ADAMTS13 Activity Measurement Using a Chemiluminescence-Based Technique. Prospective Multicenter Comparative Study of the Spanish Apheresis Group (GEA)- Spanish Society of Hematology and Hemotherapy (SEHH)

INTRODUCTION

Thrombotic thrombocytopenic purpura (TTP) is a rare and potentially life-threatening thrombotic microangiopathy (TMA) characterized by a severe deficiency of ADAMTS13 activity (<10%). A rapid and accurate estimation of ADAMTS13 activity its crucial for TTP diagnosis. The rapid technique based on chemiluminescence has been implemented in several healthcare laboratories in Spain and Portugal for the determination of ADAMTS13 activity. However, there are certain concerns about the sensitivity and specificity of this technique, as well as its correlation with other commonly used techniques, such as ELISA and/or FRETS (gold-standard).

OBJECTIVES

To evaluate the performance of the chemiluminescence technique for the determination of ADAMTS13 activity in patients with suspected TMA, in comparison to other commonly used techniques.

MATERIAL AND METHODS

A prospective, multicenter, international study has been conducted between October 2022 and January 2024, to compare the chemiluminescence-based technique (CLIA, Werfen, Barcelona, Spain )against FRETS-VWF73 and commercial ELISA (TECHNOZYM ADAMTS13 Activity ELISA Kit, Technoclone, Austria) at 6 centers in Spain and Portugal, within the project “Improvement of immunologic and molecular techniques for the diagnosis and follow-up of patients with thrombotic thrombocytopenic purpura: a collaborative study proposal of the Spanish Apheresis Group (GEA) in collaboration with the Spanish Society of Hematology and Hemotherapy (SEHH), NCT05046717.

The citrated plasma samples previously centrifuged at 2000 g for 15 minutes and frozen on the day of extraction at - 40 ºC, were thawed and processed simultaneously in the 2 corresponding techniques. Routine clinical and laboratory parameters were collected for each patient at presentation

The correlation (Lin/Passing Bablock coefficient) and bias (Bland-Altman) between CLIA and the different reference techniques were evaluated. The cut-off point of <10% activity was used to evaluate concordance (Kappa), sensitivity and specificity.The study was conducted in accordance with the principles of the Declaration of Helsinki.

RESULTS

A total of 512 samples from patients with suspected TMA (74 diagnostic and 365 follow-up samples of acquired TTP, 38 congenital TTP, 5 hemolytic uremic syndrome and 30 samples of other TMA) were analyzed by the reference techniques (ELISA or FRET) vs CLIA. The most commonly used gold standard was ELISA (84.4% ELISA vs. 15.6% FRET).

A high concordance was found between CLIA and reference techniques for the classification of patients with ADAMTS13 < 10% (kappa 0.93 for ELISA and 0.85 for FRETS), with a sensitivity of 90.5% (95% CI: 82.0 to 95.1) for ELISA and 95.5% (95% CI: 84.9 to 98.7) for FRETS and a specificity of 99.7% (95% CI: 98.4 to 100) for ELISA and 89.0% (95% CI: 74.7 to 95.6) for FRETS.

For the ADAMTS13 activity cut-off of <10%, 8 false negatives and 7 false positives were found with the CLIA technique, compared with ELISA/FRET, almost all from samples of patients with iTTP in follow-up. One of the false negatives with a borderline value was an iTTP sample at the time of diagnosis that did not influence the patient's final diagnosis." The Bland-Altman plot showed that the mean difference between FRET and CLIA was 5.4% with a limit of agreement between -28.1 and 38.9 while that between ELISA and CLIA was 11.8 with a limit of agreement between -27.2 and 50.8.

CONCLUSIONS

In this prospective study, which includes a high number of samples of suspected TMA, we demonstrate that there is a good agreement in the ADAMTS13 activity results obtained by CLIA with respect to the “gold standard” FRETS/ELISA methods. We found discrepancies mostly in some follow-up iTTP samples, which could have therapeutic implications in a small number of patients. To date, borderline results with CLIA should be confirm with a validation technique.