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Duplex qPCR for BCR-ABL1 MRD. Gareth Gerrard [email protected] 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

Duplex qPCR for BCR-ABL1 MRD

Gareth Gerrard

[email protected]

Leukaemia MRD Unit,

Centre for Haematology,

Imperial College, Hammersmith Hospital,

London.

the hammersmith problem
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

the solution
The Solution:

DUPLEX qPCR

BCR-ABL

ABL

Triplicates: 24 Samples

Average Daily Run: 2 Duplex plates

= 48 Samples fully quantitated

probe strategy
Probe Strategy

FAM

TAMRA

ENP541

BCR-ABL1 (BCR ex13 – ABL1 ex2)

TAMRA

FAM

VIC

ABL-183T

ABL1 (ABL1 ex3 – ABL1 ex4)

other changes made
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
run conditions
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

validation
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
slide10

Sx

Dx

potential issues
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?
conclusions
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!
the future
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)
acknowledgements
Acknowledgements
  • Prof Letizia Foroni
  • Katherine Mudge
  • Rebecca Stewart
  • Pierre Foskett
  • David Stevens
  • Natalie Killeen
  • Dr Richard Szydlo
ad