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Calibration against the WHO Standards of National Reference Preparations for detection of blood viruses by NAT: the Italian experience. SOGAT XXI Brussels, 28-29 May 2009. European Pharmacopoeia (I). 2.6.21 Nucleic Acid Amplification Techniques. 7.3.1 External Controls.

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Calibration against the WHO Standards of National Reference Preparations for detection of blood viruses by NAT: the Italian experience

SOGAT XXI

Brussels, 28-29 May 2009


European pharmacopoeia i
European Pharmacopoeia Preparations for detection of blood viruses by NAT: the Italian experience(I)

2.6.21 Nucleic Acid Amplification Techniques

7.3.1 External Controls


European pharmacopoeia ii
European Pharmacopoeia Preparations for detection of blood viruses by NAT: the Italian experience(II)

External Controls

Negative control: a sample of the same matrix already proven to be free of the target sequences

Extraction

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

Amplification

Detection

External Controls


External controls

Commercial 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

External Controls

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 Reference Preparations for NAT assays


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 (I)


Iss ref prep for nat assays the italian approach ii

CALIBRATION plasma donation with respect to the viral load and the genotype 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)

ISS Ref. Prep. for NAT assays: the Italian approach (II)


Example of ISS Collaborative study (HCV) (I) plasma donation with respect to the viral load and the genotype

Samples

HCV WHO International Standard

*Provided to participants (pre-diluted by

the ISS)

#Dilutions to be carried out by participants

Dil. 10-3* 10-3.5 # 10- 4 #10-4.5 #10-5#

IU/mL 100 32 10 31

HCV ISS Reference Preparation^

^Provided undiluted to participants

§Dilutions to be carried out by participants

(based on the preliminary titer)

Dil.§ 10-1.6 10-2.1 10-2.6 10-3.1 10-3.6


Example of iss collaborative study hcv ii

Testing scheme plasma donation with respect to the viral load and the genotype

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

Example of ISS Collaborative study (HCV) (II)


Example of ISS Collaborative study (HCV) (III) plasma donation with respect to the viral load and the genotypeStatistical analysis by the ISS (Probit)

0.63


HCV RNA ISS 1005 plasma donation with respect to the viral load and the genotype

ISS Last Collab. Study (2007) (I)

PARTICIPANTS

1

  • 11 Italian BTCs

  • 1 Spanish BTC

  • ISS

  • NAT assays

  • Cobas Ampliscreen (6 laboratories)

  • TMA Ultrio (7 laboratories)


Hcv rna iss 1005 iss last collab study 2007 ii
HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (II) plasma donation with respect to the viral load and the genotype

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 RNA ISS 1005 - ISS Last Collab. Study (2007) (III)


HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (IV) titre (—) and the interval of confidence (mean ± geometric coeff. of variation ( --- ))Distribution of results

HCV WHO IS

HCV ISS/1005


When a failure confirms the validity of your approach
When a failure confirms the validity titre (—) and the interval of confidence (mean ± geometric coeff. of variation ( --- ))of your approach

HBV WHO IS

HBV ISS/0905

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


What is the right concentration for titre (—) and the interval of confidence (mean ± geometric coeff. of variation ( --- ))

a positive run control?

100

95% cut-off

80

60

40

Probability %

20

0

0

1

10

100

1000

IU/ mL (log)

Positive samples/total

number tested

3-4 x 95% cut-off


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)

What is the right concentration for

an ISS reference preparation?


Ultrio Tigris (TMA-Novartis) curent ISS reference preparations (HCV, HIV, HBV):

95% DL as stated

by the manufacturer

RUN CONTROL

4 X 95% DL as suggested

by the ISS


Real Time PCR (S201-Roche) curent ISS reference preparations (HCV, HIV, HBV):

95% DL as stated

by the manufacturer

RUN CONTROL

4 X 95% DL as suggested

by the ISS


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 is the right concentration for

an ISS reference preparation?


What s next

ISS Collaborative Study 2009: curent ISS reference preparations (HCV, HIV, HBV):

HCV RNA ISS 1008 (Genotype 1)

HIV RNA ISS 0109 (Subtype F)

10 Italian transfusion centres will participate

Same approach as just described

What’s next?


Thank you for your attention
Thank you for your attention! curent ISS reference preparations (HCV, HIV, HBV):


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