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Laboratory Validation of Technofluor FXIII Activity on Ceveron s100 Instrument Rachel R Leger, Juliana Perez Botero, M.D., Dong Chen, M.D., Ph.D., Rajiv K Pruthi, M.B.B.S. Mayo Clinic, Rochester, MN, USA

Abstract

Introduction: Factor XIII (FXIII) is a transglutaminase enzyme that plays a critical role in the final stage of the coagulation cascade by stabilizing fibrin clots through the formation of covalent cross-links. In plasma, factor XIII circulates as a heterotetramer (FXIII-A₂B₂) composed of two catalytic A subunits and two carrier/regulatory B subunits.  FXIII deficiency, congenital (rare, approximately 1 per million) or acquired can manifest as severe bleeding diathesis. Traditional clot solubility assays are qualitative and only detect severe deficiencies, lacking sensitivity for mild FXIII activity reductions. This study aimed to validate a sensitive, quantitative FXIII activity assay on a Ceveron s100 analyzer. Methods: The Technofluor FXIII Activity assay was optimized on the Ceveron s100 instrument (both from Technoclone Diagnostica, Austria) using FXIII assigned calibrators and controls (Precision BioLogic Inc., NS Canada). Acceptance criteria and validation parameters included: Repeatability/within laboratory precision (CV≤5.7%/CV≤15.9%), method comparison to samples with assigned values or compared with an ammonia release FXIII activity assay (R² ≥0.900, slope 1.00 ± 0.20 and y-intercept ±5), acceptable deviation from linearity for reportable range (concentration levels ≥10% FXIII Activity ±5.6%, levels <10% FXIII Activity ±1.6) and analytical sensitivity (CV ≤20%). Repeatability and within laboratory precision studies were performed on 2 replicates, 2 runs/day for 20 days on three FXIII activity concentrations (~10, 30 and 130%) using residual waste patient plasma or diluted control plasma. Method comparison studies were performed on commercially available calibrators, controls, proficiency testing samples and waste de-identified patient samples (n=114) spanning the analytical measurement range. A linearity panel consisting of twelve concentration levels, tested in quadruplicate, from residual waste specimen diluted with FXIII assay buffer. The reference range was established following CLSI guidelines (n=120). Analytical sensitivity (limit of detection) was performed on 2 reagent lots for 3 days with 20 replicates per day on assay kit buffer and a control material diluted to ~2% FXIII activity. Results: The assay met all acceptance criteria outlined in methods section: Repeatability and within-laboratory precision were ≤5.2% and ≤7.5% CV, respectively. Method comparison (Figure 1) showed strong correlation (R² = 0.933, slope = 1.004, y-intercept = 2.36). Linearity (Figure 2) concentrations ranged from 1.7 – 237.6% and had nonlinearity of ≤4.3% on concentrations >10% FXIII activity and <0.6 FXIII nonlinearity on concentrations <10%. Analytical sensitivity was confirmed at 2% FXIII activity (15.4% CV). The normal reference range was established at ≥55% FXIII activity. Interference from hemolysis, icterus, and lipemia may falsely decrease FXIII activity results by up to 50%. This was resolved by sample dilution (1:5) with saline. Unfractionated heparin, up to 10 U/mL, did not interfere with the assay. Conclusions:  The Technofluor FXIII Activity assay on the Ceveron s100 analyzer provides sensitive and reliable detection of mild and moderate FXIII deficiencies, overcoming the limitations of traditional clot solubility assays. The validation met all performance criteria, supporting its use in clinical laboratories for improved and more clinically impactful diagnosis of FXIII deficiency.

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