FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Abstract

Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

Effect of ADAMTS13 activity turnaround time on plasma utilization for suspected thrombotic thrombocytopenic purpura

“…Our preference was the FRET assay and recently interassay nonconcordance has been observed in up to 10% of TTP samples, perhaps attributable to the presence of ADAMTS13 antibodies interfering with the performance of the collagen-binding assay and resulting in falsely high ADAMTS13 activity. 14,15 Our data show that providing ADAMTS13 activity levels in a timely fashion significantly reduces unnecessary plasma utilization in a tertiary care center and is not associated with any increase in overall mortality for patients with TTP or suspected TTP. In conjunction with previously established diagnostic criteria, widespread adoption of rapid TAT for ADAMTS13 activity may result in significant plasma avoidance, improved patient outcomes, and cost savings.…”

“…The collagen‐binding assay and the FRET assay are two widely accepted functional assays for the measurement of ADAMTS13 activity. Our preference was the FRET assay and recently interassay nonconcordance has been observed in up to 10% of TTP samples, perhaps attributable to the presence of ADAMTS13 antibodies interfering with the performance of the collagen‐binding assay and resulting in falsely high ADAMTS13 activity …”

Thrombotic thrombocytopenic purpura

“…31,58 Collagen-binding activity and FRETS-VWF73-based assays are adopted reference methods for ADAMTS13 activity measurement, 54,59 whereas FRETS-VWF73 is probably superior to collagen-binding activity assay. 60 These methods may also be time-consuming, with the shortest one (FRETS-VWF73 assay) 54 requiring several labor-intense hours for turnaround of results. As a consequence, these reference methods are limited to expert laboratories (usually 1 or 2 laboratories per country worldwide centralizing ADAMTS13 biology and networking with clinical centers involved in the management of patients with TMA).…”

ADAMTS13 and von Willebrand Factor in Thrombotic Thrombocytopenic Purpura

“…Zheng, unpublished observation). Denaturants added to the reaction, such as urea or guanidine, may dissociate ADAMTS13-autoantibody complexes, which might falsely elevate plasma ADAMTS13 activity (81). In general, most samples with severe deficiency of ADAMTS13 activity (<5%) show a good agreement among CBA, FRETS-VWF73, and chromogenic VWF73 assays, but only 83% of these samples with <11% activity and 52% of samples with 11–55% activity show agreement between the CBA and FRETS-VWF73 or chromogenic VWF73 results (82).…”

“…Such discrepant results indicate the presence of urea, bilirubin, and free hemoglobin, which may affect one assay but not another. A recent report suggests that measurement of plasma ADAMTS13 activity by the FRETS-VWF73 assay, but not a multimeric VWF-based assay in the presence of urea, more closely reflects plasma ADAMTS13 status in TTP patients (81). …”