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CC BY 4.0 · TH Open 2018; 02(03): e329-e333
DOI: 10.1055/s-0038-1672187
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Bethesda Assay for Detecting Inhibitory Anti-ADAMTS13 Antibodies in Immune-Mediated Thrombotic Thrombocytopenic Purpura

Chiara Vendramin
1   Department of Haematology, University College London Hospital, London, United Kingdom
,
Mari Thomas
2   Department of Haematology, University College London Hospital, Cardiometabolic Programme NIHR UCLH/UCL BRC, London, United Kingdom
,
John-Paul Westwood
1   Department of Haematology, University College London Hospital, London, United Kingdom
,
Marie Scully
2   Department of Haematology, University College London Hospital, Cardiometabolic Programme NIHR UCLH/UCL BRC, London, United Kingdom
› Author Affiliations
Further Information

Publication History

07 June 2018

09 August 2018

Publication Date:
26 September 2018 (online)

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Abstract

A diagnosis of thrombotic thrombocytopenic purpura (TTP) is confirmed by a severe deficiency (<10%) of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity. Autoantibodies to ADAMTS13 can be detected with a simplified enzyme-linked immunosorbent assay (ELISA). An alternative methodology is a Bethesda assay, which has never been formally assessed in TTP. This study aimed to investigate the inhibitory anti-ADAMTS13 antibody assay and determine if the Bethesda assay is advantageous compared with the ELISA, measuring total immunoglobulin G (IgG) antibodies to ADAMTS 13. The Bethesda method determines the neutralizing activity of anti-ADAMTS13 antibodies in pooled normal plasma. We selected six immune-mediated TTP (iTTP) patients with ADAMTS13 activity levels <10% and strong ADAMTS13 inhibitors by 50:50 mixing studies and analyzed anti-ADAMTS13 antibodies using the Bethesda and ELISA assays. ADAMTS13 activity was stable at room temperature, while a time-dependent decrease in activity was detected in assay conditions of 37°C. Adding 5 mM Ca2+ to citrated plasma prevented loss of ADAMTS13 activity with time. There was time dependence to the antibody-mediated inactivation, after 2-hour incubation. Two of the iTTP patients had no detectable ADAMTS13 antibodies by the Bethesda assay, but had high titer of anti-ADAMTS13 antibodies and low ADAMTS13 antigen levels. The Bethesda assay can only detect anti-ADAMTS13 antibodies that functionally inhibit ADAMTS13. The anti-ADAMTS13 IgG ELISA instead allows the rapid identification of total IgG autoantibodies, detecting both inhibitory and noninhibitory antibodies.

Keywords

ADAMTS13 protein - Human plasma - anti-ADAMTS13 inhibitors - Bethesda assay - enzyme-linked immunosorbent assay - thrombotic thrombocytopenic purpura

Authors' Contributions

C.V. analyzed the data and wrote the manuscript; M.T., J-P.W., and M.S. collected data and reviewed the manuscript.