Reliability of the Dilute Russel Viper Venom Time (dRVVT) in Determining Lupus Anticoagulant (LAC) in Anticoagulated Patients

Objectives Antiphospholipid syndrome (APS) can be complicated by thrombosis in the presence of laboratory evidence of persistent antiphospholipid antibodies, usually in the form of a LAC. The diagnostic criteria for APS/LAC require persistent prolongation of two phospholipid-dependent clotting assays, most often the dRVVT and a thromboplastin-based phospholipid-neutralization (PN) assay. When there is discrepancy between tests, LAC diagnosis is inconclusive. This testing is complicated by anticoagulation, which may yield false positive results. We investigated the impact of various anticoagulants on LAC diagnosis by evaluating the rates of dRVVT and PN discrepancy between anticoagulated and non-anticoagulated patients. Methods dRVVT and PN (LAC panel) values were examined for 1452 consecutive patients samples since April 2017, along with anticoagulant status at the time of testing. Each LAC panel was classified as positive (both dRVVT and PN abnormal), negative (both dRVVT and PN normal), or discrepant (one normal and one abnormal). The proportion of discrepant results between anticoagulated and non-anticoagulated patients was calculated and also stratified according to the class of anticoagulant. Results Discrepant results occurred in 161 of 493 anticoagulated patient samples (33%) versus 135 of 1012 non-anticoagulated patient samples (13%). The odds ratio (OR) for a discrepant result for anticoagulated patient samples was 3.15 (95% CI=2.43–4.09) compared to non-anticoagulated patient samples. When narrowed to individual anticoagulant classes, Lovenox had the lowest chance of yielding discrepant results (OR = 1.16 95% CI= 0.51–2.66), whereas Rivaroxaban (OR = 6.9 95% CI= 4.49–10.55) and Warfarin (OR = 5.4 95% CI= 3.43–8.55) had the highest odds of yielding discrepant results. Conclusions Patient anticoagulant status is an important determinant in laboratory testing for APS/LAC. The dRVVT and PN test are 3-fold more likely to yield inconclusive results when a patient is anticoagulated; the type of anticoagulant is even more critical given the large difference in rates of discrepant results for patients on Rivaroxaban and Warfarin versus patients on Lovenox. The difference in discrepancy rate is likely due to the differential impact of each class of anticoagulant on the various components of the coagulation cascade. Rivaroxaban is a direct factor X inhibitor, and Warfarin directly lowers active factor X, amongst other, levels; by contrast, Lovenox, which was not significantly discrepant, requires antithrombin to amplify inhibition of factor II and X activity.

Faster Detection of Helicobacter pylori Infection by 13 C-Urea Breath Test. Comparing Short versus Standard Sampling Time

Background & Aims: 13 C-Urea Breath Test (UBT) is a non-invasive, highly accurate and recommended test to detect Helicobacter pylori (H. pylori) infection and to confirm post-therapy eradication. However, differences exist in terms of manufacturers, dose of labelled urea, addition of citric acid, solid vs. liquid formulation, and sampling times of breath samples. In this study, we compared the diagnostic accuracy of “short” (15 minutes) vs. “standard” (30 minutes) time for a single type of liquid UBT. Methods: We compared the performance of a single UBT type (BREATHQUALITY, AB Analitica, Padua, Italy, 10 mL of 75 mg 13 C-Urea and 1.4 g citric acid) during a “short” vs. “standard” breath sampling time. Enrolled were 151 subjects requiring UBT as naïve (N=92) or post-eradication (N=59) checks. Results: UBT at 15 and 30 minutes were highly comparable, showing optimal correlation in all subsets of patients (i.e. naïve vs. post eradication, negative vs. post eradication check). One discrepant result occurred at the borderline zone of the DOB 4‰, but proved to be true positive at a later confirmation by a second UBT and stool antigen test. Conclusions: By shortening the testing time of BREATHQUALITY to 15 minutes (-50%) comparable accuracy will be maintained and in addition, it will bring some benefits to patients’ waiting lists, compliance, and hospital staff.

Multisite Evaluation of the BD Max Extended Enteric Bacterial Panel for Detection of Yersinia enterocolitica, Enterotoxigenic Escherichia coli, Vibrio, and Plesiomonas shigelloides from Stool Specimens

ABSTRACTThe purpose of this study was to perform a multisite evaluation to establish the performance characteristics of the BD Max extended enteric bacterial panel (xEBP) assay directly from unpreserved or Cary-Blair-preserved stool specimens for the detection ofYersinia enterocolitica, enterotoxigenicEscherichia coli(ETEC),Vibrio, andPlesiomonas shigelloides. The study included prospective, retrospective, and prepared contrived specimens from 6 clinical sites. BD Max xEBP results were compared to the reference method, which included standard culture techniques coupled with alternate PCR and sequencing, except for ETEC, for which the reference method was two alternate PCRs and sequencing. Alternate PCR was also used to confirm the historical results for the retrospective specimens and for discrepant result analysis. A total of 2,410 unformed, deidentified stool specimens were collected. The prevalence in the prospective samples as defined by the reference method was 1.2% ETEC, 0.1%Vibrio, 0%Y. enterocolitica, and 0%P. shigelloides. Compared to the reference method, the positive percent agreement (PPA) (95% confidence interval [CI]), negative percent agreement (NPA) (95% CI), and kappa coefficient (95% CI) for the BD Max xEBP assay for all specimens combined were as follows: ETEC, 97.6% (87.4 to 99.6), 99.8% (99.5 to 99.9), and 0.93 (0.87 to 0.99);Vibrio, 100% (96.4 to 100), 99.7% (99.4 to 99.8), and 0.96 (0.93 to 0.99);Y. enterocolitica, 99.0% (94.8 to 99.8), 99.9% (99.8 to 99.9), and 0.99 (0.98 to 1);P. shigelloides, 100% (96.4 to 100), 99.8% (99.5 to 99.9), and 0.98 (0.95 to 1), respectively. In this multicenter study, the BD Max xEBP showed a high correlation (kappa, 0.97; 95% CI, 0.95 to 0.98) with the conventional methods for the detection of ETEC,Vibrio,Y. enterocolitica, andP. shigelloidesin stool specimens from patients suspected of acute gastroenteritis, enteritis, or colitis.

Allele Drop Out Conferred by a Frequent CYP2D6 Genetic Variation For Commonly Used CYP2D6*3 Genotyping Assays

Background/Aim: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (∼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. Methods: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. Results: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. Conclusion: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.

Revisiting a Discrepant Result: A Propensity Score Analysis, the Paired Availability Design for Historical Controls, and a Meta-Analysis of Randomized Trials

There is an ongoing controversy over whether epidural analgesia for women in labor increases the probability of Caesarean section. Previous research compared results from three methods for estimating the effect of epidural analgesia on the probability of Caesarean section: a propensity score analysis, the paired availability design for historical controls, and meta-analysis of randomized trials. The propensity score analysis and a paired availability design gave substantially different results with the latter in closer agreement with results of a meta-analysis of randomized trials. We updated this investigation in three ways. First, we discussed the use of causal graphs for variable selection in the propensity score analysis. Second, we introduced new extrapolation estimates to improve generalizability for the paired availability design and the meta-analysis of randomized trials with crossovers. Third, we included the results from more recent studies. This analysis provides a window into various topics in causal inference and comparative effectiveness research.

Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2

Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

Screening for Interference in Immunoassays

Background: The presence of interfering substances in patient samples submitted for immunoassay cannot be reliably anticipated. We therefore evaluated three interference screening techniques and estimated the prevalence of interfering substances as defined by positive outcomes with these protocols. Methods: We evaluated 160 samples for the presence of substances that may interfere with four immunoassays (40 samples for each): thyroid-stimulating hormone, prostate-specific antigen, β-human chorionic gonadotropin, and cortisol. Interference was defined by nonlinear responses with serial dilution, discrepant results after pretreatment with heterophile blocking reagent (HBR), and positive reactions on a mouse-antibody-negative control reaction (Tandem ICON® ImmunoConcentration HCG). Criteria for declaring significant discrepant results were based on a Z-score computed using the assay CV. The McNemar test was used to compare the prevalence of discrepancies across the three screening techniques. The association between type of immunoassay and prevalence of discrepant results was determined by a modified Pearson χ2 statistic. Results: Five of the 160 samples [3.1%; 95% confidence interval (CI), 1.0–7.1%] screened positive with the ICON. Seventy-two of the 148 samples with informative serial dilutions (48.6%; 95% CI, 40.4–57.0%) had at least one discrepant result at higher dilutions. After pretreatment with HBR, 53 of the 140 samples (38%; 95% CI, 29.8–46%) were discrepant. Only 48 of the 140 samples with informative measurements for all three screening techniques (34%; 95% CI, 26–43%) were negative by all three. The prevalence of positive screens varied significantly by type of immunoassay (P <0.0001) for both HBR and serial dilution. Only 3% (0.8–7%) of the samples tested with HBR showed a change from normal to abnormal or the reverse after treatment. Conclusions: Introducing a protocol based on any of these three techniques into the immunochemistry laboratory to prescreen for interfering substances is not warranted. The evaluation of specimens for the presence of interfering anti-animal antibodies should be reserved for cases in which clinical history or suspicious results indicate the need.

The Effects of Brand Positioning Strategies on Consumers’ Brand and Category Perceptions: Some Insights from Schema Research

Results of four studies demonstrate that perceptions of how different a brand is from other brands in the product category affect perceptions of the brand's position within the category. Specifically, perceptions that a brand is strongly discrepant result in a subtyped (or niche) position, whereas perceptions that a brand is moderately discrepant result in a differentiated position within the general category. Perceptions of discrepancy are affected both by the extent of discrepancy on an attribute and whether the discrepant information is concentrated in a single ad for the brand or dispersed across multiple ads for the product. The effects associated with a subtyped position, in comparison with a differentiated position, are identified (study 1) and are found to increase with time (study 2). The subtyped versus differentiated distinction for a strongly versus moderately discrepant brand is validated with a sorting task (study 3). This distinction is shown to hold in the context of multiple discrepant brands that differ in their extent of discrepancy (study 4). Implications of the findings for a theoretical understanding of subtyping versus differentiation and for the application of positioning strategies in the marketplace are discussed.

Rubella-specific IgG subclass concentrations in sera using an enzyme-linked immunosorbent assay (ELISA): the effect of different sources of rubella antigen

SUMMARYFive rubella antigens were evaluated in an antiglobulin enzyme-linked immunosorbent assay for rubella-specific IgG subclass antibody. One monoclonal anti-human IgG subclass antibody was used for each of IgG1, IgG2and IgG4, but two were compared for IgG3. A total of 101 sera were tested from cases of rubella in the distant past and from cases of primary rubella, reinfection and following immunization. Only one serum gave a discrepant result for specific IgG1, being positive with only one rubella antigen, a commercially prepared antigen coated on to microtitre wells (Enzygnost; Behringwerke). No sera contained detectable specific IgG2. Only four sera contained specific IgG4, and this was detectable only with Enzygnost antigen. For specific IgG3little difference was observed between the two monoclonal anti-human IgG3subclass antibodies; only two very weakly positive sera gave discrepant results. However, varying results were obtained for specific IgG3with the different antigens. Enzygnost gave more positive results for specific IgG3with most categories of sera.It is concluded that the differences between various reports of the rubella-specific IgG subclass profile cannot be explained entirely by the use of different rubella antigens.