Flow Cytometry CD45 Gating for Immunophenotyping of Acute
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Leukemia (1997) 11, 1878–1886 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia F Lacombe, F Durrieu, A Briais, P Dumain, F Belloc, E Bascans, J Reiffers, MR Boisseau and Ph Bernard Laboratoire d’He´matologie, Service des Maladies du Sang, Hoˆpital du Haut-Le´veˆque, Pessac, France A flow cytometry method has been introduced into the routine age values within the malignant blast cell populations or investigation of whole bone marrow samples following red within the total nucleated cells present in each sample. In blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by order to give numbers of malignant cells and proportions of CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) cells within this cohort, a reliable practical discrimination and the results were compared to a conventional FSC/SSC gat- between malignant blasts and normal cell types would be ing procedure and to MGG-staining smears. The percentages required. In this paper, as previously recommended by of blast cells in these samples as defined by the morphological Borowitz et al,6 we suggest that such a discrimination can be analysis of MGG smears correlated better with the values readily facilitated by introducing primary gating for CD45 determined by CD45/SSC gating (r 5 0.94) than with the blast antigen expression and side scatter (CD45/SSC). We also sug- cell counts recorded with FSC/SSC gating (r 5 0.76). These findings were not surprising because while CD45 expression gest that this step should replace the first gating step for for- was regularly lower on leukemic blasts than on normal lymph- ward scatter and side scatter (FSC/SSC), as this latter procedure oid and monocytic cells, the FCS/SSC characteristics of these does not discriminate well between leukemic blasts, lympho- populations were overlapping. In 53 samples, the blast cell cytes and monocytes. populations were also analyzed with a panel of FITC-conju- In this paper we present the application of double and triple gated monoclonal antibodies that were utilized in double labe- ling with CD45-PE. We show that the CD45/SSC gating pro- immunofluorescence (IF) analysis of bone marrow samples cedure improved phenotypic determination of the blast cells in taken from patients during the presentation of AML. With the three ways: (1) by discriminating between leukemic blast cells systematic use of leukocyte common antigen (CD45) marker and residual normal cells; (2) by excluding normal cells from in combination with lineage-specific markers, a good dis- the phenotypic analysis of leukemic blast cells; and (3) by crimination can be achieved between the blast cell popu- identifying blast cell heterogeneity in many cases of leukemia lations and the normal cells. This discrimination is based on on the basis of different CD45 display. Moreover, this immuno- phenotyping procedure on whole bone marrow samples also the fact that the precursor cells in the bone marrow, as well allowed an efficient discrimination between the various cell lin- as the leukemias which derive from these cell types, express eages and facilitated the analysis of leukemic blasts present in low and intermediate values of CD45, while lymphocytes and low proportions. monocytes express high levels of CD45.7 Keywords: acute myeloid leukemia; immunophenotyping; CD45; We also present evidence that with conventional FSC/SSC flow cytometry gating miscalculations of blast cell percentages can occur, which can lead to an increase in both false positivity and false negativity in the percent values of leukemic blasts expressing Introduction different antigens. Furthermore, the use of CD45/SSC gating facilitates the expression of results, primarily gated on The characterization of acute leukemias is based on a multi- CD45low blast cells, in a uniform manner that provides similar parametric analysis which includes clinical features, cell mor- observations in lysed whole bone marrow and in enriched phology, genetics and immunological markers. These markers mononuclear populations following Ficoll separation. Our have been shown to be important for the diagnosis and prog- observation adds to previous findings by Borowitz et al,6 Shah nosis of acute myeloid leukemia (AML), particularly when the et al,7 Stelzer et al8 and Rainer et al9 and indicates that precise lineage of the blast cells cannot be defined by light CD45/SSC gating may provide a common platform for microscopy morphology and cytochemistry.1–3 Recently, both uniform data processing during the immunodiagnosis of AML. the European Group for the Immunological Characterization of Leukemias (EGIL)4 and the European Working Group on Flow Cytometry and Image Analysis5 have identified the most Materials and methods important lineage-specific markers for the myeloid and lymph- oid lineages. Nevertheless, there is still disagreement in two Specimens areas. First, it is not yet decided whether the blast cells should be enriched by Ficoll-density gradient centrifugation prior to Seventy-four bone marrow samples were consecutively col- phenotypic analysis or should be studied in the whole bone lected over a 12-month period from adult patients with a sus- marrow following red cell lysis. While opinions tend to favor pected diagnosis of de novo AML. Bone marrow (2–3 ml) was the study of lysed whole bone marrow, samples are still fre- aspirated into EDTA K3 tubes (Vacutainer; Becton Dickinson, quently Ficolled for cryopreservation and storage. Thus, Pont de Claix, France) and received in the laboratory within phenotypic methods which are suitable for analyzing leu- 1 h of collection. Heparinized samples were not suitable for kemic blast cells following both sample handling procedures the bone marrow lysis method used in our laboratory. could be very useful. The second related issue is that it is not yet agreed whether the results should be expressed as percent- Patient population Correspondence: F Lacombe, Laboratoire d’He´matologie, Hoˆ pital du Haut-Le´veˆque, Avenue Magellan, 33604 Pessac, France; Fax: 33 556 The patients included 38 women and 36 men and their age 55 68 09 ranged from 16 to 84 years (median 58.5). The morphological Received 7 March 1997; accepted 1 August 1997 analysis and differential count (excluding erythroid cells) were Flow cytometry immunophenotyping of AML and CD45/SSC gating F Lacombe et al 1879 performed on May–Gru¨nwald–Giemsa-stained smears by two were adjusted using a CD4-FITC/CD8-PE staining of whole experienced readers. blood lymphocytes and levels for positivity were determined Diagnosis was made according to the FAB classification,10 according to PE- and FITC-conjugated isotypic Ig control cytochemistry and immunophenotyping, as AML0 (n = 8), (IgG1-FITC and IgG1-PE from Coulter). A first step (gate A) AML1 (n = 9), AML2 (n = 23), AML3 (n = 10), AML4 (n = 9), was to exclude debris and fat cells on a FSC/SSC histogram; AML5 (n = 10), AML6 (n = 1), AML7 (n = 1), biphenotypic 10 000 cells were counted in gating A and data were recorded acute leukemia (n = 3) as defined by GEIL.11 in list mode. Immunofluorescence (IF) staining Data analysis IF staining was performed on fresh cells by direct IF as follows. List mode data on viable cells (gate A; see above) were ana- Samples were diluted 5 × 105 cells/ml with AB-positive pooled lyzed using the Coulter Elite (version 4.01) or Coulter System plasma (instead of PBS; see below) and 50 ml were incubated II programs. On the CD45/SSC histograms four gates were set for 30 min at room temperature in the dark with 5 ml of each up: gate L for lymphocytes, M for monocytes, G for granulo- FITC (fluorescein isothiocyanate)-conjugated antibody and cytes and B for blast cells (Figure 1b). As the CD45 reagent 5 ml of PE (phycoerythrin)-conjugated CD45 (Table 1). Diag- was labeled with phycoerythrin (CD45-PE) in each gate, the nosis was confirmed by cytoplasmic markers for myeloperoxi- analysis of IgG1-FITC fluorescence intensity distribution was dase, CD3c and CD22c4,5 or megakaryocytic and erythroid also performed. Fewer than 1% cells were found in the linear markers (CD61, CD41b and glycophorin-A; not shown). regions shown in each monoparametric histogram of Figures Samples were then treated using the Coulter ImmunoPrep 1c and 3. Histograms for a panel of antibodies used were ana- reagent kit and Multi-Q-Prep System (Coutronics, Margency, lyzed using the same settings (ie gate A in Figures 1a or 5a, France) according to the manufacturer’s instructions. The c and gates L, M, G, B in Figures 1b or 5b, d). An example Multi-Q-Prep worked well only in samples diluted with in Figure 1c for the CD33 antigen shows the differential plasma as the admixture of PBS caused cell aggregation. In expression of the four cell sub-populations determined in the the first 21 patients, the FSC/SSC and CD45/SSC histograms CD45/SSC histogram. Other examples from the same sample were analyzed and in the other 53 patients all additional are given in Figure 3 for the CD3, CD19, CD14 and CD7 double IF tests were also performed. antigens. In some cases, the gates could be repositioned using backgating from the staining with the FITC-labeled lineage- specific reagents (eg CD3 for T lymphocytes, CD19 for B lym- Flow cytometry phocytes, CD14 for monocytes, as shown in Figure 3). However, these adjustments were minor. In order to keep the setting and histograms uniform during the Blast cell populations were identified by using either gate studies, a Coulter XL flow cytometer was checked daily with S (Figure 1a) in the FSC/SSC histogram, or gate B in the calibration beads (DNA-Check; Coulter). With 488 nm exci- CD45/SSC histogram (Figure 1b).