Reducing Inhibition of RT-PCR in plasma samples from cadavers

1
Reducing Inhibition of RT-PCR in plasma samples
from cadavers
SoGAT XIX, Bern, Switzerland 2006

• Dr Simon Carne
• National Transfusion Microbiology Reference
  Laboratory (NTMRL)
• National Blood Service
• Colindale
• London NW9 5BG

2
Introduction

• Deceased (cadaveric) tissue donors require NAT
  screening
• Previous NAT screening system used manual QIAGEN
  extraction with LC RT-PCR- Very low Inhibition
  rate (5)
• QIAGEN MDx biorobot purchased, processes 96
  samples in 2hrs
• Extensive validation performed with variety of
  plasma samples but not cadaveric- too scarce

3
Cadaveric inhibition Jun 05
4
Initial Strategies for reducing Inhibition

• On advice from QIAGEN replaced MDx protease with
  more robust proteinase K
• During validation investigations on F/T
  cadaveric material inhibition, observed at 14
  for HCV, 34 HIV
• Freeze/thaw Proteinase K introduced

5
Cadaveric inhibition Sep/Oct 05
6
Inhibition Reduction Strategies

• Treatment with QIAGEN InhibitEX matrix
• Treatment with QIAGEN RNEasy kit
• Addition of polyvinylpyrrolidine (PVP) to lysis
  buffer

7
Inhibition Reduction Results
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Summary of Outcomes

• QIAGEN InhibitEX matrix - some limited success -
  expensive and impractical
• QIAGEN RNEasy kit - unsuccessful but results so
  unexpected further work needed
• PVP addition -limited success - inexpensive BUT
  some concerns as 2 samples changed status from
  non-inhibitory to inhibitory

9
Overall Conclusions

• No magic wand for overcoming inhibition in
  cadaveric samples
• More robust NAT assay may be required
• Some evidence that inhibition rates vary
  depending on referral centre - Inhibition-linked
  to retrieval and storage?
• Pelleting of virus by ultracentrifugation may be
  means to tackle cadaveric sample inhibition

10
Acknowledgements

• Dr. Alan Kitchen
• Patricia Lowe
• Raksha Karia
• NTMRL
• National Blood Service, Colindale NW9 5BG London,
  United Kingdom