Background/Case Studies: Screening for Human T-cell Leukemia Virus Type 1 (HTLV-1) is mandatory for all blood donors in Japan to prevent transfusion-transmitted infections. The Japanese Red Cross Society initially screens for HTLV-1/2 antibodies using a chemiluminescent immunoassay (Abbott). Specimens testing positive are confirmed using a line immunoassay (LIA; Fujirebio), but approximately 10% of LIA results remain indeterminate. To resolve these cases, a PCR assay for HTLV-1/2 proviral DNA is performed. We developed an in-house assay using the cobas omni Utility Channel tool on a cobas 5800 System (Roche) to fully automate the PCR process.
Study
Design/Methods: A real-time PCR assay was developed according to the instructions provided in the User Guide of the cobas omni Utility Channel. Primers and probes targeted the pX regions of HTLV-1 and HTLV-2, with human RNase P gene as an internal control to detect cellular genomic DNA. PCR conditions, including thermal profiles, primer and probe concentrations, dye selection, and quencher positioning, were optimized using the cobas omni Optimization Kit on the LightCycler 480 system (Roche) Optimized PCR conditions were transferred to the cobas 5800 System. A reagent cassette containing custom primers and probes was prepared, and an automated assay was performed. To assess detection sensitivity, HTLV-1- and HTLV-2-positive T-cell lines (MT-2 and MoT) suspended in whole blood from HTLV-negative individuals, as well as whole blood from HTLV-1-positive blood donors, were used.
Results/Findings: PCR performance was enhanced using methylated primers and Black Hole Quencher-labeled probes. For DNA extraction from whole blood using the cobas 5800 System, a 2.25-fold dilution with MagNA Pure External Lysis Buffer (Roche) was used as sample pretreatment to ensure stable test results. The volume of whole blood used for each assay was 223 µL, significantly higher than that of the conventional method (80 µL). The 95% limit of detection was 4.0 copies/reaction for HTLV-1 and 5.7 copies/reaction for HTLV-2, comparable to or exceeding conventional methods.
Conclusions: Fully automated HTLV-1/2 PCR testing using the cobas 5800 System is feasible, providing complete automation from sample loading to result reporting, reducing labor and processing time while improving quality control. However, testing costs are approximately three times higher than conventional methods, necessitating careful cost-benefit consideration
