NAT Detection of Blood Borne Viral Markers in Tissues from Cadaver Donors John Saldanha 1 and David Padley 2 1 Canadian Blood Services, Ottawa, Canada 2 NIBSC, South Mimms, UK SoGAT, Paris May, 2004

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

West Nile Virus Infection in an Organ Donor and Four Transplant Recipients August 2002 Organ Donor WNV PCR – IgM – Organ Donor WNV PCR + Culture + IgM – Kidney recipient WNME (fatal) Kidney recipient WNME Liver recipient WNF Heart recipient WNME Blood components from 63 donors

Regulatory Requirements?

Optimised NAT Assay for HCV RNA Qiagen DNA mini kit 200µl blood or 25mg tissue extracted –proteinase K digestion (37 o C 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

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 50 o C/30min 95 o C/15min 43 cycles: 95 o C/45s 57 o C/40s 72 o C/40s final cycle: 72 o C/5min

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

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

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

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

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

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)

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)

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

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

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

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

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

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)

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

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.

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.’ ‘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.’ ‘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.’