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Yiu-Lian Fong, Ph.D, Associate Director

June 13, 2007. Performance of Chiron Quantitative and Qualitative HBV PCR Assay and Confirmation of HBV Yield Cases. Yiu-Lian Fong, Ph.D, Associate Director. Agenda.

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Yiu-Lian Fong, Ph.D, Associate Director

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  1. June 13, 2007 Performance of Chiron Quantitative and Qualitative HBV PCR Assay and Confirmation of HBV Yield Cases Yiu-Lian Fong, Ph.D, Associate Director

  2. Agenda A qualitative and quantitative HBV DNA assay has been developed and validated in house for sensitive detection and accurate quantification of HBV DNA for confirmatory purposes. • Assay Performance • Confirming HBV Yield Cases • Issues raised for standardized calibration of various genotypes

  3. Assay Performance • Analytical Sensitivity and LOD for WHO HBV Standard • Analytical Sensitivities for DDL Genotypes A-G • Linearity, LLOQ, reproducibility and precision • Clinical Sensitivity • Analytical and Clinical Specificities

  4. Analytical Sensitivity and LOD for WHO HBV Standard % Positive HBV (IU/mL)

  5. Analytical Sensitivity and LOD for WHO HBV Standard *Procleix Ultrio PI, IN0166EN Rev. A

  6. Analytical Sensitivity for DDL HBV Genotypes

  7. Reproducibility, Linearity, Accuracy, and LLOQ R2 = 0.995 - 0.999 CV = 0.4% - 1.3% LLOQ = 3.3 IU/mL

  8. 15 days Clinical Sensitivity: Early Detection of Seroconversion Panel #6289 *Acrometrix PI. **Combined testing results from multiple sites.

  9. 23-28 days Clinical Sensitivity: Early Detection of Seroconversion Panel #11006 *Acrometrix PI. **Combined testing results from multiple sites.

  10. Clinical Sensitivity: Early Detection of Seroconversion Panel #11008 18-33 days *Acrometrix PI. **Combined testing results from multiple sites

  11. Analytical and Clinical Specificities In-house Assay Validation: • 100 random donor samples tested in triplicates: all negative • Freeze/thaw up to five cycles with 5, 20, and 5000 IU/mL WHO HBV standard • Potential Interfering Substances Tested • Anticoagulants: ACD, CPD, K2EDTA, K3EDTA, Na Citrate, Na Heparin • Protein at > 7 g/dL • Lipid/Triglyceride at > 3000 mg/dL • Hemoglobin at > 500 mg/dL • Bilirubin at >20 mg/dL • Bacteria (>105 CFU/mol) commonly seen in blood related products • Blood-born pathogens:HCV, HTLV, HAV, CMV, EBS, HSV, and HIV-1

  12. Confirming HBV Yield Cases: Chiang Mai University Study (HBsAg Negative)* *Nantachit, N. et al, in press, 2007. **Later HBsAg positive. nd, not determined.

  13. Quantitation of DDL Genotype A-G * Supplied as 30,000 Cp/mL.

  14. Summary • A sensitive qualitative and quantitative HBV DNA assay has been developed and validated in house • LOD at 3.3 IU/mL for WHO HBV standard • Detects various genotypes • Detects window phase low viremia samples • Confirms yield cases • Conclusion: This highly sensitive and reliable assay is suitable for confirmatory purposes • Remaining Issues: • How to standardize? • How to calibrate various genotypes? • Which test method to use?

  15. Acknowledgement • Dennis Madriaga • Baohe Shen • Barney Krebs

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