Congress of Laboratory Medicine and Clinical Chemistry

Evaluation of an enzymatic assay for the detection of gamma-hydroxybutyric acid in urine on a Roche Modular-P system

G Hofmann³, G Weigl¹, I Schluder¹, C Wallner², S-D Schindler², S Pauschenwein-Frantsich³, W Hübl¹

¹Inst. f. Labormedizin, Otto-Wagner-Spital, Vienna, Austria, ²Psychiatrisches Zentrum, Otto-Wagner-Spital, Vienna, Austria, ³Fachhochschule Wiener Neustadt, Wiener Neustadt, Austria

Gamma-hydroxybutyric acid (GHB) is a neurotransmitter naturally present in human metabolism. On the other hand, GHB is used as medication (Alcover^®) in the therapy of alcohol and GHB withdrawal syndromes. The aim of the present investigation was the validation of an enzymatic assay (EA) of the Bühlmann Laboratories AG for automatized GHB detection in urine on a Roche Modular-P system. The evaluation was focused on the technical suitability of the EA as well as in terms of diagnostic urine monitoring of patients following GHB medication. Regarding materials tested, 10 quality control and calibration materials provided by the reagent manufacturer and 22 urine samples of 9 GHB treated patients from the Centre of Psychiatry, Otto-Wagner-Spital, Vienna, were used for this evaluation study. Patient samples were collected between September and December 2013. In terms of technical and analytical aspects, the Bühlmann GHB EA showed a reliable performance on the Roche Modular-P system for the detection of GHB in standard materials and patient urine samples. However, the detection window of GHB in Alcover^® treated patients is short. Positive specimens were just found within two hours after GHB medications in recommended doses (p=0.0095694, fisher-exact-test). Applying the manufacturer’s recommended cut off of 5 mg/L, sensitivity and specificity were 50% and 100%, respectively. It could be shown that sensitivity increased up to 89% by adapting the cut off of the EA ≥1 mg/L, however, interferences with endogenous GHB levels have to be considered.

The tryptophan metabolite picolinic acid suppresses proliferation and metabolic activity of CD4+ T-cells by inhibition of c-Myc activation

J Prodinger¹, LJ Loacker¹, R Schmidt¹, F Ratzinger¹, G Greiner¹, G Hoermann¹, A Neunkirchner², S Jutz², P Steinberger², WF Pickl² and KG Schmetterer¹

¹ Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

² Institute of Immunology, Medical University of Vienna, Vienna, Austria

Tryptophan metabolites such as kynurenine (KYN), 3-hydroxyanthranilic acid (3-OH-AA) and picolinic acid (PA) are key mediators of immunosuppression by cells expressing the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). However, comprehensive studies on the influence of tryptophan metabolites on CD4⁺ T-cells have so far only focused on the effects of KYN and 3-OH-AA. Consequently, we assessed the influence of PA on cell viability, proliferation, cellular metabolism, cytokine secretion and production and up-regulation of surface markers following in vitro activation with agonistic anti-CD3/anti-CD28 antibodies. In contrast to KYN and 3-OH-AA, exposure of T-cells with PA did not affect cell viability, while proliferation and metabolic activity was suppressed in a dose-dependent manner. On the other hand, T-cell functions requiring protein synthesis, such as cytokine secretion and cell surface marker up-regulation, were not or only weakly inhibited by PA. Restimulation experiments revealed that PA-exposed T-cells entered a state of deep anergy which could not be overcome by the addition of exogenous IL-2. In order to define the underlying molecular mechanisms, activation of intracellular signaling pathways was examined using phospho-protein specific flow cytometry and reporter cell lines. These assays revealed that phosphorylation of c-Myc at Ser62, which is indicative of c-Myc activation, was strongly reduced in PA-exposed T-cells following activation. In contrast, more upstream signaling molecules such as the MAP-kinases ERK and p38 and the mTOR target protein S6RP were not affected by PA. Similarly, NFAT and NF-kB promoter activity in Jurkat T-cells was not influenced by exposure to PA. In conclusion, PA mediates a unique immunosuppressive program in T-cells, inhibiting cell cycle and metabolic activity while leaving protein production intact. This functional features are accompanied by reduced activation of c-Myc. It remains to be determined whether this effect is mediated by direct inhibition of ERK kinase activity or whether indirect mechanisms apply.

Constitutively active STAT3 governs tolerogenic functions in CD4+ T-cells

KG Schmetterer¹, A Neunkirchner², J Leitner², P Steinberger² and WF Pickl²

¹ Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

² Institute of Immunology, Medical University of Vienna, Vienna, Austria

STAT3 is a key integrator of signals transmitted from cell surface immune receptors. Yet, the role of STAT3 in CD4⁺ T-cells is not well established. Current research has indicated Th17-inducing but also tolerogenic roles. To assess the effects of STAT3 activation in human T-cells, a constitutively active mutant of STAT3, i.e. STAT3C, was generated and cloned into an IRES-GFP harboring retroviral vector. STAT3C-transgenic CD4⁺ T-cells showed clear-cut transgene expression and, in contrast to FOXP3-transgenic T-cells, phenotypically displayed an effector T-cell phenotype regarding the regulatory T-cell markers CD25, CD39, CD127, CTLA-4, LAG-3 and CD49b. Besides, a significant up-regulation of intracellular granzyme B levels could be observed. STAT3C⁺ T-cells were hypo-responsive following CD3/CD28 or CD3/CD2 stimulation. They further displayed a distinct cytokine secretion profile including a significant increase in IL-10 and a reduction of IFN-γ, IL-2, TNF-α, and IL-13 secretion. Of note, STAT3C⁺ T-cells activated via CD3 and CD2 significantly suppressed effector T-cell proliferation. Similar suppressive activity was observed when STAT3C⁺ T-cells were activated via CD3 and CD28, however, only if the co-stimulus was provided at a suboptimal strength. Mechanistically, suppression was dependent on the release of granzyme B and concomitant apoptosis of effector T-cells. In contrast, IL-10 and TGF-beta were not involved in the suppressive function of STAT3C⁺ T-cells. Our observations are further substantiated by the finding that peripheral blood CD4⁺CD45RA^-LAG3⁺CD49⁺ Tr1 cells reveal activation-induced hyperphosphorylation of STAT3. Furthermore, exposure of Tr1 cells to a STAT3 inhibitor could partially revert their hyporesponsiveness. This indicates a clear-cut relation between activation of STAT3 and the acquisition of a tolerogenic program, which might also be involved in the function of bona fide peripheral blood Tr1 cells.

Are Heart Failure Recommendations And Guidelines Established In Practical Laboratory Medicine In Europe, US And Canada? The CARdiac MArker Guideline Uptake in Europe (CARMAGUE)

^1,12A Hammerer-Lercher, ^2,12P Collinson, ^3,12M P van Dieijen-Visser, ^4,12K Pulkki, ^5,12J Suvisaari, ^6,12A Stavljenic-Rukavina, ^7,12H Baum, ^8,12C Duff, ^9,12KM Aakre, ^10,12MR Langlois, ^11,12S Stankovic, ^5,12P Laitinen

¹Central Institute of Medical and Chemical Laboratory Diagnostics, University Hospital Innsbruck, Austria; ²Departments of Chemical Pathology and Cardiology, St George’s Hospital, London, UK; ³Maastricht University Medical Center, the Netherlands; ⁴University of Eastern Finland and Eastern Finland Laboratory Centre, Kuopio, Finland; ⁵HUSLAB Laboratories, Department of Clinical Chemistry, Helsinki University Central Hospital, Finland; ⁶University of Zagreb, Zagreb, Croatia; ⁷Regionale Kliniken Holding RKH GmbH, Germany; ⁸Department of Clinical Biochemistry, University Hospital of North Staffordshire, Stoke-on-Trent, UK; ⁹Haukeland University Hospital, Bergen, Norway; ¹⁰Asklepios Core-lab, Department of Laboratory Medicine, AZ St-Jan Hospital Bruges and Ghent University, Ghent, Belgium; ¹¹Center for Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia; ¹²European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Cardiac Markers

Introduction: The well established heart failure (HF) markers B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are widely used by clinicians, and analytical and clinical recommendations for measurement of these biomarkers have been published. The aim of this survey (closed in August 2014) was to investigate how well these guidelines for measurement of NP have been implemented in laboratory practice in Europe and as a comparison in United States (US) and Canada.

Method: Member societies of European Federation of Clinical Chemistry and Laboratory Medicine were invited in 2013 to participate in a web-based audit-questionnaire. The questionnaire requested information e.g. on type of tests performed, reason of method choice, decision limits for HF and accreditation status.

Results: Of all participating laboratories, preliminary results show that there were 29% and 41% university laboratories in Europe and US/Canada, respectively. In Europe, 305 responders out of 494 measured NP as well as 64 out of 135 US/ Canadian responders. NT-proBNP was most widely used in Europe (67% of NP offering laboratories) and BNP slightly more (55%) in US/Canada. The reason was availability of instrument in both regions, and in Europe additionally stability and clinician preference. The preferred methods were Roche and Abbott in Europe, and in US/Canada Roche as well as Abbott and Siemens Advia Centaur. Most laboratories used ng/l or pg/ml and pmol/l was only rarely stated as unit. More than half of responders used the recommended BNP rule-out cut-off of 100 ng/l for acute HF, however, less than one third of responders used the recommended NT-proBNP cut-offs (<50 years: 450 ng/l, 50-75 years: 900 ng/l, >75 years 1800 ng/l). In Europe, age dependent decision limits for chronic HF were reported only in one third of responders and were mostly stated to be the same as for acute HF. Also in US/Canada mainly the same cut-off levels for acute and chronic HF seemed to be applied. There were still laboratories not performing external quality assurance in Europe (Europe: EQA 11%; US/Canada: EQA: 1%). In Europe, one third of laboratories were not accredited for NP, whereas in US/Canada almost all were.

Conclusion: NP became important HF markers available in more than half of the responding laboratories. However, recommended cut-off values for acute HF were still not adequately implemented. Further, accreditation is an ongoing process in Europe, and EQA concerning HF biomarkers remains an important issue to be solved in European laboratories.

Elevated Carbohydrate deficient Transferrin in abstinent Pregnant women measured with Capillary Zone Electrophoresis. Investigation for a new reference limit.

D Neumann-Richter, TK Felder, HG Mustafa

Labor Dr. Mustafa, Dr. Richter, Salzburg

Reference limits are essential in laboratory medicine. Routine tests have well established reference limits that are mostly gender-specific, age-related and method-specific. Concerning pregnant women existing reference limits sometimes need to be reviewed and if indicated have to be adjusted. Carbohydrate Deficient Transferrin (CDT) is a routinely used test with a well defined reference limits for different methods to detect increased alcohol consumption. Detection methods are Carpillary Zone electrophoresis (CZE) or High pressure liquid chromatography (HPLC). Elevated CDT-values in verifiable abstinent pregnant women have been reported by several authors. Some of them are suggesting a new reference limit in the last trimester of pregnancy. Concerning this matter there did not take place any investigation measuring CDT in pregnant women with CZE until now. The aim of this research was to reevaluate the existing reference limit of CDT for CZE and to find out if a new reference limit for pregnant women is necessary.

Methods: 330 serum samples were collected. Samples were divided into non-pregnant women (healthy group) and pregnant women. The latter group was divided into the three different trimesters of pregnancy. One part of the samples where pregnant women who where continuously examined during pregnancy. Inclusion criteria besides being female and being capable of bearing children (max age 50 years) were at least a normal gamma-Glutamyltransferase (GGT). For the group of healthy, not pregnant women additional criteria were to have a normal MCV (mean corpuscular Volume), MCH (mean corpuscular Hemoglobin), Hkt (Hematokrit), Aspartat-Aminotransferase (ASAT) and Alanin-Aminotransferase (ALAT). Blood was taken by venous puncture into serum tubes. After at least 30 min clotting they were centrifuged for 10 min with 1370 rcf (relative centrifugal force (3000U/min)). Serum was collected and stored in aliquots of minimum 2 ml at -20°C without interruption. All samples were examined with CZE Minicap (Sebia, EVRY Cedex France) and 260 samples were additionally confirmed by the currently recommended method HPLC. (Variants where excluded from statistical analysis and replaced by new samples). Differences between each of the groups were statistically analyzed. According to the guidelines of CLSI and IFCC statistically significant results were confirmed by further investigation concerning existing reference limit.

Result: Minicap (Sebia, EVRY Cedex France) reference limits are based on Schellenberg et al. Schellenberg defined a cut-off value (1,3% CDT), a “grey zone” (1,3%-1,6% CDT) and a reference limit (>1,6%). This means values >1,6% are equal to the right sided 95^th percentile and proof an alcohol consumption of 50-60g ethanol per day. Based on this existing reference limits first, second and third trimester samples were compared with the control group of non-pregnant women. p-values <0,05 were considered to be statistically significant. When comparing 1^st Trimester group with control group we were not able to find significant difference (p-value: 0,986.) Comparison of 2^nd and 3^rd trimester and the control group showed a p-value <0,0001. Data concerning 2^nd Trimester women showed a statistically significant difference but the calculated 95^th percentile (confidence interval 90) revealed a value of 1,5%. As above mentioned this value is situated in the above mentioned “grey zone” and therefore cannot be considered as a proof for alcohol consumption. In conclusion we focused on the values of the 3^rd Trimester women also in accordance with the results of previous publications. As the 95^th percentile of the first 70 3^rd trimester-samples calculated with 1,7% was above the recommended upper reference limit the number of analysis in the 3^rd trimester was then raised up to 120 patients (following the recommendations of CLSI (NCCLS)) (Fig. 1, Tbl. 1). Statistical analysis of the total of 120 patients then showed a mean 1,3% and the 95. Percentile was 1,6%. Alongside analysis of the 20 pregnant women who were tested 3 times during pregnancy showed an increasing CDT (Fig. 2). Furthermore 260 samples were controlled with HPLC. The slope coefficient was 0,743 intercept 0,007.

Conclusion: The existing CDT% reference limit can be used for pregnant women including 3^rd Trimester. However it has to be mentioned that the existing reference limit of 1,6% and the in this work determined 95^th percentile of 1,6% with median of 1,3% is very close to the existing reference limit. If pregnant women are suspicious for occult drinking additional analysis should be made in order to protect the unborn child on the one hand and to protect women from false accusation on the other hand. We also propose that the manufacturer should mention a possible alteration of CDT in pregnancy.

Interference of a widely used drug with enzymatic assays: Case report and investigation

P Würtinger, L Loacker, W Prokop, G Weigel, A Griesmacher

Institute of clinical and chemical laboratory diagnostics (ZIMCL), University Hospital Innsbruck, Innsbruck, Austria

Interferences of several organic substances with the measurement of creatinine by the Jaffe reaction (kinetic alkaline picrate method) are widely known, which is why they are generally replaced by the enzymatic method. We report two cases of interferences with common enzymatic assays like, amongst others, the enzymatic creatinine method by a frequently used drug.

Blood samples of a 69-year old male with femoral neck fracture as well as a 56-year old male suffering from lumbago were taken and several routine analyses were performed. In both patients, creatinine levels as determined by the enzymatic method were below the detection limit. However, subsequently creatinine determination by Jaffe reaction showed values in the normal range. Due to these major deviations between both methods used for creatinine determination, patient’s clinical records were investigated and revealed the administration of metamizole (Novalgin) in both patients. The structure of metamizole and its main metabolite methylaminoantipyrine are practically identical to that of aminophenazone, a substance used in several enzymatic assays as auxiliary substrate in the indicator reaction. Based on this observation, the influence of metamizole on enzymatic assays using aminophenazone was examined in mixing studies. Therefore, eleven heparin plasma spare samples of patients who gave informed consent were prepared to contain metamizole at final concentrations of 10, 25, 40 and 80 mg/l as these concentrations seem to reflect therapeutic levels. Creatinine (enzymatic assay and Jaffe reaction), triglycerides (Wahlefeld method), uric acid (modified Town method), cholesterol (Roeschlau method), lactate (lactate oxidase reaction) and glucose (hexokinase method and glucose-oxidase/peroxidase method) were determined on a Cobas 8000 analyzer with appropriate reagents (Roche Diagnostics, Mannheim, Germany). The presence of 80 mg/dl metamizole led to an underestimation between 2-12% of all analytes except creatinine by Jaffe reaction as well as glucose by the hexokinase method.

Because of the various and frequent administration of metamizole the assessment and interpretation of enzymatic assays that are in routine use in clinical chemistry should always be accomplished with awareness of this possible drug interference. Considering the pharmacological half-life of metamizole, selection of enzymatic assays containing aminophenazone as substrate might be reasonable at least several hours after metamizole administration. Higher metamizole levels, as they must be assumed in our patients, can be induced by quite often occurring mistakes during blood draw. In consequence of these findings the manufacturer initiated a re-evaluation of the affected enzymatic tests and a modification of the respective package inserts.

Oral glucose tolerance test within the scope of prenatal care: Evaluation 2010 – 2012

E Krendl, L Mustafa

Medical-chemical laboratory, Dr. Mustafa-Dr. Richter OG, Salzburg, Austria

Background: General screening for gestational diabetes mellitus (GDM) is recommended in Austria since 2010. As a result of the guidelines, pregnant women are tested between 24 and 28 weeks of gestation with the 75 g/2 h-oral glucose tolerance test (75 g/2 h-OGTT) [1]. The aim of this study was to evaluate the prevalence of GDM in our laboratory retrospectively. Furthermore, we wanted to study the pattern of abnormal 1 h- and 2 h-glucose values from 75 g/2 h-OGTTs compared with fasting plasma glucose values. Further testing of GDM patients after delivery is recommended. As a result of this issue we analyzed all follow-up screening.

Methods: Standardized 75 g/2 h-OGTTs were assessed in 3963 pregnant women. The cut-off value for fasting plasma glucose (FPG) is 5.1 mmol/L, for 1 h value 10.0 mmol/L, and for 2 h value 8.5 mmol/L. One or more abnormal values were considered as GDM, respectively.

Results: GDM was detected in 8.5% (n = 335) of the tested pregnant women. Elevated FPG values were measured in 5.1% (n = 201). These are 60% of all GDM patients. After delivery we analyzed 14 out of 335 GDM patients (4.2%) to reevaluate postpartum glucose tolerance with the standard OGTT (World Health Organization criteria).

Conclusions: GDM is a common disease, and in our study 8.5% of pregnancies were affected. When and how to screen is still a matter of discussion. Different strategies to become more cost-effective and patient friendly are in progress (for example a two-step screening algorithm including FPG measurement and a risk estimation model [2]). In postpartum follow-up, there is still considerable potential to reduce diabetes-associated illness and costs.

Kautzky-Willer A, Bancher-Todesca D, Pollak A, Repa A, Lechleitner M, Weitgasser R. Gestationsdiabetes (GDM). Wien Klin Wochenschr 2012;124:58–65.

Göbl CS, Bozkurt L, Rivic P, Schernthaner G, Weitgasser R, Pacini G, et al. A two-step screening algorithm including fasting plasma glucose measurement and a risk estimation model is an accurate strategy for detecting gestational diabetes mellitus. Diabetologia 2012;55:3173–81.

Serologic markers of autoimmunity in women with recurrent pregnancy loss

K Hefler-Frischmuth¹, B Fabian², K Walch³, L Hefler⁴, C Tempfer⁵

¹Labor für hämatologische Spezialdiagnostik, Krankenhaus der Barmherzigen Schwestern Linz, Austria

²Orgentec Diagnostika GmbH, Mainz, Germany

³Universitätsklinik für Frauenheilkunde Wien, Austria

⁴Abteilung für Gynäkologie, Krankenhaus der Barmherzigen Schwestern Linz, Austria

⁵Universititätsfrauenklinik der Ruhr-Universität Bochum, Germany

Objective: Recurrent pregnancy loss (RPL) is the loss of 3 or more spontaneous and consecutive pregnancies before 16 weeks of gestation. There are many causes, such as genetic, anatomic, hormonal, and also immunologic causes (1). Autoimmunologic processes have been hypothesized to be involved in various ovarian pathologies such as idiopathic infertility, endometriosis, and the polycystic ovary syndrome (2) and also in the pathogenesis of RPL. Regarding antiphospholipid syndrome, pregnancy losses are one of the characteristics of this autoimmune disorder. The aim of the present study was to contribute further data on autoimmunologic processes and to elucidate potential mechanisms for their interrelation in RPL investigating a panel of serologic autoimmunologic parameters in affected women.

Methods: In this prospective case-control study we investigated serum levels of IgG class antinuclear antibodies (ANAs), serum levels of IgG class autoantibodies against histones, IgG class autoantibodies against nucleosomes, IgG class autoantibodies against double-stranded (ds) DNA, IgG/IgM/IgA class autoantibodies against cardiolipin and IgG/IgM/IgA class autoantibodies against ß2-glycoprotein I in 120 women with RPL and 107 age-matched healthy controls. All parameters were measured by enzyme immunoassays (Orgentec Diagnostika, Mainz, Germany).

Results: Mean age at diagnosis was 31.9 years (SD=5.4). With respect to serum levels of anti-ß2-glykoprotein I, levels of < 10 and ≥ 10 U/ml were regarded as negative and positive tests, respectively. Women with RPL had significantly elevated quantitative serum levels of anti-ß2-glykoprotein I [4.23 (5.87) vs 3.06 (1.33) U/ml, (p=0.04)]. Hence, 7 women with RPL were tested positive for anti-ß2-glykoprotein I, whereas none of the healthy control women (p=0.01). No differences were ascertained regarding serum levels of ANAs, serum levels of antibodies against histones, antibodies against nucleosomes, antibodies against dsDNA, and antibodies against cardiolipin.

Discussion: Autoimmunological phenomens have been hypothesized to be involved in the etiology of RPL. Of note, “standard” autoimmunologic laboratory parameters presumably only measure a small minority of these processes. In our study we only ascertained an already previously known significant difference between patients with RPL and healthy controls regarding anti-ß2-glykoprotein I (3). The “standard” autoimmunologic antibodies targeting the cell nucleus were not different between cases and controls.

References:

1. Bansal AS, Bajardeen B, Shehata H, Thum MY. Recurrent miscarriage and autoimmunity. Expert Rev Clin Immunol 2011;7(1):37-44.

2. Hefler-Frischmuth K, Walch K, Huebl W, Baumuehlner K, Tempfer C, Hefler L. Serologic markers of autoimmunity in women with polycystic ovary syndrome. Fertil Steril 2010;93(7):2291-4.

3. Sater MS, Finan RR, Abu-Hijleh FM, Abu-Hijleh TM, Almawi WY. Anti-phosphatidylserine, anti-cardiolipin, anti-ß2 glycoprotein I and anti-prothrombin antibodies in recurrent miscarriage at 8-12 gestational weeks. Eur J Obstet Gynecol Reprod Biol 2012;163(2):170-4.

HEp-2 cells cut off: SLE versus non inflammatory rheumatic diseases

W Klotz, M Herold

Medical University of Innsbruck, Department of Internal Medicine VI, Austria

Indirect immunofluorescence (iIF) is the method of choice to detect antinuclear antibodies (ANA) in patients with autoimmune diseases. Since 1997 a serum dilution of 1:100 is used to discriminate patients with systemic lupus erythematodes (SLE) from healthy individuals (HI) by ANA titre (1). Recently (2) it was again recommended that each laboratory has to evaluate the cut off with respect to differences on reagents, equipment and other local factors.

Persons without any sign of an autoimmune disease (blood donors, n=29; outpatients of rheumatology unit, n=22; elderly people, n=340) were tested and compared with SLE patients (outpatients, treated; n=114).

ANA were measured using iIF on HEp-2 cells (INOVA Diagnostics, Sacramento), starting at a screening dilution of 1:40 and increasing dilution stepwise to end titre. Optimal cut off was estimated by calculation of the 95^th percentile and by visual examination of a histogram blotted against end titre dilution and determined from 114 patients suffering from SLE. If the 95^th percentile is calculated using blood donors and patients with non inflammatory rheumatic symptoms the optimal cut off is 1:80. If aged people are also included in the control group the cut off has to be raised to 1:160. A visual examination of the results drawn in a histogram also yields an optimal cut off titre of about 1:100.

In agreement with first recommendation given 1997 also in our patient population a cut off titre of about 1:100 is optimal to discriminate LE patients from persons who are not affected by systemic inflammatory autoimmune diseases.

Our results reveal two interesting points. Once, a serum dilution of 1:100 which was used in our laboratory ever since as upper normal range is right. And second, our results are identical with results found in a different laboratory many years ago. We doubt that every laboratory has to find its individual normal range.

1. Tan EM et al. Arthritis Rheum. 1997;40:1601-11.

2. Agmon-Levin N et al. Ann Rheum Dis. 2014;73(1):17-23.

Determination of antibodies to native DNA

W Klotz, M Herold

Medical University of Innsbruck, Department of Internal Medicine VI, Austria

Anti native DNA (nDNA,dsDNA) antibodies are important in the diagnosis of systemic lupus erythematodes (SLE) and included in the ­classification critheria of this diesease.

nDNA antibodies can be measured by serveral methods including indirect immunofluorescence (iIF) on crithidia luciliae (crithidia lucilia immunofluoreacence test, CLIFT), radioimmuno assay (Farr Assay) or solid phase immunoassays (ELISA). Results of nDNA assays may vary both between methods as well as between immune assays of different manufacturers.

50 samples of patients suffering from SLE, 18 sera out of an external quality control program and 24 consecutive samples sent to our laboratory for measurement of nDNA antibodies were analysed using iIF on Crithidia luciliae slides (CLIFT) of two manufacturers (INOVA Diagnostics, San Diego, USA; Menarini Diagnostics, Firenze, Italy), an automated chemiluminescent immunoassay (QUANTA Flash dsDNA, INOVA) and an automated solid phase immunoassay (EliA dsDNA, Phadia, Uppsala, Sweden).

Results of 24 consecutive samples (12 of them positive and 12 negative for nDNA antibodies) were 100% concordant between the two CLIFT assays. Agreement between the two automated assays was 82%. Accordance between CLIFT and the QUANTA Flash assay was 90%, the relative sensitivity for the QUANTA Flash assay 88% and relative specifity 91% setting CLIFT as gold standard. Accordance between CLIFT and EliA assay was 84%, the relative sensitivity for the EliA assay 90% and relative specifity 75% with CLIFT as gold standard.

In conclusion the two CLIFT assays give identical results whereas results from the two automated immunoassays may differ both between the two assays and between the automated assay and CLIFT.

Indirect Immunfluorescence – Roboter vs. Human Eye

J Hofmann

Donauspital im SMZ – Ost, Institut für Labormedizin, Wien

For many years, autoimmune diagnostic has been one of the core competences in the area of special analytics at the Institute for Laboratory Medicine of the SMZ Ost-Donauspital. This is traditionally a laboratory area with various analytical systems, an immunofluorescence microscope and several manual work steps. In the context of an increasing demand for the diagnosis of autoimmune diseases and the transfer of this area of diagnostics from other hospitals, the implementation of an automatical indirect immunfluoerscence ( IIF) system could provide a solution for the consolidated performance of these analyses.

Indirect immunofluorescence (IIF) as the primary screening method is managed bi-directionally online via the ZEN-IT software by means of primary barcode identification without any risk of mix-ups. The slides are prepared in the ZENIT UP. The automated microscope ZENIT G-SIGHT scans the slides’ individual application areas and evaluates these via the associated software. The system is able to discriminate negative samples in a comprehensive manner. This is of particular importance when these analyses are requested not only by specialised outpatient clinics, but also by departments that use autoimmune analysis for diagnosis by exclusion (with a corresponding high share of negative findings). The dilution of positive samples can be selected directly. In addition, further serological analyses can be requested.

A point to be particularly emphasised is the complete documentation of all IIF images. These are an important aid for observing the patients’ course and for discussing the findings with the referring departments.

After one year of experience, it is possible to say that an automatical IIF-system permits a considerable step forward in the area of automated processing, discrimination of negative samples and documentation of the analyses.

Recommendations to ANA Diagnostic in Austria

M Herold, W Klotz

Medical University of Innsbruck; EASI Austria, Vienna

Background: The Austrian EASI group (Ulrike Demel, Georg Endler, Ernst Forster, Andrea Griesmacher, Manfred Herold, Jörg Hoffmann, Christin Hübner, Sonja Wagner, Günter Steiner; http://www.easi-network.com/dia_templates/Page____406.aspx) decided to give recommendations to Austrian laboratories working with autoimmune diagnostics in addition to recent published international recommendations concerning the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies (ANAs; Agmon-Levin N et al. Ann Rheuma Dis 2013).

Methods: The paper was discussed, listed patterns compared to pattern descriptions used in laboratories in Austria, missing patterns were added and German wording of English phrasing was discussed.

Results: Some patterns (e.g. large speckled, some patterns related to the mitotic apparatus; some nucleolar patterns) are added. Patterns are described in German. Whether the original English patterns should be added in brackets is in discussion.

Conclusions: There is a common consensus that a standardized description of patterns is useful and necessary. Non-English speaking countries have a problem of defining words to describe patterns according to international consensus and understandable to doctors used to English descriptions. Rare patterns are easy to be seen but difficult to describe and recognize.

IgG-anti-IgA- Antibodies and Immunodeficiency

U Demel

Department of Internal Medicine, Division of Rheumatology and Immunology, Graz, Austria

Common variable Immunodeficiency (CVID) comprises a heterogenous group of diseases of unknown cause. A significant hypogammaglobulinemia is the most important laboratory sign. Men and women are affected equally, the prevalence is 1/25000 among Caucasians and 1/100000 among Asians. The major peak of onset can be in early childhood but also during second and third decade of life. Most patients suffer from recurrent bacterial infections concerning bronchitis, sinusitis, otitis and pneumonia. Chronic pulmonary damage is one of the major complications, but 25% of patients develop autoimmune phenomena (immuno thrombocytopenic purpura or autoimmune hemolytic anaemia). Splenomegaly and lymphadenopathy are presented by 40% of patients. Reasons for CVID can be due to an intrinsic B cell defect, intrinsic T cell defect or without a known genetic defect.

A curative therapy does not exist. Treatment is based on immunoglobulin replacement therapy, which can be administered intravenously or subcutaneously. The way of immunoglobulin replacement depends on patient’s situations concerning veins and the titres oft IgG-anti-IgA- antibodies.

The first description of IgG-anti IgA –antibodies was made in 1968 (Vygas et al.) when anaphylactoid reactions in patients treated with intravenous immunoglobulins occured. Even patients receiving immunoglobulins subcutaneousely were described. The significance of IgG-anti-IgA-antibodies is still a contentious issue.

Nevertheless IgG-anti-IgA-antibodies are a useful tool in patients suffering from CVID as they can help making a decision which way of immunoglobulin replacement therapy should be given.

MedUni Wien Biobank – Promoting scientific output by centralized storage facilities

H Haslacher, F Ratzinger, M Repl, E Ponweiser, O Wagner, T Perkmann

Medizinische Universität Wien, Klinisches Institut für Labormedizin, Wien

The increasing knowledge in the areas of personalized and evidence-based medicine leads to several logistic problems. On the one hand, large sample sizes are needed in order to achieve high statistical power. Since this can take several years to complete(1), the error caused by pre-analytical variance must be minimized to improve the comparability of the results(2). During the last decades, professional facilities called “Biobanks”, which are specialized in sample handling and storage have been established to face these problems at various locations(3). However, a recent survey showed a high level of fragmentation of the European Biobank landscape(4). Among the 126 of 176 participating Biobanks, governance issues as e.g. informed consent, sample coding, ownership and access were considerably different. The next goal must be to harmonize these standardized procedures in order to allow for large-scale scientific collaborations. Thus, large consortia have emerged to consolidate these Biobank activities. One of those consortia is BBMRI (Biobanks and Biomolecular Ressources Research Infrastracture), which acts as a Central Research Infrastructure comprising of national hubs. The Austrian hub, which is funded by the Federal Ministry of Economy, Research and Science, is formed by the Biobanks of the Medical Universities as well as the Alpe-Adria University of Klagenfurt and the Life Science Governance Institute. Within this large project, the most significant issues are addressed in several work packages, ranging from standardization of sample and data logistics as well as ethical and legal aspects to the implementation of quality management systems(5). The latter workpackage is operated by the MedUni Wien Biobank, which was founded in 2007 as a central facility operated by the Departments of Laboratory Medicine, Clinical Pathology and Neuropathology. Its main purpose is to assist medical investigators with logistic issues in the context of clinical studies. So far, approximately 1,000,000 liquid samples have been prospectively stored at the Department of Laboratory Medicine within more than 40 different collections. Sample quality is enhanced by standardized procedures, which are planned, monitored and perpetually improved by the Department’s quality management system according to ISO 9001 and ISO 15189. The present work will give some insight in the development of a specific biobank within the setting of a clinical laboratory and depicture the potential fundamental role of biobanks for research and innovation in the future.

References:

1. Burton PR, Hansell AL, Fortier I, Manolio TA, Khoury MJ, Little J, et al. Size matters: just how big is BIG?: Quantifying realistic sample size requirements for human genome epidemiology. Int J Epidemiol. 2009 Feb;38(1):263-73.

2. Lippi G, Guidi GC, Mattiuzzi C, Plebani M. Preanalytical variability: the dark side of the moon in laboratory testing. Clin Chem Lab Med. 2006;44(4):358-65.

3. Ollier W, Sprosen T, Peakman T. UK Biobank: from concept to reality. Pharmacogenomics. 2005 Sep;6(6):639-46.

4. Zika E PD, Schulte in den Bäumen T, Braun A, RijKers-Defrasne S, Deschênes M, Fortier I, Laage-Hellman, J SCA, Ibarreta D. Biobanks in Europe: Prospects for Harmonisation and Networking: European Commission Joint Research Centre, Institute for Prospective Technological Studies; 2010.

5. Haslacher H. Biobanking auf Europäischer Ebene. SPECTRUM Onkologie. 2014;Sonderausgabe Personalisierte Medizin.

“Enzyme-only” detected red blood cell alloantibodies in pre-transfusion testing

D Enko¹, C Habres², G Kriegshäuser¹, R Stolba¹, F Wallner¹, G Halwachs-Baumann¹

¹Department of Laboratory Medicine, Central Hospital Steyr, Steyr, Austria; ²University of Applied Sciences for Health Professions Upper Austria, Bachelor Program Biomedical Science, Central Hospital Steyr, Steyr, Austria

Background: In Austria the “blood-depositories” (hospital blood banks without donation and screening facilities) are mainly managed by specialists for laboratory medicine, transfusion medicine, anesthesiology and internal medicine. Differences of opinion exist among the “blood-depositories” regarding the use of an enzyme-test for the pre-transfusion testing. Therefore the present study was conducted to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC) alloantibodies in patients hospitalized in Austria.

Methods: In total 2420 patients underwent routine blood group testing with the RBC alloantibody screening and antibody identification during the admission to the Central Hospital in Steyr (Austria). The patients were mainly adults and in very few instances children. They were tested for the ABO blood group, the Rh antigen, and the RBC alloantibodies. The antibody screening was performed with a three-cell-panel in the low-ionic strength-saline (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) three-cell-panel fully automated on the ORTHO AutoVue^® Innova System (Ortho Clinical Diagnostics, Raritan, New Jersey). The antibody identification was carried out manually with an eleven-cell-panel in the LISS-IAT and with an enzyme-pretreated (papain) eleven-cell-panel. All patients with a positive RBC antibody screening result were tested for the Rh antigens C, c, E, and e.

Results: In total, 4.05% (n = 98) of all the patients (n = 2420) had a positive RBC antibody screening result. Of these 25.51% (25/98) showed “enzyme-only” detected RBC alloantibodies or a non-specific enzyme reaction in the antibody identification. Rhesus and Lewis system alloantibodies were found the only specificities of “enzyme-only” anti-erythrocyte alloantibodies. In total, 4.08% (4/98) were found with anti-E, 3.06% (3/98) with anti-Le^a, 3.06% (3/98) with anti-D after anti-D prophylaxis and 1.02% (1/98) were found with anti-e. All in all, 14.29% (14/98) showed a non-specific enzyme reaction.

Conclusion: In the routine testing Rhesus and Lewis system alloantibodies were found the only specificities of “enzyme-only” detected RBC alloantibodies accompanied by a high number of unwanted non-specific enzyme reactions. Compared to the existing literature, the majority of “enzyme-only” detected RBC alloantibodies are considered to play not such a clinically significant role in the etiology of hemolytic transfusion reactions compared to RBC alloantibodies detected with the IAT. Each hospital “blood-depository” should make its own pragmatic decision whether the enzyme-test is justified or not in their patient population in daily clinical practice.

Acquired von Willebrand syndrome caused by left ventricular assist devices is Adamts-13 and Platelet dependent

P Jilma¹, P Quehenberger¹, H Schima², M Stoiber², P Knöbl³, B Steinlechner⁴, S Schranz⁵, B Jilma⁵

¹Clinical Institute of Medical and Chemical Laboratory Diagnostics; ²Center for Medical Physics and Biomedical Engineering; ³Division of Haematology and Haemostasis, Department of Internal Medicine I; ⁴Division of Cardiothoracic and Vascular Anaesthesia, ⁵Clinical Pharmacology; Medical University of Vienna

The high shear rates induced by left ventricular assist devices cause acquired von Willebrand syndrome (aVWS) (1). We hypothesised that an ex vivo model could be established to study whether mechanical shear alone causes aVWS or whether this process depends also on the VWF cleavage protein ADAMTS-13 and on platelets.

Healthy volunteers and two patients with congenital ADAMTS-13 deficiency donated blood. In vitro closed extracorporeal circuits were established using medically approved left ventricular assist devices (Heartware®). VWF multimers were quantified by gel electrophoresis, VWF antigen, ristocetin cofactor activity (VWF:RCo), ADAMTS-13 levels and platelet function were assessed. The high shear stress in the extracorporeal circulation rapidly decreased VWF:RCo and thereby the VWF:RCo/VWF:Ag ratio by 47% (p<0.01) to pathologically low values. Concomitantly, high molecular weight multimers (HMWM) decreased: up to 14-15 mers were visible on the gels at baseline, which were reduced by a maximum of 6-7 mers, corresponding to an average 68% lower densitometry signal of HMWM (p<0.01). This was accompanied by a 3-fold reduction in ristocetin and ADP induced aggregation (p<0.01). In contrast, the two patients with congenital thrombocytopenic purpura with virtually complete deficiency of ADAMTS-13 activity had only a minimal or no decrease in multimers (p<0.05 vs. healthy controls). After circulation of only platelet poor plasma, no depletion of large multimers was observed. A model for LVAD associated aVWS was established, which demonstrated that ADAMTS-13 activity and platelets are essential for the depletion of HMWM of VWF that occurs in VAD-associated aVWS.

References:

1. Steinlechner B, Dworschak M, Birkenberg B, et al. Platelet dysfunction in outpatients with left ventricular assist devices. The Annals of thoracic surgery 2009;87(1):131-7.

Evaluation of a new generation of commercially available vitamin D assays

D Enko, D Rinner, E Worf¹, N Dirsch³, G Kriegshäuser¹, E Rezanka¹, R Stolba¹, G Halwachs-Baumann¹

¹Department of Laboratory Medicine, Central Hospital Steyr, Steyr, Austria; ²University of Applied Sciences for Health Professions Upper Austria, Bachelor Program Biomedical Science, Steyr, Austria; ³RECIPE Chemicals + Instruments GmbH, Munich, Germany

Background: Considering the difficulties of 25-hydroxy-vitamin D (25[OH]D) measurements in the past, this study was conducted to compare recently released new 25(OH)D assays, that are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM). Therefore the IDS-iSYS 25(OH)D (Immunodiagnostic Systems Ltd) chemiluminescence immunoassay (CLIA), the recently released follow-up IDS-iSYS 25(OH)D^S (Immunodiagnostic Systems Ltd, Boldon, United Kingdom) CLIA and the new ORGENTEC 25(OH)D3/D2 (ORGENTEC Diagnostika GmbH, Mainz, Germany) enzyme linked immunoabsorbent assay (ELISA) were compared among and with the ClinMass^® liquid chromatography-tandem mass spectrometry (LC-MS/MS) Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany).

Methods: In total 198 blood samples were parallel measured by the recalibrated IDS-iSYS 25(OH)D^S, the previous used IDS-iSYS 25(OH)D (CLIA) (Immunodiagnostic Systems Ltd), the ORGENTEC 25(OH)D3/D2 ELISA (ORGENTEC Diagnostika GmbH), and the ClinMass^®LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH). The study population mainly consisted of adults and a total of five adolescents between 15 and 19 years. No newborns or young children were included. Pearson’s correlation coefficients, Bland-Altman plots and Deming regression analyses were calculated. A p-value < 0.05 was considered statistically significant.

Results: The results of the correlation analysis graded from the highest to the lowest Pearson correlation coefficients were 0.89 (IDS-iSYS 25[OH]D versus IDS-iSYS 25[OH]D^S assay), 0.82 (IDS-iSYS 25[OH]D^S assay versus ClinMass^®LC-MS/MS Complete Kit), 0.81 (IDS-iSYS 25[OH]D assay versus ClinMass^®LC-MS/MS Complete Kit), 0.78 (ORGENTEC 25[OH]D3/D2 assay versus ClinMass^®LC-MS/MS Complete Kit), 0.75 (IDS-iSYS 25[OH]D^S versus ORGENTEC 25[OH]D3/D2 assay) and 0.72 (IDS-iSYS 25[OH]D versus ORGENTEC 25[OH]D3/D2 assay) (p < 0.001 for all comparisons). Modest systematic bias was observed ranging from a positive mean bias of 6.63 ng/mL (ORGENTEC 25[OH]D3/D2 assay versus ClinMass^®LC-MS/MS Complete Kit) to a negative mean bias of 5.77 ng/mL (IDS-iSYS 25[OH]D versus ORGENTEC 25[OH]D3/D2 assay).

Conclusion: The highly positive correlations and the low-to-moderate mean biases demonstrated among the tested 25(OH)D assays are a clear indicator for a widespread introduction of a well standardized new 25(OH)D assay generation in clinical laboratories within the next few years. The Vitamin D Standardization Program will further improve interassay-variabilities of different 25(OH)D assays used in daily clinical routine.

Comparison of immunoglobulin heavy/light chain ratio with other quantitative assays for diagnostic and disease monitoring in multiple myeloma patients

J Radek¹, V Aliskanovic¹, P Birkner¹, M Wiesholzer², P Balcke², D Trubert-Exinger¹, A C Haushofer^1,3

¹Institute of Laboratory Medicine, University Hospital St. Poelten, Austria

²First Department of Medicine, University Hospital St. Poelten, Austria

³Institute of Medical and Chemical Laboratory Diagnostics with Blood Bank, Klinikum Wels-Grieskirchen, Austria

Background: Hevylite™ is a method to identify the different light chain types of each immunoglobulin (Ig) class. The different light chain types of each immunoglobulin class (for example IgAκ and IgAλ were determined to calculate the ratio of the monoclonal/polyclonal immunoglobulin (HLCR) ¹. The aim of the study was to determine and evaluate the sensitivity of the Hevylite™ assay in patients with positive serum immunofixation electropheresis (sIFE) and to compare it with other quantitative assays which were used so far for disease monitoring². Therefor we compared the total immunoglobulin values, the total light chains values and the serum free light chains.

Patients and Methods: 134 serum samples with a positive IFE result were tested. sFLC and HLC were quantified using polyclonal antisera assays on the BN II nephelometer. Absolute values of IgG, IgA and IgM and light chains were analyzed on the BN II nephelometer. sIFE was performed using the Sebia Hydrasys 2 system.

Results: A pathological HLCR was given in 78.9% (IgG), 81% (IgM) and 90.3% (IgA) of the patients with a positive sIFE result. The values of the absolute immunoglobulin were pathological in 50.8% (IgG), 76% (IgM) and 77.4% (IgA) of the sIFE positive patients. The determination of the absolute light chains ratio was also less accurate than the HLC-ratio. There was accordance with the positive sIFE results in 69.6% (IgG), 28.6% (IgM) and 70% (IgA) of the patients. In comparison to the determination of the absolute light chains ratio the FLC-assay could detect 100 % of the sIFE positive samples.

Conclusion: The HLCR showed the highest accordance with the positive sIFE results of the patients compared to the conventional assays. We could show that the HLCR is a more sensitive method than the determination of the absolute immunoglobulin values and the absolute light chains values. We identified patients who still had normal physiological absolute immunoglobulin- and absolute light chains values whereas the HLCR was already abnormal. As previously shown this indicates that the HLCR detects the presence of residual disease or an evolving relapse earlier than the other quantifying methods³.

1. Bradwell AR, Harding SJ, Fourrier NJ, Wallis GL, Drayson MT, Carr-Smith HD et al. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios. Clin Chem 2009; 55: 1646–1655.

2. Dimopoulos M, Kyle R, Fermand JP, Rajkumar SV. San Miguel J, Chanan-Khan A et al. Consensus recommendations for standard investigative workup: report of the International Myeloma Workshop Consensus Panel 3. Blood 2011; 117: 4701–4705.

3. Ludwig H, Milosavljevic D, Zojer N, Faint JM, Bradwell AR, Hübl W, Harding SJ. Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients. Leukemia 2013;27: 213-219.

Comparison of the eSensor^® Respiratory Viral Panel to duplex real-time PCR assays for detection of viral respiratory pathogens

E Stelzl, M Bozic, V Sinner, HH Kessler

Research Unit Molecular Diagnostics, Center of Applied Biomedicine, Medical University of Graz

Background: Rapid and accurate diagnosis of upper and lower respiratory tract infections improves the patient’s outcome through avoidance of unnecessary prescription of antibiotics and hospital care. The eSensor® Respiratory Viral Panel (RVP) multiplex assay (GenMark) is a qualitative assay for simultaneous detection of 7 viruses based on multiplex reverse transcription PCR amplification of extracted nucleic acids followed by detection of amplification products on the eSensor® XT-8 instrument.

Objectives: To compare the eSensor® RVP to duplex assays routinely used in the diagnostic laboratory. The accuracy of the new assay was evaluated with panels of secondary reference material, the clinical performance was compared to that of the routinely used assays. Additionally, times-to-result including the automated and the manual times were compared.

Materials and methods: Accuracy of the new technology was determined using proficiency panels provided by an international external quality assessment program (QCMD). For evaluation of the clinical performance, 96 respiratory specimens that remained following routine clinical testing were tested. For comparison, results obtained by duplex assays of the respiratory Multi Well System (Argene/bioMerieux) were used.

Results: When accuracy was tested with 7 QCMD proficiency panels, all results were found according to the expected results. Analysis of 96 routine clinical samples confirmed results from routine analyses with the duplex standard assays in 87 samples (90.6%). Viruses detected by both methods included adeno-, human metapneumo-, influenza-, respiratory syntycial-, and rhinoviruses. Nine samples (9.4%) showed discrepant results. When times-to-result were compared, the eSensor® RVP required 5.25 h compared to 2.7 h for the duplex assays. Time required for manual steps was similar for both assays; however, two additional manual steps (exonuclease digestion and preparation for detection) were required for the eSensor® RVP.

Conclusions: The new multiplex assay showed good accuracy and clinical performance. Further automation and shorter times-to-result are required for future use in the routine diagnostic laboratory.

Clinical utility of infection biomarkers in neutropenic SIRS patients at the standard care ward

F Ratzinger¹, H Haslacher¹, T Perkmann¹, K G Schmetterer¹, WPöppl², D Mitteregger³, G Dorffner⁴, H Burgmann²

¹ Department of Laboratory Medicine, Division of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria

² Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna Austria

³ Department of Laboratory Medicine, Division of Clinical Microbiology, Medical University of Vienna, Vienna, Austria

⁴ Section for Artificial Intelligence, Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna

Background: Neutropenic patients are at high risk of developing a severe infection. In these patients, parameters with a high negative predictive value (NPV) are especially desirable to exclude infection or bacteraemia. The present study evaluated the predictive value of sepsis biomarkers for infection or bacteraemia in neutropenic patients suffering from a systemic inflammatory response syndrome (SIRS).

Material and Methods: In a prospective observational cohort study, patients with clinically suspected sepsis were screened. Clinical data and sepsis biomarkers, including procalcitonin (PCT), C-reactive protein (CRP) and lipopolysaccharide binding protein (LBP) of neutropenic SIRS patients were analysed and compared with non-neutropenic SIRS patients. For selecting an appropriate control cohort, propensity score matching was applied, balancing confounding factors between neutropenic and non-neutropenic SIRS-patients.

Results: Within 2,384 prospectively screened patients with suspected infection, 51 suffered from neutropenic SIRS. For the identification of infection, none of the assessed clinical parameters and biomarkers presented adequate discriminatory potency. To discriminate between non-bacteremic and bacteremic SIRS patients with neutropenia, LBP, CRP and PCT presented a discriminatory capacity (ROC-AUC: 0.860 (CI: 0.753-0.967), 0.781 (CI: 0.639-0.924) and 0.818 (CI: 0.683-0.913), respectively). When maximizing the sensitivity to 100%, LBP still reached 58% (57.9% (CI: 40.8%-73.7%) specificity. In neutropenic patients, LBP had a significantly better ROC-AUC than in a comparable non-neutropenic patient cohort for identifying bacteremic patients (0.606 ROC-AUC, CI: 0.442-0.769).

Conclusion: In neutropenic SIRS patients, none of the evaluated biomarkers was able to identify infection. LBP and PCT presented a good performance in identifying bacteremia. Further research is necessary to elucidate the better performance of LBP in neutropenic than in non-neutropenic SIRS patients.

Azithromycin suppresses CD4⁺ T-cell activation by direct modulation of mTOR activity

F Ratzinger¹, H Haslacher¹, W Poeppl², G Hoermann¹, J J Kovarik³, S Jutz⁴, P Steinberger⁴, H Burgmann², W F Pickl⁴ and K G Schmetterer¹

¹ Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

² Division of Infectious Diseases and Tropical Medicine, Department of Medicine I, Medical University of Vienna, Vienna, Austria

³ Clinical Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria

⁴ Institute of Immunology, Medical University of Vienna, Vienna, Austria

Background: Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4+ T-cells.

Material and Methods: Isolated CD4+ T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of different macrolides. Cell proliferation was measured using thymidine incorporation assays and fluorescent tracers. Cytokine secretion was assessed in corresponding supernatants using multiplex analysis. Cell viability was quantified by annexin V/propidium iodide staining. Intracellular signaling pathways were evaluated using Jurkat reporter cell lines, FACS analysis and immunoblotting. Furthermore, the effect of AZM on mTOR activity was evaluated in an in vitro kinase assay.

Results: AZM inhibited cell proliferation rate and cytokine secretion of CD4+ T-cells in a dose-dependent manner. At therapeutic concentrations, no reduction of cell viability was observed. In contrast, CLM showed only minor effects on T-cell function. Analysis of molecular signaling pathways revealed that treatment with AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target mTOR (mammalian target of rapamycin). In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this effect was independent of FKBP12 (FK506-binding protein 12).

Conclusion: We show for the first time that AZM acts as immunosuppressive agent on CD4+ T-cells at therapeutically relevant concentrations. Moreover, we demonstrate that this effect is mediated by inhibition of mTOR activity. Our results might have implications for the clinical use of AZM. This immunomodulatory effect of AZM should be profoundly assessed in clinical studies.

The use of T-SPOT.TB in the screening for M.tuberculosis specific immune responses in patients with rheumatoid arthritis

V Fagerer, L Mustafa

Med. chem. Labor Dr. Mustafa, Dr. Richter OG, Salzburg, Austria

Different pathways of immune response are involved in the infection with Mycobacterium tuberculosis (M.tuberculosis). These include the activation of T-cells which in turn produce interferon-γ (IFN-γ), a cytokine playing an important role in the elimination of M.tuberculosis. During chronic inflammation, such as rheumatoid arthritis, immune response is mediated primarily by the cytokine tumor necrosis factor (TNF). Individuals who are treated with TNF antagonists have been shown to be of increased risk of reactivating latent infections, especially tuberculosis, thus supporting a screening for M.tuberculosis specific immune responses prior to anti-TNF therapies. A two step approach is supported, involving a tuberculin skin test (TST, Mendel-Mantoux-Test) and an interferon-γ release assay (IGRA) (1,2,).

In this study, we evaluated the T-SPOT.TB (Oxford Immunotec, Oxford, UK) to screen individuals with rheumatoid arthritis for a latent tuberculosis infection (LTBI), who are going to undertake TNF-alpha inhibitor therapy. A total of 148 patients was tested, 89,2% being candidates for treatment with anti-TNF. Negative test results were observed in 87,8% of these patients, 4.1% patients had a positive IGRA, which was followed by treatment with Isoniazid (INH) prior to TNF-alpha inhibitor therapy. In 1 patient, who had a negative test result at the beginning of the TNF-alpha inhibitor therapy, panuveitis was reported after 3 months of therapy with Etanercept (Enbrel®). Panuveitis is known to be associated with rheumatoid disease, but it in this case it has to be considered, that panuveitis can also be consistent with the re-activation of a latent TB during anti-TNF therapies. INH therapy was started, although it was not possible to obtain clinical specimen to isolate M.tuberculosis by performing a TB culture or by PCR methods.

Reassessing, our data prove, that the T-SPOT.TB plays an important contributive role in the diagnosis or exclusion of latent tuberculosis in immunocompromised patients.

1. Tuberkulose & Biologika. Consensus statement. ÖÄZ Supplementum (2011) S.1-12

2. Use of interferon-gamma release assays in support of TB diagnosis. ECDC Guidance (2011) S. 1- 32

Phosphopeptide Enrichment on a Monolithic Column Containing Superficially Immobilized Nano TiO₂

A Fichtenbaum, T Panić-Janković, W Chen, S Seyfert, A Führer, A Pungor, Z Nagy, U Černigoj, J Vidič3, M Barut, O Wagner, G Mitulović

Medical University of Vienna, Clinical Institute of Laboratory Medicine, Vienna

Protein phosphorylation plays an important role in regulating a broad spectrum of key physiological processes like cell signaling, cell growth and cell differentiation. Prior to LC/MS analysis, enrichment of phosphopeptides is a crucial step because of their low stoichiometry in biological sample, longer retention on reversed phase columns, and lower ionization efficiency compared to non-phosphorylated peptides. The use of metal oxides, most prominently of TiO₂ enabled efficient and relatively simple phosphopeptide-enrichment. In this study a new monolithic column from BIA Separations packed with immobilized TiO₂-nanoparticles was tested for its ability to enrich phosphopeptides. The TiO₂-column was also tested for possible carryover originating from biological samples. The enrichment procedure was conducted according to an altered and optimized protocol described by Mazanek et al. (1), sample was loaded onto the column using a syringe pump and five fractions were collected. Briefly: the flow-through fraction, two wash and two elution fractions. Resulting fractions were re-injected onto the reversed phase nano separation column and detected with MS. Results show that the “blank” column was unable to discriminate between the phosphorylated and unphosphorylated peptides. In conclusion, tested monolithic TiO₂ columns show significant binding ability for phosphopeptides and are considered as suitable for phosphopeptide enrichment. Additional optimization steps will be applied for better prevention of carryover and for more efficient peptide trapping by using different loading buffers.

Acknowledgement: This work was suported by European Union Seventh Framework Programme, Grant Prot-HiSPRA # 282506

References

1. Mazanek M, Roitinger E, Hudecz O, Hutchins JR, Hegemann B, Mitulovic G, et al. A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes. Journal of chromatography B, Analytical ­technologies in the biomedical and life sciences. 2010;878(5-6):515-24. Epub 2010/01/16.

Improving Prediction of In-Vitro-Fertilization Success: Looking for Putative Biomarkers in In-Vitro-Fertilization-Media upon Embryo Cultivation

T Panić-Janković¹, A Lobas², M Gorshkov², A Fichtenabum¹,W Chen¹, D Pietrowski³, S Seyfert¹, O Wagner¹, G Mitulovic¹

¹Clinical Institute of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

²Russian Academy of Science-INEP, Moscow, Russia

³Clinical Department for Obstetrics and Gynecology, Medical University Of Vienna, Vienna, Austria

Embryo selection for transfer is the most important step of assisted reproductive technology. The determination of the viability of embryos is performed using morphological criteria like cleavage rate and blastomere symmetry (1). Because morphology alone cannot determine the molecular signature of an embryo, multiple implantations are necessary in order to increase the probability of successful fertilization. This often leads towards multiple pregnancies, thus proving to be a risk factor for both mother and offspring. Progress in proteomics allows discovery and identification of biomarkers in various diseases analyzing proteins derived from small samples. Here, we report about our efforts to analyze the secretome of human embryos used in infertility treatment. The secretome of human embryos was compared between embryos that were labeled as morphologically positiv, negativ and mixed on different days of cultivation. Sample preparation included two filtration steps with different MW cutoff followed by lysinC and trypsin digestion. Separation and detection of peptides was performed using liquid chromatography and mass spectrometry. The database search and analysis was performed using MASCOT and Scaffold. We have identified 45 proteins secreted by developing embryos. The number of identified proteins lowered with progressing embryo incubation time, irrespective of the morphological estimation of those embryos. By comparing identified proteins in the embryo secretom, the differences in the pattern of secreted proteins were observed in morphologically different embryos. For example, serotransferrin was detected only in the secretom of embryos which were labeled as morphologicaly negativ. It is known that transferrins are responsible for the transport of iron from sites of absorption and hems degradation to those of storage and utilization. Serotransferrin may also have a role in stimulating cell proliferation. So far, only one study has been published on the effect of iron on fertility, which is based on the Nurse’s Health Study. We can conclude that the proteome based analyses of embryonaly secreted proteins can provide a useful tool to understand the processes during early embryo development. It may also help to define embryo derived distinct protein profiles causative for embryo pathologies.

Acknowledgements: This research has been funded by European Union within FP7 Project Prot-HiSPRA, 282506

References

1. Rienzi L, Ubaldi F, Iacobelli M, et al. Significance of morphological attributes of the early embryo. Reprod Biomed Online 2005;10:669–81.

2. Sylvia S. Cortezzi & Jerusa S. Garcia & Christina R. Ferreira & Daniela P. A. F. Braga & Rita C. S. Figueira & Assumpto Iaconelli Jr & Gustavo H. M. F. Souza & Edson Borges Jr & Marcos N. Eberlin. Secretome of the preimplantation human embryo by bottom-up label-free proteomics. Anal Bioanal Chem (2011) 401:1331–1339

3. M.G. Katz-Jaffe, S. McReynolds, D.K. Gardner1 and W.B. Schoolcraft. The role of proteomics in defining the human embryonic secretome. Oxford Journals, Life Sciences & Medicine, MHR: Basic science of reprod. Medicine, Volume 15, Issue 5, Pp. 271-277

Fast and simplified methodology for analysis of street and novel designer drugs

KM Ostermann^1,2, A Luf ³, NM Lutsch^1,2, R Dieplinger^1,2, TP Mechtler^1,2, TF Metz^1,2, R Schmid³, G Mitulivic³, O Wagner³, David C Kasper^1,2

¹Department of Pediatrics and Adolescent Medicine, Medical University of Vienna

²Research Core Unit Pediatric Biochemistry and Analytics

³Clinical Institute of Laboratory Medicine, Medical University of Vienna

Introduction: Illicit drug markets across Europe experienced a steadily increasing number of novel designer drugs in recent years (1). The knowledge of the composition of these “bath salt” drugs is essential for drug prevention institutions and health-care professionals i.e. clinics and emergency wards. Therefore, comprehensive analytical methods for rapid analysis of street drug samples need to be developed and implemented. The Viennese addiction prevention project ‘checkit!’ offers on-site analysis of psychoactive substances for consumers anonymously and free of charge (2). The data acquired provide valuable insights into the drug market and consumption patterns.

Methods: We used Matrix-Assisted Laser Desorption/Ionisation (MALDI) High-resolution accurate mass (HR/AM) mass spectrometry (MS) for the analysis of 80 drug samples of unknown content obtained through “checkit!” at music events. We compared our method with the’checkit!’ on-site procedure, which employs four portable HPLC-UV-DAD systems including LC-MS coupling. An’in-house’ in silico reference database containing all identified substances to match acquired spectra was created using ExactFinder® software. Calibration curves were created to enable quantification of substances with MALDI-HRMS.

Results: In total, 80 street drug samples were analyzed. Subsequent data analysis identified 50 pharmacologically active compounds. The newly developed MALDI-HR/AM-MS method confirmed all substances identified by the’checkit!’ onsite screening method. In addition, 16 substances not detected during on-site analysis were identified by applying the new method. The serial dilutions of the reference substances cocaine, lysergic acid diethylamide, levamisole, and papaverine yielded a%CV of 0.2-14 and correlation coefficients of 0.95-0.99.

Conclusions: The developed analytical methods facilitate simple and rapid analysis of complex mixtures of psychoactive substances without elaborate sample preparation and present a complementary method and a valuable alternative to conventional methods. Moreover, the hereby acquired data can be used to guide health care professionals in interventions concerning drug related conditions.

References

1. EMCDDA (2014) European Drug Report, http://www.emcdda.europa.eu/publications/edr/trends-developments/2014

2. Rosenauer R., Luf A., Holy M., Freissmuth M., Schmid R., Sitte H.H. A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content. ACS Chem. Neurosci. 2013;4:182–190.

Fast Micro/Nano LC-MS/MS for Analysis of Midazolam, Fentanyl, Alfentanil, and Remifentanil, and their Metabolites in Blood

G Mitulović, O Wagner, R W Schmid

Clinical Institute of Laboratory Medicine, Medical University of Vienna and General Hospital Vienna, Wien

Administration of benzodiazepines and opiates is a successful combination when sedation and analgesia are needed for surgery and for patients’ support during their stay in an intensive care unit and during the ventilation support. In case of patients being in prolonged depressed mental state, it is of great importance to monitor the concentration of these substances in patient’s blood. Once the medication has been stopped it must be determined whether the depressed state is caused due to prolonged action of medication or because of neurological damage. Further, not only the principle substance is active, their metabolites can also enhance their potency and lead to fatal result during the surgery or during the transportation from the site of accident to the hospital [1, 2].

We have developed analytical method (micro HPLC-MS/MS) for fast analysis of multiple analytes and their metabolites in blood. Sample preparation was very simple: upon protein precipitation with acetonitrile, the supernatant was concentrated by evaporation in a vacuum centrifuge, re-dissolved in mobile phase, and injected onto the C18 micro column. Detection was performed using positive electrospray ionization mass spectrometry. All measurements were performed using single ion monitoring.

All analytes and their metabolites were separated and detected within 7 minutes by applying isocratic chromatographic run. Total time for sample preparation, chromatographic separation, and analysis was 45 minutes. This time can further reduced by adjusting sample preparation (e.g. using turbo-flow chromatography instead of protein precipitation and concentration) and using UHPLC with sub-2μm particles for fast separation.

Reference:

1. Portier, E.J.G.;K. de Blok;J.J. Butter, and C.J. van Boxtel, Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection. Journal of Chromatography B: Biomedical Sciences and Applications, 1999. 723(1–2): p. 313-318.

2. Ghassabian, S.;S.M. Moosavi;Y.G. Valero;K. Shekar;J.F. Fraser, and M.T. Smith, High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC–MS/MS. Journal of Chromatography B, 2012. 903(0): p. 126-133.

Lactose malabsorption testing: Genetic test (C/T_-13910 polymorphism) versus hydrogen/methane breath test

D Enko, G Kriegshäuser¹, E Rezanka¹, R Stolba¹, G Halwachs-Baumann¹

¹Department of Laboratory Medicine, Central Hospital Steyr, Steyr, Austria;

Background: The non-absorbable disaccharide lactose is hydrolyzed into the monosaccharides glucose and galactose by the lactase enzyme in the small intestine brush border. The primary lactose malabsorption is an inherited autosomal recessive form and results in a decline of lactase enzyme activity. A single nucleotide polymorphism (C/T_-13910) 14 kb upstream from the lactase gene (LCT) locus is associated with this most common phenotype found in humans. The secondary lactose malabsorption is an acquired and reversible form associated to gastrointestinal diseases. The present study was conducted to compare the genetic test (C/T_-13910 polymorphism) and the combined hydrogen/methane (H₂/CH₄) breath test results of lactose malabsorption testing.

Methods: All in all 263 patients were included in this retrospective study. Inclusion criteria were the parallel measurement of the combined H₂/CH₄ breath test and the genetic test (C/T_-13910 polymorphism), a twelve hour overnight fasting and abstaining from smoking. Patients who have completed antibiotic therapy were excluded for at least four weeks from the breath test. The C/T_-13910 polymorphism was performed using a melting curve analysis on the LightCycler Instrument (Roche Diagnostics, Rotkreuz, Switzerland). After the ingestion of 50 g lactose, gas-chromatography (QuinTron Model DP Plus MicroLyzer™ [QuinTron], Milwaukee, Wisconsin, US) was employed to measure the end-expiratory breath H₂ and CH₄ concentration at 15, 30, 45, 60, 75, 90 and 120 minutes. The breath test result was considered positive if the H₂ and/or the CH₄ peak was > 20 ppm over the baseline value.

Results: A total of 51 patients (19.4%) had a C/C_-13910 genotype, indicating primary lactose malabsorption. Only 19 patients (7.2%) had also a positive H₂/CH₄ breath test. In total 136 patients (51.69%) had a C/T_-13910, and 76 patients (28.91%) a T/T_-13910 genotype, indicating lactase persistence. Four patients (1.5%) with the C/T_-13910 genotype and one patient (0.4%) with the T/T_-13910 genotype had a positive H₂/CH₄ breath test result, indicating secondary lactose malabsorption. The sensitivity of the genetic test compared to the gold standard lactose breath test was 79%, the specificity 87%, the positive predictive value 60%, and the negative predictive value was 98%. Cohen’s Kappa for agreement between the two methods was 0.44.

Conclusion: Only moderate agreement between the genetic test and the breath test results was shown. Secondary lactose malabsorption as well as user-related pre-analytical deviations in handling the breath test or to instruct the patients to exhale the end-expiratory breath can contribute to discrepant results between both methods.

Iron deficiency: A comparative study between different laboratory markers

D Enko, H Wagner², G Kriegshäuser¹, C Kimbacher¹, E Rezanka¹, R Stolba¹, G Halwachs-Baumann¹

¹Department of Laboratory Medicine, Central Hospital Steyr, Steyr, Austria; ²Department of Applied Statistics, Johannes Kepler University Linz, Linz, Austria

Background: Ferritin and the transferrin saturation (TSAT) have become standard laboratory tests for the assessment of the human iron status, but both parameters are known as acute-phase dependent biomarkers. The measurement of the soluble transferrin receptor (sTfR) and the reticulocyte hemoglobin content (CHr) are considered as alternative laboratory parameters for the diagnosis of iron deficiency (ID). Therefore this study was conducted to compare the classical biomarkers ferritin and TSAT with the sTfR/log ferritin ratio and the CHr in form of the Thomas-plot in identifying patients with ID.

Methods: A total of 445 adult patients, who were admitted to the Central Hospital Steyr (Austria), were studied. Hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), reticulocytes, CHr, sTfR, ferritin, the transferrin saturation (TSAT), and the C-reactive protein (CRP) were analyzed. The CHr and the sTfR/log ferritin ratio were used in form of the Thomas-plot (quadrant 1-4). Functional ID was defined as a CHr < 28 pg. Quadrant one of the Thomas-plot indicated patients without ID, quadrant two patients with latent ID, quadrant three patients with simple functional ID without anemia of chronic disease (ACD), and quadrant four indicated patients with functional ID combined with ACD. Separate logistic regression models with potential biomarkers (sTfR/log ferritin, sTfR, ferritin, TSAT) were calculated to compare the diagnostic performance of predicting functional ID in patients with acute-phase reaction (CRP > 0.5 mg/dL) (n = 220) as well as in patients without acute-phase reaction (CRP ≤ 0.5 mg/dL) (n = 225).

Results: All in all 153 patients (34.38%) showed ID with the Thomas-plot. Of these 62 (40.52%) had latent ID (quadrant two) and 91 (59.48%) had functional ID (quadrant three and four). If ID was defined as a ferritin-value < 30 ng/mL, only 105 patients (23.59%) were identified with ID. If ID was defined as a TSAT < 20%, 215 patients (48.31%) were categorized with ID. The logistic regression models demonstrated that the sTfR/log ferritin ratio showed the best positive predictive value (PPV) to indicate functional ID in patients with acute-phase reaction (PPV: 64.41%) (n = 220) as well as in patients without acute-phase reaction (PPV: 62.50%) (n = 225) compared to the sTfR (PPV: 61.67% and 58.14%), ferritin (PPV: 32.86% and 32.50%) and the TSAT (PPV: 42.86% and 26.74%).

Conclusion: The prevalence of ID depends on the different biomarkers and definitions, which are used. The classical parameters ferritin and TSAT demonstrate weaknesses to reflect functional ID. A ferritin serum-level < 30 ng/mL is an established cut-off value to confirm ID, but the Thomas-plot as diagnostic tool should be used in combination with ferritin as single marker to increase the efficiency in identifying patients with ID.

Hepcidin-25 biomarker in iron deficiency testing

D Enko¹, H Wagner², G Kriegshäuser¹, C Kimbacher¹, E Rezanka¹, R Stolba¹, G Halwachs-Baumann¹

¹Department of Laboratory Medicine, Central Hospital Steyr, Steyr, Austria; ²Department of Applied Statistics, Johannes Kepler University Linz, Linz, Austria

Background: In the past patient studies using the hepcidin-25 serum-levels as biomarker have been hampered by the lack of validated assays. The present study was conducted to compare the hepcidin-25 measurements, performed with the recently released first European Community (CE)-marked enzyme-linked immunoabsorbent assay (ELISA) (DRG Instruments GmbH), with conventional biomarkers and diagnostic tools of iron deficiency (ID) testing.

Methods: All in all 233 hospitalized adult patients, who underwent routine blood testing for ID, were included. Hepcidin-25 serum-levels, sTfR, ferritin, transferrin, serum iron, C-reactive protein (CRP), the reticulocyte hemoglobin content (CHr), and the complete blood cell count were analyzed. The CHr and the sTfR/log ferritin ratio were used in form of the Thomas-plot (quadrant 1-4). Logistic regression models were built with potential biomarkers to compare the positive predictive values (PPVs) in view of indicating functional ID in patients with acute-phase reaction (CRP > 0.5 mg/dL) (n = 117) as well as in patients without acute-phase reaction (CRP ≤ 0.5 mg/dL) (n = 116).

Results: Patients, who were classified as a combined state of functional ID and ACD in quadrant four of the Thomas-plot (n = 12), showed the highest hepcidin-25 median value (37.18 ng/mL) compared to patients with simple functional ID (without ACD) in quadrant three (n = 35) (median: 7.16 ng/mL), patients with latent ID in quadrant two (n = 35) (median: 15.64 ng/mL), and patients categorized without ID in quadrant one (n = 151) (median: 33.95 ng/mL). Patients with acute-phase reaction (CRP > 0.5 mg/dL) (n = 117) showed a distinct higher hepcidin-25 median value (35.60 ng/mL) compared to patients without acute-phase reaction (CRP < 0.5 mg/dL) (n = 116) (18.55 ng/mL). In both subgroups the sTfR/log ferritin ratio and the sTfR presented the better PPVs (58.33% and 70.80% for the sTfR/log ferritin ratio; 60.00% and 60.00% for sTfR) to indicate functional ID compared to hepcidin-25 (PPVs: 37.74% and 42.86%) and ferritin (PPVs: 27.53% and 46.15%).

Conclusion: Patients with a combined state of functional ID and ACD in quadrant four of the Thomas-plot showed the highest hepcidin-25 median value compared to the patients in the other three quadrants. The logistic regression models demonstrated that the sTfR/log ferritin ratio and the sTfR as single markers were the better positive predictors to indicate functional ID compared to hepcidin-25 and ferritin in patients with acute-phase reaction as well as in patients without acute-phase reaction. In clinical practice, hepcidin-25 and ferritin have limitations to reflect the actual human iron status.

NADPH oxidase 4 (NOX4) derived superoxide affects actin cytoskeleton integrity in human cell lines

S Auer, M Rinnerthaler, J Bischof, R Geisberger, J Cadamuro, K Richter, C Mrazek, H Wiedemann, H Oberkofler, E Haschke-Becher and T K Felder

Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria

NADPH oxidase (NOX) enzymes transport electrons from NADPH across membranes to oxygen, forming the free-radical superoxide. NOX derived reactive oxygen species (ROS) are involved in a variety of intracellular physiological and pathophysiological signaling pathways in mammalian cells. The recently identified yeast NADPH oxidase 1 (YNO1) localizes to the endoplasmatic reticulum and demonstrates that hydrogen peroxide (H₂O₂) acts as a signaling molecule in the regulation of actin cytoskeleton dynamics. We speculated that human NOX4 could have a similar biological effect on the nucleation of filamentous F-actin. NOX4 was expressed in human liver and brain tissue samples as well as in cultured human hepatoma and neuroblastoma cell lines. Green fluorescence protein (GFP) tagged Nox4 localized to the endoplasmatic reticulum, resembling YNO1 localization. General NOX inhibition, and strikingly siRNA mediated NOX4 knockdown significantly changed the morphology of the actin cytoskeleton. Reduced expression of NOX4 decreased cellular actin cable content, insofar as the overall actin level was not altered, whereas the ratio of F-actin to globular G-actin was reduced. Additionally, NOX4 knockdown impaired cell migration in human neuroblastoma cells. The NOX4 knockdown phenotype was rescued by the addition of H₂O₂, compensating decreased superoxide levels. Here we provide the first direct link between the activity of Nox4 and the actin cytoskeleton as Nox4 derived regulation of actin nucleation impacts cell migration in human neuronal cells. Our findings unravel an additional branch of NOX4 signaling, which affects basic actin-dependent cellular processes and therefore makes the modulation of Nox4 activity an attractive therapeutic target.

Absence of interleukin-33 in mice drives prevalence of proinflammatory macrophages in adipose tissue and obesity development

^1,2S Demyanets, ¹S Amann, ¹A Jais, ²D Ritzel, ²C Kaun, ²R Pentz, ³H Saito, ⁴S Nakae, ⁵P Uhrin, ¹H Esterbauer, ²J Wojta

¹Department of Laboratory Medicine, Medical University of Vienna, Austria; ²Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Austria; ³Department of Allergy and Immunology, National Research Institute for Child Health & Development, Tokyo, Japan; ⁴Laboratory of Systems Biology, The Institute of Medical Science, University of Tokyo, Japan; ⁵Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria.

Background: Interleukin (IL)-33 is a member of the IL-1 family and the ligand of the ST2 receptor. IL-33 and ST2 levels are elevated in adipose tissue of obese humans and mice. The aim of this study was to investigate the development of obesity in IL-33 deficient (IL-33^-/-) mice.

Methods: IL-33^-/- mice and IL-33^+/+ littermates were fed either high fat diet (HFD) or low fat diet (LFD) for 18 weeks. Body weight was monitored weekly and oral glucose tolerance tests (oGTT) and insulin tolerance tests (ITT) were performed after 16 and 17 weeks on diet, respectively. Organs were weighed at the day of sacrifice, and stored for histological, mRNA and protein analyses. The stromal vascular fraction (SVF) was isolated from epididymal white adipose tissue (eWAT) and analysed by flow cytometry.

Results: IL-33^-/- mice displayed impaired glucose tolerance on both HFD and LFD (p≤0.05). Of note, 15 minute oGTT insulin levels were lower in IL-33^-/- mice on HFD compared to wild type controls (p≤0.05). After a 2 hours fast, IL-33^-/- mice on HFD displayed higher blood glucose levels that littermate controls while ITTs revealed no obvious difference in insulin sensitivity. Body weight did not differ between IL-33^-/- and IL-33^+/+ mice after 18 weeks on a particular diet. Interestingly, SVFs of obese IL-33^-/- mice contained a higher percentage of proinflammatory (M1-like) macrophages (38±2% as compared to 11±3% in IL-33^+/+) and a lower percentage of antiinflammatory M2-like macrophages (23±2% as compared to 47±3% in IL-33^+/+). Further, IL-10 mRNA levels were lower in SVF and adipocyte fractions obtained from eWAT of IL-33^-/- mice on HFD as compared to HFD fed wildtype littermates.

Conclusion: IL-33 is protective during obesity development in mice by influencing glucose tolerance as well as adipose macrophage subset distributions.

Detection of enterohaemorrhagic E. coli (EHEC) and verotoxin producing E. coli (VTEC) using the RIDA®GENE EHEC/EPEC multiplex real-time PCR assay

A Wojna, C Schausberger, D Rieder, L Mustafa

Med. chem. Labor Dr. Mustafa, Dr. Richter OG

Background: VTEC/EHEC (=VTEC plus eae gene) can cause severe foodborne disease, leading to haemolytic uraemic syndrome (HUS) in up to 10% of patients.

Objectives: To evaluate the RIDA®GENE EHEC/EPEC real-time PCR assay, and to compare it to our current culture method using CHROMagar™O157.

Methods: 68 stool samples and 22 E. coli isolates grown on ChromAgar ™O157 were tested with RIDA®GENE EHEC/EPEC. Stool samples were selected for PCR based on age up to 2 years and on Bristol Stool Chart types 5 to 7. DNA isolation, amplification and detection were performed automatically using the BDMax™ system. The PCR assay detects gene fragments specific for the virulence factors stx1/2 (shigatoxin 1/2), eae (E. coli attaching and effacing gene) and ipaH (invasion plasmid antigen H). For serotyping stool samples and/or isolates were sent to the Austrian reference laboratory.

Results: PCR and O157 agar were found positive for VTEC/EHEC in 7 and 4 cases. 5 EHEC (stx1/2 and eae positive) of 4 different serotypes (O157:H7, O157:HNM, Orough:HNM, O98:HNM) and 2 VTEC (stx1/2 positive) of 1 serotype (Orough:H21) were detected. 3 VTEC/EHEC non-O157 (2 Orough:HNM,1 O98:HNM) were cultured on O157 agar, but one EHEC O157 failed to grow. The two VTEC isolates were detected by PCR only. Furthermore 14 EPEC and 3 EIEC were found based on positivity of eae and ipaH.

Conclusion: According to the data of the Austrian reference laboratory (1), VTEC/EHEC non-O157 were 6 times more frequent than EHEC O157 in 2012. In our samples the ratio of EHEC O157 to VTEC/EHEC non-O157 was 1:2,5. Nevertheless it is clear that screening for VTEC/EHEC should be based on the detection of shigatoxins to overcome the lack of sensitivity and specifity of culture. PCR turned out to be the more rapid and sensitive tool leading to better management of patients and contact persons. 1. Austria. Jahresbericht 2012. Nationale Referenzzentrale für Escherichia coli einschließlich Verotoxin-bildender E. coli. AGES – IMED Graz; 2013.

Characterization of genetic determinants and transcription factors associated with prostate cancer antigen 3 gene expression

I Kremser¹, S Auer¹, J Dadrass¹, T Felder¹, J Cadamuro¹, S Hruby², D Colleselli², G Janetschek², E Haschke-Becher¹ and H Oberkofler¹

¹Institute of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria; ²Department of Urology, Paracelsus Medical University, Salzburg, Austria;

Objective: Prostate cancer (PCa) still remains an important cause of cancer-related death in males (1). Although PSA-based screening has led to an increase in PCa detection, it is still controversial whether it also reduces the rate of death from PCa (2). Prostate cancer antigen 3 (PCA3) has initially shown promise as novel biomarker for early diagnosis, prognosis and monitoring of PCa patients and numerous genetic variants significantly increasing PCa risk have been described (3,4). We aimed to characterize genetic determinants and transcription factors associated with PCA3 gene expression.

Methods: Bioinformatics was used to identify single nucleotide polymorphisms (SNPs) and transcription factor binding-sites in the PCA3 promoter region. The PCA3 score was determined using transcription-mediated amplification and selected SNPs were genotyped using Taqman 5’-exonuclease assays. Prostate cancer cell line DU145 was transiently co-transfected with PCA3 promoter-reporter-constructs together with expression plasmids harboring coding sequences of selected transcription factors.

Results: Six SNPs were identified in the promoter region but no significant associations with the PCA3 score were observed. Nine additional SNPs selected from various chromosomal regions associated with PCA risk were analyzed and rs4430796 in the hepatocyte nuclear factor 1 homeobox B gene at 17q12 and rs1447295 in the cancer susceptibility candidate 8 gene at 8q24.21 were associated with PCA3 expression levels in our study population. Our promoter studies indicated stimulatory effects of selected transcription factors, including USF1/2, PPARα, FOXA1 and FOXA2, on PCA3 expression.

Conclusions: Two PCa risk SNPs, in the HNF1-B and in the CASC8 gene,respectively, were identified as genetic determinants of PCA3 gene expression, whereas SNPs in the PCA3 promoter region did not affect PCA3 gene expression. Our preliminary observations on transcription factors involved in PCA3 gene regulation might provide the basis for valuable future insights into pathophysiological mechanisms underlying PCa development.

References:

1. Ruijter E van de Kaa C, Miller G, Ruiter D, Debruyne F, Schalken J. Molecular genetics and epidemiology of prostate carcinoma. Endocr Rev. 1999; 20:22-45.

2. Fradet Y. Biomarkers in prostate cancer diagnosis and prognosis: beyond prostate-specific antigen. Curr Opin Urol 2009; 19:243-246.

3. Schalken JA., Hessels D. and Verhaegh G. New targets for therapy in prostate cancer: differential display code 3 (DD3(PCA3)), a highly prostate cancer-specific gene. Urology 2003; 62:34-43.

4. Eeles R, Goh C, Castro E, Bancroft E, Guy M, Al Olama AA, Easton D, Kote-Jarai Z. The genetic epidemiology of prostate cancer and its clinical implications. Nat Rev Urol. 2014; 11:18-31.

Post-transfusion hepatitis B infection: Is donor screening for HBsAg and HBV DNA sufficient?

T Mayerhofer¹, A Höfinger², H Krucher², V Aliskanovic¹, D Trubert-Exinger¹, HH Kessler³, E Stelzl³, AC Haushofer^1,4

¹Institute of Laboratory Medicine, Landesklinikum St. Poelten, Austria

²Blood Bank, Landesklinikum St. Poelten, Austria

³Research Unit Molecular Diagnostics, IHMEM, Medical University of Graz, Austria

⁴Institute of Medical and Chemical Laboratory Diagnostics with Blood Bank, Klinikum Wels-Grieskirchen, Wels, Austria

Background: In addition to mandatory donor screening for HBsAg, blood is also screened on HBV DNA in Austria. The serological pattern of anti-HBc antibody (HBcAb) positivity without HBs antigen (HBsAg) and anti-HBs antibody (HBsAb) positivity may be present in up to 4% of Europeans. Serum HBV DNA can be detected in up to 10% of them, depending on the population studied.

Objectives: To demonstrate transmission of HBV through blood products from an HBsAg negative donor with a very low HBV load in blood.

Materials and methods: In a look-back study, 11 of 22 patients who had received erythrocyte concentrates between 2002 and 2010 from an HBsAg negative donor who was found to be positive for HBV DNA subsequently were investigated for the post transfusion hepatitis B status. Transfusion recipients were tested on HBsAg, HBcAb, and HBsAb using the Architect (Abbott, Wiesbaden, Germany) and the Ortho ECI (Ortho Clinical Diagnostics, London, UK) platforms. The serum HBV DNA load was tested by the Cobas AmpliPrep/Cobas Taqman (Roche Molecular Systems, Branchburg, NJ) assay. Sequencing of HBV was done by Sanger sequencing using the TRUGENE HBV Genotyping Kit (Siemens Siemens Healthcare Diagnostics, Tarrytown, NY). Up to 2011, donors had been screened by a qualified pool HBV PCR followed by a commercial available PCR system, the Cobas S201 (Roche).

Results: Of 11 patients, 6 did not give evidence to a serological reaction regarding HBV infection with HBV DNA being undetectable, 4 showed a serology according to past hepatitis B infection (3 of them post transfusion) with undetectable HBV DNA), and one showed HBsAg only. One of the patients had accidently been investigated on HBV DNA in 2010 and an HBV DNA load of 30500 IU/ml had been found. When the donor was retested (retain and fresh samples) with the Cobas S201 system and the Cobas AmpliPrep/Cobas Taqman assay, HBV DNA could always be detected (□20 IU/ml) and HBcAb were found. When sequences obtained from the donor’s and the recipient’s HBV strains were compared, both showed HBV genotype D and the alignment analysis of □509 nucleotides revealed two heterologies only.

Conclusions: Alignment analysis showed that at least one of the patients was most likely infected through an erythrocyte concentrate that was derived from an HBsAg negative donor with a very low HBV DNA load in blood. With introduction of the latest version of systems, the Cobas S201, including smaller pools (pools of 6 samples), HBV DNA became detectable in the sample. Testing on HBcAb may be considered as an additional valuable screening tool for identifying an HBsAg negative donor with a very low HBV DNA concentration in blood.