Standardization: Calibration of International … › Sogat › sogatcdI_presentations ›...
Transcript of Standardization: Calibration of International … › Sogat › sogatcdI_presentations ›...
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Standardization: Calibration of
International Standards,
reference preparations
and working standards
Micha Nübling, PEI
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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
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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“
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Standardization of NAT assays
WHO Working Group „SOGAT“
Representatives from...
...official medicinal control labs (OMCLs)
...plasma manufacturers
...in vitro diagnostic industry
...regulators
...universities, diagnostic labs
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Standardization of NAT assays
WHO International NAT Standards
* lyophilised preparations of biologicals
* high concentration of target
* „gold standard“ for secondary standards
and working reagents
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Standardization of NAT assays
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)
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Standardization of NAT assays
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.
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Standardization of NAT assays
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
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Standardization of NAT assays
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
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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
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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
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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)
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Biological Reference Preparation HCV-RNA(PhEurBRP)
Collaborative calibration study
* Calibration against WHO standard
* Assignation of unitage (IU/ml)
* Suitability for OMCLs‘ plasma pool testing
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Biological Reference Preparation HCV-RNA(PhEurBRP)
Collaborative calibration study
26 Participants (Europe, USA)
OMCLs (9)
plasma derivatives manufacturers (11)
diagnostic labs (6)
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Biological Reference Preparation HCV-RNA(PhEurBRP)
Collaborative calibration study
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
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10-1
10-1.5
10-2
10-2.5
10-3
10-3.5
WHO IS + + + + + + + + + + + + + + + + + + - + + - - -
PhEur + + + + + + + + - + - + - - + - - - - - - - - -
Biological Reference Preparation HCV-RNA(PhEurBRP)
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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
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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
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Biological Reference Preparation HCV-RNA(PhEurBRP)
Collaborative calibration study
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)
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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
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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
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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 ?
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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
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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
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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
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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 %