Establishment of National Standards for NAT Blood Viruses and NAT Proficiency Study Program in Japan Saeko Mizusawa, Yoshiaki Okada National Institute of Infectious Diseases, Japan SoGAT XX In Warsaw, Poland 12-13 June 2007

Study Design for National NAT Standards <Preparation of the Candidate: JRC> A candidate plasma was diluted in cryosupernatant, and stored at –80℃. <Organizer & Participants> NAT Working Group (Chaired by Dr Teruhide Yamaguchi ) Industries for Plasma Derivatives/ JRC/Proficient Labs/NIID < Assay > End-point assay 1st assay: 10-fold dilutions to determine endpoint 2nd –5th assay: 7 half-log dilutions around the endpoint <Potency of the Candidate > Calibrated against WHO International Standards Subcommittee on Safety for Plasma-Derived Products Working Group on the Establishment of National Standard for Nucleic Acid Technology Assay <Preparation of the Candidate> 2 Candidate Source Plasma (>10 6 IU/mL) The selected plasma was diluted to 105IU per mL,-80C   <Assay for Determination of the Potency > End-point assay Five independent assay on different days Five independent assays on different days 1st assay: 10-fold dilution to determine endpoint 2nd –5th assay: 7 half-log dilution around the endpoint Candidate (fresh vial ) WHO IS (reconstitute & distribute in aliquots, stored at –80C) Diluted in plasma <Estimate of the Potency for the Candidate>

Summary of Assays Used for HCV-NAT by the Participants in the Study Table1. Assays used in the collaborative study. a) R&D:Smi-test EX-R&D Amplicor: Amplicor HCV virsion 1 (Roche) QIAamp:QIAamp DNA Blood Mini Kit (QIAGEN) b) Eq. Vol. Amplified : the equivalent volume of sample that was amplified in an assay

Potency of HCV-122 Calibrated against WHO IS for HCV-RNA (96/790) Overall (a) = the overall mean potency calculated from the all laboratories. Overall (b) = the overall mean potency calculated from the data excluding laboratories 1 and 2. Overall (a) = the overall mean potency calculated from the all laboratories Overall (b) = the overall mean potency calculated from the data excluding laboratories 1 and 2.

Potency of HCV-122 Calibrated against WHO IS for HCV-RNA (96/790) Mean 0.01 (-0.204 - 0.201) Anti-log 1.02 (0.979 – 1.588)

Potency of HBV-129 Calibrated against the WHO IS for HBV-DNA (97/746) 3 4 5-1 5-2 6 7 2 -3 -2 -1 Lab. Code No. Log Rerative Potency to IS 97/746 Mean -0.352 (-0.539 - -0.165) Anti-log 0.444 ( 0.289 - 0.684) Relative Potency against WHO IS Potency for HBV-DNA (97/746) ( IU/mL) Log -0.352 ( -0.539 - -0.165) 10 5.65 Anti-log 0.444 ( -0.288 - -0.683) 4.4x10 5

Potency of HIV-00047 Calibrated against the WHO IS for HIV-DNA (97/656) 1.8 temes 10 to the fifth

National Standards for NAT, Japan International standard HCV (genotype) 1.0x105 ( 1b ) ( 1 ) HBV 4.4x105 ( C ) 1.0x106 ( A ) HIV-1 1.4x105 ( B ) IU/mL Each vial of a National Standard contains 0.5ml of positive plasma diluted incryosupernatant and should be stored at –80℃.

NAT Proficiency Program in Japan National Standard for NAT 1999 HCV 2002 HBV 2002 HIV Guideline for industry on NAT 2004 HCV, HBV, HIV detection limit: at least 100IU/mL NAT Control Surveillance 2006 HBV 2007 HCV 2007 HIV

HBV-NAT Control Surveillance 2006 Panel: 3 independent assays National Standard for HBV-NAT (Genotype C) 10000、3000、1000、300、100、30、10　IU/mL & HBV Negative Plasma Laboratories of Industries : Location No. of Industries Laboratories Japan 4 6 USA 2 EU 1 Total 7 10

Results of HBV-NAT Control Surveillance 36 assays =3 assays x 12 assay systems % of Positive results

Summary National Standard for NAT 1999 HCV 2002 HBV 2002 HIV HBV-NAT for Blood Products used in Japan Fulfills Sensitivity Required in Japan NAT Guideline

Collaboration with: Department of Bacteriology II, , NIID Yoshinobu Horiuchi Department of Blood and Safety Research, NIID Mizuochi Toshiaki Kazunari Yamaguchi Working Group on Establishment of the National Standards for Nucleic Acid　Technology Assay Working Group on NAT Control Surveillance