disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease. (Blood. 2013;121(14):2773-2784
| INTRODUC TI ONRegulation of fibrin formation through activated protein C (APC) degradation of activated factor V (FVa) and activated FVIII is a major anticoagulant process of hemostasis. In 1993, Dahlbäck et al 1 identified thrombophilic individuals whose plasma showed a poor response to APC in a coagulation assay based on activated partial thromboplastin time (APTT). This was found to be related to an arginine 506 to glutamine substitution in F5 rendering FV resistant to APC inactivation, and called FV Leiden (FVL). 2 Mutations in F5 other than FVL that confer APC resistance (APC-R) with various degrees of clinical severity have been described, although they appear to be very rare. 3 As the original assay was based on APTTs performed on undiluted plasma, it was prone to numerous interferences, so a modification was subsequently proposed that improved sensitivity and specificity by diluting test plasma in FV-deficient plasma, and remains in widespread use. 4-6 Acquired APC-R may occur in the absence of F5 mutations, and represents an independent risk factor for venous thrombosis. 7 APC-R detection has evolved since the original assay and its modification, and this is the first guideline to address clinical laboratory testing recommendations for the wider set of commonly available phenotypic assays. | PRE ANALY TIC ISSUE S | Patient selectionIndiscriminate testing for APC-R is not recommended in unselected patients with venous thromboembolism (VTE). Targeted testing is recommended for those situations in which the results may give an indication of risk of recurrence and influence treatment, as is the case for any patient undergoing evaluation for thrombophilia involving VTE. More detail is available in recent clinical guidelines. 8-10 | Sample handlingVenous blood should be collected into a one-tenth volume of 3.2% (0.105-0.109 mol/L) trisodium citrate, and double-centrifuged to ensure a residual platelet count of <10.0 × 10 9 /L. 4,11,12 As some other thrombophilia assays also require similarly effective platelet removal, this permits running of multiple assays with the same, suitably prepared, plasma sample. The white cells remaining after plasma removal can be stored for DNA extraction should molecular analysis be required, or cells from an EDTA-anticoagulated sample can be used.Plasma for APC-R assays should be tested within 4 hours of collection or frozen (at -20°C if stored for ≤2 weeks, and at −70°C or below if stored for >2 weeks) if testing is postponed. Frozen samples should be rapidly thawed in a waterbath at 37°C, and thoroughly mixed by multiple, gentle inversions prior to testing. Icterus, hemolysis and lipemia can interfere, especially in assays performed on undiluted plasma. Plasma predilution assays are less affected by platelet contamination. 3 Preliminary coagulation screening is useful to detect previously unknown factor deficiencies and anticoagulation, and, if a suitably sensitive APTT reagent is employed, lupus anticoagulants (LAs) may also be detected. | InterferencesAssay design and reag...
Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.
Background: Quantifying A disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS-13) activity enhances thrombotic thrombocytopenic purpura (TTP) diagnosis but most assays are time consuming, technically demanding, and mainly available in reference centers.Objective: Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP.Patients/Methods: Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREEN ® ADAMTS13 activity screening test in comparison with TECHNOZYM ® ADAMTS-13 activity ELISA at two centers, and in-house fluorescence resonance energy transfer assay at the third center. The screening test indicates if ADAMTS-13 activity is at one of four level-indicator points: 0, 0.1, 0.4, or 0.8 IU/mL.Results: Screen results were interpreted as binary data in that ADAMTS-13 activity was above or below the 0.1 IU/mL TTP clinical threshold. Combining all sites' data, the screen exhibited 88.7% sensitivity, 90.4% specificity, 74.6% positive predictive value, and 96.2% negative predictive value, comparable to published data for quantitative assays. Five samples with quantitative results below the threshold gave screen readings of 0.1 IU/mL and seven marginally above the threshold gave screen readings of zero. All would warrant plasma exchange while the level is quantified. Nine samples with normal/ near normal results gave screens of zero and confirmatory quantifications would prompt early treatment withdrawal, as is current practice. One sample generated screen/quantitative results of 0.4/0.00 IU/mL respectively and was the only clear false-negative. Conclusions:The screening test provides more rapid ADAMTS-13 level evaluation than most currently available assays. Its simple operation renders it suitable for adoption in routine or specialist laboratory environments.
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