Malgorzata Gorniak1, Jasmine Singh1, George Grigoriadis1, 2, Sue Morgan 1, Shaun Fleming1, 2
1Laboratory Haematology, Alfred Pathology, 2Clinical Haematology, Monash Health
Background:
The Implementation of a 10 colour Custom Designed Flow Cytometry Panel that is able to monitor minimal residual disease of Acute B-cell lymphoblastic leukaemia in adults
Aims: A single 10-colour flow cytometry assay with sensitivity up to 10-4 for detecting MRD in B-ALL
Methods: A 10-colour single tube flow cytometry assay that include Cluster of Differentiation (CD) markers able to detect B cell maturation pattern comparable to that found in litrerature:CD19, CD22, CD20, CD38, CD10, CD45 and CD34 as well as most frequent aberrant markers: CD58, CD13/33, CD66c were designed by our Laboratory’s Haematologists.
Patient samples were analysed using two gating strategy:
To achieve assay sensitivity of our assay at 10-4, all bone marrow samples were cell enriched by bulk ammonium chloride lysis to maximize cell yields with a target of 1 x 10-6
To check assay sensitivity and specificity patient samples correlated to standard of care molecular monitoring with either IG/TCR qPCR in Philadelphia negative (Ph-) disease and BCR-ABL qRT-PCR in Philadelphia positive (Ph+) disease. Statistical correlation was performed in Graphpad Prism version 7.0 for linear regression and calculation of correlation coefficient.
Results:
30 routine Bone Marrow samples showed 100% concordance with routine.
Two Case studies will be presented.
Post assay validation of 75 samples from 20 patients was tested by our Flow assay for MRD, 40 samples were able to be correlated for MRD by both molecular and flow cytometric methods.
Of the 40 correlated samples most were concordant with only 4 discordant results at low level of detection (<0.01%), all discordant results were below test’s calculated lower limit of detection (LLOD).