroid function tests Pg. 20

POC diagnosis of diabetes Pg. 26

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Phage-displayed peptides as novel reagents for norovirus detection

[12 - 13] Systematic multiplex PCR for the diagnosis of infectious gastroenteritis [14]

A blood test for checking stomach health

[16 - 19] GENETIC TESTING [16 - 17] Gene testing gets primed for the mass market [19]

Genetic testing news

[20 - 22] ASSAY INTERFERENCE Interference in thyroid function tests - problems and solutions

[23 - 24] TRACE ELEMENT MONITORING Trace elements and clinical chemistry

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6

Gastroenteritis

Phage-displayed peptides as novel reagents for norovirus detection Current methods for detecting noroviruses (NoVs) have significant limitations in sensitivity and feasibility for use in remote locations. Our group recently identified phage-displayed peptides with specific binding to NoVs and sensitivity comparable to that of existing antibodies. These reagents can be easily optimized by mutagenesis and represent promising diagnostic tools. by Amy M. Hurwitz, Prof. Robert L. Atmar and Prof. Timothy G. Palzkill

Norovirus infection and diagnosis Each year, norovirus (NoV) infections cause approximately 267 million new cases of gastroenteritis and 200,000 deaths worldwide [1]. Infection spreads rapidly in areas of close human contact, such as cruise ships and hospitals, and is treated only by rehydration, as no antiviral therapy currently exists. An infectious dose estimated to be as low as 18 virions and high environmental stability contributed to classification of NoVs as a category B biodefense agent in the U.S. Therefore, rapid, accurate and highly sensitive diagnosis is important for outbreak recognition and control, and also to guide physicians in patient management. The potential health and economic consequences that may be ameliorated by early NoV detection have led to a high demand for optimized detection reagents that can be used to develop reliable diagnostic assays with minimal requirements for expensive, bulky equipment or technical training. NoVs are divided into six different genogroups (GI–GVI) based on the amino

Figure 1. R-Biopharm’s RIDASCREEN® Norovirus 3rd Generation ELISA kit. (Photo credit: T. Palzkill).

acid sequence of the major capsid protein (VP1). These are organized further into more than 30 genotypes, and finally into numerous strains or variants [2]. The VP1 protein assembles to form an icosahedral shell with an inner shell (S) domain and outer protruding (P) domain. The P domain is on the virus surface and is the most accessible, while the S domain has the highest sequence conservation across different strains. Given the ability of NoVs to evolve rapidly to result in novel or recombinant strains, continual optimization of detection reagents may be necessary in order to recognize the majority of human-infecting strains. Strains classified into GI and GII are most relevant for human infections, and thus the focus for diagnostic assay development efforts.

Current diagnostic methods and their limitations Methods used currently for the diagnosis of norovirus infection are far from ideal as they exhibit several limitations that hinder their use for individual patient diagnoses or in rural and developing locations. The gold standard for diagnosis is reverse transcriptase (RT)-PCR, which requires multiple sets of primers to detect about 90% of human-infecting strains [3]. This method has significant equipment and expertise requirements, which are often not available outside of large institutions. Further, the expense of running multiple samples and the need for timely instrument accessibility limit the feasibility of applying RT-PCR as point-of-care applications or for preventing the rapid spread of an outbreak. Other existing methods include immune electron microscopy (IEM) and enzyme

immunoassays. IEM was the first method described for identifying NoVs and was used originally to classify viruses based on structural appearance. This method has limited sensitivity, and also requires expensive equipment and skilled expertise. Enzyme immunoassays, developed after the discovery of type-specific antibody epitopes on the NoV capsid, detect viral particles in human stool samples [4]. This method offers increased specificity and has led to the development of commercially available ELISA and lateral flow assays. Currently, the only FDA-approved antigen detection assay is an ELISA called RIDASCREEN® (3rd Generation) produced by R-Biopharm, which uses an antibody cocktail with specificity for GI and GII NoVs [Fig. 1]. Due to limitations in sensitivity, this assay is only approved for use during outbreaks and takes several hours to produce results. Several companies, including R-Biopharm, have developed rapid diagnostic assays that use lateral flow technology and have also demonstrated strong specificity for NoV GI and GII strains. However, these have similar limitations with sensitivity and thus are only recommended for preliminary screening to be confirmed by RT-PCR, and are distributed primarily outside of the United States [5]. Overall, there is a clear need for improved diagnostic methods to detect norovirus rapidly with strong specificity, high sensitivity, and with minimal equipment and expertise requirements.

Novel diagnostic phage reagents Recent studies in our laboratory have identified short, 12-mer peptide reagents with specific binding to the GI.1 NoV genotype [6]. The small size of these peptides displayed on phages offers the ability to access epitopes that may be buried in the capsid protein and not accessible to antibodies, and the potential for increased avidity through multiple linked peptide molecules. To identify peptides with specific binding to NoV, we used phage display technology to screen commercially available, large-scale libraries of randomized peptides that are fused to the gene III

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protein and expressed in five copies on one end of the phage. Rounds of biopanning were performed in which filamentous phage libraries were screened for phages displaying peptides that bind immobilized Norwalk (NV) GI.1 viruslike particles (VLPs). The phage libraries were added to VLPs and, after washing away non-binding phages, the phages displaying VLP-binding peptides were eluted with low pH [Fig. 2A]. Two to four subsequent rounds of biopanning using the resulting phage populations enriched for phages displaying peptides with the highest binding affinity for NV. DNA sequencing of individual phage clones recovered after multiple rounds of biopanning revealed three peptides, named NV-O-R5-3, NV-O-R5-6, and NV-N-R5-1, that occurred most commonly, and the phage clones displaying the peptides were further characterized for their NoV binding properties [6].

8

Gastroenteritis

Phage-based ELISAs confirmed the binding specificity of phage-displayed peptides to NV VLPs. These affinitybinding assays used NV VLP captured by immobilized rabbit polyclonal antiNV antibody in order to maintain the structural integrity of VLPs. Single phage clones were added to the captured VLPs and binding was detected using anti-M13 phage antibody that was conjugated to horseradish peroxidase to provide a signal for bound antibody [Fig. 2B]. Of the three peptide-displaying phage clones analysed, NV-N-R5-1 exhibited a dosedependent response with decreasing NV VLP concentration and the highest sensitivity with a limit of detection at 1.56 ng NV VLP. Additional phage ELISAs indicated that NV-N-R5-1 binds to the P domain of the capsid protein, which extends the furthest out from the virus, and has comparable sensitivity for NV as existing antibodies used for diagnostics

[6]. These results provide proof-of-concept and a strong lead reagent for developing novel phages displaying peptides as effective detection reagents for NoV. Further, the methods described establish a platform methodology for using phage display to identify antigen-specific binding reagents that may be applied to any pathogen with distinct surface epitopes.

Current status To develop our lead phage-displayed peptide into a commercially viable tool, we are currently optimizing its binding affinity for other genogroups of NoV in order to broaden its diagnostic applications. Phage display technology provides a simple platform for constructing collections of new mutations in a lead peptide that can be used for additional rounds of biopanning to screen for variants with optimal affinity properties [Fig. 2C]. The three phage-displayed peptides discussed above share conserved amino acid sequence motifs that likely confer binding specificity for particular epitopes on the NV capsid protein. Directed evolution through mutagenesis of amino acids surrounding these consensus sequences can enable us to improve binding affinity to NV and alter binding specificities starting with the lead phage peptide, NVN-R5-1. In particular, developing phagedisplayed peptides with optimized binding affinity for the NoV GII.4 genotype, which accounts for >80% of NoV infections worldwide [1], and other GI and GII NoV genotypes will have the greatest relevance for diagnostic applications.

Future development of bacteriophage reagents

Figure 2. Development of phage-based affinity reagents begins with the identification of a lead phage through biopanning against an antigen (A). Phage-based ELISA assays characterize the binding affinity of a lead phage to the antigen target (B). Optimization occurs through directed evolution of a lead phage by random or directed mutagenesis, screening against the antigen target, and characterizing binding until the optimal binding affinity is achieved (C). An optimized phage reagent can be produced in scaled quantities for use in diagnostic assays (D). As antigens evolve, the process may be repeated with a new or modified biopanning target.

For decades, phages have been used to identify their target bacterial strains and species in order to diagnose the cause of infections by phage typing. More recent applications have begun to leverage synthetic biology and genomic engineering strategies to customize phage specificity and reporter signals to enable ‘nearreal-time’ detection of a broader range of human pathogens [7]. Our recent work has established a methodology for the identification, characterization, and development of phage-based affinity reagents that may be applied to different pathogens and translated into diagnostic applications. The process outlined in Figure 2 demonstrates the progression from (A) identifying lead reagents against a target of interest, (B) characterizing binding affinity for the antigenic target, (C) optimizing leads through directed evolution

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Gastroenteritis

or genomic engineering strategies, and finally (D) producing scalable quantities of reagent for commercial diagnostic applications. Zou and colleagues, for example, used a similar method to identify a phage-displayed peptide reagent with specific binding to transmittable gastroenteritis virus (TGEV) that also showed potential antiviral activity [8]. Several groups have also developed phage-based reagents to detect bacterial pathogens, such as Salmonella enterica and Escherichia coli [9, 10]. In summary, the use of phage-based reagents for microbial diagnostics offers many advantages in comparison to more commonly used detection reagents, such as antibodies. Phage display technology enables rapid identification and validation of candidate phage reagents with specificity for new or evolved pathogens through biopanning of commercial or custom made phage libraries (Fig. 2A, B). Phage manipulation through directed evolution facilitates development of reagents with optimized binding affinity and specificity to a target of interest (Fig. 2B). Finally, production of large quantities of phages is accomplished rapidly and inexpensively, as simple preparation methods can produce sufficient phage for hundreds of assays

(Fig. 2D). As viral pathogens such as NoV continually evolve, the flexibility provided by phage-based reagents will be essential for developing next generation diagnostics for effective containment of outbreaks. A cocktail of phages, each of which binds to a specific target NoV genotype, may ultimately be the ideal strategy for producing an assay to detect the broadest possible range of NoVs without sacrificing specificity. Overall, phages have an enormous potential for use as detection reagents in clinical, agricultural, food, and environmental settings, and represent an underutilized resource for diagnostic development.

References 1. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunological Reviews 2008; 225(1): 190–211. 2. Kroneman A, Vega E, Vennema H, Vinjé J, White P, Hansman G, Green K, Martella V, Katayama K, Koopmans M. Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology 2013; doi:10.1007/s00705-013-1708-5. 3. Atmar RL, Estes MK. The epidemiologic and clinical importance of norovirus infection. Gastroenterology Clinics of North America 2006; 35(2): 275–290. 4. Parker TD, Kitamoto N, Tanaka T, Hutson AM, Estes, MK. Identification of Genogroup I and Genogroup II broadly reactive epitopes on the norovirus capsid. Journal of Virology 2005; 79(12): 7402–7409. 5. Ambert-Balay K, Pothier P. Evaluation of 4 immunochromatographic tests for rapid detection of norovirus in faecal samples. Journal of Clinical Virology 2013; 56(3): 194–198. 6. Rogers JD, Ajami NJ, Fryszczyn BG, Estes MK, Atmar RL, Palzkill TG. Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples. Journal of Clinical Microbiology 2013; 51(6): 1803–1808. 7. Lu TK, Bowers J, Koeris MS. Advancing bacteriophage-based microbial diagnostics with synthetic biology. Trends in Biotechnology 2013; 31(6): 325–327. 8. Zou H, Zarlenga DS, Sestak K, Suo S, Ren X. Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential. Antiviral Research 99(3): 383–390. 9. Schofield DA, Sharp NJ, Westwater C. Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage 2012; 2(2): 105–283. 10. Galikowska E, Kunikowska D, Tokarska-Pietrzak E, Dziadziuszko H, Loś JM, Golec P, Węgrzyn G, Loś M. Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents. European Journal of Clinical microbiology & Infectious Diseases 2011; 30(9): 1067–1073.

The authors Amy M. Hurwitz1 BS, Robert L. Atmar2,3 MD, Timothy G. Palzkill*2,4 PhD 1. Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA 2. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA 3. Department of Medicine, Baylor College of Medicine, Houston, Texas, USA 4. Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA *Corresponding author E-mail: [email protected]

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Gastroenteritis

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– September 2013

Systematic multiplex PCR for the diagnosis of infectious gastroenteritis Current methods for the detection of gastroenteric pathogens are insensitive, slow, and laborious. Testing directed at specific organisms misses important infections. Systematically testing all fecal samples using multiplex PCR for common viral, bacterial and parasitic pathogens allows laboratories to increase diagnostic yield, improve workflow, reduce waste and turn-around times. by Dr Gary McAuliffe

Introduction Within a single diagnostic laboratory, multiple methods are used to detect gastroenteric pathogens. Selective agar plates differentiate bacterial pathogens, whereas immunoassays are used to detect viruses and parasites such as Giardia lamblia and Cryptosporidium spp.. Laboratories perform microscopy with special stains for the detection of Entamoeba histolytica and Dientamoeba fragilis. PCR has generally been restricted to the detection of norovirus, but many studies have demonstrated its potential for the detection of other enteric pathogens. Multiplex PCR (M-PCR) panels have been shown to enhance detection of gastroenteric organisms. These panels combine several enteric pathogen targets in one or more PCR reaction vessel(s). O’ Leary et al. demonstrated 100% sensitivity compared with culture for the detection of four bacterial pathogens [1]. Wolffs et al. used a panel targeting seven viral pathogens and detected a pathogen in 97% of samples compared with 49% by conventional methods [2]. Stark et al. showed 100% sensitivity and specificity of a M-PCR panel targeting four parasites, which also reliably differentiates E. histolytica from non-pathogenic E. dispar and E. moshkovskii [3]. de Boer et al. successfully replaced bacterial culture at their institution with a molecular COMPANY

screening approach targeting four bacteria and G. lamblia [4]. Several commercial M-PCR fecal panels are available [Table 1]. These may be directed against parasites, viruses or bacteria, or contain targets from all three groups of organisms, allowing systematic testing of stool samples for all common gastrointestinal pathogens. In the author’s study, 1758 samples from community and hospital patients were tested using the Fast-Track Diagnostics bacterial, viral and parasite M-PCR panels. Pathogens were detected in 30% of samples by this systematic M-PCR approach compared with 18% using conventional testing as directed by clinician request [5][Table 2].

Advantages of a systematic M-PCR approach Studies have demonstrated enhanced detection of entero-hemorrhagic E. coli (EHEC), Clostridium difficile, G. lamblia, E. histolytica, norovirus, adenovirus and rotavirus by PCR compared with immunoassays and other conventional assays [2–4, 6, 7]. Bacterial PCR has generally performed comparably with culture, with the exception of Salmonella spp., where reduced detection by PCR has been demonstrated in several, but not all, studies [4, 5, 8]. An advantage of increased sensitivity is that

BACTERIAL

VIRAL

PARASITE

COMBINED

AUSDIAGNOSTICS

X

X

X

X

ENTERICBIO (SEROSEP)

X

FAST-TRACK DIAGNOSTICS

X

X

X

SEEPLEX (SEEGENE)

X

X

XTAG (LUMINEX)

X

X

Table 1. Examples of commercial multiplex PCR panels.

X

X

smaller amounts of feces are required for testing, and multiple samples are not required for the detection of common parasites. Clinicians should be aware that organisms such as adenovirus and norovirus can be shed in feces for several weeks following infection and that PCR detects low levels of these organisms which may not be clinically relevant. Selective bacterial media lack specificity, requiring time and further testing to discount commensal organisms. E. histolytica cysts cannot be reliably differentiated from other members of the Entamoeba complex by microscopy and staining. M-PCR generally overcomes these issues, though specificity depends on the target sequence chosen. For adenovirus, some panels target the hexon gene which does not differentiate between enteric and non-enteric serotypes, whereas others target sequences specific to enteric sub-types. Currently laboratories restrict their testing to a limited range of pathogens for which they have sensitive and affordable assays available. A number of organisms that laboratories do not commonly test for, such as astrovirus, sapovirus, Vibrio spp., and nonO157 EHEC, can be missed. M-PCR allows a wider range of organisms to be targeted. In the author’s study astrovirus was found to be the second most common cause of gastroenteritis, and non-O157 EHECs were detected in sixteen samples by targeting the stx genes. Our laboratories did not have assays for these organisms prior to the study. Laboratories may design their own panels to reflect locally and internationally important pathogens, or buy commercial panels which reflect these requirements. Rare or imported infections not targeted by the panels will not be detected, and laboratories need to decide when additional tests are required. Up to five targets may be tested in a single real-time PCR reaction vessel; therefore increasing the number of targets reduces the number of samples on a given PCR amplification run. The xTAG system (Luminex Corp.) overcomes this limitation by post-amplification analysis using microspheres with up to 100 differing spectra. Eleven targets are currently included in the xTAG gastroenteritis panel within a single PCR reaction vessel [9]. TaqMan array cards (Life technologies)

13 also overcome this restriction by allowing simultaneous real-time PCR in 384 PCR reaction wells. This platform allows up to eight samples to be run in parallel [10]. Fecal samples are usually tested for a limited range of viruses, bacteria, or parasites dependent upon clinician request and laboratory algorithms. Studies have shown that important pathogens such as G. lamblia and EHEC are missed using this approach. Testing for enteric viruses is not widely employed outside hospitals despite their prevalence. The M-PCR approach tests every sample for every target in the panels. It is less reliant on clinicians’ knowledge of the organisms that the patient has likely been exposed to, or those that may be causing the patients clinical syndrome. This systematic approach accounted for the majority of the increased diagnostic yield in the author’s study. In our laboratory it can take 3–4 days for the identification of Salmonella spp. by culture. Time to generation of results is significantly reduced with the M-PCR approach. In the author’s study, samples collected were batch tested the following day, giving results for eleven pathogens simultaneously within 24 hours of collection. Handson time is also reduced. The preparation and reading of a trichrome stain can take up to 40 minutes by an experienced operator, whereas the hands-on time for testing a sample by PCR is a quarter of this. The workflow created by the systematic M-PCR approach fits well with current ORGANISM

% SAMPLES POSITIVE CONVENTIONAL

M-PCR

4.8

4.8

C.DIFFICILE

0.9

4.0

EHEC

0.3

1.2

SALMONELLA SPP.

1.8

1.3

SHIGELLA SPP.

0

0

YERSINIA SPP.

0.1

0

ADENOVIRUS

0

2.9

ASTROVIRUS

-

5.4

NOROVIRUS GI/GII

2.1

9.7

ROTAVIRUS

1.4

0.2

0.1

0.2

E. HISTOLYTICA

0.1

0.1

G. LAMBLIA

1.3

3.4

laboratory systems; testing is performed in a single pathway rather than by several laboratory departments. Conventional fecal testing employs multiple diagnostic kits and selective media, some of which come with significant cost, short expiry times, and extensive quality control requirements. In our laboratory stool samples for bacterial culture are inoculated onto seven agar plates which need to be stored, processed, and incubated, generating a significant amount of waste. O’ Leary et al. reported that M-PCR significantly reduced this wastage [1]. M-PCR does not obviate the need for bacterial culture as it does not offer an antibiogram, but it allows focused testing of samples found to be positive for these bacterial targets. Pathogens such as Shigella spp, G. lamblia and D. fragilis are labile in stool. Delays may occur in transportation, inoculation or examination which can compromise yield. For M-PCR less initial processing is required. Specimens are inoculated into tubes containing Stool Transport and Recovery buffer (STAR) (Roche Diagnostics) which binds inhibitors, and stabilizes nucleic acids for later processing. PCR is also able to detect organisms rendered non-viable by inadequate transport, or the use of antibiotics. Feces contains high levels of bilirubin and bile salts, which can lead to inhibition of PCR amplification. Inoculating samples into STAR buffer on arrival, and using extraction methods such as the EasyMag platform (Biomerieux) can help overcome this issue. In the author’s study 1.7% of samples exhibited inhibition, but all gave adequate internal control amplification following dilution and repeat testing.

BACTERIA CAMPYLOBACTER SPP.

VIRUSES

PARASITES CRYPTOSPORIDIUM SPP.

Table 2. Pathogen detection by conventional and systematic M-PCR methods.

Cost is a major factor preventing systematic testing of fecal samples by conventional techniques in diagnostic laboratories. The costs of PCR are reducing relative to conventional tests, and batch testing of samples using the systematic M-PCR approach is becoming a viable option. In the author’s study testing a sample for bacteria, viruses and parasites was significantly cheaper by M-PCR than using equivalent conventional tests [NZ$152 (£75) versus NZ$280 (£140)]. Laboratories have successfully reported replacing their conventional methods with a molecular screening approach for bacteria [1, 4] or viruses [2]. Where the cost of replacing all traditional diagnostics with systematic testing may currently remain restrictive, laboratories may choose to replace testing for organism groups, e.g. viruses, and institute systematic testing at a later date.

– September 2013

Conclusion Current conventional methods for the detection of enteric pathogens are labour intensive, insensitive, slow, and applied piecemeal to submitted fecal samples. Testing stool samples using multiplex PCR panels which simultaneously detect all common bacteria, viruses and parasites increases the detection of gastroenteric pathogens. This approach improves turnaround time, workflow, reduces labour and waste. Costs are reducing, making systematic M-PCR testing an attractive alternative to currently used techniques.

References 1. O’Leary J, et al. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol. 2009; 47: 3449–3453. 2. Wolffs PF, et al. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. J Clin Microbiol. 2011; 49: 1926–1931. 3. Stark D, et al. Evaluation of multiplex tandem realtime PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol. 2011; 49: 257–262. 4. de Boer RF, et al. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol. 2010; 48: 4140–4146. 5. McAuliffe GN, et al. Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples. J Infect. 2013; 67(2): 122–129. 6. Costantini V, et al. Diagnostic accuracy and analytical sensitivity of IDEIA norovirus assay for routine screening of human norovirus. J Clin Microbiol. 2010; 48: 2770–2778. 7. Luna RA, et al. Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children’s hospital. J Clin Microbiol. 2011; 49: 851–857. 8. Cunningham SA, et al. Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture. J Clin Microbiol. 2010; 48:2929–2933. 9. Coste JF, et al. Microbiological diagnosis of severe diarrhea in kidney transplant recipients by use of multiplex PCR assays. J Clin Microbiol. 2013; 51(6): 1841–1849. 10. Liu J, et al. A laboratory developed TaqMan array card for simultaneous detection of nineteen enteropathogens. J Clin Microbiol. 2013; 51(2): 472–480.

The author Gary McAuliffe MBBS Microbiology Department, LabPlus Laboratory, Auckland, New Zealand E-mail: [email protected]

– September 2013

14

Gastroenteritis

A blood test for checking stomach health Helicobacter pylori (Hp) -infection and atrophic gastritis (AG) are the most important risk conditions preceding gastric cancer (GC). Following extensive research and development, a Finnish biotechnology company, Biohit Oyj, has launched the GastroPanel test, a panel of four stomach-specific biomarkers that give accurate information on both the structure and function of gastric mucosa. by Dr Kari Syrjänen

Since the risk of GC and peptic ulcer disease among individuals with healthy stomach is very low, it is essential to distinguish between subjects with healthy stomach and those with gastric disorders. With GastroPanel - a simple blood test - it is now possible to detect the patients who are at high risk for GC because they harbour either Hp -infection, AG or both in their stomach mucosa. Hp -infection alone increases the risk of GC several-fold, and this risk is over 90-fold among patients with Hp -related severe AG of both the corpus and antrum (pangastritis)[1, 3]. Another area of use for the GP test are the dyspeptic complaints, which in western countries appear in 20-40% of the population. According to most current medical practices, the assessment of these complaints should invariably include a gastroscopic examination for which the existing resources are clearly insufficient and which is actually not really necessary. The same applies to the costly and risky “test” medications with

proton-pump inhibitors (PPIs), since it is now possible to screen the patients at true risk and for whom gastroscopy is indicated by using the GastroPanel test. With this approach, approximately 40-70% of the limited and expensive endoscopy capacity can be released for colonoscopies, i.e. for screening and early detection of colorectal cancer. Because of the fact that, particularly among the elderly, dyspeptic complaints are frequently of large intestinal origin, it is cost-effective to supplement the examinations of these dyspeptic patients with colorectal screening methods like the ColonView test, a stool based detection of Hb and Hb/Hp complex, or colonoscopy. The safe and cost-effective GastroPanel test enables early detection of many different disorders, and thus helps avoiding the majority of subsequent health problems. Besides gastric and esophageal cancer, undetected AG of the corpus (acid-free stomach) can eventually also lead to malabsorption of vitamin B12, iron, magnesium, calcium,

and some drugs. AG of the antrum, in turn, increases the risk of peptic ulcer disease and GC. Concomitant AG of the antrum and corpus (pangastritis) is the single most important risk condition for GC. A minority of GCs can develop directly from HP-induced gastritis, without recognizable stages of mucosal atrophy. It is well known that vitamin B12 deficiency can lead to pernicious anemia (PA), dementia, depression, and injuries of the peripheral nervous system. Calcium deficiency, in turn, leads to osteoporosis. The absorption of many drugs is impaired in acid-free stomach. The risk of serious intestinal infections (giardiasis, malaria, Clostridium difficile and E. coli EHEC) can be increased particularly among senior citizens with AG. Within public healthcare, it is possible to achieve substantial cost savings by replacing the systematic use of gastroscopy with a simple and inexpensive first-line diagnostic tool like the GastroPanel test for all patients with dyspeptic symptoms.

References 1. Suovaniemi O. GastroPanel-tutkimus osaksi dyspepsian hoitokäytäntöä. Yleislääkäri 2007; 4:104-106. 2. Malfertheiner P, Mégraud F, O’Morain C ym. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007; 56:772-781. 3. Agreus L, Kuipers EJ, Kupcinskas L, Malfertheiner P, Di Mario F, Leja M, Mahachai V, Yaron N, van Oijen M, Perez Perez G, Rugge M, Ronkainen J, Salaspuro M, Sipponen P, Sugano K, Sung J. Rationale in diagnosis and screening of atrophic gastritis with stomach-specific plasma biomarkers. Scand J Gastroenterol 2012; 47:136-147.

The authors Prof Kari Syrjänen,* MD, PhD, FIAC, Chief Medical Director, Biohit Oyj. Lea Paloheimo PhD Director of Business Development and Quality, EurClinChem,

The GastroPanel test kit.

*Corresponding author E-mail: [email protected]

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– September 2013

16

Genetic testing

Gene testing gets primed for the mass market Questions about gene testing were highlighted dramatically this summer after Hollywood superstar Angelina Jolie announced she had undergone a preventive double mastectomy. The reason: gene tests showed she carried the breast cancer-linked BRCA1 mutation. In an Op-Ed piece in the ‘New York Times’, the actress encouraged other women, who believed they were also at risk, to also get tested. Ms. Jolie’s decision has been hailed by some, criticized by others. However, it may well mark a watershed, when gene testing began a paradigm shift to the mass market. Her announcement, for example, led to a doubling of cancer checks at top clinics in London.

US Patent ruling will bring costs down Such trends are likely to be reinforced, strongly, by a US Supreme Court ruling in June 2013 (shortly after Ms. Jolie’s announcement) that human genes cannot be patented. The decision reversed three decades of US intellectual property case law, and within days, several US labs announced they would be offering BRCA tests. The latter could previously only be tested for by a single company, Myriad Genetics. Though patent laws are national matters, it is likely that the US court ruling will make an impact elsewhere. In Europe, the EU Biotech Directive allows patenting of gene tests, while Myriad itself recently won a Federal Court ruling in Australia upholding its BRCA patents. Revenues from genetic screening were $5.9 billion in the US in 2011, according to a study by the respected Battelle Institute. To put the figure in perspective, this is about 10% of the total US clinical testing market. Globally, sales of genetic tests could be

Genetic testing has so far largely been restricted to specialist labs and top academic medical centres.

conservatively estimated at $10-$15 billion. Scores of vendors already offer a range of tests – from selective screening for some hundred-odd major disease genes to complete sequencing of a person’s genome. The once-prohibitive costs of gene tests have seen downwards pressure over the past decade. As with other consumer technology cycles, lower prices are expected to drive an expansion in affordability, in users and revenues, in a virtuous cycle. One of the key market catalysts has been direct-to-consumer testing (DTC) companies. US DTC leader 23AndMe has seen its gene tests used by about 200,000 consumers. For just $99, the company provides information on 50 carrier traits, 20 drug classes and disease risk information. 23AndMe is currently seeking FDA certification. European firms are less visible. A leading vendor, deCode Genetics, shut down its DTC service after being acquired by Amgen in late 2012. The Iceland-based firm had been offering its deCodeme personal genomic scanning service for just under $1,000, as well as screening for cardiovascular diseases and common cancers – in a package for $350. Other major DTC players in Europe are also from the US, among them Navigenics, DNADirect and Genelex. Price falls are now almost certain to accelerate after the US Supreme Court decision on gene patents. Myriad, for example, was using its monopoly on BRAC to charge $3,000 and more for a test. After the Court ruling, the test is projected to see a steep fall in its price to just $100.

Drivers of consumer tests The key reason for the growth of DTC is that genetic testing has so far largely been restricted to specialist labs and top academic medical centres. In spite of a sharp rise in the number of registered tests to over 7,500, most have yet to be translated into clinical applications. A study by United Health, the US managed health group, found 63% of physicians saying that screening provided them “the ability to diagnose conditions that would otherwise be unknown.” However, a larger number, about three of four, also noted there were patients in their practices “who would benefit from a genetic test but have not yet had one.” United Health estimates that the US testing market alone would reach about $15 to $21 billion by 2021. In Europe too, an increase in formal healthcare settings for gene testing is likely to be welcomed, given growing concerns about DTC. A recent survey of clinical geneticists found 84% of respondents expressing concern about “replacing face-to-face supervision by a medical doctor with supervision via telephone” through DTC testing firms. A little less than half the respondents said they had at least one patient make contact with them after they had undergone a DTC genetic test, and 86% said they would provide post-test counselling to such patients. The survey posed the likelihood of a ‘cascade effect’ in the future, particularly should physicians spend more time on patients with DTC test results that are not medical priorities. As a result, it seems market growth will be accompanied by the encouragement of general hospitals and physician practices to do gene testing.

The emergence of personal medicine The impact of mass gene testing will clearly be enormous. One new frontier is personal medicine, where medicine choice and dosages would be prescribed according to a patient’s specific genetic profile. Further down the horizon may be an end to several inherited diseases. In January 2009, the UK saw the birth of the first baby “tested preconceptionally for a genetic form of breast cancer.” The baby was born at University College London (UCL) Hospital, using Preimplantation Genetic Diagnosis,

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which involves undertaking an in vitro fertilization treatment cycle to have several embryos available for genetic tests. More recently, UCL announced that its scientists had developed a microchip test to analyse 35 different genetic mutations linked to cancer, and enable doctors to identify and target specific genes from a small sample of tissue. UCL Professor Charles Swanton said the test marked the beginning of tailored cancer care in the NHS.

Ethical questions remain Nevertheless, there is some way to go. One barrier consists of still-lingering questions about the ethical implications of gene tests. Here, the first issue is uncertainty. Even now, gene testing (including that for the high-profile BRCA 1 and 2) only predicts an increase in risk, not certainty of disease. This transfers the choice and responsibility for an irreversible prophylactic intervention to a patient, and to his or her best guess. It also rules out the possibility of effective, new and less-invasive surgical interventions emerging in the future. Such technology evolution challenges – of better choices becoming available – apply broadly to all genetic testing. Some tests do not (as yet) identify all possible gene mutations which lead to a particular disease, or have only limited predictive value. Finally, it remains unclear whether a mutation is not just a symptom of a disease, rather than being a cause. For example, in cystic fibrosis (CF), there is still no way to predict disease severity, even when a fetus has inherited two mutations. Parents thus face the dilemma of deciding whether to continue or end a pregnancy without full knowledge. In the meanwhile, even as data on CF mutations grows steadily by the year, promising new drug therapies are becoming available. For example, Ivacaftor (Vertex Pharmaceuticals), which addresses the G551D mutation affecting 4% of CF patients, is now being evaluated for the more prevalent F508del mutation. The above dilemmas are aggravated by the question of false positives and false negatives. In spite of being at the cutting edge of mass screening techniques for Down’s syndrome and neural tube defects, Quad tests for pregnant women still retain a 5% false positive and 20% false negative rate. Elsewhere, while metabolic genetic

It remains unclear whether a mutation is not just a symptom of a disease, rather than being a cause.

disorders such as phenylketonuria can be identified by fetal gene tests and then addressed by dietary changes, many others lack treatment options. The broader debate on gene tests and its ethics is unlikely to go away soon, but policy makers are broadly swinging to accept its inevitability. The Human Genetics Commission in Britain stated in April 2011 that there were “no ethical barriers preventing the use of genetic testing in couples before they conceive.” Within months, the German parliament enacted a law to allow testing fertilized embryos for possible life-threatening genetic defects, via Preimplantation Genetic Diagnosis (like that launched by University College London in early 2009). Critics in Germany have been especially vociferous, calling the move “a step toward designer babies.” One of the biggest concerns about genetic testing is the emergence of ‘a la carte’

health insurance, providing choice of cover and premium based on a person’s particular disease risks and (eventual) treatment requirements, rather than loading the highest-risk beneficiaries atop the lower-risk ones. In the US, resulting concerns about discrimination due to genetic testing led to the 2008 Genetic Information Nondiscrimination Act (GINA), which bars denial of health insurance or employment because of a genetic predisposition to a particular disease. In Europe, different laws and regulations in the Member States seek to address ethical questions. A major hurdle here is the lack of an “approved definition of a genetic test,” in spite of the EU-funded project EuroGentest. One of the latter’s goals was to “try to develop at least some key elements for a working definition” of a gene test.

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GENETIC TESTING NEWS Successful beta trial results for methylation status detection kit DNA methylation status plays an important role in an individual’s disease risk and likely treatment outcome. As such it is also a necessary part of the assessments required to deliver complete personalized medicine. Now, having completed a successful beta trial, the TrueMethyl kit from Cambridge Epigenetix is a step nearer to the market. As a key part of the product validation process, 13 leading epigenetics labs were provided with the kits for independent and rigorous testing using a shared panel of control samples. The trial results demonstrate that the kit delivers a reliable and consistently high performance. The oxidative bisulfite sequencing (oxBS-Seq) technology allows quantitative, single-base resolution sequencing of the modified bases hydroxymethyl cytosine (5-hmC) and methylcytosine (5-mC), enabling accurate analysis of the DNA methylome. The kit can be used with a variety of common platforms including next generation sequencing systems, methylation arrays, and targeted assays. www.cambridge-epigenetix.com

A genetic test for autism spectrum disorders?

Autism spectrum disorders (ASD) are an increasingly diagnosed group of neurodevelopmental disorders. Although heritability suggests a strong genetic component, efforts to identify genes involved have had disappointing results, and the difference in disease state between identical (monozygotic) twins points to a potential role for epigenetic factors. Two new studies have found a significant correlation between DNA methylation (DNAm) patterns and ASD traits. Wong et al. performed a genome-wide analysis of DNAm in a sample of 50 monozygotic twin pairs sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype [1]. Numerous differentially methylated regions associated with ASD were identified and

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significant correlations between DNAm and quantitatively measured autistic trait scores were reported. Ladd-Acosta et al. examined DNAm in post-mortem brain tissue from 19 autism cases and 21 unrelated controls. Over 485 000 CpG loci were measured across a diverse set of functionally relevant genomic regions and four genome-wide significant differentially methylated regions were identified [2]. 1. Wong et al. Mol Psychiatry 2013; doi: 10.1038/ mp.2013.114 (www.nature.com/mp/journal/vaop/ ncurrent/full/mp201341a.html). 2. Ladd-Acosta et al. Mol Psychiatry 2013; doi: 10.1038/mp.2013.114 (www.nature.com/mp/jour-

– September 2013

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nal/vaop/ncurrent/full/mp2013114a.htm).

Genetic diagnosis of dementia by next-generation sequencing Early diagnosis of dementia is essential for the instigation of the best treatment regime. This is, however, notoriously difficult, as changes begin occurring many years before any symptoms may be apparent. Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. In this study, the authors assessed the use of next-generation sequencing (NGS) technologies as a quick, accurate and cost effective method for determining a genetic diagnosis in EOD. Gene panel-based technologies were developed to assess 16 genes known to contain dementia-causing mutations and were combined with PCR-based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP, the prion protein gene. In a blinded study of 95 samples, very high sensitivity and specificity were shown to be achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in the amyloid precursor protein, APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. Gene panels, such as this one from the Medical Research Council, UK, and similar technologies are likely to transform the diagnosis of early onset dementia diagnosis, significantly impacting the proportion of patients in whom a genetic cause is identified.

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– September 2013

20

Assay interference

Interference in thyroid function tests – problems and solutions Interference in immunoassay is a well described phenomenon and all clinical immunoassays, including thyroid function tests, are potentially at risk. Spurious results can lead to over investigation or mismanagement if not detected, but a proactive approach by the laboratory will help to identify and resolve these problems. by Dr Olivia Bacon and Dr David J. Halsall

Background Thyroid disorders are relatively common, and are associated with long-term morbidity and mortality. Clinical signs and symptoms are often non-specific, so reliable laboratory tests are critical for diagnosis. Therefore, thyroid function tests (TFTs) are frequently requested immunoassays with around 10 million results being reported each year by UK laboratories. In the UK, TFTs typically include a high sensitivity immunoassay for thyroid stimulating hormone (TSH) with an immunoassay estimation of nonprotein bound thyroxine (fT4), either run simultaneously or added if the TSH value is outside the reference interval [1]. For the majority of tests, both results will be within the reference interval and thyroid disease can be excluded. In some patients TFTs support the diagnosis of hypothyroidism (raised TSH with fT4 low, or lownormal) or hyperthyroidism (TSH undetectable, and fT4 elevated), and

these results will confirm clinical findings. However, due to the high volume of TFTs performed, it is not unusual for the laboratorian to be faced with a set of TFTs that are either internally inconsistent, or incompatible with the clinical details provided. Many medications can affect the thyroid axis, as can other non-thyroidal pathologies; these are often transient, but can cause unusual patterns of TFT. Much rarer genetic or pituitary conditions can also cause discordant TFTs [2]. However, if drug effects are excluded, it is necessary at this stage for the laboratorian to consider that one of the TFT results is incorrect, as analytic error is at least as common as these rare thyroid conditions. As spurious TFT results can lead to over investigation, or even inappropriate treatment, it is critical, but not trivial, for the laboratory to confirm the analytical validity of the TFT results. In one study of more than 5000 samples received for TSH analysis, assay inter-

Figure 1: Architecture of two-site immunoassays and mechanisms of possible interference by endogenous antibodies. (A) In the presence of analyte, capture and detection antibodies are crosslinked and signal is generated. (B) In the absence of analyte, circulating antibodies may cross-link the reagents, generating a false positive signal. (C) Despite the presence of analyte, circulating antibodies may prevent cross-linking and signal generation, leading to a false negative result.

ference with the potential to adversely affect clinical care was detected in approximately 0.5% of patients [3]. This equates to a rather alarming 50,000 tests per annum in the UK. Although assay design is continually improving, no routine immunoassay is currently robust to interference. Technical errors with many routine chemistry methods caused by inappropriate sample collection or handling, chemical or spectral interference can be detected during result validation. However, detection of spurious TFT immunoassay results is more challenging as there is no automatic ‘flag’ from the analyser, and there is usually a wide range of plausible values for these analytes, making it difficult to question those which are ‘suspicious’. Consequently clinical validation, where results are checked for discordance with the clinical correlates and other laboratory tests, is used to detect potentially incorrect results before reporting. For TFTs this is aided by the characteristic reciprocal relationship between TSH and fT4 in patients with an intact pituitary–thyroid axis.

Mechanisms of interference in TSH assays Endogenous interfering antibodies are a well described cause of immunoassay interference [4]. In TSH assays these antibodies can have affinity for TSH itself or towards assay components. Anti-reagent antibodies can be ‘anti-animal’ antibodies, specific to the species in which the reagent antibody was raised, or weak, polyspecific ‘heterophilic’ antibodies, which may be part of the natural process of the generation of antibody diversity [5]. Anti-animal antibodies are more prevalent in animal handlers or patients treated with therapeutics based on animal immunoglobulins. Anti-reagent antibodies can interact with either the capture or detection antibodies in two-site assays, blocking the generation of signal in the presence of analyte (false negative result) or by causing antibody cross-linking in the absence of analyte (false positive result) [Fig. 1]. Anti-TSH antibodies can generate high molecular weight TSH : antibody

21 complexes (‘macro-TSH’). Depending on the exact site of the antibody–analyte interaction, false positive TSH results may occur as the macro-TSH is unlikely to be biologically active [6].

Detection of interference in TSH assays Once suspected, a robust laboratory strategy is required for confirming or excluding assay interference. Method comparison using an alternative method is often used as the first step. Most laboratories use two-site immunoassays for TSH, but assay formulations, antibody species and incubation times vary between manufacturers. Varying amounts of blocking agents, designed to prevent non-specific binding of heterophile antibodies, may be included. Significant disagreement between two TSH methods is a strong indicator of assay interference.

to dilution and immunosubtraction, and correlates with fT4 results plus clinical findings, can be used with a reasonable degree of confidence.

Mechanisms of interference in fT4 assays fT4 assays present a particular analytical challenge as >99.9% of T4 in the serum is protein bound, and the unbound T4 fraction must

– September 2013

be measured without upsetting the equilibrium between the two fractions [8]. Therefore, an abnormal T4 binding protein, or agent which affects binding protein affinity in vitro, has the potential to generate incorrect results. Most commercial fT4 assays are one-site immunoassays based on competitive principles, using either labelled T4 analogue or anti-T4 antibodies for detection. Both hetero-

phile and anti-T4 antibodies therefore also have the potential to interfere with these methods [4]. Non-esterified fatty acids (NEFAs) are a common T4 displacing agent as they can release T4 from the low affinity, high capacity T4 binding site on albumin. NEFAs can be generated in vitro, usually as a consequence of heparin therapy, which stimulates the

Dilution studies are a simple but effective tool to investigate the analytical validity of an immunoassay. Non-linearity to dilution suggests a result is unreliable. However, although a good ‘rule in’ test, linearity to dilution alone cannot be used to exclude interference [3,7]. Immunosubtraction is a useful method to confirm the presence of antibody interference. This can be done crudely using polyethylene glycol (PEG) precipitation or more specifically using anti-immunoglobulin agaroses. Proprietary heterophile blocking tubes can also be used to confirm the presence of this class of interferent [3,4]. Once assay interference is established it can still be difficult to determine the correct TSH value, as there is no ‘gold standard’ method for TSH. However, an alternative immunoassay result which gives the expected responses

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– September 2013

action of lipoprotein lipase on triglyceride. Although the measured fT4 result is genuinely high, it does not reflect the in vivo situation [9]. Familial dysalbuminaemic hyperthyroxinaemia (FDH) is a benign genetic condition where the affinity of albumin for T4 is increased, such that circulating albumin-bound T4 is elevated. Despite the high total T4 (tT4), concentrations of free hormone in vivo are unaffected due to the homeostatic regulation of the thyroid axis. However, FDH is often associated with falsely high fT4 measurements using commercial immunoassays [10] [Fig. 2]. Both the increased affinity of the variant albumin for some labelled T4 analogues, as well as potential disruption of the T4 : albumin equilibrium during the assay, are likely mechanisms. The presence of the FDH mutation can be confirmed using molecular genetic approaches.

Detecting interference in fT4 assays Despite the greater analytical challenge, confirming interference in fT4 assays can be easier than for TSH due to the availability of physical separation methods, such as equilibrium dialysis, as ‘gold standard’ assays [8]. However, these methods are technically difficult and not available in most clinical biochemistry laboratories. Also, these methods do not solve the in vitro problems of hormone displacement. Again a first approach is often method comparison, using a different immunoassay architecture. Dilution and immunosubtraction studies can also be informa-

Figure 2. Free thyroxine (fT4) measurements, using fT4 reagents from five different manufacturers, in three patients with genetically confirmed familial dysalbuminaemic hyperthyroxinaemia (FDH). The assay-specific reference ranges are indicated in a lighter colour. All patients were euthyroid with nonsuppressed thyroid-stimulating hormone (TSH), but fT4 was consistently above the reference range and varied considerably between assays.

22

Assay interference

tive, although some fT4 methods are not robust to matrix effects so careful control experiments are required. Measurement of total rather than free T4 can be useful in situations where there is a suspicion of abnormal T4 binding proteins. For example, total T4 will be elevated in the presence of anti-T4 antibodies and in FDH.

Clinical causes of aberrant TFTs As mentioned above there are well described pharmacological and pathological causes of unusual TFTs; an increased awareness of analytical artefacts should not detract from the detection of these conditions. For example thyroxine treatment, a TSH secreting pituitary tumour (TSHoma), the genetic condition thyroid hormone resistance, FDH or TFT antibody interference can give elevated fT4 results with a TSH within the reference interval. Attempts by the laboratory to exclude assay interference should complement both the diagnosis of transient and genetic thyroid conditions as well as the more common drug related effects.

Conclusions and future directions Immunoassay manufacturers have invested considerable resources into reducing the potential for antibodymediated assay interference, for example by including blocking agents, or using antibody fragments rather than intact antibodies as assay reagents. Although these measures are effective, it is worth bearing in mind that changes to assay formulations may introduce novel types of interference. We have observed negative interference in one fT4 assay which appears related to the presence of a blocking agent introduced to reduce the risk of positive interference in this method [11]. Mass spectrometric methods have been introduced to eliminate antibody interference in both fT4 and tT4 methods, but unfortunately the fT4 methods still require careful optimization to avoid interference caused by binding proteins and displacing agents. As current TFT methods remain prone to analytical interference the clinical laboratory must remain vigilant to the potential for assay interference, promote effective communication with requesting clinicians, and have procedures in place for investigation of discordant results.

References 1. Association for Clinical Biochemistry (ACB), British Thyroid Association (BTA), British

Thyroid Foundation (BTF). UK guidelines for the use of thyroid function tests.2006; www.acb. org.uk/docs/TFTguidelinefinal.pdf. 2. Gurnell M, Halsall DJ, Chatterjee VK. What should be done when thyroid function tests do not make sense? Clin Endocrinol. (Oxf) 2011; 74(6): 673–678. 3. Ismail AA, Walker PL, Barth JH, Lewandowski KC, Jones R, Burr WA. Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid-stimulating hormone and gonadotropin immunoassay results. Clin Chem. 2002; 48(11): 2023–2029. 4. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998; 44: 440–454. 5. Kaplan IV, Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem. 1999; 45: 616–618. 6. Halsall DJ, Fahie-Wilson MN, Hall SK, Barker P, Anderson J, Gama R, Chatterjee VK. Macro thyrotropin-IgG complex causes factitious increases in thyroid-stimulating hormone screening tests in a neonate and mother. Clin Chem. 2006; 52: 1968–1969. 7. Ross HA, Menheere PP, Thomas CM, Mudde AH, Kouwenberg M, Wolffenbuttel BH. Interference from heterophilic antibodies in seven current TSH assays. Ann Clin Biochem. 2008; 45: 616. 8. Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best Pract Res Clin Endocrinol Metab. 2013; in press. 9. Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab. 2009; 23(6): 753–767. 10. Cartwright D, O’Shea P, Rajanayagam O, Agostini M, Barker P, Moran C, Macchia E, Pinchera A, John R, Agha A, Ross HA, Chatterjee VK, Halsall DJ. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009; 55(5): 1044–1046. 11. Bacon O, Gillespie S, Koulouri O, Bradbury S, O’Toole A, Stuart-Thompson D, Taylor K, Pearce S, Gurnell M, Halsall DJ. A patient with multiple Roche serum immunoassay interferences including false negative serum fT4. Ann Clin Biochem. 2013; 50(Suppl 1): T50.

The authors Olivia Bacon PhD and David Halsall* PhD, FRCPath, CSci Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, Cambridge, UK *Corresponding author E-mail: [email protected]

Trace element monitoring

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Trace elements and clinical chemistry Patients are routinely monitored for levels of trace elements to investigate situations of deficiency or toxicity. This article covers some of the reasons why trace elements are investigated in the clinical setting and discusses, with examples, how the measurements are carried out using advanced analytical instrumentation. It then goes on to suggest some important new developments in the field of inorganic mass spectrometry, which could have an important impact on future clinical assays. by Dr Chris Harrington

Trace elements are defined as having a concentration of less than 100 μg/g or 100 mg/L and traditionally there are two main reasons for their measurement in a clinical setting: for the determination of deficiency or toxicity. There are about 10 inorganic micronutrients essential for human health which include the transition elements Cu, Co (as vitamin B12), Cr, Fe, Mn, Mo and Zn, the metalloid Se and the halogen elements F and I. The human body also contains As, B, Ni, Si, Sn and V, but there is no firm evidence any of these are essential for health. These elements have a number of biochemical roles, e.g. co-factors for different enzymes; constituents of important molecules, such as the thyroid hormones; and electron transport, due to their redox chemistry. The toxic effect of any trace element is dose-dependent, but there are a number which exert toxicity at low concentration and examples of these include: Hg, Ti, Pb, Cd and As. The degree of harmful toxicity will not only depend on the concentration, but also on the actual chemical form and exposure time. In the case of an element such as As, it is highly toxic when present as arsine (AsH3) because it is a gas, but when exposure is via a more complex organometallic compound such as arsenobetaine (C5H11AsO2) which is common in fish and seafood, an equivalent dose of As would be harmless. Commonly exposure to a toxic trace element is determined by analysis of a urine sample normalized to the creatinine concentration and comparison to an established guidance value such as a biological exposure index (BEI). However, clearly in the case of As, a measurement of total As in the urine will not differentiate between different chemical forms. To achieve this aim each of the separate As-containing

species will need to be determined using methods based on elemental speciation, whereby a chromatographic separation is coupled to a suitable atomic spectroscopic detector, for example HPLCICP-MS (inductively coupled plasma mass spectrometry in combination with HPLC). A more recent development in the clinical measurement of trace elements relates to the orthopedic area and the increasing use of metal alloys containing Cr, Co, Mo and Ti as the components of metal-on-metal (MoM) hip replacements. As a result of complications with the use of such implants and the potential for failure requiring revision surgery, all patients in the UK with MoM replacements are now monitored on an annual basis. The guidelines issued by the UK Medicines and Healthcare products Regulatory Agency (MHRA) in 2010 [MDA/2010/033] and subsequently updated in 2012 [MDA/2012/008 and 036] provide advice to healthcare professionals involved in the management of patients implanted with MoM hip replacements. The initial alert recommended that all patients should be followed up regularly by measurement of Co and Cr in whole blood samples and that this should be carried out most frequently on patients with symptoms consistent with high rates of failure. The medical device alert stipulates that if either element was elevated above a concentration of 7 μg/L (134 and 119 nmol/L for Cr and Co respectively), then further tests should be performed including imaging, to identify patients with potentially failing MoM hip joints. Whereas there are already action limits for these elements relating to occupational exposure, the concentration of 7 μg/L was chosen after consultation with

orthopedic clinicians and using information from the National Joint Registry for England and Wales, as a level at which the joint was not showing optimum performance. It was not set as an indication of toxicity but rather as an indicator of joint performance and is thus interpreted with this in mind. Internal quality control and external quality assurance are important prerequisites for measuring trace elements and making appropriate diagnosis or treatment decisions. A good example of this is the routine annual follow-up of patients with MoM hip-replacements, where clinicians need to make sure that their decisions are based on well controlled analytical measurements. How, for instance, can a clinician decide if an increase in concentration of Co or Cr results from a change in the particular joint and does not arise from a change in the laboratories measurement performance? We recently looked at data [1] from the UK National External Quality Assessment Scheme for trace elements (TEQAS). This supplies whole blood specimens which are spiked with known amounts of a number of trace elements including Co and Cr. The mean recovery over the samples measured in the 2011– 12 scheme year was 96.4% (SD 2.23, CV 2.3%) for Co and 96.1% (SD 3.19, CV 3.3%) for Cr. The excellent agreement between the amounts in the specimens and the mean value indicates the results are accurate, and agreement between the pools distributed on different occasions shows they are reproducible over time. This should provide the necessary confidence to the clinical decision maker that the laboratories providing the Co and Cr results are competent and the results are suitably accurate.

Analytical instrumentation The instrumental mainstay of clinical laboratories which specialise in the measurement of trace elements is inductively coupled plasma mass spectrometry (ICP-MS), which is a form of inorganic MS measuring elemental ions rather than molecular ions. Developed as a commercial analytical technique in the early 1980s it was initially used in environmental and geological laboratories, but after instrumental improvements it is now gaining popularity in the clinical

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Trace element monitoring

area. This is mainly because it is multi-elemental in nature. A review of new research and instrumental approaches in the elemental analysis of clinical and biological materials, foods and beverages is published annually as an Atomic Spectrometry Update [2]. The instrumentation itself will not be discussed as many texts [3] deal with the fundamentals of ICP-MS. However, the significant strengths of the technique include: multi-elemental detection in a single run; wide elemental coverage up to m/z 254 (UO+); high sensitivity with low limits of detection, down to sub ng/L levels (limited by purity of the reagents); fast analysis times as a result of the scanning speed of the quadrupole analyser; wide linear working range, up to 9 orders of magnitude in the same run; isotopic information, making high accuracy calibration via isotope dilution mass spectrometry available; and it can be used for specialist applications such as speciation analysis, where it is used as a chromatographic detector for HPLC, GC, CE or GE separations. The main weaknesses of the technique are: the presence of isobaric interferences on some elements, which mean the isotope of one element is at the same m/z ratio for the analyte of interest, for instance Ca has an isotope at m/z 48 which is the same as the most abundant isotope for Ti, making the measurement of Ti in clinical samples problematic; the formation of polyatomic ions from sample matrix and atmospheric ions can impinge on the m/z for the analyte of interest, an example would be the measurement of Cr at its most abundant isotope at m/z 52 which has a major interference from the formation of ArC; and the formation of doubly charged ions, for instance Gd2+ can interfere with Se at m/z 78. Luckily instrumental developments based on the use of a reaction/collision cells containing a suitable gas have been introduced to overcome the problems due to polyatomics and doubly charged ions. These work by using a reactive gas, e.g. H2 in the cell and removing the interference by reactively neutralising it, which we have recently demonstrated for the removal of the Gd2+ interference from the measurement of Se [4], or using a collision gas, e.g. He to remove the larger polyatomic ions by collision induced kinetic energy discrimination. These newer instruments are extremely robust and can rapidly deliver highly accurate measurements for multi-elements at low concentrations in difficult matrices such as whole blood, serum or urine.

Future trends and developments Over the last 5–10 years the capabilities of ICP-MS for the detection of molecules that do not contain a trace element have been investigated. By using a reagent with specificity for the analyte and which carries a metal or nanoparticle tag, the molecule of interest becomes visible for detection by ICP-MS. The reagents used are often antibodies, so the protocols often mimic those developed for immunochemical assays and, in theory, can be applied to the determination of the same analytes, including peptides, proteins and other specific biomarkers. So why would this be advantageous compared to conventional immunochemical assays? Most importantly this approach has a greater potential for multiplexing than spectroscopic methods; there are a large number of elemental tags to choose from and no overlap between them. As illustrated in Figure 1 this is not the case with fluorescence signals. Other advantages include: analyte quantification with high precision; low detection limits; large dynamic range; low

(a)

(b)

Figure 1. (a) Conventional single-cell assays by flow cytometry use fluorescent tags. Overlap of the emission spectra limit the method to about 4-plex (efforts are being made to extend this to >10-plex, but with inherent compensation problems). (b) Mass spectra of a sample of enriched lanthanide isotopes with mass cytometry. Each isotope is independent, but colour-coded here to show the corresponding elements (i.e. if tagging with naturally-abundant elements, then each of the isotopes for a given element would be present at intensities corresponding to their natural abundance).

matrix effects from other components of the biological sample (i.e. contaminating proteins in the sample have no effect on elemental analysis); low background from plastic plates (i.e. plastic containers do not cause interference on elemental detection as it can with fluorescence), and superior spectral resolution. As can be seen in Figure 1 this has generated a new approach to flow-cytometry based on ICP-MS and it’s very likely that other new approaches to other biochemical analytes will shortly become commercially available.

Acknowledgements Scott Tanner, University of Toronto for permission to use the figures from the CyTOF flow cytometer based on ICP-ToF-MS (DVS Sciences Inc).

References 1. Harrington CF, Taylor A. BMJ 2012; 344: e4017. 2. Taylor A, Day MP, Hill S, Marshall J, Patriarca M, White M. J Anal At Spectrom. 2013; 28: 425–459. 3. Inductively Coupled Plasma Mass Spectrometry Handbook. Ed. Nelms S, Blackwell 2005. 4. Harrington CF, Walter A, Nelms S, Taylor A. Ann Clin Biochem. 2013; submitted.

The author Chris Harrington PhD, MRSC SAS Trace Element Laboratory, Faculty of Health and Science, University of Surrey, Guildford, Surrey, GU2 7XH, UK E-mail: [email protected]

INDUSTRY NEWS BD and the College of American Pathologists announce strategic alliance to support laboratory quality and performance in India and China BD Diagnostics and the College of American Pathologists (CAP) have announced the launch of a new strategic alliance that will provide solutions to advance laboratory quality for improved patient outcomes in China and India. BD and CAP announced the collaboration during the American Association for Clinical Chemistry (AACC) Annual Meeting in Houston, Texas. Laboratories play a critical role in the diagnosis and treatment of disease for the more than 2.5 billion people who live in China and India. The BD/CAP Strategic Alliance will improve access to external quality assurance/proficiency testing (PT) that can have a direct and positive impact on laboratory quality, and therefore, patient outcomes. Together BD and CAP will provide education to improve awareness of global practice standards and training that will help laboratories achieve their quality improvement goals. Additionally, BD will manage PT distribution, including sales, shipping, and first-line client service. “The clinical laboratory and pathology contribute more than 70 percent of the information used to determine diagnoses and drive treatment decisions,” said CAP President Stanley J. Robboy, MD, FCAP. “CAP has all of the tools necessary to help laboratories improve and monitor efforts that drive quality performance. This strategic alliance with BD will increase access to these essential resources, helping laboratories accelerate their quality improvement journey so that they can contribute to better quality care, differentiate themselves and their institutions, and be recognized globally among the best providers of laboratory and pathology services.” CAP’s Laboratory Accreditation, Surveys, PT programmes, and other quality management resources combined with BD’s in-depth clinical knowledge of preanalytical systems provide a comprehensive, expert-based toolkit to help laboratories in China and India continuously improve the quality of the testing services they provide to patients. Through participation in CAP accreditation, laboratories in China and India can demonstrate their compliance to the most robust and comprehensive clinical laboratory testing standards in the world.

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Having operated in China since 1994 and in India since 1996 supporting public and private sector partners in enhancing laboratory standards, BD has extensive experience in deploying clinical expertise and educational resources in these regions, as well as a deep understanding of the unique needs of laboratories throughout these countries. Today in China, there are 18 CAP-accredited laboratories and nearly 100 laboratories participating in PT. In India, of the 71 laboratories participating in CAP PT, 42 have achieved CAP accreditation. BD’s access and logistics experience will support CAP PT importation and ensure more timely delivery and quality, reduce participants’ administrative work, and allow billing in local currency. Market launch of this initiative will begin in China and India in August 2013 with PT distribution initiated in January 2014. www.bd.com www.cap.org

Beckman Coulter and IRIS Diagnostics offer automated erythrocyte sedimentation rate analysis systems through distribution agreement with Alifax

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subsequent results every 35 seconds or less, versus one hour for standard ESR analysis by the reference Westergren method,” said John Blackwood, senior vice president, Product Management, Beckman Coulter Diagnostics. “The small sample volume and STAT capability are expected to reduce ER waiting times and significantly minimize the need for patient specimen redraws.” Employing patented technology, the Alifax ESR system provides fully automated, hands-off operation in a small footprint and processes the same whole blood tubes from the hematology analyser. ESR is a common blood test, and is a nonspecific measure of inflammation. Standard ESR analysis is a measure of red cells settling over time, typically one hour, and variations occur in the results, depending on the degree of specimen viscosity and presence of inflammatory proteins. Alifax’s technology measures the kinetics of red blood cell aggregation by capillary photometry and reads aggregation over a ten second period. www.beckmancoulter.com www.alifax.com

Diagnostica Stago enters into joint research agreement with Bristol-Myers Squibb

Beckman Coulter, Inc., and IRIS Diagnostics announce the launch of the Alifax automated Erythrocyte Sedimentation Rate (ESR) analysis system through a distribution agreement with Alifax. The automated ESR analysis systems are designed to fit all workloads, including small hospitals, large reference laboratories, core laboratories and satellite locations. The automated ESR system delivers increased productivity, efficiencies in laboratory labour utilization and reduced turnaround times (TAT), along with improved patient outcomes and physician satisfaction. “This patented technology is a breakthrough in turnaround time – generating

Diagnostica Stago S.A.S, a privately held company that provides reference tests and instrumentation in hemostasis, announced that the company has entered a joint research agreement with Bristol-Myers Squibb Company (BMY) to develop an assay for measuring the circulating blood concentration of the oral Factor Xa inhibitor ELIQUIS (apixaban). No commercial assay is presently available to specifically measure apixaban plasma concentration. Terms of the agreement were not disclosed. “Stago is pleased to have the opportunity to develop this test”, said Tristan Herve, Director of Pharmaceutical Development, “These oral Factor Xa inhibitors address an important unmet need for patients requiring anticoagulant therapy”. Stago has already completed prototype development and will be applying for health authority approvals of the assay worldwide. Diagnostica Stago retains 100 percent global development and commercialization rights for the assay. www.stago.com

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Point of care diagnosis

Managing and diagnosing diabetes at the point-of-care Clinical decisions need to be made at the earliest possible time to facilitate the administration of quick and accurate treatment plans. Point of care testing (POCT) enables tests to be convenient and fast, making them suitable for use with a broad range of patients, including diabetics. Glycated hemoglobin (HbA1c) is commonly tested in diabetics as it provides a reliable measure of glycemic control (Figure 1). However, the role of HbA1c in the diagnosis of diabetes has only more recently been documented. HbA1c levels reflect average circulating glucose levels over the lifespan of red blood cells (2-3 months). Once hemoglobin molecules have been glycated, they become highly stable, enabling a greater level of clinical information to be obtained from them than a single glucose measurement taken at a particular point in time.

by Gavin Jones, Diabetes Product Manager, EKF Diagnostics

By taking serial HbA1c measurements, an individual’s control over their glucose levels can be assessed in response to changes in management strategies. Measurements should be taken every 2-6 months with target HbA1c levels set individually and therapy adjusted accordingly to provide the most effective treatment (1). The target ranges of HbA1c for diabetic patients, depending on their risk of severe hypoglycemia, cardiovascular status and co-morbidities, should be set between 6.5 – 7.5% DCCT (48 – 58 mmol/mol), with the nondiabetic reference range being 4.0 – 6.0% DCCT (20 – 42 mmol/mol). One point for consideration is that HbA1c results may be affected by any condition that leads to a change in red blood cell survival. But even then, HbA1c can be used to detect trends in a patient’s glycemic control.

HbA1c in POCT-based diabetes monitoring HbA1c determination was originally based on methods such as ion exchange and affinity chromatography with

alternative affinity and immunological methods following later, taking HbA1c into the POC environment. Typically, when using laboratory-based testing, patients with existing diabetes are monitored for HbA1c every 2-6 months, requiring a visit to a nurse or phlebotomist and a follow-up appointment 1 to 2 weeks later to discuss the results. Use of POCT would mean that after just one visit, patients can leave with their results, eliminating the need for a follow-up appointment. By enabling an earlier therapeutic decision, diabetes control can be improved whilst also providing economic benefits in terms of cost and time.

Diabetes diagnosis The benefits of HbA1c in the management of diabetes can also be directly applied to the diagnosis of diabetes. Unlike glucose levels, which are affected by what has been eaten and drunk in the previous 2-3 hours, the measurement of HbA1c levels does not

require fasting. As a simple and immediate test for diabetes, POC HbA1c can support the early identification of at-risk individuals. This would rapidly enable them to make small changes to their lifestyle to significantly reduce the risk of developing type 2 diabetes. Patients diagnosed with diabetes who are able to maintain low blood HbA1c levels also have a significantly reduced chance of complications after diagnosis (2); early detection by POCT can reduce this risk even further. The ability to rapidly assess and change these risk outcomes has significant health benefits and reduces the costs associated with recurrent leg ulcers, blindness, heart disease and stroke, for example, all of which are conditions and complications commonly associated with type 2 diabetes. The World Health Organization (WHO) has recommended the use of HbA1c for the diagnosis of diabetes (3). In the UK for example, the National Institute for Clinical Excellence (NICE) has published guidelines for diabetes prevention which aim to identify people at high risk of type 2 diabetes and offer cost-effective, appropriate interventions to prevent or delay onset (4). Used in conjunction with a lifestyle health risk assessment, these guidelines advocate the monitoring of HbA1c levels to allow healthcare providers to advise individuals on treatment regimens, depending on their classification as low, moderate or high risk. Current guidance, therefore, supports the use of HbA1c in screening for type 2 diabetes, and in the management of patients with diabetes. The use of POCT could improve the management of patients with established diabetes in both primary and secondary care settings and enable earlier type 2 diabetes diagnosis.

What to look for in a POC HbA1c analyser

Figure 1: Glycated hemoglobin (HbA1c) explained.

Most POC HbA1c analysers use a single drop of blood (4-10 μL), which is applied to a reagent cartridge. The cartridge is then directly inserted into a desktop device for analysis. Time to results is generally between 3 to 10 minutes, depending on the analyzer. This quick turn-around time, in

27 the AACC set up the ’National Glycohemoglobin Standardization Program’ (NGSP) in 1996. In parallel, the International Federation of Clinical Chemistry (IFCC) developed reference methods for glycated hemoglobin. In 2006 and 2007, an international consensus between IFCC and AACC was agreed upon (5).

Figure 2. The Quo-Lab POC HbA1c analyser from EKF Diagnostics.

combination with simple operation, is key to maintaining effective POC testing.

Simplicity To minimize human error and the subsequent need for repeat testing, a POC analyser should be as easy as possible to use. Also the analyser should be highly intuitive, requiring little user training. Features that support ease-of-use include ready-to-use reagent cartridges which can be inserted straight into the analyser. The blood sample can then be added directly, without the need for premixing or pipetting. Minimizing the number of steps in the procedure not only reduces the potential for user error, but also helps to standardize results by eliminating variation from different users.

Audit trails For patient safety purposes, audit trails must be readily available. Use of barcode scanning for patient and user identification, as well as confirmation of the batch of reagents and controls used, ensures an analyser can provide such information in a timely manner. Two levels of quality controls that are recorded and held within the analyser’s memory are also ideal for auditing purposes.

Certification Certification of the analyser in order to confirm delivery of accurate, standardized results should also be a key consideration. In an effort to standardize HbA1c results,

The calibration and certification of laboratories and manufacturers to the same standards has improved the conformity of results. However, in practice, differences can still be observed among technologies and between individual systems. These observed differences arise because of heterogeneity of hemoglobins, underlying differences in technologies (e.g. ion exchange, boronate affinity, immunoassay), calibration drifts or lot to lot variability. Providing the manufacturer follows the recommendations of the IFCC and NGSP to ensure instruments and reagents are accurately aligned and traceable to the reference method, this should not be a problem.

Methodology There are POC HbA1c analysers available (e.g., the Quo-Lab, EKF Diagnostics, Cardiff, UK) (Figure 2) where results are not affected by hemoglobin variants (which do not result in reduced erythrocyte life span), labile glycated hemoglobin or hematocrit levels. Such analysers use Boronate Fluorescence Quenching Technology (BFQT) (6) (Figure 3) which is associated with simple, yet powerful multiple optical measurements. This is based on well-documented boronate affinity chromatography systems used in reference laboratories. However, as BFQT does not require chromatographic separation, the methodology allows for fast, simple and accurate POC measurement of HbA1c to deliver comparable results to chromatography-based techniques.

Summary Type 2 diabetes can be managed easily and effectively through the monitoring of HbA1c levels, as opposed to blood glucose. POC diagnosis enables early detection in higher risk patients, before any additional complications arise. POCT

Figure 3: Boronate Fluorescence Quenching Technology principle.

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therefore not only improves patient access to testing, but provides accurate diagnoses there and then. Treatment strategies can be determined immediately, eliminating the need for a follow-up visit to discuss the results. The ability for diagnosis to occur near to the patient provides greater convenience, thus increasing the likelihood of compliance. When selecting an analyser for use at the POC, users need to bear in mind that it needs to be a convenient and appropriate option. The focus should be on meeting regulatory requirements, as well as ease of use in order to ensure rapid testing, with accurate, standardized resulting data.

References: 1. Diabetes UK. HbA1c Standardization: Information for Clinical Healthcare Professionals. 2009. http://www.diabetes. org.uk/Guide-to-diabetes/Monitoring/ Blood_glucose/Glycated_haemoglobin_ HbA1c_and_fructosamine/HbA1c_ Standardisation_Information_for_Clinical_Healthcare_Professional. 2. Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of longterm complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86. 3. World Health Organization. Use of glycated hemoglobin (HbA1c) in the diagnosis of diabetes mellitus. 2011. www. who.int/diabetes/publications/reporthba1c_2011.pdf. 4. National Institute for Health and Clinical Excellence. Preventing type 2 diabetes: risk identification and interventions for individuals at high risk. 2012. www.nice.org.uk/nicemedia/ live/13791/59951/59951.pdf. 5. Geistanger A, Arends S, Berding C, Hoshino T, Jeppsson JO, Little R, Siebelder C, Weykamp C; on behalf of the IFCC Working Group on Standardization of Hemoglobin A1c. Statistical Methods for Monitoring the Relationship between the IFCC Reference Measurement Procedure for Hemoglobin A1c and the Designated Comparison Methods in the United States, Japan, and Sweden. Clin Chem. 2008 Aug;54(8):1379-85. 6. Wilson DH, Bogacz JP, Forsythe CM, Turk PJ, Lane TL, Gates RC and Brandt DR. Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection. Clinical Chemistry October 1993 vol. 39 no. 10 2090-2097

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Science news

Research findings presented at AACC hold promise for improving patient care Leaders from the medical diagnostics, laboratory medicine, and healthcare fields convened in Houston, Texas, July 28 – August 1 for the AACC annual meeting, the world’s largest diagnostics conference and expo. Over 17,000 attendees took part in the event and the exhibit totalled more than 625 companies. A selection of research papers presented in Houston are summarized below. of research showing that study patients

New biomarkers for prostate cancer device to rapidly, accurately, and quanti- who carried a specific genetic variation Dimitra Georganopoulou, PhD, Ohmx Corporation, presented results of a pilot study to find a new biomarker for prostate cancer aggressiveness. The researchers measured the enzyme activity of prostate-specific antigen (PSA), termed the “aPSA”, in patient specimens that had been removed by radical prostatectomy. They wanted to determine if this activity level could be a clue to how aggressive the cancer was. The team found that there was a significant negative correlation between prostate cancer progression and the aPSA in prostatic fluid. Patients with the least amount of aPSA (PSA activity) had the most aggressive prostate cancer. Tests for an “aggressiveness biomarker” would provide critical information for making decisions about when clinical treatment should occur or when it could be postponed. Many men might be able to avoid radical treatments if their cancer was known to be non-aggressive. Likewise, men whose cancer was too aggressive to employ the “active surveillance” or “watchful waiting” approach would have more information to help them make meaningful personal decisions with the help of their doctors about what level of treatment was right for them. The findings from this study could lead to the development of a new tool to use along with existing screening tests. PSA Enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness. Dimitra Georganopoulou, PhD, OHMX Corporation, Evanston, Ill., U.S.A.

Diagnosing cystic fibrosis at the point of care Xuan Mu from Peking Union Medical College presented test results from cystic fibrosis patients using an exciting new point-of-care method. Microfluidics and colour changes within a Band-Aid type of adhesive strip on the skin allow the new

tatively diagnose cystic fibrosis in a small amount of sweat. Detecting sweat chloride has been the gold standard in diagnosing cystic fibrosis for more than 50 years. The new test detects increased chloride in sweat using a colour change in paper on an adhesive strip when a very small amount of sweat is absorbed. The intensity of changed colour is recorded with a cell phone camera, and is then measured against a colour model. Cystic fibrosis in an inherited disease of the body’s mucus glands. Technically a rare disease, the incidence of cystic fibrosis varies around the world and by ethnic group. Different mutations in the CFTR gene cause the severity and symptoms of CF to vary considerably. Respiratory and digestive systems are affected, as well as sweat glands and reproductive systems. The new pointof-care test device can distinguish healthy people from cystic fibrosis samples and conveniently integrates the many separate steps of current sweat chloride tests whose results take several hours to obtain. Treatment advances have increased the life expectancy of cystic fibrosis patients over the past several decades from the mid-teens in the 1970s to more than 36 years today in the U.S. An early diagnosis and a comprehensive treatment plan can improve both survival and quality of life of patients. This new method demonstrates a fast and cost-effective opportunity in diagnosing cystic fibrosis. On-site colorimetric detection of sweat chloride ion for diagnosing cystic fibrosis. Xuan Mu, Peking Union Medical College, Beijing, China

Determining the safety of olanzapine for schizophrenia and bipolar disorder AACC member Werner Steimer from Munich, Germany presented the results

in an antipsychotic-metabolizing enzyme developed significantly higher serum concentrations of the drug olanzapine. The increased drug concentrations were still noteworthy even when researchers accounted for differences in the patient’s age, sex, weight, and other medications that they used. This is the first study to demonstrate that this polymorphism influences serum levels of olanzapine, and the study is extremely timely in the context of the recent FDA safety alert on the injectable form of olanzapine, an “atypical” or second generation antipsychotic medication. Under investigation are two unexplained deaths of patients who received an intramuscular injection of Zyprexa Relprevv (olanzapine pamoate) and showed very high blood levels of the drug, although they had received appropriate doses. They died 3-4 days after injection. Olanzapine is approved by the U.S. FDA for treating schizophrenia and bipolar disorder in adults and children older than 13, and is one of the most widely prescribed of the atypical antipsychotics. Olanzapine is available in tablet, injectable, and long-acting “depot” formulations. Long acting medications can be more tolerable to some patients and help them adhere to treatment. Olanzapine is metabolized in the liver by specific cytochrome P450 enzymes. Some individuals have genetic variations – polymorphisms – of cytochrome enzymes. These can impact the way that drugs are broken down and distributed throughout the body and sometimes even the strength or effectiveness of treatment. The CYP1A2*1D Polymorphism has a significant impact on Olanzapine serum concentrations. Werner Steimer, MD, Klinikum Rechts der Isar – Technische Universität München, Munich, Germany.

PRODUCT NEWS

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700 litre freezer POCT analyser The Maglumi 600 analyser applies ABEI [N-(4-Aminobutyl)N-ethyl-isoluminol] as label and uses nano magnetic microbeads as separation technology, offering a high degree of convenience in diagnostic immunoclinical analysis and greatly facilitating clinical operation because of its high throughput, excellent reliability and various operating modes. Covering the widest range, the test menu includes tumour markers, thyroid, cardiac, fertility, hepatic fibrosis, infectious diseases, drug monitoring, anemia, kidney function, immunoglobulin, bone metabolism, glycol metabolism, inflammation monitoring, TORCH, EBV and others.

SNIBE DIAGNOSTICS www.cli-online.com & search 26390

Multiparameter hepatitis C test The Multisure HCV antibody test is intended to fill the gap that exists in the current Hepatitis C diagnosis and may provide additional information about the infection profile. MP Diagnostics Multisure HCV is a qualitative multiparameter immunochromatographic assay, intended for the detection and differentiation of Hepatitis C antibodies that may be present in patients with acute or chronic Hepatitis C infection. This innovative test is developed based on the patented reverse-flow technology, giving greater sensitivity and stronger visual signals. The multiparameter platform test uses human whole blood, plasma or serum. The test has a short test-time and can detect & differentiate HCV antibodies against both structural and nonstructural proteins across all six genotypes. Multisure HCV Antibody Assay is instrument-free, easy to use and is suitable for use as Point-of-care or with automation and does not require highly trained personnel to perform the test. The test has excellent correlation with PCR, EIA & immunoblot. It allows medical professionals to get additional information about the infection profile as well as stage; patients may be able to receive earlier diagnosis, timely treatment and thereby a better disease management. MP BIOMEDICALS

With a capacity of 728 litres, the Eco VIP KM-DU73Y1E -86°C upright freezer is the most energyefficient of its size and is ideal for sample storage within hospitals, biomedical research institutes and biopharmaceutical laboratories. Energy savings lead to reduced running costs as well as environmental benefits, and so the KM-DU73Y1E provides a range of advantages to all users of ultra-low temperature freezers. In comparison with other widely used, -86°C freezers of this size, the KM-DU73Y1E can help to reduce electrical running costs by up to 50%. This efficiency is achieved with the use of natural refrigerants, advanced Panasonic refrigeration technology and VIP Plus vacuum panel insulation. As well as energy savings, VIP Plus insulation panels save 30% floor space compared to conventionally insulated upright freezers. The combination of the above features with Panasonic’s control technology provides improved cooling performance and excellent insulation characteristics and door-open recovery times. A streamlined cooling system provides even greater reliability for the security of valuable and often irreplaceable samples. PANASONIC www.cli-online.com & search 26393

DIAsource ImmunoAssays S.A.

25-Hydroxyvitamin D Total ELISA Now also FDA approved

Simple automation & 100% All-In-One*

Newly available: 1,25-Dihydroxyvitamin D ELISA FREE 25-Hydroxyvitamin D ELISA (RUO) *AIO = All-In-One: pretreatment + ELISA in same MT-PLATE www.diasource.be z [email protected] z Tel: +32 10 84 99 00

www.cli-online.com & search 26366 www.cli-online.com & search 26182

– September 2013

30

PRODUCT NEWS

Compact hematology analyser

The BC-5150 5-part hematology analyser is offered as a compact solution for clinical labs, especially those with limited spare space. Its compact footprint is a result of innovative technology improvements, including miniaturized semi-conductor laser source, highly integrated electronic boards and optimized liquid handling systems. The tri-angle laser scatter is the key technology used on BC-5150, enabling better 5-part WBC differentiation even on samples with high eosinophils. The instrument has very user-friendly software and hardware. With a 10.4-inch TFT full-colour touch screen, the need for an external PC is practically eliminated. The BC-5150 inherits its convenient and proven powerful software design from the BC-6800 & BC-3600 platforms, specifically modified for easier handling in small sized labs. Four USB ports located on the left side allow convenient data transmission as well as connections to printers, keyboard, mouse, barcode scanner, etc. The BC-5150 has 2 test modes for capillary blood sample, capillary whole blood mode and pre-diluted mode. In the capillary whole blood mode, the user can aspirate capillary blood sample directly through the sample probe. In order to provide an economical solution for labs, the liquid handling system minimizes reagent consumption during sample testing, start up and shut down procedures.

MINDRAY www.cli-online.com & search 26382

Blood collection system The Vacuette Tube-Touch is a safety product of the newest generation with an automated activation of the protective shield. This product is a further development of the already existing Vacuette Premium Safety Needle System. It was developed in close collaboration with users. The result is the Vacuette Tube-Touch Holder. The new name vividly captures the product’s functional capabilities. The safety mechanism is activated by placing the tube in the holder. In this way, needlestick injuries are automatically avoided. Apart from being easy to use, the Vacuette Tube-Touch is characterized by its newly designed protective shield, which is freely movable upon

activation. Graduation marks have been added to the protective shield in order to easily demonstrate the position of the needle in the vein for the user. These marks show how far the needle is located in the vein. During removal of the needle from the vein, the protective shield automatically encloses the needle, making the product practical, simple and safe. GREINER BIOONE www.cli-online.com & search 26370

HPV DNA microarray

for cervical carcinoma, which can then be minimized by more frequent followup examinations to detect morphological cell changes at an early stage. Based on the recommendations of the respective professional societies, HPV-negative women can forgo Pap smears for a longer time interval. The EUROArray procedure is extremely easy to perform, requiring no expertise in molecular biology. DNA from patient samples is first amplified by multiplex polymerase chain reaction (PCR). The PCR products are then incubated with Biochip microarray slides containing immobilized complementary DNA probes. Results are evaluated and interpreted fully automatically by the user-friendly EUROArrayScan software. Meticulously designed primers, ready-touse PCR components and integrated controls all contribute to the reliability of the analysis. The entire EUROArray process from sample arrival to report release is IVD validated and CE registered. EUROIMMUN AG www.cli-online.com & search 26388

Computer printable labels for liquid nitrogen storage

A new microarray based on state-ofthe-art EUROArray technology provides fast and reliable detection and typing of human papillomavirus (HPV). HPV is involved in the development of cervical carcinoma, and HPV testing plays a central role in risk assessment and early diagnosis of this cancer. In contrast to Pap examinations, HPV detection is not dependent on subjective evaluation and it offers very high sensitivity even in the early stages of infection. The HPV EUROArray is based on detection of the viral oncogenes E6/E7, which provides highest possible sensitivity. Using an extensive panel of specific primers and probes, the EUROArray detects all 30 genitally relevant HPV subtypes in one test and distinguishes between high-risk subtypes that trigger cancer and low-risk subtypes that cause benign genital warts. Multiple infections are reliably identified, and primary and persistent infections can be differentiated. A positive highrisk result indicates an increased risk

Perfect for LN2 freezing and long-term cryogenic freezer storage, the computer printable durable label for ultra low temperatures provides immediate permanent adhesion to all cyrovials, tubes and liquid straws stored down to -196ºC. Available in any format including ‘cap & tube’ label sets, they allow variable data (lot numbers, barcodes etc) to be added ‘in minutes’ straight from a standard laser, inkjet or thermal transfer printer, saving time and eliminating the risk of smudged or illegible handwritten data, providing reliable sample identification for liquid nitrogen storage. CILS INTERNATIONAL www.cli-online.com & search 26389

PRODUCT NEWS Automated stainer Offering significant improvements in workflow optimization, particularly when multiple protocols are used, the Gemini AS advanced automated stainer has been intelligently designed to be easier to use than ever, with a new touch screen interface and intuitive software. The instrument has an all-new touchscreen interface that is intuitive to use and makes day-to-day operation simplicity itself. A glance is all that is needed to check the batch stain progress. New LED lighting allows the operator a clear view of what is going on inside the staining area. It is also fully compatible with the Thermo Scientific range of coverslippers to match the workflow of modern high-throughput laboratories. To maximize throughput, up to 12 slide racks can be processed at the same time. The control software automatically arranges the reagent positions for greatest speed and performance. Furthermore, duplicate reagents can be assigned automatically according to QC usage values to reduce downtime to change reagents. Gemini AS has 26 different reagent stations, catering for both H&H and Papanicolaou stains without changing reagent pots. Once the racks are loaded and the protocols are selected, the intelligent software does the rest. Furthermore, the instrument has been configured for more application-specific staining requirements such as those for cytogenetics and andrology. The instrument’s dual-layer configuration makes the most use of laboratory space. Also featured is a built-in power supply for protection of valuable samples. Protocols are easy to prepare and readily modified, and the manufacturer’s recommended protocols are pre-installed. A USB port allows data to be downloaded for off-line processing and analysis. THERMO FISHER SCIENTIFIC www.cli-online.com & search 26401

Parvovirus B19 molecular assay Designed for use on the Liaison Iam instrument, the Iam Parvo assay is a highly sensitive, rapid quantitative molecular assay for Parvovirus B19 diagnosis. Parvovirus B19 (B19V) infection is common and occurs worldwide, with epidemics following a 3-4 year cycle. The virus is typically acquired in childhood when it causes the mild, selflimiting rash illness erythema infectiosum

31

(commonly known as ‘slapped cheek’ or ‘fifth disease’). It is transmitted through respiratory droplets and can vary in severity from asymptomatic to life-threatening illness. B19V infection during pregnancy can have severe consequences. The infection can transfer from mother to fetus across the placenta and may cause fetal death or poor outcomes (including severe neurological disease) in surviving babies. B19V is also problematic in immunocompromized patients and is the leading cause of pure red cell aplasia in AIDS patients. In individuals with sickle cell anemia, B19V infection can result in transient aplastic crisis (TAC). Chronic B19V infection can lead to persistent and severe anemia and is also associated with conditions such as arthropathy, glomerulonephritis and neurological disease. Calibrated against the WHO standard for B19V, Iam Parvo provides exceptional time-to-result benefits when compared to PCR, with equivalent specificity. The Liaison Iam instrument uses DiaSorin’s proprietary Q-LAMP technology. DIASORIN MOLECULAR DIVISION www.cli-online.com & search 26397

In vitro diagnostic reagent kits for Vitamin D A new series of reagent kits is specifically designed for in vitro diagnostic use in conjunction with mass s p e c t rom e t r y i n s t r u m e nt a tion for clinical analysis. The first kit is focused on vitamin D analysis and is CE marked to meet European regulatory requirements. Highly selective and sensitive, MS provides routine diagnostic testing laboratories in Europe with the ability to quantitate multiple trace level compounds in a single analysis with high confidence in the results. The first kit in the new SCIEX IVD-MS portfolio to harness this advanced technology for routine clinical applications is the SCIEX IVD-MS Kit for 25-OH Vitamin D Analysis. This kit is designed to help clinicians make diagnoses of vitamin D

– September 2013

deficiencies, with the ability to quantitate both 25-OH-Vitamin D2 and 25-OHVitamin D3 in a single run. Adequate levels of vitamin D have been known for decades to be essential for strong bones. Recent research has linked vitamin D to be important in the prevention of multiple common and serious diseases, such as type 1 diabetes, cancer and heart disease. The new SCIEX IVD-MS kits work with the AB SCIEX 3200MD CE-IVD series of MS systems – including the API 3200MD and 3200MD QTRAP LC/ MS/MS systems – which the company launched recently in Europe. AB SCIEX www.cli-online.com & search 26403

ELISA for the quantitation of human IgG

Now available is a specifically formulated and optimized ELISA procedure for the quantitation of intact human IgG from matrices containing non-human proteins. The availability of such an assay meets the needs of clinical, biopharmaceutical, and medical researchers alike. The quantitation of IgG in cell culture supernatant is important in the selection of high yielding clonal cell line candidates for downstream scale-up fermentation. This ELISA utilizes sheep polyclonal antibody, specific to the Fc and light chain regions of human IgG for capture and detection respectively, ensuring not only intact human IgG is quantified. Coupled with that, the assay virtually eliminates any cross reactivity to common laboratory species (i.e. mouse, rat, primate) and to bovine serum proteins ensuring the utmost in test specificity. This unique ELISA procedure, displaying a measuring range for intact human IgG from 2.5ng/mL to 810ng/mL, also features a convenient operator-oriented packaging design, with sufficient reagents to perform 96 separate tests. It also includes all necessary calibration and quality control materials and offers exceptional performance characteristics, lot-to-lot consistency and shelf life stability. THE BINDING SITE www.cli-online.com & search 26396

– September 2013

32

PRODUCT NEWS Fibrinogen reagent

Automation system The Power Express* automation system features a dynamic inlet, high-speed refrigerated stockyard and connection to the AU5800 chemistry analyser, UniCel DxI 800 immunoassay analyser, or other third-party analysers. The Power Express features radio-frequency identification (RFID) technology and is designed for high throughput via a four-lane tube processing unit. It offers a consolidation of testing disciplines including chemistry, immunochemistry and hematology. The dynamic inlet provides control of sample tube flow and offers prioritized tube handling for the centrifuge bypass and eight positions for specialized rack types – STAT, routine, centrifuge bypass and remap. The option to integrate with a 5,400 high-speed refrigerated stockyard makes it possible to automate sample tube storage, retrieval and disposal. BECKMAN COULTER www.cli-online.com & search26392

For the measurement of fibrinogen concentration in plasma, Fibrinogen by BioSystems is a turbidimetry liquid bireagent method which is calibrated with a multipoint curve (lyophilized standard) and controlled with a lyophilized control plasma. Fibrinogen is a protein that is essential for blood clot formation. It is produced by the liver and released into the circulation as needed along with several other clotting factors. The concentrations of fibrinogen are increased in case of diabetes, obesity and inflammatory syndromes and are decreased in case of disseminated intravascular coagulation (DIC), fibrinolysis and genetic deficiency. In addition, elevated concentrations of fibrinogen can be involved in the pathogenicity of different cardiovascular thrombotic events. In recent years, a large number of prospective studies have shown that the fibrinogen concentration is a predictor of a variety of cardiovascular events, including stroke, myocardial infarction, leg ischemia (in arterial disease) and postsurgical arterial re-occlusion. BIOSYSTEMS www.cli-online.com & search 26395

PRODUCT NEWS

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– September 2013

Certified solutions of thyroid hormones Certified Snap-N-Spike solutions of thyroid hormones T4, T3, and reverse T3 are now available at concentrations of 100 μg/mL in 0.1 N ammonia in methanol for each solution standard. These Certified Spiking Solutions are suitable for use as critical starting materials in preparation of calibrators, controls, or linearity standards for analytical testing including LC-MS/MS analysis of free and total thyroid hormone levels. Analytical challenges in thyroid hormone testing range from standardization of free thyroid hormone testing methods to agreement in value assignment of the calibrator concentration. With the push towards developing LC-MS/MS laboratory developed tests (LDTs), the need for highly accurate certified reference materials has significantly increased. Accuracy in the concentration of thyroid hormone calibrators and controls, for example, are complicated by the presence of residual impurities of not only water, solvent and inorganic but also other thyroid hormones in the reference material. Any significant difference in purity of the thyroid hormone reference material or its impurity profile from lot-to-lot could increase variability and impact the test’s accuracy and reproducibility. With thyroid hormones it is critical to use fully characterized compounds because some of the impurities present in each hormone are the other hormones of interest. Cerilliant Certified Spiking Solutions are prepared and certified to the highest industry standards including ISO Guide 34 and ISO 17025. Comprehensive certificates of analysis (COAs) are provided for every Cerilliant certified reference standard and include all analytical data from full neat material characterization and solution standard verification to uncertainty and traceability information for a laboratory’s regulatory requirements. COAs for the thyroid hormone Certified Spiking Solutions also identify T1, T2, T3, reverse T3 or T4 if present in the certified reference material.

Online Submission

CALL FOR POSTERS Submission deadline AUGUST 25, 2013

I00% MEDICAL BIOLOGY

13-14-15 NOV. 2013 CNIT PARIS LA DÉFENSE

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INTERNATIONAL DAYS OF BIOLOGY

CERILLIANT

58e EDITION

www.cli-online.com & search 26394

www.jib-sdbio.fr Automated workflow system Using intelligent robotics, the cobas 8100 automated workflow series automatically prepares blood samples for immediate testing and post-analytical processing, providing high-speed processing of up to 1,100 samples per hour. The system offers dynamic and efficient sample transport options and great flexibility, allowing labs to automate both routine and STAT tests. Healthcare professionals benefit from on-time, high quality diagnostic results for both routine and emergency tests, even at laboratory workload peaks, while the risk of sample mix-ups is minimized. The cobas 8100 series can easily be added onto Roche’s existing cobas analysers and its flexible, modular design allows labs to grow over time as demand increases. ROCHE DIAGNOSTICS www.cli-online.com & search 26398

200 exhibitors Innovations in lab technology presented in sneak preview Free badge (code : PNG1)

An event of

Scientific Meetings

Organised by

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– September 2013

PRODUCT NEWS

ß-hydroxybutyrate strip test The new STAT-Site M ß-HB quantitative strip-based test for ß-Hydroxybutyrate (ß-HB) provides a quick and easy assessment of diabetic ketoacidosis (DKA), delivering results in less than 80 seconds. The presence and degree of ketosis can be rapidly assessed at the point-of-care by accurately measuring blood levels of ß-HB, which accounts for 78% of the three ketone bodies, from just 10 μL of serum or plasma. This provides useful information to provide quality care when monitoring DKA in newly diagnosed patients, whilst also reducing time and costs in an intensive care unit setting. EKF DIAGNOSTICS www.cli-online.com & search 26391

Laboratory automation solution For lower- and mid-volume labs, whose budgets are often particularly constrained and where track-based automation may not be the right approach, VersaCell X3 Solution delivers several advantages. The new platform maintains its small footprint and upright design, but now connects up to three Siemens analysers through a single robotic sample interface for increased productivity gains. By supporting a customized mix of chemistry, immunoassay and/ or integrated systems and associated assay menus, VersaCell X3

Solution helps labs reduce operator sample handling, better prioritize staff work and eliminate pre- and post-analytical processing— all through one-touch sample management. A conveniently placed priority drawer on VersaCell X3 Solution, situated above existing routine sample drawers, provides eight new STAT positions and frees up 50 sample positions for routine processing, enhancing STAT management and increasing overall sample routing flexibility. Plus, modifications to VersaCell X3 Solution’s signature robotic arm also enable it to support both rounded and flat-bottom tubes, and the incorporation of a docking plate reduces service intervention time for all connected systems, increasing uptime so that labs can continue to deliver results to clinicians on time. A centralized screen on VersaCell X3 Solution provides consolidated, single-operator access to all connected analysers, and further data consolidation is achievable through a connection to Siemens’ CentraLink™ Data Management System. SIEMENS HEALTHCARE www.cli-online.com & search 26402

CALENDAR OF EVENTS Nov 3-6, 2013

Dec 5-8, 2013

Seoul, Korea

www.istanbul2014.org

CMEF Autumn 2013

European Biomedical Labora-

www.kimes.kr/eng

July 27-31, 2014

Xiamen, China

tory Science Congress

www.cmef.com.cn/en/

Athens, Greece

May 10-13, 2014

Chicago, IL

http://ebsc2013.com/

24th European Congress of

www.aacc.org

AACC 2014

Clinical Microbiology and

Nov 13-15, 2013 JIB 2013 – Journées Interna-

Jan 27-30, 2014

Infectious Diseases (ECCMID)

October 7-10, 2014

tionales de Biologie

MEDLAB at Arab Health 2014

Barcelona, Spain

EuroLabFocus

Paris, France

Dubai, UAE

www.congrex.ch/eccmid2014

Liverpool, UK

Tel. +33 1 4756 5079

www.arabhealthonline.com/

e-mail: [email protected]

en/Medlab/

www.jib-sdbio.fr

www.eurolabfocus2014.org June 20-22, 2014 XIIIth Int’l Congress of Pediat-

Feb 18-20, 2014

ric Laboratory Medicine

Nov 20–23, 2013

MEDLAB Asia Pacific 2013

Istanbul, Turkey

Medica 2013

Marina Bay Sands, Singapore

www.icplm2014.org

Düsseldorf, Germany

www.medlabasia.com

www.medica.de

June 22-26, 2014 March 13-16, 2014

IFCC WorldLab 2014

KIMES 2014

Istanbul, Turkey

For more events see: www.cli-online.com/events/ Dates and descriptions of future events have been obtained from official industrial sources. CLi cannot be held responsible for errors, changes or cancellations.

A91DX-9252-A1-4A00 Product availability varies by country. Not available for sale in the U.S. © 2012 Siemens Healthcare Diagnostics Inc.

Test patients, not your patience. With the CLINITEK Novus analyzer, Siemens answers the need for faster, more reliable urinalysis testing.

siemens.com/novus

Most would agree that urinalysis testing is labor intensive and time consuming. When labs are overworked, testing accuracy may be compromised. The CLINITEK Novus™ analyzer is changing that by addressing the relationship between the two.

Performs Albumin-to-Creatinine ratio test. Detects intact red blood cells for enhanced sensitivity in identifying kidney disease.

Building on the technology of our CLINITEK Atlas® system, the CLINITEK Novus analyzer improves workflow with cassette-based test loading and increases throughput. A color touch screen features intuitive navigation along with customizable menu options. Plus, a new optical system enhances clinical accuracy. Faster throughput. Improved workflow. Optimal patient care. When labs are under pressure, the CLINITEK Novus analyzer is up to the test. Take a self-guided tour at siemens.com/novus or contact your Siemens representative.

Answers for life. www.cli-online.com & search 26339

Bio-Rad Laboratories

HEMOGLOBIN TESTING

See the Difference Bio-Rad HPLC Lets You See the Whole Picture %LR5DG$FDOORZV\RXWRVHHSRWHQWLDOLQWHUIHULQJ VXEVWDQFHVLQFOXGLQJKHPRJORELQYDULDQWVZLWK \RXU+E$FUHVXOWV %LR5DGKDVIXOO\DXWRPDWHGVROXWLRQVWKDWSURYLGH DFFXUDWH+E$FUHVXOWVHYHQLQWKHSUHVHQFHRI +E6&'DQG(YDULDQWVDVZHOODVGHWHFWLRQ RIRWKHUFRPPRQKHPRJORELQYDULDQWV 7KH9DULDQWOO785%2WHVWLQJV\VWHPOHWV\RXVHHWKH ZKROHSLFWXUHHQVXULQJDFFXUDF\LQSDWLHQWRXWFRPHV

Bio-Rad A1cō6HHWKH'LIIHUHQFH For more information, contact your local Bio-Rad office

1-800-224-6723

www.bio-rad.com/diagnostics

www.cli-online.com & search 26325