NAT Detection of Blood Borne Viral Markers in Tissues from Cadaver Donors

1. NAT Detection of Blood Borne Viral Markers in Tissues from Cadaver Donors John Saldanha1 and David Padley2 1Canadian Blood Services, Ottawa, Canada 2NIBSC, South Mimms, UK SoGAT, Paris 26-27 May, 2004

2. Introduction • Kits used for microbiological testing in tissue banks have not been validated for testing samples from cadavers • Antibody assays  high rates of false positives • NAT assays  false negatives • Quality of samples (haemolysis, autolysis) affect reliability of results

3. Kidney recipientWNME (fatal) Kidney recipientWNME Liver recipientWNF Heart recipientWNME West Nile Virus Infection in an Organ Donor and Four Transplant RecipientsAugust 2002 Blood components from 63 donors Organ Donor Organ Donor WNV PCR +Culture +IgM – WNV PCR –IgM –

4. Regulatory Requirements?

5. Optimised NAT Assay for HCV RNA Qiagen DNA mini kit • 200µl blood or 25mg tissue extracted • proteinase K digestion (37oC 10min-2h) • absorption onto silica membrane • two washes (AW1 and AW2) • elution of nucleic acids with 60µl water • Duplicate sample spiked with HCV working reagent (71 IU/ml) extracted in parallel to control for inhibitors

6. RT/PCR RT/PCR 10µl RNA in final reaction volume of 25µl 15pmole each forward and reverse primer from 5’ non-coding region  320 bp product 50oC/30min 95oC/15min 43 cycles: 95oC/45s 57oC/40s 72oC/40s final cycle: 72oC/5min

7. Analysis of Amplified Products • Amplified products run on 2% agarose gel and stained with ethidium bromide • Positive sample produced band of 320 bp

8. Results • 36 samples from St. Thomas’ Hospital Trust • 20/36 samples HCV RNA positive • 16/36 samples HCV RNA negative • 6/16 HCV RNA negative samples inhibited (parallel spiked sample was HCV RNA negative) • Inhibition in 3/6 samples overcome by 1:5 dilution of RNA prior to RT/PCR

9. Lanes 2, 4, 6, 8, 10, 12: RT/PCR of RNA of unspiked blood samples. Lanes 3, 5, 7, 9, 11, 13: RT/PCR of RNA from blood samples spiked with 71 IU/ml HCV Lane 14: 71 IU/ml HCV Lane 16: negative control Lanes 1 and 15: PCR markers

10. Results Removal of RT/PCR inhibitors • Treatment with Qiagen AX matrix ((InhibitEX) • DNA/RNA damaging substances absorbed by matrix • RT/PCR inhibitors easily removed during subsequent purification

11. Lanes 2, 4, 6, 8, 10, 12: unspiked blood samples. Lanes 3, 5, 7, 9, 11, 13: blood samples spiked with 71 IU/ml HCV [22, 23]. Lane 15: positive control containing 71 IU/ml HCV [ Lane 16: negative control. Lanes 1 and 14: PCR marker

12. Results • Two liver samples also pre-treated with AX matrix • sample 1: positive, not inhibited • sample 2: negative and inhibited • Both samples successfully pre-treated with AX matrix • sample 1: no difference in signal • sample 2: inhibitors removed (sample shown to be negative)

13. Conclusions • Optimised method applicable to routine testing of cadaver samples (blood and liver) for HCV RNA by NAT • May also be used for testing for other viruses (HIV-1, HBV)

14. Acknowledgements • UK Department of Health • Sourcing and collection of samples – Dr. Sebastian Lucas, St Thomas’ Hospital • NIBSC Microbiology Working Group of the NIBSC Steering Group on Tissue/Cell Banking and Engineering • Dr. Morag Ferguson, Dr. Ruth Warwick

15. Acknowledgements • UK Department of Health for funding the project • Morag Ferguson & Ruth Warwick, NBS, for help and input in the design of the studies • Sourcing and collection of samples - Dr Chris Womack, Peterborough District Hospital Dr Sebastian Lucas, St Thomas’ Hospital • NAT studies - John Saldanha • Serological assays -Dr E MacMahon and Miss Helen Dunn, St Thomas’s Hospital; Dr P Taylor, Royal Brompton Hospital; staff in the Divisions of Retrovirology and Virology at NIBSC • NIBSC Microbiology Working Group of the NIBSC Steering Group on Tissue/Cell Banking and Engineering, Dr John Barbara and Professor Richard Tedder for assistance and advice in development of the study protocols

17. International Herald Tribune 6/10/02 (the New York Times) • Tissue donor transmitted Hepatitis C • 40 people received tissue or organs from Oregon man who died of HCV • 5 of six organ recipients died, remaining person has HCV • 34 received tissue form donor: 4 positive for HCV, 9 have no signs of liver disease

18. Optimisation of NAT Assay Extraction kits tested • Qiagen QIAmp DNA mini kit • Qiagen QIAmp DNA Blood mini kit • Qiagen QIAmp RNA Blood mini kit • Machery-Nagel NucleoSpin Tissue kit Samples tested • Blood • Liver • Lymph node

19. RT/PCR Two kits tested Qiagen One-Step RT-PCR kit Abgene Reverse-IT Onestep kit • Qiagen One-Step RT-PCR kit  consistent results RT/PCR 10µl RNA in final reaction volume of 25µl

20. Results • 6/16 HCV RNA negative samples inhibited • 3/6 samples - inhibition overcome by 1:5 dilution of RNA prior to RT/PCR • 6/6 samples - inhibition overcome by pre-treatment with AX matrix • All 6 samples true HCV RNA negative samples (unspiked samples, pre-treated with AX matrix remained negative)

21. Summary • Robust extraction and RT/PCR method developed for testing cadaver blood and tissue samples for HCV RNA • Extraction based on Qiagen QIAmp DNA mini kit with pre-treatment with AX matrix • One step RT/PCR using Qiagen One-Step RT-PCR kit • 36 HCV RNA positive and HCV RNA negative samples tested • Inhibitors removed from all blood samples and two liver samples by pre-treatment with AX matrix

22. Final Conclusions • False positives from serological analysis • False negatives due to NAT analysis no longer a problem • HBV positive missed by 5 separate serological kits. • Need to standardise testing of cadaveric blood and tissue.

23. Dr. Ian Williams – epidemiologist at the federal disease centers • ‘The donor’s blood was tested with a standard procedure that cannot detect the virus for weeks or even months after infection.’ • ‘A newer blood test, the viral nucleic acid assay, can pick up infections one to two weeks after they begin, but is not being used by organ or tissue banks because cadaver blood has properties that make the test inaccurate.’