Time‐ and Temperature‐Dependent Response of Acquired FV Inhibitors: Implications for Laboratory Diagnosis and Clinical Management

BACKGROUND: Acquired factor V (FV) inhibitors are rare circulating autoantibodies that may cause severe bleeding and false-negative detection without standardized incubation. This study aimed to evaluate time- and temperature-dependent FV inhibitor activity and define optimal laboratory detection conditions. METHODS: Fifteen serially diluted plasma samples from 4 FV inhibitor-positive patients were incubated at room temperature (RT) or 37°C for 0-120 min. APTT mixing assays, residual FV activity, PT mixing assays, and Bethesda inhibitor titers were detected, with inhibitor-free normal pooled plasma (NPP) as thermal degradation control. ISTH-recommended MTC index was adopted for standardized mixing result interpretation. RESULTS: At 37°C, APTT/PT mixing times were progressively prolonged and residual FV activity declined continuously, reaching a plateau at 60 min, whereas only slight changes occurred at RT. After excluding ~6% spontaneous FV thermal loss in NPP, residual FV activity at 37°C was significantly lower than RT at all time points (p < 0.05). Bethesda titers rose progressively at 37°C across all samples, confirming antibody-mediated neutralization rather than native protein decay. Pronounced inter‑individual variability in inhibitor potency was observed under equalized inhibitor concentrations. CONCLUSIONS: FV inhibitors exhibit distinct time- and temperature-dependent neutralizing activity. RT testing without sufficient 37°C incubation raises false-negative risk. Universal 2 h incubation at 37°C is recommended for routine inhibitor screening; 60 min incubation is only suitable for follow-up of confirmed FV inhibitor cases and cannot replace standard 2-h protocol for ruling out FVIII inhibitors.