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Strategies for Reducing Inhibition in RT-PCR of Cadaveric Plasma Samples

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sanne
10 diapositives · 248 Views · Aug 11, 2014

This study, presented at SoGAT XIX in Bern, Switzerland, explores the challenges of nucleic acid testing (NAT) in cadaveric plasma samples. Previous methods showed low inhibition rates (~5%), but validation on cadaveric samples revealed much higher rates (up to 34% for HIV). The research involved various strategies to reduce inhibition, such as using QIAGEN’s treatments and the addition of polyvinylpyrrolidine (PVP). Overall, no definitive solution was found, highlighting the complexity of NAT in cadaveric settings and the need for further robust assay development.

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Strategies for Reducing Inhibition in RT-PCR of Cadaveric Plasma Samples
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  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

  8. 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