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
2 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
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