Variability between laboratories performing coagulation tests with identical platforms: a nationwide evaluation study.

BACKGROUND While the assessment of analytical precision within medical laboratories has received much attention in scientific enquiry, the degree of as well as the sources causing variation between them remains incompletely understood. In this study, we quantified the variance components when performing coagulation tests with identical analytical platforms in different laboratories and computed intraclass correlations coefficients (ICC) for each coagulation test. METHODS Data from eight laboratories measuring fibrinogen twice in twenty healthy subjects with one out of 3 different platforms and single measurements of prothrombin time (PT), and coagulation factors II, V, VII, VIII, IX, X, XI and XIII were analysed. By platform, the variance components of (i) the subjects, (ii) the laboratory and the technician and (iii) the total variance were obtained for fibrinogen as well as (i) and (iii) for the remaining factors using ANOVA. RESULTS The variability for fibrinogen measurements within a laboratory ranged from 0.02 to 0.04, the variability between laboratories ranged from 0.006 to 0.097. The ICC for fibrinogen ranged from 0.37 to 0.66 and from 0.19 to 0.80 for PT between the platforms. For the remaining factors the ICC's ranged from 0.04 (FII) to 0.93 (FVIII). CONCLUSIONS Variance components that could be attributed to technicians or laboratory procedures were substantial, led to disappointingly low intraclass correlation coefficients for several factors and were pronounced for some of the platforms. Our findings call for sustained efforts to raise the level of standardization of structures and procedures involved in the quantification of coagulation factors.

[New oral anticoagulants - influence on coagulation tests].

The new oral anticoagulants (NOACs) represent alternative antithrombotic agents for prophylaxis and therapy of thromboembolic diseases. They act either by inhibition of the clotting factor Xa or IIa (thrombin). As a consequence, they influence several coagulation assays (for example prothrombin time, activated partial thromboplastin time). Because of the short half-life of these new agents, these changes show great variations in the course of 24 hours. Furthermore, there are significant differences of laboratory results depending on the used reagents. We explain the influence of apixaban, rivaroxaban (factor Xa inhibitors) and dabigatran (thrombin inhibitor) on the most commonly used coagulation assays. Besides we show that this influence depends on the way of action of the drug as well as on the principle of the coagulation assay. Being aware of this relationships helps to interpret the results of coagulation assays under influence of NOACs correctly.

Cognitive performance in high-altitude climbers: a comparative study of saccadic eye movements and neuropsychological tests

Impairment of cognitive performance during and after high-altitude climbing has been described in numerous studies and has mostly been attributed to cerebral hypoxia and resulting functional and structural cerebral alterations. To investigate the hypothesis that high-altitude climbing leads to cognitive impairment, we used of neuropsychological tests and measurements of eye movement (EM) performance during different stimulus conditions. The study was conducted in 32 mountaineers participating in an expedition to Muztagh Ata (7,546 m). Neuropsychological tests comprised figural fluency, line bisection, letter and number cancellation, and a modified pegboard task. Saccadic performance was evaluated under three stimulus conditions with varying degrees of cortical involvement: visually guided pro- and anti-saccades, and visuo-visual interaction. Typical saccade parameters (latency, mean sequence, post-saccadic stability, and error rate) were computed off-line. Measurements were taken at a baseline level of 440 m and at altitudes of 4,497, 5,533, 6,265, and again at 440 m. All subjects reached 5,533 m, and 28 reached 6,265 m. The neuropsychological test results did not reveal any cognitive impairment. Complete eye movement recordings for all stimulus conditions were obtained in 24 subjects at baseline and at least two altitudes and in 10 subjects at baseline and all altitudes. Measurements of saccade performances showed no dependence on any altitude-related parameter and were well within normal limits. Our data indicates that acclimatized climbers do not seem to suffer from significant cognitive deficits during or after climbs to altitudes above 7,500 m. We demonstrated that investigation of EMs is feasible during high-altitude expeditions.

Evaluation of the diagnostic value of the ELISA tests developed by using EgHF, Em2 and EmII/3-10 antigens in the serological diagnosis of alveolar echinococcosis

Alveolar echinococcosis (AE), caused by larva stage of Echinococcus multilocularis, is one of the lethal parasitic diseases of man and a major public health problem in many countries in the northern hemisphere. When the living conditions and habits in Turkey were considered in terms of relation with the life cycle of the parasite, it was suggested that AE has been much more common than reported mainly from the Eastern Anatolia region of Turkey. Since in vitro serologic diagnosis tests with high specificity for AE have not been used in our country, most of the cases with liver lesions were misdiagnosed by radiological investigations as malignancies. The aim of this study was to evaluate the diagnostic value of the in-house ELISA methods developed by using three different antigens (EgHF, Em2, EmII/3-10) in the serological diagnosis of AE. The study samples included a total of 100 sera provided by Bern University Parasitology Institute where samples were obtained from patients with helminthiasis and all were confirmed by clinical, parasitological and/or histopathological means. Ten samples from each of the cases infected by E.multilocularis, E.granulosus, Taenia solium, Wuchereria bancrofti, Strongyloides stercolaris, Ascaris lumbricoides, Toxocara canis, Trichinella spiralis, Fasciola hepatica and Schistosoma haematobium were studied. In the study, EgHF (E.granulosus hydatid fluid) antigens were prepared in our laboratory from the liver cyst fluids of sheeps with cystic echinococcosis, however Em2 (E.multilocularis metacestode-purified laminated layer) and EmII/3-10 (E.multilocularis recombinant protoscolex tegument) antigens were provided by Bern University Parasitology Institute. Flat bottom ELISA plates were covered with EgHF, Em2 and EmII/3-10 antigens in the concentrations of 2.5 µg, 1 µg and 0.18 µg per well, respectively, and all sera were tested by EgHF-ELISA, Em2-ELISA and EmII/3-10-ELISA methods. For each tests, the samples which were reactive above the cut-off value (mean OD of negative controls+2 SD) were accepted as positive. The sensitivity of the ELISA tests performed with EgHF, Em2 and Em2II/3-10 antigens were estimated as 100%, 90% and 90%, respectively, whereas the specificity were 63%, 91% and 91%, respectively. When Em2-ELISA and EmII/3-10-ELISA tests were evaluated together, the specificity increased to 96%. Our data indicated that the highest sensitivity (100% with EgHF-ELISA) and specificity (96% with Em2-ELISA + EmII/3-10-ELISA) for the serodiagnosis of AE can be achieved by the combined use of the ELISA tests with three different antigens. It was concluded that the early and accurate diagnosis of AE in our country which is endemic for that disease, could be supported by the use of highly specific serological tests such as Em2-ELISA ve EmII/3-10-ELISA contributing radiological data.

Multinomial goodness-of-fit: large sample tests with survey design correction and exact tests for small samples

I introduce the new mgof command to compute distributional tests for discrete (categorical, multinomial) variables. The command supports largesample tests for complex survey designs and exact tests for small samples as well as classic large-sample x2-approximation tests based on Pearson’s X2, the likelihood ratio, or any other statistic from the power-divergence family (Cressie and Read, 1984, Journal of the Royal Statistical Society, Series B (Methodological) 46: 440–464). The complex survey correction is based on the approach by Rao and Scott (1981, Journal of the American Statistical Association 76: 221–230) and parallels the survey design correction used for independence tests in svy: tabulate. mgof computes the exact tests by using Monte Carlo methods or exhaustive enumeration. mgof also provides an exact one-sample Kolmogorov–Smirnov test for discrete data.

Determinanten kognitiver Angstmanifestation während schulischer Tests: Eine Längsschnittstudie

Theoretischer Hintergrund und Fragestellung: Schulische Tests dienen der Feststellung von Wissen und Können. Wie jede Messung kann auch diese durch Störvariablen verzerrt werden. Während Tests erlebte Angst ist ein solcher potentieller Störeinfluss: Angst kann Testleistungen beinträchtigen, da sie sich hinderlich auf die Informationsverarbeitung auswirken kann (Störung des Wissensabrufs und des Denkens; Zeidner, 1998). Dieser kognitiven Angstmanifestation (Rost & Schermer, 1997) liegt die angstbedingte automatische Aufmerksamkeitsorientierung auf aufgaben-irrelevante Gedanken während der Testbearbeitung zugrunde (Eysenck, Derakshan, Santos & Calvo, 2007). Es hat sich allerdings gezeigt, dass Angst nicht grundsätzlich mit Testleistungseinbußen einhergeht (Eysenck et al., 2007). Wir gehen davon aus, dass die Kapazität zur Selbstkontrolle bzw. Aufmerksamkeitsregulation (Baumeister, Muraven & Tice, 2000; Schmeichel & Baumeister, 2010) ein Faktor ist, der bedingt, wie stark kognitive Angstmanifestation während Tests und damit zusammenhängende Leistungseinbußen auftreten. Ängstliche Lernende mit höherer Aufmerksamkeitsregulationskapazität sollten ihrer automatischen Aufmerksamkeitsorientierung auf aufgaben-irrelevante Gedanken erfolgreicher entgegensteuern und ihre Aufmerksamkeit weiterhin auf die Aufgabenbearbeitung richten können. Dem entsprechend sollten sie trotz Angst weniger kognitive Angstmanifestation während Tests erleben als ängstliche Lernende mit geringerer Aufmerksamkeitsregulationskapazität. Auch die Selbstwirksamkeitserwartung und das Selbstwertgefühl sind Variablen, die in der Vergangenheit mit der Bewältigung von Angst und Stress in Verbindung gebracht wurden (Bandura, 1977; Baumeister, Campbell, Krueger & Vohs, 2003). Daher wurden diese Variablen als weitere Prädiktoren berücksichtigt. Es wurde die Hypothese getestet, dass die dispositionelle Aufmerksamkeitsregulationskapazität über die dispositionelle Selbstwirksamkeitserwartung und das dispositionelle Selbstwertgefühl hinaus Veränderungen in der kognitiven Angstmanifestation während Mathematiktests in einer Wirtschaftsschülerstichprobe vorhersagt. Es wurde des Weiteren davon ausgegangen, dass eine indirekte Verbindung zwischen der Aufmerksamkeitsregulationskapazität und der Veränderung in den Mathematiknoten, vermittelt über die Veränderung in der kognitiven Angstmanifestation, besteht. Methode: Einhundertachtundfünfzig Wirtschaftsschüler bearbeiteten im September 2011 (T1) einen Fragebogen, der die folgenden Messungen enthielt:-Subskala Kognitive Angstmanifestation aus dem Differentiellen Leistungsangstinventar (Rost & Schermer, 1997) bezogen auf Mathematiktests (Sparfeldt, Schilling, Rost, Stelzl & Peipert, 2005); Alpha = .90; -Skala zur dispositionellen Aufmerksamkeitsregulationskapazität (Bertrams & Englert, 2013); Alpha = .88; -Skala zur Selbstwirksamkeitserwartung (Schwarzer & Jerusalem, 1995); Alpha = .83; -Skala zum Selbstwertgefühl (von Collani & Herzberg, 2003); Alpha = .83; -Angabe der letzten Mathematikzeugnisnote. Im Februar 2012 (T2), also nach 5 Monaten und kurz nach dem Erhalt des Halbjahreszeugnisses, gaben die Schüler erneut ihre kognitive Angstmanifestation während Mathematiktests (Alpha = .93) und ihre letzte Mathematikzeugnisnote an. Ergebnisse: Die Daten wurden mittels Korrelationsanalyse, multipler Regressionsanalyse und Bootstrapping ausgewertet. Die Aufmerksamkeitsregulationskapazität, die Selbstwirksamkeitserwartung und das Selbstwertgefühl (alle zu T1) waren positiv interkorreliert, r= .50/.59/.59. Diese Variablen wurden gemeinsam als Prädiktoren in ein Regressionsmodell zur Vorhersage der kognitiven Angstmanifestation zu T2 eingefügt. Gleichzeitig wurde die kognitive Angstmanifestation zu T1 konstant gehalten. Es zeigte sich, dass die Aufmerksamkeitsregulationskapazität erwartungskonform die Veränderungen in der kognitiven Angstmanifestation vorhersagte, Beta = -.21, p= .02. Das heißt, dass höhere Aufmerksamkeitsregulationskapazität zu T1 mit verringerter kognitiver Angstmanifestation zu T2 einherging. Die Selbstwirksamkeitserwartung, Beta = .12, p= .14, und das Selbstwertgefühl, Beta = .05, p= .54, hatten hingegen keinen eigenen Vorhersagewert für die Veränderungen in der kognitiven Angstmanifestation. Des Weiteren ergab eine Mediationsanalyse mittels Bootstrapping (bias-corrected bootstrap 95% confidence interval, 5000 resamples; siehe Hayes & Scharkow, in press), dass die Aufmerksamkeitsregulationskapazität (T1), vermittelt über die Veränderung in der kognitiven Angstmanifestation, indirekt mit der Veränderung in der Mathematikleistung verbunden war (d.h. das Bootstrap-Konfidenzintervall schloss nicht die Null ein; CI [0.01, 0.24]). Für die Selbstwirksamkeitserwartung und das Selbstwertgefühl fand sich keine analoge indirekte Verbindung zur Mathematikleistung. Fazit: Die Befunde verweisen auf die Bedeutsamkeit der Aufmerksamkeitsregulationskapazität für die Bewältigung kognitiver Angstreaktionen während schulischer Tests. Losgelöst von der Aufmerksamkeitsregulationskapazität scheinen positive Erwartungen und ein positives Selbstbild keine protektive Wirkung hinsichtlich der leistungsbeeinträchtigenden kognitiven Angstmanifestation während Mathematiktests zu besitzen.

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BACKGROUND Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Natural anti-FcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria.

IgG autoantibodies against the alpha-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the alpha-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti-alpha-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BACKGROUND Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(®), bioMérieux, Marcy-L'Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(®) assay (Abbott Laboratories, Sligo, Ireland). RESULTS The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. CONCLUSIONS The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.