PB0068 - Evaluation of a standardized reagent kit for functional ADAMST13 inhibitor testing

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 a reagent kit for quantitative determination of functional ADAMTS13 inhibitors based on a Bethesda principle suitable for all ADAMTS13 activity measurement protocols.

Methods: Plasma samples were heat treated for 30min at 56°C to eliminate residual ADAMTS13 activity. Heat inactivated samples were diluted in dilution buffer from the kit depending on the functional inhibitor concentration. Diluted samples were mixed with the reagent containing 1.0IU/mL ADAMTS13 and incubated for 30min. ADAMTS13 activity was then quantified by either Technozym ADAMTS13 activity ELISA (Technoclone), or Technofluor ADAMTS13 Activity (Technoclone) performed on a Ceveron s100 (Technoclone). Compared with a control mixture (1+1 mix of ADAMTS13 depleted plasma with 1.0IU/mL reagent), residual ADAMTS13 activity was calculated, and Bethesda units determined. Additionally, results were compared to the presence of anti-ADAMTS13 IgG (Technozym ADAMTS13 inhibitor ELISA) measured in these samples.

Results: More than 25 samples from patients with acquired TTP were tested. Several patients had clearing antibodies and were Bethesda-negative, only manifesting in anti-ADAMTS13 IgG ELISA. Correlation between Bethesda titre by FRETS and ELISA was Pearson r >0.95. Sensitivity was investigated with >60 inhibitor free samples, generating a mean BU of 0.06 (SD 0.08). Furthermore, the mixing with 1.0IU/mL ADAMTS13 plasma, and incubation with subsequent ADAMTS13 activity determination, can be fully automated on the Ceveron s100 using the Technofluor FRETS method.

Conclusion(s): The ADAMTS13 functional inhibitor reagent kit provides a standardized setup to determine functional inhibitors in TTP patients. Using the automated FRETS method on Ceveron s100, the procedure is fully automated and provides immediate distinction between hereditary and acquired TTP through functional inhibitory antibodies.