Page 1
CHAPTER 28
METHODS IN CELL BIOCopyright 2004, Elsevier In0091-679X/04 $35.00
Flow CytometryImmunophenotypic Characteristicsof Monocytic Population inAcute Monocytic Leukemia(AML-M5), Acute MyelomonocyticLeukemia (AML-M4), andChronic MyelomonocyticLeukemia (CMML)
Wojciech GorczycaDirector of Hematopathology/Oncology ServicesGenzyme Genetics/IMPATHNew York, New York 10019
I.
LOGc. All
I
Yrig
ntroduction
II. M aterials
III.
M ethods
, VOLhts re
A. F
. 75serve
low Cytometry Analysis
IV. A cute Monocytic Leukemia V. C hronic Myelomonocytic Leukemia
VI.
A cute Myelomonocytic Leukemia VII. D iVerential Diagnosis
VIII.
C onclusion R eferen ce s
d. 665
Page 2
666 Wojciech Gorczyca
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 nonspecific esterase (NSE) staining, but a significant subset of acute
monocytic leukemias is NSE negative. Therefore, the definite diagnosis often
requires correlation of complete blood cell (CBC) count data, cytological features,
and cytochemistry with additional techniques such as immunophenotyping by
flow cytometry (FC), cytogenetics/fluorescence 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, classification, 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 firm morphological confirmation were excluded. The
neoplasms were classified according to the World Health Organization (WHO)
classification 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.
Page 3
28. Flow Cytometry of Neoplastic Monocytes 667
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 filter (<100�m). The cells were washed in RPMI media and
centrifuged at 1500 rpm for 5 minutes. To minimize nonspecific 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 fluorescein 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 fluorescence detectors’ settings and calibration
according to manufacturer’s 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 specific antigen
expression.
Page 4
668 Wojciech Gorczyca
IV. Acute Monocytic Leukemia
Acute monocytic (monoblastic) leukemia (AML-M5) is defined 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 findings.
V. Chronic Myelomonocytic Leukemia
Chronic myelomonocytic leukemia (CMML) is mixed myelodysplastic/myelo-
proliferative disorder defined by persistent monocytosis ( >1 by 109/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 (5–19 blasts in blood and 10–19% 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
Page 5
Fig. 1 Acute monocytic leukemia (AML-M5). (A) Bone marrow aspirate shows monoblasts with
irregular nuclei. (B) Nonspecific esterase is strongly positive. (C–L) 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
Page 6
Table IComparison of Flow Cytometric Features of Monocytic Cells in Acute MonocyticLeukemia, 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
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 (M0–M2) 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
Page 7
Fig. 2 Chronic myelomonocytic leukemia (CMML) flow 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 flow cytometry and/or enumeration on fresh bone marrow aspirate.
Page 8
Fig. 3 Acute myelomonocytic leukemia (AML-M4) flow 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
Page 9
C
E
AML-M2
A
DF
CD45CD45/2D1/IgG1/PerCP
0205753 GL01.001
R2
R4R2
R3 R1 R1R4
R2
R5
R6
R4 R2
R3
R5
R6
R1R3
R5
R3
R5 R5 R5
R6
R1R4R4
R3
R2
R1R4
R3 R1
R2
R5
R6
R6
100
020
040
060
080
010
00
101 102 103 104
100
020
040
060
080
010
00
101 102 103 104 100
020
040
060
080
010
00
101 102 103 104 100
020
040
060
080
010
00
101 102 103 104
CD45
blasts
granulocytes withdecreased side scatter
Chronic myelomonocytic leukemiaAcute monocytic leukemia (AML-M5)AML, M4
Sid
e sc
atte
r-H
eigh
t
CD45
100
020
040
060
080
010
00
101 102 103 104 100
020
040
060
080
010
00
101 102 103 104
Sid
e sc
atte
r-H
eigh
t granulocytes
lymphocytes
CD45 CD45 CD45
monocytes
blasts withlow side scatter
Blastic NK-cell leukemiaBenign bone marrow
monocyticcells
blasts
monoblasts
B
blastsmonocytes
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.
Page 10
674 Wojciech Gorczyca
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 flow 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).
Page 11
Table IIDiVerential 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 �/þ � �/þ � � þCD4 þ (rare �) þ/� �/þ � þ þ/�CD7 �/þ �/rare þ �/þ � þ/very rare � �CD10 �/þ � � � � �CD11b þ/� � � � � �CD11c þ (bright) þ (dim/moderate) � � � �CD13 dim þ (moderate) þ (dim/moderate) þ (dim) � þ (dim)
CD14 subset cells þ � � � � �CD16 rare þ � � � � �CD23 �1/3 cases þ � � � � �/þCD33 100% þ (bright) þ (variable) þ (moderate/bright) þ (bright) �/very rare þ þ (bright)
CD34 � þ � � þ/� þ (occasionally only
in subset)
CD41/61 � � � þ � �CD45 100% þ (moderate/bright) þ (dim/moderate) þ (moderate) þ þ (dim/moderate) þ (moderate)
CD56 þ (86%) �/rare þ �/þ �/rare þ 100% þ �/rare þCD64 þ (dim/moderate) þ/� þ (dim) � �CD117 �/rare þ (25%) þ þ þ (dim) �/rare þTdT � þ/� � � �/þ �HLA-DR þ (96% cases) þ/rare � � þ (dim) þ (dim) �
Page 12
676 Wojciech Gorczyca
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.
References
Baumgarth, N., and Roederer, M. (2000). A practical approach to multicolor flow cytometry for
immunophenotyping. J. Immunol. Methods 243, 77–97.
Borowitz, M. J., Bray, R., Gascoyne, R., Melnick, S., Parker, J. W., Picker, L., and Stetler-Stevenson,
M. (1997). U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of
hematologic neoplasia by flow cytometry: Data analysis and interpretation. Cytometry 30, 236–244.
Gorczyca, W., Weisberger, J., Liu, Z., Tsang, P., Hossein, M., Wu, C. D., Dong, H., Wong, J. Y.,
Tugulea, S., Dee, S., Melamed, M. R., and Darzynkiewicz, Z. (2002). An approach to diagnosis of
T-cell lymphoproliferative disorders by flow cytometry. Cytometry 50, 177–190.
Harris, N. L., JaVe, E. S., Diebold, J., Flandrin, G., Muller-Hermelink, H. K., Vardiman, J., Lister,
T. A., and Bloomfield, C. D. (2000a). The World Health Organization classification of neoplasms of
the hematopoietic and lymphoid tissues: Report of the Clinical Advisory Committee meeting–Airlie
House, Virginia, November, 1997. Hematol. J. 1, 53–66.
Harris, N. L., JaVe, E. S., Diebold, J., Flandrin, G., Muller-Hermelink, H. K., Vardiman, J., Lister,
T. A., and Bloomfield, C. D. (2000b). The World Health Organization classification of neoplastic
diseases of the haematopoietic and lymphoid tissues: Report of the Clinical Advisory Committee
Meeting, Airlie House, Virginia, November 1997. Histopathology 36, 69–86.
Jennings, C. D., and Foon, K. A. (1997a). Flow cytometry: Recent advances in diagnosis and
monitoring of leukemia. Cancer Invest. 15, 384–399.
Jennings, C. D., and Foon, K. A. (1997b). Recent advances in flow cytometry: Application to the
diagnosis of hematologic malignancy. Blood 90, 2863–2892.
Knapp, W., Strobl, H., and Majdic, O. (1994). Flow cytometric analysis of cell-surface and
intracellular antigens in leukemia diagnosis. Cytometry 18, 187–198.
Kotylo, P. K., Seo, I. S., Smith, F. O., Heerema, N. A., Fineberg, N. S., Miller, K., Greene, M. E.,
Chou, P., and Orazi, A. (2000). Flow cytometric immunophenotypic characterization of pediatric
and adult minimally diVerentiated acute myeloid leukemia (AML-M0). Am. J. Clin. Pathol. 113,
193–200.
Kussick, S. J., and Wood, B. L. (2003). Using 4-color flow cytometry to identify abnormal myeloid
populations. Arch. Pathol. Lab. Med. 127, 1140–1147.
Manaloor, E. J., Neiman, R. S., Heilman, D. K., Albitar, M., Casey, T., Vattuone, T., Kotylo, P., and
Orazi, A. (2000). Immunohistochemistry can be used to subtype acute myeloid leukemia in routinely
processed bone marrow biopsy specimens. Comparison with flow cytometry. Am. J. Clin. Pathol.
113, 814–822.
Orfao, A., Chillon, M. C., Bortoluci, A. M., Lopez-Berges, M. C., Garcia-Sanz, R., Gonzalez, M.,
Tabernero, M. D., Garcia-Marcos, M. A., Rasillo, A. I., Hernandez-Rivas, J., and San Miguel, J. F.
(1999a). The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic
leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements.
Haematologica 84, 405–412.
Orfao, A., Schmitz, G., Brando, B., Ruiz-Arguelles, A., Basso, G., Braylan, R., Rothe, G., Lacombe,
F., Lanza, F., Papa, S., Lucio, P., and San Miguel, J. F. (1999b). Clinically useful information
Page 13
28. Flow Cytometry of Neoplastic Monocytes 677
provided by the flow cytometric immunophenotyping of hematological malignancies: Current status
and future directions. Clin. Chem. 45, 1708–1717.
Weir, E. G., and Borowitz, M. J. (2001). Flow cytometry in the diagnosis of acute leukemia. Semin.
Hematol. 38, 124–138.
Weisberger, J., Wu, C. D., Liu, Z., Wong, J. Y., Melamed, M. R., Darzynkiewicz, Z., and Gorczyca,
W. (2000). DiVerential diagnosis of malignant lymphomas and related disorders by specific pattern
of expression of immunophenotypic markers revealed by multiparameter flow cytometry [Review].
Int. J. Oncol. 17, 1165–1177.
