Citations

AbstractIn hemophilia B (HB), factor IX gene (F9) genotyping is used for molecular confirmation of affected individuals, for carrier testing, to facilitate the identification of those at risk for anaphylaxis/inhibitors (associated with large deletions), and to assist in assigning disease severity. Owing to test costs, optimal test utilization involves pre/post-test counseling and appropriate patient and test selection (e.g., mutation screening [F9MS] vs. known mutation [F9KM] testing). This article aims to review the trends and outcomes of F9-genotyping orders and describe the spectrum of variants identified in a sample of individuals in our reference laboratory. We performed a retrospective review of consecutive orders submitted to the Special Coagulation DNA Diagnostic Laboratory, Mayo Clinic, between 2012 and 2015. A total of 133 orders (38%) were identified for men: 118 (88%) were F9MS and 15 (12%) were F9KM. Thirteen orders (10%) were cancelled. A total of 209 orders were identified for women: 178 (85%) were F9MS and 31 (15%) were F9KM. Thirty-seven orders (18%) were cancelled and 30% of the tests performed yielded negative results. A total of 164 samples (47%) were received without clinical information. Seventeen previously unreported variants were identified. F9 genotyping provides useful information for HB management; however, 18% of our orders were cancelled and almost half were received without relevant clinical information, thus reaffirming the need for ongoing scrutiny of submitted orders. Optimal patient and test selection is important as is the accurate interpretation of variants identified. Most of the pathogenic variants identified were point mutations, with very few large deletions, consistent with the literature.

We report a 9-year-old Chinese girl with congenital thrombotic thrombocytopenic purpura found to be a compound heterozygote for 2 pathogenic variants in the ADAMTS13 gene, including a novel variation. The girl suffered from recurrent, life-threatening episodes of thrombocytopenia and hemolysis, and laboratory testing showed ADAMST13 enzyme activity of <5%. Sequencing of the ADAMTS13 gene revealed a previously reported missense variant, c.1787C>T (p.Ala596Val), and a novel duplication defined as c.1007_1025dup19 (p.Asp343Leufs*53); the duplication is predicted to result in a premature stop codon and protein truncation. We propose that this novel variant is partly responsible for the patient’s early-onset and severe phenotype.

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.

AbstractObjectivesTo analyze the economic impact of testing for activated protein C resistance (APC-R) due to factor V Leiden (FVL) mutation with APC-R with reflexive FVL genotyping (algorithmic approach) or genotyping alone.MethodsOptumLabs Data Warehouse (OLDW) data were used to assess testing approaches. Insurance claims for APC-R and FVL in 2013 were compared with the Mayo Clinic database. Centers for Medicare & Medicaid Services diagnostic fee schedules were used to assign costs.ResultsOf 19.3 million OLDW-covered individuals, 74,242 (0.385%) received 75,608 tests: APC-R, 2,265 (2.9%); FVL genotyping, 70,619 (90.1%); and both APC-R and FVL, 2,724 (7.0%). In total, 1,317 tests were performed at Mayo Clinic: APC-R with reflex FVL (1,256; 95.4%) and FVL alone (61; 4.6%). Costs per evaluated individual and per total population (person/year) in OLDW and algorithmic approach were $83.77 vs $36.38 and $0.32 vs $0.14, respectively.ConclusionsThe cost-optimized algorithmic approach reduces health care costs.

Rivaroxaban and dabigatran are among the newest anticoagulants, and measuring their concentration in patients is a new challenge for clinical laboratories. We analyzed data from the ECAT proficiency program to determine how well the assays are performing in clinical laboratories internationally. Most laboratories received a passing grade (Z score <3) for the results of their dabigatran and rivaroxaban testing. Failing Z scores were not associated with any particular method. With dabigatran, some homemade calibrators gave higher results than the commercially available calibrators. There were no significant differences among the instruments or the 5 reagents in use, but results showed inter‐laboratory variability that could have clinical significance. The 3 reagents with the lowest number of users had poor inter‐laboratory precision. Ten different anti‐Xa reagents were in use for rivaroxaban testing. One reagent gave lower results than other reagents at 100 ng/mL but not at 300 ng/mL. There were no significant differences among the different rivaroxaban calibrators or instruments. In conclusion, inter‐laboratory precision could be improved for both dabigatran and rivaroxaban assays. Homemade dabigatran calibrators differed from commercially available calibrators, and there was a statistically significant difference between some of the rivaroxaban reagents. About 10% of results received failing Z scores or passed but fell in a range that require the laboratory to investigate for bias or other inaccuracy in their method. Am. J. Hematol. 91:E464–E467, 2016. © 2016 Wiley Periodicals, Inc.