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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

12 Issues per year


IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

SCImago Journal Rank (SJR) 2016: 1.000
Source Normalized Impact per Paper (SNIP) 2016: 1.112

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1437-4331
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Volume 51, Issue 10 (Oct 2013)

Issues

Congress of Clinical Chemistry and Laboratory Medicine / 10th Annual Meeting of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL), Dresden, Germany, 23rd–26th October, 2013*)

Published Online: 2013-09-27 | DOI: https://doi.org/10.1515/cclm-2013-0737

10th Annual Meeting of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL)

Dresden, Germany, 23rd–26th October, 2013

Under the auspices of

Sabine von Schorlemer, Staatsministerin für Wissenschaft und Kunst im Freistaat Sachsen

European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)

World Association of Societies of Pathology and Laboratory Medicine (WASPaLM)

Congress President

Gabriele Siegert (Dresden, Germany)

Congress Secretary

Thomas Demant (Dresden, Germany)

Sponsor of Abstract awards

Dr. Neumann & Kindler Ltd. & Co. KG (Bochum, Germany)

Scientific Committee

Bidlingmaier, Martin (Munich), Brügel, Mathias (Munich), Ceglarek, Uta (Leipzig), Chavakis, Triantafyllos (Dresden), Conrad, Karsten (Dresden), Demant, Thomas (Dresden), Diehl, Frank (Hamburg), Eckardstein, Arnold von (Zürich/CH), Fiedler, Georg Martin (Bern/CH), Gässler, Norbert (Hildesheim), Isermann, Berend (Magdeburg), Junker, Ralf (Kiel), Kiehntopf, Michael (Jena), Kratzsch, Jürgen (Leipzig), Lackner, Karl J. (Mainz), Lange, Robert (Berlin), Luppa, Peter B. (Munich), Menschikowski, Mario (Dresden), Morawietz, Henning (Dresden), Nauck, Matthias Alexander (Greifswald), Neukammer, Jörg (Berlin), Neumaier, Michael (Mannheim), Orth, Matthias (Stuttgart), Peetz, Dirk (Berlin), Raulf-Heimsoth, Monika (Bochum), Renné, Thomas (Hamburg), Renz, Harald (Marburg), Riedl, Jürgen A. (Dordrecht/NL), Ruf, Andreas (Karlsruhe), Ruland, Jürgen (Munich), Schmidt, Michael (Bonn), Schmitz, Gerd (Regensburg), Schuff-Werner, Peter (Rostock), Schwarz, Markus J. (Munich), Seger, Christoph (Innsbruck/AT), Siegert, Gabriele (Dresden), Stauch, Thomas (Karlsruhe), Tausche-Wunderlich, Anne-Kathrin (Dresden), Teupser, Daniel (Munich), Thiery, Joachim (Leipzig), Torzewski, Michael (Stuttgart), Trauner, Michael (Wien/AT), Vogeser, Michael (Munich), Ziehr, Stephanie C. (Innsbruck/AT)

Talks

Plenarsitzung Cell Regeneration in Health, Disease and Aging

T01 – Talk Spalding

Radiocarbon analysis of cell and tissue regeneration in humans

K. Spalding1

1Karolinska Institute, Cell and Molecular Biology, Stockholm, Schweden

Objectives: Much of the impetus in regenerative medicine is fuelled by the prospect of promoting cell replacement, or blocking unwanted cell production. Without knowing, however, if a specific cell type is renewed in the healthy or pathological situation, it remains uncertain whether it may be realistic to modulate this process. Despite the importance of this information, remarkably little is known about the age of cells in many regions of the adult human brain and body – such as do humans make new neurons? How is adipose tissue mass maintained? This is largely due to difficulties in studying this process in humans. We set out to develop a novel method whereby the turnover of cells and molecules could be examined, retrospectively in human tissues.

Methods: Using recently established methodology, which integrates biomedical approaches with recent developments in nuclear physics, it is possible to establish the turnover of cells in human tissues. By measuring 14C derived from nuclear bomb tests in DNA, triglyceride and tooth enamel, it is possible to retrospectively establish the birth date of cells, lipids and humans.

Results and Conculsions: New neurons are added to the adult hippocampus at substantial levels, however no evidence for long-term stable integration of newborn neurons was found in the cortex, cerebellum or olfactory bulb. Adipocytes are exchanged in adulthood, such that approximately 50% of adipocytes are renewed every 8 years and the lipid inside the fat cells is replaced an average of 6 times during the cell lifespan. Radiocarbon dating of tooth enamel also enables a precise determination of when an individual is born, assisting police and forensic officials in making identifications of unidentified deceased individuals.

Vaskuläre Inflammation

T02 – Talk Nourshargh

Neutrophil transmigration in vivo: Mechanisms, dynamics & contribution to dissemination of systemic inflammation

S. Nourshargh1

1Centre for Microvascular Research within the William Harvey Research Institute at Queen Mary’s Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London, E1 4NS, United Kingdom

It has long been known that inappropriate, excessive or prolonged leukocyte transmigration is associated with the pathogenesis of inflammatory disorders. However despite much research there has been a disappointingly slow progress in fruitful targeting of leukocyte trafficking for development of anti-inflammatory drugs, indicating a need for better understanding of the intricacies of targeted pathways. Using an advanced confocal intravital microscopy platform we have analysed details of neutrophil-vessel wall interactions in real-time in 3D and have noted previously unreported physiological responses (eg sub-endothelial cell crawling 1) and also potential pathological events such as neutrophil reverse transendothelial cell migration, a response that we have associated with dissemination of systemic inflammation 2. Such studies have demonstrated that detailed analysis of leukocyte-vessel wall dynamics are likely to identify novel and disease-specific phenomena that could promote a change in thinking towards development of new therapeutic strategies.

1. Proebstl, J et al., Exp Med 209: 1219-1234 (2012).

2. Woodfin, A et al., Nat Immunol 12:761-769 (2011).

This work was funded by The Wellcome Trust.

Vaskuläre Inflammation

T03 – Talk Wenzel

Inflammation in vascular disease: Role of myeloid cells and involvement of coagulation

P. Wenzel1

1University Medical Center Mainz, II. Medical Department and Center for Thrombosis and Hemostasis, Mainz, Deutschland

Inflammation and atherosclerosis are causally interconnected. Recent data suggest, that inflammatory leukocytes from the myelomonocytic lineage (lysozyme M positive) are involved in the pathogenesis of vascular inflammation, endothelial dysfunction in the setting arterial hypertension as well. Vascular infiltration of monocytes, macrophages and dendritic cells, but also T-cells and NK-cells are a hallmark of angiotensin II mediated inflammatory injury. Inhibition of inflammatory cytokine pathways like IL-6, IL-17, IL-12 or T-bet/Interferon-γ, depletion of LysM+ or NK1.1+ cells or knockout of MCSF dependent signalling or RAG-1 dependent cell development have all been shown to be effective strategies in limiting vascular injury in experimental arterial hypertension. Coagulation factors like tissue factor are also mechanistically involved in vascular dysfunction. The interface between coagulation factors, circulating and infiltrating inflammatory cells and the vascular wall is currently being investigated in more detail.

Molekulare Onkologie

T04 – Talk Hahn

Bedeutung von mikroRNAs in der Onkologie am Beispiel des Kolonkarzinoms

S. Hahn1

1Ruhr-University Bochum, Molecular GI-Oncology, Bochum, Deutschland

Recently, microRNAs (miRNAs), a novel class of 18-23 nt long non-coding RNAs, have gained attention as another family of molecules involved in cancer development. Upon binding to their target RNAs, miRNAs cause post-transcriptional gene silencing by either cleaving the target mRNA or by inhibiting the translation process. To date, mis-regulation of miRNAs has been demonstrated in virtually any hematological and solid tumor entity including colon cancer and there is ample evidence that altered miRNA expression is contributing to tumor development and progression. From the diagnostic standpoint, the main advantage of miRNAs are their proven high stability in poorly preserved specimen and the availability of high specific and sensitive miRNA detection assays. Furthermore, it was shown that miRNAs originating from tumors are indeed present in the circulation and that binding to proteins or their inclusion into microvesicles are key to their protection from degradation. Therefore, they can be considered as robust clinical analytes for biomarker discovery. By virtue of their role in tumorigenesis, miRNAs were not only shown to be useful for prognostic and diagnostic biomarker discovery strategies including colon cancer but also have therapeutic potential. In fact one miRNA-based therapeutic aiming at blocking replication of the hepatitis C virus has already entered Phase 2 clinical trials. The potential utility of this approach for colon cancer awaits its proof of concept.

Molekulare Onkologie

T05 – Talk Diehl

Zirkulierende Tumor-DNA als neuer Biomarker in der Molekularen Onkologie

F. Diehl1

1Inostics GmbH, Hamburg, Deutschland

Objective: The molecular characterization of tumors has shown to be important for guiding treatment decisions in oncology. For example, defined tumor specific somatic mutations are predictors of response to targeted therapies. The analysis of free circulating DNA isolated from plasma or serum could extent the molecular characterization of tumors to situations were tissue is not available. In particular, plasma DNA testing could improve patient stratification and monitoring of therapy response as well as disease recurrence. In addition, the detection of resistance mutations arising during therapy could be facilitated by plasma DNA testing.

Methods & Results: Previous mutation detection assays have not been sufficiently specific, sensitive, and quantitative for the assessment of the clinical utility of circulating nucleic acids in oncology. Newly developed technologies based on digital PCR provide a high sensitivity and at the same time allow quantification of the fraction of mutant to normal DNA molecules. One of these approaches applied by Inostics is the BEAMing Digital PCR Technology combining emulsion based digital PCR with magnetic beads and flow cytometry. Several studies have been performed using BEAMing showing that advanced tumors of the breast and colon release sufficient amounts of tumor DNA for non-invasive testing.

Conclusion: In summary, we believe that plasma DNA testing has the potential to greatly improve patient management as well as early detection in oncology.

Joint Symposium AG LC-MS/MS der DGKL und AG TDM der AGNP Klinische Massenspektrometrie und therapeutisches Drug Monitoring - Anspruchsvolle Zusammenarbeit zwischen Klinik und Labor

T06 – Talk Eap, Talk Stingl

From therapeutic drug monitoring to genotyping

C. Eap1, J. Stingl2

1Centre for Psychiatric Neuroscience of the Department of Psychiatry-CHUV, Unit of Pharmacogenetics and Clinical Psychopharmacology (UPPC), Hospital of Cery, Prilly-Lausanne, Schweiz

2Leiterin Abteilung Forschung, Bundesinstitut für Arzneimittel und Medizinprodukte, Professorin für Translationale Pharmakologie, Universität Bonn, Bonn, Deutschland

From therapeutic drug monitoring to genotyping

S. Crettol Wavre, N. Ansermot, CB. Eap

Unit of Pharmacogenetics and Clinical Psychopharmacology, Department of Psychiatry, Lausanne University Hospital, Switzerland

Objective: To discuss the present and future use of therapeutic drug monitoring (TDM) and genotyping

Methods: Review of published studies in the field

Results: Multiple genetic factors can influence the response to treatment with somatic and psychotropic drugs. Due to the very large interindividual variability in drug pharmacokinetics, TDM can be very useful and, in some cases, even necessary. Numerous studies have tried to characterize the determinants of drug pharmacokinetic variability, many of them on the cytochrome P450 (CYP) isoforms. Discoveries on the genetic determinants of the activities of CYP isoforms have raised the possibility that CYP genotyping could be done before starting a treatment so to adapt the dose according to the patient’s metabolizing status. However, until now, with very few exceptions, there is a notable lack of generalization of pharmacogenetic tests in routine clinical practice. We will present examples of genes for which genotyping allows a good estimation of the activity of the enzyme (e.g. CYP2D6), and of those for which genotyping poorly reflect the enzyme variability (e.g. CYP1A2). For the latter, TDM remains presently the best option.

Conclusion: The present and possible future use of TDM and pharmacogenetics will be discussed, also taking into account the fast decreasing cost of genetic analysis

Joint Symposium AG LC-MS/MS der DGKL und AG TDM der AGNP Klinische Massenspektrometrie und therapeutisches Drug Monitoring - Anspruchsvolle Zusammenarbeit zwischen Klinik und Labor

T07 – Talk Schwarz

Von der Anforderung zum Befund - Welche Angaben benötigt das Labor und was beinhaltet ein hilfreicher TDM-Befund?

M. Schwarz1

1Institut für Laboratoriumsmedizin, Standort TDM-Labor Psychiatrische Klinik und AG Neurobiochemie, Klinikum der Universität München (LMU), München, Deutschland

Therapeutic Drug Monitoring – which information is needed in the lab and which comments should accompany the pure lab result?

Therapeutic Drug Monitoring (TDM) goes far beyond the mere determination of drug concentrations in blood: A helpful report of TDM analyses includes an evaluation of the measured concentrations and it gives advice to optimize pharmacotherapy in order to improve drug safety and response to treatment. Particular attention is paid to pharmacokinetic aspects.

Even in case of the classical indication - the test for toxic blood levels - high blood levels may not only be caused by exacting dosage, but may also be due to pharmacokinetic interactions or pharmacogenetic abnormalities. TDM is the quick and simple tool to gather this crucial information. In order to make a meaningful statement on pharmacokinetics, it is usually important to determine the main metabolite of the drug to be monitored. Based on the ratio of metabolite to parent compound, the result may indicate an abnormal metabolism. The possibility of a partial non-adherence to the drug has to be taken into account, as well.

Several requirements and detailed information is needed for the correct evaluation of the findings: In general, steady state has to be reached and the blood sampling should be done at trough levels. Information on daily dose of the drug and its application scheme, as well as a list of all other medications the patient is taking is essential. Further information should be provided to the pharmacokinetically relevant additional use, as well as pharmacokinetically relevant comorbidities.

This presentation will present typical cases to demonstrate how TDM can be used for individualized risk reduction.

Session der Sektion Immunologie

T08 – Talk Kirschfink

Moderne Komplementanalytik – Indikationen, Methodik, Perspektiven

M. Kirschfink1

1Institute of Immunology, University of Heidelberg, Heidelberg, Deutschland

The complement system plays a crucial role in a multitude of physiological and pathophysiological processes. Complement analysis in the clinic is usually associated with the quantification of C3 and C4, measurement of C1-inhibitor and screening for complement activity. These analyses have been available in routine diagnostic laboratories for decades. In recent years, however, the field of complement analysis has expanded considerably, with the introduction of novel assays to detect complement activation products, and expanding still further towards genetic analysis to reveal the basis of complement deficiencies, and to identify mutations and polymorphisms associated with defined diseases such as atypical haemolytic uraemic syndrome and age related macular degeneration. Thus, in recent years modern complement analysis has decisively contributed to the elucidation of pathophysiological processes of various diseases and has thus paved the way for effective new therapies. The current status of complement analysis, including assays for the quantification of complement activity and complement activation products, aiming at to detect deficiencies and deleterious hyperactivation of the system will be presented. Leading international diagnostic laboratories have now joined with the goal of quality assurance and further development of complement tools taking into account the legitimate desire for high-quality complement analysis (http://www.iuisonline.org/iuis/index.php/quality-assessment-and-standardization-committee.html).

Session der Sektion Immunologie

T09 – Talk Sack, Talk Boldt, Talk Kahlenberg

Analytik Antigen-spezifischer T-Zellen

U. Sack1, A. Boldt2, F. Kahlenberg3

1Universitätsklinikum Leipzig, Institut für Klinische Immunologie, Leipzig, Deutschland

2Institut für Klinische Immunologie, Medizinische Fakultät, Universität Leipzig, Diagnostik, Leipzig, Deutschland

3Institut für Klinische Immunologie, Medizinische Fakultät, Universität Leipzig, Diagnostik, Leipzigq, Deutschland

Objectives: The targeted identification of antigen-specific T cells in patients represents still a diagnostic challenge but has a high impact on diagnosis of infections, autoimmune diseases, allergy, and immunodeficiency.

Methods: Here, we describe detection of antigen-specific T cells by flow cytometry in an 8-tube-8-colour based staining, but also by Elispot techique, and in cell culture approaches.

Results: Dependent on clinical questions, cell culture or direct staining protocols should be chosen. Elispot and bulk culture methods mostly do not give any specific information about cell subpopulations. Flow cytometry can include detection of antigen specific T cells and allows deep analysis of subsets involved in antigen specific responses. Combinationof intracellular stainings with surface activation markers allows a detailled analysis of immune response.

Conclusion: Detection of antigen-specific T cells by flow cytometry is a promising tool for improving analysis of T-cell driven immune response. Elispot and bulk culture approaches are easier but not so specific concerning the target cell population.

Session der Sektion Immunologie

T10 – Talk Bien

Serologische Diagnostik autoimmuner Enzephalopathien

C. Bien1

1Epilepsy Centre Bethel, Krankenhaus Mara, Bielefeld, Deutschland

Auto-antibodies of the IgG class directed to neuronal CNS antigens have become an important tool for establishing the diagnosis and for pathophysiological understanding of autoimmune encephalitides in today’s neurology.

In the 1980s/1990s, antibodies to intracellular antigens were discovered in the context of paraneoplastic disorders (i.e., neurological conditions caused by immunological consequences of a cancer remote from the nervous system). These antibodies react with the tumor and the nervous system and are therefore named “onconeural antibodies”. Well-known antigenic targets are Hu, Yo, Ri, Ma1/2 or amphiphysin. These antibodies are determined by indirect immunofluorescence on rodent brain and on immuno-dot-blots. They are quite specific diagnostically. However, they are probably not the primary causes of the clinical conditions which are notoriously refractory to immunological treatment.

More recently, antibodies to antigens on the surface of neurons have been detected. The most frequent targets are the NMDA-receptor and elements of the voltage-gated potassium channel (VGKC-complex), namely LGI1 and CASPR2. For their detection, indirect immunofluorescence on HEK cells transfected with the respective antigens are used. These antibodies to surface antigens are much more frequent than those to intracellular targets. They are not only diagnostically meaningful; they also indicate neurological conditions which often respond well to immunological therapies.

Metabolische Erkankungen

T11 – Talk Biemann, Talk Fischer, Talk Navarrete Santos

Endocrine disrupting chemicals: Ursache der Adipositas Epidemie?

R. Biemann1, B. Fischer1, A. Navarrete Santos1

1Universitätsklinikum Halle, Medizinische Fakultät, Institut für Anatomie und Zellbiologie, Halle (Saale), Deutschland

Objectives: Pre- and postnatal exposure to environmental stimuli may contribute to the rising prevalence of obesity. Here we show that endocrine active food contaminants (EDC) drive the commitment of mesenchymal stem cells (MSC) to the adipogenic lineage.

Methods: Murine embryonic MSC, C3H10T1/2, were exposed to 10µM bispenol A (BPA), 100µM diethyhexylphthalate (DEHP) or 100nM tributyltin (TBT) at different stages of adipogenesis and analysed for the resulting percentage of adipocytes and the cellular triglyceride content. To investigate the mechanisms of EDC-induced adipogenic differentiation, PPARg promoter activation and expression of adipogenic and cell fate specific marker genes, such as PPARg1, PPARg2, Pref-1 and SOX9, were quantified at different stages of adipogenesis and compared with corresponding non-exposed controls.

Results: Depending on the exposure window, BPA led to an inhibition of adipocyte development, whereas DEHP and TBT enhanced adipocyte generation. The nuclear transcription factor PPARg was identified as a target of DEHP and TBT during adipogenic differentiation. Additionally, TBT promoted adipogenic cell fate commitment of MSC by a PPARg independent mechanism.

Conclusion: This study shows that EDC affect adipogenesis by altering cell fate commitment and final adipogenic differentiation by different and independent mechanisms. The impact of EDC on early cell fate commitment documents a novel mechanistic insight into predisposition for obesity.

Supported by DGKL, EU (FP7; REEF #212885) and the Wilhelm Roux Programme of the MLU Faculty of Medicine

Present address: Department of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University, Magdeburg, Germany

Metabolische Erkankungen

T12 – Talk Herzig

Weiß, Beige und Braun: Die drei Farben des Fetts

S. Herzig1

1Deutsches Krebsforschungszentrum, ZMBH, Heidelberg University, and Heidelberg University Hospital, Molekulare Stoffwechselkontrolle (A170), Heidelberg, Deutschland

Adipose tissue can be subdivided into at least two distinct categories of fat cells:

White adipocytes are specialized for the storage of chemical energy such as triglycerides, and under conditions of obesity white adipose tissue is characterized by tissue inflammation and energy overload.

In contrast, brown adipocytes dissipate energy in the form of heat (thermogenesis) by uncoupling of the mitochondrial electron transport chain from ATP formation through the specific expression of so-called uncoupling protein (UCP)-1. Lineage tracking studies in mice have demonstrated that white adipocytes are distinct from brown adipocytes during normal embryonic development. Indeed, progenitors of the brown adipocye lineage have been shown to share a common precursor with the myogenic lineage.

In addition, a third distinct brown adipocyte-like cell type has been identified (brown-in-white, BRITE). In response to cold exposure, many WAT depots develop an increased number of BRITE cells, which seems to be mediated –at least in part- through sympathetic beta3-adrenoceptor agonist action. Despite typical BAT-like functional properties, such as UCP-1 expression, the BRITE adipocytes are developmentally distinct from the “classical” brown cells and their functional contribution to systemic energy metabolism has only recently been documented.

Here, new developments and findings with respect to white vs. brown vs. BRITE adipocyte determination as well as its implication for the control of systemic energy homeostasis will be discussed.

Hämatologie

T13 – Talk Haferlach

Hämatologische Diagnostik: Von der Morphologie zum Next-Generation-Sequencing

T. Haferlach1

1MLL Münchner Leukämielabor GmbH, München, Deutschland

The diagnostic approaches in hematologic malignancies are changing. A stepwise approach is still state of the art with respect to timelines, workflow and resources of personnel and budget. Therefore, based on clinical information and morphology all other techniques should be ordered specifically. As the importance is increasing, new technologies have been introduced such as multiplex PCR and especially next-generation sequencing (NGS). The latter technique allows to investigate many genes of interest in one approach, has much higher sensitivity than standard Sanger sequencing and especially is now compatible with a workflow below three working days.

One important aspect is not only the quality of the material to be investigated but especially the read out: This means a professional help of biostatisticians is absolutely needed and without this, valid information cannot be achieved. This is true for the exclusion of copy-number variations, polymorphisms or SNPs that do not really contribute to the diagnosis.

Today it’s the ultimate goal to not only implement those assays into routine settings but also to define the role of Next-Generation Sequencing in the diagnosis of hematologic malignancies. It will be absolutely mandatory to challenge this new technology as it will make diagnoses safer, quicker and maybe even cheaper in the next future. The healthcare providers should address this important diagnostic challenge quickly to use the advantages of the next-generation sequencing for the individual patient.

Hämatologie

T14 – Talk Huber, Talk von Känel, Talk Brunner

Molekulargenetische Diagnostik der Anämien und der Hämoglobinopathien

A. Huber1, T. Von Känel2, S. Brunner2

1Kantonsspital, Center of Laboratory Medicine, Aarau, Schweiz

2Kantonsspital Aarau, Center of Laboratory Medicine, Aarau, Schweiz

Background and Objectives: Anemias are common findings through all ages. They can be caused by a variety of diseases either aquirred (malign or non-malign) or of congenital nature. While many causes are easily clarifed (e.g. iron deficiendy), in other situations it is more difficult to definitively define the underlying defect. Many different genetic disorders presenting with anemia and often other clinical features exist, yet they are of very low prevalences (orphan diseases). Finally, there is a large entity of common inherited anemias, the hemoglobinopathies and the red cell membrane defects.

Methods: We developed new algorithms in order to optimally employ different modern molecular biology methods to most efficiently and definitively define the cause of an anemia.

Results: Incorporating medical and family history, clinical presentation and features (syndromes) and initial classical laboratory (screening) assays allowed for the development of strategies on how to use different molecular biology tools such as RT-PCR, Hybridization assays, DNA chip analyses, and next generation sequencing to come to a final diagnosis.

Conclusion: The knowledge of the underlying causes of anemias has grown dramatically. Together with powerful diagnostic laboratory tools, it is nowadays possible to find a final answer to the cause of an anemia quite rapidly. We were further able to define novel diagnostic strategies to efficiently (resources) provide these services to our patients. Ultimately, it is a matter of cost and professionality to offer such diagnostics even for screening purpose in developing countries.

Labormanagement

T15 – Talk Löffert

A Pilot-Study of the Importance of Laboratory Medicine in the View of Different Professional Groups

S. Löffert1

1German Hospital Institute, Duesseldorf, Deutschland

Objectives: To determine the value of laboratory medicine for in-patient treatment and for the economic performance of clinical care providers from the viewpoint of different in-hospital professional groups.

Methods: Semi-structured in-depth interviews (n=10) and expert group discussions (n=23) were carried out with clinicians; anonymous open web-based structured questionnaires were used for collecting opinions from hospital managers (n=211), hospital physicians (n=211) and nurses (n=166). Importance vs Realization diagrams were constructed to visualize results.

Results: All professional groups rated laboratory medicine as an integral part of fast diagnoses and efficient treatment monitoring. In clinical routine expectations are usually met for treatment monitoring and to a slightly lower extent for fast diagnoses. Physicians ranked fast sample processing as most important for diagnoses and patient care according to clinical pathways. A need for ever-present (24h/7d) laboratory services was emphasized. Both physicians and nurses rated the importance of clinical sample logistics and even of mobile teams of blood sample collectors as higher than currently provided by laboratories. Short turn-around times and rapid reporting to clinicians were ranked highest for achieving a reduced length of stay and success of patient treatment. Overall, the ratings by hospital administrators were similar to those by clinicians with the exception of an expressed preference for centralized in-hospital laboratories in contrast to satellite facilities due to economical considerations.

Conclusion: This study indicates that laboratory medicine is well practiced in daily clinical routine. Further improvements can be achieved in pre- and post-analytical procedures

Labormanagement

T16 – Talk Klosson

Laborkostenkalkulation im DRG System

R. Klosson1

1Institut für Laboratoriumsmedizin/Klinikum Hanau GmbH, Hanau, Deutschland

Medical technical domains seem to be especially fit to be controlled through economic characteristic values, as there are supposedly “hard” operational data available. The laboratory cost shares published by the German InEK (institute for the remuneration sytem in the hospital) for individual DRGs (diagnosis related groups) are based on a large data base, data registration is well standardized, the results of the calculation are available to everybody and the determined values do also consider kind and seriousness of the respective DRG case.

However, the InEK data, too, do predominantly consider just the economic side of laboratory diagnostics. If the basic conditions are not duly considered and the processes behind the figures are not taken into consideration, the risk of wrong strategic decisions on future laboratory organization is rather high.

A system of characteristic values must not only capture the economic efficiency and the technical quality of laboratory diagnostics, but also the effectiveness for the patient outcome. In the future competition the so-called non-analytical services of clinical laboratories will gain a constantly rising significance for the patient outcome.

The attempts made during the last few years of controlling clinical laboratories by DRG-supported characteristic values must therefore be further developed, to enable clinical laboratories to fulfil their tasks as a central part of infrastructure within patient care also in the future.

Sektion Molekulare Diagnostik - “Nicht-codierende RNA in Pathophysiologie und Diagnostik”

T17 – Talk Stadler

The Brave New World of Non-Coding RNAs

P. Stadler1

1Professur für Bioinformatik Institut für Informatik, Universität Leipzig, Leipzig, Deutschland

A decade after the completion of the human genome we have realized that there is little, if any, junk DNA. Instead the human genome is pervasively transcribed into a complex pattern of overlapping transcriptional units, only a small minority of which codes for proteins. Although hard empirical evidence if available only for a small number of individual ncRNAs, there are very clear indications that much of the transcriptional has regulatory functions. The molecular mechanisms employed are by no means uniform and divide the realm of non-coding RNAs into a plethora of different classes.

The advent of high-throughput sequencing has allowed systematic and unbiased investigations into the structure of transcriptome and revealed an unexpectedly complex pattern of RNA processing that includes the systematic formation of fusions and circularized RNAs. In my presentation I will attempt to given on overview on the current state of the art.

Porphyrie - Klinik, Diagnostik und Therapie

T18 – Talk Doss

“Einfach kompliziert”: Akute Porphyrien „Simply complicated“: Acute Porphyrias

M. Doss1

1Consultation Porphyria, Marburg, Deutschland

Porphyrias are a heterogeneous group of metabolic diseases which are based on a genetic or acquired deficiency of one or more of the enzymes along the sequence of the heme biosynthesis. Clinical porphyrias are divided into acute and non-acute as well as pathogenetically into hepatic and erythropoietic disorders. The acute hepatic porphyrias (AHP) display a great molecular genetic heterogeneity. The majority of gene carriers (about 85%) never develop clinical symptoms or AHP manifestations.

Liver heme regulates via repression of the key enzyme 5-aminolevulinic acid synthase 1 the biosynthesis of 5-aminolevulinic acid (ALA), porphobilinogen (PBG) and porphyrins. Exogenous and endogenous factors such as drugs, fasting, hormones, alcohol and stress destabilize the regulatory heme pool in the liver causing induction of ALA synthase. This increase of enzyme activity has a compensatory counter-regulatory dynamics initiating the porphyric disease process with overproduction of porphyrin precursors and porphyrins leading to the acute clinical crisis, which includes an abdominal-neuropsychiatric-cardiovascular syndrome. Both porphyrin precursors are considered to exhibit neuropharmacological effects.

Among the AHP, acute intermittent porphyria is most common, followed by variegate porphyria, hereditary coproporphyria and a recessive form with ALA dehydratase deficiency, which is biochemically similar to lead poisoning. Thus, lead poisoning is a toxic or toxogenetic acute porphyria. The diagnosis of clinically symptomatic AHP is based on the excretory metabolite excess, in which each porphyria expresses its own specific constellation.

Generally, AHP are both under- and over-diagnosed. The interpretation of secondary asymptomatic porphyrinurias as “primary” porphyrias is a frequent error. Coproporphyrin-urias are observed in non-porphyria clinical context, in liver- and blood diseases, under the influence of drugs, nutrition and various toxic substances.

Therapy: Besides symptomatic measures the regulatory treatment by intravenous glucose and hemin is in the first line. Both glucose and exogenous heme repress the induction of hepatic ALA synthase reverting the enhanced metabolite production along the heme biosynthetic pathway. Late complications in the course of AHP are hypertension, renal insufficiency and hepatocellular carcinoma. This has to be considered in the management of patients with this kind of diseases.

Porphyrie - Klinik, Diagnostik und Therapie

T19 – Talk Stauch

Diagnose von Porphyrien: Vom Metabolit zum Gen

T. Stauch1

1Klinische Chemie und Toxikologie, -Porphyrie-Speziallabor EPNET-, MVZ Labor PD Dr. Volkmann und Kollegen GbR, Karlsruhe, Deutschland

Porphyrias are a group of rare metabolic disorders, whose major diagnostic difficulty comprises the consideration of their possible occurence in a particular case. After overcoming this important obstacle, the availability of different diagnostic tools applicable in varying matrices makes the diagnosis of disorders in the heme biosynthetic pathway an easily manageable but still multilateral task, in which biochemical and molecular genetic methods are implemented to localize the specific enzyme deficiencies associated with porphyria. The ways of approach to the solution of diagnostic questions connected with porphyria are described, starting from the clinical point of view including symptoms as well as anamnestic data proceeding to the stepwise prosecution of biochemical tests based on the results of previously performed analyses. Preanalytical aspects concerning the sample handling of urine, faeces and blood are elucidated to clarify that a clinical suspicion of porphyria does not entail a preanalytical monster demanding herculean tasks of the requesting clinician, but, in terms of work and effort, is comparable to other diagnostic procedures involving laboratory work. An analytical flow chart is presented starting from tests suitable for general screening e. g. plasmafluorescence scanning, Hoesch-Test, semi-quantitative determination of porphobilinogen in urine going towards more complex analytical procedures with differentiating and confirming character. Advantages, disadvantages and problems frequently associated with the tests and the clinical usefulness of molecular genetic sequencing are discussed.

POCT - Anwendungsfelder im Wandel

T20 – Talk Kochinsky

Sind POCT-Systeme für die Diagnostik des Gestationsdiabetes geeignet?

T. Koschinsky1

1Retiered, München, Deutschland

Are POCT-systems suitable for the diagnosis of gestational diabetes mellitus?

The diagnosis of gestational diabetes mellitus (GDM) is based on venous plasma glucose values derived from an oral glucose test according to the recent update of the GDM-guideline of the German Diabetes Association. For the first time, this guideline recommended for this purpose also selected methods of blood glucose testing at the point-of-care (POCT) using unit-use reagents as an alternative method to established laboratory methods. It has been questioned, whether such glucose-POCT-systems are available at present that match the analytical performance and the legal requirements for their quality control according to the german RiliBÄK.

Analytical performance evaluations of such glucose-POCT-systems according to the DIN EN ISO 15197:2003 protocol provide evidence that their analytical results after a succesful CE-marking process can differ over a wide range between such systems as well as beween different sensor batches of the same system. Therefore, at least 4 groups have been distinguished using the maximal acceptable deviations of the glucose results from the reference value for 95.5% (2 SD) of the examined glucose values as separating criteria: Group A) >20%; B) =20% - >15%; C) =15% - >10%; D) =10%. Only Group D) POCT-systems are suitable for GDM-diagnosis and at least 7 of these CE-marked systems are available in Germany.

To provide further evidence for their diagnostic suitability the German Diabetes Association has initiated the prospective DIAPOC-study to examine the question whether the diagnostic results of Group D) glucose-POCT-systems match those of standard laboratory methods comparing them according to the GDM-guideline under daily life conditions.

POCT - Anwendungsfelder im Wandel

T21 – Talk Petersmann

Intelligente Vernetzung von POCT

A. Petersmann1

1Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsmedizin Greifswald, Greifswald, Deutschland

Smart IT connection solutions in POCT

Objectives: In hospitals patient near testing (point of care, POCT) is an accepted part of clinical routines. In addition to transmitting measurement results data transfer to and from a POCT device includes further information like user names, lots or time stamps. The main focus for IT solutions in POCT so far, are legal and normative requirements.

Methods: Requirements for IT solutions in POCT regard more than the exchange of data between the POCT device and the respective middleware or laboratory information system. POCT can be merged into the hospital workflows.

Results: An integrated data management, for example for data regarding users of POCT devices of different manufacturers is realized in many hospitals. Merging user data in a hospital context for different departments such as patient care, technics or human resources is often prevented by data formats or legal questions on data handling. Smart IT solutions can contribute to a high availability of POCT analysis. Independent of the time of day broken devices can be replaced by non-technicians without limiting the amount or the quality of data transferred to and from the POCT device.

Conclusion: Current IT solutions in POCT register and document all important data linked to the measurement and the respective user. Smart POCT IT solutions can improve hospital workflows by making them more efficient and safer.

Präanalytik Festsymposium Prof. Walter Guder

T22 – Talk Narayanan

Perspectives on the Preanalytical phase and outlook for the future

S. Narayanan1

1Weill Medical College of Cornell University, Pathology and Laboratory Medicine, New York, USA

Perspectives on the preanalytical phase and outlook for the future-S. Narayanan, Weill Medical College of Cornell University, F-715, New York, NY 10021 USA Preanalytical issues surface in tandem with progress in instrumentation, introduction of new diagnostic markers and progress in therapy. Refinement of sample collection devices have paralled advances in instrumentation and mechanization. Contamination of stoppers from evacuated blood collection tubes were uncovered as early as 1974 in gas chromatographic analysis of therapeutic drugs. Zinc from stoppers were shown to abolish the effect of heparin on the activated partial thromboplasin (aPTT) assays, yielding a normal aPTT in patients on heparin therapy. The discovery of the presence of the organism Serratia Marcescens in non-sterile evacuated blood collection tubes in Canada in 1975 ushered in the era of sterile evacuated blood collection tubes in North America. The mid-1970s witnessed the introduction of gel-based serum separation blood collection tubes which with some modifications are still in use today. They too revealed interferences in therapeutic drug monitoring and other assays. Even in the 1st decade of the 21st century we have witnessed problems with immunoassays resulting from silicone surfactant coating that is used to coat the interior of plastic tubes to improve blood flow and hemorepellancy. Day-to-day incidents in the laboratory have exposed the limitations of the stability of specific analytes, effect of sample storage and vagaries of sample collection techniques. Widespread use of molecular biology techniques and use of herbs have uncovered a host of preanalytical issues.

Präanalytik Festsymposium Prof. Walter Guder

T23 – Talk Findeisen

präanalytische Aspekte für Anwendungen des clinical Biobanking

P. Findeisen1

1Medizinische Fakultät Mannheim der Universität Heidelberg, Institut für Klinische Chemie, Mannheim, Deutschland

Preanalytical variations do seriously affect the results of most routinely performed diagnostic assays and also have major impact on biomarker studies. However, analytical quality monitoring specifically for serum- and plasma specimens is not feasible up to now. MS-based analyses of blood samples revealed a time dependent decay of the low molecular weight fraction that is related to intrinsic proteolytic activity of serum and plasma. Accordingly, a LC-MS based method was developed to determine the preanalytical quality of blood specimens by monitoring the time dependent decay of endogenous and exogenous reporterpeptides.

First, a training set was generated with specimens that were aged under controlled conditions and a pattern recognition algorithm was built respectively. Second, an independent validation set was classified with the algorithm to confirm robustness of the method. Taken together, peptides are processed continuously ex vivo in blood specimens after blood withdrawal. This “degradation clock” can be used to estimate the preanalytical quality of blood specimens and could serve as a quality control tool for clinical biobanking. This might minimize erroneous results from biomarker discovery studies that are related to insufficient preanalytical quality of specimens.

Lipidomics

T24 – Talk Obeid, Talk Geisel, Talk Herrmann, Talk Fliser, Talk Heine

Clinical and diagnostic value of blood and urine choline biomarkers

R. Obeid1, J. Geisel1, W. Herrmann2, D. Fliser3, G. Heine4

1Univeristätsklinikum des Saarlandes, Klinische Chemie, Homburg, Deutschland

2Medical School Saarland University, Department of Clinical Chemistry, Homburg, Deutschland

3Medicine, Innere Medizin IV, Homburg/Saar, Deutschland

4of Internal Medicine IV, University Hospital of Saarland, 66421, Homburg, Homburg, Deutschland

Objective: Choline links methylation and lipid metabolism. It is oxidized to betaine, an important methyl donor and osmolyte. Choline is converted to phosphatidylcholine (PC). Phosphatidylethanolamine (PE) consumes 3 methyl groups to produce 1 mole PC. We studied the association between blood methylation markers and plasma lipids. We further investigated urinary choline markers and combined renal outcome in a 4 y follow-up study.

Methods: 246 elderly people and choline related metabolites were measured. Urinary betaine choline and acetylcholine were measured in renal patients at baseline.

Results: Diabetics treated with statins had lower cholesterol, LDL-C and HDL-C and higher TG and plasma choline compared to the controls (11.8 vs. 9.8 µM, p=0.014). Plasma choline concentrations were negatively related to HDL-C (r=-0.508) and S-adenosylmethionine/S-adenosylhomocysteine ratio (r=-0.568) and positively to SAH (r=0.681) in diabetics treated with statins. Male sex, diabetes, statin therapy and higher BMI were associated with a higher choline level. Urinary acetylcholine, betaine, and choline were all increased in patients with renal disorders and the increment predicted worse renal outcome during a median follow-up of 4y.

Conclusion: Higher plasma concentrations of choline are associated with vascular risk factors and components of the metabolic syndrome. The loss of choline and betaine in urine seems to be released to cell shrinkage or apoptosis. Betaine loss can lower the activity of BHMT in the kidney. The limited increase of dimethylglycine suggests an impaired betaine utilization as a methyl donor in renal patients.

Lipidomics

T25 – Talk Schmitz, Talk Matysik

Simultane Massenspektrometrische Analyse von Cholesterinvorstufen, Phytosterolen und Oxysterolen in der Diagnostik von Dyslipidämien

G. Schmitz1, S. Matysik1

1Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Regensburg, Regensburg, Deutschland

To evaluate the balance between cholesterol biosynthesis and intestinal absorption the analysis of cholesterol precursors, i.e. intermediates of endogenous cholesterol biosynthesis such as lanosterol and lathosterol and phytosterols, such as sitosterol, which serve as absorption markers is necessary. Ratios between sitosterol/lathosterol can be used to evaluate the major features of cholesterol metabolism and to differentiate between oversynthersizers and hyperabsorbers. By means of this individual sterol profile recommendations can be generated for hypercholesterolemia patient stratification for therapy. The analysis of sterols also includes the quantification of oxidative products of cholesterol, the oxysterols. Oxysterols play essential roles in the regulation of various biological processes and functions exerting a multitude of biological effects of potential pathophysiological relevance and can therefore be regarded as severity and progression markers of atherogenesis. Oxysterols like 24S-hydroxycholesterol are associated with Alzheimer’s disease and 7-ketocholesterol and cholestane-3b,5a,6b-triol are sensitive biomarkers for Niemann-Pick-Type C. Thus, robust analytical procedures for clinical routine are required. Gas chromatography coupled to triple quad MS offers new opportunities in the combined analysis of cholesterol precursors, phytosterols and oxysterols. Main results of recent studies revealed that (i) statin treatment reduces serum cholesterol precursors but increases serum phytosterols, (ii) cholesterol precursors are associated with Apo E genotypes, (iii) oral probiotic supplementation reduces total cholesterol and phytosterols in hypercholesterolemic adults.

Allergologie Joint-Symposium

T26 – Talk Raulf-Heimsoth

New diagnostic tools for the diagnosis of occupational allergy

M. Raulf-Heimsoth1

1Institut für Prävention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung, Institut der Ruhr-Universität Bochum (IPA), Allergologie/Immunologie, Bochum, Deutschland

More than 250 agents are identified as sensitizer of occupational asthma (OA). Therefore, an appropriate and early diagnosis of allergic occupational disorders is required to avoid the augmentation of symptoms, to minimize socio-economic costs and to prevent disability and unemployment. Identification of the allergen and the detection of a causal relationship between exposure to allergen and the occurrence of symptoms are important steps. Measurement of specific IgE (sIgE) in sera is the most important and also practical available in-vitro test. For occupational allergens the standard procedure of sIgE detection based on allergen extracts. Compared to environmental allergens only for few occupational allergens the composition is known so far. If recombinant allergens are available they offer the determination of sensitization profiles and “spiking” of the the allergen preparation with the recombinant allergen if the relevant allergen is too labile to survive all steps of extract preparation. Methods for coupling the occupational allergens to a sufficient solid phase are useful if commercial test tools are not available for individual cases. For discrimination between true allergy and co-sensitization the use of single recombinant allergens and cross-reactive carbohydrate determinates (CCDs) is meaningful. Both are included in an latex diagnostic work-flow. Especially for wood dust allergy the discrimination between peptidic and carbohydrate IgE epitopes seems to be useful. However, in some patients it is not possible to detect sIgE in serum. In these cases it is interesting to evaluate whether cellular test, capable of detecting IgE-mediated reactions of the blood basophil level, could bring some information.

Allergologie Joint-Symposium

T27 – Talk Kleine-Tebbe

Molecular diagnostic of food allergies – No more need for oral challenge tests?

J. Kleine-Tebbe1

1Allergie- und Asthma Zentrum Westend, Praxis Hanf, Ackermann & Kleine-Tebbe, Berlin, Deutschland

The knowledge about allergenic molecules in foods facilitates new diagnostic options for the dectection of allergen-specific IgE antibodies. In how far oral provocation tests – used to prove the clinical relevance of a presumed food reaction - can be omitted, cannot be generally answered, but only allergen-specifically defined.

Few molecule families of plant or animal origin consist of relevant food allergens of similar sequence and structure, carrying common IgE epitopes as the basis of cross reactions (i. e. Bet v 1-homologous PR-10 family, lipid transfer proteins (LTP), various seed storage proteins (SSP), oleosins, profilins in plant products).

Sorting the available single allergens, SSP from peanut (Ara h 2), hazelnut (Cor a 9, Cor a 14), walnut (Jug r 1) and soy beans (Gly m 5/6), LTP from peanut (Ara h 9), hazelnut (Cor a 8) and walnut (Jug r 3), PR-10 soy protein Gly m 4, peach-LTP Pru p 3 and wheat gliadin Tri a 19 represent interesting examples as risk molecules for systemic allergic reactions.

Molecular diagnostic of food allergy with improved analytical sensitivity will increase the number of positive allergen-specific IgE results; their clinical relevance is only present in case of corresponding symptoms and should by be confirmed on an individual basis.

Controlled oral challenge tests with peanut allergic subjects have demonstrated that sensitizations to peanut allergen Ara h 2 are associated with an increased risk of systemic reactions and that provocation tests can partly be omitted. Such clinical evaluations are still lacking for other foods. However, negative IgE results are of great value, indicating a lack of sensitization and therefore excluding (clinically severe) allergic reactions to the food in question.

AG Durchflusszytometrie und Mikroskopie

T28 – Talk Neukammer

Development of reference measurement procedures for the enumeration of DNA copy number, lipoprotein particles and blood cells

J. Neukammer1, M. Kammel1, H. Parkes2, C. Foy2, S. Ellison2, M. Sassi3, C. Divieto3, V. Delatour4, T. Dreo5, M. Akgöz6, J. Sahin6

1Physikalisch-Technische Bundesanstalt (PTB), Braunschweig und Berlin, Germany

2Laboratory of the Government Chemist (LGC Ltd.), Teddington, UK

3L’Istituto Nazionale di Ricerca Metrologica (INRIM), Turino, Italy

4Laboratoire National de Métrologie et d’Essais (LNE), Paris, France

5National Institute of Biology (NIB), Ljubljana, Slovenia

6Scientific and Technological Research Council of Turkey, National Metrology Institute (TÜBITAK UME), Kocaeli, Turkey

Accurate counting of biological entities is needed in healthcare to support diagnostic and therapeutic decisions. Examples include viral load monitoring in patients, platelet concentrations in transfusion medicine, circulating tumour cells in cancer, cell contamination in vaccines and blood product and the detection of lipoprotein particles for risk estimation of patients. High accuracy is indispensible to reliably classify measurement results with respect to border lines for e.g. initiating transfusions or treatment of HIV patients. Higher order reference measurement procedures and reference materials are prerequisite to derive traceability and to underpin comparability of measurements. Metrological traceability to the International System of Units, defined through a calibration hierarchy in accordance with ISO 17511, will allow to establish or improve external quality assurance schemes for routine laboratories and hence result in an impact upon accreditation and regulatory compliance. Also manufacturers benefit from the availability of reference materials when developing new diagnostic tests.

Concepts and first results for the traceability of measurement results of DNA copy number, lipoprotein particles and cells, based on digital PCR, scanning mobility particle size spectrometry and flow cytometric and microscopic cell counting will be presented.

The project is funded by the European Union within the European Metrology Research Programme (EMRP call 2012) SIB-54 Bio-SITrace.

AG Durchflusszytometrie und Mikroskopie

T29 – Talk Kammel

Standardization of measurement procedures for the determination of cell concentrations in body fluids

Martin Kammel1, Nicole Bock1, Andreas Ruf2, Jörg Neukammer1

1Physikalisch-Technische Bundesanstalt (PTB), Braunschweig und Berlin, Deutschland

2Städtisches Klinikum Karlsruhe gGmbH, ZLMT-Abteilung für Transfusionsmedizin und Hämostaseologie, Karlsruhe, Deutschland

Determination of cell concentration is one of the most frequently requested quantities to support medical diagnostic. Critical decisions are based on the results of concentration measurements, e.g. in transfusion medicine the platelet concentration is required and in HIV therapy the concentration of T-helper cells is essential. To reliably support diagnosics based on defined limits for therapy selection, the concentration of cells needs to be measured with high accuracy. National standards for the determination of cell concentrations, describing requirements for higher order or primary reference measurement procedures, are only available for platelets, red blood cells and white blood cells. However, there is an urgent need to extend such primary reference procedures to other clinically relevant target cells, in particular CD4- (T-helper) or CD34-positive stem cells. Besides highly sophisticated primary measurement procedures, simple and robust secondary reference measurement procedures are requested. This will allow manufacturers and end users on-site assessment of the performance of their instruments and validation of protocols. A secondary reference procedure on the basis of relative counting will be presented and comparisons between the primary and the secondary reference measurement procedure will be discussed with respect to the uncertainties of measured particle concentrations. The project was funded by the German Federal Ministry of Economics and Technology (BMWi).

AG Durchflusszytometrie und Mikroskopie

T30 – Talk Höckner

Flow cytometric determination of blood stem cells: comparison of dual and single platform methods

Jana Höckner2, Martin Kammel1, Jörg Neukammer1, Andreas Ruf2,

1Physikalisch-Technische Bundesanstalt (PTB), Braunschweig und Berlin, Deutschland

2Städtisches Klinikum Karlsruhe gGmbH, ZLMT-Abteilung für Transfusionsmedizin und Hämostaseologie, Karlsruhe, Deutschland

National and international guidelines suggest that for the determination of blood stem cell concentrations single platform procedures seem to be superior to dual platform methods. However, this assumption has not yet been proven by comparison with a reference device. We used a Partec CyFlow ML equipped with a high precision dosage unit developed on the PTB as reference device (RD) according to ISO 17511 which allows a direct precise cell concentration determination. In fresh and cryopreserved (cryo) peripheral blood stem cell samples CD34 cell concentrations were determined with the RD, a FacScan cytometer (Becton Dickinson BD) and a FC 500 (Beckman Coulter BC). CD 34 cells were identified according to the ISHAGE guidelines. Single platform (SP) methods were based on Trucount beads (BD) and Flow-Count beads (BC), respectively. For the dual platform (DP) methods leukocyte counts were determined by the Sysmex XN 2000. Normalised deviations of the CD34 cell concentrations of the FacScan to the RD were in: DP fresh samples (7 ± 7)%, DP cryo samples (39 ± 22)%, SP fresh samples (9 ± 5)%, SP cryo samples (13 ± 10)%. Similar results were obtained with the other cytometers. Normalised deviations of the leukocyte concentration from the Sysmex to RD were in cryo samples (26 ± 10)% and (8 ± 6)% using the anti-CD45 antibody from BD and BC, respectively. Single and dual platform procedures reveal equivalent analytic performance with respect to accuracy as long as leukocytes can be discriminated from other particles by immunological and light scatter characteristics. The study was founded by the BMWi and the DGKL.

Hämostaseologie und Inflammation

T31 – Talk Brodde, Talk Anne, Talk Speth, Talk Heilmann, Talk Loeffler, Talk Kehrel

Interaktion zwischen Thrombozyten und Leukozyten

M. Brodde1, B. Anne1, C. Speth2, C. Heilmann3, B. Loeffler4, B. Kehrel5

1UKM, Anaesthesiology, Intensive Care and Pain Therapy, Muenster, Deutschland

2Medizinische Universität Innsbruck, Hiegiene, Microbiology ans Social Medicine, Innsbruck, Österreich

3UKM, Medical Microbiology Muenster, Muenster, Deutschland

4UKM, Medical Microbiology, Muenster, Deutschland

5Universitätsklinikum Münster, Zentralklinikum, Anästhesiologie, operative Intensivmedizin und Schmerztherapie, Experimentelle Hämostaseologie, Münster, Deutschland

Thrombosis, inflammation and host defence involve complex platelet-leukocyte interactions.

Platelets influence leukocyte function and vice versa via direct cell–cell contact and/or soluble secreted mediators. Examples of the cross-talk between platelets and leukocytes will be given. Increased interactions between leukocytes and platelets occur in the circulation of patients suffering from a variety of ischemic disorders. H2O2 formed by activated leukocytes can be oxidized by myeloperoxidase (MPO) in the presence of Cl- to HOCl. HOCl alters protein structures. The modified proteins are highly potent platelet agonists, which activate via the scavenger receptor CD36, as CD36 deficient platelets were not activated. As granulocytes get activated, they secrete alpha defensin 1-3 (HNP1-3). At concentrations, which have been found under pathologic conditions, HNP1-3 are potent platelet agonists. The pore-forming Staphylococcus aureus toxin Pantone-Valentine leukocidin (PVL) induces severe necrotizing diseases and thrombus formation in vivo. The thrombus formation is a result of tight granulocyte platelet interaction, as PVL induces the secretion of HNP1-3 and MPO, which leads to strong platelet activation. Platelets and neutrophils help each other to form extracellular traps to ensnare microorganisms. Human pathogens, like Staphylococcus aureus, Candida albicans, and HIV-1 as well as platelets themselves bind to these nets.

Qualitätssicherung - von der Transplantationsmedizin zur RiliBÄK

T32 – Talk Schmidt, Talk Kaise, Talk Neumaier, Talk Thiery

Qualitätssicherung in der Lebertransplantationsdiagnostik - Ergebnisse der Lab-MELD-Score Pilotstudie External quality control for liver transplantation diagnostics – results of a lab-MELD-score pilot study

M. Schmidt1, T. Kaiser2, M. Neumaier3, J. Thiery2

1Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin e.V., Bonn, Deutschland

2Uniklinik Leipzig AöR, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Deutschland

3Klinikum Mannheim der Universität Heidelberg, Institut für Klinische Chemie, Mannheim, Deutschland

Objectives: According to current guidelines, the allocation to the group of elective patients (= 16 years) is depending on the severity of liver diseases. These measurements based on the lab-MELD score (laboratory model of end-stage liver disease). The lab-MELD score is calculated from laboratory values ??bilirubin (mg/dl), creatinine (mg/dl) and prothrombin time expressed as INR (International normalized ratio).

Aim: The aim of our study was to analyse the relevance of the different diagnostic methods for bilirubin, creatinine and INR using an external proficiency panel to calculate the lab-MELD scores.

Methods: Four medical cases were combined with clinical samples from two different collection time points. All samples were analyzed by seven participants for creatinine, bilirubin and INR, respectively and interpreted with anamnestic medical data.

Results: The measurement uncertainty was greatest for INR and smallest for bilirubin. All participants performed a correct calculation of the lab-MELD-score values ??and made correct interpretations of calculated values ??due to anamnestic data. Bilirubin had an influence on final lab-MELD-score values of one point, creatinine of two points and INR of three points.

Conclusion: The external proficiency study design was well performed by all participants. Consultants in laboratory medicine were able to made correct lab-MELD-score calculations and interpretations of valid test results in context to clinical data. To improve quality for liver transplantation new liver synthesis parameter like factor five will be investigated in next proficiency studies.

Qualitätssicherung - von der Transplantationsmedizin zur RiliBÄK

T33 – Talk Vogt

Die neue RiliBÄK - ein umfassendes System

W. Vogt1

1München, Deutschland

Die Richtlinien der Bundesärztekammer zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen, kurz RiliBÄK, waren bis 2002 im Eichgesetz verankert, seit diesem Zeitpunkt sind sie Teil des Medizinprodukterechts. Letzteres fällt in die Zuständigkeit des Bundesministeriums für Gesundheit (BMG).

2002 hat das BMG die Bundesärztekammer (BÄK) gebeten, eine umfassende Neuregelung in Angriff zu nehmen, die alle Bereiche laboratoriumsmedizinischer Untersuchungen umfasst. In sechs Arbeitsgruppen wurden in den letzten zehn Jahren ein Allgemeiner Teil A, der die Begriffsbestimmungen und die Grundsätze des Qualitätsmanagements enthält (in Kraft seit 1. 4. 2008), und fünf Spezielle Teile B intensiv beraten und fertiggestellt. Die Entwürfe wurden vor der endgültigen Verabschiedung in Anhörungsverfahren mit den betroffenen Kreisen diskutiert und abgestimmt.

Die speziellen Teile B beinhalten detaillierte Vorgaben zur regelmäßigen internen Qualitätssicherung und zur Teilnahme an Ringversuchen. Es sind dies:

  • B 1 „Quantitative laboratoriumsmedizinische Untersuchungen“ (in Kraft seit 1. 4. 2008)

  • B 2 „Qualitative laboratoriumsmedizinische Untersuchungen“ (in Kraft seit 1. 7. 2011)

  • B 3 „Direkter Nachweis und Charakterisierung von Infektionserregern“ (in Kraft seit 1. 4. 2013)

  • B 4 „Ejakulatuntersuchungen“ (in Kraft seit 1. 1. 2011)

  • B 5 „Molekular- und zytogenetische laboratoriumsmedizinische Untersuchungen“ (in Kraft seit 1. 10. 2011)

Die Anforderungen in der RiliBÄK beruhen auf § 4a Medizinproduktebetreiberverordnung. Sie sind so abgefasst, dass sie von den Laboratorien eingehalten werden können, da im Gegensatz zur freiwilligen Akkreditierung Verstöße gegen die RiliBÄK formale Sanktionen durch die Überwachungsbehörden nach sich ziehen können.

Free Talk

Metabolic Diseases

FT01 – Talk Chung

LFA-1 regulates trafficking of regulatory T cells into adipose tisse (AT) and maintains insulin sensitivity in diet induced obesity (DIO)

K. Chung1, J. Gebler1, A. Klotzsche - von Ameln1, M. Prucnal1, S. Grossklaus1, S. Bornstein2,

V. Alexaki1, T. Chavakis3

1University Dresden, Department of Internal Medicine III, Institute of Physiology, Dresden, Germany

2University Dresden, Department of Internal Medicine III, Paul-Langerhans Institute, Dresden, Germany

3University Dresden, Department of Internal Medicine III, Institute of Physiology, Institute for Clinical Chemistry and Laboratory Medicine, Paul-Langerhans Institute, Dresden, Germany

Objectives: Inflammatory cells and AT inflammation play a critical role in obesity-associated insulin resistance. However, the molecules that control inflammatory cell trafficking into the AT have not been studied.

Methods: Male WT and LFA-1 KO mice were fed with normal diet or high fat diet for 18 weeks. Glucose and insulin tolerance tests were performed and leukocyte subpopulations in the fat tissue were analyzed by flow cytometry. For T cell reconstitution assay, obese LFA-1 KO mice received isolated CD4+CD25+ regulatory T cells (Treg) intra-peritoneally. For cell trafficking assay, fluorescence labelled T cells were injected to WT obese mice.

Results: Unexpectedly, LFA-1 deficient mice displayed severe glucose intolerance and insulin resistance in both lean and obese mice. AT macrophage infiltration and inflammation were also increased, whereas Treg, as well as CD4+ and CD8+ T cells were decreased in the AT of LFA-1-deficient mice. Consistently, reconstitution of obese LFA-1-deficient mice with LFA-1-proficient CD4+CD25+ Treg rescued insulin sensitivity and reduced macrophage accumulation in the AT. In addition, isolated LFA-1-deficient Treg displayed impaired trafficking into the obese AT, as compared to LFA-1-sufficient cells.

Conclusion: LFA-1 controls Treg accumulation in the AT, thereby regulating AT inflammation and AT insulin sensitivity

Haematology

FT02 – Talk Kohlschein

Discrimination of reactive and neoplastic leukocytosis by combining the DIFF and the WPC channel of the new XN-series

P. Kohlschein1, K. Dreißiger1, P. Schuff-Werner2

1Universitätsmedizin Rostock, Institut für Klinische Chemie und Laboratoriumsmedizin, Rostock, Germany

2Ärztlicher Vorstand und Vorstandsvorsitzender, Universitätsmedizin Rostock – rechtsfähige Teilkörperschaft der Universität Rostock, Rostock, Germany

Introduction: The analytical technology of the XN series is based on the measurement of three side fluorescence signals (sFL). These channels are WNR, WDF, WPC, PLTF & RET-channel. The WDF in combination with the WPC channel is used for getting more information on white pathological cells in terms of a more detailed flagging information.

Aim of Study: Evaluation of sensitivity and specificity for distinguishing between reactive and neoplastic leukocytosis by DIFF and WPC channel of the Sysmex XN haematology system in comparison to XE2100.

Patients:Samples from 254 patients included in this study were selected from independent routine samples which were either flagged by the XE or from patients with known systemic hematological disease.

Results: The reference method (smear & immune phenotyping & diagnosis) identified 174 samples as positive for reactive or neoplastic cells. Out of these patients 51 were positive for neoplastic cells; 110 out of the 123 samples identified as “reactive” had an increased number of IG pos lymphocytes but no hematological disorder and 44 had an increased number of morphological reactive lymphocytes. From the 51 samples identified as neoplastic 19 were positive for lymphoma cells and 32 for blasts cells.

Using the XN, the positive flagged samples was reduced by around 20% (214 vs 179). There was also a overall reduction of flags (166 vs 65).

Conclusion: The comparison of the XN analyzer with the XE series resulted in a total reduction of 20% smears of false positive flagged samples without increasing the rate of false negatives samples. The XN showed a reduction of flags suspecting neoplastic cells by 41% as compared to the flags of the XE analyzer without increasing the false negatives.

Molecular Diagnostics

FT03 – Talk Schmitz

A simple method for quantifying graft cfDNA as an early biomarker of rejection

J. Beck1, S. Bierau1, S. Balzer1, R. Andag2, P. Kanzow2, J. Schmitz2, J. Gaedcke3, O. Moerer4,

J. Slotta3, P. Walson2, O. Kollmar3, M. Oellerich2, E. Schütz1

1Chronix Biomedical, Göttingen, Germany

2University Medicine, Department of Clinical Chemistry, Göttingen, Germany

3University Medicine, Department of General and Visceral Surgery, Göttingen, Germany

4University Medicine, Department of Anaesthesiology, Göttingen, Germany

Cell free DNA (cfDNA) from grafts in the circulation of transplant recipients is a promising early biomarker of rejection. Its usefulness was shown in heart transplantation (HTx), using microarrays and massive parallel sequencing of donor and recipient DNA. A digital droplet PCR (ddPCR) method was developed to quantify the percentage of graft cfDNA in organ recipients as an early rejection biomarker.

Plasma from 6 patients soon after liver transplantation (LTx), from stable LTx (n=10), stable kidney (KTx, n=9), and 8 stable HTx recipients (n=8) was used for preamplifcation, followed by a panel of 41 SNP assays on a LightCycler to define heterologous SNPs, and quantification by ddPCR (BioRad QX100).

At 2% minor allele content, the recovery was 94% with a total imprecision of 4 to 14%. Directly after re-establishing blood flow to the liver graft (n=3), the graft cfDNA was ~90%, and decreased with an approximate half-life of 24 to 48 hours. The uncomplicated LTx cases almost always had a graft cfDNA content of <15% from day 10 onwards. In the stable maintenance phase, KTx and HTx patients showed values <4%, whereas in LTx the values were <7%. In contrast, significant increase of graft cfDNA (up to >60%) preceding AST and bilirubin increase was demonstrated during liver rejection (n=2).

A novel method that allows for the rapid quantification of graft cfDNA using ddPCR at reasonable cost was developed, which for the first time can fulfill the practical needs for routine clinical use.

Cardiovascular Diseases

FT04 – Talk Mey

The influence of Vascular endothelial growth factor (VEGF) on endonuclease driven apoptosis in the vascular endothelium

L. Mey1, M. Hörmann2, Z. Kharip3, A. Hildenberg3, N. Al-Fakhri4, H. Renz5

1Philipps University Marburg, Institute of Laboratory Medicine, Marburg, Germany

2Clinic for Vascular Surgery, University Clinic of Cologne, Cologne, Germany

3Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics; Philipps University, Marburg, Germany

4Philipps University, Institute for Laboratory Medicine, Marburg, Germany

5Universität Gießen und Marburg, Institut für Labormedizin, Pathobiochemie und Molekulare Diagnostik, Marburg, Germany

Background: VEGF is a potent anti-apoptotic effector for endothelial cells (EC). Apoptosis is executed by endonucleases like caspase-activated deoxyribonuclease (CAD), but the influence of VEGF on CAD and inhibitor of caspase-activated deoxyribonuclease (ICAD) has not been shown yet. Therefore, effects of VEGF on CAD and ICAD were investigated in EC, an organ culture model and human arteriosclerosis.

Methods: EC (HUVEC, EC-lines) were incubated with VEGF-A(165) 100pg/ml-1µg/ml for 24h-6d in the presence or absence of ICAD-siRNA 10nM. Apoptosis was induced by cRGDfV 5µg/ml and measured by annexin-V-FACS, caspase-3 and endonuclease activity. ICAD was analyzed by Western blot and real-time PCR. VEGF receptors VEGFR-1, -2 and Neuropilin-1 were investigated by immunofluorescence microscopy and incubation with inhibitors. Human arteriosclerotic versus normal arteries and a vascular organ culture model were analyzed by immunohistochemistry for expression of ICAD, CAD, VEGF and VEGF-receptors.

Results: Incubation of EC with VEGF reduced the sensitivity to apoptosis and increased ICAD expression leading to reduced CAD activity. VEGF effect was demonstrated to be ICAD specific by mRNA-knockdown. VEGF signaling involved VEGFR-2 and Neuropilin-1. The relevance of in-vitro results was demonstrated in an organ culture model and in human arteriosclerosis.

Conclusion: VEGF exerts part of its anti-apoptotic effect by regulation of the endonuclease inhibitor ICAD. VEGF influences endothelial survival by interfering with the terminal stage of apoptosis execution through the inhibition of endonuclease activity. This offers a potent target for the modulation of VEGF-driven vascular processes such as arteriosclerosis.

FT05 – Talk Northoff

Identification of Acvr1c and Surf4 as novel Candidate Genes for Atherosclerosis in Mice through Genome-wide Expression Analyses and Next-Generation Sequencing

B. Northoff1, K. Sass2, A. Nicolaou1, J. Thiery3, D. Teupser1, L. Holdt1

1Ludwig-Maximilians-University Munich, Institute of Laboratory Medicine, Munich, Germany

2University Leipzig, Institute of Anatomy, Leipzig, Germany

3University Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany

Objectives: The aim of the present study was to identify potentially causative genes at two quantitative trait loci (QTL) of atherosclerosis on mouse chromosome (Chr) 2 based on a F2 intercross of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant BALB/cByJ (BALB) female mice on the LDL-receptor deficient background (Ldlr-/-).

Methods: Genome-wide expression analyses in 165 aortas and 176 livers of F2 mice were performed to identify candidate genes co-segregating with the atherosclerosis QTL by expression QTL (eQTL) mapping. Next-generation sequencing of Chr2 was performed in B6 and BALB mice to identify additional candidate genes.

Results: eQTL mapping identified Surf4 as cis-regulated candidate gene co-localizing with the proximal QTL for atherosclerosis. Surf4 mRNA was ubiquitously expressed. Immunohistochemical (IHC) staining revealed expression of Surf4 in endothelial cells of aortic roots. A nonsense-mutation was identified in Acvr1c co-localizing with the distal atherosclerosis QTL. Highest expression was detected in fat tissues, brain and aorta. Expression of Acvr1c in smooth muscle cells of aortic root was verified by IHC staining. In vitro experiments revealed a potential role of both genes in mechanisms of atherogenesis.

Conclusion: We have identified Surf4 and Acvr1c as novel candidate genes of atherosclerosis susceptibility on mouse Chr2. In current work, pathways influencing atherosclerosis susceptibility at these loci are being identified.

Biobanking

FT06 – Talk Findeisen

MS-based monitoring of proteolytic decay of synthetic reporter peptides for quality control of plasma- and serum- specimens

P. Findeisen1, J. Thumfart1, V. Costina1, R. Hofheinz2, M. Neumaier1

1Medical Faculty Mannheim of the University of Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany

2Medical Faculty Mannheim of the University of Heidelberg, III. Medical Clinic, Mannheim, Germany

Objective: Preanalytical variations have major impact on most biological assays. Specifically MS-based multiparametric proteomics analyses of blood specimens are seriously affected by limited protein stability due to high intrinsic proteolytic activity of serum and plasma. Here, we suggest determining the preanalytical quality of serum and plasma by monitoring the time dependent ex vivo decay of a synthetic reporter peptide (RP) with LC/MS.

Methods: Serum and plasma specimens were spiked with the RP and proteolytic fragments were monitored with LC/MS at different preanalytical timepoints ranging from 2h to 24h after blood withdrawal.

Results: The concentration of fragments changed in a time dependent manner and respective peptide-profiles were used to classify specimens according to their preanalytical time span. Classification accuracy was high with values always above 0.89 for areas under receiver operating characteristic curves (AUROC).

Conclusion: This ‘proteomic degradation clock’ can be used to estimate the preanalytical quality of serum and plasma and might have impact on quality control procedures of biobanking repositories.

POCT - Anwendungsfelder im Wandel

FT07 – Talk Christoph

Strategy for Quality of POCT Bilirubin-Results

J. Christoph1, E. Kattner1, F. Guthmann1

1Auf der Bult, Hannover, Germany

Introduction: POC blood gas analysers are frequently used for the measurement and management of bilirubin levels in NICU patients. Recently we observed falsely elevated POCT bilirubin readings different from central lab results.

Method: Bilirubin was tested on two ABL835 blood gas analysers (Radiometer) routinely used in our NICUs and on a Siemens Dimension in the central lab.

From 2007 to 2012 >23.000 bilirubin results from Radiometer BGA and >13.000 from Siemens Dimension are stored in our laboratory information system (LIS). In >1.500 bilirubin results the time between POCT BGA and acceptance of the same patient’s sample in the laboratory differed up to 1 hour. Our sample consists of these retrospectivly merged pairs. It is impossible to determine if both samples came from the same blood sample, as the material type (arterial / venous / capillary sample) during the investigation period was not stored in the lab computer.

Results: Bilirubin results from both ABL835 blood gas analyzers seem to shift upwards by "aging" of the hemolyzer / oximetry modules. The ratio POCT / laboratory bilirubin increased >25% over the years without any problem in daily quality controls (QC) with aqueous QC solutions. After replacing the oximetry modules on 15.02./13.03.2012, the POCT readings were not excessive compared with the central lab. Segmented regression analysis of interrupted time series showed a highly significant change in level at the moment of intervention.

Conclusion: Laboratory methods must be compared against each other in order to determine the in-house position values. Matrix effects (whole blood versus cell-free aqueous QC-fluids) can influence the accuracy of the oximetry modul of blood gas analysers.

Immunology

FT08 – Talk Lim

Developmental endothelial locus-1 (Del-1) is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination

J. Lim1, E. Choi2, S. Lee2, M. Samus1, H. Kim2, S. Grossklaus1, T. Chavakis1

1TU Dresden, Institute of physiology, Dresden, Germany

2University of Ulsan College of Medicine, Department of Biomedical Sciences and Department of Pharmacology, Seoul, Republic of Korea

Objectives: Inflammation in the central nervous tissue (CNS) and disruption of the immune privilege of the CNS are major components of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor by inhibiting LFA-1-integrin-dependent leukocyte adhesion and IL-17-dependent inflammation. Here we show that Del-1 is a homeostatic factor of the CNS that contributes to its immune privilege.

Methods: EAE disease developed by injection of MOG with complete adjuvants. Demyelination and axonal damage of spinal cord were examined by LFB and neurofilament staining to the tissue section, respectively. Flow cytometry data shown IL-17-producing T cells infiltrated into spinal cord.

Results: Del-1 in the CNS is expressed not only in endothelial cells but also in oligodendrocytes. Furthermore, Del-1 expression decreases in chronic active MS lesions, as well as in the inflamed CNS in the course of EAE. Interestingly, Del-1-deficient mice displayed increased EAE severity, accompanied by increased demyelination and axonal loss, as compared to wild-type mice. In addition, Del-1-deficient mice showed increased infiltration of Th17 cells and inflammatory myeloid cells in the spinal cord in the course of EAE, as compared to control mice. Consistent with an activation of the IL-17 pathway, increased EAE severity and inflammation due to Del-1-deficiency were reversed in mice lacking both Del-1 and IL-17 receptor.

Conclusion: Our findings suggest that Del-1 is an endogenous anti-inflammatory factor in the CNS preventing from IL-17-driven neuroinflammation and from demyelination.

Lipidomics

FT09 – Talk Gräßler

Correlation of specific lipoprotein apheresis-induced changes of plasma lipidome with indicators of oxidative stress

J. Gräßler1, J. Passauer1, S. Fischer1, K. Schuhmann2, A. Shevchenko3, S. Bornstein1, U. Julius1, S. Kopprasch1

1Universitätsklinikum Carl Gustav Carus, Medizinische Klinik und Poliklinik III, Dresden, Germany

2MPI-CBG / MK3 Uniklinikum, Dresden, Dresden, Germany

3Max-Planck-Institut für molekulare Zellbiologie und Genetik, Dresden, Germany

Objective: The aim of the study was to investigate to which extend specific changes in plasma lipidome induced by two different methods of apheresis coincided with apheresis-related oxidative stress.

Methods: Changes in plasma lipidome and oxidative balance were investigated in two groups of patients: 1: patients under lipid filtration (LF; n=8), 2: patients under dextran-sulphate-adsorption with Liposorber D (DSA, n=7). Plasma lipidome was screened by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 180 lipid species of 10 major lipid classes. Apheresis-evoked oxidative stress was characterized by leukocyte count, basal activity of NADPH-oxidase (NOX), circulating (COR) and maximum (MOR) opsonin receptor-dependent activities, and concentration of oxidized LDL (oxLDL).

Results: DSA was associated with higher reduction of LDL-cholesterol and lower reduction of HDL-cholesterol and triglycerides than LF. In contrast to LF patients on DSA revealed an increase of phosphatidylethanolamines and diacylglycerols. LF was accompanied by increased leukocyte count and COR, whereas DSA resulted in a more pronounced reduction of oxLDL. LF accompanied oxLDL decrease correlated directly with CE-, LPC-, PC-, PCO-, PEO-, and SM metabolites, which were inversely correlated with MOR and NOX. DSA associated decrease of leukocytes correlated inversely with CE-, PC-, PCO-, PE-, PEO metabolites and COR.

Conclusions: These data indicate an increased cellular stress of blood cells in patients under DSA. On the other hand, inverse correlation of lipid reduction in LF patients to NOX and MOR pointed to increased oxidative stress.

Allergologie Joint-Symposium

FT10 – Talk Junge

Recombinant allergens in the differentiation of food allergies

S. Junge1, K. Nemat2, S. Abraham3, A. Bauer3, G. Wolf1, G. Siegert1, V. Neumeister1

1Universitätsklinikum C.G.Carus an der TU Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Universitätsklinikum C.G.Carus an der TU Dresden, Klinik und Poliklinik für Kinderheilkunde, Dresden, Germany

3Universitätsklinikum C.G.Carus an der TU Dresden, Klinik und Poliklinik für Dermatologie, Dresden, Germany

Objectives: Food allergy may clinically present as oral allergy syndrome (OAS; for sensitization against PR-10/Profilin) and systemic reaction (SR; for sensitization against lipid transfer proteins (nsLTP)/storage proteins (SP)), respectively. Our aim was to compare sensitization pattern (PR-10/Profilin, nsLTP/SP) with clinical history of patients.

Methods: In 36 patient (female n=14, age 3-69y; male 22, 2-63y) serum samples with positive specific IgE (sIgE) for peanut (f13), hazelnut (f17) and pollen allergens (t3, g6, w6), we determined sIgE for recombinant allergens (PR-10: Ara h8, Cor a1, Bet v1, Pru p1, Gly m4, Api g1; Profilin: Bet v2, Pru p4, Phl p12; nsLTP: Pru p3, Cor a8, Ara h9; SP: Ara h1, Ara h2, Ara h3) using CAP system. Based on anamnestic data, patients were divided into 6 groups: no clinical reaction (I; n=4), food avoidance (II; 17), verified OAS (III; 6), suspected OAS (IV; 3), mild SR (V; 3), severe SR (VI; 3).

Results: Group I: n=4 PR-10/Profilin positive, n=0 nsLTP/SP negative; group II: 14 PR-10/Profilin positive, 10 nsLTP/SP positive; group III: all PR-10/Profilin positive and nsLTP/SP negative; group IV: all PR-10/Profilin positive and nsLTP/SP negative; group V: 2 PR-10/Profilin positive, all nsLTP/SP negative; group VI: 3 PR-10/Profilin positive, 2 nsLTP/SP positive.

Conclusion: Recombinant allergens may significantly contribute to risk assessment for systemic reaction in patients sIgE positive for peanut, hazelnut and pollen allergens.

Hämostasis

FT11 – Talk Manukyan

Human cofactor independent monoclonal anti-cardiolipin antibody triggers thrombosis in vivo by activating endosomal NADPH oxidase

D. Manukyan1, S. Jäckel2, N. Prinz1, C. Reinhardt2, U. Walter2, K. Lackner1

1University Medical Centre Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany

2University Medical Centre Mainz, Center for Thrombosis and Hemostasis (CTH), Mainz, Germany

Objectives: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and/or venous thrombosis associated with the presence of antiphospholipid antibodies (aPL). Currently, it is assumed that only aPL binding to β2GPI may induce thrombosis in vivo. We aimed to investigate the potential of human cofactor independent monoclonal anti-cardiolipin antibody HL5B to induce thrombus formation in vivo.

Methods: Intravital fluorescence microscopy has been used to visualise thrombus formation in living mice. In anesthetised animals thrombus formation was estimated in carotid artery and in vena cava. In addition, Rotem thrombelastography has been used to evaluate the fibrin formation in whole blood ex vivo.

Results: Thrombus formation in vena cava was dramatically enhanced 3 h after intravenous injection of HL5B but not by control antibody. Thrombus formation was abolished by injection of an inhibitory anti-tissue factor (TF) antibody. Moreover, HL5B did not trigger thrombus formation in NADPH oxidase knockout mice. In addition, HL5B increased fibrin formation but not platelet adhesion in the mouse carotid artery. Injection of HL5B in mice shortened the clotting time of whole blood ex vivo.

Conclusions: Our results indicate for the first time that the human cofactor independent monoclonal aPL HL5B promotes arterial and venous thrombus/fibrin formation via NADPH oxidase mediated TF pathway in living mice. This opens new insights into the complex pathogenesis of APS.

FT11a – Talk Vogt

Mechanisms of TNF Tolerance in Human Monocytes

*N. Vogt1, J. Günther1, K. Hampel1, R. Bikker1, S. Page1, B. Müller1, J. Kandemir1, M. Kracht2, O. Dittrich-Breiholz3, R. Huber1, K. Brand1

1Medizinische Hochschule Hannover, Institut für Klinische Chemie, Hannover, Germany

2Biomedizinisches Forschungszentrum Seltersberg, Rudolf-Buchheim-Institut für Pharmakologie, Gießen, Germany

3Hannover Medical School, Institute of Physiological Chemistry, Hannover, Germany

Objective: TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon which is referred to as tolerance.

TNF tolerance is considered as protective in sepsis but may also represent a paradigm for immunoparalysis. To date, the molecular basis of TNF tolerance is poorly understood.

Methods: We used primary human monocytes isolated from whole blood (n=5) and analysed the respective RNA expression using an array-based genome wide approach. Mechanisms were further analysed by siRNA, (phospho)western blot, inhibitor and overexpression experiments.

Results: In human monocytes, we detected 2 forms of TNF refractoriness: absolute tolerance was selective, dose-dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Under high-dose tolerance, we observed blockade of I?B-a proteolysis/phosphorylation and reduced basal I?B-a levels. TNF-induced phosphorylation of p65-Ser536, p38, and c-jun was inhibited and basal p65-Ser468 phosphorylation was increased. Low-dose tolerance is mediated by GSK3, whereas high-dose tolerance is regulated by A20/GSK3 and PP1 phosphatase-dependent mechanisms.

Conclusions: We present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially shifts the kinase/phosphatase balance and may play an important role in resolution of inflammation.

Metabolomics

FT12 – Talk Kopprasch

Cholesteryl ester and ether-linked phosphatidylcholine deficiency in prediabetic and type 2 diabetic individuals – association with insulin sensitivity indices

S. Kopprasch1, K. Schuhmann2, P. Schwarz1, S. Bornstein1, A. Shevchenko2, J. Graessler1

1Carl Gustav Carus Medical School, Department of Internal Medicine 3, Dresden, Germany

2Max Planck Institut für Molekulare Zellbiologie und Genetik, Dresden, Germany

Objective: Glucolipotoxicity is a major pathophysiological mechanism in the development of insulin resistance and type 2 diabetes. We aimed to detect subtle changes in the individual circulating lipid profile by lipidomic shotgun analysis and to correlate them with different insulin sensitivity indices.

Research design and methods: The cross-sectional study comprised 56 men with a broad range of insulin sensitivity including normal glucose tolerance (NGT, n=20), impaired glucose tolerance (IGT, n=20) and newly detected type 2 diabetes (T2D, n=16). Plasma was obtained and quantitatively analyzed for 172 lipid molecular species from 12 different lipid classes.

Results: Subjects with IGT and T2D exhibited significant decreased circulating cholesteryl esters (CE, total content and five subspecies) and ether-linked phosphatidylcholines (PC-O, total content and six subspecies) when compared to NGT individuals even after adjustment for age and BMI. CE and PC-O lipid species showed a close correlation with HOMA, glucose sensitivity index, and insulin sensitivity index.

Conclusion: The pronounced differences in plasma levels of CE and PC-O species between NGT, IGT, and T2D individuals and their close association to distinct insulin sensitivity indices may prove useful these lipid species as new potential circulating disease biomarkers.

FT13 – Talk Hoene

Stable isotope-assisted lipidomic and metabolomic analysis reveals distinct lipid and palmitate utilization patterns in oxidative and glycolytic muscles of mice

M. Hoene1, C. Weigert1, G. Xu2, E. Schleicher1, R. Lehmann1

1Universitätsklinikum Tübingen, Medizinische Klinik IV, Klinische Chemie und Pathobiochemie, Tübingen, Germany

2Dalian Institute of Chemical Physics, Chinese Academy of Sciences, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian, China

Objectives: Mice are common model organisms to study the (patho)physiology of lipid metabolism in different tissues. We hypothesized that the glycolytic Gastrocnemius (Gast.) and the oxidative Soleus (Sol.) muscle have different lipid and acylcarnitine profiles and palmitate utilization.

Methods: Fed, exercised (1h treadmill at moderate speed) or fasted (16h) mice (male C57Bl/6, 12 weeks, n=6 per group) received an i.v. bolus of U13C-palmitate. After 10 min., liver and muscles were prepared for lipidomics, acylcarnitine (carn.) and FFA analyses by UHPLC-MS.

Results: More than 40 acylcarn., 30 FFA and 200 lipid species from 10 lipid classes could be detected in a muscle-specific pattern, as shown by multivariate analysis. Labelled palmitate and its metabolites were also detected in a tissue- and metabolic state-dependent fashion. While the highest total amount of tracer was found in hepatic lipids independent of the metabolic state, labelled phosphatidylcholine and triacylglycerol species were also detected in the muscles, where levels were lower after fasting and tended to be higher in Sol. The amounts of and exercise-induced increases in labelled acetyl- and hydroxybutyrylcarn. were also higher in Sol. than Gast.

Conclusion: Using stable isotope-assisted lipidomics and metabolomics, we could show that lipid and acylcarnitine profiles as well as palmitate metabolization and response to exercise and fasting clearly differ between mouse Sol. and Gast. muscles. The observed differences are in agreement with the higher oxidative fibre content of the Sol. muscle and might aid in selecting the appropriate muscle to study lipid metabolism in mouse models.

Endokrinologische Diagnostik bei Störungen des Knochenstoffwechsels

FT14 – Talk Hocher

Relationship of oxidized to non-oxidized PTH in Children with Chronic Renal Failure, Adult Patients on Hemodialysis and Kidney Transplant Recipients

B. Hocher1, D. Oberthür2, T. Slowinski3, U. Querfeld4, F. Schäfer5, A. Doyon5, M. Tepel6, H. Roth7, H. Grön8, C. Betzel2, F. Armbruster8

1Institute of Nutritional Science, University of Potsdam, Potsdam, Germany

2Institute of Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany

3Department of Nephrology, Charité-Mitte, Berlin, Germany

4Department of Pediatric Nephrology, Charité-Mitte, Berlin, Germany

5Department of Pediatric Nephrology, University of Heidelberg, Heidelberg, Germany

6Odense University Hospital, Department of Nephrology, and Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

7Department of Endocrinology/Oncology, Limbach Laboratory, Heidelberg, Germany

8Immundiagnostik AG, Bensheim, Germany

Background: Oxidized PTH (oxPTH) loses its PTH receptor stimulating properties, whereas non-oxidized PTH (n-oxPTH) is a full agonist of the receptor. This was described in more than 20 well published studies in the 70th and 80th of the last century. However, PTH oxidation was ignored during development of PTH assays for clinical use. Even the nowadays used third generation assay systems do not consider oxidation of PTH. We recently developed an assay to differentiate between oxPTH and n-oxPTH.

Methods: We established normal values for this assay system.

Furthermore, we compare the ratio of oxPTH to n-oxPTH in different population with chronic renal failure: 620 children with renal failure stage 2-4 of the 4C study, 342 adult patients on dialysis, and 602 kidney transplant recipients.

Results: Children had the highest mean as well as maximum n-oxPTH concentrations as compared to adult patients (both patients on dialysis as well as kidney transplant recipients). The relationship between oxPTH and n-oxPTH of individual patients varied substantially in all three populations with renal impairment. The analysis of n-oxPTH in 89 healthy control subjects revealed that n-oxPTH concentrations in patient with renal failure were higher as compared to healthy adult controls (2.25-fold in children with renal failure, 1.53-fold in adult dialysis patients, and 1.56-fold in kidney transplant recipients, respectively).

Conclusion: A huge proportion of circulating PTH measured by current state of the art assay systems is oxidized and not biologically active. The relationship between oxPTH and n-oxPTH of individual patients varied substantially. Non-oxidized PTH concentrations are 1.5 -2.25 fold higher in patients with renal failure as compared to health controls

Poster

Immunology / Allergologie Poster I

P001

CD63 and CD203c expression during specific immunotherapy (SIT) for wasp venom allergy using Basophile Activation Test (BAT)

*N. Reiß1, P. Spornraft-Ragaller2, B. Hauswald3, K. Nemat1, D. Koschel4, A. Bauer2, G. Wolf1, G. Siegert1, V. Neumeister1

1Universitätsklinikum C.G.Carus an der TU Dresden, Klinik und Poliklinik für Kinderheilkunde, Dresden, Germany

2Klinik und Poliklinik für Dermatologie, Universitätsklinikum C.G.Carus an der TU Dresden, Dresden, Germany

3Universitätsklinikum C.G.Carus an der TU Dresden, Klinik und Poliklinik für HNO-Heilkunde, Dresden, Germany

4Zentrum für Pneumologie, Fachkrankenhaus Coswig GmbH, Coswig, Germany

Objectives: SIT is an established therapy for wasp venom allergy. The aim of our work is to investigate the progression of surface antigen CD63 and CD203c expression during SIT using BAT.

Methods: We report on 45 adult patients with SIT against wasp venom. Blood samples were collected before (baseline), 6 months (6m), 1 year (1y) and 1.5 years (1.5y) after SIT start. CD63 and CD203c expression was determined using BAT after stimulation with various wasp venom concentrations. The relative proportion of activated basophile granulocytes at 57µg/l venom concentration and the calculated concentration c50 to stimulate 50% of total activatable basophile granulocytes was compared against baseline. The study was approved by the institutional ethical review board.

Results: CD63 expression (6m(n=36) / 1y(27) / 1.5y(9)) decreased in 22/12/5 and increased in 9/6/2 patients, while it was constant in 5/9/2 cases. CD63 expression after 1 year was significantly different from baseline (p<0.05). CD203c expression (6m(45) / 1y(30) / 1.5y(12)) decreased in 23/16/6, increased in 16/12/2 and did not change in 6/2/4 patients. No significant differences from baseline could be observed.

Conclusion: Expression of CD63 and CD203c in BAT do not show a uniform behavior during the investigated time. Significant differences can be demonstrated for CD63 expression, but not for CD203c. Further work is required to gain inside into long-term stimulation behavior in BAT and correlation with sting challenge.

P002

Connective tissue growth factor (CTGF/CCN2) in serum serves as an indirect marker for neoplastic transformation of cirrhosis in patients with chronic liver disease

*O. Gressner1, H. Li2, M. Fang2, A. Gressner3, C. Gao2

1Laboratoriumsmedizin Köln, Dres. Wisplinghoff und Kollegen, Köln, Germany

2Eastern Hepatobiliary Surgical Hospital, Department of Laboratory Medicine, Shanghai, China

3Laboratoriumsmedizin Berlin, Berlin, Germany

Introduction: Previously, we have shown that connective tissue growth factor (CTGF/CCN2) in serum might be a promising candidate non-invasive biomarker of liver fibrogenesis, which is extended in the present study to cirrhosis and liver cell carcinoma (HCC).

Methods: Healthy (n=32), fibrotic (n=77), cirrhotic (n=17), and HCC-patients (n=72), clinically, biochemically and histopathologically well characterized, were tested for CTGF serum concentrations using a CTGF-ELISA (EIA#5195, DRG, Marburg, Germany) and data correlated.

Results: Following an increase of serum CTGF during different stages of fibrogenesis, ranging from 15.9 (S0), 20.3 (S1/2) to 36.9 (S3/4), we found highest CTGF-concentrations in cirrhotic patients (43.6 µg/L), compared to healthy controls (17.7 µg/L), followed by a significant decrease in HCC-patients (38.5 µg/L; p=0.001). No association between CTGF and parameters of liver injury were noticed but CTGF correlated negatively with liver function parameters such as albumin (p=0.007) or platelet number (p=0.0032). In HCC patients, CTGF concentrations decreased with advanced HCC TNM stage II (30.5 µg/L) and stage III (33.6 µg/L) (compared to TNM stage I [41.6 µg/L]).

Conclusion: Our data suggest a valuable diagnostic impact of CTGF in serum for follow-up of fibrotic patients suffering from chronic liver diseases at risk to develop HCC.

References:

Gressner et al., Clin Chem (2006) 52: 1815-7

P003

The neurotrophin NGF and the neurosteroid DHEA: new modulators of microglia-mediated inflammation

*V. Alexaki1, I. Charalampopoulos2, B. Soehnnichsen3, C. Echeverri3, C. Mund1, S. Grossklaus1, C. Tsatsanis4, A. Gravanis2, T. Chavakis1

1Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

2Medical School, University of Crete, Department of Pharmacology, Heraklion, Greece

3Cenix Bioscience, GmbH, BIOZ, Dresden, Germany

4Medical School, University of Crete, Department of Clinical Chemistry, Heraklion, Greece

Objectives: The Nerve Growth Factor (NGF) is a crucial factor for neuronal growth. However, its role in non-neuronal cells has been rather neglected.

Methods: In our work examined the role of NGF and its receptors in microglia inflammatory responses in vitro and in vivo experimental settings.

Results: We provide evidence that NGF plays an important immunomodulatory role in the CNS by decreasing the inflammatory responses of microglia to TLR4 stimuli, such as LPS. This effect is mediated by its high affinity receptor, TrkA and activation of the Akt pathway, and not by its low affinity receptor p75NTR (TNFR2). Recently we have identified the neurosteroid hormone dehydroepiandrosterone (DHEA) as a novel ligand of the TrkA receptor and we have synthesized a DHEA analogue, called BNN27 that cannot be converted to estrogen or androgen. DHEA and BNN27, as NGF, induce in microglia TrkA phosphorylation, dampen cytokine secretion (IL-6, TNF-α, IL-23) and induce the expression of M2-related genes (Arg1, Ym1, Fizz1, Klf-4, IL-4) in vitro and in vivo in LPS-induced brain inflammation, while TrkA deletion completely reverses the anti-inflammatory effect of NGF or DHEA. Furthermore, NGF and DHEA increase the expression of miR-146, a micro-RNA that negatively feedbacks on LPS signaling by targeting IRAK1 and TRAF6 mRNAs.

Conclusion: These data support a modulatory role of the duo NGF-TrkA in microglia-mediated inflammation, a finding that gives new insights in understanding the mechanisms of resolution of CNS inflammation.

P004

Urinary excretion of tubular and glomerular marker proteins during lysine induced blockage of tubular reabsorption

*A. Regeniter1, M. Dickenmann2, F. Brunner2, M. Mayr2, D. Tsinalis2, W. Boesken3, W. Siede4, J. Steiger2

1Basel University Hospital, Laboratory Medicine, Basel, Switzerland

2Basel University Hospital, Nephrology, Basel, Switzerland

3Nephrology, Freiburg, Germany

4Klinikum Lippe Detmold, Institut für Labormedizin, Detmold, Germany

Background: We analyzed the effect of lysine induced blockage of tubular reabsorption on the excretion of glomerular and tubular marker proteins to better quantify and qualify proteinuria.

Methods: 6 healthy volunteers (5 male, 1 female) were infused with 0.25 mol/l L-lysine chloride for 360 minutes, followed by a 120 minutes control phase. Blood and urine samples were taken every 30 minutes.

Results: The excretion of small molecular weight proteins tubular indicator proteins (alpha-1-microglobulin=A1M; retinol binding protein=RbP, β-2-microglobulin=B2M) exceeded by far the excretion of larger glomerular indicator proteins (albumin, transferrin, IgG) after lysine induced complete blockage of tubular reabsorption . This effect became most visible, when the values were expressed as multiple of the upper reference range. B2M (MG 11) and RbP (MG 21) behaved comparable, reached a maximum after 90 to 150 minutes (multiple of the upper reference range; MuR 115.0/127) and decreased by 29% (MuR 91.6) at the end of maintenance phase. A1M (MG 33) reached a maximum after 90 minutes (MuR 40.7), but decreased after 240 minutes to 58% (MuR 23.5, end of maintenance phase). All proteins returned to normal in the control phase.

Conclusions: Complete blockage of tubular reabsorption presented with RbP or B2M elevations of approximately 120 fold. Approximately 60 fold MuR indicated that 50%, and approximately 30 MuR indicated that roughly 25%, of the maximum tubular reabsorption capacity has been lost, while the increase of A1M reached only one third of these concentrations.

P005

TLR-2 and -5 activate leukocyte integrin-mediated cell adhesion in a manner dependent on Rap1a and cytohesin1

*K. Chung1, J. Gebler1, G. Siegert2, T. Chavakis3

1University Dresden, Department of Internal Medicine III, Institute of Physiology, Dresden, Germany

2University Dresden, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany

3University Dresden, Department of Internal Medicine III, Institute of Physiology, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany

Objectives LFA-1 is a major beta2-integrin on hematopoietic cells that mediates leukocyte-endothelial interactions in immunity and inflammation. Whether beta2-integrin-dependent adhesion is activated by Toll-like receptors (TLRs) has been poorly studied.

Methods: Human monocytic cells (THP-1) were stimulated with different TLR ligands and cell adhesion to ICAM-1 and fibronectin was studied. Activation of beta2-integrin affinity was analyzed by flow cytometry. The involvement of signalling pathways such as Rap1a and cytohesin-1 were performed by siRNA-mediated knockdown. The protein interactions between cytohesin1 and CD18 were assessed by co-immunoprecipitation.

Results From different TLR-ligands tested, TLR-2 or -5 stimulation activated rapidly adhesion of monocytic cells to ICAM-1 and fibronectin. This effect was accompanied by a TLR-2 and -5-mediated increase in Rap1a activity and in LFA-1 affinity. Moreover, the activation of LFA-1 and of LFA-1-dependent cell adhesion to ICAM-1 was reversed by Rap1a knockdown. In addition, TLR-2 and -5 ligation stimulated the interaction between cytohesin-1 and the beta2-chain. Consistently, knockdown or inhibition of cytohesin-1 reversed the TLR-mediated activation of LFA-1 and of integrin-dependent cell adhesion to ICAM-1 and fibronectin.

Conclusion: TLR-2 and -5 activate beta2-integrin LFA-1-dependent cell adhesion of leukocytes through Rap1a activation and increased cytohesin-1 interaction with the beta2-chain.

P007

Chip based detection of arthritis associated antibodies using a microfluidic approach

*J. Herrmann1, C. Färber1, R. Kneusel2, T. Simon2, T. Brandstetter3, C. Gärtner4, C. Carstens1, R. Elbracht1

1Praxis für Labormedizin, Naumburg, Germany

2diarect AG, Freiburg, Germany

3Institut für Mikrosystem Technik (IMTEK), Freiburg, Germany

4microfluidic ChipShop GmbH, Jena, Germany

RHEUMA-CHIP is a project developing an automated and miniaturized fluidic system. One drop of blood should be sufficient to detect and discriminate between rheumatoid and post infectious arthritis. Regarding the massive loss of working days thereby, simplified test devices are of high economic interest. A sequence of small channels and reaction chambers in a disposable microfluidic chip builds the core unit of the test set up. Following plasma separation the fluidic structure guides the sample into the serial dilution area. The diluted sample is conducted through the fluidic network, thus passing the binding cavities. Enhancement of binding efficiency is achieved using capturing molecules embedded in a three dimensional gel matrix. In a first approach, detection of bound antibodies are obtained using a standard immuno assay with precipitating dyes. Immuno assay procedures were successfully transformed into a variant fitting the fluidic requirements. Patient sera were used to define the usefulness of antigenic structures, antigen preparations, printing techniques and plastic surface influences.

P008

Phagocytosis of IE-associated S. gallolyticus subsp. gallolyticus strains: evaluation of potential pathomechanisms in a macrophage cell culture system

*M. Weinstock1, I. Grimm1, J. Dreier1, C. Knabbe1, T. Vollmer1

1Herz- und Diabeteszentrum Nordrhein-Westfalen, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Introduction: Streptococcus gallolyticus subsp. gallolyticus (SGG) is the causative pathogen in approximately 20% of all streptococcal-caused infective endocarditis (IE) cases. The clearance of blood stream bacteria is an important factor in the progression of IE. In this study, a macrophage cell culture model was evaluated to analyze phagocytosis of SGG-strains.

Methods: THP-1 cells were differentiated into macrophages with DMEM supplemented with 50 ng/ml PMA. Phagocytic uptake of SGG (105-106 CFU/mL) was enabled for 30 min, extracellular bacteria were removed and cells were subsequently incubated with DMEM with antibiotics for 48 h. Cells were lysed after several time points and bacteria quantified by plating assay.

Results: The percentage of phagocytic uptake differs between SGG strains ranging from 1.4% - 29.4%. Additionally, the course of bacterial reduction in macrophages differed between strains. One isolate was devitalized in progress of phagocytosis after 36 h, other Isolates were detectable with constantly decreasing titers. In contrast, another isolate showed a constant titer in macrophages from 8 h to 16 h.

Conclusions: The observed differences regarding the phagocytic uptake and time-course of bacterial killing could possibly influence the IE-development remarkably. Current investigations focus on the correlation of these capabilities with bacterial surface (e.g. capsule). This work was supported by Stiftung für Pathobiochemie und Molekulare Diagnostik of the DGKL.

Therapeutisches Drug Monitoring Poster

P009

Simultaneous measurement of six antiepileptic drugs in serum and plasma using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry

*J. Kuhn1, P. Kuzaj1, C. Prante1, D. Hendig1, C. Knabbe1

1Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinikum der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Objectives: We describe a rapid assay to measure the antiepileptic drugs lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide.

Methods: After the addition of internal standards and protein precipitation of patient serum or plasma, 1 µl of supernatant sample was injected onto an Acquity UPLC system which was directly coupled to a tandem mass spectrometer. The chromatographic separation was performed on a 2.1 X 50 mm reverse phase column. Analytes were then ionized and detected by ESI mass spectrometry in MRM mode. Runtime was 2.5 min per injection. Ion suppression was characterized using post-column infusion.

Results: The calibration curves of the 6 antiepileptic drugs were linear over the working range between 0.05 and 50 mg/L (r >0.99). LOD, as well as the lower LLOQ of all drugs measured in the assay was <0.05 mg/L. The intraassay and interassay CV for all compounds were <15% for very low concentration (0.1 mg/L) and <8% in the clinically relevant concentration range (>1.0 mg/L). Mean recoveries were between 85.0 and 110.7% for all drugs. No significant ion suppressions were detected at the elution times of the analytes. The mean differences between serum and plasma values were less than 6% for the 6 antiepileptic drugs.

Conclusion: In summary, a specific and sensitive stable isotope dilution UPLC-MS/MS method was developed and validated for routine clinical monitoring of lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide.

P010

Evaluation of Phospho-Flow Cytometry for the Pharmacodynamic Monitoring of Everolimus and Sirolimus in Transplant Patients

*M. Shipkova1, M. Kabakchiev1, C. Klett1, E. Wieland1

1Klinikum Stuttgart, Zentralinsitut für Klinische Chemie und Laboratoriumsmedizin, Stuttgart, Germany

Direct monitoring of the pharmacodynamic effect of the mTOR inhibitors sirolimus and everolimus may be a promising complementary tool to drug monitoring for individualizing the immunosuppressive therapy. Phosphorylation of the S6 ribosomal protein (p-S6RP) by p70S kinase (downstream mTOR effector) can be assessed by phospho-flow cytometry (PF) (1). The applicability of a commercial kit (Beckman Coulter) to detect intracellular p-S6RP in T cells was evaluated.

PF analysis was performed as proposed by the manufacturer, mitogen stimulation of whole blood was developed in our lab. T cells (CD3+) and subsets (CD4+, CD8+) of healthy persons (HP), transplant patients (TxP) with and without mTOR inhibitor and dialysis patients were investigated.

Optimal stimulation was achieved with15 µg/L Phorbol 12-Myristate 13-acetate for 6 min, 37°C. Method imprecision was ≤17% in TxP but ≤27% in HP. Attenuation of phosphorylation within 24h at room temperature was observed. p-S6RP was comparable in TxP without mTOR inhibitor, HP, and dialysis patients. Median p-S6RP was significantly decreased in pre-dose samples of patients with sirolimus, but not with everolimus. There was a very broad inter-patient variation in p-S6RP particularly with everolimus, despite similar blood drug concentrations.

The protocol seems to be promising for pharmacodynamic monitoring of mTOR inhibition in TxP when used with fresh samples and duplicate measurements.

1. Chow S. et al. Exp. Hematol 2006;34:1182.

P011

Evaluation of two assays for determination of Rivaroxaban (Xarelto®)

*A. Grotevendt1, C. Wasner1, A. Petersmann1, M. Nauck1

1University Medicine Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

Objectives: An increasing number of patients is treated with new oral anticoagulants. In acute bleeding episodes determination of these anticoagulants is crucial. Commercially available assays differ in measurement range and tolerance towards other anti-Xa anticoagulants used for therapeutic bridging.

Methods: We evaluated two chromogenic Rivaroxaban assays (A: Coamatic Heparin; B: BIOPHEN DiXa-I) on the BCS XP. Measurement ranges were 0.01 – 175 µg/l and 0.01 – 500 µg/l for A and B, respectively. For assay B it is described that other anti-Xa anticoagulants in common therapeutic range do not interfere with the Rivaroxaban determination. Samples containing Rivaroxaban were artificially contaminated with low molecular weight heparin (LMH) in different concentrations to mimic this interference. Also, we compared the two assays.

Results: Coefficient of variation of both assay were comparable, ranging from 1.6 – 8.6% at different concentrations. Regression analysis showed a good correlation (r= 0.959, N= 32). LMH (target concentration 0.64 U/ml) was added to samples containing 100 µg/l Rivaroxaban. A showed a 39% increase in the measured Rivaroxaban concentration, no deviation was observed in B.

Conclusion: Both assays showed a comparable performance. B offers a wider measurement range and proves to be in-sensitive towards other anti-Xa anticoagulants in therapeutic concentration. We consider B more appropriate to meet clinical needs in the treatment of patients anticoagulated with Rivaroxaban.

P012

Development of a new immunoassay for the accurate determination of anti-infliximab antibodies in inflammatory bowel disease

*J. Semmler1, A. Pilch2, F. Armbruster1, A. Dignass3, J. Stein4

1Immundiagnostik AG, Bensheim, Germany

2Immundiagnostik AG and Crohn Colitis Centre, Bensheim / Frankfurt, Germany

3Crohn Colitis Centre and Agaplesion Markus Krankenhaus, Frankfurt, Germany

4Crohn Colitis Centre Rhein-Main and Krankenhaus Sachsenhausen, Frankfurt, Germany

Introduction: Formation of antibodies to infliximab (ATIs) is associated with loss of response to infliximab (IFX) in patients with inflammatory bowel disease (IBD). In therapeutic drug monitoring, serum ATI levels have been assessed by immunoassays based on double-antigen format, in which ATIs were captured by immobilized IFX or protein A on a carrier. The presence of IFX can interfere with labeled IFX binding to captured ATI, causing false-negative results. Here, we developed a novel immunoassay to measure ATIs in presence of IFX.

Methods: The new assay dissociates immune complexes between IFX and ATI at low pH and prevents re-formation of immune complexes using biotinylated and peroxidase labeled IFX. ATI presence in samples was confirmed by Western blot analysis. ATI levels were measured by novel and conventional immunoassays in 29 patients with Crohn’s disease (CD) under IFX therapy. Serum IFX trough levels were determined by enzyme-linked immunosorbent assay.

Results: ATIs were detected in 7/29 patients (24.1%) by the new method. The conventional assay detected 1/29 (3.4%). In the new method, addition of IFX to samples dose-dependently blocked detection of ATIs. Patients positive for ATIs had significantly lower serum trough levels of IFX (p <0.01) and higher clinical activity scores (p <0.001) than patients negative for ATI.

Conclusion: The new method allows measurement of serum ATI levels in presence of IFX and anti-adalimumab antibodies (data not shown), allowing optimal management of IBD patients with loss of response to TNF? antibodies.

Reference(s)

Bendtzen et al. Monitoring of bioavailability, pharmacokinetics and immunogenicity of anti-tumour necrosis factor-a antibodies. Scan. J. Gastroenterol, 2009, 44: 774–781.

P013

Thiopurine treatment: Influence on proteome and phosphoproteome of human cells with different TPMT status

*S. Ziegler1, M. Misdaq1, N. von Ahsen2, M. Oellerich1, A. Asif1

1Universitätsmedizin Göttingen, Klinische Chemie, Göttingen, Germany

2Medizinisches Labor Bremen, Bremen, Germany

Objectives: Thiopurines are extensively used as immunosuppressive and anti-leukemic drugs. Polymorphism of thiopurine S-methyltransferase (TPMT) influences the metabolism of thiopurines and therapy outcome. We used (among others) a TPMT knockdown model of human Jurkat T-lymphocytes to study proteome and phosphoproteome regulation w and w/o thiopurine treatment.

Methods: Cells with normal and low TPMT expression were treated with thiopurines and regulated proteins were identified by two-dimensional gel electrophoresis (2-DE) and mass spectrometric approach. A reactive oxygen species (ROS) assay was done to confirm the regulated oxidative stress markers observed in 2-DE results.

Results: Thirteen proteins with altered expression and nine proteins with altered phosphorylation signal were identified as regulated in different groups - including proteins, which are important for cellular oxidative stress response or DNA damage response. Furthermore, the ROS assay confirmed a significant induction of oxidative stress by TPMT knockdown and thiopurine treatment.

Conclusion: We observed that thiopurine treatment of human cells with differential TPMT expression can affect important DNA damage response proteins and oxidative stress targets. Further characterization of knockdown models of these targets including mismatch repair genes may help to understand the complex relationships between metabolism and mechanisms of action of thiopurines in patients with gene polymorphisms.

P014

A rapid LC-MS/MS based method for linezolid quantification in serum of critically ill patients

*J. Zander1, B. Maier1, Z. Michael2, M. Brügel1, D. Teupser1, M. Vogeser1

1Ludwig-Maximilians-University Munich, Institute of Laboratory Medicine, Muenchen, Germany

2Ludwig-Maximilians-University Munich, Department of Anaesthesiology, Muenchen, Germany

Objectives: Linezolid serum concentrations were shown to be highly variable in critically ill patients often reaching inadequate drug levels. However, commercially available tests for linezolid quantification in routine diagnostics are not available. We therefore developed and validated a rapid and simple liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of linezolid in human serum.

Methods: An LC-MS/MS method was developed and validated on two different LC-MS/MS systems. Sample preparation was based on protein precipitation and online solid phase extraction. Linezolid d3 was used as internal standard ensuring coelution with the analyte.

Results: The method showed high sensitivity and robustness. It was characterized by short hands-on time and short run-times of 4 min. Validation revealed good analytical performance with inaccuracy ± 3% and imprecision of 2.5 – 7.3 % CV (interassay, n = 5) for three quality control samples (0.5 – 16.0 mg/l). Comparative measurements on two LC-MS/MS systems revealed close agreement.

Conclusion: This LC-MS/MS method is a simple, fast and robust method which can be routinely applied using different LC-MS/MS systems. Using this method we are currently investigating linezolid serum concentrations in critically ill patients.

P015

Monitoring of Clopidogrel active metabolite in human plasma by LC-MS/MS.

*P. Findeisen1, E. Jäger1, S. Mindt1, M. Neumaier1

1Medical Faculty Mannheim of the University of Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany

Objective: Clopidogrel (Plavix™) is a potent antiplateled pro-drug that is widely used for prevention of vascular thrombotic events in patients at risk. However, there is a broad interindividual variability of therapeutic effects as hepatic biotransformation is necessary to generate the active metabolite (AM). The quantification of AM might help to identify patients with genuine resistance to Clopidogrel due to insufficient metabolic activation of the prodrug.

Methods: AM contains a thiol group, thus requires stabilization in biological samples with an alkylatin agent. Accordingly, 0.5M 2-bromo-3-methoxyacetophenon was added to EDTA plasma tubes prior to blood withdrawal. Both, Clopidogrel and AM were quantified using LC-MS/MS using Ticlopidin as internal standard. Mass transitions for Clopidogrel and AM were 322→212 and 504→354 respectively.

Results: The Clopidogrel calibration curve was linear in the range of 0.1-60 ng/ml. Clopidogrel and AM were detected simultaneously and the respective ratio of AM and Clopidogrel showed good stability between 60-120 minutes after oral administration of the drug.

Conclusion: A decreased ratio of AM and Clopidogrel might enable the identification of patients that show insufficient inactivation of thrombocyte function. Our preliminary results have to be combined with further pharmacogenetic data (CYP2C19) and analysis of thrombocytic function.

Endokrinologie Poster I

P016

Biochemical diagnosis of pheochromocytoma using plasma free normetanephrine, metanephrine and methoxytyramine: Importance of supine sampling under fasting conditions

*R. Därr1, P. Christina1, M. Peitzsch2, K. Miehle3, A. Prejbisz4, D. Weismann5, F. Beuschlein6, R. Sinnott7, S. Bornstein1, H. Neumann8, A. Januszewicz4, J. Lenders9, G. Eisenhofer2

1University Hospital Dresden, Department of Medicine III, Dresden, Germany

2Uniklinikum Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

3University Hospital Leipzig, Leipzig, Germany

4Institute of Cardiology, Warsaw, Poland

5University Hospital Wurzburg, Wurzburg, Germany

6Medical Clinic Innenstadt - LMU, München, Germany

7University of Melbourne, Melbourne, Australia

8University Hospital Freiburg, Freiburg, Germany

9Radboud University Nijmegen Medical Center, HB Nijmegen, Netherlands

Objective: To document the influences of blood sampling under supine fasting versus seated non-fasting conditions on diagnosis of pheochromocytomas and paragangliomas (PPGL) using plasma concentrations of normetanephrine, metanephrine and methoxytyramine (P-NMN/MN/MTY).

Design and Methods: Biochemical testing for PPGL was carried out in 765 patients at six centers, two of which complied with requirements for supine sampling after an overnight fast and four of which did not. PPGL were found in 130 patients, 67 non-compliant (NoComp)) and 63 compliant (Comp), and were not found in 635 patients, 195 NoComp, 440 Comp.

Results: Plasma concentrations of NMN and MTY did not differ between Comp and NoComp sampling conditions in patients with PPGL, but were 50% higher in patients without PPGL sampled under NoComp compared to Comp conditions. Upper cutoffs were also higher under NoComp compared to Comp conditions for NMN (237 vs 144 pg/mL), MN (96 vs 80 pg/ml) and MTY (71 vs 31 pg/mL). Use of upper-cut-offs established from NoComp compared to Comp conditions resulted in substantially decreased diagnostic sensitivity (98% vs 85%). In contrast, use of upper-cut-offs established from Comp conditions resulted in a substantial decrease in diagnostic specificity for testing under NoComp compared to Comp conditions (94% vs 71%).

Conclusions: High diagnostic sensitivity of P-NMN/MN/MTY for detection of PPGL can only be guaranteed using upper-cut-offs of reference intervals established with blood sampling under supine fasting conditions. With such cut-offs, sampling under seated non-fasting conditions can lead to a 5-fold increase in false-positive results necessitating repeat sampling under supine fasting conditions.

P017

Reference Intervals for Serum Concentrations of Three Bone Turnover Markers (PINP, BAP, CTX) in Male and Female Adults

*J. Michelsen1, H. Wallaschofski1, N. Friedrich1, C. Spielhagen1, R. Rettig2, T. Ittermann3, M. Nauck1, A. Hannemann1

1Universitätsmedizin Greifswald, Institut für Klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany

2Universitätsmedizin Greifswald, Institute of Physiology, Karlsburg, Germany

3Universitätsmedizin Greifswald, Insitut für Community Medicine, Greifswald, Germany

Objectives: Bone turnover markers (BTMs) reflect the metabolic activity of bone and can be used to assess fracture risk and monitor treatment of osteoporosis. To adequately interpret BTMs, method-specific reference intervals are needed. We aimed to determine reference intervals for serum concentrations of two bone formation markers: procollagen type 1 N-terminal propepetide (PINP), and bone-specific alkaline phosphatase (BAP), and one bone resorption marker: C-terminal telopeptides of type 1 collagen (CTX).

Methods: We established a healthy reference population from the participants of the first follow-up of the Study of Health in Pomerania. Serum PINP, BAP, and CTX concentrations were measured on the IDS-iSYS Automated System (Immunodiagnostic Systems, Frankfurt am Main, Germany). The reference interval was defined as the central 95% range. We determined age-specific reference intervals for PINP and CTX in men by quantile regression. Age-independent reference intervals were determined for BAP in men and for all three BTMs in pre- and postmenopausal women.

Results: In 1107 men, upper and lower reference limits for PINP and CTX decreased over the observed age range of 25-79 years. In 544 premenopausal women, the reference limits were lower than in 498 postmenopausal women. Women taking sex hormones for contraception or menopausal hormone therapy had lower BTMs than women not taking sex hormones. There was no seasonal variation in the three BTMs and only a small influence of daytime of blood sampling on CTX concentrations but not on PINP or BAP concentrations.

Conclusion: We present adult reference intervals for PINP, BAP, and CTX measured on the IDS-iSYS Automated System.

P018

A comparison of six 25-hydroxy vitamin D assays in the assessment of vitamin D deficiency and response to treatment

*L. Saleh1, M. Daniel1, S. Eduard2, K. Spanaus1, A. von Eckardstein1, H. Bischoff-Ferrari3

1University Hospital of Zurich, Institute for Clinical Chemistry, Zurich, Switzerland

2University Hospital Zurich, Center on Aging and Mobility, Zurich, Switzerland

3University Hospital Zurich and City Hospital Waid, Centre on Aging and Mobility, Zurich, Switzerland

In this study, we investigated the performance of 25(OH)D quantification in serum by a radioimmunoassay (RIA DiaSorin), 3 automated immunoassays (Roche Elecsys, Abbott Architect, and DiaSorin Liaison), and 2 liquid chromatography-tandem mass spectrometry (LC MS/MS) methods one of which was calibrated using standards traceable to NIST SRM972 standards and therefore defined as the reference method. Serum samples (n=481) were obtained from 35 healthy postmenopausal women who were participating in a double-blind clinical trial evaluating different vitamin D supplementation strategies. All methods showed good correlations with the reference method (R>0.96), but in each case the mean values differed significantly (P<0.05) from those obtained with the ICC-LC MS/MS reference method. Median 25(OH)D concentrations were 22 ng/mL using either LC MS/MS method, 18 ng/mL using either automated immunoassay, and 24 ng/mL using the RIA. The systematic bias ranged from -1.1 ng/mL (Liaison) to 1.8 ng/mL (Roche), and the proportional bias ranged from 0.75 (Roche) to 1.07 (RIA). All assays showed significant (P<0.001) increases in 25(OH)D concentrations following vitamin D supplementation. Compared with the ICC-LC MS/MS, the change during the course of the study was larger if assessed with the RIA but smaller if assessed by the automated immunoassays. The proportion of patients classified as vitamin D deficient at the end of follow-up was 14.3% as assessed by ICC-LC MS/MS, but whereas the corresponding proportions with the other methods ranged from 5.7% (RIA) to 48.6% (Roche). In conclusion, the accuracy of commercially available 25(OH)D assays varies in a clinically meaningful manner by under- or over-diagnosing vitamin D deficiency.

P020

Validation of an Enzyme Activity Assay of 11b-HSD1 and 11b-HSD2

*K. Heussner1, M. Rauh1

1Universitätsklinikum Erlangen, Kinder- und Jugendklinik, Erlangen, Germany

Objectives: 11b-hydroxysteroid dehydrogenase (11b-HSD) is responsible for the interconversion of the active 11b-hydroxysteroid cortisol into its inactive form cortisone and reverse. For this, isoform 11b-HSD1 predominantly catalyzes the activation of cortisol in vivo whereas 11b-HSD2 operates as oxidase converting cortisol to cortisone. The aim of our study is to evaluate and validate an enzyme activity assay for both isoforms.

Methods: 11b-HSD enzyme activity of human liver microsomes was analysed. Samples were prepared with different concentrations of substrate (55-830 nM) or 11b-HSD containing microsomes (6-300 ng/mL protein) while the other parameters remained constant.

For an inhibition assay different concentrations (0.1-100 µM) of a new inhibitor (Epherix Consulting & Development e.U.) were used. All samples were measured for cortisol and cortisone on LC-MS/MS using APCI source. For further experiments the pure enzyme 11b-HSD1 was produced through baculoviral gene transfer in SF9 insect cells.

Results: Enzyme activity assay of 11b-HSD2 showed a KM-value of 7 µM whereas the apparent KM-value for the reductase activity of 11b-HSD1 is 600 nM.

Western Blot of the recombinant enzyme 11b-HSD1 showed the expected bands at ~64 kDa for the dimer and ~32 kDa for the monomer.

Conclusions: LC-MS/MS offers high specificity even in complex sample matrices and therefore allows accurate enzyme activity measurement in tissue samples.

P021

Utility of urinary free catecholamines and their O-methylated metabolites for diagnosis of pheochromocytoma in comparison to total urinary metanephrines

*M. Peitzsch1, D. Pelzel1, S. Glöckner1, A. Prejbisz2, M. Fassnacht3, F. Beuschlein4, A. Januszewicz2, G. Siegert1, G. Eisenhofer1

1University Hospital Carl Gustav Carus at the Technical University Dresden, Institute of Clinical Chemistry and Laboratory Medicine, Dresden, Germany

2Institute of Cardiology, Warsaw, Poland

3University Hospital Würzburg, Department of Internal Medicine I, Würzburg, Germany

4Medical Clinic Innenstadt - LMU, München, Germany

Objective: A novel LC-MS/MS method for simultaneous analysis of urinary free metanephrines and their precursor catecholamines is introduced and their diagnostic utility compared to total urinary metanephrines.

Methods: A method validation was performed. From urine samples of 138 healthy normotensive and primary hypertensive volunteers and 53 patients with confirmed pheochromocytoma analytes were simultaneously extracted by a solid phase extraction procedure and measured by LC-MS/MS.

Results: Analyte recoveries ranged from 60% to 96% and intra- and inter-assay coefficients of variation from 2.7 to 13.2%. The method showed excellent linearity over 3 orders of magnitude with analytical sensitivity sufficient to measure to 1.2 nmol/L. Free metanephrines were excreted at less than 20% the rates of total fractionated metabolites, but were easily measurable. Increases in urinary free normetanephrine in patients with pheochromocytoma were 10-fold higher relative to the volunteer group compared to 5.5-fold and 3.7-fold increases for total normetanephrine and noradrenaline, respectively. In contrast, relative increases in urinary free vs. total metanephrine and 3-methoxytyramine did not differ, but for 3-methoxytyramine relative increases were larger than for dopamine.

Conclusion: Measurements of urinary catecholamines and their free O-methylated metabolites provide potential advantages over urinary total fractionated metanephrines for diagnosis of pheochromocytoma.

P022

Testosterone and cardiometabolic risk in the general population - the impact of measurement method on risk associations: a comparative study between immunoassay and mass spectrometry

*R. Haring1, S. Baumeister2, M. Nauck1, H. Völzke2, B. Keevil3, G. Brabant3, H. Wallaschofski1

1Universitätsmedizin Greifswald, Institut für Klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany

2Institute for Commuity Medicine, Greifswald, Germany

3University Hospital South Manchester, Department of Clinical Chemistry, Manchester, United Kingdom

Objective: Low total testosterone (TT) serum concentrations in men have been associated with various cardiometabolic risk factors. But given error-prone immunoassays used for TT assessment, upcoming mass spectrometry methods question the validity of these risk associations. Thus, we performed the first comparative study quantifying potential differences in the association of TT with cardiometabolic risk factors between the two methods.

Methods: We used data from 1,512 men aged 20-81 years, recruited for the cross-sectional population-based Study of Health in Pomerania (SHIP). TT was repeatedly measured by chemiluminescent immunoassay (CLIA, Immulite 2500) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We tested for significant differences between coefficients from CLIA- and LC-MS/MS-based multiple linear regression models associating TT with major cardiometabolic risk factors.

Results: TT measurements by CLIA and LC-MS/MS yielded a Pearson correlation coefficient of 0.84. Only three of the ten tested associations for TT with cardiometabolic risk factor showed significant differences between the two measurement methods: in comparison to LC-MS/MS, CLIA-based TT assessment significantly underestimated risk associations of TT with waist circumference (ß: -0.54 vs. -0.63), body mass index (ß: -0.19 vs. -0.22), and glucose levels (ß: -0.006 vs. -0.008).

Conclusion: In the present comparative study, the CLIA platform showed a reasonable measurement error and yielded comparable risk associations, providing little support to measure TT concentrations in men from the general population exclusively by LC-MS/MS.

P023

A fast and fully automated chemiluminescence immunoassay for the determination of cortisol in serum, plasma, urine and saliva

*P. Grimminger1, A. Osswald1, M. Reincke1, C. Roffe2, M. Bidlingmaier1

1Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, München, Germany

2Immunodiagnostic Systems, Boldon / Tyne and Wear, United Kingdom

Objectives: Abnormal cortisol levels due to hypothalamic, pituitary or adrenal malfunction, such as in Cushing’s syndrome and Addison’s disease, can lead to severe metabolic imbalance. We present here a competitive immunoassay for the rapid measurement of cortisol on a chemiluminescence analyzer (IDS-iSYS) in various matrices.

Methods: Cortisol of the sample competes with biotinylated cortisol for binding to a monoclonal anti-cortisol-acridinium conjugate in a first incubation. After further incubation with streptavidin-coated magnetic particles and washing, the bound anti-cortisol-acridinium is measured whereby the signal is inversely proportional to the concentration of cortisol. Total time to first result is 8 minutes.

Results: The assay range covering all matrices is 0.05-75µg/dL with an analytical sensitivity of 0.02µg/dL. The results for serum and plasma (test samples of UK NEQAS, DGKL and patient samples) were correlated against commercially available assays for serum and plasma (Diasorin Liaison, Roche Cobas). They showed also excellent correlation to mass spectrometry values of the DGKL samples. For urine and saliva a good correlation to the IBL assays was observed.

Conclusion: The fully automated IDS-iSYS cortisol assay potentially presents a new, accurate and reliable immunoassay for all commonly used sample types. The extremely short time to first result makes the assay suitable also for use during diagnostic procedures like adrenal vein sampling.

P024

Determination of aldosterone by LC-MS/MS and its application in pseudohypoaldosteronism

*A. Kulle1, M. Welzel1, F. Riepe1, P. Holterhus1

1Devision of Pediatrics, Endocrinology and Diabetes, University of Schleswig-Holstein, Kiel, Kiel, Germany

Objectives: To develop a reliable assay for the detection of aldosterone. The measurement of aldosterone is important for the clinical evaluation of pseudohypoaldosteronism, hypoaldosteronism and hyperaldosteronism.

Methods: 0.1 mL sample was extracted by solid phase extraction and analyzed using LC-MS/MS. We combined the assay with our multisteroid assay [1].

Results: The calibration curve was linear and reproducible in the range of 50-2000 pmol/L aldosterone. The limit of detection was 10 pmol/L and the limit of quantification was 30 pmol/L. We compared our assay with RIA and ELISA assays for aldosterone (DRG-Diagnostics). The coefficients of determination were 0.87 for RIA vs LC-MS/MS and 0.81 for ELISA vs LC-MS/MS. The Aldosterone concentration was about 1.3 times higher if analyzed by non-extractive RIA and 20% lower if analyzed by ELISA compared to LC–MS/MS. We found no significant differences between serum and plasma samples. Having this validated assay, preliminary reference ranges for children aged 0-18 years were measured. As an example for its clinical application plasma from patients with pseudohypoaldosteronism was measured revealing 15-fold to 45-fold multiples of the median of the aldosterone reference value.

Conclusion: The detection limit and the sensitivity of this assay allow measurement of aldosterone simultaneously with 11 other steroid hormones in one injection in parallel and overcome antibody related problems of the established immunoassays. 1 Kulle AE, Welzel M, Holterhus PM, Riepe FG: Implementation of a liquid chromatography tandem mass spectrometry assay for eight adrenal c-21 steroids and pediatric reference data. Hormone Research in Paediatrics 2013;79:22-31.

Hämatologie / Hämostaseologie Poster I

P025

Developmental endothelial locus-1 (Del-1) is a negative regulator of GCSF-dependent hematopoietic stem cell mobilization

*L.-S. Chen1, I. Mitroulis2, G. Siegert2, C. Waskow3, G. Hajishengallis4, T. Chavakis2

1Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Physiologie, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

3Center for Regenerative Therapies Dresden, Dresden, Germany

4School of Dental Medicine, University of Pennsylvania, Microbiology, Philadelphia, USA

Hematopoietic stem cell (HSC) mobilization from the bone marrow to the peripheral blood is important in the course of bone marrow transplantation. HSC mobilization can be stimulated by granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonism (by AMD3100), which are broadly used in the clinical setting. Adhesive interactions, like VLA-4/VCAM-1 and LFA-1/ICAM-1 between the HSCs and bone marrow stromal cells regulate HSC retention in the bone marrow and thus modulate HSC mobilization. Del-1 is an endothelial-derived endogenous inhibitor of inflammatory cell recruitment by antagonizing LFA-1-dependent leukocyte adhesion to endothelial ICAM-1 (Choi et al., Science, 2008; Eskan et al., Nat Immunol, 2012). Here we found that Del-1 is expressed in the bone marrow, in both mesenchymal stem cells and endothelial cells. Interestingly, Del-1 KO mice showed higher numbers of HSC in the peripheral blood after G-CSF but not after AMD3100 stimulation. Upon G-CSF administration, the level of Del-1 in the endosteal bone is decreased, which is reminiscent of the reduced GCSF-mediated expression of CXCL12, Kit ligand and osteopontin, which are important factors for HSC retention. Moreover, bone marrow mononuclear cells (BMMNC) from Del-1-proficient mice can bind to immobilized Del-1 through LFA-1 in vitro, indicating that Del-1 could contribute to HSC retention directly. Our results suggest that Del-1 in the bone marrow is a negative regulator of HSC mobilization, and further studies are required to reveal the underlying mechanism.

P026

HIF-prolyl hydroxylase-2 (PHD2) is a master regulator during erythropoiesis

K. Franke1, R. Singh1, J. Kalucka1, S. Mamlouk1, S. Bergmann2, T. Chavakis3, *B. Wielockx1

1Pathology / Medical faculty Carl Gustav Carus, Dresden, Germany

2TU Dresden Universitätsklinikum, IKL, Dresden, Germany

3Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

Objectives: The production of red blood cells (erythropoiesis) must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs), inhibitors of the alpha subunit of HIF, are crucial regulators. With this research project, we sought to understand the complex role of PHD2 during erythropoiesis.

Methods: Our work is primarily based on conditional single (PHD2) (Singh et al., Blood, 2013) as well as double deficient mouse lines in erythropoietin (EPO) producing cells. Analyses were mainly focused on survival, blood picture parameters, histology, IHC, and gene profiling.

Results: We developed a new mouse line deficient for PHD2 in renal EPO producing cells, neurons, and astrocytes. Consequently, these mice display extreme hematocrits up to 86%, thrombocytopenia, and splenomegaly, directly related to the overexpression of EPO. Mice double deficient for PHD2 and either HIF1α or HIF2α, revealed that the erythrocytosis phenotype is exclusively driven by HIF2α whereas HIF1α serves as a protective factor. Consequently, conditional PHD2/HIF1α deficient KO mice showed premature lethality confined to inflammation induced neuro-degeneration, at least in part dependent on a reduced PHD3 expression.

Conclusion: Taken together, we demonstrated for the first time that PHD2 is a central oxygen sensor controlling a tight balance between HIF1α and HIF2α during erythropoiesis (Franke et al., Blood 2013).

P027

HIF-Prolyl Hydroxylase 2 (PHD2) is a Critical Regulator of Hematopoietic Stem Cell Maintenance during Steady-State and Stress

R. Singh1, K. Franke1, J. Kalucka1, S. Mamlouk1, *K. Anastassiadis2, F. Stewart2, C. Waskow3,

T. Chavakis4, B. Wielockx5

1Pathology / Medical faculty, Dresden, Germany

2Bioinovation zentrum, Dresden, Germany

3Center for Regenerative Therapies Dresden, Dresden, Germany

4Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

5University Dresden, Institute for Pathology, Dresden, Germany

Objectives: Hypoxia is a prominent feature in maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia inducible factor (HIF1a) plays a central role in this aspect and is primarily regulated by HIF-prolyl hydroxylase domain proteins (PHDs). In the present study we explored the role of PHD2 in HSC maintenance during two unique conditions of steady-state and stress.

Methods: We developed mouse lines with conditional loss of PHD2 or PHD2 and HIF1a in very early hematopoietic progenitors (Singh et al., Blood, 2013). PHD2 functionality in steady-state and stress was assessed by analyzing the bone marrow (BM) for HSC compartment or by performing bone marrow chimeras respectively. Main methods involved were flow-cytometry based analysis and sorting along with gene profiling.

Results: Under physiological conditions, loss of PHD2 in very early hematopoietic progenitors results in increased proliferation of multipotent progenitors (MPP) in a HIF1a and SMAD7-dependent manner. Whereas during stress of competitive BM transplantation PHD2 deficient progenitors exhibit a decreased peripheral and central chimerism, but not of the very primitive precursors. Conversely, in whole BM transfer, PHD2 mutant HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity, reliant on HIF1a.

Conclusion: Taken together, lack of PHD2 enhances the proliferation of MPPs during steady state and results in out-competition of hematopoietic precursors by their WT counterparts whilst promoting the self renewal of very primitive progenitors (Singh et al., Blood, 2013).

P028

Behaviour of Immature Leukocytes during Standard Orthopaedic Procedures

*T. Schumann1, O. Tiebel1, J. Lützner2, K. Günther2, G. Siegert1, B. Arneth1

1Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Klinik für Orthopädie, Dresden, Germany

Introduction: With new hematological analyzers, it is possible to count complete blood cells, immature granulocytes (IGs), and high-fluorescing lymphocytes (HFLCs). To determine the validity of these new parameters, increases in immature cells were compared with total white cell count and C-reactive protein (CRP) during standard operating procedures.

Materials and methods: As part of routine blood tests, IGs and HFLCs were analyzed in 160 patients in our orthopedic clinic who underwent standard operations (hip and total knee replacement) from May 2012 to February 2013 on an XE 5000 (Sysmex, Kobe, Japan); CRP was measured on a Modular (Roche, Mannheim, Germany).

Results: Before surgery, patients had an average IG, HFLC, CRP, and leukocyte levels of 0.19 Gpt/L, 0.03 Gpt/L, and 3 mg/L, and 6 Gpt/L, respectively. On the day of surgery, IG, HFLC, and leukocyte levels were 0.37 Gpt/L, 0.03 Gpt/L, and 10.5 Gpt/L, respectively, compared with 0.21 Gpt/L, 0.03 Gpt/L, and 6.5 Gpt/L 1 day after surgery; 5 days after surgery, the levels were 0.38 Gpt/L, 0.09 Gpt/L, and 5.7 Gpt/L, and CRP was 52 mg/L.

Discussion: Minimal increases in IGs and HFLCs were detected throughout the surgical procedures. The leukocyte increases are similar to the IGs, whereas CRP increased significantly 5 days after surgery.

Conclusion: Based on their minimal changes during operating conditions, immature cells provide good predictive values for postoperative complications.

P029

Immature Granulocytes and Highly Fluorescent Lymphocytes in Critical Ill Patients

K. Hommel1, O. Tiebel1, M. Menschikowski1, M. Ragaller2, G. Siegert1, *B. Arneth1

1Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Klinik und Poliklinik für Anästhesiologie und Intensivtherapie, Dresden, Germany

Introduction: The latest-generation XE5000 hematology analyzer records immature cell populations, such as immature granulocytes (IGs), and high-fluorescing lymphocytes (HFLCs). In this study, the value of these new parameters was examined with regard to the detection of immature cells in patients of an intensive care unit.

Methods: In 124 patients of our anesthesia intensive care unit, the new markers were measured in early morning whole blood samples using a Sysmex XE5000 (Kobe, Japan). C reactive protein (CRP) and procalcitonine (PCT) were measured on a modular (Roche, Mannheim, Germany). Patients are grouped into three groups: Group 1: 17 patients without or with prophylactic antibiosis (AB) only, Group 2: 71 patients with one continuously AB, Group 3: 36 patients with a switch in AB therapy.

Results: IG and HFLC medians continuously increased from group 1 over 2 to group 3, while PCT and CRP medians did not. Statistical density plots show that PCT, neutrophils, total WBC count and IG clearly discriminate critical ill patients (SIRS and/or sepsis) from non-critical patients while HFLC and CRP values don’t discriminate. ROC curves for the detection of sepsis using IGs and HFLCs were generated and had a high AUC for absolute IG counts.

Conclusion: Absolute IG number is a useful marker for the presence of sepsis in critically ill patients. Currently sepsis is often diagnosed and treated relatively late. Using the new IG marker, sepsis patients could be treated earlier.

P030

New Surface Plasmon Resonance (SPR) Biosensor for the Detection of FVIII-Antibodies

*C. Kocot1, M. Spannagl2, P. Luppa3

1Klinikum rechts der Isar TU München, Institut für Klinische Chemie, München, Germany

2Klinikum der Universität München, München, Germany

3Klinikum rechts der Isar, Institut für Klinische Chemie, München, Germany

Purpose: Hemophilia A is an X-linked bleeding disorder. Patients are treated with factor VIII (FVIII). Approximately 25% of patients, however, develop antibodies to these FVIII concentrates. To improve the analytical detection of the inhibitory antibodies, a biosensor system was established. We applied a SPR-sensor because SPR allows a real-time, label-free measurement of interaction between ligand (FVIII) and analyte (Anti-FVIII) and determination of kinetic parameters.

Method: Gold chips with a polycarboxylate surface are activated via EDC/NHS, and two different FVIII proteins are covalently immobilized with preservation of antigenicity. (A): recombinant FVIII without B-domain, and (B): recombinant FVIII with B domain. To exclude physiological protein interactions, the antibodies in patient´s plasma are purified using protein A columns.

Result: We designed a reusable chip, stable over 70 cycles. 30 patients and 30 healthy controls were measured. The FVIII (A) without B domain has a sensitivity of 96% and specificity of 55%. However, FVIII (B) has a sensitivity of 86% and specificity of 90%. These results show that the full length FVIII (B) is more specific while maintaining adequate sensitivity. Kinetic measurements are in progress.

Conclusion: The biosensor allows sensitive detection of FVIII antibodies with high specificity and sensitivity. Kinetic parameters can also be determined, allowing statements about anti-FVIII affinity. With regard to patients’ treatment, this information can be used. Upon occurrence of clinically relevant, highly affine antibodies during the treatment with a specific FVIII preparation, a targeted preparations change according to the biosensor results could be considered.

P031

Comparison of the immunoassays Freelite and N Latex FLC in the detection of monoclonal free light chains

*L. Eichhorn1, B. Stoffel-Wagner2, B. Zur2

1Universityhospital Bonn, Anesthesiology, Bonn, Germany

2University Hospital Bonn, Clinical Chemistry and Clinical Pharmacology, Bonn, Germany

Objectives: Measurement of free light chains in serum is an important tool in the detection of monoclonal gammopathy. We compared a polyclonal reagents Freelite immunoassay (®Binding Site Group LTD., UK) with the new monoclonal antibody N Latex FLC (®Siemens, Germany).

Methods: In 322 patient samples were measured by N Latex FLC (by Siemens) kits and a Freelite assay (by Binding Site). In the event of disparity between clinical diagnosis and immunoassays, we additionally performed immunofixation of serum with Hydrasis (Sebia) or Bence-Jones proteins detection. These results were correlated to the results from the two immunoassays.

Results: Of 322 samples, Freelite detected 147 positive and 175 negative, while N Latex FLC detected 190 negative samples and 132 positives. In 29 samples deviations were found between Freelite and N Latex FLC.

Conclusion: The two assays showed no agreement in about 9% of measurements. Compared to the gold standard IFE, Freelite showed better agreement than N Latex FLC. Thus, the Freelite immunoassay appears to be better suited despite the fact that it still produces some false negative results. Therefore, if monoclonal gammopathy is clinically suspected, IFE as well as Benje-Jones protein electrophoresis should be continued to be performed or the immunoassay test should be repeated. Further studies are required to support these recommendations.

P032

Simultaneous measurement of erythrocyte protoporphyrin IX and zinc protoporphyrin IX from unwashed whole blood by dual-wavelength excitation

G. Hennig1, *M. Vogeser2, D. Teupser2, H. Stepp3, C. Gruber1, G. Brittenham4

1Klinikum der Universität München, Laser-Forschungslabor, München, Germany

2Klinikum der Universität München, Institut für Laboratoriumsmedizin, München, Germany

3Klinikum der Universität München, Laser Forschungszentrum, München, Germany

4Columbia University, College of Physicians and Surgeons, Children′s Hospital of New York, New York, New York, USA

Objectives: Measurements of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX) are useful in the diagnosis of disorders that restrict or disrupt the biosynthesis of heme. We describe a reagent-free fluorescence spectroscopic method that can measure simultaneously both ZnPP and PPIX from unwashed whole blood.

Methods: ZnPP and PPIX from 35 EDTA blood samples were determined by a commercially available HPLC kit (Immundiagnostik AG). Fluorescence of diluted unwashed blood samples was measured by a custom front-face illumination device. The dual-wavelength excitation method determines the difference between fluorescence spectra excited at 425nm and 407nm to virtually eliminate background autofluorescence while retaining characteristic porphyrin peaks at 593nm (ZnPP) and 627nm (PPIX).

Results: Over a wide range of values, the ZnPP and PPIX fluorescence intensities evaluated by the dual-wavelength excitation method were closely correlated with the reference HPLC method (Spearman’s rho rs = 0.943 and rs = 0.959, respectively; p < 0.0001 for both).

Conclusion: Simultaneous dual-wavelength excitation fluorescence spectroscopy may provide a means for reagent-free quantification of ZnPP and PPIX in the routine clinical laboratory that would be useful in the diagnostic evalulation of iron deficiency, iron-restricted erythropoiesis, certain porphyrias, lead intoxication, and a variety of sideroblastic and inherited microcytic anemias.

P033

Diagnostic accuracy of zinc protoporphyrin/haem ratio for screening of iron deficiency anaemia in patients with inflammatory bowel disease

*M. Wiesenthal1, D. Jacobsen1, F. Hartmann2, A. Dignass3, J. Stein4

1Krankenhaus Sachsenhausen, Frankfurt, Germany

2Crohn Colitis Centre, Frankfurt, Germany

3Agaplesion Markus Krankenhaus, Frankfurt, Germany

4Crohn Colitis Centre Rhein-Main, Frankfurt, Germany

Background: Iron deficiency anaemia (IDA) is commonly measured by multiple indicators. Choice of a single iron biomarker for IDA screening at population level is controversial. Zinc protoporphyrin (ZPP) has proved a sensitive and specific marker for functional iron deficiency. Our study evaluated for the first time the diagnostic value of ZPP in IDA and differential diagnosis of IDA/anaemia of chronic disease (ACD).

Methods: The study included 116 patients with IBD (age, 36.40 ± 13.14 years, 41 % male), who consecutively attended the Crohn Colitis Centre Frankfurt for routine evaluation between 2009 and 2012. Blood count, transferrin saturation (TSAT), serum ferritin (SF), C-reactive protein and ZPP were determined by routine assay. Anaemia was defined in accordance with WHO criteria as haemoglobin (Hb) concentration of < 13 g/dl/12 g/dl (m/f). For the multiple-criteria model, ID was considered present when =2 values from SF (<30 µg/l), TSAT (<20%), and ZPP (<40 µmol/mol Hb)1were abnormal. Anaemia was classified as IDA, ACD or a mixed form (IDA/ACD)2.

Results: Unlike SF, ZPP significantly correlated with Hb. Receiver operating characteristic (ROC) curves for the multiple markers used to diagnose IDA indicated a superior diagnostic accuracy of ZPP. Crucially ZPP is elevated even in ACD, indicating iron-deficient erythropoesis (fig. 1).

Conclusion: ZPP is not a simple ID parameter but a marker of iron-deficient erythropoesis. Our results confirm the utility of ZPP in detection of ID in IBD patients with inflammation and anaemia, even in ACD.

1 Grant KE et al. Comparison of indicators of iron deficiency in Kenyan children. Am J Clin Nutr 2012;95:1231–7

2 Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005;352:1011–23

Kardiovaskuläre Erkrankungen Poster I

P034

Characterization of cellular pyrophosphate homeostasis in dermal fibroblasts derived from Pseudoxanthoma elasticum (PXE) patients

*M. Dabisch-Ruthe1, P. Kuzaj1, C. Knabbe1, D. Hendig1

1Herz- und Diabeteszentrum NRW, Universitätsklinik der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Objectives: PXE is a rare hereditary disorder characterized by progressive calcification of skin, eyes and cardiovascular system. We analyzed the cellular pyrophosphate (PPi) homeostasis of dermal fibroblasts from healthy controls (NHDF) and PXE patients.

Methods: Gene expression of alkaline phosphatase (ALP), ectonucleotide pyrophosphatase 1 (ENPP1) and ankylosis (ANKH) were determined by quantitative real-time PCR after an incubation up to 21 days with Na2HPO4. Extra- (e) and intracellular (i) PPi levels were measured by an enzyme-linked bioluminescence assay and eALP activity spectrophotometrically.

Results: Supplementation of Na2HPO4 induced a marked calcification in PXE fibroblasts compared to NHDF. ALP and ANKH expression increased in calcifying fibroblasts up to 4-fold, whereupon significantly higher eALP activities (2-fold) were measured in PXE fibroblasts. In the early days of the calcification process ALP transcript levels were strongly reduced in PXE fibroblasts. NHDF showed a 0.2-fold decrease in ENPP1 mRNA expression, whereas ENPP1 levels were reduced by 0.5-fold in PXE fibroblasts. ePPi levels in NHDF were found to be 50-fold higher compared to PXE fibroblasts by day 6. During calcification ePPi levels decreased in NHDF as well as in PXE fibroblasts. We observed an increase in iPPi in treated NHDF on day 12, whereas iPPi level of treated PXE fibroblasts further declined.

Conclusions: We show for the first time a disturbed cellular PPi homeostasis in PXE.

P035

The role of ER stress in endothelial inflammation

*A. Ziogas1, S. David1, T. Chavakis2

1Institute for Clinical Chemistry and Laboratory Medicine, University Dresden, Department of Medicine III, Division of Vascular Inflammation, Diabetes and kidney, Dresden, Germany

2Department of Internal Medicine III, University Dresden, Division of Vascular Inflammation, Diabetes and Kidney, Dresden, Germany

Objectives: X-box binding protein 1 (XBP1) is a multitasking transcription factor that is a key component of the ER stress response. In the current study we examined the potential role of ER stress, and especially XBP1, in endothelial cells for the regulation of vascular inflammation. To address this, we tested the impact of pharmaceutical induction of ER stress as well as XBP1 knockdown in the regulation of pro-inflammatory genes.

Methods: bend3 cells were treated with the ER stress inducer tunicamycin, as well as with siRNAs against XBP1. The expression levels of different genes were tested with RT-PCR. The leukocyte adhesion determined with in vitro leukocyte adhesion assay.

Results: Endothelial cells lacking XBP1 present decreased expression levels of Thrombomodulin and Endothelial Protein C Receptor (EPCR) but higher constitutive expression levels of adhesion molecules ICAM1, VCAM1, E-Selectin and cytokines IL-6, MCP1, CXCL2. In contrast, TNF-induced endothelial adhesion molecules and cytokine expression is alleviated in endothelial cells upon XBP1 knockdown or ER stress induction. In agreement with these results, endothelial cells lacking XBP1 or preconditioned by tunicamycin displayed less TNF-induced leukocyte adhesion.

Conclusion: These results suggest that XBP1 is involved in inflammation of the endothelium and we are currently elucidating the underlying mechanisms.

P036

Lipoprotein apheresis of hypercholesterolemic patients mediates vasoprotective gene expression in human endothelial cells

*H. Morawietz1, W. Goettsch1, M. Brux1, M. Reimann2, S. Bornstein3, U. Julius3, T. Ziemssen2

1Bereich Gefäßendothel / Mikrozirkulation, Medizinische Klinik und Poliklinik III, Universitätsklinikum Carl Gustav Carus, an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

2UKD, Department of Neurology, Autonomic and Neuroendocrinological Laboratory, Center of Clinical Neuroscience, Dresden, Germany

3University Hospital Dresden, Dresden, Germany

Hypercholesterolemia is an important risk factor of cardiovascular diseases. Lipoprotein apheresis is an efficient strategy to reduce the serum low-density lipoprotein (LDL)-cholesterol and lipoprotein(a) levels and cardiovascular complications in patients with severe hypercholesterolemia. The underlying molecular mechanisms are not well-understood. In this study, we analyzed the impact of lipoprotein apheresis on gene expression in human endothelial cells. Human endothelial cells were stimulated with serum of hypercholesterolemic patients before and after lipoprotein apheresis. The expression of endothelial lipoprotein receptors, nitric oxide (NO) synthase and adhesion molecules was quantified by real-time PCR and Western blot. Lipoprotein apheresis reduced the expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Low-density lipoprotein (LDL) receptor expression remained unchanged. The mRNA expression of the endothelial nitric oxide synthase (eNOS) was increased with serum of hypercholesterolemic patients after lipoprotein apheresis. In contrast, endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) was reduced in response to serum after lipoprotein apheresis. In conclusion, lipoprotein apheresis reduced the expression of the proatherosclerotic oxLDL receptor LOX-1 and adhesion molecule VCAM-1 and increased the expression of vasoprotective and NO generating eNOS in human endothelial cells in response to serum of hypercholesterolemic patients. These novel molecular mechanisms may account for the antiatherosclerotic and vasoprotective potential of lipoprotein apheresis in patients with hypercholesterolemia.

P037

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

*F. Cheng1, M. Torzewski2, A. Degreif1, H. Rossmann1, A. Canisius1, K. Lackner1

1University Medical Center Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany

2Robert Bosch-Hospital, Department of Laboratory Medicine, Stuttgart, Germany

The antioxidant enzyme glutathione peroxidase-1 (GPx-1) plays a protective role in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE-/- mice. Accordingly, the aims of our study were: to analyze which cells express GPx-1 within atherosclerotic lesions and to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both In situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE-/- mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent - smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of macrophages. The MCSF- and oxLDL-induced proliferation of macrophages from GPx-1-/-ApoE-/- mice was mediated by the ERK1/2 (extracellular-signal regulated kinase 1/2; p44/42 MAPK) signaling pathway as demonstrated by ERK1/2 pathways inhibitors, western blots on cell lysates and immunohistochemistry of mice atherosclerotic lesions with antibodies against MAPKs (mitogen-activated protein kinases). Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. In summary, GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK pathway encouraging further studies on new therapeutic strategies against atherosclerosis.

P038

Novel Galectin-3 immunoassay: performance characteristics on the ARCHITECT system

*J. Dreier1, C. Prante2, C. Knabbe3

1Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Bad Oeynhausen, Germany

2Herz- und Diabeteszentrum NRW Universitätsklinik der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

3Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinikum der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Objectives: Galectin-3 is a novel cardiovascular biomarker which has shown to be useful in assessing the prognosis of patients with heart failure. We describe the analytical validation of an automated, microparticle-based chemiluminescent immunoassay method designed to measure Galectin-3 in human serum and EDTA plasma.

Methods: Assay performance characteristics such as precision, analytical sensitivity and specificity were measured following the protocols by the Clinical Laboratory Standards Institute (CLSI). Samples from apparently healthy blood donors were included in the normal range study (n=130). Method comparison against Galectin-3 ELISA from BG Medicine (BGM) was evaluated using surplus clinical specimens from patients with normal and elevated BNP levels.

Results: The detection limit for this assay was 0.58 ng/ml, the limit of quantitation was <6 ng/mL. In the precision study using the assay controls total coefficient of variation was below 6%. The assay was linear upon dilution. In apparently normal blood donors 95 percentile was 17.5 ng/mL with women having higher values than men (95 percentile: 17.9 ng/mL versus 15.4 ng/mL). Comparison with the BGM Galectin-3 assay yielded a linear correlation coefficient of 0.96. Passing Bablok fit was found to be y=1.23x+0.95, n=151.

Conclusion: The ARCHITECT Galectin-3 assay measures Galectin-3 rapidly, accurately, and precisely in human serum and EDTA plasma. It can provide useful improvements in assessing the prognosis from patients with chronic heart failure.

P039

Defining the differential effects of novel anticoagulants: fXa versus fIIa inhibition on coagulation and protease dependent cell signaling

*K. Shahzad1, F. Bock1, H. Wang1, S. Ranjan1, J. Wolter1, I. Thielmann2, B. Nieswandt2, M. Thati1, B. Isermann1

1Institut of Clinical Chemistry und Pathobiochemistry, Universitätsklinikum Magdeburg, Germany

2Rudolf Virchow Center; DFG Research Center for Experimental Biomedicine University Hospital, University of Würzburg, Germany

Introduction: Thrombin is the key protease in regard to thrombus formation; the same is not true in regard to protease dependent signaling. Hence, we postulate that inhibition of either fXa or fIIa may have comparable effects in regard to coagulation, but convey different effects in regard to receptor dependent regulation of cellular effects.

Methods: WT mice were either treated with low and high dose of dabigatran or Rivaroxaban for 1 week and then were analyzed for tail bleeding assay or for arterial injury induced thrombosis formation. Wild type (WT/ApoE-/-) mice and hypercoagulable (TMPro/Pro/ApoE-/-) mice (age 8 weeks) were fed a high fat diet (HFD) for 20 weeks with or without these drugs.

Results: Rivaroxaban and dabigatran have comparable dose-dependent effects in regard to bleeding and thrombosis in in vivo models. However, while fIIa inhibition abrogates the anti-inflammatory effect of fIIa already at low dosages, e.g. at dosages at which bleeding time is already prolonged, this is not the case when using the fXa inhibitor Rivaroxaban. In addition we observed smaller and more stable atherosclerotic plaques in fXa inhibition group, while in fIIa inhibition group; plaques were also smaller but less stable. Immunohistochemical analysis revealed more smooth muscle cells and less macrophages in RR treated mice, but on contrary more macrophages and less SMC within the plaques of DG treated mice.

Conclusion: Taken together, these results strongly support that inhibition of fIIa and fXa have similar profiles in regard to the regulation of hemostasis, but differ in their ability to modulate the inflammatory response, with fXa inhibition being superior.

P040

Predictive value of high sensitivity CRP (hsCRP) and lipoprotein associated phospholipase A2 (LpPLA2) in smokers and nonsmokers of the Ludwigshafen Risk and Cardiovascular Health Study (LURIC)

M. Kleber1, *R. Siekmeier1, G. Delgado1, T. Grammer1, B. Winkelmann2, H. Scharnagl3, B. Böhm4, W. März1

1Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Institute of Public Health, Social and Preventive Medicine, Mannheim, Germany

2Cardiology Group, Frankfurt, Germany

3Medical University Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria

4University Hospital, Ulm, Division of Endocrinology, Department of Medicine, Ulm, Germany

Objectives: Cardiovascular disease (CVD) is an important cause of mortality and smoking is an avoidable cause for CVD. Analysis of predictive factors (e. g. plasma lipids, smoking) allows individual risk estimation. Measurement of hsCRP and LpPLA2 provides information on inflammation and plaque stability. Aim of our study was the analysis of both in active smokers (S) and life-time nonsmokers (NS) of the Ludwigshafen Risk and Cardiovascular Health Study (LURIC).

Methods: 3316 patients were included of which 777 were S and 1178 NS. Within the observation time (10 years, median) 995 died (221 S, 302 NS). hsCRP and LpPLA2 were measured in all patients (N High Sensitivity CRP, Dade Behring, Germany; LpPLA2, diaDexus Inc., USA).

Results: LpPLA2 and CRP were higher in S than in NS (S vs. NS; LpPLA2, 424.2 (293.7–630.2) vs. 383.8 (272.3–533.5) ng/ml, p<0.001; hsCRP, 4.9 (1.8–10.3) vs. 2.7 (1.2–7.0) ng/ml, p<0.001) and correlated (rS=0.132, p<0.001). In 125 (16.3 %) S LpPLA2 was above the median whereas hsCRP was below. Vice versa, hsCRP was above the median in 179 (23.3 %) S whereas LpPLA2 was below (NS: 276 (23.6 %) and 208 (17.8 %), respectively). Mortality was higher in S than in NS, highest in patients with elevated hsCRP and LpPLA2and lowest in patients with hsCRP and LpPLA2 below the median.

Conclusion: Our data confirm the value of hsCRP and LpPLA2 for estimation of individual risk. Both parameters should be determined at least in high risk patients (e. g. smokers).

Klinische Massenspektronomie Poster I

P041

Quantification of vancomycin in human serum by LC-MS/MS

*K. König1, U. Kobold2, G. Fink2, A. Leinenbach2, T. Dülffer2, R. Thiele2, J. Zander1, D. Teupser1, M. Vogeser1

1Hospital of the University of Munich, Institute of Laboratory Medicine, München, Germany

2Roche Diagnostics GmbH, Penzberg, Germany

Objectives: The aim of our work was to develop and to validate a reliable LC-MS/MS based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and to harmonize therapeutic drug monitoring of this compound using routine assays.

Methods: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionisation by matrix constituents extended chromatographic separation was applied. Measurement was done by HPLC-ESI-MS/MS. For internal standardisation the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte.

Results: In a bi-center validation total CVs of less than 4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was +/- 4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement.

Conclusions: Employing a closely related homologue molecule for internal standardisation and the use of MS/MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

P042

Mass Spectrometry and Ion Mobility Spectrometry – Complementary Analytical Tools for Metabolomics and Lipidomics

*W. Vautz1

1Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Systemanalyse, Dortmund, Germany

Objectives: The detection of characteristic metabolites or lipids for diagnosis, medication and therapy control is well-established but in general tied to time consuming and expensive laboratory analysis. Therefore, rapid on-site and in the ideal case non-invasive analysis would give significant support for clinical analysis.

Methods: Ion mobility spectrometry (IMS) is an analytical method for the detection of gas phase traces. The sample is ionised and the ions separated by their size and shape in an electric field, thus enabling the selective and sensitive identification even of isomeres. Coupled to rapid GC pre-separation, the analysis of complex mixtures such as human breath for relevant metabolites in less than 10 minutes is possible. However, mass spectrometry is required as complementary tool for the identification of unknowns and to establish a data bases for the IMS. The application of electro-spray ionisation furthermore enables the extension to the liquid phase for the rapid detection of non-volatiles in e.g. salvia, sweat, urine or blood samples.

Results: It could be demonstrated successfully that IMS is capable for diagnosis and therapy control e.g. in nephrology as well as for the quantification of medicaments such as Propofol in human breath with non-invasive sampling. Furthermore, investigations of sweat and urine were carried out with promising results.

Conclusion: Ion mobility spectrometry combines rapid results with high selectivity and sensitivity, thus demonstration high potential for clinical applications. However, more work has to be done on specific applications.

P043

Quantification of plasma proteins by mass spectrometry in clinical chemistry - State of the art

*H. Schlüter1

1Universitätsklinikum Hamburg-Eppemdorf, Inst. f. Klin. Chemie, Hamburg, Germany

Mass spectrometric quantification of small organic molecules such as drugs like sirolimus in blood is common now in many clinical chemistry laboratories. In contrast quantification of plasma proteins by mass spectrometry is not yet well established. In comparison to broadly applied immunological methods mass spectrometric approaches offer the opportunity to quantify multiple proteins in parallel. Furthermore costs for development of a mass spectrometric assay are much lower than costs for development of antibody based assays. Several reasons may be responsible for slow progress of the integration of mass spectrometry for quantification of proteins into the routine diagnostic laboratory. Sample collection, storage and processing are more critical for proteins than for small organic molecules. Most striking is the aspect that a protein derived from a single defined gene in every individuum usually exists in many forms (isoforms, protein species respectively proteoforms), often resulting in a mass shift not only for the protein but also for tryptic peptides yielding false negative results e.g. in SRM assays. In addition genetic variability (inter-individual variability) increases the mass shift problem. Therefore future mass spectrometric protocols for quantification of proteins have to take into account inter- and intra-individual variations of proteins. A new promising approach is top-down mass spectrometry. In top-down mass spectrometry intact proteins are transferred into the mass spectrometer. Beside the determination of the mass information about the composition of a protein is acquired by additional fragmention experiments thus giving more confidence about its identity and its exact chemical composition.

P044

AbsoluteIDQ® Stero17 Kit: Performance on triple quadrupole mass spectrometers from various manufacturers

*H. Pham-Tuan1, D. Kirchberg1, M. Langsdorf1, A. Kania1, C. Röhring1, T. Koal1, R. Zahn1

1BIOCRATES Life Sciences AG, Innsbruck, Austria

Objectives: The Absolute IDQ® Stero17 Kit has recently been developed to address the need for standardized analysis of steroid hormones in clinical environment, and also for research and drug discovery studies. Our kit represents a ready-to-use solution for triple quadrupole MS platforms using MRM in ESI+ mode from AB Sciex (API4000, 4000QTRAP, 5500QTRAP), Waters (Xevo TQ, Xevo TQ-S), and Thermo Scientific (TSQ Vantage). The 17 target steroids analysed are: aldosterone, androstendione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, DHEA, DHEAS, DHT, E2, E1, etiocholanolone, 17α-hydroxyprogesterone, progesterone and testosterone. Proficiency tests (DGKL, RfB) have been carried out quarterly.

Methods: The Stero17 Kit was developed and validated on API4000, 4000QTRAP MS from AB Sciex. For other MS platforms MRM parameters were re-optimized. Partial validation process has been carried out focusing on selectivity, matrix effect, inter- and intraday accuracy and precision.

Results: Lowest LOD of steroid hormones in real human samples is achieved with Waters Xevo TQ-S, e.g. 0.2 ng/L androstenedione, 11-deoxycortisol and testosterone (500 µL sample). For E2 the LOD could be reduced ten-fold (5.6 ng/L on API4000to 0.6 ng/L on Xevo TQ-S).

Conclusions: The general sensitivity for steroid hormones analysis can be graded as follows: Xevo TQ < API4000 = 4000QTRAP < TSQ Vantage < 5500QTRAP < Xevo TQ-S. Because nitrogen is being used for all gases, the instruments from Waters and Thermo are less susceptible to contamination influencing steroid analysis. DHT is valid only on the TSQ Vantage, most likely due to lower matrix effect.

P045

Discovering novel proteolytic angiotensin I-processing activities in blood plasma by a mass spectrometry assisted enzyme screening system

*D. Hildebrand1, H. Schlüter2

1University Medical Centre Hamburg-Eppendorf, Institute of Clinical Chemistry, Mass spectrometry and proteome analysis, Hamburg, Germany

2Universitätsklinikum Hamburg-Eppemdorf, Inst. f. Klin. Chemie, Hamburg, Germany

Objectives: Angiotensin I (ANG-1-10) is a prohormone and part of the renin-angiotensin-system (RAS). In mammalian species, except humans, N-terminal processing of ANG-1-10 was reported. We hypothesized that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans.

Methods: The MES assay involves the covalent immobilization of proteins (body fluids or recombinant proteases) onto an affinity chromatography material, followed by incubation of the proteins with ANG-1-10. Aliquots taken after defined incubation times were analyzed by MALDI-MS and SRM–coupled LC-QQQ-MS. Angiotensin-generating proteases were characterized by addition of different protease inhibitors during the incubation experiments. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay.

Results: We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The plasma concentration of ANG-4-10 (8pg/ml), ANG-5-10 (53pg/ml) and ANG-6-10 (7ng/ml) was determined. Generation of these peptides was carried out by antipain sensitive cysteine/serine proteases as well as by edta-sensitive metalloproteases. Recombinant aminopeptidase N and ACE-1 were also able to hydrolyze ANG-2-10, -4-10 and -5-10.

Conclusion: By a well working combination of MALDI-MES and SRM-based MES-methods we demonstrated the generation of a couple of novel N-terminal angiotensin-peptides by ANG-1-10-processing protease activities in human plasma. This gives new insights into the generation of angiotensins and the understanding of the complex RAS.

P046

TICE™ – an innovative, easy-to-automate extraction technique for small molecule analytes from biological fluids for LC-MS/MS analysis

*S. Bächer1, R. Geyer2, C. Lehmann2, D. Teupser1, M. Vogeser1

1Hospital of the University of Munich, Institute of Laboratory Medicine, Munich, Germany

2Tecan Schweiz AG, Männedorf, Switzerland

Objectives: Tecan Immobilized Coating Extraction (TICE™) – a sample preparation technique prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis – uses the commercially available, innovative 96 well plate format extraction device AC Extraction Plate (Tecan) for automated sample extraction of small molecule analytes from biological fluids for routine analysis.

Methods: AC Extraction Plate was used for 25-hydroxyvitamin D (25OHD) extraction from serum together with a robotic liquid handling system Freedom EVO® (Tecan). An ultra-performance (UP) LC-MS/MS method was developed for 25OHD3/2 quantitation. To assess assay performance for routine analysis the TICE™ UPLC-MS/MS method was evaluated and compared to a method combining protein precipitation (PPT) and two-dimensional (2D) UPLC-MS/MS.

Results: TICE™ UPLC-MS/MS method was linear over the range 4.3 – 65.8 ng/mL for 25OHD3 and 14.1 – 54.7 ng/mL for 25OHD2 (r2 > 0.994). Lower limit of quantification was 4.3 ng/mL for 25OHD3 and 14.1 ng/mL for 25OHD2. Coefficients of variation for the intra-day and inter-day precision were <6% for 25OHD3 and <7% for 25OHD2. Accuracy ranged between 96.9% – 102.2% for 25OHD3 and 95.4% – 107.3% for 25OHD2. TICE™ UPLC-MS/MS method agreed well with PPT 2D UPLC-MS/MS method (r = 0.988, n=73).

Conclusion: TICE™ enables the implementation of a robust and reliable method for 25OHD analysis, allowing complete automation highly applicable for routine analysis. Evaluation of the applicability for other analytes appears appropriate.

P047

Characterization of polyunsaturated fatty acids and eicosanoids in isolated human lipoprotein fractions and plasma by LC-MS/MS

*J. Dorow1, L. Kortz1, J. Thiery1, U. Ceglarek1

1LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany

Objectives: A disturbed lipid metabolism is associated with the pathogenesis of atherosclerosis. Here, we investigated the distribution of polyunsaturated fatty acids (PUFAs) and eicosanoids in very low, low and high density lipoproteins (VLDL, LDL, HDL) compared to plasma in healthy donors.

Methods: Density-gradient ultracentrifugation was applied for the isolation of lipoproteins of six healthy volunteers. After on-line solid phase extraction 6 PUFAs and 94 eicosanoids were analyzed by liquid chromatography combined with tandem mass spectrometry.

Results: In lipoprotein fractions, 5 PUFAs, 9 eicosanoids and 2 other PUFA metabolites could be found. The percentual distribution of PUFAs was comparable between lipoproteins. Among hydroxyeicosatetraenoic acids (HETE), 5-, 11-, and 12-HETE were present in most VLDL, LDL and HDL fractions, while 13- and 9-hydroxyoctadecadinoic acid were present in each fraction. In some LDL and HDL fractions, 11β-prostaglandin E2 (PGE2) and 5-hydroxyeicosapentaenoic acid (HEPE) were also found. However, 5-HETE, 11β-PGE2 and 5-HEPE were absent in the plasma of these donors. In contrast, most dihydroxyeicosatrinoic acids of plasma samples were not present in lipoproteins.

Conclusion: Interestingly, oxidized pro- and anti-inflammatory PUFA metabolites were found in lipoproteins and bring up the question of the constitution of malfunctioning lipoproteins, which might appear in patients with a metabolic syndrome, especially LDL, which can be easily oxidized.

P048

Standardized Sample Pretreatment for the Simultaneous Quantification of Seven Apolipoproteins in Human Plasma via LC-QTrap MS

*J. Dittrich1, S. Becker1, F. Baumann2, L. Kortz1, J. Thiery1, U. Ceglarek1

1Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, LIFE –Leipzig Research Center for Civilization Diseases, Leipzig University, Leipzig, Germany

2Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

Objectives: Apolipoproteins are predictors of cardiovascular disease. Recently, LC-MS/MS emerged as an alternative to immunological protein determination methods. We investigated different sample pretreatment strategies and developed a standardized protocol for the simultaneous quantification of seven apolipoproteins in human plasma via proteotypic peptides by LC-QTrap MS.

Methods: Denaturation, reduction, alkylation and tryptic digestion including microwave and ultrasound assistance were investigated. Quantification and peptide confirmation were performed by micro-liquid chromatography coupled to a quadrupole-linear ion trap mass spectrometer. For 500 human plasma samples method comparison with an immunoassay was performed for Apo A-I and Apo B.

Results: Tryptic digestion times varied between 5 min (Apo A-I, Apo E, Apo A-IV) and 16 h (Apo A-II). Digestion yield was not improved by application of ultrasound or microwave radiation. Linearity was approved between 0.1 nmol/L - 100 mmol/L. The lower limits of quantification were ≤0.4 µmol/L (Apo A-I, Apo A-IV, Apo B-100, Apo CI, Apo C-III, Apo E) and <1.4 µmol/L (Apo A-II). Variabilities <13 % were determined. Method comparison with immunoassays showed a good agreement for Apo A-I and Apo B.

Conclusion: Using the validated pre-analytical protocol the simultaneous analysis of seven apolipoproteins in human plasma can be applied in clinical studies for reliable investigations of apolipoprotein distributions in cardiovascular disease.

Labormanagement und Qualitätskontrolle Poster I

P049

Development of an Excel® Add-in for Method Validations and Internal Quality Control in the Clinical Laboratory

*J. Pum1

1Abacus Validation Systems, Jena, Germany

Objectives: Method validations are indispensable in any accredited laboratory for the purpose of demonstrating fittness-for-use of newly implemented analytical methods. This served as the impetus for creating an Excel® add-in (“Abacus”) for the clinical-analytical laboratory, with the specific purpose of providing standardized protocols for method validations, internal quality control and laboratory-relevant statistical functions.

Methods: Abacus is coded entirely in VBA and VB .NET. The “FURPS” principle (Functionality, Usability, Realiability, Performance and Supportability) was actively adhered to during the entire development and programming phase. Guidelines implemented for the method validation- and quality-control protocols included current recommendations of relevant associations and expert panels (e.g. CLSI, IFCC, CAP, CLIA 88, FDA, ICH, EURACHEM, RiliBÄK and DIN), as well as peer-reviewed publications.

Results: The add-in adds a total of 52 modules in three categories (method validation, quality control and general statistics) to the basic functions of Excel®. Its seamless integration in Excel® allows data processing in a well-known working-environment and the formatted reports satisfy legal and regulatory requirements. Editable internal databases, containing target values for bias, precision and total error, are used for automatic result interpretations. The comprehensive help file, example reports and the data entry assistant ensure a flat learning curve.

Conclusion: Abacus constitutes a useful tool for clinical-analytical laboratories and is already implemented by a wide range of academic and private medical laboratories.

P050

Implementation of an external quality assurance programme for bacterial detection in platelets

*T. Vollmer1, V. Schottstedt2, L. Pichl2, M. Hourfar3, W. Sireis3, M. Schmidt4, J. Dreier1

1Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

2German Red Cross Blood Transfusion Service West, Central Laboratory, Hagen, Germany

3Institute of Transfusion Medicine and Immunohematology, German Red Cross, Frankfurt, Germany

4Reference Institute for Bioanalytics, Bonn, Germany

Background: In Germany, the maximum shelf-life for platelets was reduced from 5 days to 4 days to increase blood product safety regarding bacterial contamination of PCs. Alternatively, application of rapid screening methods allows the extension of platelet shelf-life again to 5 days, but validation of these methods represents a complicated process.

Methods: The aim of this study is the development of a collaborative trial for the detection of bacteria in PCs using rapid screening methods such as Bactiflow (BF), bacteria specific NAT, PGD and BacTx. PCs were spiked with bacterial titers at two concentrations 10E+03/10E+05 CFU/ml and were shipped temperature controlled to participants. Time schedule for testing will be limited at 6 h, data were implemented into a web based platform.

Results: Participants will pass the proficiency panel if negative controls had negative results, samples spiked with bacteria in the range of 10E+05 CFU/ml must have a positive screening result, samples with 10E+03 CFU/ml should have positive results, depending on the rapid method used. Testing under collaborative study conditions with a quality control panel of 6 different bacteria showed a good performance using the two rapid methods BF and NAT.

Conslusion: The planned proficiency panel enables the verification of the analytical sensitivity of rapid bacterial detection systems under controlled routine conditions and represents an important contribution for blood product safety. The first pilot collaborative testing will be performed in June 2013.

P051

Interlaboratory reproducibility of 34 routine clinical chemistry parameters on the Beckman Coulter AU5800, Roche Cobas 8000, Roche Cobas 6000, Roche Modular PE, or Siemens ADVIA 2400 analytical systems

*O. Gressner1, U. Schallenberg1, C. Knauff1, F. Wisplinghoff1

1Laboratoriumsmedizin Köln, Dres. Wisplinghoff und Kollegen, Köln, Germany

Introduction: A lack of reproducibility of results obtained in different medical laboratories frequently hinders a cost-efficient patient care. Therefore we performed a comparison of results of routine biochemical parameters between 5 different laboratories using different analytical systems.

Methods: 2,040 anonymized serum samples submitted to our laboratory were frozen in aliquots, analyzed in parallel in 5 different laboratories (23,193 individual analyses), using either the Beckman Coulter AU5800, Roche Cobas 8000, Cobas 6000, and Modular PE, or Siemens ADVIA 2400 analyzers and results statistically evaluated.

Results: We found a very good concordance between the Roche Systems themselves, closely followed by the Roche Modular PE or Cobas 6000 systems and the AU5800, however. The comparison of Modular PE and AU5800 demonstrated the least variance of all calculated parameter-specific correlation coefficients, indicating a largely method- and parameter-independent reproducibility of results between these systems, being generally best in enzymatic photometry and worst in immunoturbidimetry. Comparing the Siemens ADVIA 2400 with all Roche analyzers and the Beckman Coulter AU5800, poor correlation results were obtained.

Conclusion: In conclusion, the Beckman Coulter AU5800 demonstrated the best reproducibility with all other systems and, compared with the other systems tested, this reproducibility was largely independent from the tested parameter and method used.

P052

Method Comparison in Clinical chemistry using two automated immuno-luminometric Immunoassays

J. Steglich1, B. Pötschke2, S. Neumann2, *C. Günther3, K. Borgwardt4, C. Hintze4, H. Welle-Scharmann4, R. Findeisen2

1Hochschule Zittau/Görlitz, Zittau, Germany

2Oberlausitz-Kliniken gGmbH, Bautzen, Germany

3Oberlausitz-Kliniken gGmbH, Bautzen, Germany

4DiaSorin Deutschland GmbH, Dietzenbach, Germany

Introduction: Quantitative automated luminometric immunoassays for the determination of Cancer Antigen 125 (CA125), Alpha-Fetoprotein (AFP), Total Prostate-Stimulating Antigen (t-PSA), Ferritin, Prolactine, Parathormone (PTH), Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were compared with a Architecht i2000SR (ABBOTT GmbH & Co. KG Max-Planck-Ring 2, Wiesbaden, Germany) and a LIAISON XL (DiaSorin Deutschland GmbH Von-Hevesy-Str. 3, Dietzenbach, Germany).

Material and Methods: The commercially available luminometric immunoassays were performed as described by the manufacturer. The luminometric immunoassays are monoclonal two-site immunoluminometric methods (Sandwich principle). Antibody-coated tubes serve as solid phase. The light signal is directly proportional to the concentration of the measuring sample. The whole measuring procedure consists of pipeting of samples, incubation period, washing cycles, and measurements. Twenty patient samples for each method have been measured parallel. Person`s Correlation Coefficient and Passing-Bablok-Correlation were used to evaluate the results mathematically.

Results: Passing-Bablok-Regression and Pearson`s Correlation (R) were calculated for the following methods: t-PSA (Y=0,95x+0,03, R=0,9999); AFP (Y=1,13x+0,16; R=0,9942); Ferritin (Y=0,84x+53,12; R=0,9340); Prolactine (Y=0,70x+1,39; R=0,9997); PTH (Y=0,62x+0,18; R=0,9915); LH (Y=1,20x+0,68; R=0,9824); FSH (Y=1,26x+1,23; R=0,8875); CA125 (Y=1,14+1,62; R=0,9962).

Conclusions: The results showed a high degree of correlation and regression. The immunoassays at LIAISON XL can be accurately measured using the automated method which can be recommended.

P053

Valid serum creatinine measurement in face of severe icteric interference for reliable MELD Scoring

*C. Gnewuch1, S. Reichl1, G. Schmitz1

1University Hospital Regensburg, Institute for Clinical Chemistry and Laboratory Medicine, Regensburg, Germany

Objectives: Invalid serum creatinine owing to extreme bilirubin concentrations is a major problem in MELD Score calculation for liver transplant allocation. Even with the less susceptible enzymatic measurement procedures falsely reduced creatinine levels are possible. We evaluated if a simple additional dilution step prior to enzymatic reaction in the clinical chemistry autoanalyzer sufficiently eliminates icteric interference yielding valid creatinine values.

Methods: Serum Creatinine, total bilirubin and icteric index of 51 serial two-month cases with creatinine values, that were commented as invalid in the lab report due to severe icteric interference, were re-measured on a Siemens Dimension Vista® 1500 analyzer after manual 1:2 dilution with 0.9% NaCl.

Results: In all but two cases sample dilution yielded bilirubin concentrations that fell below the critical threshold of 20 mg/dL where serum creatinine measurements become invalid. Pre-diluted creatinine values (CV < 6%) were higher than the invalid original values at an average of 16% with an optimal linear correlation (r = 0,9969).

Conclusion: More than 90% of invalid creatinine measurements in severe icteric samples can easily be prevented by a moderate 1:2 pre-dilution step. This leads to more reliable MELD Score calculation and in most cases to a higher priority in liver transplant allocation.

P054

Pitfalls during the implementation of a reference measurement procedure for the quantification of electrolytes in serum, plasma and urine

*D. Grote-Koska1, R. Klauke1, K. Brand1, G. Schumann1

1Institute of Clinical Chemistry / Medizinische Hochschule Hannover, Hannover, Deutschland

Objectives: Elektrolyte concentrations in patient samples play a great role in laboratory medicine. They reflect the electrolyte-water balance of the body and bring further physiological aspects about depending on the kind of substance. Standardisation of the clinical analytics is mandatory and requires higher order reference systems. Here, arising pitfalls during the implementation of an ICP-OES reference measurement procedure are presented.

Methods: ICP-OES analyses have been carried out for the investigation of different potential influences on measurement results. Namely the complex organic matrix must be digested prior to analysis and contaminations, interactions between different ions present in the sample or a fluctuating energy of the plasma must be considered. Proper wavelengths for the detection and the application of a suitable internal standard material need to be figured out. Further, the complex measurement system requires a defined control procedure and standard materials, which contain a preferably low content of interfering substances.

Results: Different parameters have been optimized to finally present a metrologically traceable reference system for the determination of electrolytes in patient samples. Reference measurement values with an expanded measurement uncertainty of 1.1 % (k = 2) are obtained.

Conclusion: Implementation of higher order reference systems for standardization requires detailed investigation and optimisation of the individual measurement parameters.

P055

Quality of customer informations in product problems of reagents for infection testing published by BfArM 2005 - 2012

*J. Hannig1, R. Siekmeier1

1Pharmaceutical Institute, University Bonn, Germany, Drug Regulatory Affairs, Bonn, Germany

Objectives: The European Directive 98/79/EC on In-vitro Diagnostics (IVD) regulates marketing of IVD in Europe. In cases of incidents and field safety corrective actions (FSCA) manufacturers have to inform the responsible Competent Authority (CA) and the public by field safety notices (FSN). We analysed FSN of IVD for reagents for infection testing.

Methods: FSCA and FSN published by BfArM 2005-2012 were analysed in respect to the MEDDEV 2.12-1 rev 8.

Results: 150 FSCA for the studied IVD were published. German/English FSN were found in 142/138 cases. FSN were characterized as FSN in 121/128 cases and product names were provided in 139/114 cases. Lot-No. and other information for product characterization were available in 113/113 and 99/111 cases. Information on FSCA and product malfunction was found in 142/137 and 130/124 cases. Information on product related risks with previous use of affected IVD was provided in 97/99 cases. In 138/136 cases manufacturers provided information for mitigation of product risks including retesting in 62/70 cases. Requests to pass FSN to persons needing awareness and contact data were found in 91/72 and 109/117 cases. Confirmation regarding CA information and included customer information were found in 32/34 and 116/100 cases.

Conclusion: Most FSN fulfil the MEDDEV criteria but differences between German and English FSN and deficiencies were observed. Due to the importance for risk reduction type and content of FSN should be improved.

Metabolische Erkrankungen Poster I

P056

Hif2alpha in myeloid cells regulates angiogenesis in proliferative retinopathy

*A. Klotzsche - von Ameln1, I. Korovina1, M. Economopoulou2, G. Siegert3, B. Wielockx4, T. Chavakis1

1Department of Internal Medicine III, University Dresden, Division of Vascular Inflammation, Diabetes and Kidney, Dresden, Germany

2University Dresden, Department of Ophthalmology, Dresden, Germany

3University Dresden, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany

4University Dresden, Institute for Pathology, Dresden, Germany

Retinal hypoxia is the main trigger for pathological neovascularization (NV) in proliferative retinopathies (PR). Synergisms between hypoxia, angiogenesis and innate immune response have been suggested, which could be integrated at the level of the hypoxia-inducible factors (HIF). HIF2α is expressed highly in endothelial and myeloid cells, e.g. macrophages. Thus we studied the role of myeloid cell HIF2α for pathologic NV in the course of PR; we engaged myeloid specific HIF2α ko mice in the model of Oxygen Induced Retinopathy (OIR), which mimics the PR in diabetes. Seven-day old mice were exposed to 75% O2 for 5 days, and then returned to normoxic conditions. Eyes were collected at P17 and NV was quantified by counting of epiretinal nuclei in PAS stained histological sections. IHC for CD11b and Iba1 was done in retinal cross sections. In the OIR model we observed significantly reduced NV due to myeloid HIF2α deletion. IHC revealed more CD11b and Iba1 staining in myeloid HIF2α deficient mice, indicating a higher amount of activated microglia. In primary mouse BMDM and microglia proangiogenic M2-polarization markers (Arg1, Fizz1 and Ym1) were diminished by HIF2α deficiency, which might reduce NV in the OIR. In contrast, physiological retinal development was not influenced by myeloid HIF2α deletion. Our study indicates that myeloid HIF2α deficiency influences myeloid cell polarization in the retina, thereby modulating pathological NV in PR without affecting normal angiogenesis.

P057

Mast cell protease-4 impairs insulin signaling and mediates obesity-related metabolic dysregulation

*J. Chmelar1, A. Chatzigeorgiou2, T. Chavakis1

1Technical University Dresden, Department of Internal Medicine III, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany

2Technical University of Dresden, Department of Internal Medicine III, Paul-Langerhans Institute, Dresden, Germany

Objectives: Mast cells were recently found as key players in obesity-related metabolic dysregulation. Our study addresses the role of mast cell protease-4 (the mouse counterpart of human chymase, which is an important mast cell secreted protease) in obesity and insulin resistance development.

Methods: Mouse mast cell protease-4 deficient mice (Mcpt4-/-) and control mice were fed a high fat diet in diet-induced obesity (DIO). The content of inflammatory cells in adipose tissue and the expression of metabolic and inflammatory genes in liver, subcutaneous and epidydimal adipose tissue were estimated. Insulin resistance, glucose tolerance and insulin signaling were studied in vivo. 3T3-L1 adipocytes and mouse primary hepatocytes were used for mechanistic studies in vitro.

Results: Although Mcpt4-deficiency was associated with increased weight gain and Mcpt4-/- mice had increased adipose tissue and liver weight in DIO, they displayed improved glucose tolerance and were protected from insulin resistance. Moreover, Mcpt4-/- mice showed improved insulin signaling, as insulin-induced Akt phosphorylation was higher due to Mcpt4-deficiency. In addition, Akt was cleaved after incubation of 3T3-L1 cells with chymase. No difference in inflammatory cell content was observed after 21 weeks on high fat diet between Mcpt4-deficient and –sufficient mice.

Conclusion: Mast cell chymase contributes to metabolic dysfunction in DIO by inhibiting insulin signaling.

P058

Modern diagnosis of chronic and acute porphyrias

*H. Ertl1, J. Hartleb1, W. Schmidt2

1Labor Lademannbogen, Analytische Chemie, Hamburg, Germany

2Labor Lademannbogen, Humangenetik, Hamburg, Germany

Advances in understanding the porphyrias and in DNA testing technology may enable improvements in both diagnosis and therapy. Both should benefit since more is known about the genetic mutations involved and specific gene tests are employed. For the diagnosis of clinically expressed porphyrias a logical stepwise approach was adapted. Baseline of this approach is the biochemical analysis of the porphyrines and precursors in 24-hour urine, including Kopro-Isomere ratio. Using this approach it is possible to recover or exclude all types of clinically expressed porphyria (acute, chronic or erythropoietic as well as secondary porphyrias like lead intoxication) in a short period of time. This approach is particularly useful regarding outpatients and/or when clinical symptoms were not provided to the laboratory, which still often occurs. The second step depends on the results of the first step and often may consist of the analysis of porphyrines in stool or blood. In some cases a third step is reasonable, which is the molecular genetic test. Genetic tests offer a maximum of clearness and in particular are useful in facilitating correct treatment and for the screening of family members at risk. Nevertheless, it should be pointed out that not all gene carriers do show (or even will show) a clinical porphyria, and also some porphyrias occur as an acquired disease. A logical stepwise approach is preferred for the diagnosis of porphyria. Biochemical urine analysis remains the baseline.

P059

Comparison of a novel microbiological assay with standard HPLC determination of vitamin B6 in plasma

*T. Dschietzig1, S. Loitsch2, J. Stein2

1Immundiagnostik AG, Medical Sciences, Berlin, Germany

2Institute of Pharmaceutical Chemistry, Frankfurt, Germany

Higher plasma homocysteine is associated with higher cardio-vascular risk. Apart from renal insufficiency, deficiencies of vitamins B6, B12, and of folic acid pose the most common causes of moderately elevated homocysteine. We have developed novel, micro-titre plate-based turbidimetric kits (ID-VitR) to determine biologically active B6, B12 and folic acid by measuring bacterial growth in factor-deficient media. The novel ID-VitR for vitamin B6 in human plasma uses micro-titre plates pre-coated with lyophilized Saccharomyces cerevisiae. It was compared with the gold standard, a HPLC assay, in 170 healthy individuals and in 68 patients with coronary artery disease (CAD). Homocysteine in CAD patients was measured by HPLC. The new ID-VitR correlates well with HPLC (r= 0.89; p<0.0001). A Bland-Altman analysis revealed good agreement between the results of both methods, with a bias of -0.9 ng/ml (HPLC- ID-VitR ) and 95% of all values grouping within the lines of agreement (mean ± 2SD of differences HPLC- ID-VitR). In CAD patients, homocysteine values did not differ between stable CAD and acute coronary syndrome (14.4 ± 3.4 vs. 14.0 ± 2.7 µmol/l) and were not elevated. Thirty four % of CAD patients had elevated serum creatinine. Neither HPLC nor ID-VitR values for B6 correlated with homocysteine levels. We conclude that the ID-VitR and the HPLC standard assay are in good agreement. The new assay can easily be automated and is less laborious than common microbiological assays. The lack of correlation between B6 vitamin and homocysteine can be accounted for by the fact that homocysteine in our CAD patients was in the high-normal range and that a relevant percentage of patients had impaired renal function.

P060

Glucose measurements in diagnosis and monitoring of patients with diabetes mellitus: Comparison of assay performance of Patient near testing and Core-lab methods

*A. Petersmann1, A. Kallner2, M. Blaurock1, M. Nauck1

1University Medicine Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

2Karolinska University Hospital, Institute for Molecular Medicine and surgery, Stockholm, Sweden

Objectives: RiLiBAEK imposes the same analytical limits for glucose determinations in patient near testing and core-lab tests. Whereas core-lab methods can be used for both diagnosis and monitoring of patients with diabetes mellitus, the use of patient near testing is limited by guidelines to monitoring of patients, even though both comply with RiLiBAEK.

Methods: Imprecision is generally determined as a coefficient of variation (CV). Imprecision can be translated into the “minimal difference” (MD): the difference in concentration by which two results must be apart to be regarded as different. For monitoring diabetes mellitus patients the MD includes the variance of two consecutive results. For diagnosis, based on cut-offs, the MD contains only the variance of the result since the cut-off is without uncertainty. Accordingly the demand of precision is higher for monitoring than for diagnosis.

Results: We compared glucose determinations of Dimension Vista, YSI and StatStrip. Venous samples were collected from 55 patients undergoing oGTT. Samples were taken under basic conditions and 120 minutes after glucose administration. Li-heparin plasma was measured in duplicates on each instrument. Correlations and CVs were calculated from these duplicate results. The MD for diagnoses (monitoring) ranged from 0.24 to 0.46 mmol/L (0.33 to 0.66 mmol/L).

Conclusion: The MD is a useful measure to assess clinical performance and make statistical terms practical for professionals involved in patient care.

P061

Metagenomic sequencing of the human gut microbiome before and after bariatric surgery in obese patients with type 2 diabetes: Correlation with inflammatory and metabolic parameters

*J. Gräßler1, Y. Qin2, H. Zhong2, J. Licinio3, T. Chavakis4, T. Lohmann5, T. Wolf5, S. Bornstein6

1Universitätsklinikum Carl Gustav Carus, Medizinische Klinik und Poliklinik III, Dresden, Germany

2BGI Shenzhen, Shenzhen, China

3John Curtin School of Medical Research, Department of Translational Medicine, Canberra, Australia

4Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

5Krankenhaus Dresden-Neustadt, Medizinische Klinik I, Dresden, Germany

6University Hospital Dresden, Dresden, Germany

Objectives: The aim of the present study was to characterize intra-individual changes of gut microbial composition before and 3 months after Roux-en-Y gastric bypass (RYGB) by metagenomic sequencing in morbidly obese patients (BMI>40 kg/m2) with T2D and its correlation with clinical indices.

Methods: RYGB included the creation of a small stomach pouch and its connection to the distal small intestine. The upper part of the small intestine was reattached in a Y-shaped configuration. DNA isolation from faecal samples was performed after disrupting cells using a Retsch mixer mill MM400 with glass beads. DNA library construction and sequencing were performed following the manufacturer’s instruction (Illumina). Comparisons between pre- and post-operative abundances were performed by ANOVA.

Results: Based on gene relative abundance profile 1,061 species, 729 genera, and 44 phyla were identified. The RYGB-induced shift was characterized by a reduction of Firmicutes and Bacteroidetes and an increase of Proteobacteria. 22 microbial species and 11 genera were significantly altered by RYGB. Using PCA two common components have been defined. Component 1 included Proteobacterium E. cancerogenus, F.prausnitzii, and C. comes which were associated with BMI and C-reactive protein. Spearmen’s Rank correlation indicated an association of 10 species with plasma total- or LDL-cholesterol, 5 species with triglycerides. Faecalibacterium prausnitzii was directly correlated to fasting blood glucose.

Conclusion: These data demonstrate profound and specific intra-individual modification of gut microbial composition and show a clear correlation of microbiome composition with metabolic and inflammatory parameters.

Molekulare Diagnostik Poster I

P062

Identification of a Novel HMBS Gene Mutation in a Young German Woman with Acute Intermittent Porphyria

*W. Schmidt1, J. Hartleb2, H. Ertl2, T. Bäumer3, S. Rosenkranz3

1Labor Lademannbogen, Humangenetik, Hamburg, Germany

2Labor Lademannbogen, Analytische Chemie, Hamburg, Germany

3Universitätsklinikum Hamburg-Eppendorf, Neurozentrum, Klinik für Neurologie, Hamburg, Germany

Objectives: Acute Intermittent Porphyria (AIP). the most common acute hepatic porphyria, is an autosomal dominant disorder with incomplete penetrance that results from partial deficiency of hydroxymethylbilane synthase. Attacks in AIP are characterized by abdominal pain, neurological disturbances and psychiatric disorders. Diagnosis is often delayed because the clinical symptoms are nonspecific. The case of a 26-year old female German patient with a novel mutation in the HMBS gene as a cause of acute intermittent porphyria is presented to illustrate the clinical manifestations and difficulties in management. The results of the genetic analysis are discussed in the context of the clinical findings.

Methods: We describe clinical, biochemical and genetic features of a 26-year old female German patient, who was hospitalized during an attack. The patient presented first with symptoms of acute porphyria mainly followed by an uncommon prolonged period of hallocinations and desorientation.

Results: Sequencing the HMBS gene revealed a novel G deletion in exon 10, c.628delG (p.Glu210Asnfs*45); reference sequence NM_000190.3), which predicts a frameshift and a premature stop codon. The amino acid position 210 is located in an evolutionarily highly conserved sequence motif across many species boundaries.

Conclusion: Clinicians should suspect acute porphyrias in patients presenting with variable neuropsychiatric symptoms and unexplained pain. Proper identification can lead to less iatrogenicity associated with porphyrinogenic agents, appropriate management, and a better patient outcome.

P063

Performance evaluation of hepatitis E virus real-time PCR assays for blood donor screening compared to single donation antigen testing

*T. Vollmer1, C. Knabbe1, J. Dreier1

1Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Background: The risk of transfusion-transmitted HEV infections by contaminated blood products currently remains unknown, amongst others due to the absence of available NAT screening methods. Here, the sensitivity and performance of four different HEV RNA amplification systems and one HEV antigen test were evaluated for (a) blood donor pool screening, (b) individuals with acute or chronic infections.

Methods: The analytical sensitivity and the precision of the 3 commercially available assays and one in-house RT-PCR assay were determined using a twofold dilution series of HEV RNA positive plasma. RNA was extracted with a high volume extraction protocol (4.8 ml, chemagic Viral 5K) for blood donor pool screening and the Nuclisens easyMAG for ID-NAT. HEV antigen screening was performed using the Axiom HEV antigen test.

Results: Assays provided a good analytical sensitivity ranging from 4.7 to 91.2 IU/ml (pool screening, 96 pool: 451 - 8755 IU/ml individual donation) and 37.8 to 180.1 IU/ml (ID-NAT). Intra- and interassay variation coefficients were below 3%. HEV antigen was only detectable in four out of ten samples, with viremias ranging from 1.92E+03 to 2.19E+05 IU/mL. Viremias in antigen-negative donors ranged from 1.86E+01 to 4.74E+04 IU/mL.

Conslusion: Evaluated methods present powerful tools providing sensitive possibilities for viral testing comprehending further investigative studies. Application of HEV antigen screening is not an option, due the decreased sensitivity compared to NAT methods.

P064

A microfluidic unit for fast determination of bacterial DNA or resistance genes

*S. Hoffmeier1, K. Löwe1, N. Hlawatsch2, C. Gärtner2, T. Brandstetter3, C. Carstens1, R. Elbracht1

1Praxis für Labormedizin, Naumburg, Germany

2microfluidic ChipShop, Jena, Germany

3Institut für Mikrosystem Technik (IMTEK), Freiburg, Germany

Vision of project INLINE is a flow-through system for DNA analysis. The name INLINE describes the processing and assay readout in an unbraced fluidic channel. In order to transfer idea to prototype, functionality was proven first in single modules for DNA isolation, PCR and detection. Artificial bacterial mixtures and clinical samples pass a sequence of reaction chambers and microfluidic structures realized in plastic disposables. After isolation of bacterial DNA is performed, fluorescent amplicons created in subsequent PCR hybridize to 3-dimensional hydrogel areas with capturing nucleotides. Signals obtained by captured fluorescent dyes are visualized. For prototype realization, functional partial modules were combined and connected to an operation unit. Fluidic transport needed for buffer dilution, supply with reactive components and sample flow is directed automatically by program-driven valves, magnets and pumps. To prove its value in a clinical context, the integrated system was tested in Neisseria meningitidis detection. Connecting the modules enhanced efficiency, although new parameter settings were required for functional linking. Working ability ranged from 102 to 109 colony forming units. Test sensitivity increased from 200 bacteria in the modular set-up to less than 50 in identical working conditions. System specificity was investigated with isolates of clinical importance and reference strain panels. No interfering cross reactivity was found for 17 gram negative, or 13 gram positive species, for related neisseriaceae or two candida species; but functional extension was successfully demonstrated with specific primers for group B streptococci or ESBL bacteria.

P065

Rapid and reliable first-line detection of possible mutation combination in various autoinflammatory disease genes by Pyrosequencing

*F. Häuser1, R. Hassoun1, C. Neukirch1, P. Lohse2, J. Bickmann1, K. Lackner1, H. Rossmann1

1University Medical Center Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany

2Clinic of the Ludwig-Maximilians-University, Munich, Institute of Clinical Chemistry, München, Germany

Objective: Periodic episodes of fever and inflammation might have a genetic origin. Today, in the majority of cases the identification of the causative genetic variants allows molecular genetic confirmation of the clinical diagnosis. This enables approaches with specific drug treatment and improves patient compliance as well as genetic counselling. No fast and cost effective first level testing for a panel of different hereditary periodic fever syndromes (HPF) is available yet. We have established a two-step screening for the molecular diagnostic of HPF with innovative cost effective and rapid methods, complemented with standard procedures of mutation detection.

Methods: We designed a two-step assay-system, the first-line screening bases on published mutation frequencies and Pyrosequencing (PSQ) technology, followed by conventional sequencing of other candidate genes by standard capillary electrophoresis.

Results: The current test system of PSQ assays for the target mutations in MEFV-gene, MVK-gene and TNFRS1A-gene enables the composition of a high sensitive first line ethnically independent screen (sensitivity up to 70%), assumed that the mutation frequencies are known and similar in the studied ethnic groups. The following second level test (complete sequencing of the respective genes) increases the sensitivity of our assay-system and is used if one or no mutations can be found combined with a strong clinical suspicion.

Conclusions: PSQ-based HPF mutation screening is a reliable, convenient, highly flexible, and inexpensive alternative to the few and incomplete conventional methods for first-level testing of HPF, facilitating flexible adaptation of the analyzed mutation panel to the clinical symptoms.

P066

Peptide microarrays as diagnostic tool for cancer associated protease profiling

*E. Dyrcz1, P. Findeisen1

1University Hospital Mannheim, Institute for Clinical Chemistry, Mannheim, Germany

Objectives: Proteases are known to play an important role in progression and metastasis of cancer. Some of them are secreted from the tumor and can be found in the bloodstream. Functional protease profiling aims at discovering tumor associated protease activity in clinical specimens for diagnostic use. Therefore, synthetic reporter peptides, which are exclusively cleaved by tumor associated proteases, are added to the blood specimens ex vivo and respective cleavage is monitored. In order to identify relevant reporter peptides we established a new array-based peptide assay for screening of high complex peptide libraries.

Methods: The on-chip synthesis of the peptides was directly performed on coated glass-slides using a proprietary laser printing technique. The peptides were then labeled with a fluorescent dye for further enzymatic digestion and final readout. In parallel real-time digestion experiments and readout were performed.

Results: The optimization of the assay platform comprised the improvement of fluorochrome labeling efficiency-, and background to noise ratio. Furthermore various linkers were tested to minimize sterical hindrance. For fluorochrome labeling TAMRA-dyes showed the most promising efficiencies so far. With trypsin as model protease the real-time measurements revealed, that the slope for fluorescent decay for most of the reporter peptides had a maximum within the first hour.

Conclusion: First results of the real-time experiments showed promising data that lead to the conclusion that peptide microarrays might be a useful tool in protease profiling. This technology might improve the laboratory based diagnosis of malignant disease.

P067

Glass chip development for microarray measurements of antiphospholipid antibodies using reflectometric interference spectroscopy

*A. Schindler1, P. Luppa1

1Klinikum rechts der Isar, Institut für Klinische Chemie, München, Germany

Antiphospholipid syndrome (APS) is an autoimmune disease that results in thrombophilia and causes recurrent fetal loss. Antiphospholipid antibodies (aPL) target anionic phospholipids, mainly cardiolipin, phospholipid-binding plasma proteins, such as β2-glycoprotein I (β2-GPI) or prothrombin (PT) and phospholipid-protein complexes. Using reflectometric interference spectroscopy (RIfS) a novel, label-free microarray shall be developed to record the interactions of aPL with APS antigens in serum.

The APS antigen β2-GPI was immobilized by applying six different strategies. First, glass chips were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS), PEG and AMD respectively. Subsequently, the antigen was immobilized either directly to the modified surfaces or as biotinylated variant to a covalently bound streptavidin moiety. To compare selectivity and sensitivity of those diverse surfaces, sera of APS patients and healthy controls were analyzed. We designed a chip surface that allows immobilization of β2-GPI without compromising its antigenicity. Measurements with APS patient sera showed a good sensitivity and selectivity for the detection of anti-β2-GPI antibodies with the β2-GPI directly coupled to 11-AUTMS. In contrast, the PEG-β2-GPI surface provided only for moderate discrimination of APS patients and healthy controls. All three streptavidin-biotin immobilization variants did not allow the detection of aPL. β2-GPI immobilized on 11-AUTMS allowed for a clear differentiation between aPL positive sera and healthy controls. Thus, microarray chips with PT, annexin V, cardiolipin, phosphatidylserine and phosphatidylethanolamine are now under development.

P068

Diagnostic Potential of Combined Analysis of Anti-PLA2R Antibodies with HLA-DQA1/ PLA2R Genotyping in Diagnosis of Idiopathic Membranous Nephropathy

*P. Eichhorn1, U. Schoenermarck2, M. Fischereder2, T. Sitter2, V. Vielhauer2, M. Weiss3, W. Wilfert1, L. Hempel2, E. Hirsch2, D. Teupser1, M. Bruegel1, L. Holdt1

1Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany

2Med. Klinik Polyklinik IV, Nephrologisches Zentrum, Ludwig-Maximilians-University Munich, Munich, Germany

3Department of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany

Objectives: Idiopathic membranous nephropathy (IMN) is the most common diagnosis in adults with nephrotic syndrome. Recently, 2 single nucleotide polymorphisms in HLA-DQA1 (rs2187668) and PLA2R (rs4664308) were associated with prevalence of IMN. The aim of the current study was to investigate a diagnostic potential of genetic testing in IMN.

Patients and Methods: SNPs were genotyped with a high-throughput melting-curve assay in DNA isolated from serum of patients with bioptically investigated renal disease (RD). IMN associated serum phospholipase A(2) receptor antibodies (anti-PLA2R) were analyzed by indirect immunofluorescence.

Results: Until now, 131 serum samples from patients with (I) exclusion of RD (n=14), (II) non-immunogenic RD, anti-PLA2R negative (n=65), (III) immunogenic RD, anti-PLA2R negative (n=12), (IV) IMN, anti-PLA2R negative (n=22), (V) IMN, anti-PLA2R positive (n=18) were analyzed. 14% of group I (n=2) and II (n=9) patients were carriers of risk alleles at rs2187668 and rs4664308. One patient of group III (8%) revealed carrier status. In contrast, risk alleles were detected in 32% of group IV (n=7) and 44% of group V (n=8) patients.

Conclusion: Patients with bioptically proven IMN showed significantly higher prevalence of HLA-DQA1/PLA2R risk alleles compared to control groups irrespecitve of presence of anti-PLA2R antibodies at time of diagnosis. Thus, genotyping of DQA1/PLA2R might have additional diagnostic value in IMN.

Neue Biomarker in Metabolomics und Lipidomics Poster I

P069

Supplementation of folic acid (500 µg/d) over 1 year lowers homocysteine and increases serum and whole blood 5-methyltetrahydrofolate

*S. Kirsch1, W. Herrmann1, V. Kruse1, R. Eckert2, S. Gräber3, J. Geisel1, R. Obeid1

1Saarland University Hospital, Clinical Chemistry and Laboratory Medicine - Central Laboratory, Homburg/Saar, Germany

2Marienkrankenhaus, Geriatric Rehabilitation Center, St. Wendel, Germany

3Saarland University Hospital, Department of Medical Biometry, Epidemiology, and Medical Informatics, Homburg, Germany

Objectives: Folate deficiency and elevated total homocysteine (tHcy) are risk factors for age-related diseases. We aimed to investigate the effect of supplementation with 500 µg/d folic acid (FA) over 1 year on serum and whole blood (WB) folate forms in older Germans in a randomized double-blind trial.

Methods: Older German adults (>50 years) were divided into two groups to receive either FA (500 µg), vitamin B12 (500 µg), vitamin B6 (50 mg), vitamin D3 (1,200 IU), and CaCO3 (456 mg) (group A, n=31) or vitamin D3 and CaCO3 (group B, n=28). Folate forms in fasting serum and WB were determined at baseline, after 6 months, and after 1 year using ultra-performance liquid chromatography tandem mass spectrometry.

Results: Mean 5-methyltetrahydrofolate levels were significantly increased in serum (baseline: 16.4; 6 months: 46.7; 1 year: 41.9 nmol/L) and WB (baseline: 481; 6 months: 1,270; 1 year: 1,169 nmol/L) after supplementation. A steady state was not reached after 6 months. The incidence of unmetabolized FA increased after 6 months of FA intake but remained stable thereafter. Elevated tHcy levels at baseline (mean 11.8 µmol/L) were normalized after supplementation with B vitamins for 6 months (mean 9.6 µmol/L) and remained low after 1 year (mean 8.9 µmol/L).

Conclusion: The dosage used in this study was similar to the daily intake under fortification. 500 µg FA/d increased serum and WB folate and lowered tHcy. The steady state in WB was not reached within 6 months.

P070

Interactions of mast cell tryptases with protease-activated receptors

*L.-A. Fratila1, M. Grundhuber1, E. Falk1, D. Teupser2, C. Sommerhoff1

1Institut für Laboratoriumsmedizin, AG Klinische Chemie / Proteolyse, München, Germany

2Universitätsklinikum der LMU, Institut für Klinische Chemie, München, Germany

Objectives: Protease-activated receptors (PARs) are G protein-coupled receptors that play an important role in inflammatory disorders of the cardiovascular, respiratory and central nervous systems. The four known PARs are activated by trypsin-like serine proteases such as thrombin (PAR1, 3, 4) or trypsin and mast cell tryptase ß (PAR2) that cleave the N-terminal extracellular receptor domain and unmask a tethered ligand. Whether any of the other tryptases expressed by mast cells (i.e. a, ?, and d) can activate PAR1 or 2 has not been studied so far.

Methods: To determine whether tryptases activate PARs we have used a model system of human keratinocytes (HaCaT) that express PAR1 and PAR2 in a ratio of approx. 1:3. Activation of cells was determined by following the change of intracellular Ca2+ levels using FURA-2 and FLUO-4.

Results: As expected thrombin and trypsin as well as synthetic peptides that mimic the tethered ligand domains of PARs activate PAR1 and PAR2 in a dose-dependent manner. Compared to tryptase ß that activates PAR2 in the nanomolar range all other tryptases are only weak agonists or inactive in this model system.

Conclusion: Based on these in vitro-studies tryptase ß likely is the predominant protease responsible for the PAR2-activation seen after mast cell degranulation in vivo.

Supported by DFG Priority Program 1394 “Mast Cells – Promoters of Health and Modulators of Disease”

P071

Comparative Characterization of Human Mast Cell Tryptases

*M. Grundhuber1, L. Fratila1, S. Simon1, D. Teupser2, C. Sommerhoff1

1Universitätsklinikum der LMU, Institut für Klinische Chemie, München, Germany

2Klinikum der Ludwig-Maximilians-Universität, Institut für Laboratoriumsmedizin, Munich, Germany

Objectives: Tryptases are mast cell-specific proteases that are thought to be involved in innate and adaptive immune responses as well as disorders such as allergy and asthma. Among the four human tryptases (a - d) so far only the closely related tryptases a and ß have been characterized. The pathophysiological functions of tryptases ? and d, however, and their usefulness as biomarkers of mast cell activation remain to be determined.

Methods: For structural and functional comparisons we have expressed tryptase a, ß, d and soluble forms of ? as well as their zymogens in E. coli and Pichia pastoris. If necessary, mutations were introduced to increase the solubility and yields of the recombinant proteins.

Results: Functional studies showed that tryptase ß and ? are enzymatically active with trypsin-like substrate specificity whereas a and d are virtually inactive. The active tryptases differ, however, in the pH optimum, substrate and inhibitor profiles and quaternary structure. To further localize and quantify all tryptases polyclonal antisera are currently raised that may discriminate between the four proteases.

Conclusion: These results suggest distinct but overlapping functions of the active tryptases.

Supported by DFG Priority Program 1394 “Mast Cells – Promoters of Health and Modulators of Disease”

P072

Eicosanoid quantitation with a multi-targeted LC/sMRM method in stimulated human blood culture

*T. Vielhaber1, Y. Schober1, H. Renz1, W. Nockher1

1Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps University Marburg, Marburg, Germany

Objectives: Eicosanoids such as leukotriens, prostaglandins, resolvines and protectins, are part of the complex, multi-layered signalling network of the immune system and prone to act as pro- or anti-inflammatory lipid mediators. However, differential synthesis of these mediators may vary inter-individually and is thought to influence the immune response. TruCulture® Culture System (Myriad RBM) employs a single self-contained tube for blood collection and culture. In multi-centre studies, it simplifies patient specific blood cell culture and can be easily applied in standardized work-flows. This system was used in an initial study (n=10) to compare the kinetic of stimulated eicosanoid release.

Methods: Blood samples were collected into TruCulture® tubes containing lipopolysaccharide, zymosan or aCD3-/aCD28-antibodies as different stimuli for granulocytes and/or lymphocytes. After incubation for defined time periods, the supernatant was separated from the cells. Before analysis, analytes were concentrated with solid phase extraction and separated from contaminants. Quantitative analysis was carried out by liquid chromatography coupled to tandem mass spectrometry utilizing scheduled multiple reaction monitoring (LC/sMRM).

Results: LC/sMRM has been established for identification and quantitation of eicosanoids. The kinetic of mediator release during 48 h culture varied between different eicosanoids. Due to the cascade of enzyme activities and subsequent metabolite formation, samples should be cultured for 4 h and 48 h in TruCulture® tubes.

Conclusion: A robust LC/sMRM method was established to analyze over 130 lipid mediators, mostly eicosanoids, metabolized in stimulated blood.

P073

Metabolite profiles in sepsis: developing prognostic tools based on the type of infection

*S. Neugebauer1, E. Giamarellos-Bourboulis2, A. Pelekanou2, A. Marioli3, F. Baziaka2, I. Tsangaris4, M. Bauer5, M. Kiehntopf1

1Universitätsklinikum Jena, Institut für klinische Chemie und Laboratoriumsdiagnostik, Jena, Germany

2University of Athens, Medical School, 4th Department of Internal Medicine, Athens, Greece

3Sismanogleion General Hospital, 2nd Department of Internal Medicine, Maroussi, Greece

4University of Athens, Medical School, 2nd Department of Critical Care Medicine, Athens, Greece

5Department of Anesthesiology and Intensive Care, Jena University Hospital, Jena, Germany

Objectives: Currently used biomarkers cannot safely discriminate between patients with SIRS (systemic inflammatory response syndrome) of non-infectious origin and sepsis. The aim of the study was to identify surrogate markers for sepsis taking into account the underlying type of infection using a targeted metabolomics approach.

Methods: A total of 186 metabolites comprising six analyte classes were determined via LC-MS/MS in 372 patients. Patients were divided into one test cohort (n= 248) and into one confirmation cohort (n= 124) that run in parallel at different hospitals.

Results: Serum concentrations of most metabolites were significantly decreased in sepsis compared to SIRS. A classification model comprising five markers resulted in 75.9%, 95.5% and 99.4% sensitivity, specificity and positive predictive value, respectively. Changes of the metabolites between sepsis and severe sepsis/septic shock varied greatly according to the underlying type of infection. This analysis allowed identification of metabolites predictive for unfavorable outcome in community-acquired pneumonia; intraabdominal infections; and primary Gram-negative bacteremia.

Conclusion: Starting from a metabolomics approach, single metabolites are depicted to develop a score for sepsis diagnosis and prognosis taking into consideration the underlying infection.

Onkologie Poster I

P074

Methylation of the Plasminogen Activator Inhibitor 1 Gene in Serum DNA as a Potential Biomarker for Prostate Cancer

B. Nacke1, A. Hagelgans1, S. Fuessel2, M. Wirth2, G. Siegert1, *M. Menschikowski1

1Uniklinikum Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Department of Urology, Dresden, Germany

Objective: Epigenetic modifications such as DNA methylation are common in malignant tissues. Here we report the analysis of plasminogen activator inhibitor 1 (PAI1) gene methylation in patients with prostate cancer.

Methods: The degree of PAI1 methylation in prostate tissues and free circulating serum DNA was analyzed using methylation sensitive high resolution melt analysis after bisulfite modification of DNA and methylation sensitive restriction enzyme based quantitative PCR (MSRE-qPCR) without previous bisulfite modification, respectively.

Results: A hypermethylation of the PAI1 gene was observed in malignant tissue samples of 20 prostate cancer patients with bone and lymph node metastases when compared to those of non-malignant adjacent tissues (20.0±2.1% versus 4.3±1.0%, p<0.001). On the basis of this finding, isolated DNA from serum of 29 prostate cancer patients and 22 healthy individuals was analyzed using MSRE-qPCR. Median age of prostate cancer patients was 75 years (range 51-99 years) and tumor stages varied between 2 and 4. The methylation of the PAI1 gene averaged 13.3% in cancer patients and 2.4% in the group of healthy individuals. In comparison, the methylations of the glutathione-s-transferase (GSTP1) gene were 57.0% and 5.9% in the cancer and control groups, respectively. A combination of PAI1 and GSTP1 methylations as serum markers resulted in a better diagnostic performance with a sensitivity of 90% and a specificity of 96% than the single markers.

Conclusions: The study shows that the PAI1 gene is hypermethylated in malignant prostate tissues and that the measurement of the methylation degree in serum samples may have potential in the diagnosis of prostate cancer.

P075

Detection of monoclonal gammopathies by HIL measurements of clinical chemistry analysers: a retrospective and a prospective study

*A. Grundt1, S. Schneider1, P. Findeisen1, M. Neumaier1

1Uniklinikum Mannheim, Institut für Klinische Chemie, Mannheim, Deutschland

Objectives: Monoclonal gammopathies (mG) are caused by proliferation of a single B-cell or plasma cell secreting immunoglobulin and, in the case of malignant disease are accompanied by cellular infiltration of lymphatic nodes, spleen, bone marrow or blood. Patients with mG are usually identified in late stages of disease in the course of clarification of abnormalities in total protein and/or albumin concentrations followed by serum electrophoresis, immunofixation and quantification of the paraproteins.

Methods: As mG are known to influence the spectral-photometric characteristics of serum or plasma we investigated in a retrospective study 636 samples with immunofixation result and in a prospective study 60,000 random patients the HIL testing as a pre-screening tool for the detection of mG.

Results: The Retrospective analysis of non-lipemic patient samples with known serum electrophoresis and immunofixation results show that false-positive lipemia-flags indicate mG with a diagnostic sensitivity of 25%. The highest sensitivity is found for IgM mG (56%), while IgG mG is detected in only 12% of cases. The prospective study of 60,000 patients demonstrates the high specificity of the assay which was calculated to be of 99,8% for the detection of mG and 99,8% for the detection of IgM mG and IgG mG.

Conclusions: Our data demonstrate that spectrophotometric pre-screening of serum samples is a powerful tool for the early detection of monoclonal gammopathies, especially of IgM mG with an PPV 33,6% and a NPV of 99,9%.

P076

Dihydrouracil/uracil ratio variability in healthy and cancer patient populations in relation to genetic variation in the dihydropyrimidine dehydrogenase

*B. Büchel1, D. Kummer1, T. Froehlich1, J. Sistonen1, C. Largiadèr1

1Universitätsinstitut für Klinische Chemie / Inselspital Bern, Bern, Switzerland

Objectives: The use of 5-fluorouracil (5FU) to treat solid cancers is compromised by 10-15% of patients developing severe toxicity. While it has been estimated that 50-75% of the 5FU toxicities are caused by reduced 5FU catabolism mediated by the dihydropyrimidine dehydrogenase (DPD), only a third of these toxicities can be explained by variation in the DPD gene (DPYD). Since the endogenous DPD product-substrate ratio (i.e. dihydrouracil/uracil, UH2/U) has been proposed as a surrogate marker for DPD activity, we aimed to characterize the ratio with respect to DPYD genotype in healthy and cancer patient populations and to evaluate its potential to predict 5-FU toxicity.

Methods: Samples were collected from 320 healthy volunteers and 28 cancer patients and genotyped for DPYD variants. Plasma U and UH2 concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: Although DPYD risk variant (c.1129-5923C>G, c.1679T>G, c.1905+1G>A, or c.2846A>T) carriers showed lower UH2/U ratios compared to wild-type carriers (P = 0.005), there was a large overlap in the values. The UH2/U ratio was strongly dependent on the U or the 5FU levels with most pronounced differences between DPYD genotypes observed at high substrate levels.

Conclusion: While the baseline UH2/U ratio is a poor predictor of 5FU toxicity, the predictive power of a loading test (i.e. administering the natural DPD substrate to patients prior to the therapy) should be further evaluated.

P077

Immunoglobulin subtypes: A supplement to M-protein quantification by serum protein electrophoresis (SPEP)

*J. Freund1, C. Röllig2, S. Richter2, B. Arneth1, U. Platzbecker2, G. Siegert1, H. Kostka1

1Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Medizinische Klinik und Poliklinik I, Dresden, Germany

Objectives: To monitor the therapy for multiple myeloma (MM), the monoclonal protein (M-protein) has been determined by SPEP quantification. The M-protein of IgA-type often shifts into the beta-fraction, and the definition of an M-gradient becomes difficult to establish. Further, SPEP is only a semi-quantitative method. Here, we investigated whether the turbidimetric determination of the IgA kappa (IgAK) and IgA lambda (IgAL) subtypes reflects the proportion of monoclonal IgA in a patient with a definable M-gradient in SPEP.

Methods: During therapy (July 2011 to October 2012) of a female patient with MM (IgA lambda-type) immunofixation (IFE), SPEP (Hydrasys, Sebia, Lisses, France), IgAK and IgAL (Hevylite, SPAplus, The Binding Site GmbH, Schwetzingen, Germany) were examined in parallel (n=18). The monoclonal portion of IgA lambda-type was calculated mIgAL=IgAL-(IgAK/1.18) and compared with the M-protein (SPEP).

Results: The M-protein (SPEP) in this patient was clear defined and ranged from 0.8-37.1 g/L. In four samples no M-protein was detected. Two samples were negative and two samples were positive by IFE. The IgAK/IgAL ratios in these four samples were in the normal range. The M-protein correlated with mIgAL in this patient during the entire treatment period mIgAL=1.31 x (M-protein) + 0.301, r2=0.997.

Conclusion: The determination of immunoglobulin subtypes is an alternative to the quantification of M-protein, especially for M-proteins with beta-mobility by SPEP.

P078

Co-expression of Hypoxia-inducible factors (HIF) in patients with circulating tumor cells in colorectal cancer

*D. Ernst1, A. Hauburger2, J. Boese-Landgraf3

1Klinikum Chemnitz gGmbH, Klinik für Allgemein- u. Viszeralchirurgie, Chemnitz, Deutschland

2Zentrum für Diagnostik GmbH am Klinikum Chemnitz, Molekularbiologie, Chemnitz, Deutschland

3Klinikum Chemnitz gGmbH, Klinik für Allgemein- u. Viszeralchirurgie, Chemnitz, Deutschland

Objectives: Analysis of circulating tumor cells (CTC) is a promising tool in oncology. The aim of this study was to evaluate the co-expression of Hypoxia-inducible factors (HIF) in the presence of CTC in peripheral blood of Patients with colorectal cancer (CRC) before treatment.

Methods: CTCs were evaluated in 49 CRC patients. After immunomagnetic enrichment using the antibodies BM7 (targeting mucin1) und VU1D9 (targeting epithelial cell adhesion molecule) CTCs were characterized by real-time RT-PCR of the marker genes KRT19, MUC1, EPCAM, CEACAM5 BIRC5. In addition marker genes of HIF were analyzed.

Results: CTC analysis revealed that 55% of CRC patients were CTC positive before treatment. After stratification into groups positive for CTCs (at least one marker gene was positive) and negative for CTC detection, the positive group showed a significant higher co-expression of HIF (40% vs.18%).

Conclusion: As it is known by now the detection of CTCs itself is liked to a poorer outcome. Co-expression of HIF in CRC- patients with detected CTCs is significant higher than in CTC-negative patients. Therefore expression of HIF might not alone be linked to the presence of tumor cells, but might be a general finding in patients suffering from advanced cancer disease. Further studies are required to show to which degree this affects the prognosis of patients with colorectal cancer.

Immunology / Allergologie Poster II

P079

Detection of deletion 13q14 and abberant CD56 expression in plasma cells of a patient with autoimmune hepatitis and lupus erythematosus

S. Tuve1, U. Sommer2, H. Kostka3, B. Mohr1, *U. Oelschlägel1, J. Middeke1, M. Berning1, J. Fantana4, A. Erler4, M. Aringer4, M. Bornhäuser1, J. Babatz1

1University Hospital Dresden, Medical Clinic I, Dresden, Germany

2University Hospital Dresden, Institute of Pathology, Dresden, Germany

3University Hospital Dresden, Institute of Laboratory Medicine, Dresden, Germany

4University Hospital Dresden, Medical Clinic III, Dresden, Germany

An important mechanism of autoimmunity is the evolvement of autoreactive plasma cell clones which produce autoantibodies. The molecular basis of this process is poorly understood. Here we report the first evidence that genomic changes and abberant surface marker expression may occur in autoreactive plasma cell clones. We show in a patient with newly diagnosed autoimmune hepatitis and lupus erythematosus the presence of a plasma cell subset with deletion 13q14 and abberant CD56 expression in the bone marrow in the absence of a monoclonal proliferative plasma cell disorder. Deletion 13q14 has been shown to enable proliferation due to loss of MIR15A/MIR16A microRNA expression in malignant plasma cell disorders. The plasma cells of this patient showed polyclonal antibody production, in particular of anti-dsANA and anti-LKM antibodies. No evidence for monoclonal immunoglobulins production was detected using serum immunofixation electrophoresis and high sensitive subdifferentiation of immunglobulins according to kappa and lamda-restriction (HevyLite™assay). Immunosuppressive treatment with prednisolone, azathioprine and quensyl resulted in reduction of plasma cell frequency, suppression of autoantibody production as well as complete clinical remission in this patient.

P080

Inflammasome activators induce the secretion of IL-1a via two distinct pathways displaying differential requirement for a protease activity-independent function of Caspase-1

*O. Groß1, C. Thomas1, J. Tschopp2

1Klinikum rechts der Isar, Institut für Klinische Chemie und Pathobiochemie, München, Germany

2University of Lausanne, Department of Biochemistry, Epalinges, Switzerland

Through their capacity to sense danger signals and to generate active IL-1β, inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1β, little is known about the regulation of IL-1α activity. Unexpectedly, we find that inflammasome activators also induce the regulated secretion of IL-1α, thus leading to the co-secretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α is inflammasome/caspase-1-dependent or –independent. Unlike for IL-1β, Calcium influx, induced by the opening of cation channels such as TRPV2, suffices for the inflammasome-independent IL-1α activation. In both cases IL-1α is released primarily in a processed form, caused by a calpain-like protease. Remarkably, caspase-1-dependent release of IL-1α and IL-1β is independent of caspase-1 catalytic activity, defining a novel function for caspase-1. Considering the involvement of inflammasomes in chronic inflammatory diseases such as gout and diabetes, the use of IL-1α antagonists may be beneficial in the treatment of these disorders.

P081

Inhibition of complement-dependent phagocytosis by Del-1

*I. Mitroulis1, G. Siegert2, E. Choi3, T. Chavakis4

1University of Dresden, Department of medicine and institute for clinical chemistry and laboratory medicine, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

3University of Ulsan college of Medicine, Ulsan, Democratic People′s Republic of Korea

4Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

Objectives: Developmental endothelial locus-1 (Del-1) has been recently identified as a negative regulator of inflammatory cell recruitment, through interactions with the β2-integrin LFA-1 (1). Herein, we study the effect of Del-1 in complement-driven phagocytosis via protein interactions with another β2-integrin family member, Mac-1.

Methods: Binding of Del-1 to Mac-1 and LFA-1 was studied using a solid-phase binding assay. Adhesion of Chinese hamster ovary (CHO) cells stably transfected with aM and b2 chains (aMb2=Mac-1= complement receptor-3) to immobilized Del-1 was studied using a static adhesion assay. Phagocytosis of complement opsonised sheep RBCs by RAW 264.7 cells was used to investigate the effect of Del-1 in Mac-1-dependent phagocytosis.

Results: Del-1 bound not only to LFA-1 but also to Mac-1, as assessed in the solid-phase binding assay. Additionally, increased adhesion of CHO cells expressing Mac-1 to Del-1 compared to non-transfected cells was demonstrated. Finally, Del-1 significantly diminished Mac-1 mediated phagocytosis of complement-opsonized RBC by RAW 264.7 cells.

Conclusion: Del-1 may regulate complement-driven phagocytosis. We propose that direct protein interactions between Del-1 and the β2-integrin Mac-1 are involved in this effect.

1. Choi EY et al, Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science. 2008;322:1101-4

P082

Cellular Immune Surveillance in Human Central Nervous System: Revealed in Cerebrospinal Fluid (CSF) with the Marburg CSF Model

*T. Kleine1

1Universitätsklinikum Giessen und Marburg Standort Marburg, Institut f. Laboratoriumsmedizin u. Pathobiochemie, Molekulare Diagnostik, Referenzlabor, Marburg, Germany

Objectives: In mice immune surveillance is carried out with sensitised (activated) T lymphocytes (RM Zinkernagel, PC Doherty: Nature 1974; 248:701-702; 251:547-548). Question: In humans do HLA-DR+ activated T lymphocytes - independently of their specific activation - pass from blood into CSF under physiological conditions?

Methods: CD3+, CD4+, CD8+ lymphocytes, natural killer cells, B lymphocytes are detected with specific fluorescent antibodies by flow cytometry in CSF, blood, lymph of human controls.

Results: In lumbar CSF ca. 90% of T lymphocytes are non-activated, refluxed with ca. 0.3 ml thoracic duct lymph; ca. 10% represent HLA-DR+ activated T, not detected in duct lymph. For blood cells blood-brain barrier is bolt with large tight junctions in brain capillaries; tight junctions + basement membrane lock blood-CSF barrier in choroid plexus epithelium. Only in circumventricular organ CVO, capillaries are fenestrated in area postrema, organum vasculosum of lamina terminalis, subfornical organ: Here, leucocytes of ca. 1 µl blood are pumped with blood pressure into ventricular CSF to produce suboccipital (SOP) CSF with ca. 10% activated blood lymphocytes; SOP CSF flows into lumbar and arachnoidal CSF.

Conclusion: In human brain, immune surveillance is performed via CSF with HLA-DR+ activated blood T lymphocytes which enter from blood through CVO capillaries into ventricular CSF that flows into external CSF spaces; from CSF activated T cells can migrate through the brain.

P083

Cytokine gene-expression in a co-cultivation of monocytes and endothelial cells in response to Streptococcus gallolyticus subsp. gallolyticus

*M. Weinstock1, I. Grimm1, J. Dreier1, C. Knabbe1, T. Vollmer1

1Herz- und Diabeteszentrum Nordrhein-Westfalen, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Introduction: Streptococcus gallolyticus (SGG) is the causative agent in about 20% of streptococcal caused infectious endocarditis (IE) cases. In this study, a co-cultivation model of monocyte and endothelial cells was analyzed to elucidate the inflammation in SGG-IE.

Methods: Co-cultivation of monocyte THP-1 and endothelial EA.hy926 cells was performed in two models: a) direct cell-contact b) separation of cell types in compartments. Cells were also cultivated in mono-culture. All culture models were inoculated with SGG (2 h, 6 h) and gene expression of cytokines was relative quantified by real-time PCR.

Results: In contrast to endothelial cells, the monocyte mono-culture demonstrates the stimulation potential of SGG. The co-cultivation in one compartment with cell-contact revealed an increase in IL-1β gene expressions compared to mono-cultures in case of one Isolate (e.g. 6 h: THP-1: 2.9, EA.hy926: 2.2; co-culture: 11.2). Co-culture in separated compartments in general showed gene expressions comparable to mono-cultured cells. However an increase in endothelial IL-8 gene expression compared to the mono-culture was demonstrated.

Conclusions: This model provides insights into the importance of cell-cell-interaction to defend or enable the development of SGG-IE. Further work will focus on the characterization of the direct interaction between monocytes and endothelial cells. This work was supported by Stiftung für Pathobiochemie und Molekulare Diagnostik of the DGKL.

P084

Lower Motor Neuron Disease Caused by Biclonal IgM-Paraproteinemia (GM1-IgM and GD1B-IgM Antibodies)

*L. Klingelhöfer1, H. Kostka2, K. Conrad3, U. Schuler4, H. Reichmann1, J. Schäfer1

1Technical University Dresden, Department of Neurology, Dresden, Deutschland

2Uniklinikum an der TUD, Institut für Klinische Chemie und Labormedizin, Dresden, Germany

3Institut für Immunologie, Medizinische Fakultät "Carl Gustav Carus" der Technischen Universität Dresden, Dresden, Germany

4Technical University Dresden, Department of Internal Medicine I, Dresden, Germany

Objectives: Polyneuropathies can be caused by paraproteinemia and may be associated with anti-ganglioside antibodies. In contrast, a paraproteinemic or paraneoplastic cause of motor neuron disease (MND) is discussed controversially. Increased titers of antibodies and monoclonal gammopathies associated with MND have been described previously, but their origin has not been elucidated.

Case history: The patient, a 59 year old man, has a two year history of progressive, right and arm accentuated motor tetraparesis (flail arm syndrome) with severe muscle atrophy, areflexia and functional disability. Neurophysiological and imaging investigations indicated damage of the lower motor neuron and no conduction blocks. A biclonal IgM-gammopathy was detected. The IgM-peaks were identified as massively elevated anti-GM1-IgM (1:100.000) and anti-GD1B-IgM antibodies (>1:1 million). A paraneoplastic cause was excluded. In line with the established therapy of multifocal motor neuropathy we prescribed IVIG`s which had a positive effect on muscle power and resulted in decreased levels of anti-GM1-IgM (1:1.000) and anti-GD1B-IgM (1:10.000). We therefore escalated the therapy to Rituximab.

Conclusion: The positive effects of IVIG support a paraproteinemic etiology of the lower motor neuron disease in this patient. To our knowledge, this is the first report of a paraproteinemic etiology of lower motor neuron disease caused by extremely high titers of neuronal specific ganglioside antibodies.

P085

Optimization of laboratory workflow for measurement of mHLA-DR on monocytes

*O. Frey1, M. Kiehntopf1

1Institut für Klinische Chemie und Laboratoriumsdiagnostik, Universitätsklinikum Jena, Jena, Deutschland

Objectives: Quantification of monocytic HLA-DR (mHLA-DR) expression by flow cytometry is commonly used for immunomonitoring in sepsis. However, it is well known that HLA-DR expression is stable only for less than four hours. The aim of our work was to establish a workflow that allows HLA-DR quantification at 7/24 beyond the normal working hours of the flow cytometry laboratory.

Methods: mHLA-DR was measured using flow cytometry. We compared different sample stabilization tubes and protocols for sample preparation.

Results: Sample stabilization using stabilizing fixatives reduced mHLA-DR staining. This effect was not reversible, since several washing steps could not restore the fluorescence signal. However, in samples treated with the stabilizing fixative mHLA-DR expression remained constant for three days. As an alternative approach we tested the stability of freshly stained samples that were subsequently fixed using an automated sample preparation system. This approach yielded reasonable data, showing a stabile mHLA-DR signal for up to 48 hours.

Conclusion: While the use of stabilizing fixatives might be useful in clinical studies, it cannot be used for routine purposes. Immediate staining of the samples using an automated system and subsequent storage until measurement is a possible way to overcome the low stability of mHLA-DR expression and to make this parameter feasible for routine diagnostics.

P086

HBS AG, ANTI HCV, ANTI HIV, TPHA AT THE BLOOD DONORS IN KOSOVO

*S. Tahiraj-Lokaj1

1University Clinical Centre of Kosovo, Medical Biochemistry, Pristina, Kosovo

Objectives: Blood donation besides saving lives, under special circumstances might be fatal to a patient.Many infectious diseases can be transmitted through blood transfusion viral, bacterial or parasitic diseases. The determining of these parameters is obligatory in blood donors.

Methods: Samples were analyzed via Elisa method on the Abott Tecan, Abott Axsym apparatus.

Results: In the period 2001– 2009, were tested 114092 blood samples of blood donors, from which 113638 males, and 454 females, of the age range 18 to 60, of different nationalities and social structures. HBsAg positive donors (twice confirmed) were 3469, namely 3.04%. From them 3066 were male (2.69%) and 403 female (0.35%). 2001 – 3.93%, 2002 – 5.41%, 2003 – 3.40%; 2004 – 2.94%, 2005 – 2.77%, 2006 – 2.64%; 2007 – 2.18%, 2008 – 2.06%, 2009 – 2.07%. Anti HCV positive donors were 233, namely 0.20%. From them 19 were male (0.17%) and 42 females (0.03%). TPHA positive were 78, namely 0.07%. From them 64 were male (0.06%) and 14 female (0.01%). Anti HIV positive was only 1 case in ’02, female. Statistical methods, T-Test have depicted differences in transmissible diseases incidence between these years and based on trend measurements it seems that the presence of HBSAg, HVC, TPHA, HIV positive has a reducing tendency.

Conclusion: In the early post-war years, the number of patients with positive markers in Kosovo was high, while in the last years it has dropped significantly and has a reducing trend .All this as a result of people hard living conditions in Kosovo.

P087

Immunogenicity testing of recombinant hGH – methodological challenges and clinical relevance

*M. Schaab1, J. Thiery1, J. Kratzsch1

1Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

Objectives: Substitution with rhGH is used to treat growth hormone deficiency (GHD). During treatment anti-drug antibodies (ADAs) can be induced and may exert neutralizing activity thereby reducing treatment efficacy. Therefore, determination and in depth characterisation of ADAs could be of clinical importance in GHD treatment. We here present pitfalls of rhGH immunogenicity testing and discuss the clinical relevance of anti-rhGH antibody determinations.

Methods: In a rhGH treated group of pediatric and adult patients with clinically suspected GH-insensitivity, ADAs were assessed and further characterised by titer determinations and a neutralization assay. Anti-rhGH antibodies were determined by an in-house RIA (Radio Immunoprecipitation Assay) or ECLIA (ElectroChemiLuminescence ImmunoAssay). The neutralizing potential of ADAs was investigated with a cell-based bioassay.

Results: In pediatric patients with GHD anti-hGH antibodies apparently disrupt the circadian secretion pattern of endogenous hGH through complex formation. Furthermore high binding antibody activity often went along with an increased neutralizing capacity. Interestingly, in a 56 year old male patient we could still detect a low titer of anti-hGH antibodies 50 years after the last treatment which was boosted due to a resumption of rhGH therapy.

Conclusion: ADA determination and characterisation is helpful to exclude antibody-related causes of reduced growth velocity during rhGH treatment and can be supportive for a clinician’s decision to change treatment regime.

Endokrinologie Poster II

P088

An intact MyD88 expression in inflammatory but not in adrenal steroid-producing cells is crucial for LPS-mediated adrenal inflammation and the HPA axis activation.

*W. Kanczkowski1, U. Lehnert1, S. Grossklaus2, T. Chavakis2, S. Bornstein1

1Technical University Dresden, Department of Internal Medicine III, Dresden, Germany

2Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav C, Department of Internal Medicine III, Dresden, Germany

Background: Inflammation in the course of systemic inflammatory response syndrome (SIRS) or sepsis often results in dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis; however, the underlying mechanisms are not well understood. In the present study, we studied whether an intact TLR-signaling in adrenocortical cells or in myeloid cells is crucial for the regulation of adrenal gland (dys-)function during LPS-mediated systemic inflammation.

Material and Methods: Mice with myeloid-differentiation factor 88 “MyD88”-deficiency either in adrenocortical cells (Akr1b7-Cre-MyD88-flox/flox) or in hematopoietic cells (MX1-Cre MyD88 flox/flox) were generated. LPS-mediated adrenal gland inflammation (levels of IL1 b, IL6 and TNF cytokines) and the HPA axis activation were assessed using quantitative PCR and commercial ELISA tests, respectively.

Results: Our results demonstrate that systemic administration of LPS strongly induced expression of IL1 beta, IL6 and TNF, locally the adrenal glands and systemically in the plasma. However, LPS-mediated adrenal gland inflammation was significantly reduced in mice with MyD88 deficiency in hematopoietic cells but not in adrenocortical cells, as compared to control animals. Interestingly, reduced inflammation found in MX1-Cre-MyD88-flox/flox animals was associated with decreased activity of the HPA axis, as demonstrated by decreased corticosterone and ACTH levels 3h post LPS administration.

Conclusions: Together, these data suggest that although an intact TLR signaling in the hematopoietic but not adrenocortical cells is crucial in the LPS-mediated adrenal gland inflammation, it is however required for the proper HPA axis regulation.

P089

LC-MS/MS based screening of Pheochromocytoma and Paraganglioma using Plasma and Urinary Metanephrines: Additional Utility of Methoxytyramine

*M. Peitzsch1, A. Prejbisz2, F. Beuschlein3, M. Fassnacht4, A. Januszewicz2, G. Siegert1, G. Eisenhofer1

1University Hospital Carl Gustav Carus at the Technical University Dresden, Institute of Clinical Chemistry and Laboratory Medicine, Dresden, Germany

2Institute of Cardiology, Warsaw, Poland

3Medical Clinic Innenstadt - LMU, München, Germany

4University Hospital Würzburg, Department of Internal Medicine I, Würzburg, Germany

Objective: Comparison of performances of plasma free metanephrines (PFM) with urinary free and deconjugated metanephrines for diagnosis of pheochromocytomas and paragangliomas (PPGLs). Examination of diagnostic utility of additional measurements of plasma and urinary 3-methoxytyramine, the O-methylated metabolite of dopamine.

Methods: Plasma and urine samples from 63 patients with confirmed PPGLs and 323 patients without evidence of PPGLs were biochemically characterized by LC-MS/MS.

Results: ROC curve analysis indicated optimal respective diagnostic sensitivities and specificities of 95% and 97% for PFM, 89% and 98% for urinary free metanephrines, 90% and 97% for urinary deconjugated metanephrines and 87% and 85%, for urinary catecholamines. With introduction of 3-methoxytyramine, sensitivity and specificity respectively increased to 100% and 98% for PFM and to 95% and 98%for urinary free metanephrines. In contrast, no increase in diagnostic performances were observed for deconjugated metabolites upon inclusion of 3-methoxytyramine, nor for urinary catecholamines upon inclusion of dopamine into testing regimes.

Conclusion: Combined measurements of free metanephrines and 3-methoxytyramine in plasma or urine provide potential advantages over traditional measurements of plasma free or urinary deconjugated metanephrines for diagnosis of PPGLs.

P090

Circulating very low density lipoproteins from subjects with impaired glucose tolerance accelerate adrenocortical cortisol and aldosterone synthesis

*S. Kopprasch1, S. Saha1, P. Schwarz1, S. Bergmann2, S. Bornstein1, J. Graessler1

1Carl Gustav Carus Medical School, Department of Internal Medicine 3, Dresden, Germany

2Carl Gustav Carus Medical School, Institute of Clinical Chemistry and Laboratory Medicine, Dresden, Germany

Objective: Glycoxidative modifications of lipoproteins are increasingly being recognized as etiological factor for increased cardiovascular morbidity and mortality in prediabetic individuals. However, the causative relationship between lipoprotein modifications and steroidogenesis in impaired glucose tolerance (IGT) is not well defined. We aimed to investigate the impact of in vivo modified lipoproteins isolated from subjects with normal glucose tolerance (NGT) and IGT individuals on aldosterone and cortisol release from human adrenocortical cells (NCI H295R).

Methods: 20 NGT and 20 IGT subjects were randomly selected from the ongoing Dresden PRAEDIAS prevention study and plasma lipoproteins were isolated individually from each subject. H295R cells were incubated with HDL, LDL (both 100 µg/ml) or VLDL (50 µg/ml) for 24 h. Following incubation, hormone release into supernatants was measured.

Results: Compared to HDL and LDL, VLDL induced greater stimulating effects on adrenocortical hormone release. Moreover, VLDL isolated from IGT subjects evoked significantly higher effects (p<0.05) on hormone release than VLDL from NGT subjects.

Conclusion: In vivo modified VLDL are able to promote prediabetic hormonal dysregulation by enhancing adrenal aldosterone and cortisol release.

P091

The thrombomodulin protein C system modulates mitochondrial function and cardiolipin synthesis during experimental autoimmune encephalomyelitis (EAE)

*J. Wolter1, M. Thati1, K. Shahzad1, F. Bock1, L. Schild1, B. Isermann1

1Otto-von-Guericke-Universität Magdeburg, Institute for Clinical Chemistry and Pathobiochemistry, Magdeburg, Germany

Objectives: ROS (reactive oxygen species) is increased in CNS of EAE mice. Inhibition of ROS and mitochondrial dysfunction reduces EAE severity. The thrombomodulin (TM) - protein C (PC) system ameliorates the disease course. We hypothesized that the anticoagulant and cytoprotective TM-PC modulates EAE through regulation of mitochondrial function and ROS-formation.

Methods: EAE was induced in wild-type (Wt), ThbdP/P (low levels of protein C) and ThbdP/P x hPChigh mice (restored levels of aPC). EAE scores were determined and tissues collected for ex vivo analyses. O2 consumption of mitochondria was analysed by a respirometra. The Cardiolipin (CL) content was determined by HPLC-MS/MS.

Results: In ThbdP/P mice EAE was aggravated as compared to Wt. In ThbdP/P x hPChigh mice the aggravated disease course was partially corrected. Spinal cord analyses demonstrated enhanced myelin loss in ThbdP/P EAE mice as compared to Wt. Restored levels of aPC reduced myelin loss and ROS. Isolated brain mitochondria revealed a decreased respiration rate and a complex II deficiency in Wt EAE mice. ThbdP/P EAE mice showed an additional defect in complex I. The CL content was also decreased in ThbdP/P EAE mice. Depleting the redox-enzyme p66Shc partially reversed the disease course of EAE in ThbdP/Pmice.

Conclusion: These studies strongly suggest that Thbd-dependent PC activation modulates EAE. Further, aPC ameliorates EAE by inhibiting the activity of the redox enzyme p66Shc. These results provide novel insight into the neuroprotective effects of aPC.

P092

Proinsulin Values in Normal Oral Glucose Tolerance Test: Biomarker for Hidden Risk of Prediabetes ?

*S. Bergmann1, P. Schwarz2, J. Graessler2

1TU Dresden Universitätsklinikum, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

2Carl Gustav Carus Medical School, Department of Internal Medicine 3, Dresden, Germany

The aim of the present study was to define reference values for insulin (DiaSorin), C-Peptide (DiaSorin) and Proinsulin (ZenTech) at 0, 60, and 120 minutes of a standard oral glucose tolerance test (75g oGTT). From the ongoing Dresden Prediabetes study (PRAEDIAS) 39 men and 80 women with normal oGTT were included. The inclusion criteria were: age <85 years (mean 68 years) ; HbA1c< 6,1%; fasting glucose < 6.1 and 120min post challenge glucose <7.8 mmol/l, normal CRP as well as normal fasting concentrations of insulin, proinsulin and C-Peptide. Inter 95%-percentiles were [4.25-5.87 mmol/l] (fasting glucose); [2.98-7.31 mmol/l] (120min glucose) and [27-43mmol/mol] (HbA1c). There was no age-dependence for glucose or hormones at baseline. Following inter95-perzentiles were calculated during oGTT: insulin: [0.01-0.12 nmol/l](0min), [0.15-1.85 nmol/l](60min), [0.09-0.82 nmol/l](120min); C-Peptide: [0.39-1.38 nmol/l], [1.89-6.18 nmol/l],[1.52-5.29 nmol/l]; proinsulin: [0.6-5.4pmol/l], [3.0-30.2pmol/l], [4.5-50.8pmol/l]. There was an age-dependence of 120min post challenge values of glucose and C-Peptide. Striking high values of proinsulin were observed in some participants at 60 and 120 min. Proinsulin values were highly correlated with fasting glucose. Significantly higher concentrations of 60- and 120min- proinsulin were observed among participants with fasting glucose in the upper quintile (>5.35 mmol/l) as compared with the lowest quintile (<4.56). Taken together, these data indicate that even among individuals with normal glucose tolerance there is a subgroup of individuals with increased risk for prediabetes as indicated by increased values of proinsulin measured at 60 and 120 min of oGTT.

P093

Performance evaluation of the IGF-I Assay on the LIAISON® XL Analyser

*E. Rauhut1, R. Schlett1, G. Markowitz1, D. Schell1

1DiaSorin Deutschland GmbH, Dietzenbach, Germany

Background: Circulating IGF-I levels are growth hormone dependent and are also impacted by age, gender, nutritional status and disease. The majority of circulating IGF-I is associated with high affinity insulin like growth factor binding proteins (IGFBPs), making accurate and precise measurements of total IGF-I concentrations in biological matrices technically challenging. The aim of the present study was to evaluate the LIAISON® IGF-I Assay on the LIAISON® XL analyzer against the well established LIAISON® platform and to compare the LIAISON® IGF-I Assay with the IDS iSYS IGF-I Assay.

Methods: Comparison: To verify the dose correlation between the two analysers and the IDS iSYS IGF-I Assay a panel of 121 patient samples spanning the assay range was tested. Precision: 11 samples were tested on2 reagent integral lots in two replicates per run, 2 runs per day for 20 operating days for a total of 160 replicate results per sample.

Results: Intra-Assay CVs were in the range 3.7-4.9% and inter-Assay CVs in the range 5,3 – 8,3 %. The dose correlation across the range is:

LIAISON® = 0,998(LIAISON XL) + 0,649 r2= 0,997 LIAISON® = 0,92(iSYS) + 2 r2= 0,9448.

Conclusion: The performance of the LIAISON® IGF-I Assay was demonstrated to be comparable between the two platforms and the IDS iSYS IGF-1 Assay.

P094

Neuroendocrine stress responses to an admission interview: significant sex differences and better non-technical performance in non-responders

*M. Haensel1, M. Huebler2, S. Kern3, S. Bergmann4, K. Weidner1, T. Koch2

1TU Dresden Universitätsklinikum, Psychotherapie und Psychosomatik, Dresden, Germany

2TU Dresden Universitätsklinikum, Anästhesiologie, Dresden, Germany

3TU Dresden Universitätsklinikum, Neurologie MS-Zentrum, Dresden, Germany

4TU Dresden Universitätsklinikum, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

Objectives: Standardized stress alters cognitive performance. This well-known effect is hardly investigated in real-life situations. Our study evaluated the association between individual stress response and the performance in a real-life admission interview.

Methods: 202 young healthy subjects underwent four standardized interviews as selection routine for admission at the Medical Faculty Dresden. Salivary cortisol levels as marker of hypothalamus–pituitary–adrenal activity were measured at six time points: T1, 30 min before the first interview; T2-T5, between the interviews; T6, 30 min after the last interview. Data regarding interview performances and performance in preliminary medical exams were collected.

Results: Cortisol levels at T1 were significantly higher than at T6 in all subjects. Changes in cortisol levels over time were significantly different when comparing the two sexes, whereas men showed a larger increase in the area under the curve. Non-responders had significantly higher cortisol levels than responders at T1. Further, non-responders performed significantly better at the interviews focusing on situative skills. No additional group difference was found.

Conclusion: Cortisol responses in real-life stress differ between men and women. Non-responders show significantly better performance in psychological skills, for which preparation was not possible.

P095

Non-oxidized, biologically active parathyroid hormone determines mortality in hemodialysis patients

M. Tepel1, F. Armbruster2, H. Grön2, A. Scholze3, C. Reichetzeder4, H. Roth5, *B. Hocher4

1Odense University Hospital, Department of Nephrology, and Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

2Immundiagnostik AG, Bensheim, Germany

3Odense University Hospital, Department of Nephrology, and Institute of Clinical Research, University of Southern Denmark, Odense, Denmark

4Institute of Nutritional Science, University of Potsdam, Potsdam-Rehbrücke, Germany

5Department of Endocrinology/Oncology, Limbach Laboratory, Heidelberg, Germany

Background: It was shown that non-oxidized parathyroid hormone (n-oxPTH) is bioactive, whereas oxidated PTH at methionine residues results in loss of biological activity.

Methods: Now, we analyzed the association of n-oxPTH on mortality in hemodialysis patients using a recently developed assay system.

Results: Hemodialysis patients (224 men/116 women) had a median age of 66 years. 170 patients (50%) died during the follow up time of 5 years. Median n-ox-PTH levels were higher in survivors (7.2 ng/L) compared to deceased patients (5.0 ng/L; p=0.002). Survival analysis showed an increased survival in the highest n-ox-PTH tertile compared to the lowest n-oxPTH tertile (Chi square 14.3; p=0.0008). Median survival was 1702 days in the highest n-ox-PTH tertile, whereas it was only 453 days in the lowest n-oxPTH tertile. Multivariable-adjusted Cox regression showed that higher age increased odds for death, whereas higher n-oxPTH reduced the odds for death. Another model analyzing a subgroup of patients with iPTH concentrations at baseline above the upper normal range of the iPTH assay (70 ng/L) revealed that mortality in this subgroup was associated with oxidized PTH but not with n-oxPTH levels.

Conclusions: The predictive power of n-oxPTH and iPTH on mortality of hemodialysis patients differs substantially. Measurements of n-oxPTH may reflect the hormone status more precise. The iPTH associated mortality is most likely describing oxidative stress relatd mortality.

P096

Mild hypercalcitoninemia in patients with benign diseases of the thyroid and calcium homeostasis

*J. Kratzsch1, S. Karger2, F. Raue3, J. Feldkamp4, S. Scheel4, J. Thiery1, G. Kahaly5

1University Hospital Leipzig, Institute of Laboratory Diagnostics, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany

2University Hospital Leipzig, Clinic of Endocrinology and Nephrology, Department of Internal Medicine, Neurology and Dermatology, Leipzig, Germany

3Praxis Prof.Raue & colleagues, Heidelberg, Germany

4Klinikum Bielefeld, Bielefeld, Germany

5Gutenberg University Medical Center, Department of Medicine I, Mainz, Germany

Objective: Calcitonin (CT) is a biomarker for diagnosis and therapeutic monitoring of patients with medullary thyroid carcinoma. We systematically evaluated a new immunoassay for the determination of CT in sera from patients with different benign diseases of the thyroid and of the calcium homeostasis.

Methods: Serum was withdrawn from the following groups of patients in a multi-centric study: apparently healthy adult subjects (females: n=166; males: n=160), patients with thyroid nodules (PTN; n=181), Graves disease (GD; n=54); Hashimoto thyreoiditis (HT; n=87); non-Graves hyperthyroidism (NGHT; n=18) and diseases of the calcium homeostasis (DCH; n=25). CT was measured by the new Cobas assay (Roche). Intra- and Interassay coefficient of variation were below 6.2% for concentrations between 1-20 pg/mL.

Results: The upper reference limit (97.5 percentile) was defined at 6.40 pg/mL for females and 9.52 pg/mL CT for males by data of apparently healthy subjects. The same statistical limit for patients with PTN was found at 13.3 pg/mL. Maximal CT concentrations of patients with GD, HT, NGHT and DCH were 19.9, 10.1; 13.3 and 19.1 pg/mL.

Conclusion: Benign diseases of the thyroid and calcium homeostasis may cause a mild hypercalcitonemia if values are compared with reference limits of apparently healthy subjects.The detailed clinical history of patients has to be considered for the correct interpretation of CT results to avoid unwarranted further diagnostic efforts.

Hämatologie / Hämostaseologie Poster II

P097

The anti-oxidative defence system of erythrocytes during storage in blood bags and after simulated extracorporal circulation with a heart-lung machine model

L. Krampen1, H. Roth2, A. Grebe3, S. Fennrich2, R. Handgretinger1, *G. Bruchelt1, H. Wendel4

1Children′s University Hospital, Tuebingen, Germany

2University Hospital, Dept. of Thoriac, Cardiac and Vascular Surgery, Tuebingen, Germany

3Children′s University Hospital, Tuebingen, Germany

4University Hospital, Dept. of Thoriac, Cardiac and Vascular Surgery, Tuebingen, Germany

Objective: During storage of erythrocytes in blood bags lesions may occur, e.g. by iron release from haemoglobin, followed by formation of reactive oxygen species (ROS). We asked whether iron chelators may stabilize erythrocytes.

Methods: Erythrocytes were stored at 4°C in absence and presence of the iron chelators Desferal or Exjade. At different times several parameters were measured, especially those of the anti-oxidative defence system. In a second approach, erythrocytes previously stored for 1, 14 and 28 days were pumped through a heart-lung machine for up to 6 h in the absence or presence of Desferal / Trolox.

Results: Iron chelators did not influence erythrocytes during storage. Indeed, a time-dependent metHb increase was observed during the 6 hours heart-lung machine treatment. This effect was highly developed the longer the erythrocytes had been stored at 4°C before. MetHb formation is associated with ROS generation, but the anti-oxidative system of erythrocytes remained stable during storage; nevertheless some lipid peroxidation (LPO) occurs. LPO induced by iron/H2O2 was significantly reduced in the presence of Desferal + Trolox.

Conclusion: Less than 1% hemolysis is allowed before blood transfusion, but up to 25% of the transfused erythrocytes are degraded within the first 24 hours in the circulation. We conclude that usage of iron chelators in combination with anti-oxidative substances before transfusion may be protective, especially when older erythrocytes are used.

P098

Schistocytes as a useful parameter for STEC-HUS diagnostic.

*M. Nassour1, J. Doulgere1, H. Rohde2, B. Otto1, C. Wagener1, T. Streichert3

1University Medical Center Hamburg-Eppendorf, Department of Clinical Chemistry, Hamburg, Germany

2University Medical Center Hamburg-Eppendorf, Department of Medical Microbiology, Virology and Hygiene, Hamburg, Germany

3University Medical Center Cologne, Department of Clinical Chemistry, Köln, Germany

Objectives: In 2011 North Germany experienced one of the largest STEC outbreaks worldwide involving more than 3000 patients. We questioned the significance of schistocytes as a diagnostic parameter for hemolytic uremic syndrome (HUS) at admission. We examined three patient groups: Patients suffering from STEC infection plus developing HUS (STEC-HUS), STEC patients without HUS (STEC) and non-STEC patients exhibiting bloody diarrhoea (control group).

Methods: Classification of STEC-patients was performed via microbiological evidence. Distinction between STEC and STEC-HUS patients was defined by the triade of thrombocytopenia, anaemia and renal failure. We counted the number of schistocytes in the blood smears and determined number of platelets and concentrations of haemoglobin and creatinine.

Results: In our analysis we included 75 STEC patients, 74 STEC-HUS patients and 69 control patients. The coefficients of correlation between the different investigators were in the range of r=0.713 to r=0.779. We observed a clear elevation of schistocyte counts (median=0,8%) in STEC-HUS patients in comparison to STEC patients (median=0,1%) and control patients (median=0,1%) at admission.

Conclusions: We investigated the so far largest STEC-HUS patient cohort and report a clear potential of schistocyte counts for the differentiation between STEC-HUS patients and STEC patients as well as control patients.

P099

Effective estimation of correct platelet counts in pseudothrombocytopenia using magnesium salt as anticoagulant (ThromboexaktR)

*P. Kohlschein1, K. Dreissiger1, P. Schuff-Werner1

1Rostock University Medical Center, Institute of Clinical Chemistry and Laboratory Medicine, Rostock, Germany

Pseudothrombocytopenia remains a challenge in the diagnostic haematological laboratory. The pre-analytical problem that platelets tend to easily aggregate in vitro, giving rise to lower platelet counts, has been known since EDTA and automated platelet counting procedures were introduced in the haematological laboratory. Patients with unexpectedly low platelet counts or when reports were flagged for suspected aggregates were selected and blood smears were prepared to verify the presence of platelet aggregates. If platelet clumping was microscopically confirmed, the patients were asked to consent to the taking of an additional sample of blood anti-coagulated with an magnesium additive; in some patients citrate samples could be used for platelet count in parallel. Using this approach, we documented 48 patients with pseudothrombocytopenia. In all cases platelet counts were markedly higher in samples anti-coagulated with the magnesium containing anticoagulant when compared to EDTA-anticoagulated blood samples. The absolute and relative increase in platelet numbers ranged from 134 to 468*103/µL or 1.5 - to 25-fold, respectively. Five patients were identified with combined pseudothrombocytopenia demonstrating low platelet counts in EDTA and citrate anticoagulated blood samples. We conclude that in patients with known or suspected pseudothrombocytopenia magnesium-anticoagulant blood samples may be recommended for platelet counting.

P100

Evaluation of new BD ESR instrumentation in comparison to the standard manual Westergren method

G. Esmaeil Pourmahram1, *K. Schlueter2, C. Casadio3, C. Severini4, S. Church1

1BD Diagnostics - Preanalytical Systems, Oxford, OX4 4DQ, United Kingdom

2BD Diagnostics - Preanalytical Systems, Heidelberg, Germany

3Laboratory Department, Vital Diagnostics S.r.l, Forlì, Italia

4Laboratorio Analisi, Polo Sanitario Opera Santa Teresa del Bambino Gesù, Ravenna, Italia

The manual Westergren method published by the International Committee for Standardization in Hematology (1973) is considered the reference method for evaluation of Erythrocyte Sedimentation Rate (ESR). Automated instrumentation and closed evacuated blood collection systems have led to significant advances in the measurement of ESR such as standardization, workflow efficiency and safer working practices. To evaluate the new instrumentation system: BD Sedi-20 (with external mixer) and BD Sedi-40 (with internal mixer), using BD Vacutainer® Seditainer™ Glass Citrate blood collection tubes (4:1), a correlation study was conducted between one hour ESR results obtained using the Westergren method and the new instrumentation system. After obtaining informed consent, samples were collected from 100 patients, from each patient 2 tubes were drawn: 1 tube for manual method, 1 BD Seditainer™tube for the BD Sedi-20 and BD Sedi-40 instruments. All tubes were mixed, prior to analysis for 20 minutes using a Duo-Mix external mixer. Results were recorded after 30 minutes for the automated systems and 60 minutes for the Westergren method. All analysis was conducted within 4 hours of sample collection. Normal and abnormal samples were collected across the measurement range (N): 0-30 mm/hr (55), 31-60 mm/hr (25), 61-90 mm/hr (10), >91 mm/hr (10). Acceptable correlation (R2 ≥0.96) was demonstrated between the BD Sedi-20 and the Westergren method (R2=0.968), and between the BD Sedi-40 and the Westergren (R2=0.968). This demonstrates that the new ESR Instrumentation, BD Sedi-20 and BD Sedi-40, using BD Vacutainer®Seditainer™ Blood Collection Tubes, produce equivalent results to the manual Westergren method.

P101

Evaluation of five routine hematology analyzers in a clinical setting

*M. Brügel1, D. Nagel1, M. Neumeier1, P. Fuhrmann1, D. Teupser1

1Ludwig-Maximilians-Universität München (LMU), Institut für Laboratoriumsmedizin, München, Germany

Objectives: The aim of the present study was to compare diagnostic performances of current routine hematology analyzers in a clinical setting.

Methods: A 5-way comparison among Abbott Diagnostics Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000 and Sysmex XN hematology analyzers was performed. A total of 465 samples were analyzed side by side on all 5 instruments. Numeric results from each analyzer were compared, each instrument´s white blood count differential was further compared with a reference 200-cell manual differential. Evaluation of automated flaggings was performed by verification of abnormalities on peripheral blood smear.

Results: Interinstrument comparison revealed correlation coefficients (r) greater than 0.97 for standard blood count (SBC) and from 0.1 to 0.95 for differential blood count (DBC). Comparison with manual differentiation revealed coefficients from 0.23 to 0.95. Sensitivity/specificity (%) of flagging for blasts, variant lymphoids and immature granulocytes were 80/93, 60/99 and 58/91 for Cell-Dyn Sapphire, 69/96, 67/97 and 69/93 for DxH 800, 69/95, 70/93 and 43/96 for Advia 2120i, 67/97, 73/98 and 78/92 for XE-5000 and 96/95, 77/98 and 91/87 for Sysmex XN.

Conclusion: A comparable diagnostic performance of investigated analyzers could be demonstrated for SBC, however marked differences could be shown for DBC analysis and flagging efficiency.

P102

Analysis of storage-induced changes of the cytosolic red blood cell proteome

*K. Walpurgis1, M. Thevis1, F. Wenzel2

1German Sport University Cologne, Köln, Germany

2Medical Center of University Düsseldorf, Düsseldorf, Germany

Background: The storage of packed red blood cells (RBCs) is associated with the development of morphological and biochemical changes. Within this study, two-dimensional gel electrophoresis analysis (2D DIGE) and nano-liquid chromatography high-resolution mass spectrometry (LC-MS) were used to analyze the storage-induced changes of the cytosolic RBC proteome and identify characteristic protein patterns and potential marker proteins for the assessment of RBC storage lesions.

Material and Methods: Leukodepleted RBC concentrates of healthy volunteers (n = 12, six female and six male) were stored according to standard blood bank conditions for 0, 7, 14, 28 and 42 days. After hemoglobin removal the RBC proteins were labeled by DIGE technique and separated using SDS-PAGE gel electrophoresis. The identification of the proteins were performed by in-gel tryptic digestion and LC-MS.

Results: A total of 189 different RBC proteins were identified comprising metabolic enzymes, chaperones and components of the cytoskeleton as well as proteins of the cellular defense or the ubiquitin-proteasome-system. Following statistical evaluation, a total of 14 protein spots were found to be significantly altered after 42 days of ex vivo storage. Especially, three proteins could be selected to be potentially useful as biomarkers for RBC aging comprising transglutaminase 2, beta actin and copper chaperone for superoxide dismutase (additionally validated by western blot analysis).

Conclusions: The observed alterations of RBC proteins during storage were only modest, but it was possible to identify three potential marker proteins for the assessment of RBC storage lesions serving as a basis for the development of a respective screening assay.

P103

The influence of the „new“ anticoagulants Rivaroxaban and Dabigatran on non-routine coagulation assays.

*G. Steinbach1

1Uniklinik Ulm, Klinische Chemie, Ulm, Germany

Objectives: The influence of the so called “new” anticoagulants upon routine coagulation assays is well described. Little is known about the influence of these anticoagulants on non-routine assays like APCR or Lupus-Inhibitor testing. Especially the activity assays are prone to be influenced by the “new” anticoagulants. Since more and more patients are treated with these anticoagulants interpretation of these assays may be flawed.

Methods: We used the commercially available calibrator series for Dabigatran (Hyphen®) and Rivaroxaban (Technoclone®) in 1:1 dilution with normal plasma as sample in our regularly assayed non-routine test panels. E.g.: Protein-S and Protein-C activity and concentration, APCR, AT-III, DVV, LCA, KCT, D-Dimer, coagulation factors II, V, VII, VIII, IX, X, XI and XII, vWF-activity.

Results: Dabigatran and Rivaroxanban interfere severely with functional non-routine coagulation assays. Assays relying on antigen binding are not disturbed. Especially the APCR and determination of Lupus-Inhibitors are disturbed by low concentrations of the “new” anticoagulants.

Conclusion: Sampling for non-routine coagulation assays should be performed at least 12 hours, better 24 hours, after the last dose of the “new” anticoagulants. The determination of thrombin-time should be performed alongside the non-routine coagulation assay to rule out the influence of Dabigatran.

P104

Inhibition and Regulation of Blood Coagulation by Poly-P

*B. Arneth1, T. Chavakis1, G. Siegert1

1Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

Introduction: Poly-P is a large inorganic molecule present in the alpha granules of platelets. Recent studies have described the blood coagulation cascade initiated by poly-P by activating factor XII and pre-kalligrenin. Based on these findings, we wanted to determine the influence of poly-P on the activation of blood coagulation factor V, because the latter is also expressed in the granules of platelets.

Material and methods Standard plasma (unical) was incubated with poly- P (n=45; n=700) or diluent (10min, 37°C) and subsequently used in one-stage factor clotting assays for factors II, V, VII, and X (Stago/ Roche, Mannheim, Germany) and in a thrombin generation assay (TGA, technoclone, Vienna, Austria).

Results and Discussion: Poly-P inhibited factor V in the factor V clotting assay. We noted concentration-dependent inhibition of only coagulation factor V by poly-P - not of factors II, VII, or X. Consistent with the inhibition of factor V by poly-P, the area under the curve (AUC) in the TGA declined significantly dose depended when poly-P was added to unical plasma. The effect was reversible by overnight digestion of poly-P with phosphatase.

Conculsion: Poly-P inhibits coagulation factor V and the degree of thrombin generation decreases substantially in the presence of poly-P. Thus, platelet poly-P has inhibiting and regulatory effects on the blood coagulation system that are mediated through inhibition of coagulation factor V.

P105

p45-NFE2 potentially regulates syncytiotrophoblast formation in Human Placenta

*S. Kohli1, X. Liu1, J. Hoffmann1, K. Shahzad1, F. Bock1, S. Ranjan1, M. Thati1, L. Luley2, F. Jensen2, A. Zenclussen2, B. Isermann1

1Otto-von-Guericke-Universität Magdeburg, Institute for Clinical Chemistry and Pathobiochemistry, Magdeburg, Germany

2Universitätsfrauenklinik, Magdeburg, Experimental Obstetrics and Gynecology, Magdeburg, Germany

Objectives: p45-NFE2 transcription factor, known to regulate megakaryocyte maturation, has been recently found to be required for normal syncytiotrophoblast formation and embryonic growth in mice. We aim to study the role of NFE2 in in human placenta.

Methods: Human Choriocarcinoma Cell line, Bewo was stimulated with Forskolin to induce syncytiotrophoblast formation which was evaluated by studying the secreted levels of hCG-β and expression of marker genes, Gcm1 and hCG-β. Human placenta sections from healthy controls and patients with pregnancy complications were compared for syncytial knot formation and studied for marker gene expression. p45-NFE2 expression was studied after forskolin treatment in Bewo cells and placenta tissues.

Results: Forskolin treatment elevated Gcm1 and hCG-β expression levels along with an increased secreted level of hCG-β. In parallel, it lowered the expression levels of NFE2 indicating that it could play a role in modulating syncytiotrophoblast formation. The diseased placenta samples showed higher number of syncytial knots, elevated marker genes expression indicating enhanced synctiotrophoblast formation, and reduces expression of p45-NFE2.

Conclusion: The current observation in human trophoblasts matches those made in mice, suggesting that placental p45-NFE2 prevents excess syncytiotrophoblast formation in humans. Reduced p45-NFE2 expression in placenta may be an early cause and/or marker of placental dysfunction.

P106

Influence of the new anticoagulants on platelet function tests

*T. Eller1, J. Busse2, C. Knabbe2, C. Brandenburger2, M. Dittrich3, T. Flieder2, S. Alban4, I. Birschmann2

1Johannes Wesling Klinikum Minden, Gerinnungsambulanz, Institut für Laboratoriumsmedizin, Minden, Germany

2Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinikum der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

3Biocenter, University of Würzburg, Department of Bioinformatics, Wuerzburg, Germany

4Christian-Albrechts-Universität zu Kiel, Pharmazeutisches Institut, Kiel, Germany

Objectives: For over 50 years phenprocoumon is traditionally used as main drug for anticoagulation, but long-term treatment is hampered with several problems. Therefore alternative anticoagulants with favorable pharmacodynamic and pharmacokinetic traits, such as the direct thrombin inhibitors argatroban and dabigatran and the factor Xa inhibitors rivaroxaban, apixaban and fondaparinux have been developed. Due to their increasing clinical usage it is important to evaluate their interferences with haemostaseological assays. Here we investigate these anticoagulants primarily focusing on their influence on platelet function.

Methods: For analyzing platelet function we used PFA-200® (col/epi, col/ADP, P2Y) and light transmission aggregometry (LTA). Activation of platelets (LTA) was induced by ADP, arachidonic acid, epinephrine, collagen, ristocetin, A23187, PAF C-16, PMA, TRAP, U46619 and thrombin.

Results: Our measurements by LTA revealed a distinct decrease of platelet aggregation after in vitro incubation with supratherapeutic concentrations of argatroban compared to control. Neither therapeutic nor supratherapeutic concentrations of dabigatran, rivaroxaban, apixaban and fondaparinux had a comparable effect on platelet function. The PFA analysis revealed no influence on aggregation as well.

Conclusions: Based on our results, it seems that platelet aggregation remains unaffected by the investigated drugs under therapeutic conditions.

Kardiovaskuläre Erkrankungen Poster II

P107

Involvement of the LRC family member OSCAR in atherosclerosis and vascular inflammation

*N. Al-Fakhri1, K. Nemeth1, L. Mey1, L. Hofbauer2, M. Schoppet3

1Philipps University, Institute for Laboratory Medicine, Marburg, Germany

2TU Dresden, Clinic for Internal Medicine, Endocrinology, Dresden, Germany

3Philipps University Marburg, Clinic for Cardiology, Marburg, Germany

Objectives: The LRC family member Osteoclast-associated receptor (OSCAR) was previously demonstrated on endothelial cells (EC) and vascular smooth muscle cells. This study characterized the functional role of OSCAR on EC for atherosclerosis development and inflammation.

Methods: WT, OSCAR-siRNA treated and -overexpressing HUVEC were stimulated with VEGF, bFGF, TGF-beta, TNF-alpha, INF-alpha and -gamma. EC were analyzed with BrdU- and wound-scratch-assay for proliferation and migration. OSCAR expression was determined by immunofluorescence, Western blot and real-time RT-PCR. In binding assays the adhesion of beta2-integrin-expressing and wildtype HEK and CHO cells and of soluble OSCAR to LFA-1 and Mac-1 were analyzed. ApoE -/- and WT mice were analyzed for OSCAR expression.

Results: All cytokines and high concentrations of bFGF, but not VEGF or TGF-beta increased OSCAR expression on EC. OSCAR expression was demonstrated on the cell surface and in adherence junctions of EC. Enhanced or deleted OSCAR expression reduced the migratory and proliferative capacity of EC, INF-alpha and -gamma augmented these effects in OSCAR-overexpressing cells. OSCAR bound specifically to leukocyte integrins in ligand and cell based assays. OSCAR expression was increased in ApoE -/- mice.

Conclusion: OSCAR is a surface and junctional receptor for leukocytes on EC induced by cytokines. Endothelial OSCAR expression could play a role in leukocyte recruitment in atherosclerosis and inflammation.

P108

Changes in coagulation and fibrinolysis in smokers and nonsmokers of the Ludwigshafen Risk and Cardiovascular Health Study (LURIC)

*M. Kleber1, R. Siekmeier1, G. Delgado1, T. Grammer1, B. Winkelmann2, B. Böhm3, W. März1

1Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Institute of Public Health, Social and Preventive Medicine, Mannheim, Germany

2Cardiology Group, Frankfurt, Germany

3University Hospital, Ulm, Division of Endocrinology, Department of Medicine, Ulm, Germany

Objectives: Cardiovascular disease (CVD) is an important cause of mortality and smoking is an avoidable cause for CVD. Analysis of predictive factors (e. g. plasma lipids, smoking) allows individual risk estimation. Measurement of coagulation and fibrinolysis provides information on the risk for thrombus formation. Aim of our study was the analysis of markers for coagulation and fibrinolysis in active smokers (S) and life-time nonsmokers (NS) of the Ludwigshafen Risk and Cardiovascular Health Study (LURIC).

Methods: 3316 patients were included of which 777 were S and 1178 NS. Within the observation time (10 years, median) 995 died (221 S, 302 NS). Protein C, factor V, protein S, fibrinogen, prothrombin fragment 1 and 2, D-dimer, plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI) and endogenous thrombin potential (ETP) were determined.

Results: Fibrinogen (S vs. NS, 417.0±113.9 vs. 384.1±99.7 mg/dl, p<0.001), ETP (99.1±24.5 vs. 94.4±28.5 %, p<0.001), protein S (120.2±35.7 vs. 114.4±32.8, p=0.027) and TFPI (1.30±0.37 vs. 1.23±0.37 µg/l, p<0.001) were higher in S whereas protein C (106.7±25.3 vs. 112.1±25.1 %, p=0.006 was lower. No differences were found for the results of the other parameters.

Conclusion: Our data show an increased thrombogenic potential in S compared to NS. Therefore, parameters of coagulation and fibrinolysis should be determined at least in high risk patients (e. g. S).

P109

Differential Eicosanoid Response in Whole Blood from Patients with or without Coronary Artery Disease

*A. Kleinhempel1, L. Holdt2, D. Nagel2, U. Ceglarek1, F. Beutner1, L. Kortz1, J. Thiery1, D. Teupser2, M. Bruegel2

1Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

2Institut für Laboratoriumsmedizin, Ludwig-Maximilians-Universität München (LMU), München, Germany

Objectives: Based on the hypothesis that differential intensity of the eicosanoid response may affect coronary risk, we aimed to investigate the individual eicosanoid response as potential marker of coronary artery disease (CAD).

Methods: Whole blood from patients without (n=40) or with (n=20/32) CAD (<50%/>50% stenosis) was incubated with or without LPS (100ng/mL) for 4h and 24h. RNA was isolated and target genes of the arachidonic acid (AA) pathway (cyclooxygenase (COX) 1 and 2, prostaglandin E synthase (PGES) and 5-lipoxygenase activating protein (FLAP)) were analyzed by quantitative RT-PCRs. Corresponding metabolites (AA and hydroxy eicosatetraenoic acids (HETEs)) were analyzed in supernatants using LC-MS/MS.

Results: Patients with CAD>50% showed an increased inducibility of COX-1 and FLAP expression after 24h and a reduced inducibility of COX-2 expression after 4h LPS activation (P<0.05). Patients with CAD revealed a reduced release of AA (P<0.01), 12-HETE and 5-HETE (P<0.05) after 4 and 24h LPS activation. A ROC curve analysis of differentially regulated mediators and target genes revealed an area under the curve of 0.78 for combined FLAP expression and AA release after 24h LPS activation.

Conclusion: Differentially regulated target genes and mediators of AA metabolism in CAD patients suggest that individual regulation of eicosanoid response may represent a marker of atherosclerotic risk.

P110

Pla2g12a and Elovl6 as Novel Candidate Genes of Atherosclerosis Susceptibility in Mouse Chromosome 3

*A. Nicolaou1, K. Sass2, B. Northoff1, J. Thiery3, D. Teupser1, L. Holdt1

1Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany

2University Leipzig, Institute of Anatomy, Leipzig, Germany

3Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics Leipzig, Leipzig, Germany

Objectives: In a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3, Pla2g12a and Elovl6 were identified as potential candidate genes. The aim of the current study was to investigate their role in mechanisms of atherosclerosis.

Methods: Single Nucleotid Polymorphism (SNP) genotyping was performed with a homogenous melting-curve based assay to fine-map Chr3. Expression patterns in different tissues were investigated by qRT-PCR, Western blot analysis and immunohistochemical staining (IHC). siRNA knock-down and over-expression experiments were performed in RAW cells.

Results: Fine-mapping showed co-segregation of Elovl6 and Pla2g12a mRNA expression with the atherosclerosis LOD score peak suggesting a causal role of these genes in atherogenesis. qRT-PCR in different tissues showed high expression of Elovl6 in fat and liver and high expression of Pla2g12a in muscle and fat. Immunohistochemical staining of Elovl6 in aortic roots indicated high expression in plaque and predominantly in endothelial cells. Western blot analysis showed 4 times higher protein expression of Pla2g12a in FVB than in B6 tisssue. siRNA knockdown of Elovl6 was associated with higher adhesion and higher apoptosis. The impact of Pla2g12a knock-down is currently investigated.

Conclusion: Elovl6 and Pla2g12a represent candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to understand the influence of these two genes in atherosclerosis development.

P111

Chronic Q fever a bloodculture negative endocarditis – a neglected entity

B. Hermann1, *K. Boden2

1Institute of Medical Microbiology, University Hospital Jena, Jena, Germany

2Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Jena, Jena, Germany

Objectives: The objective was to determine the impact of chronic Q fever in blood culture negative endocarditis in our hospital.

Methods: In our routine diagnostic all specimens from heart valve surgery with suspected endocarditis were collected from October 2007 to December 2012. We screened all patients with available sera for Coxiella burnetii-specific antibodies by ELISA (Virion/Serion) and performed an IFT (Focus) for confirmation and quantification of Phase 1-IgG. We also took into accounts the results from routine diagnostic. Heart valves from patients with serological constellation of chronic Q fever were further investigated by real time PCR and ACCM2 culture for Coxiella burnetii.

Results: We screened 215 sera for C. burnetii-specific Phase 2-IgG-antibodies. We detected 8 patients with positive and 11 patients with borderline antibody levels. 1/8 patients revealed a Phase 1- IgG titer (IFT) above 1:64 000 corresponding to chronic Q fever. We could confirm our finding by a positive PCR and culture. A second case was detected in routine diagnostic with a Phase 1- IgG titer of 1: 8192 in serum. We couldn`t obtain the native heart valve for PCR and culture testing.

Conclusion: Chronic Q fever is accountable for a small number of blood culture negative endocarditis. As mortality is high and combination therapy according to the guidelines takes 18 months it is crucial to correctly identify all possible cases. This work was supported by the Federal Ministry of Education and Research Germany (Grant 01 KI 0735 and 01 KI 1001C).

P112

Pianp and Eno2 – Two New Candidate Genes of Atherosclerosis Susceptibility at the Brachiocephalic Artery

*P. Harmel1, L. Holdt2, P. Wolf2, R. Baber1, J. Thiery1, D. Teupser2

1Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

2Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany

Objectives: Previous studies in two independent crosses of inbred mouse strains C57BL/6xFVB/N (C57xFVB) and C57BL/6xBALB/cByJ (C57xBALB) revealed a locus of atherosclerosis at the brachiocephalic artery (BCA) on mouse chromosome 6 (Chr6). The aim of the current study was to identify genes responsible for different atherosclerosis susceptibility.

Methods: Genome-wide expression analyses revealed 10 differentially expressed genes (fold change > ±20%, P < 0.05) in aortas and livers of Chr6 congenic (L6F) and FVB male mice (n=4/4). Differential expression of Pianp and Eno2 was verified by TaqMan quantitative RT-PCR in aortas and livers of L6F and FVB males (n=6/6). Both genes were subsequently characterized in cell culture studies concerning atherosclerosis related functions.

Results: Expression quantitative locus mapping (eQTL) analyses in livers of male C57xFVB and C57xBALB F2 mice (n=232/189) revealed co-segregation of the Chr6 atherosclerosis QTL. siRNA-mediated knock-down of Pianp and Eno2 in RAW cells led to decreased proliferation and apoptosis whereas no effects on adhesion were noted. Sequencing of Pianp and Eno2 promoter regions revealed previously unknown SNPs modifying potential transcription factor binding sites. Functional relevance of the promoter SNPs was validated by reporter gene assays.

Conclusion: Pianp and Eno2 are plausible candidate genes for the Chr6 mouse atherosclerosis locus. Current work focusses on the translation of finding in the human situation.

P113

ATP-binding cassette transporter G1 (ABCG1) prevents accumulation of atherogenic oxysterols

*T. Engel1, M. Fobker2, A. Schürmann3, J. Nofer2, U. Seedorf4

1Leibniz-Institute for Arteriosclerosis Research at the Westphalian Wilhelms-University, Münster, Germany

2Center for Laboratory Medicine, University Hospital Muenster, Münster, Germany

3German Institute of Human Nutrition, Department of Experimental Diabetology, Potsdam-Rehbrücke, Germany

4Leibniz-Institute for Arteriosclerosis Research at the Westphalian Wilhelms-University, Münster, Germany

Objectives: Oxysterols are oxidized derivatives of sterols that have cytotoxic effects. Efficient oxysterol removal by the sub-family G member 1 of the ATP-binding cassette transporters (ABCG1) is essential for cell survival. However, the specific role of ABCG1 in the transport of various oxysterol species has been not systematically investigated to date.

Methods: We examined the involvement of ABCG1 in oxysterol metabolism by studying oxysterol tissue levels in a mouse model of Abcg1-deficiency by gas-liquid chromatography coupled to mass spectrometry (GC-MS) analysis. For functional analysis our stable regulable ABCG1 expressing cell lines were used.

Results: Analysis of lung tissue of Abcg1-/- mice on a standard diet revealed that 3β,5a,6β-cholestanetriol (CT) and 25-hydroxycholesterol (HC) accumulated at more than 100-fold higher levels in comparison to wild-type mice. A [3H]-labeled assay employing regulable ABCG1-expressing HeLa cell lines revealed that 25-HC export to albumin was dependent on functional ABCG1 expression and could be blocked by an excess of unlabeled 25-HC or 27-HC. In this cell line, 25-HC at low doses triggered mitochondrial membrane potential, and reactive oxygen species production, which are both indirect indicators of cellular energy expenditure.

Conclusion: Our results suggest that 25-HC and CT are physiologic substrates for ABCG1 mediated export from cells and that primary function of ABCG1 is protection against oxysterol-induced cell death, an anti-atherosclerotic property.

Klinische Massenspektronomie Poster II

P114

Rapid and high confident analysis of fatty acids in human erythrocyte membrane by chemical ionization-gas chromatography-tandem mass spectrometry

*Y. Schober1, T. Vielhaber1, W. Nockher1, H. Renz1

1Philipps-Universität Marburg, Institut für Laboratoriumsmedizin und Pathobiochemie, Molekulare Diagnostik, Marburg, Germany

Objectives: Mediators of fatty acid (FA) metabolism are known to influence inflammatory and cardiovascular diseases. Therefore the quantitative analysis of FA in human body fluids is of growing interest in the clinical laboratory. Classical techniques are gas chromatography (GC) combined with flame ionization or single mass spectrometry detection. These methods require long chromatographic run times to achieve an adequate chromatographic separation of the analytes. Tandem mass spectrometry advances analyte identification and has the potential to simplify the GC separation.

Method: After hexane extraction of FA from blood erythrocyte membranes, FA were esterified with tetramethylguanidine. FA methyl esters were analyzed using a TripleQ-GC-Tandem-MS system, equipped with a chemical ionization (CI) source. Ammonia was used as reactant gas.

Results: CI with ammonia generated specific quasi molecular ions (QMI) which were further subjected to analyte specific fragmentation. The QMI were used for quantification, their specific fragment ions were additionally used as qualifier to ensure the identification. The runtime is <20 minutes, less than the half time compared to our previous method (Boecking et al, 2010). In total up to 43 FA were analyzed, among them almost unnoticed fatty acids like docosapentaenoic acid.

Conclusions: Using a CI-GC-Tandem-MS system a rapid, sensitive and high confident method is now available for the quantitative analysis of FA in the clinical laboratory.

P115

Identification of Isolates of Mycobacterium species by MALDI Biotyper Analysis

E. Richter1, S. Rüsch-Gerdes1, M. Timke2, B. Wegemann2, *M. Kostrzewa2

1National Reference Center for Mycobacteria; Research Center Borstel, Borstel, Germany

2Bruker Daltonik GmbH, Bremen, Germany

Objectives: Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is widely used for identification of microorganisms in clinical diagnostics. However, some Mycobacterium species show low-quality mass spectra with standard preparation methods. Therefore a zirconia/silica beads based method was applied to 196 blinded Mycobacterium spp. isolates.

Methods: Mass spectra were recorded with a microflex LT and compared to Mycobacteria Library 2.0 reference spectra using MALDI Biotyper 3.1 software (Bruker Daltonik, Germany). Reference method for study isolates was 16S rRNA gene, ITS, and rpoB sequencing analysis, GenoType® Mycobacterium CM and MTBC.

Results: High (≥ 2.0) MALDI Biotyper log(score) values and correct species identification was obtained for 179 (91.3%) isolates. Correct species was assigned to six isolates (3.1%) but with a lower confidence (log(score) value (1.7 – 1.999). It has to be stressed that all MTBC isolates (n = 51) were reliably identified as members of this complex.

Conclusion: Results indicate the high potential of MALDI-TOF MS for fast and reliable identification of M. tuberculosis complex and nontuberculous mycobacteria isolates.

P116

Evaluation of rapid MS-based antibiotic resistance screening to assess beta lactamase activity in clinical routine analysis

*M. Peer1, K. Sparbier1, C. Lange1, B. Wegemann1, M. Kostrzewa1

1Bruker Daltonik GmbH, Bremen, Germany

Objectives: Microbial resistance against antibiotics has become an issue in the clinical environment. We recently introduced the MALDI-Biotyper-Spectrum-Beta-Lactamase (MSBL) workflow, a rapid and easy to use mass spectrometry based assay for robust prediction/identification of beta-lactamase activity in clinical specimens. Minimal hands-on time and an automatable workflow enables easy integration in the laboratory. Here we compare the adoption of this workflow to the 108mazon speed LC-ESI-MSn ion trap, demonstrating the workflows superior specificity, robustness and usability in clinical routine laboratories also in an UHPLC-ESI-MS(n) environment.

Methods: The resistance assay is based on the analysis of beta-lactam hydrolysis. In brief, antibiotics were incubated separately with fresh overnight cultures of isolated bacteria and control strains for 2 h. Hydrolysis was analyzed using MALDI-TOF or in a 4.5 min LC-ESI-MSn multiplex analysis. Automated data analysis and reporting was conducted using a novel software interface.

Results: The bacterial hydrolysis of four beta-lactam antibiotics (ampicillin, ertapenem, cefotaxime and ceftazidime) was analyzed in eight bacterial strains. Strains resistant against beta-lactam antibiotics were identified correctly. The results confirm accurate, reproducible analysis and robust, equivalent classification of bacterial resistance on different mass spectrometric systems.

Conclusion: Our robust and specific high throughput mass spectrometry based beta-lactamase resistance workflow enables routine laboratories to rapidly determine the bacterial resistance profile allowing individual adjustment of therapeutic treatment.

P117

Development and Validation of a LC-MS/MS method for quantification of Voriconazole

*B. Maier1, D. Teupser1, M. Vogeser1

1Universitätsklinikum der LMU, Institut für Laboratoriumsmedizin, München, Deutschland

Objectives: Voriconazole shows high inter-individual variability in serum concentrations. The aim of our work was to develop and to validate a convenient and reliable LC-MS/MS method for the routine quantification of voriconazole in serum.

Methods: Sample preparation was based on protein precipitation followed by on-line solid phase extraction. Voriconazole-d3 was used as the internal standard; three MRM-transitions were recorded for target analyte and internal standard, respectively. The method was validated on two different LC-MS/MS instruments.

Results: Intra- and inter-assay precision in three different concentration levels were below 8%, and accuracy was +/- 6%. Comparative measurement of 30 samples using two instruments demonstrated close agreement. Reliable control of ion suppression was observed.

Conclusions: The method for quantification of voriconazole in serum was found to be fit for purpose.

P118

Adopting a screening approach to specific needs in the lab: Screening for psychotropics in serum using an ion trap MS

J. Kempf1, J. Traber1, *M. Meyer2, L. Huppertz1

1Universitätsklinikum Freiburg, Institut für Rechtsmedizin, Freiburg, Germany

2Bruker Daltonik GmbH, Bremen, Germany

Objectives: Various types of instruments are used to develop an ultimate comprehensive screening to identify as many compounds as possible in a single run. But in routine work, the question for the detection of a dedicated set of substances arises very often. We present a screening method for psychotropics based on the Toxtyper™ open library concept.

Methods: A library with data from the Toxtyper and spectral data of additionally available psychhotropics was set up. For ionization a conventional ESI- and an ionBooster™ source were used. All other parameters were adopted from the Toxtyper approach. Samples were analysed on a Dionex RSLC coupled to a Bruker Daltonics oniza speed. Drug-free serum samples fortified with different mixes of psychotropics at 2 low therapeutic concentrations and several authentic samples were analysed.

Results: The method contains the precursor mass and retention time of 105 psychotropic drugs. Two sets of 12 mixes were analysed using both onization techniques and the original screening approach. The customized method led to a higher rate of positive findings (92 %), especially at low concentration levels, than the comprehensive screening approach (87%). Two analytes couldn’t be detected, while 5 compounds could only be identified when using the ionBooster. Results from routine analysis of cases with known intake of psychotropic drugs were confirmed with positive findings, if the concentration range was above or around the assumed LOD from this evaluation study.

Conclusion: The presented method is a fast and robust screening tool for the detection and identification of 105 psychotropics in serum. Evaluation in spiked human samples showed detection of low therapeutic levels for the majority of compounds.

P119

High-throughput shotgun lipidomics of blood plasma lipoproteins

*K. Schuhmann1, J. Gräßler2, S. Bornstein2, A. Shevchenko3

1MPI-CBG / MK3 Universitätsklinikum, Dresden, Germany

2Universitätsklinikum Carl Gustav Carus, Medizinische Klinik und Poliklinik III, Dresden, Germany

3Max-Planck-Institut für molekulare Zellbiologie und Genetik, Dresden, Germany

Top-down shotgun lipidomics relies on the direct infusion of total lipid extracts into a high resolution tandem mass spectrometer and is a powerful technology for high-throughput clinical screens. In the recent study we were able to quantify 222 species from 15 major lipid classes in human blood plasma in less than 4 min. Here we show how shotgun lipidomics can be extended to characterizing lipoproteins. Blood plasma was fractionated by preparative ultracentrifugation in D2O and Ficoll media into very low, low, and high density lipoproteins as well as plasma proteins. These 4 fractions were one-step extracted by methyl-tert butyl ether/methanol and analyzed on a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa robotic nanoflow ion source (Advion Bioscience). The analysis of all plasma fractions was completed in 30 min with the quantification error < 10 %. The proteomics and lipidomics profiles of lipoprotein fractions corroborated the reference profiles obtained by conventional NaCl/NaBr density gradient centrifugation. The combination of MS-compatible lipoprotein fractionation, one-step extraction and shotgun lipidomics represents a valuable tool for studying metabolic disorders and therapies that affect the lipoprotein composition.

P120

The Utilization of Novel Platform in a LC-MS/MS Workflow for the Analysis of Vitamin D, Testosterone, Immunosuppressants, Chemotherapeutics and Cortisol

*B. Duretz1, D. Argoti2, K. Hassel2, S. Fair2, J. Hermann2

1ThermoFisher, LSMS, Courtaboeuf, France

2ThermoFisher, Franklin, USA

Introduction: The development of a rugged system for the sample preparation and chromatographic analysis prior to MS detection is essential in a clinical environment. In this work, we present the application of a new LC platform for the development of faster and more reproducible LC/MS methods.

Methods: All samples were vortex mixed and centrifuged after the addition of an internal standard solution. Supernatant was transferred into the LC-MS/MS instrument. On-line sample clean-up and chromatographic separations of 25-OH-D2, 25-OH-D3, immunosuppressants, chemotherapeutics, and cortisol were performed using turboflow chromatography with a new platform equipped with a 0.5x50 mm ThermoScientific HTLC-Cyclone-P column, and a 50x2.1mm, 2.6 µm particle size ThermoScientific Accucore PFP analytical column. The detector for the system was a TSQ Vantage triple quadrupole mass spectrometer with HESI-II ionization probe in positive mode.

Results: The interday and intraday accuracy and precision for 25-OH-D2 and 25-OH-D3 was done at a range of 2-100 ng/mL and 2-100ng/mL, respectively. For testosterone the evaluated range was 0.02-10 ng/m. Immunosuppressants and chemotherapeutics were evaluated at a range of 1-2000 ng/mL and cortisol from 3.62 - 362 ng/mL. The assay precision were less than 15.0% for all compounds. Additionally, accuracy was ±15% of the theoretical value for all the assays. The correlation coefficient values for all the compounds ranged from 0.991 to 0.999. Recoveries were all above 90%.

Conclusions: A novel platform for LC/MS analyses consistently performed successful on-line sample clean-up for the compounds of interest using less than 3 mL of mobile phase per sample in under 4 min total run time.

P121

Rapid Quantification of Salivary Cortisol, Cortisone, Dexamethasone and Prednisolone via Liquid Chromatography – Tandem Mass Spectrometry

*A. Gaudl1, J. Stäker1, J. Thiery1, J. Kratzsch1, U. Ceglarek1

1Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, LIFE – Leipzig Research Center for Civilization Diseases, Leipzig University, Leipzig, Germany

Objectives: Reliable determination of salivary cortisol (CL) concentration is of interest in the assessment of adrenocortical functioning. Liquid chromatography coupled to mass spectrometry enables the rapid simultaneous quantification of endogenous and exogenous glucocorticoids avoiding known issues of commonly used immunoassays like cross reactivity.

Method: Chromatographic separation and purification via online SPE is performed on a Shimadzu Prominence® UFLC system equipped with a Merck Chromolith® SpeedROD and an Applied Biosystems Poros® as analytical and SPE column respectively. The mobile phase consisted of 50% A (H2O/MeOH 97/3 v/v + 0.1% formic acid) and 50% B (H2O/MeOH 3/97 v/v + 0.1% formic acid). It was adjusted to 100% B in 5 min and reequilibrated for 1 min. Eluent C (H2O/MeOH 90/10 v/v) was used for loading the SPE column. Detection via mass spectrometry occurred on an AB SCIEX QTRAP® 5500 through positive atmospheric pressure ionization and multiple reaction monitoring.

Results: Mean coefficients of variation determined in intra assay experiments (n=10) were 2-3% for CL and cortisone (CN) and 6-9% for prednisolone (PN) and dexamethasone (DN). Linearity was verified for 0.3-120 ng/ml for CL and CN, 0.1248 ng/ml for DN and 1.5-600 ng/ml for PN.

Conclusion: We present a rapid, specific and reliable quantification method to assess salivary glucocorticoid concentrations which is of interest for clinical routine laboratories and in various areas of research.

P122

INVESTIGATION OF FREE OXYSTEROLS DERIVED FROM REACTIVE OXYGEN SPECIES AND FREE CHOLESTEROL IN HUMAN PLASMA AND CAROTID PLAQUE

*C. Helmschrodt1, S. Becker1, J. Schröter1, J. Thiery1, G. Aust2, U. Ceglarek1

1Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Leipzig, LIFE –Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany

2Department of Surgery, Research Laboratories, Universität Leipzig, Leipzig, Germany

Objectives: From in vitro experiments it is known that reactive oxygen species (ROS) derived oxysterols play a role in atherogenesis. Oxysterols are able to accumulate in circulating LDL and atherosclerotic plaques, show inflammatory, apoptotic, necrotic and pro-oxidative effects.

Methods: Concentrations of free oxysterols and cholesterol were analyzed in plaque and plasma samples from 65 patients with advanced internal carotid artery (ICA) stenosis. The quantitative analysis of the oxysterols 7-keto- (7-KC), 7-hydroxy- (7-OHC), 5,6a-epoxy- (5,6a-EC), and 5,6ß-epoxycholesterol (5,6ß-EC) was performed using a monolithic column coupled to an API 4000 (AB SCIEX) mass spectrometer. Plasma and plaque sample preparation strategies were validated in relation to artificial autoxidation of cholesterol.

Results: Oxysterol concentrations were normalized to cholesterol and had the following distribution in plaque: 7-KC 50.4% > 5,6ß-EC 19.7% > 7-OHC 17.6% > 5,6a-EC 12.3%. The investigation of plasma showed the same pattern: 7-KC 43.5% > 5,6ß-EC 24.1% > 7-OHC 18.7% > 5,6a-EC 13.8% that is about 40-60 times lower concentrated. However, no significant correlations were found between both compartments for the individual patient.

Conclusion: Our data suggest that plaque oxysterols accumulate from plasma and are subject to in situ transformation processes in plaque. Therefore, it is essential to determine oxysterols in both compartments to obtain information about these transformation processes.

Labormanagement und Qualitätskontrolle Poster II

P123

Quality of customer informations in product problems of reagents for coagulation testing published by BfArM 2005 - 2012

*J. Hannig1, R. Siekmeier1

1Pharmaceutical Institute, University Bonn, Germany, Drug Regulatory Affairs, Bonn, Germany

Objectives: The European Directive 98/79/EC on In-vitro Diagnostics (IVD) regulates marketing of IVD in Europe. In cases of incidents and field safety corrective actions (FSCA) manufacturers have to inform the responsible Competent Authority (CA) and the public by field safety notices (FSN). We analysed FSN of IVD for reagents for coagulation testing.

Methods: FSCA and FSN published by BfArM 2005-2012 were analysed in respect to the MEDDEV 2.12-1 rev 8.

Results: 61 FSCA for the studied IVD were published. German/English FSN were found in 47/60 cases. FSN were characterized as FSN in 46/60 cases and product names were provided in 47/60 cases. Lot-No. and other information for product characterization were available in 39/51 and 40/53 cases. Information on FSCA and product malfunction was found in 47/60 and 47/60 cases. Information on product related risks with previous use of affected IVD was provided in 33/55 cases. In 47/60 cases manufacturers provided information for mitigation of product risks including retesting in 20/29 cases. Requests to pass FSN to persons needing awareness and contact data were found in 24/41 and 37/43 cases. Confirmation regarding CA information and included customer information were found in 14/15 and 37/44 cases.

Conclusion: Most FSN fulfil the MEDDEV criteria but differences between German and English FSN and deficiencies were observed. Due to the importance for risk reduction type and content of FSN should be improved.

P124

Ferritin in Cerobrospinal fluid– The Dimension Vista LOCI® assay as an alternative to a nephpolmetric assay

*A. Petersmann1, E. Thein2, H. Preez1, M. Nauck1, A. Dressel2

1University Medicine Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

2University Medicine Greifswald, Department of Neurology, Greifswald, Germany

Objectives: Ferritin in cerebrospinal fluid (CSF) can serve as a sensitive marker for subarachnoid haemorrhage that occurred more than 12 hours ago. We compared the CSF ferritin assay on the BN ProSpec with the LOCI ferritin assay on the Dimension Vista (both Siemens Healthcare Diagnostics, Eschborn, Germany).

Methods: Routine CSF samples were used after completion of requested tests. For the method comparison as well as the calculation of the coefficient variation (CV) and minimal difference (MD) ferritin was measured in both assays in duplicates. The MD describes the minimal analytical difference which is needed to distinguish between two different results. An imprecision profile was measured twice a day for five days using three CSF pools (11, 17 and 20 µg/L). Reference intervals were established as the central 95th percentile for both assays (inclusion criteria: <5 erythrocytes, <5 leucocytes, normal lactate).

Results: Both assays correlated well (r=0.955) but the LOCI assay gave about 20 % lower results. Likewise the reference intervals were lower. The CV of the BN ProSpec assay ranged from 7.1 % to 9.4 % and for the Vista from 2.7 % to 3.0 %, respectively. The MD for the BN ProSpec was 2.3 µg/L and 0.9 µg/L for the Vista at the decision limit.

Conclusions: We therefore suggest the LOCI ferritin Vista assay as an alternative for ferritin concentration measurements in CSF.

P125

Sample flow optimization in a complex laboratory automation – special emphasis on centrifugation conditions of coagulation samples

J. Suchsland1, A. Grotevendt1, N. Friedrich1, J. Lüdemann1, M. Nauck2, *A. Petersmann1

1University Medicine Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

2Universitätsmedizin, Institut für Klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany

Objectives: To decrease turn around time in laboratory automation system (LAS) centrifugation time of clinical chemistry samples can be limited to 5 min if g is increased to 3300. Integrating coagulation into LAS is limited by pre-analytical requirements. CLSI H21-A5 guideline advises platelet counts (plt) <200 Gpt/l for prothrombin time (PT) as well as activated partial thromboplastin time (APTT) and <10 Gpt/l for other coagulation assays.

Methods: We evaluate centrifugation conditions of coagulation samples by using short routine centrifugation conditions in single and consecutive run.

  • We compared two centrifugation conditions:

  • A: 5 min; 3300g (routine clinical chemistry condition)

  • B: 2 x 5 min; 3300g

  • by analyzing plt, PT, APTT, factor VIII and protein S using random routine samples.

Results: Plt <200 Gpt/l was achieved in all samples by applying centrifugation conditions used for routine clinical chemistry samples (A). A reduction of plt <10 Gpt/L was obtained in 75% of the samples when using these conditions twice (B). Regression analyses showed an equal performance of PT (r=0.987, N=175), APTT (r=0.967, N=171) and factor VIII (r=0.981, N=66) after first (A) and second (B) centrifugation. Protein S (r=0.796, N=54) displayed a poorer correlation, but no systematic deviation.

Conclusion: Our data support integration of coagulation samples into the workflow of a LAS using short centrifugation for routine and consecutive centrifugation conditions for Platelet sensitive coagulation assays.

P126

Evolution to simplicity (Why POCT systems for practical use will have to be a lot simpler than most published results)

*K. Drese1

1Institut für Mikrotechnik Mainz GmbH, Mainz, Germany

In the future POCT systems will spread not only over the stations and satellite labs of hospitals furthermore the usage in the doctor’s office will be very likely. This will definitely also have a big impact concerning the role of central diagnostic labs and the test reimbursement. The spendings in healthcare are always a critical issue and these changes will lead to a direct competition between mobile POCT units and the central laboratory. For these reasons commercial success of Lab-on-a-chip (LoC) based diagnostic systems for point of care testing is determined by the manufacturing costs of consumables and instrument and the resulting price per LoC test. This resembles a big challenge, because for example nucleic acids based tests are having a very long sample processing chain until the final result. If such a process is just directly transferred into a Lab on a Chip system, the manufacturing costs will just explode. The consequence of this is that future POCT systems need to have a simple and cost effective design which corresponds to the required clinical sensitivity of a given assay (and nothing more). This presentation will point to problems and solutions regarding the new technologies, concepts and assay designs. In addition, advices how to develop simple Lab-on-a-chip consumables with a good chance for commercial success will be given.

P127

Retrospective comparison of urine electrolyte-creatinine ratios in a childrens hospital with published reference ranges

*J. Christoph1, E. Kattner1, F. Guthmann1

1Auf der Bult, Hannover, Germany

Question: Is the distribution of the molar urinary electrolyte-creatinine ratio of Na, K, Ca, Cl, and phosphate in children (tested in clinical routine) comparable with published reference ranges?

Materials and Methods: From 2004 to 2012 >2,000 tests of sodium, potassium and chloride, >4,000 of calcium and phosphate as well as >8,000 of creatinine in urine were examined on a Siemens dimension. The 5th, 50th and 90th Percentiles were calculated for the molar electrolyte / creatinine ratio of each parameter in different age groups (Na, K, Cl: N=1326; Ca: N=3915; P N=4053).

Results: The 90th percentile of Na & Cl / creatinine quotient in routine clinical practice is in all age groups outside the reference intervals published by Mersin / Bianchetti 1995 or Bogaru / Guignard 2002. Values from the first month of life were compared with the data of Trotter/Pohlandt.

Our 90th percentiles of Ca & P-creatinine ratios in urine are higher than the values reported by Guignard or Pohlandt. The 5 percentiles are not substantially different from the published reference values.

Conclusion: According to DIN EN ISO 15189:2013-03 5.5.2 the laboratory should define biological reference ranges or clinical decision values for the population served. Results of tests from hospitalized patients can be used to calculate reference intervals (Hoffmann RG: Statistics in the practice of medicine, JAMA 185 (1963), 864-873; cited in Soldin SJ: Pediatric Reference Intervals, AACC Press, Washington DC, 7th Edition, 2011, Preface XIV), especially if a sufficient number of normal values are not available. Clinical results should be compared with published data but they might be affected e.g. by diuretic therapy.

P128

Examination of laboratory value ranges in healthy stem cell donors

*M. Becker1, G. Giers1, F. Wenzel1

1Medical Center of University Düsseldorf, Düsseldorf, Germany

Introduction: The determination of laboratory value ranges is dependent on the examined control group. In allogenic stem cell donation, respective donors are selected by an extensive health questionnaire before invitation to the health check. Therefore the laboratory results of these assumed healthy donors were compared with the established routine laboratory ranges.

Material and Methods: From 200 healthy stem cell donors (n = 100 male, n = 100 female) 179 laboratory parameters (e.g. Hb, MCV, MCH, cholesterol, triglycerides, etc.) per donor were determined using a routine laboratory equipment (haematological parameters were determined by a Cell-Dyn Ruby (Abbott, USA), clinical diagnostic parameters by a Hitachi Chemistry Analyzer (Roche, Switzerland)).

Results: Regarding the haematological values, MCH was correlated to MCV resulting in high MCH values regularly accompanied by high MCV values. In contrast, decreased MCV was accompanied by low ferritin levels in about 5% of the donors. Regarding clinical diagnostics, nearly 50% of the donors showed elevated cholesterol values (235 ± 28 mg/dl, normal range < 200 mg/dl). Additionally these donors had an increased proportion of elevated triglyceride levels (28%) in comparison to the donors showing a normal cholesterol level (10%). In conclusion, the regular correlations of haematological parameters could be clearly reconstructed by the examined results of the healthy stem cell donors enabling the identification of latent iron deficiencies. In clinical diagnostics, the incidence of elevated cholesterol values in combination with elevated triglyceride values may suppose a high proportion of disturbed lipid metabolism in healthy individuals.

P129

Impact of different measurement method of creatinine on patients listed to liver transplantation

*J. Thiery1

1Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

Objectives: The aim of the study was to evaluate the impact of measurement method for creatinine on the result of the MELD-Score and therefore on organ allocation. For most of the patients listed to liver transplantation organ allocation is based on the MELD-Score (Model for End-stage Liver Disease). Since 2006 the MELD-Score has replaced the partially clinical based organ allocation. The MELD-Score consists of three laboratory parameters serum bilirubin, serum creatinine and the INR (international normalized ratio) that are calculated with a formula and rounding in whole numbers.

Methods: In 187 samples of patients taken for MELD diagnostic and messaged to Eurotransplant the MELD-Score was calculated. In each case creatinine measurement happened parallel by the enzymatic method and by Jaffé method. Outcomes of the MELD-Scores measured by the enzymatic method were orderly compared to outcomes measured by Jaffé method. Depending on the MELD-Score the sample collective was divided into the two groups MELD<25 (152 samples) and MELD=25 (35 samples) for statistical analysis.

Results: Outcomes showed that the MELD-Score in the collective MELD=25 varied significantly depending on the measurement method of creatinine. In 22 of 35 samples the calculated MELD-Score differed. In detached cases the difference is up to four points. In the group of MELD<25 the correlation with each other was over all acceptable.

Conclusion: The measurement method of creatinine has an impact on the MELD-Score. In patients with a MELD-Score=25 measurement method of creatinine greatly influences the MELD-Score and results in significant higher values by using Jaffé method.

Metabolische Erkrankungen Poster II

P130

The complement anaphylatoxin C5a receptor (C5aR) contributes to obese adipose tissue (AT) inflammation and insulin resistance

*J. Phieler1, K. Chung1, A. Klotzsche-von Ameln1, R. Garcia-Martin1, A. Chatzigeorgiou2, J. Lambris3, T. Chavakis4

1University Clinic Dresden, TU Dresden, Division of Vascular Inflammation, Diabetes and Kidney, Department of Medicine III, Dresden, Germany

2Mediziniche Klinik III, Universitätsklinikum Carl Gustav Carus, TUD, Dresden, Germany

3University of Pennsylvania School of Medicine, Department of Pathology & Laboratory Medicine, Philadelphia, USA

4Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Anstalt des öffentlichen Rechts des Freistaates Sachsen, Dresden, Germany

Objectives: C5aR has been implicated as regulator of macrophage activation and polarization. However, the role of C5aR on obesity and AT inflammation has not been addressed so far.

Methods: We performed the model of diet-induced obesity (DIO) in mice sufficient or deficient in C5aR. Therefore, 8-9 week old mice were fed a high fat (HFD) or control normal diet (ND) for up to 28 weeks. Insulin resistance development was tested by i.p. glucose tolerance test (GTT) and i.p. insulin tolerance test (ITT). Subcutaneous and peri-gonadal fat pads were excised and the stromal vascular fraction (SVF) was analyzed by fluorescence activated cell sorting (FACS). In addition, excised AT was analyzed by immunohistology and western blot.

Results: In DIO, expression of C5aR was significantly upregulated in the obese AT, as compared to the lean AT. Although lean and obese C5aR-deficient mice had increased adipocyte size obese C5aR-/- mice displayed improved systemic and AT insulin sensitivity, but not hepatic insulin sensitivity. Improved AT insulin sensitivity in C5aR-deficiecy was associated with reduced accumulation of total and pro-inflammatory M1 macrophages as well as decreased intermediate M1/M2 macrophages in the obese AT, increased expression of IL-10 and decreased AT fibrosis.

Conclusion: These results suggest that the C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.

P131

The impact of B7.1-B7.2 double deficiency in obesity-related adipose tissue (AT) inflammation and liver steatosis

*A. Chatzigeorgiou1, K. Chung1, R. Garcia-Martin1, J. Phieler1, S. Grossklaus2, T. Tzanavari3, G. Siegert4, S. Bornstein5, K. Karalis3, V. Alexaki2, T. Chavakis2

1Dresden University of Technology, Department of Internal Medicine III, Dresden, Germany

2MTZ, Carl Gustav Carus, TU Dresden, Department of Internal Medicine, Institute of Clinical Chemistry and Laboratory Medicine, Dresden, Germany

3Biomedical Research Foundation of the Academy of Athens, Athens, Greece

4Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

5University Hospital Dresden, Dresden, Germany

Objectives: Macrophages and lymphocytes are important mediators of AT inflammation and liver steatosis in obesity. The B7.1-B7.2 costimulatory molecules regulate T-cell activation; however, their contribution in obesity-related metabolic deregulation is poorly characterized.

Methods: B7.1 and B7.2-deficient (double knockouts, Dko) or -sufficient mice were fed a high or low-fat diet for 18 weeks. Metabolic blood parameters were measured and GTT and ITT tests were performed. Stromal vascular fraction (SVF) from gonadal and subcutaneous AT was analyzed by FACS and inflammatory gene expression by qPCR. Liver steatosis was investigated by measuring triglyceride levels and liver non-parenchymal cells were isolated and analysed by FACS.

Results: Dko mice showed higher glucose, leptin and cholesterol and impaired GTT when fed a HFD compared to the B7.1/B7.2-sufficient mice. Both subcutaneous and gonadal AT displayed a downregulation of Tregs, CD4 and CD8 lymphocytes and a parallel upregulation of pro-inflammatory M1 macrophages. Dko mice displayed also increased hepatosteatosis, linked with increased M1-like inflammatory macrophages and decreased levels of Tregs in the liver. Higher levels of inflammation and fat accumulation genes in AT and liver respectively were observed after qPCR analysis.

Conclusion: B7.1 and B7.2 significantly regulate adipose tissue inflammation and liver steatosis in the course of obesity.

P132

Medical Diagnosis of Hyperornthinemia-Hyperammonemia-Homocitrullinuria Syndrome

*M. Stopsack1, M. Lee-Kirsch2, M. Smitka2

1TU Dresden / Faculty of Medicine Carl Gustav Carus, Clinical Chemistry and Laboratory Medicine, Dresden, Germany

2University Hospital Carl Gustav Carus at the TU Dresden, Pediatrics, Dresden, Germany

Objectives: Prior to school enrollment a 6 years old boy with a mild mental and statomotoric retardation and multiple generalized tonic-clonic seizures was referred for further diagnostic workup. Both, magnetic resonance imaging of the brain and electroencephalography revealed no pathologic findings. The metabolic work-up yielded a hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH-syndrome; OMIM#238970).

Methods: Amino acid analysis in plasma and urine (Ion exchange chromatography), organic acids (GCMS), Orotic acid (HPLC) and Homocitrulline HCIT (LCMSMS) in urine.

Results: Plasma amino acids revealed clearly elevated Ornithine and high-normal Glutamine. Ammonia concentration once only reached 59.9 µmol/L. Urine analysis additional showed elevated Methionine. As this is possible due to co-elution of HCIT with Methionine, an independent measurement of HCIT established noticeable amounts of this pathognomonic metabolite. Considerable amounts of Uracil and Orotic acid in urine justified suspicion diagnosis HHH-syndrome. This urea cycle defect due to impaired ornithine transportation into the mitochondria was confirmed compound heterocygous for mutations in the SLC25A15 gene, which encodes the mitochondrial ornithine transporter.

Conclusion: The HHH-syndrome is a very rare autosomal-recessive error of metabolism with a wide and variable clinical spectrum. Early diagnosis and consecutive therapeutic interventions can prevent or decelerate further deterioration. This emphasizes the relevance of metabolic testing in patients with mental retardation and epileptic seizures.

P133

Activation of the inflammasome in glomerular cells aggravates experimental diabetic nephropathy

K. Shahzad1, *F. Bock2, C. Wacker1, H. Wang1, S. Ranjan1, J. Wolter1, S. Stoyanov3, K. Reymann3, M. Thati1, B. Isermann1

1Otto-von-Guericke-Universität Magdeburg, Institute for Clinical Chemistry and Pathobiochemistry, Magdeburg, Germany

2University Hospital Heidelberg, Internal Medicine I and Clinical Chemistry, Heidelberg, Germany

3Deutsches Zentrum für Neurodegenerative Erkrankungen, Magdeburg, Magdeburg, Germany

Introduction: Activation of the NLRP3-Inflammosome has been implicated in many diseases including diabetes. Whether inflammosome activation is causally involved in the pathogenesis of diabetic nephropathy (dNP) and whether resident or bone marrow derived inflammatory cells are involved remains to be elucidated.

Methods: Murine models of type 2 and type 1 diabetes were analyzed. dNP was quantified based on albuminuria and histological changes. Expression levels of cl. caspase-1 and IL1b was determined by immunoblot. A group of mice was injected with IL-1 receptor antagonist. In addition bone marrow transplantation experiments were conducted. In vitro markers of inflammosome activation were analyzed in high-glucose stimulated endothelial cells/podocytes at different time points.

Results: In renal cortex extracts of diabetic mice increases of Nlrp3 expression and maturation of IL-1b were observed. NLRP3-/- or caspase1-/- mice as well as anakinra treated mice were protected against dNP. Confocal immunofluorescence labeling revealed that inflammasome activation is enhanced in glomerular cells in mice and humans with DN. The severity of dNP was unchanged in bone marrow chimeras (Nlrp3-/- > db/db, wt > db/db). In vitro we observed time dependent activation of the inflammasome pathway upon glucose stimulation in podocytes and endothelial cells.

Conclusion: Taken together, these results strongly support that activation of the inflammasome in residual glomerular cells contributes to dNP.

P134

Evaluation of the new HEMOGLOBIN A1c assay on the ARCHITECT c8000 instrument

*J. Lotz1, R. Lott1, L. Lennartz2, K. Pick2, K. Lackner1

1Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

2Abbott GmbH & Co. KG Diagnostika, Wiesbaden, Germany

Diagnosis of diabetes mellitus (DM) is based on either fasting plasma glucose or oral glucose tolerance test. Recommendations now include Hemoglobin A1c (HbA1c) value of ≥ 48 mmol/mol as an additional test to diagnose DM. The new ARCHITECT HEMOGLOBIN A1c assay has been developed to fulfill the strict requirements for diagnosing DM. Objective of the study was to evaluate the assay performance and compare results to the routine BioRad HPLC method. The ARCHITECT HbA1c measures glycated and total hemoglobin to determine the percent HbA1c or hemoglobin fraction in mmol/mol (IFCC units) in whole blood (WB). Assay uses an enzymatic method that specifically measures N-terminal fructosyl dipeptides of the HbA1c β-chain after an automatic on-board lysis and proteolytic degradation. Precision assay robustness and method comparison to the routine BioRad Variant HPLC method were performed using surplus WB samples on the routine ARCHITECT c8000 system. The total %CV was 2.2% for sample concentration of 27.4 mmol/mol and 0.5% for 74.6 mmol/mol. For 123 WB samples (31 to 102.4 mmol/mol) linear regression correlation between first versus second ARCHITECT HbA1c replicate was y=1x+0.068 nmol/mol, R=0.999, for BioRad HPLC y=0.998x-2.798 nmol/mol, R=0.989. Less than 2.7 %CV was seen when testing immediately and after 1, 2, 3, 4, 5 hours. The new ARCHITECT HbA1c is a robust, automated method allowing the accurate and precise measurement of HbA1c on the ARCHITECT c8000 instrument.

P135

Age-dependent changes in cerebrocortical insulin response and in insulin transport into the central nervous system

*A. Peter1, T. Sartorius1, M. Heni1, A. Fritsche1, W. Maetzler2, H. Häring1, A. Hennige3

1Institute for Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich at the University of Tuebingen, Member of the German Center for Diabetes Research (DZD), University of Tuebingen, Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry and, Tübingen, Germany

2German Center for Neurodegenerative Diseases (DZNE), Tübingen, Department of Neurodegenerative Diseases and Hertie Institute for Clinical Brain Research, University of Tübingen and, Tübingen, Germany

3Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, Tübingen, Germany

Objectives: Insulin action in the brain determines food intake, physical activity and finally glucose homeostasis. In particular, insulin improved brain activity and promoted locomotion in lean humans and mice while obese individuals are characterized by physical inactivity. In this study we aimed to investigate the effect of aging on the transport of insulin into the central nervous system and the impact on insulin-dependent brain function and locomotion.

Methods: Insulin and glucose concentrations were determined in 162 paired human serum and cerebrospinal fluid (CSF) samples covering a broad range of age. Additionally, insulin was applied for four days by subcutaneous injection in young and aged mice, and assessment of cortical activity and locomotion were performed by radiotelemetry. Analogously, insulin was applied intracerebroventricularly to circumvent the blood brain barrier.

Results: In humans, not only glucose but also CSF insulin concentrations were tightly correlated with the respective plasma concentrations. In contrast to the CSF/serum ratio for albumin which increased with age, the CSF/serum ratio for insulin was reduced in aging subjects, suggesting an impaired transport of insulin into the CSF. As a surrogate for insulin action in the brain, brain activity and locomotion instantly increased in young mice treated with subcutaneous insulin injections. In contrast, the increase in cortical activity and locomotion was delayed in aged mice. Interestingly, when insulin was intracerebroventricularly applied into aged animals, brain activity and locomotion were readily improved.

Conclusion: These data suggest that insulin transport into the central nervous system is impaired in ageing subjects and mice while insulin action in the brain remains intact. Impaired insulin transport into the central nervous system may therefore contribute to cerebral insulin resistance in elderly people.

Molekulare Diagnostik Poster II

P136

Human xylosyltransferases are regulated by miRNA-34a and miRNA-18a

*I. Faust1, K. Böker1, J. Kuhn1, C. Knabbe1, D. Hendig1

1Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinikum der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany

Objectives: microRNAs (miRNAs) and changes in their expression pattern were shown to play emerging roles in the onset and progress of diseases. The aim of this study was to find out whether human xylosyltransferases (XT), catalyzing the initial and rate-limiting step in proteoglycan biosynthesis, are regulated by miRNAs.

Methods: miRNAs predicted to target human xylosyltransferase I and II were selected by in silico analysis. miRNAs miR-34a and miR-18a were chosen for transfection of human dermal fibroblasts for 48h, 72h, 96h and 144h. Relative mRNA transcript levels of human XYLT1 and XYLT2 as well as extracellular matrix proteins were monitored by quantitative real-time PCR. The enzymatic XT activity was determined by a radioactive enzyme assay.

Results: 48h after transfection, we observed a specific knockdown of XYLT1 mRNA expression by miR-34a (20 nM) and of XYLT2 mRNA expression by miR-18a (20 nM) up to 75 %. However, XT activity only slightly decreased after 48h, whereby the effect strengthened with increasing time. A decrease of collagen type I alpha 1 mRNA expression up to 67 % was detected after transfection of miR-34a and miR-18a, respectively.

Conclusion: We demonstrate for the first time a specific downregulation of human xylosyltransferases by miRNAs. It will be of high interest whether the in vivo expression levels of these miRNAs are dysregulated in diseases being referred to an increased XT activity, e.g. fibrosis.

P137

PREOPERATIVE GENOTYPING OF CYP3A5 AND PXR VARIANTS MAY GUIDE POSTOPERATIVE TACROLIMUS DOSING IN HEART TRANSPLANTATION

*D. Lesche1, V. Sigurdardottir2, R. Setoud2, M. Oberhänsli2, T. Carrel3, C. Largiadèr1, P. Mohacsi2, J. Sistonen1

1Institute of Clinical Chemistry, Inselspital Bern, Bern, Switzerland

2Department of Cardiology, Swiss Cardiovascular Centre Bern, Inselspital Bern, Bern, Switzerland

3Department of Cardiovascular Surgery, Inselspital Bern, Bern, Switzerland

Objectives: Identification of biomarkers that allow optimization of immunosuppressive therapy may lead to improved long-term outcome after heart transplantation (HTx). We assessed the role of seven genetic variants in CYP3A4, CYP3A5, POR, PXR, and ABCB1 acting jointly in immunosuppressive drug pathways in treatment response.

Methods: Associations of clinical and genetic factors with tacrolimus (TAC), cyclosporine A (CSA), and everolimus (ERL) dose requirement, as well as with allograft rejection and renal function were retrospectively evaluated in 104 patients at different time points after HTx.

Results: Compared to CYP3A5 non-expressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1) required 2-3-fold higher daily TAC dose to reach the target blood trough (C0) level at all studied time points (1 month to mean 7.6 years after HTx; P≤0.001). In 85% of the CYP3A5 expressors, TAC was withdrawn or switched to a combination therapy with an mTOR inhibitor due to kidney dysfunction or rejection compared to 30% of the non-expressors (P<0.001). The PXR g.-25385C>T variant was additionally shown to decrease the TAC dose requirement in CYP3A5 non-expressors during high corticosteroid exposure (P=0.032). No significant associations between genetic variants and CSA or ERL response were observed.

Conclusions: Inexpensive preoperative genotyping of CYP3A5*3 and PXR g.-25385C>T variants may guide postoperative TAC dosing and prevent unnecessary delay of reaching the adequate TAC-C0 levels.

P138

Detailed genetical analysis of a multidrug resistant hospital pathogen in Leipzig

*K. Finstermeier1, T. Kaiser1, M. Häntzsch1, M. Siegemund1, J. Thiery1, A. Rodloff2

1Uniklinik Leipzig AöR, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

2Uniklinik Leipzig AöR, Institut für Medizinische Mikrobiologie und Infektionsepidemiologie, Leipzig, Germany

Objective: Since July 2010 a continuous outbreak of a multiresistant strain of Klebsiella pneumoniae (KPC-2) appeared at the Leipzig University Hospital. The first patient with an identified infection was transfered from Rhodos (Greece) with a pneumonia. Based on pulsed field gel electrophoresis the strain was identified as subtype 2-5. Initial shotgun sequencing however gave hints of incompatible genomic structures compared to available reference genomes from Genbank.

Methods: The first isolate was shotgun sequenced on the 454 (Roche) and the Pacific Bioscience RS platform with 24.9 and 156.8 fold coverage respectively. Platform specific de-novo assemblies were combined to construct 109 contigs, which were annotated using BASYS and compared to reference genomes.

Results: 5742 genomic and 831 plasmic genes could be found, 1.4% less non-hypothetical genomic genes than the closest related reference K. pneumoniae hs11286. 78 genomic genes are unique to the strain in Leipzig mainly coding for resistance/defense, biosynthesis and metabolism. Large rearrangements within continuous contigs could be identified in plasmids and the genome.

Conclusion: Even with the newest generation of sequencing platforms and available software tools a complete automated assembly of a K. pneumoniae genome is currently too difficult. This persistent strain consists of a huge set of unique genes probably to increase adaptation and resistance to the hospital environment.

P139

4.8 kDa Alpha-1-Antitrypsin peptide – A promising biomarker for sepsis?

N. Blaurock1, *D. Schmerler1, K. Ludewig2, F. Brunkhorst2, E. Giamarellos-Bourboulis3, M. Kiehntopf1

1Universitätsklinikum Jena, Institut für Klinische Chemie und Laboratoriumsdiagnostik, Jena, Germany

2Universitätsklinikum Jena, Institut für Anästhesiologie und Intensivmedizin, Jena, Germany

3University of Athens, Medical School, 4th Department of Internal Medicine, Athens, Greece

Objectives: Systemic inflammatory response syndrome (SIRS) is a leading cause of mortality in the critically ill. Discrimination of sepsis from non-infectious etiologies of SIRS is difficult, because both conditions display identical clinical symptoms. Many biomarkers have been proposed, however most of them have not been validated for clinical routine use or fail to reliable discriminate between infectious and non-infectious SIRS. We identified a proteolytic fragment of alpha-1-antitrypsin (4.8 kDa AAT) as potential discriminatory sepsis biomarker and developed an LC-MS/MS method for its specific quantification.

Methods: 4.8 kDa AAT concentrations were determined in 102 plasma samples of SIRS-, sepsis- and HIV patients as well as in 40 healthy controls by LC-MS/MS. Results were validated in an independent cohort of 120 patients with different sepsis etiologies.

Results: Concentrations of the 4.8 kDa AAT were significantly higher in sepsis patients of different etiologies compared to patients with non-infectious SIRS. No significant differences between the sepsis etiologies were observed.

Conclusion: This study confirms that the 4.8 kDa AAT peptide is a promising biomarker for the differential diagnosis of SIRS and sepsis. In ongoing research peptide concentrations in the time course of sepsis will be evaluated.

P140

Application of the next- generation sequencing technique to diagnostic sequence analysis of the VWF gene. Evaluation and development of protocols.

*S. Sollfrank1, K. Lackner1, H. Rossmann1

1University Medical Center Mainz, Institute for Clinical Chemistry and Laboratory Medicine, Mainz, Germany

Objectives: We designed and validated a 454 sequencing (NGS) assay for mutation detection in patients with suspected Von Wilebrand diesease (VWD) to improve diagnosis and classification.

Methods: A multiplex- based NGS- and a conventional amplicon sequencing- approach for the 52 exons and the promoter region of the VWF gene was established. MLPA (MRC Holland) was used to check for gross deletions of the VWF gene. The blood group 0 allele c.261delG was analysed by a pyrosequencing assay and laboratory specific reference ranges for routine VWD assays were determined for G/- and -/- individuals.

Results: 46 biochemically pre-characterized patients were subjected to molecular genetic testing. NGS results were in agreement with conventional sequencing results for all detected variants. In patients 19 different VWD causing mutations were detected (6 of them not previously described) within 25 individuals. 2 additional variants found in patients are still of unknown clinical significance. In 2 out of those 25 patients the second, compound heterozygous mutation is still missing. Moderately decreased VWF levels in 5 patients were sufficiently explained by blood group 0.

Conclusion: NGS is a reliable, convenient and cost efficient alternative to conventional sequencing of the VWF- gene. c.261delG genotyping and the application of adapted reference ranges to functional assays reduces the sequencing load significantly. Transferring the mentioned VWF- NGS assay to Illumina´s MiSeq was performed in order to compare the yield and the quality of the resulting sequencing data, without altering the current assay. The resulting sequencing data still has to be analyzed with regard to the performance of the different NGS platforms.

P141

Applicability of high-resolution multicapillary electrophoresis for molecular characterization of immune gene rearrangement profiles in acute lymphoblastic leukemia (ALL)

*H. Trautmann1, M. Kozulic2, A. Kruse1, M. Kneba1, M. Brüggemann1

1UK-S-H, Campus Kiel, II. Med. Klinik, Kiel, Germany

2QIAGEN Instruments AG, Hombrechtikon, Switzerland

Objectives: Analysis of clonally rearranged immunoglobulin- (IG) and T-cell receptor (TCR) genes is used to identify markers for minimal residual disease (MRD) diagnostics in ALL. Therefore, products of standardized IG/TCR PCR assays are applied to GeneScan, dHPLC and/or heteroduplex analysis to distinguish polyclonal from clonal rearrangements. In this setting, we tested the applicability of an automated high-resolution capillary electrophoresis, the QIAxcel Advanced system (QIAGEN), as alternative option for fragment analysis.

Methods: The performance of QIAxcel for analysis of IG/TCR PCR products was compared to GeneScan and dHPLC. A panel of 15 multiplex PCRs (BIOMED2), targeting different immune gene rearrangements (6 IGH, -L, -K and 9 TCRB, -G, -D PCRs) was applied to 10 B-lineage-ALL samples resulting in a total of 150 PCR products that were analyzed by QIAxcel, GeneScan and dHPLC. IG/TCR profiles were validated by sequence analysis. Additional sensitivity testing was performed via dilution experiments with patient samples in polyclonal DNA.

Results: Just as dHPLC and GeneScan, QIAxcel accurately detected all amplifiable clonal IG- and TCR-rearrangements (61/150) as well as all polyclonal signals (89/150). In addition, depending on the individual PCR target, the detection limit was 10-5% of clonal cells in a polyclonal background.

Conclusion: The applicability of QIAxcel to identify IG/TCR markers in ALL is on a par with dHPLC and GeneScan. In addition, it reduces manual handling errors, eliminates the need for gel preparation, and ensures electronic documentation of data. Our results indicate that it is a reliable, cost-effective and accurate high-throughput tool for IG/TCR marker characterization in ALL.

P142

Differentially Regulated Candidate Genes at the Chr21q22.11 Locus of Human Atherosclerosis

*B. Kazak1, G. Gäbel2, M. Scholz3, F. Beutner4, J. Thiery4, D. Teupser5, L. Holdt6

1Ludwig-Maximilians-University Munich, Institute of Laboratory Medicine, München, Germany

2Division of Vascular and Endovascular Surgery, Ludwig-Maximilians-University Munich, Munich, Germany

3Institute for Medical Informatics, Statistics and Epidemiology, University Leipzig, Leipzig, Germany

4Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

5Universitätsklinikum der LMU, Institut für Klinische Chemie, München, Germany

6Institut für Laboratoriumsmedizin, Klinikum der Universität München (LMU), München, Germany

Objectives: The Chr21q22.11 locus of human atherosclerosis was first discovered in genome-wide association studies in 2009 and has been replicated several times. The haplotype block spans 134 kbp and contains no protein-coding genes except for transcripts of a long non-coding RNA. In close proximity, protein-coding genes MRPS6, SLC5A3, KCNE1/2 are located. Thus, it was the aim of the current study to unravel potential molecular mechanisms at this locus.

Methods: Genotyping of lead SNP rs9982601 was performed in 2280 subjects of the Leipzig LIFE Heart Study and in 318 endarterectomy specimens of an independent cohort of patients undergoing vascular surgery. Expression of mRNA transcripts was detected with quantitative RT-PCRs detecting specific exon-exon boundaries.

Results: The Chr21q22.11 locus was replicated in the Leipzig LIFE Heart study (P = 0.001). Expression of the ncRNA was directly correlated with the extent of coronary atherosclerosis (P = 0.01). The risk allele at rs9982601 led to increased expression of ncRNA transcripts in human atherosclerotic plaque (P = 0.008). Furthermore, expression of adjacent protein-coding genes was investigated and in part regulated by the Chr21q22.11 genotype (P = 0.04).

Conclusion: We have identified a long non-coding RNA at Chr21q22.11 as novel candidate gene of human atherosclerosis. Further work will be necessary to understand the molecular mechanisms leading to increased atherosclerosis susceptibility.

Neue Biomarker in Metabolomics und Lipidomics Poster II

P143

Kynurenine Pathway: Analytical and preanalytical aspects

*G. Schütze1, M. Schwarz1

1Institut für Laboratoriumsmedizin, Standort TDM-Labor Psychiatrische Klinik und AG Neurobiochemie, Klinikum der Universität München (LMU), München, Germany

The Kynurenine pathway of the tryptophan metabolism comprises several highly important key interfaces in the functional interplay between immune system and neurotransmitter system. The first step in this pathway is mediated by two enzymes: TDO and IDO; the activity of TDO is regulated by glucocorticoides, while distinct cytokines are inducers or inhibitors of IDO. T cell tolerance is under control of IDO and the activity of several cells of both, the innate and the adaptive immune system is regulated by the KYN metabolism. On the other hand, several KYN-pathway intermediates are neuroactive, e.g. kynurenic acid is the only known endogenous antagonist at the NMDAR, while quinolinic acid is an NMDAR agonist and plays a crucial role in the pathophysiology of Alzheimers disease. Therefore, we developed a method for the detection of Tryptophan and twelve metabolites in serum, cerebrospinal fluid, tissue homogenate or cell culture supernatant. The sample preparation is a very cost-effective double-stage protein precipitation. To increase sensitivity, the metabolites with extremely low concentration undergo an additional derivatisation process. Part of the validation of this method was the determination of diverse preanalytical confounding factors. There were significant dietary effects. Moreover different intermediates of the Trp metabolism showed a distinct circadian rhythm. Altogether we established and validated a highly sensitive and specific method for quantitative analyses of both arms of the Trp metabolism. Since previous studies have indicated the potential role of different Trp katabolites in several disease entities, our method will contribute to future biomarker research.

P144

3-Hydroxykynurenine serum concentrations as possible biomarkers Alzheimer’s disease

*M.J. Schwarz1, G. Guillemin2, K. Bürger3, H. Hampel4, S. Teipel5

1Institut für Laboratoriumsmedizin, Standort TDM-Labor Psychiatrische Klinik und AG Neurobiochemie, Klinikum der Universität München (LMU), München, Germany

2Macquarie University, The Australian School of Advanced Medicine, Sydney, Australia

3Klinikum der Universität München, Institute for Stroke and Dementia Research, München, Germany

4Klinikum der Universität München, Klinik für Psychiatrie und Psychotherapie, München, Germany

5Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Rostock, Germany

Increased degradation of tryptophan (TRP) through the kynurenine (KYN) pathway (KP) is discussed to be involved in the molecular mechanisms resulting in the neuropathogenesis of Alzheimer’s disease (AD). Activation of the KP leads to the production of neurotoxic metabolites 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) by immune cells and neuroprotective derivates kynurenic acid (KYNA) and picolinic acid (PIC) by astrocytes and neurons. We therefore investigated whether an imbalance between neurotoxic and neuroprotective kynurenine metabolites could be detected in patients with AD. We measured serum levels of TRP, KYNA, 3-HK, PIC and QUIN in 20 patients with AD and for comparison in 20 patients with major depression, and 19 subjectively cognitive impaired (SCI) subjects. Serum levels of 3-HK were markedly increased in AD patients compared to the comparison groups (p<0.0001). Serum levels of the other KP metabolites were not significantly different between groups. Our data indicate an increased production of the neurotoxic KP metabolite 3-HK in AD. In contrast to its down-stream metabolites QUIN and PIC, 3-HK can cross the blood-brain barrier via an active transport process. Our data therefore indicate an enhanced availability of 3-HK in the brain of AD patients, which may be related to the previously reported higher production of QUIN in AD brains. With a sensitivity of 80% at a specificity of 85% in the ROC analysis, the ratio of serum 3-HK to TRP levels could become useful as a much-sought non-invasive peripheral diagnostic biomarker for AD.

P145

„Added Value“-Determination in Multi-Class Settings

*A. Leichtle1, U. Ceglarek2, P. Weinert3, C. Nakas4, J. Nuoffer1, J. Kase5, T. Conrad6, H. Witzigmann7, J. Thiery8, G. Fiedler9

1Inselspital—Bern University Hospital, Center of Laboratory Medicine, University Institute of Clinical Chemistry, Bern, Switzerland

2Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

3Leibniz Supercomputing Centre, Bavarian Academy of Sciences and Humanities, Garching, Germany

4Laboratory of Biometry, University of Thessaly, Magnesia, Greece

5Charité—Universitätsmedizin Berlin, Department of Hematology, Oncology and Tumor Immunology, Campus Virchow Clinic, Berlin, Germany

6Free University of Berlin, Department of Mathematics, Berlin, Germany

7University Hospital Leipzig, Clinic of Visceral Surgery, Leipzig, Germany

8Uniklinik Leipzig AöR, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany

9Center of Laboratory Medicine, University Institute of Clinical Chemistry, Inselspital—Bern University Hospital, Bern, Switzerland

Objectives: The determination of the “added value” of a new biomarker model compared to an established marker (eg. conventional tumor marker) is a frequent problem and likewise an intricate task. Since even a “significant” difference e.g. in ROC curves might not be “relevant”, several methods of added value determination have been proposed. Whereas these techniques might be favorable and provide insight in a binomial classification setting, they are usually not applicable in multi-class environments. In a clinical three-class example of pancreatic carcinoma, pancreatitis, and health we evaluated the added value of an amino acid model over CA19-9 alone by a resampling-Δ[VUS] approach.

Methods: To determine the added value a combined multinomial logistic model of amino acids and CA19-9 over CA19-9 alone, we determined the three-dimensional volume under ROC surface (VUS) as an equivalent of the 2-dimensional AUROC. By resampling the Δ[VUS] we determined its confidence intervals and visualized them in a forest plot.

Results: With our resampling-Δ[VUS]approach we could show that our combined multinomial logistic model of amino acids and CA19-9 was superior (90%CI: 49-87% Δ[VUS]) to the conventional marker CA19-9 alone (set to 0% ± 5% δ [predefined equivalence range]).

Conclusion: We evaluated the added value of a marker model by a resampling-Δ[VUS]approach in a three-class setting and suggest this technique for comparisons including more than two classes.

P146

Definition of Preanalytical Factors for the Analysis of Sphingoid Bases via Tandem Mass Spectrometry in Human Plasma

*S. Becker1, J. Dittrich2, J. Thiery1, U. Ceglarek1

1Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Universitätsklinik Leipzig, LIFE –Leipzig Forschungszentrum für Zivilisationserkankungen, Universität Leipzig, Deutschland, Leipzig, Germany

2Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Universitätsklinik Leipzig, Leipzig, Germany

Objectives: Sphingolipids are promising biomarkers for the diagnosis and therapy of civilization diseases such as diabetes, coronary heart disease and cancer. For a reliable quantification of sphingosine (SPH), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) we thoroughly investigated the stability and preanalytical factors of these analytes in whole blood, plasma and processed samples under various conditions.

Methods: EDTA plasma was processed by methanolic protein precipitation prior to chromatographic separation on a ZIC-HILIC column (Merck). Tandem mass spectrometric detection was carried out on an API 4000 LC/MS/MS (AB SCIEX) with positive electrospray ionization and multiple reaction monitoring.

Results: Our investigation of preanalytical influences indicated that a storage temperature of 4 °C should be favored for EDTA whole blood and plasma until centrifugation and sample preparation, respectively. Concentration changes for S1P ( 18 %) and SPH (+40 %) have already been observed after the first freeze-thaw cycle, while SA1P remained stable over five cycles. Storage of EDTA plasma should be preferred at -130 °C compared to -80 °C. Processed samples are stable up to 11 hours at room temperature and over five freeze-thaw cycles.

Conclusion: We developed a standardized protocol for the reliable LC-MS/MS analysis of SPH, S1P and SA1P, which takes account of preanalytical factors and is applicable to clinical studies.

P147

Production and release of acylcarnitines by primary human myotubes reflect the differences in fasting fat oxidation of the donors.

*M. Wolf1, S. Chen2, X. Zhao2, M. Scheler3, M. Irmler3, H. Staiger1, J. Beckers3, M. Hrabé de Angelis3, A. Fritsche1, H. Häring1, E. Schleicher1, G. Xu2, R. Lehmann1, C. Weigert1

1University of Tübingen, Department of Internal Medicine, Division of Endocrinology, Diabetology, Angiology, Nephrology, Pathobiochemistry and Clinical Chemistry, Tübingen, Germany

2Chinese Academy of Sciences, Dalian Institute of Chemical Physics, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian, China

3Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Institute of Experimental Genetics, Neuherberg, Germany

Objectives: Acylcarnitines are biomarkers of incomplete β-oxidation but indicate also high rates of mitochondrial fatty acid oxidation (FAO). Our objective was to quantify the acylcarnitine production in primary human myotubes obtained from lean, metabolically healthy subjects with low and high fasting respiratory quotient (RQ).

Methods: Fasting RQ was determined by indirect calorimetry. Muscle biopsies were taken from 6 subjects with low (mean 0.79 ± 0.03) and 6 with high fasting RQ (0.90 ± 0.03), and satellite cells were isolated, cultured, and differentiated to myotubes. Myotubes were cultivated with 125 μM 13C-labeled palmitate for 30 min, 4 and 24 h. Quantitative acylcarnitine profiling was performed by stable isotope dilution-based metabolomics analysis by UHPLC-ESI-qTOF-MS.

Results: Myotubes from donors with high fasting RQ produced and released significant higher amounts of medium-chain acylcarnitines. High extracellular 13C8 and 13C10 acylcarnitine levels correlated with high fasting RQ. The decreased expression of medium-chain acyl-CoA dehydrogenase (MCAD) in these myotubes can explain the higher rate of incomplete FAO. A lower intracellular [13C]acetylcarnitine to carnitine and lower intracellular 13C16/13C18 acylcarnitine to carnitine ratio indicate reduced FAO capacity in these myotubes. Mitochondrial DNA content was not different.

Conclusion: Acylcarnitine production and release from primary myotubes of donors with high fasting RQ indicate a reduced FAO capacity and a higher rate of incomplete FAO. Thus, quantitative profiling of acylcarnitine production in human myotubes can be a suitable tool to identify muscular determinants of fat oxidation in vivo.

Onkologie Poster II

P148

Metastatic (Bone Marrow) Alveolar Rhabdomyosarcoma with Troponin T as a Tumor Marker – a Case Report

*S. Gehrisch1, S. Richter2, M. Zogbaum3, G. Siegert4

1Institut für Klinische Chemie und Laboratoriumsmedizin, Medizinische Fakultät, TU Dresden, Dresden, Germany

2Medizinische Klinik I, Universitätsklinikum Dresden, Dresden, Germany

3Institut für Klinische Chemie und Laboratoriumsmedizin, Stadtkrankenhaus Dresden-Friedrichstadt, Dresden, Germany

4Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, Dresden, Germany

Objectives: Troponin (trop) T and I are highly specific for myocardial cell injuries. However, a patient with progressive metastatic alveolar rhabdomyosarcoma (AR) and no signs of myocardial damages was described with elevated trop T but normal trop I serum levels (Isotalo et al). Here we report a 32 years old man with AR of the left sinus maxillaris and bone marrow metastasis where trop T could be used as a marker of disease progression.

Methods: Serum levels of trop T, CK, CK-MB and CK mass were quantified with test from Roche Diag. (Mannheim). Trop I was analysed with a CMI-assay from Abbott (Wiesbaden).

Results: After an AR diagnosis and a neoadjuvant chemotherapy (epirubicin and ifosamide) a surgery including neck dissection followed by adjuvant radiotherapy was performed. At the end of treatment levels of trop T and CK were almost normal (15 ng/l and 1,0 µmo/l*s). Two months later bone marrow metastasis was diagnosed and a 2nd line chemotherapy was administered. After further disease progression a strongly elevated trop T (400 ng/l) level was measured for the first time. During clinical progression a dramatic increase of trop T level up to 2146 ng/l followed by a rapid decrease to 215 ng/l after chemotherapy could be observed. Levels of trop I were below the cut-off of 28 ng/l at three occasions at this time. CK, CK-MB activity and CK mass reflected the course of trop T. Any myocardial involvement was excluded by myocardial diagnostics including cardio magnetic resonance imaging.

Conclusion: The report shows that in patients with AR the monitoring of trop T and CK serum levels including CK-MB and CK mass can be used as markers for the course of the AR disease.

Ref.: Isotalo PA et al. Clin Chem. 1999;45:1576-8.

P149

Reduced susceptibility to genotoxic stress found in individuals with colorectal hyperplastic polyps

*S. Nittka1, C. Lopez Pinto1, M. Löhr2, M. Neumaier1

1Medical Faculty Mannheim of the University of Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany;

2Karolinska Institutet, Stockholm, Department of Clinical Science, Intervention and Technology, Stockholm, Sweden

Objective: Loss of apoptosis is a hallmark of pre-neoplastic lesions, e.g. of the human colon. We hypothesize that individuals with genuinely reduced susceptibility to apoptotic responses might have an increased risk of developing pre-neoplastic and hyperplastic lesions prior to colorectal cancer. Consequently, genotoxic stress towards peripheral blood mononuclear cells (PBMCs) should indicate individuals with inherently reduced apoptotic susceptibility.

Methods: In a routine clinical setting, we have investigated individuals with (n=23) or without (n=16) endoscopically and histopathologically confirmed, apoptosis deficient hyperplastic colorectal lesions. PBMCs of both groups were subjected to five different apoptotic stimuli including g-irradiation-induced DNA damage and were subsequently analyzed by annexin-V staining and alkaline Comet assay, respectively.

Results: PBMCs from patients with hyperplastic lesions showed reduced apoptosis compared to controls when challenged with camptothecin (p=0.0233) and etoposide (p=0.0031). The initial DNA damage (N0’) induced by g-irradiation in the hyperplastic polyp group was significantly diminished (p=0.0006), although DNA damage repair capacity was unaffected.

Conclusion: Our results strongly suggest that individuals with hyperplastic polyps show a reduced inherent susceptibility to apoptosis induced by genotoxic stress.

P150

Pancreatic stellate cells promote the hapto-migration of pancreatic cancer cells through collagen I-mediated activation of a2ß1 integrin-FAK pathway

*J. Lu1, S. Zhou1, M. Siech2, H. Habisch1, T. Seufferlein3, M. Bachem1

1University of Ulm, Department of Clinical Chemistry, Ulm, Germany

2Ostalb Hospital, Department of General Surgery, Aalen, Germany

3University of Ulm, Department of Internal Medicine, Ulm, Germany

Objective: To investigate the role of pancreatic stellate cells (PSCs) in pancreatic cancer cell (PCC) migration and to identify the underlying mechanisms.

Methods: The effects of PSC supernatant (PSC-SN) and adhesive molecules on trans-migration, adhesion and motility of Panc1 and UlaPaCa cells were investigated by modified Boyden chamber assay, adhesion assay and single cell tracking assay, respectively. Integrin expression and focal adhesion kinase (FAK) phosphorylation were assessed by Western blot. Anti-integrin α2/β1 antibodies and FAK inhibitor (PF-573228) were used to verify the role of collagen I in PCC migration and the intracellular pathway.

Results: PSC-SN dose-dependently induced PCC trans-migration, mainly by improving adhesion and motility. PSC-SN-mediated adhesion was a prerequisite for the stimulation on PCC migration. As pure chemoattractant, PSC-SN was not sufficient to stimulate trans-migration or motility of PCCs. In contrast to poly-L-lysine or fibronectin, collagen I alone showed resembling effects to PSC-SN on PCCs, including polarized morphology, facilitated adhesion, accelerated motility and trans-migration. Both PSC-SN and collagen I induced haptokinesis of Panc1 and haptotaxis of UlaPaCa. Blocking antibodies against integrin α2/β1 subunits significantly attenuated PSC-SN/collagen I-promoted PCC trans-migration and adhesion. PSC-SN and collagen I constantly enhanced FAK phosphorylation (Tyr397) in PCCs. PF-573228 significantly diminished PSC-SN/collagen I-induced PCC migration.

Conclusion: Collagen I is the major mediator for PSC-SN-induced hapto-migration of Panc1 and UlaPaCa cells by activating integrin α2β1-FAK signaling pathway.

P151

In depth re-sequencing of the 5-fluorouracil target in cancer patients: new insights into the thymidylate synthase gene using Sanger and next-generation sequencing

*T.K. Froehlich1, J. Sistonen1, F. Buechler1, S. Aebi2, M. Joerger3, G. Andrey-Zürcher1, C. Largiadèr4

1University Institute of Clinical Chemistry / Inselspital Bern, Bern, Switzerland

2Cantonal Hospital, Department of Medical Oncology, Luzern, Switzerland

3Cantonal Hospital, St. Gallen, Department of Oncology, St. Gallen, Switzerland

4University Institute of Clinical Chemistry / Inselspital Bern, Bern, Switzerland

The fluoropyrimidine 5-fluorouracil (5FU) is among the most commonly used chemotherapeutic agents for the treatment of solid carcinomas. The major target of 5FU is the thymidylate synthase. To date, a comprehensive screening of its gene (TYMS) for variants related to 5FU toxicity is lacking probably due to its challenging structure. Previous association studies have focused on a 28bp variable number of tandem repeats (VNTR) in the TYMS enhancer region (TSER) of the 5’UTR with contradictory results. We re-sequenced the entire genetic region of TYMS in 23 cancer patients treated with 5FU using Sanger sequencing, including 12 patients experiencing severe toxicity with the aim to infer the haplotype block structure of the TYMS and to study the association of the genetic variation with toxicity. Three patients were re-sequenced with next-generation 454 pyrosequencing on a Roche Junior platform and results were compared to data obtained in the framework of the 1000 Genomes Project using other next-generation sequencing techniques. No genetic variation was observed in the exonic regions of the TYMS. A total of 127 intronic variants were detected: 114 SNPs and 13 insertion-deletion polymorphisms, of which 7 SNPs and 5 InDels were novel, and 42 SNPs and 13 InDels had not been characterized by the 1000 Genomes Project. A total of 118 variants with a minor allele frequency ≥0.05 were included in linkage disequilibrium and haplotype block estimation, which revealed two major recombination events: 300 bp upstream of the TYMS initiation codon, and in intron 1. Furthermore, the re-sequencing of the TSER-VNTR in a cohort of 500 patients revealed a different, more polymorphic, TSER structure as so far published in studies using RFLP approaches.

P152

Identification of CEACAM1 isotypes in neoexpressing Malignant Melanoma

*M. Melcher1, B. Hill1, S. Nittka1, M. Neumaier1

1Universitätsmedizin Mannheim, Medical Faculty Mannheim of the University Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany

Objectives: The CEACAM1 (CC1) neoexpression in primary cutaneous malignant melanoma has been reported to be correlated to metastatic progression of the disease. However, there are various structural differences between the 11 known CC1 isotypes (1-4 extracellular domains, transmembrane/soluble and cytoplasmatic ITIM motif or not). This study aimed to analyze melanoma cells for their CC1 isotype expression to clarify conflicting reports concerning the CC1 function.

Methods: A panel of melanoma cell lines from the Mannheim Melanoma Cell Collection (MaMel), a kind gift from Prof. Schadendorf (DKFZ Heidelberg/Mannheim, now Essen) together with well known melanoma cell lines was screened by PCR, Flow Cytometry and SDS-PAGE/Western Blotting for CC1/5/6 expression and the results were compared with recombinant expressing cells and deglycosylated cell lysate to verify the expressed isotypes.

Results: We detected two CC1 isotypes in melanomas, one of them CC1-4L. No sings of CC5 or 6 were found. Comparison with colon cancer cell lines, a tumor entity known to downregulate CC1, showed that the expressed CEACAMs and isotypes are highly different. The overwhelming majority of CC1 positive cells were nodular or amelanotic melanomas (the most aggressive forms).

Conclusion: Our results fit well with those of earlier studies and should additionally help to shed some light on the function of CC1 isotypes as well as their expression in different tissues and tumor entities.

About the article

Published Online: 2013-09-27

Published in Print: 2013-10-01


These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax and usage are the authors’ responsibility.


Citation Information: Clinical Chemistry and Laboratory Medicine, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2013-0737.

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