High-throughput extraction, amplification, and detection (HEAD) of HCV-RNA in individual blood donations.

Abstract

High-throughput nucleic acid amplification techniques (NATs) are required for the detection of viral genomes in individual blood donations and might be helpful in any virological laboratory. To develop and automate a method for the detection of hepatitis C virus RNA in individual blood donations, compatible with the time schedule of routine blood bank screening an product release. The viral RNA was isolated with the use of target specific capture oligonucleotides and magnetic beads. This extraction method was combined with reverse transcription/amplification (RT/PCR) and fluorescence detection. We adapted our method on a pipetting robot and pipetted all steps in a single room. When the pipetting was completed, microtiter plates were heat-sealed with foils and placed into a thermocycler. Positive reactions were detected with a fluorescent dye in a second room. Aerosols were avoided with programmed slow pipetting steps and with a special device constructed for the removal of the used disposable tips. During a 7 month period, we used this method in routine testing of individual donations prior to the release of all blood components. The total number of 11,700 individual donations including platelet concentrates were analysed. We tested up to 192 specimens in one run within 7 h. The frequency of cross-contamination using the automated procedure was 0.1%. Five specimens have been found repeatedly reactive for HCV-RNA, four of these were anti-HCV positive, one sample from a repeat donor was negative in anti-HCV assays. A seroconversion was detectable at his next presentation, 6 months later. In this pilot study, we demonstrate that automated HCV-RT-PCR testing is practicable for individual donations in high-throughput. Additionally, the described PCR approach could easily be adapted to the detection of other viral genomes by the use of specific primers.