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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


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


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