A-163 Validation of a new semi-automated ADAMTS13 activity assay for prompt evaluation of presumptive thrombotic thrombocytopenic patients

Abstract Background The ADAMTS13 activity assay is considered a diagnostic test for confirmation of clinical diagnosis of thrombotic thrombocytopenia purpura (TTP). Nonetheless, most common methodologies involve ELISA-based assays that are manual and time consuming, limiting the availability and turnaround time (TAT). A faster, semi-automated assay in a coagulation analyzer can help expedite cases and improve the TAT. Objective: To validate the ADAMTS13 activity and inhibitor assay in the Ceveron-s100 coagulation analyzer (DiaPharma) in comparison with the ELISA-like assay (ACTIFLUOR™ ADAMTS13 Activity Kit from BioMedica Diagnostics). Both methods are based on Fluorescence resonance energy transfer (FRET), thus allowing a direct comparison of the TAT. Methods The inter-, intra-assay precision, accuracy, linearity, interferences and verification of the reference range in the Ceveron were validated in comparison to the ELISA-like assay. The TAT was monitored before and after go-live with the Ceveron. Results 28 samples were tested for the ADAMTS13 activity and 29 for the inhibitor assay to establish accuracy and performance. Results were compared between the original ELISA results and each individual Ceveron analyzer. The correlation between the two methods was good (R = 0.95, and slope = 0.9 ± 0.3). The inter- and intra-assay precision for both instruments achieved <10% CV. To verify the linearity, we ran several calibrator dilutions from the ACTIFLUOR™ ADAMTS13 Activity Kit, achieving an R2 greater than 0.990 when both results from both Ceverons were compared to the ELISA results. To establish the limit of detection, heat-inactivated Normal Pooled Plasma was tested 10 times on the analyzer. The limit of detection was < 1% activity (mean ±3SD). We verified the thresholds for interferences and validated a 1:5 dilution for hemolysis of 3+ and bilirubin >10 mg/dl. To verify the reference range, 25 different normal donor samples were tested in the Ceveron instruments resulting in an activity mean ±2SD of 54-133%. 29 samples were tested for the inhibitor assay in the Ceveron compared to the original results from the ELISA. 94% of samples had a CV less than 20%. We monitored the TAT one month before go-live using the ELISA-like assay and it compared to the first month after go-live with the Ceverons. The average TAT improved to 12.3 hrs. from 34 hrs. and the median improved to 3.7 hrs. compared to 25.7 hrs. the month prior. Conclusion In conclusion the semi-automated Ceveron ADAMTS13 activity is comparable to the ELISA-like FRET assay. The Ceveron offers several advantages including decreased sample preparation, minimal cleaning and maintenance, lot-stable monthly calibration curve, individual sample testing allowing for fast TAT with potential 24/7 availability. Lastly a cost-effective reflex algorithm was established wherein only samples with a low ADAMTS13 activity are sequentially tested for an inhibitor.