title: Validation of the NucliSens Extractor combined with the AmpliScreen HIV version 1.5 and HCV version 2.0 test for application in NAT minipool screening.
abstract: BACKGROUND
  Routine HCV NAT minipool screening (48 donations) of all blood donations was implemented in July 1999 and was combined with HIV NAT in November 2000. This report describes the validation of the NAT methods and the results of quality control testing.
  
  
  METHODS
  Nucleic acid was extracted from 2-mL plasma samples by using an automated silica-based extraction method (NucliSens Extractor, Organon Teknika). Eluates were tested with RT-PCR (AmpliScreen HIV-1 version 1.5 and AmpliScreen HCV version 2.0 test, Roche Diagnostic Systems). HIV-1 and HCV RNA reference panels and run controls (PeliCheck and PeliSpy, respectively, Sanquin-CLB) and human plasma minipools were used for NAT validation.
  
  
  RESULTS
  The 95-percent detection limit (and 95% CI) for HIV-1 RNA genotype B, HIV-1 RNA genotype E, and HCV RNA genotype 1 was 32 (19-76), 30 (17-72), and 21 (13-44) genome equivalents (geq) per mL, respectively. During initial validation, 2332 samples for HIV-1 RNA and 2644 samples for HCV RNA were analyzed, with 13 (0.56%) and 12 (0.45%) invalid test results, respectively. Thereafter, over 19,600 samples (minipools and run controls) were analyzed during the first 11 months of routine screening. Invalid test results for HIV-1 RNA and HCV RNA were found in 1.1 and 1.07 percent of the samples tested, respectively. HIV-1 RNA minipool testing resulted in 27 (0.16%) initial false-positive results and 3 (0.02%) confirmed positive results. HCV RNA minipool testing resulted in four (0.02%) initial false-positive results and five (0.02%) confirmed positive results.
  
  
  CONCLUSIONS
  Routine HIV and HCV NAT minipool screening using the NucliSens Extractor, AmpliScreen HIV-1 version 1.5, and AmpliScreen HCV version 2.0 meets the sensitivity criteria set by the regulatory bodies and provides sufficient specificity and robustness for timely release of blood donations.
authors.affiliation: Sanquin Blood Bank Nortwest, Utrecht, the Netherlands. jmjongerius@hotmail.com
authors.forename: John M
authors.initials: JM
authors.lastname: Jongerius
authors.forename: Margret
authors.initials: M
authors.lastname: Sjerps
authors.forename: H Theo M
authors.initials: HT
authors.lastname: Cuijpers
authors.forename: Harry A J
authors.initials: HA
authors.lastname: van Drimmelen
authors.forename: Cees L
authors.initials: CL
authors.lastname: van der Poel
authors.forename: Henk W
authors.initials: HW
authors.lastname: Reesink
authors.forename: M Hans J
authors.initials: MH
authors.lastname: Molijn
authors.forename: Geert A H
authors.initials: GA
authors.lastname: Peeters
authors.forename: Ton P W
authors.initials: TP
authors.lastname: Peeters
authors.forename: P Nico
authors.initials: PN
authors.lastname: Lelie
mesh_terms: D000327:Adsorption; D001782:Blood Donors; D005189:False Positive Reactions; D005838:Genotype; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008403:Mass Screening / Q000295:instrumentation* / Q000379:methods; D009426:Netherlands; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D014766:Viremia / Q000097:blood / Q000175:diagnosis*
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide