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Duplex qPCR for BCR-ABL1 MRD

Duplex qPCR for BCR-ABL1 MRD. Gareth Gerrard ggerrard@imperial.ac.uk Leukaemia MRD Unit, Centre for Haematology, Imperial College, Hammersmith Hospital, London. EAC MRD qPCR. The Hammersmith Problem:. ABL. BCR-ABL. Duplicates: 36 Samples. Triplicates: 22 Samples.

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Duplex qPCR for BCR-ABL1 MRD

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  1. Duplex qPCR for BCR-ABL1 MRD Gareth Gerrard ggerrard@imperial.ac.uk Leukaemia MRD Unit, Centre for Haematology, Imperial College, Hammersmith Hospital, London.

  2. EAC MRD qPCR

  3. The Hammersmith Problem: ABL BCR-ABL Duplicates: 36 Samples Triplicates: 22 Samples Average Daily Run: 1 ABL1 plate & 2 BCR-ABL1 plates = 44 Samples per day fully quantitated

  4. The Solution: DUPLEX qPCR BCR-ABL ABL Triplicates: 24 Samples Average Daily Run: 2 Duplex plates = 48 Samples fully quantitated

  5. Probe Strategy FAM TAMRA ENP541 BCR-ABL1 (BCR ex13 – ABL1 ex2) TAMRA FAM VIC ABL-183T ABL1 (ABL1 ex3 – ABL1 ex4)

  6. Other Changes Made • Changed to ABI FAST plates: • Cheaper • Slightly faster run time in STANDARD mode • Changed Master Mix from ABI Universal MM to ABI Gene Expression MM: • Cheaper • Optimised for duplex

  7. Run Conditions • Pre-made single-use aliquots of primer/probe mix: • ENF501 – 300nM • ENR561 – 300nM • ENP541-FAM – 150nM • ABL-146F – 300nM • ABL-240R – 300nM • ABL-183T-VIC – 200nM • Mixed with x2 GE MM • 25μl reactions (22 μl + 3 μl cDNA) • Standard ABI Run conditions / 45 cycles Optimised to achieve equivalent Cts for both targets

  8. Validation • 4 Duplex plates • 8 Simplex plates • 4 ABL1 & 4 BCR-ABL1 • In parallel over 4 consecutive days • 1 Dx, 1 Sx-ABL1 and 1 Sx BCR-ABL1 per day • Analysed separately and as a meta-analysis

  9. Sx Dx

  10. Potential Issues • Increased sensitivity leads to slightly higher background and a greater probability of false positives • May need to run occasional Sx confirmation for crucial samples (eg, post SCT) • In reality, not much of a problem • Use intercept Ct+1 cut-off for positivity • Slight increase in replicate Ct deltas for ABL1 • Operationally significant?

  11. Conclusions: • Duplex qPCR for BCR-ABL1 MRD requires only a small change to: • Greatly increase throughput • Considerable cost-reduction • No loss of sensitivity • After validation and consultation our lab went live for the duplex assay at the beginning of June • Reviewed data after 1 month – all good!

  12. The Future: • MGB / NFQ probes • Reduce noise / spectral overlap • Triplex: especially for p190/e1a2 (Ph+ALL) • BCR-ABL1 • ABL1 • GUSB • Low Density arrays (ABI) • Nano-Fluidic Arrays (Fluidigm) • GeneXpert (Cepheid)

  13. Acknowledgements • Prof Letizia Foroni • Katherine Mudge • Rebecca Stewart • Pierre Foskett • David Stevens • Natalie Killeen • Dr Richard Szydlo

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