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

Abstract

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. 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. 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. 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.