Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia

Abstract

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38- leukemic stem cells.

Keywords: acute myeloid leukemia (AML); leukemia associated immunophenotypes (LAIPs); leukemic stem cells (LSCs); measurable residual disease (MRD); multiparameter flow cytometry (MFC).

Figure 1

Gating white blood cells. (A‐C) Gating of the white blood cells. For a detailed description of the gating steps see Basic Protocol 1, step 15a. (D)The final population of white blood cells is shown in blue in D.

Figure 2

Gating lymphocytes. (A) Gating of the lymphocytes. (B‐D) Lymphocytes are shown in green in B‐D. See Basic Protocol 1, step 15b for a detailed description of the gating steps.

Figure 3

Gating immature blasts. (A‐B) Gating of the immature blast cells. See Basic Protocol 1, step 15c for a detailed description of these gating steps. (C) The final population of immature blast cells is shown in dark blue.

Figure 4

LAIP detection on immature blasts at diagnosis. (A‐B) Gating of LAIP positive cells. For a detailed description of the gating steps see Basic Protocol 1, step 16. (C) LAIP positive immature blasts are shown in red. LAIP, leukemia associated immunophenotype.

Figure 5

LAIP detection on mature blasts at diagnosis. (A‐B) Gating of mature blast cells. (C) The population of mature blast cells is shown in orange. (D‐E) Gating of LAIP positive cells. See Basic Protocol 1, step 16 for a detailed description of these gating steps. (F) LAIP positive mature blasts are shown in red. LAIP, leukemia associated immunophenotype.

Figure 6

LAIP detection at follow‐up. (A‐B) Gating of LAIP positive cells in follow‐up BM. See Basic Protocol 1, step 17 for a detailed description of these gating steps. (C) LAIP positive blasts are shown in red. LAIP, leukemia associated immunophenotype; BM, bone marrow.

Figure 7

Gating of CD34+CD38− cells. (A‐C) Gating of the CD34+CD38− cells. For a detailed description of the gating steps see Basic Protocol 2, step 6. (D) The population of CD34+CD38− stem cells is shown in azure blue.

Figure 8

Gating of CD34+CD38− LSC cells using CD45RA as a stem cell marker. (A) This figure shows gating of the CD34+CD38− cells using CD45RA. (B‐C) Backgating of both the CD45RA positive and negative cell fractions in a FSC/SSC plot is shown in B and C, respectively. (D) Differences in CD34 expression between presumed LSC and HSC. (E) Final LSC results based on CD45RA as a stem cell marker. (F) Overview of the different secondary gating parameters is shown. A more detailed description of this figure can be found in Basic Protocol 2, step 7. In more detail: CD45RA negative and positive cell fractions were quite clearly separated (highlighted with a frame in A). Secondary gating parameters (FSC/SSC/CD34/CD45) were used to establish whether the presumed LSC and HSC populations were pure (this is further elucidated in the Critical Parameters and Troubleshooting section). B shows backgating of the CD45RA positive stem cells in a FSC/SSC plot. This shows that two populations with a different FSC and SSC can be discriminated within the CD45RA positive cell fraction. Backgating of the CD45RA positive FSC^low cells (marked in gray, B) in a CD34/CD38 plot shows that these cells have relatively low CD34 expression and are defined as a‐specific. C shows backgating of the CD45RA negative stem cells in an FSC/SSC plot. This backgating shows a pure CD45RA negative clustered FSC^low population, implying that there is little/no contamination with LSCs. D shows differences in CD34 expression between the LSC (in red), the presumed HSC (in green), and the earlier defined a‐specific events (in gray). Subsequently, the a‐specific events were removed from further analyses and E shows the final CD45RA expression results. F shows the results for all secondary gating used (FSC/SSC, CD34/SSC, and CD45/SSC), also showing that the CD45/SSC parameter does not contribute in this particular AML case. LSC, leukemic stem cell; HSC, hematopoietic stem cells; AML, acute myeloid leukemia.

Figure 9

Gating of the CD34+CD38− LSC cells using the Combi‐6 marker. (A) This figure shows gating of the CD34+CD38− cells using the Combi‐6 marker. (B‐C) Backgating of both the Combi‐6 positive and negative cell fractions in a FSC/SSC plot is shown in B and C, respectively. (D) Differences in CD34 expression between presumed LSC and HSC. (E) Final results of Combi‐6 as a stem cell marker including FSC as a secondary gating parameter. (F) Overview of the different secondary gating parameters. A more detailed description of this figure can be found in Basic Protocol 2, step 7. In more detail: An example of LSC gating in the same AML using the Combi‐6 marker is shown in A. In contrast to CD45RA there is no clear separation between putative LSC and HSC. Possible Combi‐6 positive and Combi‐6 negative cells were globally defined in A (highlighted with a frame). B shows backgating of the Combi‐6 positive stem cells in a FSC/SSC plot. This shows that two populations with a different FSC and SSC can be discriminated within the Combi‐6 positive cell fraction. Similar to CD45RA, backgating of the Combi‐6 positive FSC^low cells (marked in gray, B) in a CD34/CD38 plot shows that these marker‐positive FSC^low cells have relatively low CD34 expression and are defined as a‐specific events. C shows backgating of the Combi‐6 negative stem cells in an FSC/SSC plot. In contrast to CD45RA, this backgating shows a Combi‐6 negative FSC^low population and a tiny Combi‐6 negative FSC^high population. Based on the FSC characteristics of the Combi‐6 positive stem cells (dotted line between B and C), there is a tiny fraction which should be defined as leukemic based on FSC as a secondary gating parameter (Terwijn et al., 2014). D shows the differences in CD34 expression between the LSC (in red), the presumed HSC (in green), and the earlier defined a‐specific events (in gray). Subsequently, the a‐specific events were removed from further analyses and E shows the final Combi‐6 expression results. F shows the results for all secondary gating used (FSC/SSC, CD34/SSC, and CD45/SSC), also showing that the CD45/SSC parameter does not contribute in this particular AML case. So, despite the initial poor separation between LSC and HSC (A), LSC and HSC can be fairly well distinguished using secondary gating parameters resulting in similar calculated LSC frequencies: 0.012% in both CD45RA and Combi‐6 analyses which is below the previously defined cut‐off of 0.03% (% of WBCs) for positivity at diagnosis (Zeijlemaker et al., 2019). Also, quite similar LSC/HSC ratios in the total CD34+CD38− compartment were found: 37:63 for CD45RA and 34:66 for Combi‐6. LSC, leukemic stem cell; HSC, hematopoietic stem cells.