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EXPERIENCE WITH IN-HOUSE ASSAY: VALIDATION OF AN IN- HOUSE NAT ASSAY FOR THE DETECTION OF DIFFERENT GENOTYPES OF vB19 Marta José, Instituto Grifols S.A.,

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Presentation on theme: "EXPERIENCE WITH IN-HOUSE ASSAY: VALIDATION OF AN IN- HOUSE NAT ASSAY FOR THE DETECTION OF DIFFERENT GENOTYPES OF vB19 Marta José, Instituto Grifols S.A.,"— Presentation transcript:

1 EXPERIENCE WITH IN-HOUSE ASSAY: VALIDATION OF AN IN- HOUSE NAT ASSAY FOR THE DETECTION OF DIFFERENT GENOTYPES OF vB19 Marta José, Instituto Grifols S.A., Barcelona, SPAIN SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

2 IN-HOUSE vB19 METHOD DESIGN Automatic DNA extraction using the BioRobot 9604 (QIAGEN). Subsequent amplification by PCR of a conserved sequence of VP1 capsid structural protein region. Detection by means of a specific capture probe in a liquid medium and colorimetric reaction (ELISA-DIG Detection, Roche). The internal control (from the extraction) is a plasmid which is amplified with the same primers as vB19 without competition, and detected with a different probe. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

3 IN-HOUSE vB19 METHOD DESIGN and VALIDATION The method is designed to work in the exponential phase of the PCR reaction in order to use the same procedure as qualitative or quantitative assays. The method is validated according to current guidelines for the following parameters: detection limit, quantitation limit, linearity, range, accuracy, precision, specificity and robustness. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

4 IN-HOUSE vB19 METHOD VALIDATION: DETECTION LIMIT WHO IS 99/800 Dilution1/ /10 2 1/ / / TitreIU/ml Positives vs replicates Total24/2423/2420/2415/249/24 % % Detection limit 9.2 x 10 3 IU/ml (CI 95 %: 6.6 X 10 3 – 1.8 X 10 4 IU/ml) 50 % Detection limit 2.4 x 10 3 IU/ml (CI 95 %: 1.6 X 10 3 – 3.0 X 10 3 IU/ml) This detection limit is applicable when the method is used to give qualitative results. The detection limit at 95 % and 50 % was established by Probit test using the WHO IS for vB19 DNA NAT assays code 99/800. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

5 IN-HOUSE vB19 METHOD VALIDATION: QUANTITATION LIMIT Titre (IU/ml)OD Mean* This quantitation limit (QL) is applicable when the method is used to give quantitative results. To calculate the quantitation limit, different dilutions of WHO IS, near the detection limit were assayed. A regression test between the titre (IU/ml) and the OD mean of each concentration was applied. REGRESSION TEST Slope9 x Standard error of the interception (δ) R2R QL (IU/ml) (2.97 log 10 ) *Mean of 3 independent assays in triplicate SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

6 IN-HOUSE vB19 METHOD VALIDATION: QUANTITATIVE METHOD Linearity: The method is linear in the range studied, between 1.8x10 3 and 10 5 IU/ml ( log 10 IU/ml) with a coefficient of regression 0.95 and a coefficient of determination Accuracy: The method shows a correct accuracy in the interval from 3.25 to 4.75 log 10 IU/ml with a recovery within the interval 100 ± 10% for each concentration. Precision: In the same interval the method shows a correct precision, with a coefficient of variation of 10%, for both repeatability and intermediate precision studies. Range: The method is accurate and precise in the concentration range between 3.25 and 4.75 log 10 IU/ml. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

7 IN-HOUSE vB19 METHOD VALIDATION: GENERAL PARAMETERS ROBUSTNESS No cross-reactivity was observed with the presence of other blood borne viruses (HCV, HIV-1, HBV, HAV, GBV-C). No interference was detected by the presence of these viruses. SPECIFICITY I SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007 Cross-contamination: 20 samples of negative plasma spiked with vB19 DNA at approximate final concentration of 10 6 IU/ml, mixed alternately with the same samples without spiking, were tested. The results showed that the measures taken to prevent cross-contamination are efficacious. The method shows no operating differences neither with the extraction kit batch nor with the thermocycler employed.

8 IN-HOUSE vB19 METHOD VALIDATION: SPECIFICITY II.- Genotype 2 detection A6 virus DNA sample (genotype 2) from European Pharmacopoeia panel was analysed at titres of 10 4, 10 5 and 10 6 IU/ml and detected as positive using the qualitative method with a 95 % detection limit of 9.2 x 10 3 IU/ml. Two samples of the same donor analysed during the validation of the method were identified as genotype 2. QUANTITATION RESULTS SampleGRIFOLS methodLab 1Lab 2 Sample A1.90x10 7 IU/ml1.14x10 7 IU/mlNT Sample B3.71x10 9 IU/ml>5.1x10 8 IU/ml1x10 10 IU/ml SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007 N.T.: Not Tested

9 IN-HOUSE vB19 METHOD VALIDATION: SPECIFICITY III.- Genotypes 3 and 1 detection SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007 The vB19 genotype 1 detection was demonstrated during the method validation using different samples (IS, NIBSC, working reagents and in-house calibrated controls). Genotype 3 Genotype 1 A vB19 genotype 3 sample with a previous titre established in 10 5 IU/ml was analysed at titres between 10 4 and IU/ml and found positive with our qualitative method. The titre established by our quantitative method was also of 10 5 IU/ml.

10 IN-HOUSE vB19 METHOD VALIDATION: CONCLUSIONS I Qualitative method: The detection limit at 95 % has been established in 9.2 x 10 3 IU/ml. Quantitative method: The quantitation limit has been established in IU/ml. The method is linear in the range studied, between 3.25 and 5 log 10 IU/ml. The method shows correct accuracy and precision in the concentration range between 3.25 and 4.75 log 10 IU/ml. Qualitative and quantitative method: The measures taken to prevent cross-contamination are efficacious. The method shows no operating differences neither with the extraction kit batch nor with the thermocycler employed. Neither cross-reactivity nor interference was observed with the presence of other blood borne viruses (HCV, HIV-1, HBV, HAV, GBV-C). SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

11 IN-HOUSE vB19 METHOD VALIDATION: CONCLUSIONS II Qualitative and quantitative method: The method detects and quantitates samples of genotypes 1, 2 and 3 of vB19 with approximately the same efficacy. We developed and validated an in-house PCR method for detection and/or quantitation of vB19 DNA that could detect samples from the three described genotypes. SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007

12 ACKNOWLEDGEMENTS INSTITUTO GRIFOLS: Araceli Maya Mireia Prat Dr. Rodrigo Gajardo Dr. Juan I. Jorquera BIOMAT (GRIFOLS): Margarita Estrada Dr. Dolors Xairó NATIONAL BLOOD SERVICE CAMBRIDGE UNIVERSITY, UK: Dr. Daniel Candotti Dr. Jean Pierre Allain SANQUIN, NETHERLANDS: Dr. Marco Koppelman SoGAT Standardisation of Parvovirus B19 Genotypes, March 2007


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