Background: Acquired ADAMTS13 deficiency due to inhibitory or clearing antibodies leads to thrombotic thrombocytopenic purpura (TTP). Rapid discrimination of TTP from similar disorders using laboratory assays improves clinical outcomes.

Aims: Evaluate rapid screening and quantification assays for inhibitory ADAMTS13 antibodies.

Methods: Technofluour ADAMTS13 FRET Activity (Technoclone) was performed on a Ceveron s100 (Technoclone) automate. The screen assayed ADAMTS13 activity on a 1:1 mixture of index:normal pooled plasma (NPP) to calculate the ADAMTS13 Inhibitor Mix (AIM) Ratio; [mix activity/(activity of 1:1 mix of NPP + ADAMTS13 depleted plasma)]. Instead of merely assessing if a mixing test returns to normal or not, which is not always reliable, AIM Ratio is a quantitative value derived from the mix result as a function of the measured activity of the NPP in which it is mixed, interpreted against an objective cut-off. The FRET assay was used in a 30 min incubation Bethesda assay using heat treated index plasma, to quantify inhibitory antibodies. Results were compared to Technozym ADAMTS13 inhibitor ELISA (Technoclone), detecting both inhibitor types but not discriminating, and a Bethesda with Technozym ADAMTS13 activity ELISA (Technoclone). Four plasmas were tested with FRET-based Bethesda assays at different incubation times.

Results: Table 1 shows comparative results from 13 acquired TTPs. Patients 4/10/12 had clearing antibodies and were thus AIM Ratio- and Bethesda-negative. Correlations for FRET/ELISA Bethesdas, FRET Bethesda/ELISA Inhibitor, and FRET Bethesda/AIM Ratio were Pearson R 0.993/0.710/-0.870 respectively. Patients 2/13 had low Bethesdas relative to ELISA, suggesting presence of clearing and inhibitory antibodies. Patient 11 had borderline ELISA, negative Bethesdas, but weak-positive AIM Ratio, suggesting the latter detects low titre inhibitory antibodies. The Bethesda incubation experiment suggests prolonged incubation is unnecessary (Table 2).

Conclusion(s): Fully automated ADAMTS13 activity and AIM Ratio assayed simultaneously provide immediate distinction between hereditary and acquired TTP through inhibitory antibodies. Prolonged incubation for subsequent Bethesdas is unnecessary.

Table 1.

Comparative ADAMTS13 assay data

Table 2.

Bethesda values at different incubation times

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/diagnostic-potential-of-rapid-automated-adamts13-inhibitor-screening-and-quantification/