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 Ca 2+ 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.