PB1093 - Diagnostic Potential of Rapid Automated ADAMTS13 Inhibitor Screening and Quantification

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.