June 13, 2007
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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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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

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


Assay performance

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


Analytical sensitivity and lod for who hbv standard

Analytical Sensitivity and LOD for WHO HBV Standard

% Positive

HBV (IU/mL)


Yiu lian fong ph d associate director

Analytical Sensitivity and LOD for WHO HBV Standard

*Procleix Ultrio PI, IN0166EN Rev. A


Analytical sensitivity for ddl hbv genotypes

Analytical Sensitivity for DDL HBV Genotypes


Reproducibility linearity accuracy and lloq

Reproducibility, Linearity, Accuracy, and LLOQ

R2 = 0.995 - 0.999

CV = 0.4% - 1.3%

LLOQ = 3.3 IU/mL


Clinical sensitivity early detection of seroconversion panel 6289

15 days

Clinical Sensitivity: Early Detection of Seroconversion Panel #6289

*Acrometrix PI. **Combined testing results from multiple sites.


Clinical sensitivity early detection of seroconversion panel 11006

23-28 days

Clinical Sensitivity: Early Detection of Seroconversion Panel #11006

*Acrometrix PI. **Combined testing results from multiple sites.


Clinical sensitivity early detection of seroconversion panel 11008

Clinical Sensitivity: Early Detection of Seroconversion Panel #11008

18-33 days

*Acrometrix PI. **Combined testing results from multiple sites


Analytical and clinical specificities

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


Confirming hbv yield cases chiang mai university study hbsag negative

Confirming HBV Yield Cases: Chiang Mai University Study (HBsAg Negative)*

*Nantachit, N. et al, in press, 2007.

**Later HBsAg positive.

nd, not determined.


Quantitation of ddl genotype a g

Quantitation of DDL Genotype A-G

* Supplied as 30,000 Cp/mL.


Summary

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?


Acknowledgement

Acknowledgement

  • Dennis Madriaga

  • Baohe Shen

  • Barney Krebs


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