BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: 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.