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Establishment of the 1st WHO International Standard for Detection of Antibodies to Hepatitis B Virus Core Antigen (anti-HBc) SoGAT XXI 29 May 2009 Dr. Heiner Scheiblauer Paul-Ehrlich-Institut, Langen/Germany PEI-IVD. Why Anti-HBc?.

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Establishment of the 1st WHO International Standard for Detection of Antibodies to Hepatitis B Virus Core Antigen (anti-HBc)SoGAT XXI29 May 2009Dr. Heiner ScheiblauerPaul-Ehrlich-Institut, Langen/GermanyPEI-IVD

why anti hbc
Why Anti-HBc?
  • Anti-HBc are the first antibodies after Hepatitis B virus infection and persist trough out life
  • Anti-HBc can detect a persistent HBV viraemia, that is not easy detectable by HBsAg or HBV DNA
  • Anti-HBc can detect HBV infections with HBV escape mutants or genotypes which are not detectable by certain HBsAg or HBV DNA test kits
  • Anti-HBc screening can prevent transfusion-transmitted HBV infections
  • Anti-HBc has a regulatory impact: requirement for obligatory screening in some countries worldwide
who anti hbc standard demand
WHO Anti-HBc StandardDemand
  • Estimation of the analytical sensitivity for anti-HBc tests
  • Calibration of anti-HBc test systems by manufacturers
  • Accuracy and reproducibility verification in batch testing
  • Current national PEI anti-HBc standard (no 82, serum, 100 PEI U/ml) has been used widely
  • Sensitivity of assays and anti-HBc content of samples expressed as PEI-U/ml
  • PEI anti-HBc standard stocks declined, replacement preparation needed
who anti hbc collaborative study study design
WHO Anti-HBc Collaborative StudyStudy design
  • Analytical sensitivity of Samples A (NIBSC 95/522) and B (PEI 82) at the intercept with the assay´s cut-off (endpoint titer, detection limit in U/ml)
  • Potency of NIBSC 95/522 relative to PEI 82 (U/ml)
    • GMV ratio of the detection limit
    • Parallel line model
  • Correlation between analytical sensitivity for NIBSC 95/522 and anti-HBc detection in Samples C (PEI 108166) and D (CBER Panel #11)
  • Variability in results between assay methods (repeatability), and between laboratories (reproducibility)  Geometric coefficient of variation (GCV%)
  • Statistical significance of differences between methods and laboratories
  • Stability
who anti hbc collaborative study participants
WHO Anti-HBc Collaborative StudyParticipants
  • Hemocentro de Sao Paulo, Brazil
  • CBER, USA
  • INTS, France
  • Abbott Laboratories, USA
  • KFDA, Seoul, Republic of Korea
  • American Red Cross, Gaithersburg, USA
  • National Institute of Infectious Diseases, Tokyo, Japan
  • NIBSC, Hertsfordshire, UK
  • AFSSAPS, France
  • National Institute for the Control of Pharmaceutical and Biological Products Beijing Zhongyuan, China
  • NRL, Australia
  • VQC Laboratory Sanquin-CLB Diagnostics, Netherlands
  • Paul-Ehrlich-Institut, Germany
who anti hbc collaborative study test kits
ADVIA Centaur HBcT

Architect Anti-HBc

AxSYM Core

Bioelisa anti-HBc

Corzyme

Elecsys Anti-HBc

Enzygnost Anti-HBc monoclonal

Genedia Anti-HBc ELISA Plus

Hepanostika anti-HBc Uniform

Immulite 2000 anti-HBc

Immuncomb II HBc IgG

IMx Core

Anti-HBc EIA Lumipuls

Presto HBc Ab-N

Monolisa anti-HBc Plus

Murex anti-HBc

Ortho anti-HBc

PRISM HBCore

ST AIA-Pack HBcAb

Architect Anti-HBc II (ex-US) / Architect Core (US)

WHO Anti-HBc Collaborative StudyTest kits
collaborative study suitability for variety of tests mfcts labs geography
Collaborative Study - Suitability for variety of tests, mfcts, labs, geography
  • 20 anti-HBc test kits included in the study from 15 different manufacturers and from 7 different countries
  • Tested in 10 different countries (Australia, Brazil, China, France, Japan, Germany, Korea, Netherlands, UK, USA)
  • Including the current available anti-HBc test technologies
    • Competitive test format (n=14)
    • Indirect test format (n=5)
    • Sandwich test format (n=1)
    • Reductant pre-treatment (e.g. DTT) with (n=5) or without (n=15)
    • Manually conducted (n=9)
    • Automated (n=10)
    • Rapid assay (n=1)
  • All anti-HBc test kits coated with recombinant HBc antigen
who anti hbc collaborative study potency of nibsc 95 522 relative to pei 82

-

PEI-U/ml

NIBSC 95/522

0.08

0.16

0.31

0.63

1.25

2.50

5.00

10

9

9

8

PEI-82

NIBSC 95/522

8

7

7

6

6

5

5

S/Co

S/Co

4

4

3

3

2

2

1

1

0

0

0.03

0.06

0.13

0.25

0.50

1.00

2.00

-

PEI-U/ml

PEI 82

WHO Anti-HBc Collaborative StudyPotency of NIBSC 95/522 relative to PEI 82

Dilution ranges for NIBSC 95/522 (sample A) was in the dynamic measuring range of assays

Within an individual assay, Sample A and B (PEI 82) gave comparable dose responses

who anti hbc collaborative study endpoint titers

Dilution equivalent to cut-off – NIBSC 95/522

1/1024

Endpoint dilution

1/512

1/256

1/128

1/64

1/32

7

12

1

0

12

13

1

7

12

12

1

4

7

12

9

12

7

5

12

2

6

12

10

8

2

5

Laboratory

7

11

12

5

9

12

4

12

1

3

4

12

8

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Assay

WHO Anti-HBc Collaborative Study Endpoint titers
who anti hbc collaborative study overall potency
WHO Anti-HBc Collaborative Study Overall Potency
  • Overall potency was 49.8 U/ml (95%-CI 44.2 - 56.1 U/m)
  • Assay 1 had a potency of 22.8 U/ml (mean of 2 labs)
  • Assay 8 had a potency of 73.3 U/ml
  • Overall potency excluding assays 1 and 8 was 51.1 U/ml (95%-CI 45.3 - 57.5 U/ml)
who anti hbc collaborative study commutability
WHO Anti-HBc Collaborative Study Commutability

Analytical sensitivity (low detection limits) in Sample A (NIBSC 95/522) correlated with positive score for anti-HBc in Samples C (PEI 108166) and D (CBER panel #11)

One assay of the study did not follow this correlation

who anti hbc collaborative study stability

NIBSC 95/522 Stability after 4 years tested with Architect anti-HBc

90

fresh

+4°C

+20°C

37°C

45°C

80

70

60

50

Endpoint titer

40

30

20

10

0

0

1

2

3

4

5

6

7

8

9

10

Replicates

WHO Anti-HBc Collaborative Study Stability
  • The proposed anti-HBc standard NIBSC 95/522 is likely to be highly stable when stored at the recommended temperature of -20°C
1st international anti hbc standard conclusions
1st International Anti-HBc Standard Conclusions
  • NIBSC 95/522 1st International Standard (IS) for detection of anti-HBc
  • Assigned unitage of 50 IU per ampoule (ml)
  • The IU provides continuity with the currently used PEI units/ml
  • Analytical sensitivity for anti-HBc was effective for estimation of sensitivity performance  the lower the detection limit with the IS, the higher the positive score for anti-HBc concentration in other samples
  • The proposed International anti-HBc Standard was found suitable for
    • estimation of analytical sensitivity for anti-HBc detection
    • calibration of anti-HBc test kit sensitivity by manufacturers
    • to calibrate secondary standards
    • for quality control procedures, e.g. in batch release testing
1st international anti hbc standard acknowledgements
1st International Anti-HBc Standard Acknowledgements
  • NIBSC: Dr. Morag Ferguson
  • CBER: Dr. Robin Biswas
  • PEI: Dr. Peter Volkers

Dr. Sigrid Nick

Dr. Micha Nübling

  • WHO: Dr. Ana Padilla