sogat xxi brussels, 28-29 may 2009

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


European PharmacopoeiaEuropean Pharmacopoeia (I)

2.6.21 Nucleic Acid Amplification Techniques

7.3.1 External Controls


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


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


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)


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)


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)


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)


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


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


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


HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (II)

Approx. 5700 IU/mL


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 ( --- ))


HCV WHO IS

HCV ISS/1005

HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (IV)

Distribution of results


When a failure confirms the validity of your approach

HBV ISS/0905

HBV ISS/0905: not suitable as a reference preparation!

HBV WHO IS


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


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)


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


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


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


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


Thank you for your attention!

sogat xxi brussels, 28-29 may 2009

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