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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