Measurement of von Willebrand factor cleaving protease (ADAMTS‐13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas

Abstract

Turecek PL, Mannucci PM. Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas. J Thromb Haemost 2004; 2: 1601-09.Summary. Background: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. Design: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. Results: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from <10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. Conclusions: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

Thrombotic Thrombocytopenic Purpura: A Thrombotic Disorder Caused by ADAMTS13 Deficiency

“…The detection methodology ranges from gel analysis to ELISA to FRET (fluorescnce resonance energy transfer). An international collaborative study find that not infrequently the assays produce variable results [77]. Thus, interpretation of ADAMTS13 activity assay results should be individualized according to the experience of the laboratories.…”

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

“…10 The occurrence of photo-bleaching and/or substrate exhaustion for samples with an ADAMTS13 activity of 30% or less in the standard assay was excluded by an extended run of 120 cycles and an increase in the number of light flashes from 10 to 100 per cycle; (iii) a flow-based assay, as described in detail elsewhere. [11][12][13] In this assay, the extent of cleavage of platelet-covered, endothelial cell-derived ultra-large VWF strings by ADAMTS13 is evaluated under fluid shear conditions. Mixtures of normal human plasma and heat-inactivated normal human plasma corresponding to ADAMTS13 activities of 100%, 50%, 25%, and 10% were used for semi-quantitative calibration.…”

ADAMTS13 is expressed in hepatic stellate cells

“…10 Low ADAMTS13 activity levels have been described in patients with several conditions, including liver disease and sepsis. [11][12][13] Nevertheless, the mechanisms for the decreased ADAMTS13 activity levels in these conditions are not clear and except for circulating inhibitors or mutations affecting the ADAMTS13 gene, the factors regulating plasma ADAMTS13 levels remain poorly understood. The lack of characterization of the cells that synthesize ADAMTS13 has limited the progress of investigation.…”