Immunophenotypic Features of Granulocytes, Monocytes, and Blasts in Myelodysplastic Syndromes

Hee Won Moon; Jung Won Huh; Miae Lee; Ki Sook Hong; Wha Soon Chung

doi:10.3343/kjlm.2010.30.2.97

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Moon, Huh, Lee, Hong, and Chung: Immunophenotypic Features of Granulocytes, Monocytes, and Blasts in Myelodysplastic Syndromes

Original Article

Korean J Leg Med 2010;30(2):97-104.

Published online: 14 January 2010

DOI: https://doi.org/10.3343/kjlm.2010.30.2.97

Immunophenotypic Features of Granulocytes, Monocytes, and Blasts in Myelodysplastic Syndromes

Hee Won Moon, M.D.¹, Jung Won Huh, M.D.², Miae Lee, M.D.², Ki Sook Hong, M.D.², Wha Soon Chung, M.D.²

¹Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, Korea

²Department of Laboratory Medicine, Ewha Womans University School of Medicine, Seoul, Korea

Corresponding author: Wha Soon Chung, M.D. Department of Laboratory Medicine, Ewha Womans University School of Medicine, 911-1 Mok-dong, Yangcheon-gu, Seoul 158-056, Korea Tel: +82-2-2650-5043 Fax: +82-2-2650-5091 E-mail: wschung@ewha.ac.kr

Revised 17 July 200910 February 2010 Accepted 12 February 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

Despite the diagnostic utility of immunophenotyping for myelodysplastic syndromes (MDS), it has not been widely performed, and reports on this are absent in Korea. We aimed to evaluate the immunophenotypic features of non-blastic granulocytes, monocytes, and blasts in patients with MDS and non-clonal disorders using routine flow cytometry (FCM). Moreover, we evaluated the phenotypic abnormalities of mature cells in leukemic patients.

Methods:

Marrow aspirates from 60 patients, including 18 with MDS, 18 with leukemia, and 24 with non-clonal disorders (control group), were analyzed using FCM. Blasts, non-blast myeloid cells, and monocytes were gated based on CD45 expression and side scatter (SSC). The phenotypes were then compared among the 3 groups.

Results:

Compared to non-clonal disorders, the granulocytic lineages of MDS showed decreased SSC (P=0.005), increased CD45 intensity (P=0.020), decreased CD10-positive granulocytes (P= 0.030), and a higher CD56-positive rate (P=0.005). It is noteworthy that similar results were obtained in the leukemia group, and these findings were not related to the phenotypes of the leukemic cells. Using blast and monocytic gating, useful parameters for generating a differential diagnosis were not found.

Conclusions:

Gating the granulocytic region is a relatively easy method for MDS immunophenotyping. Among the parameters studied, SSC, CD10, and CD56 were the most useful for differentiating MDS from non-clonal disorders. While immunophenotypic changes in MDS appear to be useful for differentiating MDS from non-clonal disorders, these changes were also noted in the mature cells of leukemic patients.

Keywords: Immunophenotyping, MDS, Granulocyte, Monocyte, Blast

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Fig. 1.

Flow cytometric gating of bone marrow cells: Gating of sub-populations were based on CD45 intensities and side scatter (S-SC). Blasts (A, CD45^intermediateSSC^low), granulocytes (B, CD45^intermediate SSC^high), and monocytes (C, CD45^highSSC^{low-intermediate}).

Fig. 2.

The distributions of granulocyte SSC in non-clonal disorders (A), MDS (B), and leukemia (C). Compared to non-clonal disorders, granulocyte SSC was significantly lower in the MDS and leukemia groups (P=0.005 and 0.000, respectively). Values are mean±2 SD of the SSC in each group.

Abbreviation: SSC, side scatter.

Fig. 3.

Examples of immunophenotypes of granulocytes in MDS (A) and non-clonal disorders (B). In a patient with MDS, decreased SSC, absence of the CD10-positive population, and CD56 expression were noted (A) whereas CD10-positive cells were present, and CD56 was not expressed in patients with non-clonal disorders (B).

Abbreviation: SSC, side scatter.

Table 1.

Characteristics of non-clonal disorders, MDS, and leukemia

	Non-clonal disorders (N=24)	MDS (N=18)	Leukemia (N=18)
Age (yr)	37 (3-85)	69 (3-80)∗	39 (1-76)
Sex (M:F)	12:12	8:10	9:9
Peripheral blood count
WBC (×10⁹/L)	5.3 (2.7-14.6)	3.7 (0.5-58.9)	17.1 (0.6-170.8)∗¹
Hb (g/dL)	11.9 (7.3-15.7)	8.9 (5.9-12.5)∗	9.2 (4.8-16.0)∗
PLT (×10⁹/L)	162 (6-505)	67 (4-741)	53 (8-192)∗
Bone marrow
Blast (%)	1.6 (0.8-4.4)	1.5 (0.2-14.8)	60 (14-92.8)∗^†
Granulocyte	47.2 (21.8-72.4)	41.5 (7.2-66.4)	13.2 (0.2-52.0)∗^†
lineage (%)
Monocyte	1.5 (0.4-14.3)	1.6 (0.4-33.0)	1.6 (0.0-10.8)
lineage (%)
Cytogenetics
Normal (%)	22/22 (100)	11/16 (69)∗	11/18 (61)∗
Abnormal (%)	0	5/16 (31)	7/18 (39)

Data are median (range) except sex (male:female) and cytogenetics (case number/total cases with available data). ∗P<0.05 when compared to non-clonal disorders;

^† P<0.05 when compared to MDS.

Abbreviations: M, male; F, female; WBC, white blood cell; PLT, platelet.

Table 2.

Combinations of monoclonal antibodies

FITC	PE	PC5
CD10	CD19	CD45
CD20	CD5	CD45
CD3	CD22	CD45
CD7	CD33	CD45
HLA-DR	CD13	CD45
CD14	CD34	CD45
	CD56	CD45

Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PC5, phycoerythrin-cyanin 5.

Table 3.

Immunophenotypic features of non-blast, myeloid cells

	Non-clonal disorders (N=24)	MDS (N=18)	AML (N=9)	All leukemia (N=18)
% of gated cells	51.8 (12.8-66.9)	42.0 (6.0-70.3)∗	12.2 (2.4-50.8)∗	12.2 (2.4-50.8)∗
SSC, MFI	627 (491-707)	543 (346-780)∗	408 (323-658)∗^†	504 (323-772)∗
CD45, MFI	12.8 (6.0-25.2)	16.8 (9.1-43.0)∗	17.1 (0.9-25.2)	13.8 (0.9-25.2)
CD33
Positive rate (%)	22/24 (91.6)	16/18 (88.9)	8/9 (88.8)	13/18 (72.2)
% of positive cells	58.7 (11.8-92.4)	63.0 (10.6-93.6)	76.3 (4.7-93.7)	58.8 (4.3-93.7)
MFI	1.2 (0.7-2.2)	1.4 (0.7-6.0)	2.0 (1.4-5.3)∗	1.0 (0.6-5.2)∗
CD13
Positive rate (%)	24/24 (100.0)	18/18 (100.0)	9/9 (100.0)	18/18 (100.0)
% of positive cells	72.6 (28.5-90.2)	75.7 (41.4-92.4)	84.4 (64.8-94.8)∗	82.9 (58.7-99.6)∗
MFI	2.0 (1.5-4.3)	1.9 (1.2-7.7)	3.4 (1.3-5.5)∗	2.8 (1.2-6.1)∗
CD10
Positive rate (%)^‡	8/24 (33.3)	1/18 (5.5)∗	0/9 (0.0)∗	1/18 (5.6)∗
% of positive cells	13.2 (8.7-50.1)	-	-	81.5
MFI	1.4 (1.2-2.0)	-	-	2.12
CD56, positive rate (%)	1/24 (4.1)	7/18 (38.9)∗	4/9 (44.4)∗	6/18 (33.3)∗
CD14, positive rate (%)	0/24 (0.0)	2/18 (11.1)	1/9 (11.1)	1/18 (5.6)

Data are median (range), except for positive rate (positive case/total cases with available data).

Statistical significance analysis was performed for comparison of non-clonal disorders vs other groups and MDS vs AML or All leukemia.

^∗ P<0.05 compared to non-clonal disorders;

^† P<0.05 compared to MDS;

^‡ Presence of positive subpopulation.

Abbreviations: SSC, side scatter; MFI, mean fluorescence intensity.

Table 4.

Characteristics of leukemic patients with CD56-positive granulocytes

Patient no.	Sex/age	Diagnosis	Phenotypes of leukemic blast	Phenotypes of granulocytes	Karyotypes
1	F/1	AML	CD13, CD33, CD34, HLA-DR	CD13, CD33, CD56	46, XX[20]
2	M/23	AML	CD13, CD33, CD34, HLA-DR	CD13, CD33, CD56	46, XY[20]
3	M/64	ALL	CD19, CD20, HLA-DR	CD13, CD33, CD56	46, XY[20]
4	F/41	ANKL	CD13, HLA-DR, CD56	CD13, CD33, CD56	47-51, XX, +3, +6, del(6)(q12q16)×2,+8, +9, +19[cp18]/48, XX,+3,der(8)t(X;8)(p11.2;p11.2),der(11)t(1;11)(q21;q23), +19[2]
5	F/40	AML	CD13, CD33, CD7, CD56, HLA-DR, CD34	CD13, CD33, CD56	No metaphase
6	M/66	AML	CD13, CD33, CD7, CD56, HLA-DR, CD34	CD13, CD33, CD56	46, XX, t(4;12)(p16;q13)[20]

Abbreviations: ANKL, aggressive natural killer-cell leukemia.

Table 5.

Immunophenotypic features of monocytic cells

	Non-clonal disorders (N=24)	MDS (N=16)	Leukemia (N=6)
Percentage of gated cells	4.4 (1.3-7.6)	7.0 (1.9-35.7)∗	6.9 (2.2-33.6)∗
SSC, MFI	148 (120-186)	157 (126-204)∗	152 (96-184)
CD45, MFI	51.5 (36.7-95.5)	61.9 (40.5-121.0)	57.1 (3.4-64.9)^†
CD33
Positive rate (%)	24/24 (100)	16/16 (100.0)	6/6 (100.0)
% of positive cells	83.2 (63.7-94.8)	81.9 (53.6-96.5)	92.4 (81.0-94.5)
MFI	3.7 (1.3-8.0)	4.0 (0.8-5.2)	4.1 (2.4-5.5)
CD13
Positive rate (%)	24/24 (100.0)	16/16 (100.0)	6/6 (100.0)
% of positive cells	81.0 (33.0-96.2)	81.6 (50.6-96.5)	92.6 (77.3-97.0)
MFI	3.4 (1.6-11.1)	2.9 (1.4-8.3)	5.1 (2.4-12)
CD14
Positive rate (%)	24/24 (100.0)	16/16 (100.0)	6/6 (100.0)
Percentage of positive cells	69.6 (36.1-84.4)	62.9 (27.6-95.8)	82.3 (72.3-93.7)
MFI	5.9 (2.3-14.9)	8.2 (4.5-11.3)	12 (6.6-14)
CD56, positive rate	0/24 (0.0)	1/16 (6.3)	0/6 (0.0)

Data are median (range), except positive rate (positive case/total cases with available data).

Statistical significance analysis was performed for comparison of non-clonal disorders vs other groups and MDS vs leukemia.

^∗ P<0.05 compared to non-clonal disorders;

^† P<0.05 compared to MDS.

Abbreviations: See Table 3.

Table 6.

Immunophenotypic features of blast gating

	Non-clonal disorders (N=19)	MDS (N=14)
Percentage of gated cells	4.6 (0.5-6.7)	4.1 (0.2-9.4)
SSC, MFI	130 (45-164)	105 (46.3-149.0)
CD45, MFI	10.2 (7.3-18.7)	12.4 (6.9-21.7)
CD33
Positive rate (%)	16/19 (84.2)	11/14 (78.6)
% of positive cells	30.8 (0.0-53.7)	36.1 (0.0-78.4)
MFI	2.9 (1.3-5.2)	2.6 (0.8-3.6)
CD13
Positive rate (%)	17/19 (89.5)	12/14 (85.7)
% of positive cells	53.7 (0.0-79.0)	52 (0.0-71.6)
MFI	3.0 (1.6-7.0)	2.2 (1.4-3.1)∗
CD34
Positive rate (%)	18/19 (94.7)	13/14 (92.9)
% of positive cells	30.8 (14.8-70.3)	33.2 (12.9-81.9)
MFI	3.1 (1.9-3.9)	3.4 (1.7-6.4)
HLA-DR
Positive rate (%)	19/19 (100.0)	14/14 (100.0)
% of positive cells	50.0 (26.0-82.7)	58.4 (30.5-90.5)
MFI	7.2 (3.9-13.5)	6.0 (2.0-13.2)
CD19
Positive rate (%)^†	14/19 (73.6)	11/14 (78.6)
% of positive cells	20 (6.3-60.0)	13.3 (4.5-81.6)
CD56, Positive rate (%)	0/19 (0.0)	3/14 (21.4)∗
CD5, Positive rate (%)	0/19 (0.0)	1/14 (7.1)

Data are median (range), except positive rate (positive case/tested cases).

^∗ P<0.05, when compared with non-clonal disorders;

^† Presence of a positive subpopulation.

Abbreviations: See Table 3.

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