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Standardization: Calibration of

International Standards,

reference preparations

and working standards

Micha Nübling, PEI

Paul-Ehrlich-InstitutGovernmental Authority

for biological medicinal

products

Blood and blood products

Assessment of IVDs for

CE marking (NB)

IVD batch release testing

WHO CC: IVDs +

Blood Products

Standardization of NAT assays

WHO Working Group „SOGAT“

„Standardisation of Genome Amplification Techniques

for the Safety Testing of Blood, Tissues and Organs

for Blood Borne Pathagens“


WHO Working Group „SOGAT“

Representatives from...

...official medicinal control labs (OMCLs)

...plasma manufacturers

...in vitro diagnostic industry

...regulators

...universities, diagnostic labs


WHO International NAT Standards

* lyophilised preparations of biologicals

* high concentration of target

* „gold standard“ for secondary standards

and working reagents


NAT standards: Desired Features

* suitable for all NAT assays: PCR, TMA, NASBA,

branched DNA, ....

* reflect measuring range of quantitative NATs

* reflect target material

* comparability with conventional IVDs,

e.g. HIV: RNA / p24

* one unitage (IU / ml)


WHO NAT International Standards (WHO IS)

* definition of candidate materials

* collaborative study defining „mean titre“ estimate

* assignation of (arbitrary) unitage (IU / ml)

* stability studies (real-time, elevated temperatures)

* report to WHO Expert Committee on Biol. Stand.


WHO International Standards

NIBSC

Target Concentration 1 IU = code

HCV RNA 105 IU/ml 3 - 8 cps 96/798

HBV DNA 106 IU/ml 2 - 7 cps 97/746

HIV-1 RNA 106,1 IU/ml 0.5 cps 97/650

B19 DNA 106 IU/ml 0.8 cps 99/800

HAV RNA 105 IU/ml >10 cps 00/560


WHO / (NIBSC / PEI) Genotype / Subtype Reference Panels

Target Geno-/Subtypes

HCV RNA 1, 2, 3, 4, 5, 6

HBV DNA A, B, C, D, E, F (in preparation, PEI WHO CC)

HIV-1 RNA A, B, C, D, AE, F, G, AG-GH, N, O

B19 DNA 1, 2, 3a, 3b (in preparation, FDA, NIBSC WHO CCs)

HAV RNA

Secondary standards

Working Reagents

* to control individual runs

e.g. Assay Run Control: 3 x 95% pos c/o

* to control sensitivity requirements

e.g. 100 IU HCV-RNA/ml (plasma pool

testing)

* calibrated against WHO standards, if available

Secondary standards

Biological Reference Preparation for HCV-RNA

(PhEurBRP)

Background

CPMH requirement for plasma pool HCV-NAT

OMCLs confirm manufacturer‘s testing (batch release)

Need for a common reference material

Biological Reference Preparation HCV-RNA(PhEurBRP)

WHO IS (96/790) BRP Candidate Material

HCV-RNA pos plasma

HCV genotype I

antiHCV pos antiHCV neg

diluted in neg pool

100.000 IU/ml 4.000 - 10.000 gEq/ml

freeze dried (0.5 ml)


Collaborative calibration study

* Calibration against WHO standard

* Assignation of unitage (IU/ml)

* Suitability for OMCLs‘ plasma pool testing



26 Participants (Europe, USA)

OMCLs (9)

plasma derivatives manufacturers (11)

diagnostic labs (6)



Protocol

* 4 vials of candidate material and WHO IS

* routine NAT

* dilution series (0.5 log10) around end point

* statistical evaluation, potency calculation

* independant from methodical sensitivities

10-1

10-1.5

10-2

10-2.5

10-3

10-3.5

WHO IS + + + + + + + + + + + + + + + + + + - + + - - -

PhEur + + + + + + + + - + - + - - + - - - - - - - - -


PCR Assay (logit method) for data in:

log10(dose)

#p

ositiv

e/#

tria

ls

-6 -5 -4 -3 -2 -1 0

0

0.25

0.5

0.75

0.95

1

se

pa

rate

re

gre

ssio

n lin

es:

test

(x)

sta

nd

ard

(+

)

euro.hcv[lab == 1, -1]

PEI, Tue Mar 9 15:04:02 1999

WHO ISPhEurBRP

Estimated potency:

103.55 IU / ml

105 IU/ml

PCR Assay (logit method) for data in:

log10(dose)

#p

ositiv

e/#

tria

ls

-6 -5 -4 -3 -2 -1

0

0.25

0.5

0.75

0.95

1

se

pa

rate

re

gre

ssio

n lin

es:

test

(x)

sta

nd

ard

(+

)

euro.hcv[lab == 17, -1]

PEI, Tue Mar 9 15:10:31 1999

WHO IS PhEurBRPEstimated potency:

102.85 IU / ml105 IU/ml



Results

* 26 „valid“ data sets

* 23 par allel curves, 3 non-parallel curves

* mean estimated potency 102.97 IU / ml for PhEurBRP

(independant from inclusion of non-parallel curves)

2.22.42.62.83.03.23.43.6

02

46

810

histogram of all laboratories (Eur.Ph.)

potency [log10 IU/ml]

frequency

204

9

21

12

22

24

25

2

3

7

10

11

14

18

17*

19*

16

13*

26

6

23

1

5

8

15

Mean Potency:

102.97 IU / ml

= 1.000 IU / ml

Ph. Eur. BRP HCV-RNAStability at Different Storage Temperatures

1

10

100

1.000

10.000

0 10 20 30 40 50 60 70 80 90

Weeks

HC

V-R

NA

(g

eq

/ml)

37°C

- 20°C

RT

4°C

Secondary standards

more recent calibration studies:

•preferential use of quantitative NATs

•relative quantitation of WHO IS and candidate material

•similar relation on different platforms / by different assays ?

Secondary standards

Ph. Eur. BRP HCV-RNA 103 IU/ml

Ph. Eur. BRP B19-DNA 105,8 IU/ml

PEI Ref Prep HCV-RNA 104,9 IU/ml

PEI-Ref Prep HIV1-RNA 104,9 IU/ml

PEI Ref Prep HBV-DNA 104,9 IU/ml

PEI Ref Prep WNV-RNA 109,8 cps/ml

Role of WHO IS and Secondary Standards

regulations for blood and blood products

therapy guidelines

standardization of assays

proficiency testing schemes

calibration of working reagents

Limiting DilutionEstimation of „NAT-titers“, „copies“, „genome equivalents“

infectious

non-infectious

Probability for a negative test result:

p(-) = e -cv c = virus concentration

v = test volume

PCR Assay for data in:

log2(dose)

#posi

tive

/#tr

ials

0 2 4 6 8

0

0.25

0.5

0.75

0.95

1 xx

x

x

x

x

x

++

+

+

+

hcv.pcr

s

epara

te r

egre

ssi

on li

nes:

test

(x)

st

andard

(+

)

PEI, Tue Sep 15 15:58:32 1998

in-house HCV-NAT

Pr95: 24.41517 =21.3 IU/ml Pr95: 27.36196= 164.5 IU/ml

Detection limits of different HCV-NATs(limiting dilution of WHO standard HCV-RNA, Probit analysis)

63 %

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