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CHAPTER 28

Flow Cytometry
Immunophenotypic Characteristics
of Monocytic Population in
Acute Monocytic Leukemia
(AML-M5), Acute Myelomonocytic
Leukemia (AML-M4), and
Chronic Myelomonocytic
Leukemia (CMML)
Wojciech Gorczyca
Director of Hematopathology/Oncology Services
Genzyme Genetics/IMPATH
New York, New York 10019
I. Introduction
II. Materials
III. Methods
A. Flow Cytometry Analysis
IV. Acute Monocytic Leukemia
V. Chronic Myelomonocytic Leukemia
VI. Acute Myelomonocytic Leukemia
VII. DiVerential Diagnosis
VIII. Conclusion
References
METHODS IN CELL BIOLOGY, VOL. 75
Copyright 2004, Elsevier Inc. All rights reserved. 665
0091-679X/04 $35.00
I. Introduction
Monocytic proliferations comprise a heterogeneous group of disorders ranging
from reactive monocytosis to acute monocytic leukemia. Based on the cytomor-
phological and phenotypic features, diVerential diagnosis includes acute promye-
locytic leukemia (especially microgranular variant), acute myeloid leukemia
(AML) without maturation, minimally diVerentiated AML, chronic myelomono-
cytic leukemia (CMML), acute megakaryocytic leukemia, and acute myelo-
monocytic leukemia (AML-M4). Extramedullary myeloid tumors with monocytic
diVerentiation (monoblastic sarcoma) may be mistaken for large cell lymphoma,
carcinoma, or sarcoma. Well-prepared fresh bone marrow aspirate with myelo-
peroxidase staining is helpful in diVerential diagnosis. Myeloblasts and abnormal
promyelocytes are strongly MPO positive, whereas the monocytes are either
weakly positive or negative. The monoblasts and promonocytes usually are posi-
tive with nonspecic esterase (NSE) staining, but a signicant subset of acute
monocytic leukemias is NSE negative. Therefore, the denite diagnosis often
requires correlation of complete blood cell (CBC) count data, cytological features,
and cytochemistry with additional techniques such as immunophenotyping by
ow cytometry (FC), cytogenetics/uorescence in situ hybridization (FISH), and
molecular tests (e.g., polymerase chain reaction [PCR]). FC immunophenotyping
is an accurate method for quantitative and qualitative evaluation of hematopoietic
cells. It plays important role in diagnosis, classication, and monitoring of
hematopoietic neoplasms, including acute leukemias (Baumgarth and Roederer,
2000; Borowitz et al., 1997; Gorczyca et al., 2002; Jennings and Foon, 1997a,b;
Knapp et al., 1994; Kotylo et al., 2000; Kussick and Wood, 2003; Manaloor et al.,
2000; Orfao et al., 1999a, 1999b; Weir and Borowitz, 2001; Weisberger et al.,
2000). This chapter presents the phenotypic characteristic of monocytic popula-
tions from acute monocytic leukemia (AML-M5), CMML and AML-M4.
II. Materials
Flow cytometric samples from IMPATH, Incorporated (New York division),
containing abnormal monocytic populations were submitted for this study. FC
data were reanalyzed and correlated with cytomorphology and/or bone marrow
studies. All cases without rm morphological conrmation were excluded. The
neoplasms were classied according to the World Health Organization (WHO)
classication of hematopoietic neoplasms (Harris et al., 2000a,b). There were 28
cases of AML-M5, 20 cases of CMML, and 15 cases of AML-M4.
666 Wojciech Gorczyca
III. Methods
A. Flow Cytometry Analysis
We used heparinized bone marrow aspirate, peripheral blood, and fresh
tissue specimens for FC analysis and processed the specimens within 24 hours of
collection. We obtained a leukocyte cell suspension from peripheral blood and
bone marrow specimens after red blood cell (RBC) lysis with ammonium chloride
lysing solution, followed by 5 minutes of centrifugation. The cell pellet was
suspended with an appropriate amount of RPMI 1640 (GIBCO, New York).
Fresh tissue samples were disaggregated with a sterile blade, followed by passage
through a mesh lter (<100 m). The cells were washed in RPMI media and
centrifuged at 1500 rpm for 5 minutes. To minimize nonspecic binding of
antibodies, we incubated the cells in RPMI media supplemented with 10%
heat-inactivated fetal bovine serum (FBS) in a 37

C water bath for 30 minutes.


We then washed the samples with 0.1% sodium azide/10% FBS (phosphate-
buVered saline [PBS] buVer) and assessed viability using both trypan blue and
7-aminoactinomycin D (Sigma Chemical Co., St. Louis, Missouri) exclusion
assays.
Immunophenotypic analysis was performed on FACSCalibur System Instru-
ments equipped with a 15-mW 488-nm air-cooled argon-ion laser supplemented
with a 635-nm red-diode laser (Becton Dickinson Immunocytometry System, San
Jose, California). Three- and four-color directly labeled antibody combinations
consisting of uorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-
chlorophyl (PerCP), and allophycocyanin (APC) were used for surface staining of
fresh tissue, bone marrow, and peripheral blood cell suspensions. Internal negative
controls within each tube and isotype controls for immunoglobulin G1 (IgG1),
IgG2a, and IgG2b were used as negative controls.
We monitored the instrument uorescence detectors settings and calibration
according to manufacturers recommendations, using Calibrite Beads (Becton
Dickinson) and evaluated the system linearity using Sphero Rainbow Beads
(Pharmingen). FC data was collected in list mode and analyzed using CellQuest
and Cell Quest Pro software (Becton Dickinson). A six-gate strategy was em-
ployed, using CD45 PerCP versus side scatter to characterize the lymphocyte,
monocyte, granulocyte, blast, hematogone, and nucleated red cell precursor
(erythroid) populations. Five- to six-parameter analysis (forward-scatter channel
[FSC], side-scatter channel [SSC], FL1, FL2, FL3, and FL4) or multiparameter
data analysis of antibody staining patterns was used to assess specic antigen
expression.
28. Flow Cytometry of Neoplastic Monocytes 667
IV. Acute Monocytic Leukemia
Acute monocytic (monoblastic) leukemia (AML-M5) is dened as myeloid leu-
kemia in which 80% or more of the leukemic cells are of monocytic lineage
(monoblasts, promonocytes, and monocytes). In this series (28 cases), patients
ranged in age from 17 to 85 years. Figure 1 presents typical cytologic and
phenotypic features of AML-M5. Leukemic cells have abundant cytoplasm that
may show irregular borders with pseudopod and cytoplasmic vacuoles. NSE is
positive in most cases, although it may be weak. All AML-M5 are positive for
pan-myeloid markers, CD33 (bright expression), CD11c (moderate or bright), and
CD45 (moderate or bright). Most cases are positive for human leukocyte antigen-
DR (HLA-DR) (96%), CD4 (93%), CD11b (75%), CD13 (78%, usually dim
expression), CD56 (86%), and CD64 (89%). A subset of cases show positive
expression of CD2, CD7, CD10, CD23, and CD117. Table I presents details of
FC immunophenotypic ndings.
V. Chronic Myelomonocytic Leukemia
Chronic myelomonocytic leukemia (CMML) is mixed myelodysplastic/myelo-
proliferative disorder dened by persistent monocytosis ( >1 by 10
9
/L) in periph-
eral blood, fewer than 20% blasts, and dysplastic features in one or more myeloid
lineages. Molecular/cytogenetic study results are negative for bcr-abl fusion gene
(Philadelphia chromosome). The monocytes are usually mature with focal nuclear
or cytoplasmic atypia. Based on the number of blasts, CMML is divided into two
categories: CMML-1 ( <5% blasts in blood, <10% blasts in bone marrow) and
CMML-2 (519 blasts in blood and 1019% blasts in bone marrow). Figure 2
presents FC analysis of CMML-2. The neoplastic monocytes have the phenotype
resembling normal monocytes; they are always positive for CD11b (bright expres-
sion), CD11c (bright expression), CD14 (bright expression), CD33 (bright expres-
sion), CD45 (bright expression), and CD64 (bright expression). Most cases express
CD13 (95%), HLA-DR (71%), and CD4 (76%). CD56 is present in 53% of cases.
Lack of HLA-DR and CD13 and presence of aberrant expression of CD10,
CD16, CD23, CD56, and CD117 distinguished CMML from reactive monocyto-
sis. Table I presents phenotypic data of all analyzed cases.
VI. Acute Myelomonocytic Leukemia
Acute myelomonocytic leukemia (AML-M5) is an acute leukemia characterized
by the proliferation of both neutrophil and monocyte precursors with 20% or
more myeloblasts in the bone marrow. Both monocytic and granulocytic lineages
must comprise at least 20% of marrow cells. FC analysis from AML-M4 cases
shows distinct populations of blasts, monocytes, and residual (maturing) myeloid
668 Wojciech Gorczyca
Fig. 1 Acute monocytic leukemia (AML-M5). (A) Bone marrow aspirate shows monoblasts with
irregular nuclei. (B) Nonspecic esterase is strongly positive. (CL) Flow cytometry immunophenotyp-
ing. (C) Monoblasts are brightly positive for CD45 and have increased side scatter (blue dots). They are
negative for CD34 (D) and CD117 (E) and positive for HLA-DR (F), CD33 (H), CD56 (I), CD14 (J),
and CD11c (L). CD13 (G) and CD64 (K) are partially expressed.
28. Flow Cytometry of Neoplastic Monocytes 669
cells (Fig. 3). Monocytic cells in AML-M4 are always positive for CD4, CD11b,
CD11c, CD13, CD14, CD33, CD45, CD64, and HLA-DR. A subset of leukemias
show expression of CD2, CD7, CD34, and CD56 (Table I).
VII. DiVerential Diagnosis
Figure 4 presents the expression of CD45 versus side scatter in diVerent types
of leukemia. Myeloblasts in AMLs (M0M2) display moderate expression of
CD45 and low side scatter (Fig. 4B). Blasts usually predominate and there are
no or few other elements like granulocytes, monocytes, and lymphocytes (compare
with normal marrow, Fig. 4A). Monoblasts in AML-M5 are characterized
by bright CD45 and increased side scatter (blue dots, Fig. 4E). Note rare myelo-
blasts (green dots) and paucity of other bone marrow elements (especially granu-
locytes). CMML shows predominance of monocytes (Fig. 4F), with granulocytes
displaying decreased side scatter (dysgranulopoiesis). Blasts may be present but
Table I
Comparison of Flow Cytometric Features of Monocytic Cells in Acute Monocytic
Leukemia, Chronic Myelomonocytic Leukemia, and Acute Myelomonocytic Leukemia
Marker
Acute monocytic
leukemia (AML-M5)
(% positive)
Chronic
myelomonocytic
leukemia (% positive)
Monocytic population
in acute myelomonocytic
leukemia (AML-M4)
(% positive)
CD2 14 34 40
CD4 93 76 100
CD7 21 9 40
CD10 7 28 0
CD11b 75 100 100
CD11c 100 100 100
CD13 78 95 100
CD14 18 (additional 36%
shows positive
expression in
small subset)
100 100
CD16 4 29 0
CD23 32 9 0
CD33 100 100 100
CD34 14 0 26
CD45 100 (moderate/bright) 100 (bright) 100 (bright)
CD56 86 53 26
CD64 89 100 100
CD117 25 5 0
HLA-DR 96 71 100
670 Wojciech Gorczyca
Fig. 2 Chronic myelomonocytic leukemia (CMML) ow cytometry. Atypical monocytes (blue dots) are positive for CD45 (A), CD14 and CD64 (B),
CD56 (C), CD13 (D), CD33 (E), CD4 (F), HLA-DR (G), CD11b (H), and CD11c (I). Granulocytes (gray dots) and lymphocytes (red dots) are also
present. Blasts (green dots) do not exceed 20% by ow cytometry and/or enumeration on fresh bone marrow aspirate.
Fig. 3 Acute myelomonocytic leukemia (AML-M4) ow cytometry. Two distinct populations are
noted: blasts (A, green dots) and monocytic cells (A, blue dots). Granulocytes (gray dots) and
lymphocytes (red dots) are also present. Myeloblasts are positive for CD117 (B), CD33 (C), and CD34
(D), whereas monocytic cells are positive for CD33 (C), CD34 (D), and CD64 and CD14 (E).
672 Wojciech Gorczyca
C
E
AML-M2
A
D
F
CD45
CD45/2D1/IgG1/PerCP
0205753 GL01.001
R2
R4
R2
R3 R1
R1
R4
R2
R5
R6
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R2
R3
R5
R6
R1
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R5
R3
R5 R5 R5
R6
R1
R4 R4
R3
R2
R1
R4
R3
R1
R2
R5
R6
R6
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0
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CD45
blasts
granulocytes with
decreased side
scatter
Chronic myelomonocytic leukemia Acute monocytic leukemia (AML-M5) AML, M4
S
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r
-
H
e
i
g
h
t
granulocytes
lymphocytes
CD45 CD45 CD45
monocytes
blasts with
low side
scatter
Blastic NK-cell leukemia Benign bone marrow
monocytic
cells
blasts
monoblasts
B
blasts
monocytes
Fig. 4 Comparison of CD45 versus side scatter in acute leukemias. (A) Normal (benign) bone marrow. Note predominance of granulocytes (gray
dots) and lymphocytes (red dots). Rare monocytes (blue dots) are also present. (B) Acute myeloid leukemia with diVerentiation (AML-M2). Myeloblasts
(green dots) predominate. They have low side scatter and moderate CD45 expression. (C) Blastic natural killer cell leukemia/lymphoma. Blasts have low
side scatter and moderate CD45. (D) Acute myelomonocytic leukemia (AML-M4). Two populations are present: myeloblasts (green dots) and
monocytic cells (blue dots). Granulocytes are present (gray dots). (E) Acute monocytic leukemia (AML-M5). Monoblasts with increased side scatter and
bright CD45 expression predominate (blue dots). Only rare myeloblasts (green dots) and very few granulocytes (gray dots) are present. (F) Chronic
myelomonocytic leukemia (CMML). There is predominance of monocytic cells (blue dots), but lymphocytes, myeloblasts, and granulocytes are also
present. Note decreased side scatter of granulocytes.
are less than 20%. AML-M4 (Fig. 4D) shows two distinct populations of
blasts (green dots) and monocytes (blue dots). The proportion of myeloblasts,
monoblasts/monocytes, granulocytes, and lymphocytes in AML-M4, CMML,
and AML-M4 is presented in Fig. 5. Neoplastic monocytes diVer from benign
monocytes by expression of CD16 (most often in CMML), CD23 (most often in
AML-M5), CD34 (AML-M4.AML-M5), CD56 (AML-M5>CMML>AML-
M4), and CD117 (AML-M5) and lack of HLA-DR (most often seen in CMML),
CD64 (AML-M5), CD11b (AML-M5), CD14 (AML-M5), and CD64 (AML-M5)
(Table I).
Table II summarizes ow cytometric phenotypic characteristics of diVerent
types of acute leukemias, which fall into diVerential diagnosis with AML-M5.
Positive expression of HLA-DR diVerentiates AML-M5 from acute promyelocy-
tic leukemia (APL, AML-M3). Positive expression of CD56 is seen most com-
monly in AML-M5 and blastic natural killer cell lymphoma/leukemia. Expression
of CD11b and CD11c is most typical for AML-M5 (CD11c is often seen also in
AML-M0, AML-M1, and AML-M2). CD64 is slightly more brightly expressed in
AML-M5 than in megakaryocytic or promyelocytic leukemias. CD23 can be
expressed on a subset of AML-M5 and microgranular variants of APL. CD34
expression is negative in hypergranular APL and megakaryocytic leukemia. CD13
is never expressed in blastic natural killer cell lymphoma/leukemia, shows moder-
ate expression in AML-M0 through AML-M2, and dim expression in AML-M5,
APL, AML-M6, and AML-M7.
Fig. 5 Comparison of proportion of myeloblasts, monocytic cells, granulocytes, and lymphocytes in
acute monocytic leukemia (AML-M5), chronic myelomonocytic leukemia, and acute myelomonocytic
leukemia (AML-M4).
674 Wojciech Gorczyca
Table II
DiVerential Diagnosis of Acute Leukemias Based on Flow Cytometric Phenotypic Features
Marker
Acute monocytic
leukemia
(AML-M5)
Acute myeloid
leukemia
(AML-M2)
Acute promyelocytic
leukemia
(hypergranular)
Acute
megakaryocytic
leukemia
Blastic natural
killer cell
lymphoma/leukemia
Acute promyelocytic
leukemia
(microgranular)
CD2 / /
CD4 (rare ) / / /
CD7 / /rare / /very rare
CD10 /
CD11b /
CD11c (bright) (dim/moderate)
CD13 dim (moderate) (dim/moderate) (dim) (dim)
CD14 subset cells
CD16 rare
CD23 1/3 cases /
CD33 100% (bright) (variable) (moderate/bright) (bright) /very rare (bright)
CD34 / (occasionally only
in subset)
CD41/61
CD45 100% (moderate/bright) (dim/moderate) (moderate) (dim/moderate) (moderate)
CD56 (86%) /rare / /rare 100% /rare
CD64 (dim/moderate) / (dim)
CD117 /rare (25%) (dim) /rare
TdT / /
HLA-DR (96% cases) /rare (dim) (dim)
VIII. Conclusion
FC, particularly CD45 versus side scatter and expression of CD2, CD7, CD10,
CD11b, CD11c, CD14, CD16, CD23, CD33, CD34, CD45, CD56, CD64, CD117,
and HLA-DR, can identify and diVerentiate abnormal monocytes and com-
plements cytomorphology and cytochemical staining in diagnosis of myeloid
proliferations with monocytic diVerentiation.
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