Tuesday, 29 March 2011 14:34

Working group – a Portrait: Clinical Research Laboratory Featured

Starting with this article, InvitroJobs will present scientists and their innovative research in a regular feature called “Working Group – a Portrait”. We will focus on newly developed methods, their evaluation and their potential for reducing and where possible replacing animal experimentation according to the 3R principle of Russel & Burch (reduce, refine, replace). We start with two research groups from the Clinical Research Laboratory of the Paediatric Cardiac Surgery Department of Tübingen University Hospital, “Haemocompatibility” and “Pyrogen assay”. The introduction is followed by an interview with Dr. med. Stefan Fennrich.


Working group – a Portrait:
Clinical Research Laboratory of the

Paediatric Cardiac Surgery Department of
Tübingen University Hospital

We start with two research groups from the Clinical Research Laboratory of the Paediatric Cardiac Surgery Department of Tübingen University Hospital, “Haemocompatibility” and “Pyrogen assay”. The two two main emphases of applied research, development and service is described below.

1.  Development and testing for haemocompatibility of blood-contacting implants.
Implantable medical devices which come into contact with the blood (circulation) have to be tested for compatibility of blood components. This can be implemented by using in vitro models in which the interaction between the implant and the haemostasis or immune system is tested. In ISO 10993, part 4, these tests are regulated internationally.

2. Testing medical products for pyrogen occurrence:
The in vitro pyrogen test PyroDetect was developed for testing injectable pharmaceuticals for pyrogen occurrence as a complete replacement of animal experiments. Instead of inducing fever in mammal organism, the fever reaction is simulated in a test tube. Fever signal substances (endogen fever molecules) are detected in human blood in vitro. Blood contacting medical products (injection, infusion or solids (implants)) can carry thermostable bacterial residues (pyrogens) which can cause fever, a decrease in blood pressure, multi-organ dysfunction or death to humans. For this reason, legislation in every country demands testing for pyrogen occurrence. The possible applications for PyroDetect are manifold. It can be used for pyrogen detection of material surfaces of implants as well as for measuring air quality parameters.
The leader of the research group “Pyrogen assay”, Dr. med. Stefan Fennrich, gave an interview to InVitroJobs on the status and outlook of this test system.

Working group In vitro Pyrogen test. From left to right: Prof. Dr. Hans Peter Wendel, Deborah Lutz, Marcell Post, Ulrike Hennig und Dr. Stefan Fennrich. Photo: Jan Niederländer.

The initial point of discussion in our interview is the development of the so-called PyroDetect method, which was implemented as a Monocyte Activation Assay (MAT) in the European Pharmacopoeia in 2010 (EP test specification 2.6.30). The use of the assay has the potential to reduce the number of rabbits used for pyrogen testing by approximately 200,000 per year in Europe alone. The MAT has already been included in the European Pharmacopoeia as a fully adequate alternative to the use of in vivo tests. According to requirements of international law, animal-free alternatives are to be given priority for ethical reasons. This means that from now on the rabbit test must be replaced entirely. Delays can arise due to the fact that many users have yet to establish the MAT assay in their facilities, and because the approval of medical products requires that international quality standards be observed. Developers of new medical products must now state very good reasons for the use of rabbits for pyrogen testing. Researchers who report an in vivo pyrogen test to the authority can expect an official objection. So far, the final approval of the United States and Japan is still pending. The American ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods), which participated in the validation of the MAT, advocates official approval.

Photo: Biotest AG

The in-vitro-pyrogen test was originally developed by Prof. Dr. Albrecht Wendel, former head of the Department of Biochemical Pharmacology in Constance and currently director of the Interfaculty Centre of Pharmacogenomics and Drug Research (IZEPHA, Tübingen University Hospital), in collaboration with Prof. Dr. Dr. Thomas Hartung, former director of ECVAM, now incumbent of the Doerenkamp-Zbinden Chair in Baltimore and director of the Center for Alternatives to Animal Testing (CAAT) at the John Hopkins University. Both scientists have been honoured for the development of the test, for example with the European Animal Protection Award of the Fédération Internationale pour la Substitution des Experiments Animaux in 1994 and the International Animal Protection Award of the Doerenkamp-Zbinden-Foundation in 1996.

Dr. Stefan Fennrich was a member of Wendel’s and Hartung’s team. Between 1997 and 2005 he supervised the development team “in-vitro-pyrogen test” in Prof. Albrecht Wendel’s department at the University of Constance. Dr. Fennrich now has his own research team in the clinical research laboratory of the paediatric cardiac surgery department of the Tübingen University Hospital, where he can introduce and further develop his expertise with the new in vitro Pyrogen assay.

From left to right: Prof. Dr. Hans Peter Wendel, Prof. Dr. Albrecht Wendel, Dr. Stefan Fennrich
Photo: Wendel.

Professor Dr. Albrecht Wendel (ICEPHA, UKT), Professor Dr. Hans Peter Wendel and Dr. Stefan Fennrich just obtained the award „Ausgewählter Ort 2011“ by the initiative „Deutschland - Land der Ideen“  for the topic  „PyroDetect - Innovatives Medikamententestverfahren“. Now the workgroup may call itself „Ausgewählter Ort 2011“. For more information: http://www.land-der-ideen.de/

Research group “Haemocompatibility” and “Pyrogen Assay”
The research laboratory for paediatric cardiac surgery at the Tübingen University Hospital comprises five working groups. Director of the research lab with a staff of between twenty and thirty is Prof. Dr. Hans Peter Wendel. Amongst other things, they are engaged with the solution of issues which occur during the usage of blood contacting systems (for example implants, cardiopulmonary bypass components, artificial heart models, stents, artificial blood vessels) in the area of cardiac and vascular surgery. However, there is also a focus on other scientific questions, such as wound healing disorders. A further approach is the self-colonisation of transplants by the patient’s own endothelial cells, the goal being that the new organ can grow physiologically with the organism (child), without causing detrimental tissue changes. The research group’s work includes early stages of fundamental and pre-clinical (application oriented) research. For example, they envision providing people undergoing bypass surgery, who nowadays require autografted veins, with self-colonising prosthetic vessels.

The use of oxygenators for cardiopulmonary bypass systems, the application of cardiac support systems or dialysis can provoke haemal defence reactions. Therefore, new methods of investigation and application are being developed in order to improvement of blood compatibility of applied materials.

Of particular importance here is the use of in vitro blood as a “sensor” in the areas of both haemocompatibility and pyrogenicity. These are excellent models relevant to applications safe for humans.
Also an interesting and important field is research on aptamers. These are artificially produced short DNA fragments with a length of 25 to 73 base pairs, which can bind to certain surface structures due to their three-dimensional structure, similar to that of antibodies. The idea is to use them to produce “captor molecules” capable of fishing out blood building-stem cells from the surrounding region (blood cells derive from stem cells from bone marrow). Carrier materials of vascular prosthetics could be coated with these aptamers in order to achieve a self-colonisation with the body’s own vascular cells.

Due to its expertise, the laboratory was GLP-certified (good laboratory practises) for the testing of blood-contacting medical products according to DIN EN ISO 10993-4, the highest laboratory standard worldwide.

Between 1997 and 2005, Dr. med. Stefan Fennrich was director of the development team at Prof. Dr. Albrecht Wendel’s Department of Biochemical Pharmacology in Constance. He is now director of studies and projects leader in Prof. Dr. Hans Peter Wendel’s clinical paediatric cardiac surgery research laboratory at the University Hospital in Tübingen. He received many awards with his previous working group “In Vitro Pyrogen Test”, and most recently, as part of the Tübingen group alongside Prof. Dr. Albrecht Wendel and Prof. Dr. Hans Peter Wendel, he has been awarded the distinction “Ausgewählter Ort 2011” (“Selected Location 2011”) in the initiative “Deutschland – Land der Ideen” (“Germany – Country of Ideas”) for the topic “PyroDetect – innovative methods for testing pharmaceuticals” as part of the competition “365 Orte im Land der Ideen” (“365 Locations in the Country of Ideas”). In the clinical laboratory, the scientists emphasise the usage of methods relevant to humans which allow an evaluation of the research results specific to humans. They therefore abandon animal use wherever possible, giving preference to in vitro methods.

Graphic: Stefan Fennrich.

In the PyroDetect system, an innate immune response of humans to so-called pyrogens is used. Pyrogens are bacterial substances, for example cell wall fragments, yeast particles, fungi or viruses. As foreign bodies they trigger a defence reaction in human body, accompanied by a fever reaction.

For the test procedure, human whole blood is used in which the white blood cell type monocyt / macrophage releases cytokines, specific glycoproteins which trigger the immune response, if pyrogens are present. One of these inflammatory cytokines is named interleukin 1ß (IL-1ß). It is present in minute, untraceable amounts in the human body. In the in vitro method, IL-1ß is particularly suited for detecting pyrogen reactions.

The test solution is brought into contact with the blood sample and incubated at 37 °C (body temperature). If pyrogens are present, the monocytes/macrophages produce IL-1ß in the blood sample. Using the so-called ELISA test procedure, the IL-1ß is then bound to an antibody, which can be rendered visible by a colour reaction. The pyrogen concentration can then be determined.

The PyroDetect verifies a pyrogen spectrum comparable to that of the rabbit test and considerably broader than that of the Limulus Amoebocyte Lysate (LAL) Assay, which can only used for the limited detection of lipopolysaccharides (endotoxins) of Gram-negative bacteria.

Easy Handling of the PyroDetect:
(1) Withdrawal of kryoblood for blood incubation,
(2) Mix of kryoblood with the sample, additionally incubation by 37° degreee for 18 hours (if pyrogens occure the fever signalling substance interleukion 1ß is distributed),
(3) After incubation, the reaction comprising is transferred to a microtiter plate which is coated with antibodies which bin the interleukin 1ß. These reaction leads to a blue colour reaction (bright blue: less, dark blue: a lot of interleukin 1ß). After 10 minutes, the colour reaction is terminated, the blue colour turns to yellow (bright yellow less, dark yellow a lot of interleukin 1ß),
(4) The intensity of yellow tone is measured photometric by an ELISA-Reader.
Photos: Biotest AG

Comprehensive implementation of PyroDetect

InVitroJobs asked Dr. Stefan Fennrich about the current status of research, difficulties regarding the recognition of replacement methods for animal testing, and prospects and perspectives for future research.

InVitroJobs: Dr. Fennrich: Once PyroDetect was developed, what was the approval procedure?

Dr. Stefan Fennrich:
The in vitro pyrogen assay (PyroDetect) went through a lot of stages between the idea and its approval. The idea came from Prof. Albrecht Wendel, Chair of Biochemical Pharmacology at the University of Constance, where it was developed and prepared for validation, and where initial areas of application were investigated. After international validation, also involving variations of the in vitro method, followed a phase in which the validation results were assessed by a panel of experts. Once that was successfully completed, the regulatory implementation was drawn up by an international think tank. This implementation has now been put into practice and since 2010 has been published in the European Pharmacopoeia 6.7, chapter 2.6.30 under the name monocyte-activation test (MAT).

What is the validation process like, and how long does it take?

Dr. Stefan Fennrich:
The validation process itself took several years and was conducted throughout Europe by the European Centre for the Validation of Alternative Methods (ECVAM). Based on the concept of the human fever reaction, in vitro-test versions were validated, in which monocytoid cells had been activated by pyrogens. In a blind study using prototypical parenteralia (pharmaceutical preparations for injection), six test versions were validated in ten laboratories. Four of these test versions fulfilled the criteria for pyrogen detection, making them viable alternatives to pyrogen tests on rabbits.

Now that the test is stipulated by the Pharmacopoeia, is it also applied outside of Europe, or laid down in American or other regulations?

Dr. Stefan Fennrich:
Regardless of the regulation of the method in the Pharmacopoeia, the in vitro method can also be used in non-regulated areas, for instance in many development or research projects or in process control, where the absence of pyrogens plays an important part. The American authorities were involved in the validation process from the onset and propone a product-specific validation using the in vitro methods. The implementation process as an ISO standard has been already initiated, e.g. for testing medical products (implants) (ISO TC 194/WG 16, Working group Pyrogenicity, biological assessment of medical devices).

Does your working group have any other developments for replacing animal testing?

Dr. Stefan Fennrich:
We provide an in vitro method according to ISO 10993-4 for testing the haemocompatability of medical products, for which we are GLP-certified (see glossary). For this purpose we also use the “sensor blood” in vitro, in both static and dynamic flux models, as well as on the cardiopulmonary bypass model.

The PyroDetect is also intended to be applied to “other areas of application”. What could these be?

Dr. Stefan Fennrich:
The in vitro pyrogen test (PyroDetect) has proven to be suitable for testing both liquid samples (parenteralia) and surfaces which come into direct contact (e. g. transplants) for pyrogenic activity, and also for testing cellular therapeutics. In the area of occupational medicine, air quality plays an important role, for example in farming, industrial production processes, biotechnology and all other facilities with air conditioning systems.

What is the current development status?

Dr. Stefan Fennrich:
Both surface tests on implant models and air contamination tests were successfully subjected to feasibility studies, which have also already been published.

At the moment we are working on models with which we can use PyroDetect to assess human-specific health risks arising from industrial workplace air pollution. In direct cooperation with professional associations and occupational physicians, we are developing processes relevant to both authorities and legislators within the context of occupational safety and health.

Monitoring air quality is important for occupational associations, as in many workplaces the air inhaled can be contaminated in a variety of ways. That is obviously the case on farms, but also in metalworking plants. For example, aerosolised cutting fluids can enter the air and be inhaled. Currently we are developing technologies and methods for human-specific testing and evaluation of such kinds of air contamination.

What difficulties are involved in development?

Dr. Stefan Fennrich:
Because we are entering new areas of application, the challenge lies in facing new technology and scientific questions. Every day we find ourselves in a learning process, in which interdisciplinary thinking determines our day-to-day work. This affects the collaboration between different scientific disciplines, as well as the implementation of research and development in industrial and application-oriented methods and products.

Do you receive enough support for your project work? What is the situation on the “research funds marketplace” like?

Dr. Stefan Fennrich:
Research and development are financed by third-party funding or by industry within the framework of joint research. Funding is limited, and what would be desirable is a financial backing with which groundbreaking project ideas and pilot studies could be investigated and existing concepts further developed. An example of an important project is the implementation of PyroDetect subject to GMP quality standards, because only that way can the method successfully be provided and implemented as a replacement for the in vivo rabbit test. That is exactly what we are currently intend to do, as it is my desire to be able to provide the in vitro method as a blanket alternative to unnecessary animal tests. That means the mandatory replacement of pyrogen tests on rabbits with PyroDetect wherever these are still compulsory or conducted, and wherever other tests are conducted on other animals, reducing or replacing the animal use with in vitro methods.

In order to receive research funds, a respective research project application is necessary, which must first be reviewed and can only then be granted with a certain degree probability. Such applications are laborious, and the allocation of funds cannot be foreseen. Such applications also involve initiating collaborative projects, as my work often involves interdisciplinary projects which require the special expertise of cooperation partners. This is a daily challenge, as all the employees of my working group must be financed in this fashion. Continuity of staff is an important issue for me, as it directly influences the success of a project. Likewise, thesis work (bachelor, master, diploma and doctoral theses) is an important pillar for research projects.

What motivates students/graduates? Are they interested in replacement methods?

Dr. Stefan Fennrich:
The motivation I see in young students and graduates to work on developing replacement methods is fundamentally high. In my lectures I explain what it means to work in our team. So far there have always been students interested in thesis work.

What are the training prerequisites for graduates/young scientists? Must more be done for education in the area of replacement methods?

Dr. Stefan Fennrich:
I believe that a lot of research and development is done without identifying or even recognizing the potential of the replacement method. I regard pointing this out and integrating it into education as a good chance for raising awareness and understanding of the topic and to encourage thought.

What is your perception of the current efficacy of replacement methods?

Dr. Stefan Fennrich:
Fundamentally, in vitro methods compete with in vivo methods, especially where legally regulated experiments are concerned. Seen in historical terms, the in vivo methods have become a so-called “gold standard”, according to which the replacement method is judged, regardless of however deficient the standard may be. PyroDetect as an in vitro pyrogen assay has a historical chance to replace the rabbit test in the foreseeable future, making history with regard to drug safety and animal protection.

What is your opinion on the financing of replacement methods, if the boundaries between pure science and applied research no longer exist?

Dr. Stefan Fennrich:
We shouldn’t think within limits and stake our claims! Fundamental research is necessary and leads sooner or later to applications. Each success begins with a first step, no matter how small it be or how long the road. So if boundaries between fundamental and applied research are dwindling, that means that successful applied developments such as replacement methods become more and more probable. Replacement methods should be funded generously.

Dr. Fennrich, thank you for the interview.


GMP (Good Manufacturing Practice) is a set of guidelines for quality assurance in drug and pharmaceutical production processes and settings, but also in foodstuff and animal feed production. In pharmaceutical manufacture, quality assurance plays a central part, as quality deviations can directly affect consumer health. A GMP standard quality management system can be used to guarantee product quality and the fulfilment of mandatory health authority requirements for marketing such products (clearance).

GLP is a quality system which includes the organisational process of tests, the framework conditions according to which the tests are planned, carried out and monitored, as well as documentation, reporting and archiving of these tests. GLP is required for examinations of toxicity, ecotoxicity, mutagenity, environmental reactions and bioaccumulation, residues, effects on mesocosms and natural ecosystems, physiochemical and analytical chemical properties, as well as other properties subject to precise definition. GLP was first introduced in 1978 by the United States for animal experiments mandatory for drug approval. Today, GLP guidelines are prescribed worldwide and must be applied at least for tests for the approval of drugs, medical devices, and industrial and agricultural chemicals.

GLP certification
If a laboratory has integrated all required elements according to GLP and also applies these elements, it can be certified. It is then published in the list of GLP laboratories, both domestically and within the OECD.

Limulus amoebocyte lysate assay (LAL)
In the seventies of the twentieth century, a first in vitro assay for proof of endotoxins and thermostable cell wall fragments of Gram-negative bacteria and blue green algae was developed. The test is based on the observation that an infection of a horseshoe crab (Limulus polyphemus) with gram negative bacteria leads to blood clotting. This is the result of a reaction between the bacterial endotoxin and a gel-forming protein in the animal’s blood cells.
For LAL-testing, a lysate must be gained from the blue blood of the horseshoe crab. The lysate carries the gel-forming protein as an inactive precursor (inactive proenzyme). The addition of the endotoxin induces the transformation of the precursor to the active enzyme. The active enzyme reacts with a special colourless synthetic substrate (peptide) which then changes to yellow. Subsequently, the enzyme activity can be determined by a photometer.

From an ethical point of view, the LAL assay must be judged critically. In the United States, 30 000 horseshoe crabs die each year for the test. Sampling from nature leads to a decline of the natural population, resulting in an ecological imbalance within its natural habitat.

Bachinski, R. et al. (2010): Criticism of the LimulusAmoebocyte Lysate (LAL) test as an replacement method of the rabbit pyrogen test (RPT) and environmental health implicyations. Altex Abstractband ESTIV - EUSAAT- Tagung Linz 2010.

Also so referring to 3R-Principles: