Karen Cristiano Biologicals Unit, CRIVIB Calibration against the WHO Standards of National Reference...
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Transcript of Karen Cristiano Biologicals Unit, CRIVIB Calibration against the WHO Standards of National Reference...
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
Calibration against the WHO Standards of National Reference
Preparations for detection of blood
viruses by NAT: the Italian experience
SOGAT XXIBrussels, 28-29 May 2009
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
European PharmacopoeiaEuropean Pharmacopoeia (I)
2.6.21 Nucleic Acid Amplification Techniques
7.3.1 External Controls
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
European PharmacopoeiaEuropean Pharmacopoeia (II)
External Controls
Extraction
Amplification
Detection
Negative control: a sample of the same matrix already proven to be free of the target sequences
Positive control: this contains a defined number of target-sequence copies, the number being determined individually for each assay system and indicated as a multiple of the positive cut-off value of the test system
External Controls
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
External ControlsCommercial NAT test kits include positive and negative external controls for the assay validation.
However, for the positive controls there are some drawbacks: viral load unknown (in some cases too high compared to the
detection limit) integrity of the virus (?) in some cases (e.g. Ampliscreen) the control does not cover
the whole NAT method
Additional external controls: in-house positive run controls
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
ISS Reference Preparations for NAT assays
A total of 7500 vials of ISS Reference Preparations have been distributed since 1998:
3500 for HCV (3 batches) 1500 for HIV (2 batches) 2500 for HBV (3 batches)
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
ISS Ref. Prep. for NAT assays: the Italian approach (I)
Selection and characterisation of an appropriate positive plasma donation with respect to the viral load and the genotype
Small scale production in order to evaluate which dilution of the positive sample is adequate (viral load: 3000-5000 IU/ml)
Large scale preparation: dilution of the positive plasma, filling into vials…
Homogeneity test on the positive preparation (at least 10 assays in duplicate). Result of this test also used for stability studies (T=0)
Stability studies (RT 24 h, 4°C 1 week, -80°C 6 month-intervals)
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
ISS Ref. Prep. for NAT assays: the Italian approach (II)
CALIBRATION through a mini collaborative study (10-14 participants):
50% laboratories using NAT assay A (e.g. TMA§) 50% laboratories using NAT assay B (e.g. PCR*)
§now automated on Tigris platform*now automated on COBAS S201 system (Real Time PCR)
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV WHO International Standard
Example of ISS Collaborative study (HCV) (I)
Samples
Dil. 10-3* 10-3.5 # 10- 4 # 10-4.5 # 10-5#
IU/mL 100 32 10 3 1
HCV ISS Reference Preparation^
Dil.§ 10-1.6 10-2.1 10-2.6 10-3.1 10-3.6
*Provided to participants (pre-diluted by the ISS)
#Dilutions to be carried out by participants
^Provided undiluted to participants
§Dilutions to be carried out by participants (based on the preliminary titer)
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
Example of ISS Collaborative study (HCV) (II)
Testing scheme
Four indipendent dilutions of both the WHO International Standard (IS) and the ISS reference preparation are tested in four separate runs
Results are sent to the ISS for statistical analysis
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV ISS Ref. Prep. vs WHO IS
0
0.5
1
1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00
Dilution ( log. scale )
Pro
bab
ility
% ● Real data WHO IS
▲ Real data ISS Ref Prep
WHO IS --------- ISS Ref Prep
Example of ISS Collaborative study (HCV) (III) Statistical analysis by the ISS (Probit)
0.63
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV RNA ISS 1005 ISS Last Collab. Study (2007) (I)
PARTICIPANTS
NAT assays
Cobas Ampliscreen (6 laboratories)
TMA Ultrio (7 laboratories)
1 11 Italian BTCs 1 Spanish BTC ISS
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (II)
Approx. 5700 IU/mL
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (III)
Deviation of each estimated value with respect to the mean titre (—) and the interval of confidence (mean ± geometric
coeff. of variation ( --- ))
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
HCV WHO IS
HCV ISS/1005
HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (IV)
Distribution of results
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
When a failure confirms the validity of your approach
HBV ISS/0905
HBV ISS/0905: not suitable as a reference preparation!
HBV WHO IS
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
What is the right concentration for a positive run control?
Pro
bab
ilit
y %
0
20
40
60
80
100
IU/ mL (log)
0 1 10 100 1000
95% cut-off
3-4 x 95% cut-offPositive samples/total number tested
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
What is the right concentration for an ISS reference preparation?
On-going pilot study to determine the correct use of the curent ISS reference preparations (HCV, HIV, HBV):
Each laboratory receives a standard protocol to dilute the ISS reference preparations to obtain a concentration 4 x the 95% cut-off of the NAT assay (TMA or Real Time PCR)
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
Ultrio Tigris (TMA-Novartis)
HCV RNA HIV RNA HBV DNA
~ 3 UI/mL ~ 20 UI/mL ~ 10 UI/mL
~ 12 UI/mL
~ 80 UI/mL
~ 40 UI/mL
95% DL as stated by the manufacturer
RUN CONTROL4 X 95% DL as suggested
by the ISS
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
95% DL as stated by the manufacturer
RUN CONTROL4 X 95% DL as suggested
by the ISS
Real Time PCR (S201-Roche)
HCV RNA HIV RNA HBV DNA
~ 11UI/mL ~ 49 UI/mL ~ 4 UI/mL
~ 45 UI/mL
~ 200 UI/mL
~ 16 UI/mL
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
What is the right concentration for an ISS reference preparation?
On-going pilot study to determine the correct use of the curent ISS reference preparations (HCV, HIV, HBV):
Run controls are to be used with each run on a routine basis
All the results, recorded in an Excel file, are sent to the ISS on a monthly basis
Based on the results, collected up to June 2009, we will decide whether to use or not the 4 x the 95% cut-off
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
What’s next?
ISS Collaborative Study 2009:
HCV RNA ISS 1008 (Genotype 1) HIV RNA ISS 0109 (Subtype F) 10 Italian transfusion centres will participate Same approach as just described
Karen CristianoKaren CristianoKaren CristianoKaren CristianoBiologicals Unit, CRIVIBBiologicals Unit, CRIVIB
Thank you for your attention!