Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami.

UNLABELLED: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. SUMMARY: Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.

Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results.

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or <40 U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73 U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20 U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0 U/dL). For specimens with FVII >50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant.

Lupus anticoagulant detection: out of control?

INTRODUCTION: Expert guidelines indicate that normalised ratios are preferred to clotting times for lupus anticoagulant (LA) assays to mitigate analytical variation. We investigated the effects of deriving normalised ratios from the reference interval (RI) mean or different normal pooled plasmas (NPP). METHODS: Screen, confirm and mixing tests for dilute Russell's viper venom time and APTT were converted to normalised ratios and interpreted for LA. RESULTS: Of 1000 clinical samples, 824 generated identical interpretations using RI mean or NPP-derived ratios and 57 identified LAs in one or both assays via either denominator. Separate RIs were applied for normalised ratios derived from the NPP or RI mean. Applying percentage correction index (PCI) to screen and confirm assays irrespective of screen elevation increased agreement to 92.5%. Two frozen and one lyophilised NPP were then used to derive ratios for 204 samples and 130 generated identical interpretations with all NPPs, 14 had overall interpretation parity and 19 overall agreement via PCI, giving 79.9% overall agreement. The results derived from each NPP were interpreted against RIs derived from RI means to reflect differences resulting from NPPs with clotting times dissimilar to RI means. CONCLUSIONS: Disparities were largely a function of closeness of NPP clotting times to test RI means and not owing to clotting factor level differences and likely related to manufacturing variables. Diagnostic benefit of normalised ratios can be maximised by matching NPP values to RI means. If RI mean is employed, and it likely requires re-establishing with new reagent batches.