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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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slide1

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(I)

2.6.21 Nucleic Acid Amplification Techniques

7.3.1 External Controls

european pharmacopoeia ii
European Pharmacopoeia(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 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)
slide8

Example of ISS Collaborative study (HCV) (I)

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

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)
slide11

HCV RNA ISS 1005

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 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)
slide14

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

HCV WHO IS

HCV ISS/1005

when a failure confirms the validity of your approach
When a failure confirms the validity of your approach

HBV WHO IS

HBV ISS/0905

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

slide16

What is the right concentration for

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

slide17
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?

slide18

Ultrio Tigris (TMA-Novartis)

95% DL as stated

by the manufacturer

RUN CONTROL

4 X 95% DL as suggested

by the ISS

slide19

Real Time PCR (S201-Roche)

95% DL as stated

by the manufacturer

RUN CONTROL

4 X 95% DL as suggested

by the ISS

slide20
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:

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?
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