Implementation of a fully automated ADAMTS13 activity assay utilizing fluorescence resonance energy transfer with a practical approach to address high background fluorescence interference

Objective This study evaluates an automated fluorescence resonant energy transfer (FRET)–based ADAMTS13 activity assay on the Ceveron S100 instrument for the diagnosis of thrombotic thrombocytopenic purpura. It addresses the challenge of high background fluorescence (HBF), a known concern from our manual FRET assay, and proposes strategies to minimize erroneous results. Methods We compared FRET-Ceveron results with FRET-Manual (n = 100) and Technozym (Technoclone) enzyme-linked immunosorbent assay (ELISA) (n = 52) using retrospective and prospective patient samples collected throughout 2024, alongside proficiency samples and standards with assigned values (n = 24). We analyzed 7 spiked samples with HBF and 14 patient samples exhibiting HBF while exploring predilution methods. Over 200 FRET-Ceveron reactions were examined to identify abnormal patterns and establish thresholds for HBF interference. Results The FRET-Ceveron assay demonstrated a strong correlation (r² > 0.97) with Technozym ELISA, FRET-Manual, and target results. It successfully detected critically low ADAMTS13 levels (<10%) across various sample types (n = 15). While HBF affected both FRET methods, FRET-Ceveron displayed greater tolerance to HBF. No significant difference was found in FRET-Ceveron result accuracy for initial carbon nanotubes (CNTs) up to 1100 (P = .39), but significant differences were observed when CNTs exceeded 1100 (P = .02). Predilution effectively reduced HBF (P < .05), validating the results confirmed by Technozym ELISA. Conclusions The fully automated FRET-Ceveron assay is a rapid and accurate method for ADAMTS13 testing, and it is particularly effective when a normal reaction pattern is observed (initial CNTs ≤1000 with a good linearity in reaction tracing during 7- to 22-minute measurements). New sample collection is preferred in the presence of HBF, with predilution as a viable option.