Flow Cytometric Minimal Residual Disease Analysis in Acute Leukemia: Current Status

Abstract

Minimal residual disease (MRD) analysis for patients of acute leukemia has evolved as a significant prognostic factor. Based on the MRD results, the cases are risk-stratified after induction chemotherapy, and an alteration in further management is made to yield maximal therapeutic benefits. The two primary methodologies for MRD detection are multi-parameter flow cytometry (MFC) and polymerase chain reaction. MFC identifies the MRD based on characteristic 'leukemia-associated immunophenotypes' on the residual leukemia cells. MRD analysis by MFC is most frequently done at the post-induction stage of treatment and often can achieve a sensitivity of detecting one leukemic cell in 10,000 normal cells, or even higher at times. This review outlines the technical aspects and provides inputs on standard antibody panels used for MRD detection in B-, T-lineage acute lymphoblastic leukemias, and acute myeloid leukemia.

Keywords: Immunophenotyping; Minimal residual disease (MRD); Multi-parameter flow cytometry (MFC).

Fig. 1

Representative plots from a case of B-Acute Lymphoblastic Leukemia (post-induction bone marrow aspirate). a Singlets are gated on forward scatter-area versus peak. b Viable events are then gated on forward scatter-area versus side scatter-area. c Mononuclear viable cell population is gated on CD38/side scatter plot. d CD19 positive events are then gated for further analysis. e CD38/CD10 plot is showing an abnormal cell population which is CD38^dim/CD10^bright compared to plasma cells and hematogones (a LAIP). The abnormal cell population is expressing CD34 (plot f), CD58 (plot g) and CD86^bright/CD81^neg (plot h). All these are LAIPs. There is also slight expression of CD123 (plot j) and CD20^pos/CD38^dim. Once, these LAIPs are recognized, the discrete cell population of abnormal cells is gated on forward and side scatter-area. The calculated MRD is shown on right

Fig. 2

Representative plots from a case of T-Acute Lymphoblastic Leukemia (post-induction bone marrow aspirate). a Singlets are gated on forward scatter-area versus height. b Viable events are then gated on forward scatter-area versus side scatter-area. c Plot shows gating of viable CD7 positive cells (representing T-cells). d Finally, discrete T-cell population was gated with CD7/cCD3. NK cells are excluded based on CD16-CD56^pos/CD38^bright population (not shown). e On CD4/CD8 plot, some dual negative cells are present apart from normal CD4 and CD8 positive T-cells. These dual negative cells when gated (plot g) shows CD5 positivity (plot h), To note that these cells have CD5 expression dimmer than the normal T-cells (a LAIP). i Plot shows the T-cells with TdT and CD34 positivity and this abnormal population constitutes MRD, which when gated (orange) has been highlighted on CD7/cCD3 plot (plot i) with a brighter CD7 compared to normal T-cells (in blue). This population is also highlighted on CD7/SSC-A plot (k). Finally, MRD is calculated from CD45/SSC-A plot (l) as a discrete cluster. The hierarchy of all the gated cells and number of events are provided and MRD calculation is shown on right (color figure online)

Fig. 3

Representative plots from a case of Acute Myeloid Leukemia (post-induction bone marrow aspirate). a Singlets are gated on forward scatter-area versus height. b Viable events are then gated on forward scatter-area versus side scatter-area. c This plot shows the distribution of viable leukocytes based on CD45 expression versus side scatter-area. The cells in the precursor region, labelled as ‘P’ (low SSC-A and CD45^dim) are gated for further analysis. d Plot shows gating of myeloid precursors which are CD117^pos and/or HLA-DR^pos. These precursors are then analysed to look for LAIPs (exclusion of normal populations in CD45^dim gate, like hematogones, early erythroid precursors, mast cells, basophils, plasmacytoid dendritic cells, immature NK cells and promyelocytes is explained in the text). f Plot shows under-expression of CD13 and CD33 on myeloid precursors (in pink) compared to that of mature neutrophils (green). g Plot shows another LAIP in the form of aberrant expression of CD56 on myeloid precursors (pink) and these are also positive for CD34 (plot h). Finally, the discreet MRD population was gated on forward and side scatter-area plot for calculation (red). The hierarchy of all the gated cells and number of events are provided and MRD calculation is shown on right (color figure online)