NAT Detection of Blood Borne Viral Markers in Tissues from Cadaver Donors
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NAT Detection of Blood Borne NAT Detection of Blood Borne Viral Markers in Tissues from Viral Markers in Tissues from
Cadaver DonorsCadaver Donors
John Saldanha1 and David Padley2
1Canadian Blood Services, Ottawa, Canada2NIBSC, South Mimms, UK
SoGAT, Paris 26-27 May, 2004
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IntroductionIntroduction
• 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
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West Nile Virus Infection in an Organ Donor West Nile Virus Infection in an Organ Donor and Four Transplant Recipientsand Four Transplant Recipients
August 2002August 2002
Organ Donor
WNV PCR –IgM –
Organ Donor
WNV PCR +
Culture +IgM –
Kidney recipientWNME (fatal)
Kidney recipientWNME
Liver recipientWNF
Heart recipientWNME
Blood Blood components components
from 63 donorsfrom 63 donors
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Regulatory Requirements?Regulatory Requirements?
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Optimised NAT Assay for HCV RNA 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
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RT/PCRRT/PCR
RT/PCR10µl RNA in final reaction volume of 25µl
15pmole each forward and reverse primer from 5’
non-coding region 320 bp product50oC/30min
95oC/15min
43 cycles:95oC/45s
57oC/40s
72oC/40s
final cycle:72oC/5min
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Analysis of Amplified ProductsAnalysis of Amplified Products
• Amplified products run on 2% agarose gel and stained with ethidium bromide
• Positive sample produced band of 320 bp
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ResultsResults
• 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
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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 HCVLane 14: 71 IU/ml HCVLane 16: negative controlLanes 1 and 15: PCR markers
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ResultsResults
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
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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
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ResultsResults
• 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)
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ConclusionsConclusions
• 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)
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AcknowledgementsAcknowledgements
• 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
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AcknowledgementsAcknowledgements• 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
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International Herald Tribune International Herald Tribune 6/10/02 6/10/02
(the New York Times)(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
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Optimisation of NAT AssayOptimisation of NAT AssayExtraction 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
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RT/PCRRT/PCR
Two kits testedQiagen One-Step RT-PCR kit
Abgene Reverse-IT Onestep kit
• Qiagen One-Step RT-PCR kit consistent results
RT/PCR10µl RNA in final reaction volume of 25µl
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ResultsResults• 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)
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SummarySummary• 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
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Final ConclusionsFinal 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.
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Dr. Ian Williams – epidemiologist at the Dr. Ian Williams – epidemiologist at the federal disease centersfederal disease centers
‘‘The donor’s blood was tested with a The donor’s blood was tested with a standard procedure that cannot detect standard procedure that cannot detect the virus for weeks or even months the virus for weeks or even months after infection.’after infection.’
‘‘A newer blood test, the viral nucleic A newer blood test, the viral nucleic acid assay, can pick up infections one to acid assay, can pick up infections one to two weeks after they begin, two weeks after they begin, but is not but is not being used by organ or tissue banks being used by organ or tissue banks because cadaver blood has properties because cadaver blood has properties that make the test inaccurate.’that make the test inaccurate.’
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