Transcript Performance of Chiron Quantitative and Qualitative HBV PCR Assay and

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
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
2 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
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
3 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Analytical Sensitivity and LOD for WHO HBV
Standard
100
90
80
% Positive
% Positive
70
60
50
40
30
20
Observed Data
10
50% Detection Level
Fitted Probit Model
95% Detection Level
0
0.31
0.6
1.25
2.5
5
HBV (IU/mL)
Concentration
(IU/mL)
F:\BT \va\HBV_Alt\QntAltHBV\SAS\hbv_plot.sas
DAT E: 19MAY07 00:45 SAS.9.1
ED95
10
20
HBV # Positive
%
IU/mL / # 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
0
3.26 (2.21 – 6.53) IU/mL
4 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Analytical Sensitivity and LOD for WHO HBV
Standard
Expected Dose (range), IU/mL
Detection
Rate
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)
*Procleix Ultrio PI, IN0166EN Rev. A
5 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Analytical Sensitivity for DDL HBV Genotypes
HBV Genotype Genotype Genotype Genotype Genotype Genotype Genotype
Cp/mL
A
B
C
D
E
F
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
6 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Reproducibility, Linearity, Accuracy, and LLOQ
Dilution Panels
42
Run - 1
40
Run - 2
38
Run - 3
36
Run - 4
R2 = 0.995 - 0.999
CV = 0.4% - 1.3%
LLOQ = 3.3 IU/mL
CT
34
32
30
28
26
24
22
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
HBV (IU/mL)
7 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Clinical Sensitivity: Early Detection of
Seroconversion Panel #6289
Chiron QPCR Results
**Ultrio
*ABBOTT
Days
PRISM HBsAg #Positive / #Tested #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
*Acrometrix PI. **Combined testing results from multiple sites.
8 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
15 days
Clinical Sensitivity: Early Detection of
Seroconversion Panel #11006
Chiron QPCR Results
**Ultrio
*ABBOTT
Days
PRISM HBsAg #Positive / #Tested #Positive / #Tested IU/mL
0/17
0/5
0
0.26
NR
3
7
10
14
19
21
26
28
35
37
42
0.16
0.27
0.24
0.24
0.22
0.26
0.27
0.25
0.45
0.36
1.15
0/18
0/18
0/16
2/18
5/18
8/18
8/16
18/18
18/18
16/16
18/18
0/5
0/5
0/5
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
*Acrometrix PI. **Combined testing results from multiple sites.
9 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
NR
NR
NR
< 3.3
53.2
3.3
9.3
23-28 days
27.0
133.8
85.7
910.0
Clinical Sensitivity: Early Detection of
Seroconversion Panel #11008
Days
0
7
28
31
36
38
43
48
51
55
58
62
65
69
**Ultrio
Chiron QPCR Results
*ABBOTT
PRISM HBsAg #Positive / #Tested #Positive / #Tested IU/mL
0/10
0/3
NR
0.32
0/9
0/3
NR
0.17
0/10
0/3
NR
0.18
1/10
0/3
NR
0.28
0/10
2/3
< 3.3
0.25
0/10
1/3
< 3.3
0.22
1/10
1/3
< 3.3
0.27
1/10
1/3
4.7
0.23
5/9
3/3
9.9
0.24
6/9
3/3
5.4
0.30
9/10
3/3
38.8 18-33 days
0.29
10/10
3/3
196.2
0.39
10/10
3/3
344.1
0.61
10/10
1.35
2/2*
193.4*
*Acrometrix PI. **Combined testing results from multiple sites
10 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
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
11 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Confirming HBV Yield Cases: Chiang Mai
University Study (HBsAg Negative)*
ID
043
072
670
340
646
Date
6/19/05
7/11/05
9/29/05
8/23/05
10/4/05
9/5/05
10/4/05**
9/15/05
10/25/05
Anti-HBc
IgG
IgM
AntiHBs
+
+
+
+
+
+
+
nd
nd
nd
nd
+
nd
nd
nd
nd
nd
-
Ultrio
HBV/DNA
+
+
+
+
+
+
-
Chiron HBV QPCR
# Positive / # Tested
IU/mL
1/2
3/3
nd
3/3
nd
3/3
nd
3/3
nd
2.0
5.2
nd
6.7
nd
397.6
nd
71.8
nd
*Nantachit, N. et al, in press, 2007.
**Later HBsAg positive.
nd, not determined.
12 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Quantitation of DDL Genotype A-G
DDL
Bayer bDNA3.0 Chiron QPCR NGI SuperQuant Bayer/Chiron NGI/Chiron
Genotype*
Cp/mL
IU/mL
Cp/mL
Cp/IU
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.
13 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
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?
14 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)
Acknowledgement
 Dennis Madriaga
 Baohe Shen
 Barney Krebs
15 | Presentation Title | Presenter Name | Date | Subject | Confidentiality Level (Strictly Confidential, Confidential, Internal Use Only)