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’Hématologie, Service des Maladies du Sang, Hôpital du Haut-Lévêque, Pessac, France
A flow cytometry method has been introduced into the routine
investigation of whole bone marrow samples following red
blood cell lysis on the basis of a primary CD45/side scatter
(SSC) gating procedure. Blast cells were first identified by
CD45/SSC gating in 74 cases of acute myeloid leukemia (AML)
and the results were compared to a conventional FSC/SSC gat-
ing procedure and to MGG-staining smears. The percentages
of blast cells in these samples as defined by the morphological
analysis of MGG smears correlated better with the values
determined by CD45/SSC gating (r 5 0.94) than with the blast
cell counts recorded with FSC/SSC gating (r 5 0.76). These
findings were not surprising because while CD45 expression
was regularly lower on leukemic blasts than on normal lymph-
oid and monocytic cells, the FCS/SSC characteristics of these
populations were overlapping. In 53 samples, the blast cell
populations were also analyzed with a panel of FITC-conju-
gated monoclonal antibodies that were utilized in double labe-
ling with CD45-PE. We show that the CD45/SSC gating pro-
cedure improved phenotypic determination of the blast cells in
three ways: (1) by discriminating between leukemic blast cells
and residual normal cells; (2) by excluding normal cells from
the phenotypic analysis of leukemic blast cells; and (3) by
identifying blast cell heterogeneity in many cases of leukemia
on the basis of different CD45 display. Moreover, this immuno-
phenotyping procedure on whole bone marrow samples also
allowed an efficient discrimination between the various cell lin-
eages and facilitated the analysis of leukemic blasts present in
low proportions.
Keywords: acute myeloid leukemia; immunophenotyping; CD45;
flow cytometry
Introduction
The characterization of acute leukemias is based on a multi-
parametric analysis which includes clinical features, cell mor-
phology, genetics and immunological markers. These markers
have been shown to be important for the diagnosis and prog-
nosis of acute myeloid leukemia (AML), particularly when the
precise lineage of the blast cells cannot be defined by light
microscopy morphology and cytochemistry. 1–3 Recently, both
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
important lineage-specific markers for the myeloid and lymph-
oid lineages. Nevertheless, there is still disagreement in two
areas. First, it is not yet decided whether the blast cells should
be enriched by Ficoll-density gradient centrifugation prior to
phenotypic analysis or should be studied in the whole bone
marrow following red cell lysis. While opinions tend to favor
the study of lysed whole bone marrow, samples are still fre-
quently Ficolled for cryopreservation and storage. Thus,
phenotypic methods which are suitable for analyzing leu-
kemic blast cells following both sample handling procedures
could be very useful. The second related issue is that it is not
yet agreed whether the results should be expressed as percent-
Correspondence: F Lacombe, Laboratoire d’Hématologie, Hôpital du
Haut-Lévêque, Avenue Magellan, 33604 Pessac, France; Fax: 33 556
55 68 09
Received 7 March 1997; accepted 1 August 1997
age values within the malignant blast cell populations or
within the total nucleated cells present in each sample. In
order to give numbers of malignant cells and proportions of
cells within this cohort, a reliable practical discrimination
between malignant blasts and normal cell types would be
required. In this paper, as previously recommended by
Borowitz et al,6 we suggest that such a discrimination can be
readily facilitated by introducing primary gating for CD45
antigen expression and side scatter (CD45/SSC). We also sug-
gest that this step should replace the first gating step for for-
ward scatter and side scatter (FSC/SSC), as this latter procedure
does not discriminate well between leukemic blasts, lympho-
cytes and monocytes.
In this paper we present the application of double and triple
immunofluorescence (IF) analysis of bone marrow samples
taken from patients during the presentation of AML. With the
systematic use of leukocyte common antigen (CD45) marker
in combination with lineage-specific markers, a good dis-
crimination can be achieved between the blast cell popu-
lations and the normal cells. This discrimination is based on
the fact that the precursor cells in the bone marrow, as well
as the leukemias which derive from these cell types, express
low and intermediate values of CD45, while lymphocytes and
monocytes express high levels of CD45.7
We also present evidence that with conventional FSC/SSC
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
different antigens. Furthermore, the use of CD45/SSC gating
facilitates the expression of results, primarily gated on
CD45low blast cells, in a uniform manner that provides similar
observations in lysed whole bone marrow and in enriched
mononuclear populations following Ficoll separation. Our
observation adds to previous findings by Borowitz et al,6 Shah
et al,7 Stelzer et al8 and Rainer et al 9 and indicates that
CD45/SSC gating may provide a common platform for
uniform data processing during the immunodiagnosis of AML.
Materials and methods
Specimens
Seventy-four bone marrow samples were consecutively col-
lected over a 12-month period from adult patients with a sus-
pected diagnosis of de novo AML. Bone marrow (2–3 ml) was
aspirated into EDTA K3 tubes (Vacutainer; Becton Dickinson,
Pont de Claix, France) and received in the laboratory within
1 h of collection. Heparinized samples were not suitable for
the bone marrow lysis method used in our laboratory.
Patient population
The patients included 38 women and 36 men and their age
ranged from 16 to 84 years (median 58.5). The morphological
analysis and differential count (excluding erythroid cells) were
 Flow cytometry immunophenotyping of AML and CD45/SSC gating
F Lacombe et al

performed on May–Grünwald–Giemsa-stained smears by two
experienced readers.
Diagnosis was made according to the FAB classification,10
cytochemistry and immunophenotyping, as AML0 (n = 8),
AML1 (n = 9), AML2 (n = 23), AML3 (n = 10), AML4 (n = 9),
AML5 (n = 10), AML6 (n = 1), AML7 (n = 1), biphenotypic
acute leukemia (n = 3) as defined by GEIL.11
1879
were adjusted using a CD4-FITC/CD8-PE staining of whole
blood lymphocytes and levels for positivity were determined
according to PE- and FITC-conjugated isotypic Ig control
(IgG1-FITC and IgG1-PE from Coulter). A first step (gate A)
was to exclude debris and fat cells on a FSC/SSC histogram;
10 000 cells were counted in gating A and data were recorded
in list mode.

Immunofluorescence (IF) staining Data analysis

IF staining was performed on fresh cells by direct IF as follows.
Samples were diluted 5 × 105 cells/ml with AB-positive pooled
plasma (instead of PBS; see below) and 50 ml were incubated
for 30 min at room temperature in the dark with 5 ml of each
FITC (fluorescein isothiocyanate)-conjugated antibody and
5 ml of PE (phycoerythrin)-conjugated CD45 (Table 1). Diag-
nosis was confirmed by cytoplasmic markers for myeloperoxi-
dase, CD3c and CD22c4,5 or megakaryocytic and erythroid
markers (CD61, CD41b and glycophorin-A; not shown).
Samples were then treated using the Coulter ImmunoPrep
reagent kit and Multi-Q-Prep System (Coutronics, Margency,
France) according to the manufacturer’s instructions. The
Multi-Q-Prep worked well only in samples diluted with
plasma as the admixture of PBS caused cell aggregation. In
the first 21 patients, the FSC/SSC and CD45/SSC histograms
were analyzed and in the other 53 patients all additional
double IF tests were also performed.
Flow cytometry
In order to keep the setting and histograms uniform during the
studies, a Coulter XL flow cytometer was checked daily with
calibration beads (DNA-Check; Coulter). With 488 nm exci-
tation, FITC green fluorescence was measured through a
525 PB filter and orange PE fluorescence through a 575 PB
filter. The four parameters simultaneously collected were lin-
ear forward-angle light scatter (FSC), linear side scatter (SSC),
log FITC and log PE. Fluorochrome compensation settings
List mode data on viable cells (gate A; see above) were ana-
lyzed using the Coulter Elite (version 4.01) or Coulter System
II programs. On the CD45/SSC histograms four gates were set
up: gate L for lymphocytes, M for monocytes, G for granulo-
cytes and B for blast cells (Figure 1b). As the CD45 reagent
was labeled with phycoerythrin (CD45-PE) in each gate, the
analysis of IgG1-FITC fluorescence intensity distribution was
also performed. Fewer than 1% cells were found in the linear
regions shown in each monoparametric histogram of Figures
1c and 3. Histograms for a panel of antibodies used were ana-
lyzed using the same settings (ie gate A in Figures 1a or 5a,
c and gates L, M, G, B in Figures 1b or 5b, d). An example
in Figure 1c for the CD33 antigen shows the differential
expression of the four cell sub-populations determined in the
CD45/SSC histogram. Other examples from the same sample
are given in Figure 3 for the CD3, CD19, CD14 and CD7
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-
phocytes, CD14 for monocytes, as shown in Figure 3).
However, these adjustments were minor.
Blast cell populations were identified by using either gate
S (Figure 1a) in the FSC/SSC histogram, or gate B in the
CD45/SSC histogram (Figure 1b). CD antigen expression on
the blast cell population was compared according to these
two gating procedures, and more than 20% positive blasts
were recorded as positive.
Results

Table 1 Panel of monoclonal antibodies used for acute leukemia Clustering of bone marrow cell lineages
immunophenotyping in this series
Figure 1 shows the advantage of the CD45/SSC gating pro-
CD number Reactivity Source
cedure over FSC/SSC for identifying leukemic cells. When the
FSC/SSC gate is used without an immunological marker, the
CD3, FITC T lineage Ortho
blast cells in AML partially overlap with the gates for normal
CD4, FITC T lineage Coulter
CD5, FITC T lineage Dako lymphocytes and monocytes (Figure 1a). As a contrast,
CD7, FITC T lineage Coulter CD45/SSC gating clearly separates the four cell categories of
CD8, PE T lineage Coulter lymphocytes, monocytes, granulocytes and blast cells (Figure
CD10, FITC B lineage Coulter 1b). Mature lymphocytes show the highest CD45 fluorescence
CD19, FITC B lineage Immunotech intensity and the lowest SSC signal (gate L). Mature monocytes
CD20, FITC B lineage Coulter
express slightly lower but still high amounts of CD45 and are
CD22, FITC B lineage Dako
HLA-DR, FITC Ortho easily distinguished from lymphocytes by their higher SSC sig-
CD11b, FITC Granulocytes, monocytes, Ortho nal (gate M). Granulocytic lineage expresses low CD45 and
NK cells very high SSC (gate G). In a few samples within gate G, two
CD13, FITC Myeloid cells, monocytes Dako populations could be seen: the CD45low population rep-
CD14, FITC Monocytes Coulter resenting immature granulocytes and the CD45medium popu-
CD33, FITC Myeloid cells, monocytes Immunotech
lation exhibiting mature features.8 Blasts cells had the lowest
CD34, FITC Immature precursors Immunotech
CD45, PE Pan-leukocyte Coulter CD45 intensity and a low SSC, reflecting features of normal
immature bone marrow precursor cells (gate B). Erythrocytes
Ortho, Ortho Diagnostic System, Raritan, NJ, USA; Coulter, Coul- and platelets, which do not express CD45 antigen, are
tronics, Margency, France; Dako, Glostrup, Denmark; Immunotech, excluded.
Marseille, France. In 74 consecutive samples, the morphological bone marrow
 Flow cytometry immunophenotyping of AML and CD45/SSC gating
F Lacombe et al

1880

Figure 1 Representation of flow cytometry histograms of an acute myeloid leukemia. (a) Bivariate histogram FSC/SSC with two gates: gate
A to eliminate debris and fat cells, gate S on presumed blast cells. Two gates (dotted lines) show the impossibility of defining a specific area
for monocytes and blast cells. (b) Bivariate histogram CD45/SSC with four gates: gate G including granulocytes, gate M monocytes, gate L
lymphocytes and gate B blast cells. Corresponding cell percentages are indicated (the sum is not 100% since rare cells are found outside the
gates). (c) Univariate histograms of CD33 expression intensity conditioned on gate L (histogram 4 of lymphocytes), gate M (histogram 5 of
monocytes), gate G (histogram 6 of granulocytes), gate B (histogram 7 of blast cells defined in (b), gate S (histogram 8 of ‘blast cells’ defined
in (a). Corresponding light microscopic counts are indicated.

differential counts were compared with the results of the
CD45/SSC gating procedure. A strong positive correlation was
found for the four cell subpopulations (Figure 2b–e): blast cells
(r = 0.94), lymphocytes (r = 0.94), neutrophils (r = 0.90) and
monocytes (r = 0.95). By contrast, a similar comparison
between the morphological blast cell count and their corre-
sponding count performed on the FSC/SSC histogram showed
a greater spread of results (r = 0.76; Figure 2a). Moreover, the
analysis of lysed whole bone marrow by the CD45/SSC gating
showed an excellent correlation with the immunological
identification of normal and malignant cells (Figure 3). The
cells were first gated on gate A to exclude red cells, debris
and fat cells (Figure 1a). Then it was confirmed that the cells
classified as lymphocytes, monocytes, granulocytes and blast
Flow cytometry immunophenotyping of AML and CD45/SSC gating
F Lacombe et al

1881

Figure 2 Correlations between a light microscopic bone marrow

cell differential and the flow differential for hematopoietic cell evalu-
ation. (a) FSC/SSC gating for blast cells (b–e) CD45/SSC gating for (b)
blast cells (c) lymphocytes (d) granulocytes (e) monocytes.
 Flow cytometry immunophenotyping of AML and CD45/SSC gating
F Lacombe et al

1882

Figure 3 Univariate histograms of some CD expression in lymphocytes, monocytes and blast cells from the same sample as in Figure 1.
Histograms are all conditioned on gate A. From top to bottom, the distributions of fluorescence are shown for: CD3, CD19, CD14, CD7.
Histograms (a–d) are conditioned on gates L or M of Figure 1b. Histograms (e–h) are conditioned on gate S of Figure 1a and histograms (i–l)
on gate B of Figure 1b. Arrows in histograms e, f and h show the contamination of lymphocytes in blast cell immunophenotyping. Arrow in
histogram l shows the CD7+ blast cell population.
 Flow cytometry immunophenotyping of AML and CD45/SSC gating
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1883
cells (Figure 1b) did indeed express the appropriate immuno-
logical markers (Figure 3). While monocytes were positive for
CD33 (Figure 1c) and CD14 (Figure 3c), this particular case
of AML expressed only CD33 (Figure 1c) and was negative
for CD3 (Figure 3i), CD19 (Figure 3j) and CD14 (Figure 3k) if
blast cells were determined on gate B of the CD45/SSC histo-
gram. By contrast, the contamination of the blast cell popu-
lation by T lymphocytes (Figure 3e) and B lymphocytes (Figure
3f) is obvious if gate S on the FSC/SSC histogram is chosen to
isolate blast cells. The sensitivity of the method is documented
with a study of CD7 antigen. T lymphocytes were CD7+, as
expected (Figure 3d). The blast cells had two populations: one
CD7-negative and one CD7-positive (Figure 3l). The analysis
of CD7 positivity based on a FCS/SSC gating alone would
have led to the erroneous interpretation that CD7 positivity is
due to lymphocyte contamination. Moreover, this example
also illustrates the trivial error introduced by rigidly applying
an artificial value of 20% threshold positivity, since in this
case CD7 expression was present in low proportions of AML
blast cells.

Determination of surface antigens of blast cell

populations

We used a double labeling method with CD45-PE against

each CD-FITC antigen in the second part of the study where
53 consecutive samples were included in order to appreciate
the gain of information in blast cell immunophenotyping. The
positivity of CD-FITC antigen expression on blast cells was
evaluated using FSC/SSC and CD45/SSC gating. Since in the
first part of the analysis, CD45/SSC gating procedure showed
a better determination of blast cells, we have chosen this pro-
tocol as the reference procedure. If the positivity threshold for
each CD/FITC antigen expression was strictly fixed at 20%,
false positive (FP) samples would have been recorded in
samples which were positive (CD expression >20%) with the
FSC/SSC gating but negative (CD ,20%) using CD45/SSC gat-
ing. By contrast, false negative (FN) samples were also seen
negative using FSC/SSC gating but positive with CD45/SSC
gating. In Figure 4a all FP cases were found for lymphoid
(CD10, CD3, CD5, CD7) and monocytic (CD13, CD14,
CD11b) antigens. In every case the FP was due to lymphocytic
or monocytic contamination of samples by peripheral blood.
On the other hand, FN cases were found only for myeloid
(CD33, CD13, CD11b), DR and CD34 antigens. Among these
samples, most leukemias with myeloid and/or monocytic
maturation have been cases of AML2 (n = 9), AML4 or AML5
(n = 7).
Even when FP and FN cases were absent, in many leukem-
ias significant differences were noted in the degree of CD-FITC
antigen expression when the CD45/SSC and FSC/SSC gating
procedures were compared. Thus, for each CD-FITC antigen
expression, we accepted the value of antigen expression when
the difference using the two gating procedures was below an
arbitrary value of 15% (Figure 4b). The discrepancies with
.15% difference were mostly seen in the myeloid antigenic
determination of blast cells. Frequently the intensity of CD-
FITC antigen expression was lower using FSC/SSC gating than
CD45/SSC gating for CD13 (38.5%), CD33 (26.3%) and CD34
(23.3%). This was mostly due to the contamination of bone
marrow samples by peripheral blood cells which were present
within the FSC/SSC blast cell gate. On the other hand, in many
cases we also frequently found an increased expression of DR
(35%) and CD11b (40%) (Figure 4b) when studied with
Figure 4 Antigenic expression discrepancies between FSC/SSC
and CD45/SSC gating. CD45/SSC gating was considered as the refer-
ence procedure (see text). (a) Histograms of false positive/negative
results given by the FSC/SSC procedure. A threshold value of 20%
gave the antigen positivity. (b) For positive samples only (ie with
>20% positivity), histograms of CD expression discrepancies .15%
between FSC/SSC and CD45/SSC gating. Numbers of positive cases
are indicated for each CD.
CD45/SSC gating. These findings are illustrated in Figure 1c
where differences in blast cell CD33 positivity are seen
between CD45/SSC gating (60% positivity) and FSC/SSC gat-
ing (40% positivity). With CD45/SSC gating the counts of
negative cells decreased (the upper arrow on Figure 1c) and
the CD33 expression blasts were properly discriminated
(lower arrow).
Thus, CD45/SSC gating excludes peripheral blood cell con-
tamination from blast cell antigen analysis, especially in cases
of heterogeneous bone marrow sub-populations. Moreover, in
the present series, 27 samples (36%) contained fewer than
50% of blast cells and these would not have been properly
immunophenotyped without the help of CD45/SSC gating. No
correlation was found between morphological count and
FSC/SSC gating of the blast cells (r = 0.1), whereas an excellent
correlation remained when CD45/SSC gating was applied (r
= 0.92).
Utility of CD45/SSC gating in frozen samples
When Ficoll gradient preparations are needed for storage of
frozen samples for cytogenetics or molecular biology,
CD45/SSC gating remains a valuable and important step to
 Flow cytometry immunophenotyping of AML and CD45/SSC gating
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1884

Figure 5 Representation of flow cytometry histograms of the same case of AML2. (a and b) Bivariate histograms of the fresh sample with
gates similar to Figure 1. (c and d) Bivariate histograms of the frozen sample after Ficoll preparation. Corresponding cell percentages are indicated.

identify blast cells. An example of AML2 shown in Figure 5 of CD45low blast cells in the fresh sample and the correspond-
demonstrates the better separation of cell populations using ing frozen one are very similar, showing the reliability of the
the CD45/SSC gating when compared to FSC/SSC gating. It CD45/SSC gating approach. It can also be noted that, in
is clear that after freezing the conservation of CD45 antigen frozen samples, Multi-Q Prep can only be reliably applied
expression is excellent. Table 2 indicates that the percentages after washings, and thus its methodological advantage over
other methods is lost.
Table 2 Percentages of CD+ blast cells in the same case of AML2
(fresh and frozen samples) using the CD45/SSC gating
Discussion
CD expression Fresh sample Frozen sample
In this study, we demonstrate the usefulness of a simple and
reliable flow cytometry method to guide the classification of
CD34 78 75
CD117 77 74 AML. In AML, peripheral blood or bone marrow blast cells
CD7 77 76 are frequently admixed to other cell lineages and the blast
CD13 77 83 cells themselves are also heterogeneous. To avoid some of
CD33 37 30 these problems, Ficoll density gradient centrifugation is fre-
quently used to enrich samples in leukemic cells. Neverthe-

less, these steps have three major disadvantages: (1) a differen-
tial count of the leukemic sub-populations becomes unreliable
and diverges from the differentials seen on smears;
(2) valuable information on the granulocytic cells is lost; and
(3) the selective elimination of granulocytes from the sample
also prevents their use as internal controls for staining mature
myeloid cells. Thus, we prefer to proceed with whole bone
marrow samples and to preserve all the nucleated cells in the
sample by performing an erythrocyte lysis with the Multi-Q-
Prep method (Coulter). This technique, originally rec-
ommended for peripheral blood cell immunophenotyping, is
adaptable to bone marrow immunophenotyping. The results
are reproducible and time is saved since no further washings
or centrifugations are needed. Two limitations to the use of
these methods have, however, been noted: (1) immature
erythroid cells are damaged and cannot be accurately ana-
lyzed for glycophorin-A; and (2) debris and fat cells need to
be excluded by FSC/SSC pre-gating (gate A) in Figure 1a.
We confirm earlier reports suggesting that the blast cell
population can be clearly identified by virtue of their low anti-
gen membrane CD45 antigen expression and their light scatter
properties.6–9,12,13 Terstappen et al12,13 have already emphas-
ized that, when analyzed by their FSC and SSC light proper-
ties, blast cells cannot be properly distinguished from lympho-
cytes and monocytes. Other groups have recommended
CD45/SSC gating to provide a better definition of all the cell
sub-populations of the samples in normal bone marrow analy-
sis7–9 as well as in acute leukemia.6 CD45 was selected as the
basis for the gating procedure because it is found in different
amounts in immature and mature hematopoietic cells.7 In
combination with CD45, SSC makes it possible to discrimi-
nate all major lineages in separate populations in a single
bivariate analysis. Moreover, debris and mature erythrocytes
with low FSC signals are excluded from a CD451 gate.7,8 And
finally, the combination of CD45 expression and propidium
iodide staining has also been found to be a good technique
to evaluate peripheral blood contamination in the bone
marrow.14
In this work, we used PE-conjugated CD45 combined with
other FITC-conjugated antibodies in parallel samples to define
the exact phenotypic expression of AML blast cells. One of
the main findings of this paper is that this technique gives an
excellent correlation with the light microscopic bone marrow
differential counts in smears (r = 0.94 in Figure 2). Further-
more, there are similarly high correlations between the per-
centages of lymphocytes, granulocytes and monocytes and the
corresponding populations defined by the CD45/SSC gates (r
= 0.94, 0.90 and 0.95, respectively, in Figure 2). We then
used this technique to compare the antigenic expression of
the blast cells determined both on FSC/SSC gating and
CD45/SSC gating.
In the majority of leukemias with intraclonal heterogeneity
such as AML2, 4 and 5, the evaluation of blast cell popu-
lations would have been significantly interfered with by the
FSC/SSC primary gating because the positively gated popu-
lations would have included contaminating normal CD45high
lymphocytes and monocytes (Figure 3 e–l). Furthermore, as
has also been pointed out by Rothe et al5 and Borowitz et al,6
3-colour IF analysis using CD45 as the third colour is even
more discriminating than 2-color IF; the clone J-33 (labeled
with cyanine-5y-5) performs well in this respect (Figure 5). We
therefore conclude that the most informative reference histo-
gram for blast cell identification is the CD45/SSC that can be
optimally combined with two additional reagents in 3-colour
IF for precise leukemia diagnosis.
Flow cytometry immunophenotyping of AML and CD45/SSC gating
F Lacombe et al
1885
The introduction of CD45/SSC primary gating helps define
the prognostic values of other antigens without the danger of
counting normal populations within the leukemic gate.15
Consequently inter-laboratory variations are reduced and the
misdiagnosis of biphenotypic leukemias is avoided where this
result was due to the presence of normal T and B cells in the
leukemic gate. The increased sensitivity of the CD45/SSC gat-
ing inevitably leads to a suggestion that cut-off values (eg arbi-
trary 20%) lose their significance as aberrant blast cell popu-
lations can now be identified at lower detection limits (,5%).
In addition, the sensitivity of detecting minimal residual dis-
ease is improved. In this context is is essential to emphasize
that normal precursor cells are also CD45low, and therefore
CD45/SSC per se does not discriminate between normal and
malignant cells.16,17 On the other hand, the CD45/SSC gate
focuses attention on the strict normal vs malignant cell com-
parison utilizing the other two channels of antibody labe-
ling.18 These reagents may include CD7 and CD19 in ident-
ifying aberrantly expressing AML blasts while documenting
that normal precursors are CD7−, CD19−. In these gated popu-
lations the features of normal precursor cells guide, as internal
controls, the quantitative characterization of aberrant marker
expression on the residual leukemic blasts.
This technique applied to AML in our series can be used in
other acute leukemias such as acute lymphoblastic leukemia.6
The real ease of defining blast cells with CD45/SSC also led
us to test this gating procedure in myelodysplasia, which often
presents low percentages of blast cells. Preliminary results are
showing the reliability of this approach (data not shown).
We are finding therefore that CD45/SSC gating is the most
reliable and easy way to perform acute leukemia immuno-
phenotyping, especially for samples with low proportions of
blast cells. This question could well become a point for debate
in consensus conferences. We suggest that CD45/SSC gating
should replace FSC/SSC gating, and that the cost of tests could
be decreased as the systematic use of this method is a practi-
cal way to decrease the number of monoclonal antibodies
necessary to diagnose a leukemia, since the blast cell popu-
lation is definitively isolated in most cases. This could contrib-
ute to decreasing costs without affecting diagnostic quality.
In conclusion, a clear discrimination of blast cells improves
the measurement of their antigenic expression and their separ-
ation from the accompanying hematopoietic cells: CD45/SSC
gating seems to be the method of choice. A full understanding
of the leukemic process calls upon the global immunological
appreciation of all the cells present in the leukemic sample
by using a lysed whole bone marrow analysis. Such was the
case when the FAB classification was set up.10 In the future,
the method described here might become a useful technique
for the immunophenotyping of acute leukemia at diagnosis
and in treatment monitoring.
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