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

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

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

Regulatory Requirements?Regulatory Requirements?

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

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

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

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

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

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

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

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)

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)

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

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

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

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

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

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)

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

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.

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.’