The particles were counted in the venous line using a Hiarc/Royco 9703 ... Hiarc/Royco 9703 particle counting system. ..... Int J Artif Organs, 17 (11), 595-602.
Jens Hartmann
DISSERTATION
Procedural aspects in extracorporeal blood purification
angestrebter akademischer Grad
Doktor der Naturwissenschaften (Dr. rer. nat.)
Verfasser:
Mag. Jens Hartmann
Matrikel-Nummer:
8800391
Dissertationsgebiet:
Zoologie (A 091 439)
Betreuer:
Univ.-Prof. Dr. Dieter Falkenhagen
Wien, am 17. August 2007
Jens Hartmann
Acknowledgement I would like to thank my advisor Prof. Falkenhagen for giving me the possibility to work in his department on the interesting topics. I appreciate all his feedback and the suggestive discussions regarding the different topics of this thesis. Furthermore, many thanks go to my colleagues at the Center for Biomedical Technology, Danube University Krems, for the outstanding cooperation. Especially I want to thank Dr. Martin Brandl for our excellent cooperation in the field of medical technology and during the ongoing development of the MDS and its components as well as the citrate anticoagulation monitor. Special thanks go to Dipl. MTA Ute Fichtinger and Dipl. MTA Claudia Schildböck for their superior work in the laboratories. Moreover I want to thank Mag. Karin Strobl who always had a helping hand in the laboratory as well as in data processing. And last but not least, thanks to my family Claudia, Nick, Lukas and Alexa to bear with me during the stressful time of this thesis.
The projects within which this thesis was developed were financed by the government of Lower Austria, the Christian Doppler Society (Christian Doppler Laboratory for Specific Adsorption Technologies in Medicine) and/or the European Commission.
Jens Hartmann
Contents Zusammenfassung .............................................................................................................................. 4 Abstract................................................................................................................................................ 6
1.
Introduction ..................................................................................................8 1.1.
Overview of current blood purification systems.................................................. 8
1.1.1. Hemodialysis ........................................................................................................................... 8 1.1.2. Peritoneal dialysis.................................................................................................................... 9 1.1.3. Hemofiltration ........................................................................................................................ 10 1.1.4. Hemodiafiltration.................................................................................................................... 10 1.1.5. Plasmapheresis ..................................................................................................................... 11 1.1.6. Hemoperfusion ...................................................................................................................... 11 1.1.7. Liver support systems............................................................................................................ 12
2.
1.1.7.1.
The HemoCleanseTM-DT & HemoCleanseTM-PF ......................................................... 15
1.1.7.2.
The MARS® .................................................................................................................. 16
1.1.7.3.
The Prometheus® system ............................................................................................ 18
1.1.7.4.
The MDS ...................................................................................................................... 19
1.1.7.5.
Other systems and techniques .................................................................................... 21
Technical aspects of extracorporeal blood purification .........................25 2.1.
Use of microparticles in blood purification ........................................................ 25
2.2.
Anticoagulation ..................................................................................................... 27
2.2.1. Heparins ................................................................................................................................ 28 2.2.2. Heparinoids ........................................................................................................................... 30 2.2.3. Hirudin ................................................................................................................................... 30 2.2.4. Fondaparinux Sodium (Arixtra®)............................................................................................ 30 2.2.5. Argatroban............................................................................................................................. 31 2.2.6. Nafamostat mesilate.............................................................................................................. 31 2.2.7. Citrate .................................................................................................................................... 32
3.
Publications ................................................................................................34 3.1.
Abstracts and Lectures ........................................................................................ 34
3.1.1. Membrane leakage detection based on Micro-Particles marked with fluorescent dyes ....... 34 3.1.2. Microparticles in Extracorporeal Blood Purification – A Novel Device for Highly Sensitive Leakage Detection................................................................................................. 36 3.1.3. A Novel device for Highly Sensitive Leakage Detection for Microparticle Suspension Based Blood Purification Systems ........................................................................................ 37
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3.1.4. In-vitro Dialysability of Sugammadex, a Selective Relaxant Binding Agent for Reversal of Neuromuscular Block Induced by Rocuronium ..................................................................... 39 3.1.5. Citrate anticoagulation and activation of the complement system ........................................ 42 3.1.6. Optimization of Citrate Anticoagulation ................................................................................. 45 3.1.7. Encapsulation of C3A cells into alginate microcapsules for the potential use in a bioartificial liver device........................................................................................................... 47
3.2.
Peer reviewed publications & patents................................................................. 48
3.2.1. Particle Leakage in Extracorporeal Blood Purification Systems Based on Microparticle Suspensions .......................................................................................................................... 48 3.2.2. Detection of Fluorescently Labeled Microparticles in Blood.................................................. 53 3.2.3. New Methods for Hemoglobin Detection in a Microparticle-plasma Suspension.................. 61 3.2.4. Fluidized Bed Adsorbent Systems for Extracorporeal Blood Purification ............................. 70 3.2.5. Verfahren und Vorrichtung zur Detektion von Markierten Mikropartikeln (Patent) ............... 76 3.2.6. Efficient Adsorption of Tumor Necrosis Factor with an in vitro Set-Up of the Microspheres Based Detoxification System .......................................................................... 88 3.2.7. In vitro investigations of citrate clearance with different dialysis filters ................................. 94
4.
Literature...................................................................................................116 Curriculum Vitae .............................................................................................................................. 124 Lebenslauf ....................................................................................................................................... 125
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Figures Figure 1: Flow scheme of the Biologic-DT® system. .................................................................. 15 Figure 2: Flow scheme of the MARS®. ....................................................................................... 17 Figure 3: Flow scheme of the Prometheus® system................................................................... 18 Figure 4: Flow scheme of the MDS. ........................................................................................... 20 Figure 5: Flow scheme of the HepatAssistTM.............................................................................. 23 Figure 6: Percentage of accessible particle volume against diffusion depth for three different particle sizes.................................................................................................. 26 Figure 7: The coagulation cascade and the effects of anticoagulants. ...................................... 28
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Jens Hartmann
Zusammenfassung Im Vergleich zur weitgehend optimierten und standardisierten Dialysebehandlung stehen komplexere Verfahren wie Leberunterstützung, Behandlung von Sepsis und Behandlung von Autoimmunerkrankungen mittels extrakorporaler Blutreinigung noch am
Anfang
der
verfahrenstechnische
Entwicklungen. Aspekte
der
Diese
Arbeit
extrakorporalen
behandelt
ausgewählte
Blutreinigung,
insbesondere
Methoden der Antikoagulation mit dem Schwerpunkt Zitrat-Antikoagulation sowie adsorptive Elimination von Toxinen mittels Mikropartikeln. Beim
Einsatz
von
Mikropartikeln
in
der
extrakorporalen
Blutreinigung
muss
sichergestellt werden, dass im Erstfehlerfall, wie z. B. im Fall einer defekten Filtermembran, keine Partikel in den Patienten gelangen (first fault safety). Im Rahmen dieser Arbeit wurde ermittelt, welche Partikelmengen im Fehlerfall ohne ein Detektionssystem
in
den
Patienten
gelangen
würden.
Weiters
wurde
ein
Sicherheitssystem entwickelt, welches im Fehlerfall geringste Mengen an Mikropartikeln in Blut detektiert und eine sichere Behandlung ermöglicht. Einige der für die adsorptive Elimination von geladenen Toxinen eingesetzten, vorrangig kationisch geladenen Adsorber entfernen auch Heparin aus dem extrakorporalen Blutkreislauf. In diesen Fällen, aber auch aus Gründen der Verträglichkeit und der Eignung für Langzeitbehandlungen, bietet daher die Antikoagulation mit Zitrat wesentliche Vorteile. Da die Antikoagulation in enger Beziehung mit dem Komplementsystem steht, ist eine Berücksichtigung des Komplementsystems und dessen Unterdrückung von größerer Bedeutung als bisher angenommen. Ein weiterer Schwerpunkt dieser Arbeit war daher, durch in vitro Versuche an Vollblut die für eine weitgehende Unterdrückung des Komplementsystems sowie für eine ausreichende Antikoagulation erforderliche Zitratkonzentration zu bestimmen. Da Zitrat nicht in die Blutzellen eindringt, sondern sich hauptsächlich im Plasma des Patienten verteilt, hat der Hämatokrit des Patienten einen wesentlichen Einfluss auf die Wirkung von Zitrat als Antikoagulans. Dies wurde in bisherigen Empfehlungen zur Zitrat-Dosierung nicht oder kaum berücksichtigt. In dieser Arbeit wird die Abhängigkeit 4
Jens Hartmann
der für eine ausreichende Antikoagulation notwendigen Zitratmenge vom Hämatokrit des Patienten anhand von in vitro Untersuchungen ermittelt. Während der Metabolismus von Zitrat beim gesunden wie beim leberkranken Patienten weitgehend bekannt ist, existieren nur sehr wenige Daten über die Elimination von Zitrat mittels Dialyse. Im Rahmen dieser Arbeit wurde die Dialysierbarkeit von Zitrat in Abhängigkeit des eingesetzten Filtertyps sowie bei unterschiedlichen Dialysat- und Plasmaflussraten untersucht. Die neuen Erkenntnisse zur Zitratantikoagulation, welche in den Untersuchungen dieser Arbeit gewonnen wurden, stellen die Basis für die Entwicklung eines weitgehend automatisierten
Zitrat-Antikoagulationssystems
am
Zentrum
für
Biomedizinische
Technologie der Donau-Universität Krems dar.
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Jens Hartmann
Abstract While dialysis treatment became standardized and widely optimized in the past decades, newer and more complex extracorporeal treatments such as adsorptive liver support, treatment of sepsis or autoimmune diseases are still under development or at least not optimized yet. This thesis covers selected procedural aspects in these extracorporeal blood purification techniques, with its main focus on microparticles in blood purification and citrate anticoagulation. For the application of microparticles in extracorporeal blood purification, particle transfer in the case of a ruptured hollow fiber membrane has to be considered. Therefore, in vitro studies were performed to quantify the particle leakage in a defective system. Furthermore, a highly sensitive leakage detector was developed to enable first fault safety for the treatment of patients. Heparin is a broadly used anticoagulant in blood purification. In microparticle-based adsorption systems, anionic exchangers are used for the removal of charged toxins such as bilirubin. Commonly, the anionic exchangers eliminate heparin as well. Therefore, citrate anticoagulation offers fundamental advantages in these systems. Due to the close connection between the anticoagulation and the complement cascade, the inhibition of the complement system has to be considered as an important contribution to an adequate anticoagulation as well. Aim of this thesis was to evaluate the impact of citrate on the complement cascade and to find the correct citrate concentration to inhibit coagulation as well as complement activation during the treatment. Since citrate distributes in the plasma and does not enter blood cells, the patient’s hematocrit plays an essential role for citrate dosage to achieve adequate anticoagulation. In this thesis, in vitro experiments were conducted to elucidate the connection between patient’s hematocrit and the effect of citrate on the coagulation cascade. While citrate metabolism in healthy patients as well as in patients suffering from liver failure is well explored, there is still a lack of information on the dialysability of citrate, especially regarding different dialysis filters and different dialysate flow rates. Another 6
Jens Hartmann
aim of this thesis was, therefore, to determine the clearance of citrate for high flux and low flux filters of different manufacturers as well as the clearance for different dialysate and plasma flow rates. The expertise of the research activities in the field of citrate anticoagulation will be the base for a newly designed citrate anticoagulation monitor at the Center for Biomedical Technology, Danube University Krems.
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Jens Hartmann
1. Introduction 1.1.
Overview of current blood purification systems
1.1.1. Hemodialysis Hemodialysis was clinically introduced as a therapeutic method to treat patients suffering from kidney failure in 1924 by the German physician Georg Haas. A major pioneer was W. J. Kolff, who developed the first effective dialysis device which could be used clinically in 1945. The principle of dialysis is the following: the patient’s blood is pumped through a semipermeable membrane. On the other side of the membrane, dialysis fluid consisting of an aqueous solution with physiologic concentrations of ions and buffer is (re)circulated by another pump. Diffusive and convective transports through the membrane enable the removal of water soluble toxins as well as the balance of essential ions in the patient’s blood. While ions and small molecules like urea and creatinine are mainly removed by diffusive transport, larger molecules with molar masses of more than 500 (middle molecules), i.e. cytokines or ß2-microglobulin, are mainly removed by convective transport. Protein-bound toxins and substances with a molar mass over ~20.000 can neither be removed adequately by dialysis nor by convective transport due to the restrictions of the membrane and the limited solubility of the toxins. Pore sizes, pore size distribution and hydrophilicity of the membrane material have an important effect on the efficiency of the dialysis treatment. Different dialysis techniques such as low flux dialysis and high flux dialysis are used according to the needs of the patient. For both high flux and low flux dialysis, hollow fiber filters are used as semipermeable membranes. Typical
materials
for
hemodialysis
membranes
are
cellulose
derivatives
(cellulosediacetate, cellulosetriacetate, cuprophane), polysulfone or polyethersulfone, while there are several advantages and disadvantages for the different materials, especially with respect to hemocompatibility (complement activation, activation of the coagulation cascade), hydrophilicity, pressure tolerance and reusability.
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Jens Hartmann
In low flux dialysis, membranes with a low molar mass cut-off in the range of approximately 10.000 and an ultrafiltration coefficient below 10 ml/mmHg·m² are used. A membrane’s cut-off is defined as its size exclusion limit. Low Flux dialysis is the conventional way of hemodialysis. For the effective removal of middle molecules, a higher convective transport is needed. This can be achieved by using high flux dialysis membranes, which offer as a result of larger pores a higher cut-off in the range of up to 50.000 Dalton and an ultrafiltration coefficient higher than 10 ml/mmHg·m². Therefore, with high flux dialysis, higher ultrafiltration rates, i.e. higher volume removal during treatment, can be achieved. The major advantages of high flux dialysis are the suitability for long term treatments due to better biocompatibility, and the effective removal of middle molecules. One disadvantage of high flux dialysis is the higher risk of endotoxin contamination of the extracorporeal circuit via the dialysate circuit due to the larger pores of the membrane, which can be avoided by filtering the dialysate before entering the dialyzer or by using endotoxin removing dialyzers [1].
1.1.2. Peritoneal dialysis In peritoneal dialysis, the patient’s peritoneum is used as the semipermeable membrane for the removal of uremic toxins. About 1.5 - 2 L of hyperosmolar dialysis fluid is introduced to the peritoneal space, and the concentration gradient of toxins and the high osmolarity of the dialysis fluid are the driving forces for the diffusive and convective transport through the peritoneum, respectively. After several hours, the dialysate is removed and substituted by fresh dialysate. For peritoneal dialysis, no extracorporeal blood circuit is needed. Therefore, this method of dialysis is suitable for elderly patients, especially during the first 2-4 years of dialysis treatment, or patients with low blood volume or blood pressure. Compared to synthetic dialysis membranes, the peritoneum has a higher porosity. This leads to marginal protein loss during treatment. Peritoneal dialysis can be applied at home, leading to a higher quality of life for dialysis patients. However, after several years of regular treatment the permeability of the
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peritoneum degrades and the treatment becomes inefficient. Furthermore, peritoneal dialysis retrieves the risk of peritonitis and catheter infections.
1.1.3. Hemofiltration Hemofiltration is a technology which removes uraemic substances via convective transport. The relatively high volumes that are removed via a hemofilter are substituted to the extracorporeal blood circuit. The substitution can be performed upstream of the filter (predilution) or downstream of the filter (postdilution). With hemofiltration, especially middle molecules can effectively be removed compared to standard dialysis which predominantly removes smaller molecules. Hemofiltration does not need water treatment via reverse osmosis. Therefore, it has the logistical advantage of the possibility to be used outside dialysis centers. On the other hand, sterile ultrafiltration fluid in considerable amounts (15-20 L) is necessary which complicates the handling when reverse osmosis is not available.
1.1.4. Hemodiafiltration Hemodiafiltration is a combination of hemodialysis and hemofiltration, leading to a very effective removal of both small and middle molecules. Therefore, hemodiafiltration is the most effective treatment for dialysis patients. Before the first systems for liver support entered the market, hemodiafiltration was one of the options to treat patients with liver failure. Using dialysate for substitution offers a very economical way of this efficient treatment option and enables high rates of substitution up to 15-20 L per treatment. This mode of treatment is called online hemodiafiltration. However, rather poor results with all systems with small membrane pore size initiated the development of systems based on membrane/adsorption technologies using larger pore sizes for a better separation of larger as well as hydrophobic substances.
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1.1.5. Plasmapheresis In plasmapheresis, plasma is separated from patient’s blood by centrifugation or by a plasma filter. The separated plasma is discarded and substituted by human albumin solution (HSA), fresh frozen plasma (FFP) or a mixture of these substitution fluids. Due to the plasma filter’s high cut-off, also very large molecules such as immune complexes can be removed with plasmapheresis. Disadvantages of plasmapheresis are the high costs, especially for HSA, allergic reactions as well as lung edema due to the substitution fluid, which also can be contaminated by HIV or HBV in the case of FFP.
1.1.6. Hemoperfusion In hemoperfusion, anticoagulated whole blood is perfused through an adsorption column to remove target molecules, especially lipophilic substances which cannot be removed via dialysis, such as LDL (low density lipoproteins) or toxins in the case of acute intoxications. Since there is no need for a separating filter (plasma or albumin filter), the procedure is easy and the extracorporeal volume is low in comparison to technologies based on plasma or albumin separation. Because blood and especially blood cells come in direct contact with the adsorbent material, special attention has to be paid to the biocompatibility of the adsorbent materials to avoid complement activation, clotting or release of cytokines. To optimize biocompatibility, the used materials, usually activated charcoal or ion exchangers, are coated with a biologically inert material. An example for hemoperfusion is the Fresenius DALI (direct adsorption of lipoproteins) system. It was designed to remove LDL from patients suffering from familiar hypercholesterinemia. Other examples are the Asahi CH-350 column which is based on coated activated charcoal, and the Toraymyxin cartridge based on polymyxin B adsorbents for the effective removal of endotoxins in sepsis or endotoxemia [2].
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1.1.7. Liver support systems None of the technologies which were described above, except plasmapheresis and hemoperfusion, are capable for an efficient removal of protein bound, hydrophobic toxins, which are the target substances for liver failure. Due to high standards of biocompatibility,
clinical
compatibility
and
economic
considerations,
expanded
technologies have to be used for the removal of these substances. In the following chapters, different liver support systems are described. All of them are extracorporeal blood purification systems in which blood from the patient is pumped through a tubing system to the device, where the purification procedure based on different techniques take place. After toxin elimination, the blood is pumped back into the patient. Other than dialysis devices, all these systems are used in the intensive care unit only. Patients with liver failure usually show comatose episodes at various severities depending on the grade and duration of liver failure. Before the today’s liver support systems were developed, other techniques have been applied. In the late 1950’s, blood exchange transfusion was used for the treatment of acute liver failure patients [3]. Some years later, experiments with extracorporeal cross circulation and extracorporeal primate, pig or bovine liver perfusion partly led to promising results, but were not continued because of ethical reasons and the complicated handling in the clinic [4]. Another procedure which was applied is the therapeutic plasma exchange, where plasma is separated by a membrane or by centrifugation and substituted by fresh frozen plasma (FFP). However, also this method shows several disadvantages such as lung edema, arrhythmia or acute respiratory distress syndrome (ARDS) [5]. Finally, the most recent among the conventional methods for the treatment of liver failure, i.e. plasmaperfusion [6], hemoperfusion [7] [8] and plasma sorption [9] led to the development of the today’s liver support systems. Main targets for the removal of toxins in liver failure are bilirubin, aromatic amino acids, cholic acids and phenol. Bilirubin is a product of hemoglobin catabolism. Haem, the central chromophore of hemoglobin, is oxidized to biliverdin, a blue-green colored intermediate which is
12
Jens Hartmann
converted by biliverdin reductase to unconjugated bilirubin (bilirubin IXα). Unconjugated bilirubin is highly hydrophobic and bound to albumin. In healthy patients, the albumin bound bilirubin is conjugated to UDP-glucuronyltransferase, leading to conjugated bilirubin which is hydrophilic and can be excreted via the bile. Patients with liver failure frequently show impaired conjugation of bilirubin, therefore reduced excretion and high serum bilirubin. Although bilirubin per se has no or only low toxic impact, at high concentrations in serum, the albumin is almost completely occupied and therefore the transport function of albumin is disturbed which further deteriorates the patient’s condition. Aromatic amino acids (tryptophan, phenylalanine and tyrosine) are mainly metabolized by the liver, while branched chain amino acids (leucine, isoleucine and valine) are mainly metabolized by the muscles. In patients with chronic liver failure, the serum level of aromatic amino acids is raised, while due to a higher metabolism of branched chain amino acids the serum level of the latter is decreased. The ratio of aromatic amino acids to branched chain amino acids is called fisher ratio (fisher index) and is used as an indicator for the severity of chronic liver failure. Aromatic amino acids are the precursors of neurotransmitters, and at high concentrations it is believed that imbalanced amino acid concentrations in brain lead to production of false neurotransmitters which cause hepatic encephalopathy [10]. High concentrations of bile acids can indicate cholestatis or lipid metabolism disorders. Bile acids are thought to be at least one reason for pruritus in liver failure. Since the liver takes over the metabolism and detoxification of aromatic substances, especially via the cytochrome P450 monooxygenase system, those substances accumulate in patients with liver failure. Therefore, phenol or similar aromatic substances are commonly used as a target substance in the development of adsorbents for devices for liver support. Liver failure can develop after virus infection (especially HBV virus), trauma, drug abuse, intoxications or as a consequence of sepsis. Liver support systems are used primarily for bridging to transplantation in acute or acute on chronic liver failure, which means to treat the patient to overcome the waiting period for a compatible allograft. Without adequate detoxification, the patient’s survival time is limited, depending on the severity, to a few days. Although the survival time is prolonged with liver support 13
Jens Hartmann
treatment, the maximum treatment time is still limited, since many other liver functions such as carbohydrate-, protein- and fatty acid metabolism cannot be taken over by these systems. Due to the fact that many patients suffering from acute liver failure die after 2-3 weeks from infections despite the treatment with an artificial support system, it seems to be clear that also the immune regulative function of the liver is severely disturbed. Another application of artificial liver devices is bridging to liver cell regeneration to enable the liver’s recovery to normal function. Furthermore, liver support is applied after liver transplantation to bridge the time gap until the transplanted liver reaches sufficient function. Other than in dialysis, where there are standards like Kt/V to define the treatment dosage, in liver support mainly the removal from pre-treatment to post-treatment values of various substances have been quoted. The investigation of the removed amount of toxins in liver failure is more difficult, manly due to adsorptive removal and a mostly unknown generation of these substances during treatment. However, there are attempts for kinetic modeling for the quantification of the removed toxins in liver failure [11]. For acute and chronic liver disease, no reliable statistical data about the worldwide number and distribution of patients are available. In the US, approximately 150.000 patients suffering from liver failure are treated in the intensive care units every year, and about 27.000 patients die from chronic liver disease or cirrhosis per year (AASLD, American Association for the Study of Liver Diseases, 2005). An extrapolation of these numbers to the global market points out the urgent needs for liver support systems.
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1.1.7.1. The HemoCleanseTM-DT & HemoCleanseTM-PF The development of these two systems started with their predecessor, the Biologic-DT® system [12] [13] [14] (HemoCleanse, Inc., Lafayette, IN, USA). In this system, the extracorporeal blood circuit is separated by a plate dialyzer from an adsorbent suspension. By rhythmic movement of the plate dialyzer and the corresponding opening and closing of the occluders, blood flow and filtration through the dialyzer is maintained. For adsorptive removal of toxins, a mixture of activated charcoal and a cation exchanger is used in the filtrate circuit. An accumulator in the filtrate circuit acts as a volume compensator. To provide first fault safety in the case of a broken filter, an infrared-based particle detection unit is installed in the blood circuit. Figure 1 shows a flow scheme of the Biologic-DT® system.
Pump Accumulator Occluder
Occluder
Occluder
Dialyzer
Occluder
Patient
Sorbent bag
Figure 1: Flow scheme of the Biologic-DT® system.
The main disadvantage of the Biologic-DT® is the relatively low molecular weight cut-off of the plate dialyzer in the range of 5000. The membrane is based of cuprophane as a flat sheet membrane made from complement activating regenerated cellulose. Removal 15
Jens Hartmann
of substances with higher molecular weight or protein bound toxins such as bilirubin is limited. Therefore, the system was upgraded with an additional adsorbent suspension circuit downstream from the dialyzer, which is separated from blood stream by a plasmafilter (Biologic-DTPF system). The Biologic-DT® got the FDA approval for treatment of intoxications and chronic liver failure and has shown promising results in treatments of patients of liver failure [15]. Further development of the system resulted in the second generation devices called HemocleanseTM-DT and HemoCleanseTM-PF. Main difference of the new devices is their smaller size and their function as an add-on module to conventional dialysis machines.
1.1.7.2. The MARS® The MARS system [16] (Molecular Adsorbents Recirculating System) was originally developed for the company Teraklin, which was taken over by Gambro in 2004. In contrast to membrane/adsorption based systems like the MDS or the Prometheus® system (chapter 1.1.7.3 and 1.1.7.4), MARS® is based on albumin dialysis. The patient’s blood circuit is separated by a high flux dialyzer from a closed-loop circuit containing 600 ml of 20 % HSA solution. Since the dialyzer’s low cut-off does not allow for an exchange of albumin molecules between the two circuits, the dialyzer is coated with albumin and is claimed by the inventors of MARS to enable the albumin-bound toxins to be released from patient’s albumin and pass the membrane to bind to the albumin in the secondary circuit. The albumin in the secondary circuit is regenerated by adsorbent columns which contain activated charcoal and an anion exchanging resin. For removal of hydrophilic substances, a dialyzer is introduced into the albumin circuit. Figure 2 shows the flow scheme of the MARS®. Apart from the high costs for the HSA, the use of HSA implies another disadvantage. Commercially available HSA contains N-acetyl-tryptophan and/or octanoate as stabilizers, which are strongly bound to albumin. The binding constant of the stabilizers is close to the binding constant for bilirubin. This means low bilirubin binding capacity. 16
Jens Hartmann
Furthermore, the stabilizers could be released from albumin and enter the patient’s blood circuit. Main disadvantage of the MARS technology is its ineffective removal of strongly albumin bound substances due to the impermeability of albumin through the membrane, which was demonstrated in in-vitro studies [20] as well as in clinical trials [26] [27].
Blood pump
Filtrate pump
Dialyzer High Flux Dialyzer
Anion exchanger
Activated charcoal
Patient Blood circuit
Albumin circuit
Figure 2: Flow scheme of the MARS®.
So far, more than 10000 treatments have been performed with MARS®, and the device is approved by the FDA (data from 04/2007; personal communication with S. Mitzner). Although there are some promising results [17] [18], the efficiency of MARS® was controversially discussed [19], and studies that demonstrate a clear benefit for the patient are still pending.
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1.1.7.3. The Prometheus® system The Prometheus® system is a combined membrane-adsorption system for the treatment of acute or acute on chronic liver failure based on the FPSA technology developed by Falkenhagen et al [20]. Figure 3 shows a flow scheme of the system. Patient’s blood is pumped through the Albuflow® filter, which is a hollow fiber membrane especially designed for the Prometheus® system. The Albuflow® filter has a sieving coefficient of 0.65 for albumin. Since many metabolic toxins that are of relevance in liver failure are strongly bound to albumin, all those substances are able to pass the membrane and enter the secondary (filtrate) circuit. The albumin-rich fractionated plasma is recirculated in the closed secondary circuit at flow rates of 300-500 ml/min through two adsorbent columns. The first column is a neutral resin for adsorptive removal of hydrophobic substances, especially aromatic amino acids (tryptophan), cholic acid and phenol. For removal of bilirubin, another column with an anion exchanger resin is used. Each adsorbent column has a volume of approximately 330 ml. The particle size of the adsorbents is in the range of 0.2 - 0.8 mm. Since many patients suffering from liver failure show also impaired renal function, downstream from the Albuflow® filter a dialysis filter takes over the conventional renal support in the blood circuit.
Filtrate pump
Blood pump
Neutral resin
Filtrate circuit Blood circuit
Albumin filter
Anion exchanger
Dialysis filter Patient
Figure 3: Flow scheme of the Prometheus® system.
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The Prometheus® system was introduced to the market in the year 2001. Meanwhile, the system is installed in more than 65 intensive care units in 26 countries all over the world. About 700 patients have been treated in approximately 3000 treatment session up to date [data from 03/2007, personal communication with Fresenius Medical Care, Bad Homburg, Germany]. Treatment with Prometheus® led to promising results in several studies [21] [22] [23] [24]. Compared to the MARS®, it shows superior elimination of bilirubin (especially unconjugated bilirubin), ammonia and urea [22] [25] [26] [27]. Main disadvantage of the Prometheus® system is its relatively high extracorporeal volume which can cause temporary blood pressure drop during treatment [22].
1.1.7.4. The MDS The Microspheres Based Detoxification System (MDS) is, similar to the Prometheus® system, a combined membrane-adsorption system. Instead of the conventional use of adsorption columns, a microparticle suspension is used in the filtrate circuit [28]. In consequence of its high flexibility it can be applied for different applications, i.e. acute dialysis, liver support [29], autoimmune diseases, LDL-apheresis [30] and sepsis [31]. The MDS was patented in Austria (1994), Japan (1997) and in the US (1999) [32]. Figure 4 shows a flow scheme of the MDS. Blood, drawn from the patient, is pumped through a hollow fiber albumin filter. The filter separates the blood circuit (primary circuit) from a plasma circuit (secondary circuit, filtrate circuit). A pressure gradient between the two circuits is the driving force for filtration and back-filtration of plasma components (fractionated plasma). The fractionated plasma is recirculated at flow rates between 500 and 4000 ml/min and forms, together with microadsorbents ranging from 1-30 µm in diameter, a dense microparticle suspension. In the secondary circuit, the elimination of toxins of pathophysiologic relevance takes place. Downstream from the albumin filter, a dialysis filter takes over the conventional renal support. Due to its high flexibility, the MDS can be used for several disease patterns. In standard dialysis, acute dialysis, peritoneal dialysis or hemofiltration, hydrophobic protein-bound substances are removed inadequately. For the removal of these 19
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substances, i.e. p-cresolsulfate, indoxyl sulfate, hippuric acid, 3-carboxy-4-methyl-5propyl-2-furanpropionic acid (urofuranic acid) or homocystein [33] [34] [35] [36], selective adsorbents can be used. For the treatment of liver failure, the MDS can be used analogically for the efficient removal of the hydrophobic toxins bilirubin, bile acids, phenols and aromatic amino acids. A mixture of a neutral resin and an anionic exchanger adsorbent can be used to remove hydrophobic and anionic toxins, respectively [28] [29] [37]. Filtrate pump
Blood pump
Blood circuit
Albumin filter
Filtrate circuit
Dialysis filter Patient
Figure 4: Flow scheme of the MDS.
Sepsis is known as an infection with a very complicated course starting with a proinflammatory phase which is followed by an anti-inflammatory phase. The development of sepsis is specific for each patient. Therefore, a standard-treatment is difficult. The MDS offers a very flexible base for the treatment of sepsis, where different antibodybased adsorbents can be applied depending on the patient’s needs to remove proand/or anti-inflammatory cytokines like TNF-a, IL-1 or IL-6 [28] [31] [38]. In a similar way, rheumatoid arthritis can be treated with the MDS using adsorbents based on immobilized IgG antibodies.
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In comparison with most of the other liver support systems, the MDS shows several major advantages. The MDS has a considerably lower extracorporeal volume resulting potentially in a better tolerance of the treatment, especially in terms of the patient’s blood pressure. Furthermore, the microparticles of the MDS can offer a very high surface area of more than 1000 sqm/g, leading to excellent adsorption kinetics and to short treatment times. Especially in the intensive care unit, the simple setup as well as the easy handling of the system should lead to a broad acceptance. The tremendous advantage of the low diffusion distances due to the use of microparticles is discussed in chapter 2.1. The MDS offers a high flexibility by the possibility of using more than one adsorbent either at the same time or in a chronological sequence. To avoid particle transfer into the patient in case of one or more broken hollow fibers, a highly sensitive leakage detector based on fluorescently labeled microparticles has been developed [39] [40] [chapter 3]. Since the MDS is still under development, it is not commercially available yet.
1.1.7.5. Other systems and techniques SPAD System The Single Pass Albumin Dialysis (SPAD) system is similar to the MARS® [41]. It is based on albumin dialysis where the albumin solution is not recirculated but pumped in single pass through the dialysis compartment of a high flux filter. Like in the MARS, its detoxification efficiency for protein bound substances is limited because of the low albumin transfer through a high flux dialysis filter. The SPAD is a non-commercial system and at the time of this publication there are no clinical data based on randomized controlled studies available.
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SEPETTM System Selective Plasma exchange therapy (SEPETTM) is a liver assist device from Arbios Systems, Inc., USA, which is currently under development. In this system, similar to the systems based on the Albuflow® filter, a filter with a cut-off that is lower than the cut-off from a plasmafilter is used to separate fractionated plasma. The fractionated plasma is discarded and substituted by fresh human albumin solution. In other words, the treatment is a fractionated plasma exchange and similar to standard plasmapheresis. In contrast to standard plasmapheresis, essential molecules like fibrinogen, growths factors, immunoglobulins and others are not removed.
Bioartificial Liver systems and Hybrid systems Bioartificial Liver systems are based on liver cells (porcine liver cells, HepG2 or C3A cells) which are enclosed in a bioreactor. A semipermeable membrane in the bioreactor separates the liver cells from the patient’s blood or plasma. The main advantage of these types of liver support systems is the possibility to support also the synthetic activity of the liver, especially the synthesis of proteins that are pathophysiologically important (albumin, coagulation factors, growths factors). Disadvantages of bioartificial liver systems are the need of high cell volumes, the danger of virus infection (PERV in systems using porcine hepatocytes) and the complicated procedure for the preparation of the system in the intensive care unit (growing, feeding, and activating the cells) [42]. The HepatAssistTM (Arbios Systems, Inc) is a hybrid system that combines conventional adsorption to charcoal with a bioartificial module based on porcine hepatocytes attached to a semi-porous membrane in a cartridge. Figure 5 shows a flow scheme of the HepatAssistTM.
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Plasma reservoir
Pump Charcoal column
Plasmapheresis device
Bioartificial Liver cartridge
Oxygenator
Patient
Figure 5: Flow scheme of the HepatAssistTM.
HepatAssistTM is not yet commercially available. However, clinical trials based on a prospective randomized controlled study show promising results for the treatment of fulminant liver failure [43]. For other bioartificial systems like the ELAD® (Extracorporeal Liver Assist Device, Vital Therapies, Inc., San Diego, CA), which is based on the human tumor liver cell line C3A, the AMC-BAL (AMC bioartificial liver, AMC liver center, The Netherlands) [44] [45], or the
MELS
CellModule
(Modular
Extracorporeal
Liver
Support;
Charité
Universitätsmedizin Berlin) [45], not enough data are available to proof that there is a significant positive effect on the patients’ condition. Due to the above mentioned disadvantages, bioartificial liver systems might not be applied successfully in the near future. However, systems that introduce adsorption and bioartificial liver support in one system (hybrid liver systems) could be a promising combination of adsorptive toxin removal and the metabolism of liver cells. Although the amount of cells in these systems is not enough to substitute all metabolic liver functions completely, production of albumin and other important proteins can improve the patient’s condition, and production of important factors such as growth factors might 23
Jens Hartmann
stimulate liver regeneration. Another option of a (hybrid) bioartificial liver is demonstrated in chapter 3.1.7.
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2. Technical aspects of extracorporeal blood purification 2.1.
Use of microparticles in blood purification
Blood purification systems based on adsorption to microparticles with a diameter in the range of 1-50 µm offer several substantial advantages over systems based on particles in the range of 200 µm to 1 mm. The total particle surface which is available for adsorption is the sum of inner and outer surface. Considering only the outer surface or particles based on non-porous materials, for a given particle volume, particles with a diameter of 1 µm have a 1000-fold higher surface than particles with a diameter of 1 mm (surface per volume ratio). As adsorption takes place at the surface of the particles, microparticles offer a much higher adsorption capacity per volume than macroparticles. But also the inner volume has to be considered. To increase the total surface area, usually very porous polymers or charcoals are used. These materials offer surface areas of more than 1000 sqm/g. The efficiency of adsorption to the inner surface of the particles for a given time mainly depends on the material’s pore size and the diffusion coefficient of the substance. This means that when macroparticles are used, not the whole volume of the particles is accessible. In other words, when using macroparticles, adsorption takes place only at the outer spherical shell due to the limited penetration depth of the substances, and the inner part of the particles remains inaccessible. The percentage of accessible volume depends on the diameter of the particles and the diffusion depth of the substances and is shown in Figure 6. Furthermore, the kinetics of adsorption processes is much faster in case of microparticles in comparison to larger particles due to the fact that diffusion time is a function which is proportional to the length of diffusion. To optimize adsorption, the particle diameter has to be equal or smaller than the double diffusion depth. However, as mentioned above, additional total surface increase can be reached by increasing the surface per volume ratio. Unfortunately, microparticles offer some disadvantages. Particles with very low diameters in the range of 1 µm and below often have a high surface energy (zeta potential) and tend to agglutinate. As a side effect, the high surface energy can cause 25
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unspecific adsorption of substances which are not removed by the same material when it is used in the form of macroparticles. In nanotechnology it is a well known fact that substances might change their characteristics when they are extremely reduced in size. To avoid or at least to reduce these properties of microparticles, suitable materials have to be chosen and should be thoroughly tested.
100 % 90 % 80 % 70 % 60 % 50 % 40 % 30 % particle diameter 1 mm
20 %
particle diameter 500 µm particle diameter 100 µm
10 % 0% 0
100
200
300
400
500
diffusion depth [µm]
Figure 6: Percentage of accessible particle volume against diffusion depth for three different particle sizes.
One of the main issues regarding patient safety in the Biologic-DT system, the Biologic DTPF system as well as in the MDS is the particle transfer to the patient in the case of a broken membrane. Medical devices have to offer first fault safety. Therefore, either an additional barrier between the patient and the particle circuit or a particle detector is mandatory. For the Biologic-DT/DTPF systems, this problem was solved by introducing a particle detection unit based on infrared, offering a detection limit of 1/104 to 1/105 (v/v). The extracorporeal blood circuit is monitored by an infrared emitting light source and a corresponding detection unit. In the case of a broken membrane, charcoal
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microparticles will be transferred to the blood circuit. As soon as they pass the detection unit, the light will be absorbed by the charcoal and the detector will give an alarm. In the MDS, a patented fluorescence- & magnetism-based particle detector is used. The advantages and the functionality of this technique are discussed in chapter 3.
2.2.
Anticoagulation
Whenever blood comes in contact with foreign surfaces, the intrinsic pathway of blood coagulation is activated (Figure 7). In extracorporeal blood purification, this happens due to the contact to the tubing system, the dialysis and/or plasma separation filters and air bubbles. Another trigger for the initiation of blood coagulation are the passages where the speed of blood flow changes within short distances due to a change in the diameter in the disposable system, leading to high shear stress. These passages are especially the filter inlet and outlet, but also narrow points such as kinked tubing systems. Although there are some records about anticoagulant-free treatment options, the usual way to prevent clotting in the extracorporeal circuit is the use of an anticoagulant. As Figure 7 shows, the coagulation cascade is a very complex sequence of proteolytic reactions, and many cofactors inhibit or stimulate different steps within the whole cascade. This enables many options of anticoagulation by inhibiting one or several steps of the cascade by the use of different anticoagulants. Although there is a very broad spectrum of available anticoagulants, in the succeeding chapters only the most important anticoagulants for extracorporeal blood purification will be mentioned, with special focus on citrate anticoagulation and its advantageous inhibitory effect on complement activation.
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Intrinsic pathway
Extrinsic pathway
(contact activation)
(injury, trauma)
XIIa
XII
Ca2+
III (tissue factor)
XIa
XI Ca2+
IX
IXa
TFPI
VIIa
VII
Nafamostat ATIII TFPI
VIIIa, PL,
Ca2+
X Heparinoids, Arixtra®
Heparin
PL, Ca2+
Xa
X
ATIII ATIII
V
Va
Xa, Va, PL, Ca2+
Heparin
II (Prothrombin) XIII Ia (Fibrin)
Ca2+
prothrombin activator complex
Hirudin IIa (Thrombin) Argatroban Nafamostat ATIII Heparin XIIIa
Fibrin monomers
Ca2+
I (Fibrinogen)
Figure 7: The coagulation cascade and the effects of anticoagulants. Citrate inhibits all steps where Ca2+ has an activating effect. ATIII … antithrombin III, PL … phospholipids, TFPI … tissue factor pathway inhibitor. Bold arrows for inhibiting influences, dashed arrows for activating influences on the reactions (normal arrows).
2.2.1. Heparins Heparins are negatively charged mucopolysaccharides that are physiologically produced by the liver and the intestine mucosa. It is a commonly used anticoagulant in dialysis. One essential disadvantage of heparin is HIT (heparin induced thrombocytopenia). It is an allergic reaction to heparin where antibodies against the heparin-PF4 (platelet factor 4) are produced, ending up in life-threatening blood clotting. With a prevalence of approximately 4-5 % [46], HIT is a frequent exclusion criterion for heparin anticoagulation. The two main types of HIT are mentioned in chapter 3.2.7.
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There are two different types of heparin, unfractionated heparin (UFH) and low molecular weight heparin (LMWH, fractionated heparin), and both have advantages as well as disadvantages when used in extracorporeal blood purification [47]. UFH is a mixture of different heparin fractions with a molar mass of approximately 400025000. There are several anticoagulative effects of UFH. Its main mechanism in anticoagulation is the about 1000-fold amplification of the effect from antithrombin III (AT III) on thrombin. Furthermore, the low molecular weight fraction of UFH inhibits factor Xa, which plays a central role in the coagulation cascade. Since AT III interacts with several other factors in the coagulation cascade, the effect of UFH is distributed to a lot of steps within the complex coagulation cascade, which leads to an effective inhibition of blood coagulation. The half life of UFH in healthy patients is in the range of 1-2 hours. LMW heparin only inhibits factor Xa and shows less unspecific binding to other proteins, resulting in a more predictable effect [47] [48]. Advantages are the longer half life in the range of 2-4 hours, enabling the possibility of single dose treatment even for long treatment sessions [49], and the lower incidence of HIT. In the case of renal failure, the longer half-life can be a disadvantage due to higher bleeding risk. Higher costs are the major disadvantage of LMWH. Since adsorbents which are used in extracorporeal blood purification for the removal of bilirubin are based on anion exchangers, these adsorbents are well known for their unwanted but effective removal of heparin. For a safe treatment over several hours, accurate monitoring of the anticoagulation status is mandatory, and either continuous infusion or intermittent bolus injections of heparin are necessary, rendering the treatment complicated and expensive. Furthermore, the adsorption capacity for the target toxins can be reduced due to competition for binding sites. Especially in membrane/adsorption based purification systems, the use of LMWH has a major disadvantage due to its high sieving coefficient of the membrane used in high flux dialysis as well as in systems based on the Albuflow® filter (Prometheus®, MDS) and the fast removal via the adsorbent (unpublished results, see also chapter 2.2.4). Effective and widely used antidotes of UFH are protamin hydrochloride and protamin sulphate.
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2.2.2. Heparinoids Heparinoids are heparin-like anionic polysaccharides with anticoagulative properties, which mainly act via inactivation of factor Xa. They are less likely to cause HIT. The main disadvantage of heparinoids is the lack of an effective antidote, which is the reason that heparinoids are rarely used in extracorporeal blood purification.
2.2.3. Hirudin Hirudin is a polypeptide which was originally extracted from the salivary gland of the medical leech Hirudo medicinalis. It acts by selectively inhibiting thrombin. Since it is mainly excreted by the kidneys, its half life is significantly prolonged in renal failure. Although hirudin is meanwhile produced synthetically, it is one of the most expensive anticoagulants. Furthermore, no effective antidote is known. Therefore, hirudin is rarely used in extracorporeal blood purification. In patients with HIT, hirudin is an alternative way of anticoagulation to prevent further complications. However, a very thorough monitoring of the patient to avoid overdosage is mandatory [50].
2.2.4. Fondaparinux Sodium (Arixtra®) Fondaparinux Sodium (brand name Arixtra®) is a synthetic heparin-like pentasaccharide with a molar mass of 1728. It was developed by Sanofi-Synthelabo and Organon and was introduced to the market in 2002. By binding to antithrombin, it selectively inhibits factor Xa, the central factor of the coagulation cascade, and therefore is able to block both the intrinsic as well as the extrinsic pathway (Figure 7). Similar to hirudin, in healthy patients fondaparinux is mainly eliminated by the kidneys. This results in an impaired removal of fondaparinux and a significantly prolonged halflife in patients with renal failure.
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Due to its low molar mass, the sieving coefficient with albumin and plasma filters is close to 1. Therefore, in membrane/adsorption based blood purification systems, especially when anion exchanger resins are used, fondaparinux is removed very efficiently (unpublished results). This should be considered when fondaparinux is used in membrane/adsorption based systems.
2.2.5. Argatroban Argatroban is a synthetic substance with a molar mass of 527. It selectively inhibits thrombin, independently from antithrombin III. It can be monitored with aPTT, ACT and ECT. However, since there is no specific antidote, limited experience for dosage and not enough data for the removal rates via the different blood purification techniques, it is currently rather rarely used.
2.2.6. Nafamostat mesilate Nafamostat mesilate is a synthetic, non-selective serine protease inhibitor with a molecular weight of 540. In addition to its inhibitory effects on thrombin, factor Xa and other factors of the clotting system, it has an inhibitory effect on several factors of the complement system (C1s, C1r, factor B, factor D). Therefore, it could theoretically be used similar to citrate to inhibit both the clotting system as well as the complement system. Disadvantages of nafamostat mesilate arise from reports about thrombus formation or the inadequate reduction of thrombus formation and its inhibitory effect on fibrinolysis [51] as well as its adsorption to polyacrylonitrile dialysis membranes.
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2.2.7. Citrate Ionized calcium is an important factor in several steps of the coagulation cascade (Figure 7). As citrate reacts with calcium ions by forming a chelate complex, the concentration of ionized calcium in blood can be decreased by infusion of citrate. Depending on the amount of citrate, this leads to widely or completely blocked blood coagulation. In the clinic, citrate anticoagulation is carried out by infusion of trisodium citrate or a mixture of trisodium citrate and citric acid (ACD-A solution). In comparison with conventional anticoagulation methods, citrate anticoagulation offers several essential advantages. Anticoagulation with citrate is limited to the extracorporeal circuit, leaving the patient’s coagulation system unchanged. This is achieved by infusion of citrate to the arterial line and calcium chloride solution to the venous line of the extracorporeal circuit. Furthermore, in this context the impaired production of coagulation factors of liver patients has to be taken into consideration. Especially for long-term treatments such as liver support or acute dialysis, citrate anticoagulation offers the tremendous advantage of longer filter durability compared to conventional anticoagulation with heparin [52]. However, some issues have to be considered when citrate anticoagulation is applied. For each mmol of citrate, three mmol of sodium is infused into the patient when trisodium citrate is used. Therefore, especially for long term treatments, dialysate with reduced sodium concentration should be used to avoid hypernatraemia. In patients with intact liver function, about 50% of the citrate metabolism is taken over by the liver [53] [54]. Rapid metabolism of citrate might lead to an excess of bicarbonate, resulting in metabolic alkalosis. A partial substitution of citrate by citric acid in the infusion solution can reduce the risk of alkalosis as well as hypernatraemia. In hemodialysis, the bicarbonate concentration in the dialysate should be lowered when citrate anticoagulation is applied. Furthermore, the infusion of calcium chloride solution has to be monitored to avoid hyper- and hypocalcaemia.
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Another important characteristic of citrate, namely its effect on the complement system via Ca2+-reduction, has been underestimated and is only discussed in more recent publications [55]. There is a close connection between the cascades of the coagulation and the complement system, and a reciprocal effect between both is known [56] [57]. To avoid an adverse effect of one cascade on the other, the inhibition of both has to be assured. All these facts show that a close monitoring during and after treatment of patients treated with citrate anticoagulation is mandatory to avoid citrate accumulation, hypernatraemia, hyper/hypocalcaemia and bleeding. The ratio between citrate and calcium was controversially discussed [58] [59], and there are no reliable data regarding the ratio of citrate to calcium in the chelate complex. Therefore, an approach by a mathematical model for the needed infusion rates to achieve the target value of ionized calcium is difficult, and the needed amount of citrate to inhibit both the coagulation as well as the complement cascade has to be investigated by in vitro experiments. The results of the in vitro experiments, the advantages of citrate anticoagulation as well as its clearance rates in dialysis are discussed in the publications in chapters 3.1.5, 3.1.6 and 3.2.7.
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3. Publications 3.1.
Abstracts and Lectures
3.1.1. Membrane leakage detection based on Micro-Particles marked with fluorescent dyes Poster presentation at the European Society for Artificial Organs (ESAO), Lausanne, Switzerland 2000. Abstract: Background: The use of microadsorbents smaller than 5 µm for blood purification causes some problems in the safety for the patients. Microspheres, on the other hand, offer a very efficient method for blood purification due to their high surface/volume ratio. The aim of the study was to develop a safety system to detect microadsorbents in the primary circuit of the MDS (Microspheres Based Detoxification System) in case of leakage in the plasmafilter. The particledetector should be able to detect microparticles in the blood circuit at a concentration of at least 10-5. Method: Therefore we used adsorbents which have been marked with a fluorescence dye. The emitted fluorescence was measured using a photomultiplier tube in combination with a lock in-amplifier to pick the very low signal out of noise. The spectral selectivity was reached by using a special arrangement of optical lenses and filters for excitation and for emission to prevent the influence of cross light. Results: The use of the sensor system enables the detection of particle concentrations of 10-5 in blood circuits for particles smaller than 5 µm. An easy to handle module of the fluorescence based detector for the MDS was developed. Conclusions: The successful development of the particledetector makes the clinical use of the MDS possible. The new technology also offers possibilities to control membrane filtration processes, especially in aqueous systems, getting a detection rate of at least 10 8.
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Poster:
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3.1.2. Microparticles in Extracorporeal Blood Purification – A Novel Device for Highly Sensitive Leakage Detection Lecture at the Congress of the European Society for Artificial Organs (ESAO), Warsaw, 2004.
Aim: Extracorporeal blood purification systems based on microadsorbent suspensions offer several advantages in comparison to conventional systems based on adsorption columns, particularly in adsorption kinetics, in the flexibility and the lower extracorporeal volume. Aim of this study was the in vitro evaluation of the amount of mircoparticle transfer in case of a cut hollow fiber, and the development of a highly sensitive device for microparticle detection.
Methods: Particle transfer studies were carried out by using filters with one single cut hollow fiber. The particles were counted in the venous line using a Hiarc/Royco 9703 particle counting system. The particle detection device is based on fluorescently labeled cellulose microspheres with a magnetic ferrite core and a magnetic trap using neodynium magnets.
Results: Under worst case conditions, one single cut hollow fiber leads to particle leakage into the patient of more than 30 ml during a 10 hour treatment. In contrast, our newly developed sensitive detection device is able to detect 5 µl of fluorescently labeled magnetic microparticles.
Conclusion: In this study we show that in case of a defective filter significant amounts of microparticles enter the patient’s blood circuit. Therefore, microparticle systems need an additional device to enable first fault safety protection. Our microparticle detection unit is able to detect very low particle leakages caused by defective filters and provides first fault safety for blood purification systems based on microadsorbent suspensions.
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3.1.3. A Novel device for Highly Sensitive Leakage Detection for Microparticle Suspension Based Blood Purification Systems Lecture at the 5th World Congress of the International Society for Apheresis (ISFA), Rostock/Warnemünde, Germany, 2005.
Abstract:
Aim: The
Microspheres
Based
Detoxification
System
(MDS)
is
a
combined
membrane/adsorption blood purification system based on adsorption to microparticles. Compared to conventional systems for blood purification which use adsorbent columns, in the MDS the microparticles are held in suspension by means of high flow rates of 0.5 – 1.5 l/min. Therefore, the MDS provides advantages in usability, flexibility, adsorption kinetics and extracorporeal volume. However, the lack of first fault safety necessitates an additional device for microparticle detection to avoid particle infusion into the patient. Aim of this study was the in vitro evaluation of the amount of mircoparticle transfer in the case of a broken hollow fiber, and the development of a highly sensitive device for microparticle detection.
Methods: The filters for the particle transfer studies were prepared by cutting one single hollow fiber. Samples were drawn at the venous line, particle counts were performed using a Hiarc/Royco 9703 particle counting system. The microparticle detection device uses neodynium magnets as a trap for fluorescently labeled cellulose microspheres with a magnetic ferrite core. To optimize the signal strength, the magnets were positioned at an angle of 60° around the standard tubing system. An assembly with a LED, optical filters and lenses focuses the emission light at the tubing system to the area where the particles are collected. For the production of the fluorescently labeled microparticles, we used cellulose beads as a matrix. The magnetic core of the particles was obtained by precipitating ion salts inside the cellulose particles under alkaline conditions. After several washing procedures, the ferromagnetic particles were activated with tosyl and cresyl violet was
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covalently bound to get fluorescently labeled particles for the extinction/emission wavelength of 590/620 nm.
Results: Our particle transfer study points out that during a 10 hour MDS-treatment up to 30 ml adsorbent may be transferred to the patient. The amount depends on the region of the membrane defect and the membrane type. The detection limit of the device is as low as 5 µl of fluorescently labeled magnetic microparticles and therefore, a total amount of 500 µl mircroadsorbent can be detected when adding the microparticles in a ratio of 1:100 to the adsorbent suspension.
Conclusion: A particle detection device is necessary for blood purification systems that are based on microparticle suspensions. The detection device is very sensitive and ensures first fault safety for the MDS and other microparticle-based blood purification systems by preventing a significant amount of the adsorbent to be infused into the patient.
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3.1.4. In-vitro Dialysability of Sugammadex, a Selective Relaxant Binding Agent for Reversal of Neuromuscular Block Induced by Rocuronium Lecture and Poster Presentation at the annual Meeting of the European Society of Anaesthesiology (ESA), June 2006, Madrid, Spain.
Abstract:
Background and Goal of Study: Sugammadex (Org 25969), a selective relaxant binding agent, forms a complex with rocuronium, thereby reversing neuromuscular block. Sugammadex and its rocuronium complex are almost exclusively cleared via the kidney so decreased clearance is expected with renal impairment. We investigated the dialysability of sugammadex.
Materials and Methods: An in-vitro bicarbonate hemodialysis set-up was used with a 900 mL pool of heparinized human plasma and a bicarbonate buffer as dialysate. Plasma flow was 200 mL/min and was recirculated. Temperature was 37ºC. High- and low-flux membranes were used. Sugammadex and rocuronium were added to the plasma pool to concentrations of 100 µg/mL and 30 µg/mL, respectively, for a molar stoichiometry of 1:1. Plasma samples were taken before entering the membrane at 0, 5, 10, 15, 30, 60, 120 and 180 mins. Sugammadex and rocuronium concentrations were determined using validated liquid chromatographic assays with mass spectrometric detection. A control experiment without dialysate flow evaluated adsorption of the drugs to the polysulfone membrane.
Results and Discussions: Clearance of sugammadex and rocuronium was higher with the high-flux membrane than the low-flux membrane. Negligible clearance was observed in the absence of dialysate flow.
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Experiment
N Analyte
CL, mean (range), mL/min
High-flux
3
Sugammadex
86.3 (79.3-94.8)
Rocuronium
89.0 (83.5-95.0)
Sugammadex
3.9 (2.4-4.9)
Rocuronium
6.3 (5.6-7.2)
3
Sugammadex
6.0 (5.4-6.7)
High-flux, no 1
Sugammadex
0.2
dialysate flow
Rocuronium
0.2
Low flux Low-flux
3
Conclusion(s): Sugammadex in the presence of rocuronium can be efficiently removed from plasma by dialysis using a high-flux membrane, but not a low-flux membrane.
Acknowledgements: Sponsored by NV Organon, Oss, The Netherlands.
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Poster:
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3.1.5. Citrate anticoagulation and activation of the complement system Poster presentation at the annual meeting of the European Society for Artificial Organs (ESAO), Umea 2006, Sweden.
Abstract:
Background: Citrate anticoagulation is a promising way of anticoagulation in extracorporeal blood purification. The limitation of the anticoagulation to the extracorporeal circuit, but also the better biocompatibility, offer important advantages over conventional anticoagulation methods based on heparin or heparinoids. With respect to literature there are different results regarding inhibition of complement activation.
Aim: The aim of this in vitro study was to find the optimal concentration for citrate with respect to ionized Calcium in the extracorporeal blood line to inhibit complement activation as well as coagulation.
Material and Methods: The experiments were carried out firstly in batch tests and secondly in systeminvestigations using a dialysis machine. In all experiments both trisodiumcitrate and ACD-A solution were used for anticoagulation of fresh frozen plasma or fresh blood. Citrate was analyzed with a photometric test kit, ionized Calcium and Magnesium were analyzed with an ionometer (Nova biomedical) and total Calcium and Magnesium were quantified with AAS. Complement activation was measured using ELISA tests.
Results: The results show that adding of citrate to a final concentration of 5-6 mmol/L after stimulation with endotoxins at a concentration of 1 ng/ml results in a Ca2+ concentration below 0.2 mmol/L and a decrease of complement activation down to about 10 %, 42
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whereas anticoagulation can be achieved by decreasing Ca2+ to 0.4 mmol/L. The mathematical calculations taking in account the stoechiometric balance between Calcium and Citrate as well as binding constants are in agreement with the measured results of the in vitro experiments.
Conclusion: These results provide important data for an easy and safe use of citrate anticoagulation in dialysis, liver support and other fields of extracorporeal blood purification and will be the basis of a newly developed automated Citrate Calcium anticoagulation monitor. In acute intensive care patients, inactivation of the complement system by keeping the Ca2+ concentration below 0.2 mmol/L is worthwhile.
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Poster:
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3.1.6. Optimization of Citrate Anticoagulation Poster Presentation at the annual meeting of the European Society for Artificial Organs (ESAO), Krems 2007, Austria. Accepted.
Abstract:
Aim: Citrate anticoagulation offers several advantages under intensive care conditions due to its ability to block coagulation via reduction of Ca2+. Citrate induced Ca2+- as well as Mg2+-reduction also influences complement activation. There are different reports which have not clarified the influence of citrate anticoagulation on the complement system. Aim of the study was to find the optimal concentration of Ca2+ during citrate anticoagulation with respect to the suppression of coagulation as well as the complement cascade.
Methods: Our investigations were performed under in-vitro conditions using heparinized fresh blood (3 IU heparin/ml) spiked with different amounts of citrate (max. 8 mmol/l). Stimulation of complement activation was performed using 1.0 and 0.1 ng LPS /ml blood. For complement activation, C3a-desarg was measured after 15, 60 and 120 min. Ca2+ and Mg2+ were analyzed by ion selective electrodes. For coagulation, thrombin time was measured.
Results: Addition of citrate between 3-6 mmol/l results in a reduction of the physiological Ca2+ concentration to lower than 0.4 and 0.2 mmol/l, respectively. The Ca2+ concentrations correlate with the Mg2+ concentrations (p < 0.01, n = 19). The patients’ Hct has an impact on the needed amount of citrate to achieve the target value of Ca2+. In unstimulated heparinized fresh blood, C3a-desarg could be reduced from 528 ng/ml to 242 ng/ml with corresponding values of Ca2+ from 1.14 mmol/l to 0.15 mmol/l. In citrate-free blood which was stimulated with 1 ng LPS/ml, C3a-desarg-levels reach about 7×104 ng/ml, while C3a-desarg is reduced to less than 5 % by the decrease of 45
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Ca2+ from 1.13 to 0.13 mmol/l. The C3a-desarg-levels are similar after 60 and 120 minutes. In the case of blood stimulated with 0.1 ng LPS/ml, the C3a-levels could be reduced from 1000 ng/ml at a Ca2+ concentration of 0.2 mmol/l down to 350 ng/ml at a Ca2+ level of 0.13 mmol/l. Anticoagulation improves with increasing citrate concentration up to 8 mmol/l.
Conclusions: Citrate anticoagulation can be optimized by achieving Ca2+-concentrations lower than 0.2 mmol/l. Normograms involving Hct-values for citrate-infusion management should be provided in order to handle an optimized citrate anticoagulation.
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3.1.7. Encapsulation of C3A cells into alginate microcapsules for the potential use in a bioartificial liver device Poster Presentation at the annual meeting of the European Society for Artificial Organs (ESAO), Krems 2007, Austria. Accepted.
Aim: Aim of this study was the encapsulation of C3A liver cells into alginate microcapsules at a diameter of approximately 150 µm which can be recirculated at high flow rates in a bioartificial liver device based on the MDS (Microspheres based Detoxification System) technology. The microcapsules have to be permeable for essential proteins such as albumin and other metabolites.
Methods: The encapsulation was conducted with the Innotech IE-50R encapsulation device. C3Acells were encapsulated by using a 0.6 % alginate cell suspension and CaCl2 solution for precipitation. The mechanical stability was tested by recirculation of the microcapsule suspension using a roller pump and a centrifugal pump. Permeability of the microcapsules was tested with size exclusion chromatography.
Results: The diameter of the microcapsules was minimized to 160 micrometers. This enables the possibility to recirculate a microcapsule suspension through a hollow fiber filter with a centrifugal pump. As the results of the SEC show, the microcapsules are permeable for albumin and other important proteins. Therefore, nutrition of the cells as well as diffusion of metabolites through the microcapsules is possible.
Conclusion: Alginate microcapsules which contain C3A cells could be used to produce albumin and growth factors in a bioartificial or hybrid liver support system. Due to the small diameter, it is possible to recirculate the particles in suspension in the MDS which leads to optimal diffusive exchange of nutrients as well as metabolites.
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Peer reviewed publications & patents
3.2.1. Particle Leakage in Extracorporeal Blood Purification Systems Based on Microparticle Suspensions
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3.2.2. Detection of Fluorescently Labeled Microparticles in Blood
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3.2.3. New Methods for Hemoglobin Detection in a Microparticleplasma Suspension
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3.2.4. Fluidized Bed Adsorbent Systems for Extracorporeal Blood Purification
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3.2.5. Verfahren und Vorrichtung zur Detektion von Markierten Mikropartikeln (Patent)
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3.2.6. Efficient Adsorption of Tumor Necrosis Factor with an in vitro Set-Up of the Microspheres Based Detoxification System
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3.2.7. In vitro investigations of citrate clearance with different dialysis filters Hartmann J, Strobl K, Fichtinger U, Schildböck C, Brandl M and Falkenhagen D Submitted for publication in Kidney International
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Falkenhagen D, Schmitt E, Schneider P, Behm E, Tessenow W, Klinkmann H (1983):
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[17]
Stefoni S, Coli L, Bolondi L, Donati G, Ruggeri G, Feliciangeli G, Piscaglia F, Silvagni E, Sirri M, Donati G, Baraldi O, Soverini ML, Cianciolo G, Boni P, Patrono D, Ramazzotti E, Motta R, Roda A, Simoni P, Magliulo M, Borgnino LC, Ricci D, Mezzopane D, Cappuccilli ML (2006): Molecular adsorbent recirculating system (MARS) application in liver failure: clinical and hemodepurative results in 22 patients. Int J Artif Organs, 29 (2): 207-218.
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Chiu A, Fan ST (2006): MARS in the treatment of liver failure: controversies and evidence. Int J Artif Organs, 29 (7), 660-667.
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Falkenhagen D, Strobl W, Vogt G, Schrefl A, Linsberger I, Gerner FJ, Schoenhofen M (1999): Fractionated plasma separation and adsorption system: a novel system for blood purification to remove albumin bound substances. Artif Organs, 23 (1), 81-86.
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Evenepoel P, Laleman W, Wilmer A, Claes K, Maes B, Kuypers D, Bammens B, Nevens F, Vanrenterghem Y (2005): Detoxifying capacity and kinetics of Prometheus - a new extracorporeal system for the treatment of liver failure. Blood Purif, 23 (5): 349-358.
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[24]
Kramer L, Bauer E, Schenk P, Steininger R, Vigl M, Mallek R (2003): Successful treatment of refractory cerebral oedema in ecstasy/cocaine-induced fulminant hepatic failure using a new high-efficacy liver detoxification device (FPSAPrometheus). Wien Klin Wochenschr, 15; 115 (15-16): 599-603.
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Evenepoel P, Laleman W, Wilmer A, Claes K, Kuypers D, Bammens B, Nevens F, Vanrenterghem Y (2006): Prometheus versus molecular adsorbents recirculating system: comparison of efficiency in two different liver detoxification devices. Artif Organs, 30 (4): 276-284.
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Krisper P, Haditsch B, Stauber R, Jung A, Stadlbauer V, Trauner M, Holzer H, Schneditz D (2005): In vivo quantification of liver dialysis: comparison of albumin dialysis and fractionated plasma separation. J Hepatol, 43 (3): 451-457.
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Krisper P, Stauber RE (2007): Technology insight: artificial extracorporeal liver support - how does Prometheus compare with MARS? Nat Clin Pract Nephrol, 3 (5): 267-276.
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Falkenhagen D, Weber C, Schima H, Loth F, Ranjoch C, Vogt G, Moser H, Mitzner S (1994): A new technology of simultaneous filtration/adsorption based on a "high-speed" recirculation of nano- and microspheres for extracorporeal blood purification. Biomedizinische Technik, 39, 105-108.
[29]
Weber C, Rajnoch C, Loth F, Schima H, Falkenhagen D (1994): The Microspheres based Detoxification System (MDS). A new extracorporeal blood purification technology based on recirculated microspherical adsorbent particles. Int J Artif Organs, 17 (11), 595-602.
[30]
Vogt G, Schrefl A, Strobl S, Falkenhagen D (1997): A novel LDL-apheresis system based on the Microspheres Based Detoxification System – control and safety systems. Proceedings - 19th International Conference IEEE/EMBS, Chicago, IL, USA: 2431-2433.
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[31]
Weber V, Hartmann J, Linsberger I, Falkenhagen D (2007): Efficient adsorption of tumor necrosis factor with an in vitro set-up of the Microspheres-Based Detoxification System. Blood Purif, 25 (2), 169-174.
[32]
D. Falkenhagen, H. Schima, F. Loth (1999): Arrangement for Removing Substances from Liquids in particluar blood. Europäisches Patent: 94 926 169.7, Japanisches Patent-No. 501083/97, US-Patent: 5.855.782 / 5. Jan. 99.
[33]
Vanholder R, Argiles A, Baurmeister U, Brunet P, Clark W, Cohen G, De Deyn PP, Deppisch R, Descamps-Latscha B, Henle T, Jorres A, Massy ZA, Rodriguez M, Stegmayr B, Stenvinkel P, Wratten ML (2001): Uremic toxicity: present state of the art. Int J Artif Organs, 24 (10): 695-725.
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Bammens B, Evenepoel P, Keuleers H, Verbeke K, Vanrenterghem Y (2006): Free serum concentrations of the protein-bound retention solute p-cresol predict mortality in hemodialysis patients. Kidney Int, 69 (6): 1081-1087.
[36]
M Ketteler (2006): Kidney failure and the gut: p-cresol and the dangers from within. Kidney Int, 69 (6): 952-953.
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Von Appen K, Weber C, Losert U, Schima H, Gurland HJ, Falkenhagen D (1996): Microspheres based detoxification system: a new method in convective blood purification. Artif Organs, 20 (5), 420-425.
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Weber V, Linsberger I, Ettenauer M, Loth F, Hoyhtya M, Falkenhagen D (2005): Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility. Biomacromolecules, 6 (4), 1864-1870.
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[39]
Hartmann J, Schildböck C, Brandl M, Falkenhagen D (2005): Particle leakage in extracorporeal blood purification systems based on microparticle suspensions. Blood Purif, 23 (4), 282-286.
[40]
Brandl M, Hartmann J, Posnicek T, Aussenegg F R, Leitner A, Falkenhagen D (2005): Detection of fluorescently labeled microparticles in blood. Blood Purif, 23 (3), 181-189.
[41]
Sauer IM, Goetz M, Steffen I, Walter G, Kehr DC, Schwartlander R, Hwang YJ, Pascher A, Gerlach JC, Neuhaus P (2004): In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD). Hepatology, 39 (5), 1408-1414.
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van de Kerkhove MP, Poyck PP, Deurholt T, Hoekstra R, Chamuleau RA, van Gulik TM (2005): Liver support therapy: an overview of the AMC-bioartificial liver research. Dig Surg, 22 (4): 254-64.
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Poyck PP, Pless G, Hoekstra R, Roth S, Van Wijk AC, Schwartlander R, Van Gulik TM, Sauer IM, Chamuleau RA (2007): In vitro comparison of two bioartificial liver support systems: MELS CellModule and AMC-BAL. Int J Artif Organs, 30 (3): 183-191.
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[46]
Yamamoto S, Koide M, Matsuo M, Suzuki S, Ohtaka M, Saika S, Matsuo T (1996): Heparin-induced thrombocytopenia in hemodialysis patients. Am J Kidney Dis, 28 (1):82-85.
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Kassimatis TI, Apostolou T, Theodoridis T, El Ali M, Hadjiconstantinou V (2006): The use of lepirudin in haemodialysis complicated with heparin-induced thrombocytopenia type II (HIT II)--dosage monitoring. Nephrol Dial Transplant, 21 (11): 3341-3342.
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Apsner R, Schwarzenhofer M, Derfler K, Zauner C, Ratheiser K, Kranz A (1997): Impairment of Citrate metabolism in acute hepatic failure. Wien Klin Wochenschr, 109 (4), 123-127.
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Opatrny K, Richtrova P, Polanska K, Wirth J, Sefrna F, Brandl M, Falkenhagen D (2007): Citrate anticoagulation control by ionized calcium levels does not prevent hemostasis and complement activation during hemodialysis. Artif Organs, 31 (3): 200-207.
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Hennig
W,
Theopold
W
(1951):
Über
Citronensäure-Calcium-
Komplexverbindungen. Zeitschrift für Kinderheilkunde, 69, 55-61. [59]
Klinke K, Schillert B (1952): Calciumstoffwechsel und Citronensäure. Zeitschrift für Kinderheilkunde, 70, 345-353.
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Curriculum Vitae PERSONAL DATA: Name: Date/place of birth: Martial status: Address:
Jens HARTMANN, M.Sc. November 14, 1969, Nürnberg, Germany married, 3 children 3511 Furth, Stoitznergasse 499, Austria
EDUCATION: 09/80 - 06/88 10/88-03/99
Gymnasium with focus on natural sciences University Vienna, Zoology & Ecology, M.Sc. Diploma thesis at the Institute for Tumor Biology and Cancer Research, Vienna, Austria.
EMPLOYMENT: since 1999
Research associate at the Center for Biomedical Technology, Danube University Krems, Austria.
08/1999-09/2003
6 weeks research trip to the Institute for Biocybernetics and Biomedical Engineering, Polish Academy of Science, Warsaw.
12/2000 - 02/2001 Project “Tecnet AREA Biotechnology” for the government of Lower Austria since 08/2001
Head of the special field process engineering at the Center for Biomedical Technology, Danube University Krems, Austria.
2003
Quality representative training.
since 2003
Quality representative at the Center for Biomedical Technology, Danube University Krems, Austria.
FURTHER QUALIFICATIONS: Foreign languages: English Computer literacy: very good
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Lebenslauf PERSÖNLICHE DATEN: Name: geboren am: Familienstand: Adresse:
Mag. Jens HARTMANN 14.11.1969 in Nürnberg verheiratet, 3 Kinder 3511 Furth, Stoitznergasse 499
SCHULAUSBILDUNG: 09/80 - 06/88
naturwissenschaftliches Realgymnasium, 3500 Krems
STUDIUM: 10/88-03/99
Zoologie und Ökologie an der Universität Wien. Diplomarbeit am Institut für Tumorbiologie und Krebsforschung, Wien.
PRÄSENZDIENST: 04/97 - 11/97
Kanzleischreiber
BERUFLICHER WERDEGANG: seit 1999
Wissenschaftlicher Mitarbeiter am Zentrum für Biomedizinische Technologie (ZBMT) der Donau-Universität Krems, Leitung diverser Forschungsprojekte.
08/1999-09/2003
6 Wochen Forschungsaufenthalt am Institute for Biocybernetics and Biomedical Engineering, Polish Academy of Science, Warschau.
12/2000 - 02/2001 Projekt “Tecnet AREA Biotechnologie” im Auftrag der NÖ Landesregierung seit 08/2001
Leitung des Fachbereiches Verfahrenstechnik des Zentrums für Biomedizinische Technologie
Jän./Feb. 2003
Ausbildung zum QM-Beauftragten (QMS & QMSA) beim ÖVQ
seit 2003
Qualitätsbeauftragter des Zentrums Technologie der Donau-Universität Krems
für
Biomedizinische
WEITERE QUALIFIKATIONEN: Fremdsprachen: EDV:
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PUBLICATIONS / PUBLIKATIONEN: J. Hartmann (1999): Wirkung von ß-Naphthoflavon als Substitut für ubiquitäre Umweltgifte an zwei Arten von Schmetterlingsraupen. Diplomarbeit. W. Strobl, I. Linsberger, J. Hartmann, G. Vogt, M. Schoenhofen, E. Sabrowski, F. Loth and D, Falkenhagen: FPSA-System: Effective in Continuous Removal of Protein bound and High Molecular Weight toxins From Blood. Proceeding of the EMBEC 99, Part I. D. Falkenhagen, W. Strobl, J. Hartmann, A. Schrefl, I. Linsberger, F. Aussenegg, A. Leitner: Patient safety technology for microspheres adsorbent suspension technologies; ASAIO-Journal 46, 2, 214, 2000 (abstract). Poster presentation ASAIO 2000. D. Falkenhagen, W. Strobl, A. Schrefl, J. Hartmann, F. Aussenegg, A. Leitner, A. Krause, M. Schoenhofen (2000): Alternative Behandlungsmöglichkeiten für die extrakorporale Blutreinigung. In: Extrakorporaler Blutkreislauf und Dialyse - Symbiose von Medizin und Technik, S. 163-174, Pabst Verlag. J. Hartmann, A. Schrefl, W. Strobl, Ingrid Linsberger, K.-H. Kellner, F. Aussenegg, A. Leitner and D. Falkenhagen (2000): Membrane Leakage detection based on microparticles marked with fluorescent dyes. Int. J. of Art. Organs, 23 (8). Abstract. Poster presentation ESAO 2000. W. Strobl, I. Linsberger, J. Hartmann, E. Sabrowski, D. Falkenhagen (2000): Decreasing particle size improves Adsorption in Extracorporeal Blood Purification. Polymers in Medicine and Surgery (PIMS), IOM Communications Ltd., S. 447-453. ISBN 1-86125-126-2. D. Falkenhagen, W. Strobl, J. Hartmann, A. Schrefl, I. Linsberger, K-H. Kellner, F. Aussenegg, A. Leitner (2002): Patient Safety Technology for Microadsorbent Systems in Extracorporeal Blood Purification. Artif Organs, 26 (2): 84-90. A. Schrefl, K.H. Kellner, J. Hartmann, W. Strobl, D. Falkenhagen (2001): A Novel Monitor for Anticoagulation with Citrate. Int. Journ. Artif. Organs 24 (8), 535 (abstract). W. Strobl, J. Hartmann, I. Linsberger, D. Pfeiffer, V. Weber, D. Falkenhagen (2001): Comparison of Membrane/Adsorbent-based Blood Purification Devices in Liver Failure in vitro. Int. Journ. Artif. Organs 24 (8), 556 (abstract). D. Schwanzer-Pfeiffer, A. Ciechanowska, A. Józwiak, J. Hartmann, S. Sabalinska, D. Falkenhagen, J. Wójcicki (2002): Hodowla komòrek sródblonka na polisulfonowych membranach pólprzepuszczalnych plaskich i kapilarnych. XII Krajowa Konferencja Naukowa Biocybernetyka i Inzynieria Biomedyczna. (Proceedings of the XII conference of Biocybernetics and Biomedical Engineering). Warsaw, 2002. ISBN 83-901334-5-8. A. Ciechanowska, S. Sabalinska, C. Wojciechowski, A. Jozwiak, E. Rossmanith, J. Hartmann, K. Hellevuo, D. Falkenhagen, J.M. Wojcicki (2003): A Capillary Bioreactor with Endothelial Cells – Peliminary Study. Proceedings of 13th National Scientific Conference on Biocybernetic and Biomedical Engineering. Gdansk, Sept 10-13, 2003, 139-144.
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A. Ciechanowska, D. Schwanzer-Pfeiffer, E. Rossmanith, S. Sabalinska, C. Wojciechowski, J. Hartmann, K. Hellevuo, A. Chwojnowski, P. Foltynski, D. Falkenhagen, J.M. Wojcicki (2004): Artificial Vessel as a basis for Disease Related Cell Culture Model. Medicon 2004, Neapel, Italien. Abstract. E. Rossmanith, A. Ciechanowska, S. Sabalinska, D. Schwanzer-Pfeiffer, C. Wojciechowski, J. Hartmann, K. Hellevuo, A. Chwojnowski, P. Foltynski, D. Falkenhagen, J.M. Wojcicki (2004): Development of an Endothelian Cell Culture Model for Studies on Vascular Pathophysiology in Sepsis. ESAO congress 2004, Warschau, Polen. Abstract. M. Brandl, T. Posnicek, J. Hartmann, D. Falkenhagen (2003): Flowdynamic Microparticle Separation for Extracorporeal Haemofiltration Systems. ESAO congress 2003, Abstract. J. Hartmann, M. Brandl, F. Loth, C. Kendl, D. Falkenhagen (2004): Microparticles in Extracorporeal Blood Purification – A Novel Device for Highly Sensitive Leakage Detection. ESAO 2004, Warschau, Polen. Abstract (Vortrag). M. Brandl, J. Hartmann, D. Falkenhagen (2004): Verfahren und Vorrichtung zur Detektion von markierten Mikropartikeln. Österreichische Patentanmeldung AZ 431/2004. J. Hartmann, M. Brandl, F. Loth, V. Weber, C. Schildböck, M. Ettenauer, D. Falkenhagen (2005): A Novel Device for Highly Sensitive Leakage Detection for Microparticle Suspension Based Blood Purification Systems. ISFA congress 2005, Abstract (Vortrag). V. Weber, J. Hartmann, I. Linsberger, C. Schildböck, F. Loth, D. Falkenhagen (2005): Development of Specific Microadsorbents for Pathophysiological Mediators of Sepsis and Multi-Organ Failure. ISFA congress 2005, Abstract. Ciechanowska, D. Schwanzer-Pfeiffer, E. Rossmanith, S. Sabalinska, C. Wojciechowski, J. Hartmann, K. Hellevuo, A. Chwojnowski, P. Foltynski, D. Falkenhagen, J. Wojcicki (2004): Artificial Vessel as a Basis for Disease Related Cell Culture Model. IFMBE Proceedings, 6, 2004. M. Brandl, J. Hartmann, T. Posnicek, F. R. Aussenegg, A. Leitner, D. Falkenhagen (2005): Detection of Fluorescently Labeled Microparticles in Blood. Blood Purif, 23 (3), 181-189. J. Hartmann, C. Schildböck, M. Brandl, D. Falkenhagen (2005): Particle Leakage in Extracorporeal Blood Purification Systems Based on Microparticle Suspensions. Blood Purif, 23 (4): 282-286. Hartmann J, Smeets JMW, de Zwart MAH, Peters SMA (2006): In vitro dialysability of sugammadex, a selective relaxant binding agent for reversal of neuromuscular block induced by rocuronium. ESA congress 2006, Madrid. Abstract, Poster, Vortrag.
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