Performance of Chiron Quantitative and Qualitative HBV PCR Assay and

Confirmation of HBV Yield Cases

Yiu-Lian Fong, Ph.D, Associate Director

June 13, 2007

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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

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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

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Analytical Sensitivity and LOD for WHO HBV Standard

HBV IU/mL

# Positive / # Tested

% Positive

20 20/20 100

10 20/20 100

5 20/20 100

2.50 18/20 90

1.25 13/20 65

0.63 8/20 40

0.31 3/20 15

0 0/16 0DAT E: 19MAY07 00:45 SAS.9.1F:\BT \va\HBV_Alt\QntAltHBV\SAS\hbv_plot.sas

Observed Data

Fitted Probit Model

50% Detection Level

95% Detection Level

% P

ositiv

e

0

10

20

30

40

50

60

70

80

90

100

Concentration (IU/mL)

0.31 0.6 1.25 2.5 5 10 20

HBV (IU/mL)

% P

osi

tive

ED95 3.26 (2.21 – 6.53) IU/mL

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Detection Rate

Expected Dose (range), IU/mL

Chiron QPCR Ultrio* Ultrio dHBV*

ED95 3.3 (2.2–6.5) 8.0 (7.1–9.3) 6.8 (6.0–7.7)

ED50 0.8 (0.6–1.0) 3.3 (3.0–3.8) 3.0 (2.7–3.4)

Analytical Sensitivity and LOD for WHO HBV Standard

*Procleix Ultrio PI, IN0166EN Rev. A

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Analytical Sensitivity for DDL HBV Genotypes

HBV

Cp/mL

Genotype

A

Genotype

B

Genotype

C

Genotype

D

Genotype

E

Genotype

F

Genotype

G

1000 100 100 100 100 100 100 100

300 100 100 100 100 100 100 100

100 100 100 100 100 100 100 100

50 100 100 75 82 83 100 100

30 100 75 92 64 50 83 100

10 92 33 33 8 17 42 92

3 27 33 33 20 33 17 90

0 0 0 0 0 0 0 0

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Reproducibility, Linearity, Accuracy, and LLOQ

R2 = 0.995 - 0.999CV = 0.4% - 1.3%LLOQ = 3.3 IU/mL

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Clinical Sensitivity: Early Detection of Seroconversion Panel #6289

Days*ABBOTT

PRISM HBsAg**Ultrio

#Positive / #Tested

Chiron QPCR Results

#Positive / #Tested IU/mL

0 0.95 0/16 0/3 NR

2 0.20 0/17 0/3 NR

9 0.56 0/17 2/3 < 3.3

11 0.50 2/18 0/3 NR

16 0.21 12/17 3/3 6.2

18 0.22 16/17 3/3 14.1

31 2.15 18/18 3/3 962.9

36 4.94 18/18 3/3 6212.7

15 days

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

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Clinical Sensitivity: Early Detection of Seroconversion Panel #11006

Days*ABBOTT

PRISM HBsAg**Ultrio

#Positive / #Tested

Chiron QPCR Results

#Positive / #Tested IU/mL

0 0.26 0/17 0/5 NR

3 0.16 0/18 0/5 NR7 0.27 0/18 0/5 NR10 0.24 0/16 0/5 NR14 0.24 2/18 2/3 < 3.319 0.22 5/18 3/3 53.221 0.26 8/18 3/3 3.326 0.27 8/16 3/3 9.328 0.25 18/18 3/3 27.035 0.45 18/18 3/3 133.837 0.36 16/16 3/3 85.742 1.15 18/18 3/3 910.0

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

23-28 days

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Clinical Sensitivity: Early Detection of Seroconversion Panel #11008

Days*ABBOTT

PRISM HBsAg**Ultrio

#Positive / #Tested

Chiron QPCR Results

#Positive / #Tested IU/mL

0 0.32 0/10 0/3 NR7 0.17 0/9 0/3 NR

28 0.18 0/10 0/3 NR

31 0.28 1/10 0/3 NR36 0.25 0/10 2/3 < 3.3

38 0.22 0/10 1/3 < 3.3

43 0.27 1/10 1/3 < 3.348 0.23 1/10 1/3 4.751 0.24 5/9 3/3 9.9

55 0.30 6/9 3/3 5.4

58 0.29 9/10 3/3 38.862 0.39 10/10 3/3 196.2

65 0.61 10/10 3/3 344.169 1.35 10/10 2/2* 193.4*

18-33 days

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

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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

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

Anti-HBc Chiron HBV QPCR ID Date

IgG IgM

Anti-HBs

Ultrio HBV/DNA # Positive / # Tested IU/mL

043 6/19/05 + - nd + 1/2 2.0 7/11/05 + nd - + 3/3 5.2 072 9/29/05 + nd - nd nd 8/23/05 + nd - + 3/3 6.7 670 10/4/05 + nd - - nd nd 9/5/05 - - nd + 3/3 397.6 340

10/4/05** - + nd + nd nd 9/15/05 + nd - + 3/3 71.8 646

10/25/05 + nd - - nd nd *Nantachit, N. et al, in press, 2007.

**Later HBsAg positive.nd, not determined.

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Quantitation of DDL Genotype A-G

DDL Genotype*

Bayer bDNA3.0 Cp/mL

Chiron QPCR IU/mL

NGI SuperQuant Cp/mL

Bayer/Chiron Cp/IU

NGI/Chiron Cp/IU

A 38367 1997 9179 1078 28000 4.2 3.1

B 33171 2831 3107 214 4100 10.7 1.3

C 32811 1002 2843 250 33000 11.5 11.6

D 40197 6429 3328 170 77000 12.1 23.1

E 41079 527 2108 141 30000 19.5 14.2

F 49087 12332 4955 789 26000 9.9 5.2

G 37464 1558 27003 2278 38000 1.4 1.4

* Supplied as 30,000 Cp/mL.

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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?

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Acknowledgement

Dennis Madriaga

Baohe Shen

Barney Krebs