Serological screening in blood banks is a crucial step to ensure transfusion safety by preventing the transmission of infectious agents to recipients. Among the pathogens screened, human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) are included due to their potential for transfusion transmission and their possible association with neurological and hematological conditions, such as Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (TSP/HAM) and adult T-cell Leukemia/Lymphoma (ATLL). Although the immunoassays employed in initial screening are highly sensitive, they may yield false-positive results, which can lead to the unnecessary exclusion of eligible donors, increased psychological burden, and higher costs due to confirmatory testing. Therefore, evaluating the frequency and underlying factors associated with false-positive HTLV-1/2 results is essential for refining screening strategies that balance transfusion safety with donor retention.

This study aimed to determine the optimal Signal-to-Cutoff (S/CO) threshold in a chemiluminescent immunoassay to improve the discrimination of true HTLV-1/2 positivity in blood donors.

A retrospective cross-sectional analysis was conducted on 220,340 donations, among which 407 samples (0.18%) were initially reactive for anti-HTLV-1/2 antibodies using a chemiluminescent immunoassay. Of these, 223 samples (55.0%) were available for further evaluation with a confirmatory assay (Inno-Lia HTLV-1/2–Fujirebio®) and real-time PCR. A Receiver Operating Characteristic (ROC) curve was constructed to identify the optimal cutoff point.

Among the 223 retested samples, 63 (28.3%) were confirmed positive, 112 (50.0%) were negative, and 48 (21.7%) had indeterminate results by confirmatory testing. HTLV DNA was detected in 56 (25.1%) samples by real-time PCR. ROC curve analysis indicated that a S/CO ratio ≥ 4.0 provided the best discriminatory power, increasing the positive predictive value (PPV) from 47.6% to 66.1%.

Adjusting the S/CO threshold ≥ 4.0 in chemiluminescent immunoassays significantly enhances the accuracy of HTLV-1/2 screening in blood donors. This refinement reduces false-positives rates, prevents unnecessary discard of blood components, and optimizes the donor counseling and notification process. The implementation of evidence-based cut-off adjustments contributes to enhance transfusion safety, especially in low-prevalence settings, such as blood banks.