Experience with Standardisation of Blood Virology NAT. Clare Morris Division of Retrovirology National Institute for Biological Standards and Control

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1 Experience with Standardisation of Blood Virology NAT Clare Morris Division of Retrovirology National Institute for Biological Standards and Control

2 Background of Blood Virology Standardisation In the early 1990 s it became apparent there was a need to establish NAT assays to detect viral contamination in blood and blood products. In 1994 in the US a reported outbreak of HCV infection was traced to IV immunoglobulin administration. CBER then introduced mandatory HCV NAT screening, this move was followed by the European Committee for Proprietary Medicinal Products (CPMP) If NAT testing was to be successful, assays needed to be standardised therefore the availability of reference materials to validate assays runs was necessary.

3 Standardisation first acknowledged in 1916 by MRC: the existing absence of official standards and control is discreditable in our national position in the world of science and a grave danger to the community. The establishment of a Government laboratory for biological standardization is needed.

4 Why use NAT? Window period time between infection and production of antibodies by the host. Virus Window period (WP) (days) Reduction in WP by use of NAT (days) HCV HIV HBV Risk of transmission during window period can be reduced by 45-72% by NAT screening

5 HIV markers during early infection HIV RNA (plasma) HIV Antibody HIV p24 Ag Theoretical Infectivity Day 0 HIV RNA Day 11 HIV p24 Ag Day 16 HIV Antibody Day 22 5 Days 6 Days

6 HCV markers during early infection HCV RNA Anti-HCV Infection Day 0 HCV RNA Day 12 HCV Antibody Day 82

7 How to control newly emerging assays. Assay standardisation can be achieved by the use of well characterised working materials. Several proficiency studies carried out in early 1990 s demonstrated significant variation in sensitivity and specificity assays for HIV and HCV. Studies conducted by both the Eurohep group and NIBSC examined the variation in NAT assays results for HCV RNA and HIV DNA. Conclusions showed: Results were variable Few laboratories correctly identified all the samples Many reported false negative or false positive results.

8 How to control newly emerging assays..cont In 1992 the first HIV-1 DNA panel was made by Janet Bootman at NIBSC ,000 molecules of DNA/vial 26 international laboratories participated in study 54% reported incorrect results, many reporting false negatives. Variation between control authorities. Control authority N o of pools tested % positive for HCV RNA Paul Ehrlich Institut NIBSC 95 5 Real differences? Reflection of different assay sensitivity? based on data from Saldanha et al Clearly a need for standardisation through the establishment of reference materials.

9 How to control newly emerging assays..a proficiency study.

10 Establishing Working Reagents. Working Reagents:- National or laboratory reference materials Working (secondary) reference materials for day-to-day use Calibrated against International Standard (in IU) May be freeze-dried or frozen liquid ready for use Development of working reagents based on serial dilutions of virus spiked into a plasma based diluent Considerations for working reagent production: What is the aim of the working reagent. Will viral copy number to make reagent fit for purpose. Will the diluent be compatible with most assays. The volume in each vial. Strain of virus used.

11 Establishing Working Reagents cont.. Candidate working reagents are then assessed in a collaborative study. Participants typically include: Blood product manufacturers Kit manufacturers OMCL s Reference laboratories Considerations for the study: Type of participants invited to participate. Types of assay that will be represented. Inter-laboratory variation in sensitivity is identified. Working reagent identified based on the highest dilution of sample detected in each run.

12 Collaborative Studies at NIBSC Date Working Reagents Study 1990 HIV-1 DNA (study 1) 1992 HIV-1 DNA (study 2) 1994 HCV RNA 1996 HIV-1 RNA 1996 B19 DNA 1996 HAV RNA International Standards 1997 HCV RNA International standard 1998 HBV DNA International standard 1998 HIV-1 RNA International standard However, several working reagents have been developed by various control laboratories and are expressed in different unitages: copies/ml, genome equivalent/ml, PCR detectable units

13 Establishing an International Standard International Standard:- It is a preparation by means of which the WHO defines an International Unit after an international study has been completed. International Units are arbitrary units An International Unitage is assigned to the 1st International Standard. Replacement standards are calibrated against the previous standard. Usually freeze-dried to ensure greater long term stability. WHO ECBS establishes International Standards and Reference Reagents. Appropriate starting material is identified may follow on from a working reagent. Samples are coded to ensure a blind study

14 Characteristics of HIV-1 Candidate Standard Criteria Candidate XX Candidate YY Candidate ZZ Virus Primary Isolate Plasmapheresis Field Isolate Donation Subtype B B B Propagated PBMC s No T cell line Copy number High Medium Low (219K) (56K) (15K) Presentation Freeze-dried Freeze-dried Frozen Diluent Cryosupernatant Defirinated Plasma pool plasma Volume 1ml 1ml 0.5ml Stability Good Good Not tested

15 Evaluation of 1 st International Standard for HIV-1 RNA Materials evaluated in a collaborative study Participants asked to test samples in 3 independent assays. Initially at 10-fold dilutions Subsequently at half log dilutions around the end point Results: Twenty seven data sets returned 20 (74%) were in a quantitative format 7 (20%) were in a qualitative format 21 sets of results from commercial assays 6 were from In-house assays. Average copy numbers for each candidate: XX ~219K YY ~ 56K ZZ ~ 15K

16 Evaluation of 1 st International Standard for HIV-1 RNA cont.. RNA Copies/ml (Log10) Candidate YY (NIBSC Code 97/656) Log Dilution METHOD a b3 ih m nu

17 When the 1 st standard runs out! 2 nd International Standard for HIV-1 RNA SoGAT recommended that sample XX from first collaborative study be re-evaluated as candidate 2 nd International Standard (IS) Characteristics: HIV-1 subtype B low passage field isolate (CCR5/NSI) from Edinburgh (P Simmonds) Propagated on human PBMCs - stock stored under LN Virus diluted in human cryosupernatant to suitable concentration 2200 vials of material freeze-dried Fill CV = 0.2%

18 Establishing 2 nd International Standard for HIV-1 RNA Aims of study:- Calibrate XX against 1st International Standard (1 st IS) Confirm stability of XX Determine values in current assays 10 laboratories participated Candidate XX and the 1 st International Standard recoded (samples 1 and 2 respectively) Participants asked to test samples in 3 independent assays. Initially at 10-fold dilutions Subsequently at half log dilutions around the end point 8 data sets received, 5 from quantitative assays and 3 from qualitative assays NIBSC collated and analysed data

19 Establishing 2 nd International Standard for HIV-1 RNA cont M 7 2 M M NU M M NU IH IH NU M B3 IH M IH B3 B3 M IH MQ IH AS B3 A M Estimated RNA Copies (log10/ml) Monitor assaysnuclisens bdna 3 Abbott LCx In-House Ampliscreen Original Collaborative Study 1st International Standard for HIV- 1 RNA (YY) NU 9 5 M 8 24 M 7 2 M 6 18 M M M NU IH M IH MQ B3 IH IH M B3 B IH IH AS B3 A M Estimated RNA Copies (log10/ml) Sample XX Monitor assays Nuclisens bdna 3 Abbott LCx In-House Ampliscreen

20 Establishing 2 nd International Standard for HIV-1 RNA cont T 3 72 P 2 4 NU IH B3 IH M A Estimated RNA Copies (log10/ml) Current Collaborative Study 1 st IS (coded Sample 2) 10 Monitr assays Nlisens bdna 3 Procilex In-House Amplicore TMA P 3 4 NU 2 71 IH IH B3 T M A Estimated RNA Copies (log10/ml) Monitor assays Nuclisens bdna 3 Procilex In-House Amplicore TMA Sample XX in Current Study (coded Sample 1)

21 Copies/ml (Log10) Degradation studies Long term stability is important in an International Standard. Accelerated degradation: Real time degradation: Real time degradation: Week 1 Week 3 Week 6 Insert graph here.. Week 7 Week 9 Week 13 Week 15 Week 19 Week 26 Week 39 Week 52 Week

22 International Standards at NIBSC 1997: 1 st International Standard for HCV RNA (96/790) 1999: 1 st International Standard for HIV-1 RNA (97/650) 1999: 1 st international Standard for HBV DNA (97/746) 2000: 1 st International Standard for B19 DNA (99/800) 2002: 1 st International Standard for HAV RNA (00/560) 2003: 1 st International Reference Panel for HIV-1 Genotypes (01/466) 2003: 2 nd International Standard for HCV RNA (96/798) 2005: 2 nd International Standard for HIV RNA (97/650) 2007: 2nd International Standard for HBV DNA (97/750) 2007: 3rd International Standard for HCV RNA (06/100)

23 Calibration of working standards. Once an International Standard for a viral marker is established it is possible to calibrate working standards against it. HCV working reagent calibration 6 HCV RNA working reagents from international laboratories have been calibrated against the 1 st HCV RNA IS. Collaborative study set up and managed in the same as as previously described. 19 laboratories participated. Titers of working reagents and IS were determined in parallel by end point dilution assays

24 HCV working reagent calibration cont.. Reagent LOG10 PCR units LOG 10 IU/ml IU/ml International Standard PEI reference NIBSC 96/ CLB Pelispy ISS CBER panel member

25 Working Reagents at NIBSC HIV-1 RNA: *99/634 *99/636 05/158 *HCV RNA (02/264) HBV DNA (05/148) HAV RNA (01/488) B19 DNA (99/736) Multiplex (HCV, HBV, HIV, HAV, B19) (99/732) HCV Genotype Panel (genotypes 1-6) (02/202) *CE marked

26 Conclusions: A variety of working reagents and international standards for blood virology have been produced over the last 10 years. Availability of such reagents has been vital in the standardisation of new NAT assays. International standards have allowed the meaningful comparison of data between laboratories. Use of calibrated working reagents has allowed a significant improvement in the performance of testing. Important to adapt to changing needs of users.

27 Acknowledgements: NIBSC (past and present): Harvey Holmes Jacqueline Fryer Dave Padley Sally Baylis John Saldanha International Collaborators CBER/FDA PEI NRL Sanquin Collaborative study groups