Application of Piezo-Based Measuring System for Evaluation of Nucleic Acid-Based Drugs Influencing the Coagulation.

During open-heart surgery, the status of hemostasis has to be constantly monitored to quickly and reliably detect bleeding or coagulation disorders. In this study, a novel optimized piezo-based measuring system (PIEZ) for rheological monitoring of hemostasis was established. The applicability of the PIEZ for the evaluation of nucleic acid-based drugs influencing coagulation was analyzed. Thrombin aptamers such as NU172 might be used during extracorporeal circulation (ECC) in combination with a reduced heparin concentration or for patients with heparin-induced thrombocytopenia (HIT). Therefore, the effect of the coagulation inhibiting thrombin aptamer NU172 and the abrogation by its complementary antidote sequence (AD) were investigated by this rheological PIEZ system. After the addition of different NU172 concentrations, the coagulation of fresh human blood was analyzed under static conditions and using an in vitro rotation model under dynamic conditions (simulating ECC). The clotting times (CTs) detected by PIEZ were compared to those obtained with a medical reference device, a ball coagulometer. Additionally, after the circulation of blood samples for 30 min at 37 °C, blood cell numbers, thrombin markers (thrombin-antithrombin III (TAT) and fibrinopeptide A (FPA)) and a platelet activation marker (ß-thromboglobulin (ß-TG)) were analyzed by enzyme-linked immunosorbent assays (ELISAs). The increase of NU172 concentration resulted in prolonged CTs, which were comparable between the reference ball coagulometer and the PIEZ, demonstrating the reliability of the new measuring system. Moreover, by looking at the slope of the linear regression of the viscous and elastic components, PIEZ also could provide information on the kinetics of the coagulation reaction. The shear viscosity at the end of the measurements (after 300 s) was indicative of clot firmness. Furthermore, the PIEZ was able to detect the abrogation of coagulation inhibition after the equimolar addition of NU172 aptamer´s AD. The obtained results showed that the established PIEZ is capable to dynamically measure the hemostasis status in whole blood and can be applied to analyze nucleic acid-based drugs influencing the coagulation.

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).

Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1ß, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1ß and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd.

More than 70 years of pyrogen detection: Current state and future perspectives.

In the quality assurance of medical products, tests for sterility are essential. For parenteral pharmaceuticals, avoiding the presence of pyrogens is crucial. These fever-inducing substances (endotoxins and non-endotoxins) are not eliminated by standard sterilisation processes, and are biologically active once in the bloodstream, causing risks to human health, ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory. Over the years, various pyrogen testing methods have been introduced, namely: in the 1940s, the rabbit pyrogen test, which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test, which is an in vitro test (with the haemolymph of the horseshoe crab) that specifically detects endotoxin; and in 2010, the Monocyte-Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test. Due to the ubiquity and biological significance of pyrogens, we are currently further developing the MAT so that it can be used for other applications. More specifically, our focus is on the detection of pyrogenic contamination on medical devices, as well as on the measurement of air quality. In addition, further improvements to permit the use of cryopreserved blood in the MAT, to overcome the limitations in the availability of freshly-drawn blood from human donors, are ongoing.

Hemocompatibility testing according to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic activation.

Next to good hemocompatibility performance of new medical devices, which has to be tested according to the ISO 10993-4, the detection of pyrogen-contaminated devices plays a pivotal role for safe device application. During blood contact with pyrogen-contaminated devices, intense inflammatory and hemostatic reactions are feared. The aim of our study was to investigate the influence of pyrogenic contaminations on stents according to the ISO 10993-4. The pyrogens of different origins like lipopolysaccharides (LPS), purified lipoteichoic acid (LTA) or zymosan were used. These pyrogens were dried on stents or dissolved and circulated in a Chandler-loop model for 90 min at 37°C with human blood. Before and after circulation, parameters of the hemostatic system including coagulation, platelets, complement and leukocyte activation were investigated. The complement system was activated by LPS isolated from Klebsiella pneumoniae and Pseudomonas aeruginosa and by LTA. Leukocyte activation was triggered by LPS isolated from K. pneumoniae, LTA and zymosan, whereas coagulation and platelet activation were only slightly influenced. Our data indicate that pyrogen-contaminated devices lead to an alteration in the hemostatic response when compared to depyrogenized devices. Therefore, pyrogenicity testing should be performed prior to hemocompatibility tests according to ISO 10993-4 in order to exclude hemostatic activation induced by pyrogen contaminations.

Highly sensitive pyrogen detection on medical devices by the monocyte activation test.

Pyrogens are components of microorganisms, like bacteria, viruses or fungi, which can induce a complex inflammatory response in the human body. Pyrogen contamination on medical devices prior operation is still critical and associated with severe complications for the patients. The aim of our study was to develop a reliable test, which allows detection of pyrogen contamination on the surface of medical devices. After in vitro pyrogen contamination of different medical devices and incubation in a rotation model, the human whole blood monocyte activation test (MAT), which is based on an IL-1ß-specific ELISA, was employed. Our results show that when combining a modified MAT protocol and a dynamic incubation system, even smallest amounts of pyrogens can be directly detected on the surface of medical devices. Therefore, screening of medical devices prior clinical application using our novel assay, has the potential to significantly reduce complications associated with pyrogen-contaminated medical devices.

Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test.

Threats of pyrogenicity were discovered more than a century ago. Measures to determine the safety of parenterals and, more recently, medical devices and cell therapies for human use have been in place for 70 years. Currently, there are three testing possibilities available: the Rabbit Pyrogen Test, the Limulus Amebocyte Lysate test (Bacterial Endotoxin Test), and test systems using human whole blood or human monocytes, called Monocyte Activation Test (MAT). The MAT is based on the human fever reaction and thus most closely reflects the human situation. Unfortunately, regulations and testing guidelines are not fully harmonized, despite formal international validation. Furthermore, data showing that the MAT is capable of covering the totality of possible pyrogens relevant to humans were not included in the MAT validations of the last decade. For this review we collate evidence from published literature, unpublished data of our own, and results from the international validation study to show that there is overwhelming scientific evidence to conclude that the whole blood MAT reliably detects non-endotoxin pyrogens. Therefore, further validation exercises do not seem warranted.

Fever in the test tube--towards a human(e) pyrogen test.

The human whole blood IL-1 test exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after incubation. In order to understand any differences between the pyrogenic activity in this test and the existing live rabbit test (species differences versus aberrant response of the particular blood sample), the rabbit whole blood test was developed. This approach could also help to avoid the use of putatively infectious human blood for pyrogen testing in vitro.

A new method to measure air-borne pyrogens based on human whole blood cytokine response.

Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness." We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA. The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood. The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

Cryopreservation of human whole blood for pyrogenicity testing.

Human whole blood assays are increasingly employed to test immune function or detect pyrogenic contamination, since they offer advantages, such as ease of performance, few preparation artifacts and a physiological cell environment. However, the approach is often limited by the availability of freshly drawn blood, putative safety concerns in the case of infected donors and interindividual donor differences. To overcome these limitations, a method was developed and optimized to produce batches of cryopreserved blood that can be used directly after thawing without any washing steps. Mononuclear cells remained intact as shown by FACS analysis. Cytokine release could be induced by a variety of immunological stimuli. The cell preparation released higher amounts of interleukin-1beta (IL-1beta) and IL-6 compared to fresh blood, but no TNF. These differences could be attributed to the presence of the cryoprotectant dimethylsulfoxide (DMSO) alone by addition of DMSO to fresh blood. Large batches of cryopreserved blood could be produced by mixing blood donations of up to 10 donors, independent of differing blood groups. The detection limit for the World Health Organization (WHO) lipopolysaccharides (endotoxin, LPS) reference preparation (EC-6) with regard to the induction of IL-1beta release was at least 0.5 endotoxin equivalent units (EU)/ml. Endotoxin spikes at the limit concentrations prescribed in the European Pharmacopoeia could be detected in a series of drugs, showing that the In vitro Pyrogen Test (IPT) can also be run with cryopreserved blood. Further possible applications include high-throughput screening for immunomodulators or toxins as well as preservation of patient samples for later analysis of cell functions.

Comparison of the reactivity of human and rabbit blood towards pyrogenic stimuli.

A comparison between humans and rabbits was performed based on stimulation of whole blood with well-known pyrogens from Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The reactivity was measured as release of IL-1 beta and IL-8 by ELISA. The reactivity of the two species towards LPS was found to be similar, whereas their reactivity towards LTA differed considerably. Differences between the levels of IL-1 beta and IL-8 release were observed in both species. This finding suggests that the In vitro Pyrogen Test (IPT) which uses human blood to detect contaminations, e.g. of injectable drugs, might predict the human reaction to the contamination better than the "gold standard" rabbit pyrogen test.

[Evaluation and further development of a pyrogenicity assay based on human whole blood]

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever inducing contaminations) they release mediators transmitting the fever reaction within the organism. A new pyrogen test exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. In case of pyrogen contamination, the formation of interleukin-1 is induced, which is determined by ELISA. According to the various pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. In contrast to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the blood assay is not restricted to endotoxins of Gram-negative bacteria and is not to the same extent disturbed by endotoxin-binding blood proteins. Here, interim results of the ongoing optimisation and prevalidation are demonstrated. Preliminary data of the evaluation for biological and pharmaceutical drugs are presented.

[Development and evaluation of a pyrogen test based on human whole blood]

When cells of the immune system, especially blood monocytes and macrophages, come into contact with pyrogenic (fever-inducing) contaminations, they secrete messenger molecules which initiate an hyperthermic reaction in the organism. Of this group of endogenous pyrogens, most is known about interleukin-1 (IL-1). A new pyrogen test makes use of this reaction as a system for detection: The substances which are to be screened are incubated with a small volume of blood from a healthy donor. Any pyrogens present induce the production of IL-1 which can be detected by ELISA. This test has a higher sensitivity and is more economical than the conventional pyrogen test in rabbits and furthermore reflects the reaction of the relevant species. In contrast to the customary alternative method, the Limulus amoebocyte lysate test (LAL), this test is not restricted to endotoxins from Gram-negative bacteria and is also not hindered by substances which bind endotoxins, such as blood proteins, to the same extent. Consequently, more than 50 non-endotoxin pyrogens have already been traced by this test. The whole blood test is even superior to the LAL in regard to the detection of endotoxins: in a comparison of about 60 endotoxins, there was a correlation of the potency of the individual endotoxins between the whole blood test and the pyrogen test in rabbits, but neither test correlated with the LAL test. In some cases, endotoxins with equal effects in the LAL test differed in potency in the human blood model by a factor of 10 000. A method has been developed by which cryopreserved blood can be put to use in the test. In this way, blood donations from a donor can be pre-tested so that uniform material may be employed in the test. This test opens up entirely new perspectives on pyrogen testing for Gram-positive or fungal pyrogens as well as in medicinal products. In addition, it could fill the dangerous security gap which might result from the limitations of testing medications and blood products with LAL only. The project was supported by ZEBET, D-Berlin, BMBF, D-Bonn, and set, D-Mainz.