|Year : 2013 | Volume
| Issue : 4 | Page : 141-148
Contributions of flow cytometry in the evaluation of myelodysplastic syndrome patients
Amany H. Mansour1, Eman Kassm2, Tawfik Elkhodary3, Samar Taha2
1 Department of Clinical Pathology, Mansoura University, Mansoura, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
3 Oncology Center, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||12-Mar-2013|
|Date of Acceptance||16-Jul-2013|
|Date of Web Publication||19-Jun-2014|
Amany H. Mansour
MD, Department of Clinical Pathology, Faculty of Medicine, Mansoura University, 60, El Gomhoria Street, El Mansoura, 35516 Mansoura
Source of Support: None, Conflict of Interest: None
Myelodysplastic syndromes (MDS) are a group of malignant myeloid hematopoietic disorders. The diagnosis of MDS can be difficult, especially in cases with a low blast count and a normal karyotype. Flow cytometry has been used to distinguish MDS from nonclonal cytopenias. No one single simple flow cytometric parameter has been proposed to be diagnostic of MDS.
The aim of the present study was to evaluate immunophenotypic alterations in typical MDS patients and whether these abnormalities help in the differentiation process between MDS with nonclonal disorder and leukemic patients.
Materials and methods
Marrow aspirates from 29 patients, including 13 with MDS, 16 with acute myeloid leukemia, and 18 with nonclonal disorders (normal controls), were examined in this study. Their immunophenotypes were analyzed using flow cytometry. Blasts, nonblast myeloid cells, and monocytes were gated on the basis of CD45 expression and side scatter (SSC).
Comparison among the three groups showed that the granulocytic lineages of MDS showed decreased SSC compared with the controls (P<0.005 and P<0.000, respectively), altered CD45 intensity (P<0.004), decreased CD10-positive granulocytes (P<0.02), and a higher CD56 positive expression in the MDS and leukemic group (P<0.05 and P<0.001, respectively). Also, decreased intensity of CD11b (P<0.03) was observed in the MDS group. The expression rate of CD123+ was significantly higher in MDS patients than that in normal controls (P<0.0001).
Gating of the granulocytic region is a relatively easy method for MDS immunophenotyping. Among the parameters studied, SSC, CD10, CD123, and CD56 were the most useful for differentiating MDS from nonclonal disorders, whereas immunophenotypic changes in MDS appear to be useful for differentiating MDS from nonclonal disorders.
Keywords: blast, immunophenotyping, leukemia, monocyte, myelodysplastic syndromes
|How to cite this article:|
Mansour AH, Kassm E, Elkhodary T, Taha S. Contributions of flow cytometry in the evaluation of myelodysplastic syndrome patients. Egypt J Haematol 2013;38:141-8
|How to cite this URL:|
Mansour AH, Kassm E, Elkhodary T, Taha S. Contributions of flow cytometry in the evaluation of myelodysplastic syndrome patients. Egypt J Haematol [serial online] 2013 [cited 2018 Mar 18];38:141-8. Available from: http://www.ehj.eg.net/text.asp?2013/38/4/141/134781
| Introduction|| |
Myelodysplastic syndromes (MDS) comprise a complex, heterogeneous group of hematopoietic stem cell disorders. Classification and prognostic indicators include objective parameters such as cytogenetic findings and number of cytopenias in addition to morphologic assessment of lineage dysplasia and quantification of myeloblast 1; only limited data have been presented on the diagnostic and prognostic significance of flow cytometric immunophenotyping in patients with MDS 2. With an understanding of normal antigenic expression during hematopoietic development as determined by multidimensional flow cytometry (FCM), the dysregulation of hematopoietic observed in MDS could be characterized by deviations from the normal patterns 3. The diagnosis of MDS is made on the basis of a combination of clinical history, the morphological features of the peripheral blood and bone marrow (BM), cytogenetic data, and ruling out other diseases 4. Peripheral blood findings included the following: cytopenias; granulocyte abnormalities such as Pelger–Huët anomaly, hyper segmentation, bizarre nuclear shape (e.g. rings or large lobes), increased chromatin clumping, hypogranular or agranular cells, persistence of basophilia in mature cells, and blasts; platelet abnormalities, including giant platelets and hypogranulation; and erythroid abnormalities, for example nucleated red cells that show dyserythropoietic changes or megaloblastic features 5. BM aspiration is required for the assessment of dyserythropoiesis, dysgranulopoiesis, and dysmegakaryocytopoiesis. Examples of dysgranulopoiesis include abnormalities in primary granules such as decreased staining or large granules, decreased or absent secondary granules, nuclear. Dysmegakaryocytopoiesis was considered present when the following was observed: megakaryocytic dysplasia was characterized by the presence of abnormalities (e.g. micromegakaryocytes, nuclear hypolobulation, and large mononuclear cells) involving more than 40% of all megakaryocytic cells 6. Erythroid and neutrophil dysplasia was defined by the presence of more than 10% BM cells with morphological abnormalities and was also considered a diagnostic sign for dysplasia 7. More than 15% ringed sideroblasts, nuclear fragments, multiple nuclei, nuclear lobation, internuclear bridging, megaloblastic erythropoiesis, macronormoblastic erythropoiesis, irregular cytoplasm staining, and fewer than 5% erythroid cells were considered indicative of dyserythropoiesis. The diagnosis of MDS may be particularly difficult in patients with a normal karyotype or noninformative cytogenetics who do not have morphological markers, such as ringed sideroblasts or excess of blasts 8.
Histopathology, immunohistochemistry, and FCM may provide complementary information. The most common use is the assessment of blasts through immunophenotyping of CD34+ cells; although discrepancies between this approach and morphological evaluation have been reported 9, the FCM approach appears particularly useful for serial assessments in the individual patient. Pattern recognition strategies may complement morphological evaluation of dysgranulopoiesis 10. Cytogenetic abnormalities play a major role in the diagnosis of MDS and in risk assessment. Fluorescence in-situ hybridization (FISH) should complement conventional cytogenetics in particular cases. Specifically, FISH may improve the detection of deletion 5q31–q32 in patients with MDS without cytogenetic evidence of del(5q) 11. The aim of the present study was to evaluate immunophenotypic alterations in typical MDS patients, whether abnormalities help in the differentiation process between MDS with nonclonal disorder and leukemic patients.
| Materials and methods|| |
Selection of patients
The present study was carried out from September 2009 to January 2011 on 29 adult patients selected from those attending the outpatient clinics of King Fahad Hospital, Kingdom of Saudi Arabia. They were subdivided into the following groups: group I included 13 patients with MDS, group II included 16 patients with acute myeloid leukemia (AML), and group III (control group) included 18 patients with nonclonal disorders, such as immune thrombocytopenic purpura, lymphoma without BM involvement, anemia without hematological malignancy, and individuals undergoing orthopedic surgery.
The results of immunophenotyping, cytogenetics, and hematologic analysis in MDS patients were interpreted and patients were classified according to the WHO classification 12. The MDS group I included three patients with refractory anemia, two with refractory neutropenia, four with refractory anemia with ringed sideroblasts, one patient with refractory cytopenia and multilineage dysplasia, and three patients with refractory anemia and excess blasts.
All BM samples were studied systematically within the first 18 h after they were aspirated. A written consent was signed by each patient according to the recommendations of the local ethical committee.
- Full clinical history and clinical examination.
- Laboratory investigations.
All patients were subjected to the following:
- Complete blood picture: using a Coulter Beckman Counter (Beckman Coulter, California, USA).
- BM aspiration and biopsy examination.
- FCM studies of BM specimens: BM cells of the patients were aspirated into a heparinized syringe; nucleated cells were counted and stained with antibodies. Then, samples were treated using the standard ammonium chloride method at room temperature to lyse erythrocytes and then washed with phosphate-buffered saline (PBS) (2 ml/tube) and resuspended in 0.5 ml PBS. After a two-cycle wash with PBS, the cell pellet was resuspended in PBS and a cell count was performed. One million cells were incubated with an appropriate amount of the monoclonal antibody (MoAb) combinations (15 min at 48°C). Antibody staining was performed as follows: 100 μl of a cell aliquot containing 5–8×105 nucleated cells were placed into each tube and stained with three antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or peridin chlorophyll (PerCP). In this panel, the CD45 was tested in each combination to allow a primary gating of BM cell subsets on the basis of CD45 antigen expression and side scatter (SSC). Combinations of the three antibodies used in this study include FITC-conjugated CD16, CD10, HLA-R, CD15, CD38, CD14,CD71, CD123, and PE-conjugated CD11b, CD13, CD33, CD34, CD19, CD56, and CD117 and PerCP-conjugated CD45 (Becton Dickinson Biosciences, San Jose, California, USA). Biological negative control cells within each tube (e.g. B cells in a tube stained for T cells) and cells stained with isotypic controls for IgG1-FITC, IgG2-PE, and IgG2a-PerCP were used as negative controls. Data (collected in list mode) were analyzed using Lysis II and CellQuest software (Becton Dickinson Biosciences) 13.
- Histopathology: Hematoxylin and eosin (H&E) and reticulin stains (Gomori’s Silver stain) in combination with immunostaining were used to study MDS. BM cores were optimally fixed, decalcified, embedded in paraffin and stained with H&E and reticulin stains. Stained sections were examined for the estimation of cellularity and the evaluation of histoarchitectural displacement of progenies. In order to confirm the blastic nature of the cells, other sections were prepared on charged slides for immunohistochemistry using antibody to human CD34 (Mouse Anti-Human CD34 Antigen, code K4004; Dako and Clone: QBEnd-10, Carpinteria, California, USA). In normal BM, reticulin fibers exist as a few fine networks, primarily perivascular and periendosteal. When increased abnormally, it appears as coarse fibers including collagen fibrosis [Figure 1]b.
|Figure 1: Pathology findings in the myelodysplastic syndrome group. (a) Hypercellular bone marrow (H and E, ×400), (b) Increased marrow fibrosis (Reticulin stain, ×100), (c) poorly formed erythroid groups to endosteal surface (H and E, ×400), (d) Relocated myeloid precursors ((H and E, ×400)|
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- Immunohistochemistry: Immunohistochemical analysis for CD34 using a labeled streptavidin–biotin–peroxidase complex technique was performed on BM paraffin sections. Sections were deparaffinized and rehydrated through graded alcohols to water; the avidin–biotin–peroxidase method and 3,3-diaminobenzidine chromogen were applied for immunohistochemical analysis. Endogenous peroxidase activity was blocked with 0.6% H2O2. After blocking, sections were incubated at room temperature for 60 min with antibodies to human CD34. BM vasculature was used as an internal positive control. Negative control was performed by omission of the primary antibody. Examination of slides was carried out on an Olympus CX31 light microscope. Pictures were obtained using a PC-driven digital camera (Olympus E-620). The computer software (Cell; Olympus Soft Imaging Solution GmbH, Münster, German) allowed morphometric analysis to be carried out.
- Conventional cytogenetic: We carried out a conventional cytogenetic analysis on heparinized whole-BM samples using standard protocols and analyzed the samples following Giemsa banding. Twenty metaphases were evaluated for each sample.
The statistical analysis among different groups for the variables studied was carried out using the χ2-test and Mann–Whitney’s U-test. Statistical analysis was carried out using SPSS software, version 15.0 (SPSS Inc., Chicago, Illinois, USA). P values of less than 0.05 were considered statistically significant.
| Results|| |
The patients in the MDS group were significantly older than the patients with nonclonal disorders but age showed no statistical significance [Table 1]. The leukocyte count was higher in the leukemic patients compared with the other group. Both the MDS and the leukemia groups had lower hemoglobin levels than the patients with nonclonal disorders. Cytogenetic abnormalities were more frequent in the MDS and leukemia group compared with the nonclonal disorders group. There was no statistically significant difference in cytogenetic analysis between the studied groups.
Flow cytometric immunophenotyping results
- A lowered SSC (hypogranularity) in the granulocytic gate was found in the MDS and leukemia groups compared with the control group (P<0.05 and P<0.001, respectively).
- A significant decrease in CD10-positive granulocytes was observed in the MDS group compared with the control group (P<0.006).
- An abnormal decrease in CD45 expression in the MDS group was observed compared with the control group (P<0.001; [Figure 2]a and b. Also, CD45 expression on immature blast cells was relatively lower compared with their maturing progeny [Figure 2]b.
|Figure 2: CD45/side scatter expression in (a) the control and (b) myelodysplastic syndrome groups. Lymphocytes (grey), monocytes (green), myeloid cells (blue) and blast cells (red). Arrow in fig. 2b shows side scatted shift of myeloid cells in MDS group.|
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- Abnormal expression of CD11b and CD16 was observed on maturing myeloid cells and monocytes, in the form of decreased intensities of CD11b, in the MDS group compared with the control group (P=0.003; [Figure 3]. A similar finding was obtained in the leukemia group (P<0.001).
|Figure 3: Relationships between CD11b and CD16 in maturing myeloid cells. (a) Myelodysplastic syndrome and (b) control groups.|
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- Abnormal pattern of CD13 expression during granulocytic maturation: overexpression of this antigen on promyelocytes and myelocytes (convex pattern; [Figure 4]a] was observed in the MDS group. Also, an abnormal relationship was found between CD13 and CD16 expressions in the maturing myeloid compartment compared with the control groups [Figure 4].
|Figure 4: Relationships between CD13 and CD16 in maturing myeloid cells. (a) Myelodysplastic syndrome and (b) control groups.|
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- Altered CD33 expression: Lack of CD33 expression on maturing myeloid cells or monocytes with increased expression on immature cells. CD33 expression on gated monocytes was higher in the MDS and leukemia groups compared with the control group (P<0.020 and P<0.018, respectively).
- Expression of CD56 on maturing myeloid cells or monocytes. CD56 showed higher positivity in the MDS and leukemia group compared with the control group (P<0.05 and P<0.001, respectively).
- CD34 overexpression was observed on maturing myeloid cells or monocytes. CD34 is an indicator of early myeloid precursors and is not observed on normal maturing myeloid cells or monocytes (P<0.002).
- Presence of abnormal myeloblasts: Criteria such as percentage of CD34 cells, position by CD45 and SSC, and presence/absence of myeloid antigens were taken into consideration when defining a myeloblast. Normal myeloblasts identified by the heterogeneous expression of CD34 also show HLA-DR, CD13, and CD33 at high levels, but do not express other markers of mature neutrophils such as CD11b, CD15, and CD16. Normal myeloblasts are intermediate in size by FSC but have low SSC. The progression to promyelocytes is indicated by the loss of CD34 and HLA-DR expression, acquisition of high levels of CD15, and a marked increase in SSC, but without expression of CD11b.
We measured the expression of CD123 on MDS patients and found that the expression of CD123 was significantly higher in MDS than that in normal controls (P<0.0001). We found that the level of CD123 was significantly correlated with the proportion of BM blasts, which might suggest that the expression of CD123 probably confers a proliferation advantage to malignant cells.
Assessment of erythroid precursors: higher proportions of immature cells and decreased CD71 expression were observed. Analysis of the expression of CD71 and CD117 indicated increased expression of CD71and CD117 in MDS [Figure 5]a compared with the control group (P<0.001; [Figure 5]b.
|Figure 5: (a) Myelodysplastic syndrome and (b) control group. Increased blast cells in bone marrow of MDS group (red box) compared to the control group. Increased CD117 expression within blast gate in myelodysplastic syndrome group.|
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In general, hyperplastic BMs predominate in MDS [Figure 1], although in some patients hypoplastic marrows were observed. These changes consist of displacement of poorly formed erythroid groups to the endosteal surface and, conversely, relocation of myeloid cells to centromedullary spaces [Figure 1]a, c, and d. In addition, dysmorphic megakaryocytes (hypolobated, microforms, and atypical large cells) were observed frequently [Figure 1]a and d] in MDS adopting a paratrabecular position.
Membranous staining of CD34-positive cells was evaluated. BMs show CD34+ cells more than 5% and form aggregates or clusters (i.e. three or more CD34+ cells) and have been described as having ‘multifocal accumulations of CD34+ progenitor cells’ [Figure 1]c and d.
| Discussion|| |
As found previously by other investigators, no single immunophenotypic parameter could differentiate accurately between MDS and other pathological conditions. Therefore, we carried out discriminate analyses using several immunophenotypic parameters. Moreover, flow cytometric analysis has also been shown to be sensitive in a high proportion of cases with no diagnostic morphology. Finally, the quantitative evaluation provided by the discriminate functions reflected the severity of marrow dysplasia 14. This study evaluated the immunophenotypic features of MDS, acute leukemia, and nonclonal disorders. The evaluations were based on gating by SSC and CD45 intensity, respectively. The SSC of granulocytes in MDS was significantly lower than that in nonclonal disorders. The degree of decreased SSC in MDS as compared with nonclonal disorders was more evident in the mature granulocytes than in the immature blasts. The decreased SSC noted in patients with MDS has been described previously in BM studies and likely represents the morphologically appreciated hypogranularity of polymorphnuclear leucocytes in patients with MDS. Patients with early MDS appear to have accelerated apoptosis of myeloid cells, whereas patients with more advanced categories of disease have a decrease in apoptotic activity and an increase in cell proliferation 15. Decreased CD10 expression was found in the neutrophils of patients with MDS, and this finding is in concordance with Chang and Cleveland 16. They also explained that CD10/neutral end peptidase plays an important role in the control of chemotaxis and the inflammatory suggested contributing to infection susceptibility 17. This may reflect abnormal apoptosis because CD10 may be a marker of apoptosis 18. This may be because of an increased phagocytosis associated with ineffective hemopoiesis both in MDS and in nonclonal disorders 19,20. We also found decreased CD11b-positive granulocytes in MDS and leukemia patients. Kiyoyuki et al. 21 found that decreases in intensities of CD11b with other immunophenotypic evidence of a left shift were considered in MDS. Abnormalities in CD11b and HLA-DR relationships led to abnormal maturational patterns; also, abnormal expression of CD11b versus CD16 occurred simultaneously with CD35 deficiency in MDS patients. Mittelman et al. 22 confirmed the presence of a decreased expression of CD11b in granulocytes, and also observed abnormal chemotaxis and superoxide release 23. In our study, we found that CD123 is highly expressed in MDS than nonclonal disorder. In recent years, it has been found that CD123 is highly expressed in hematological malignant diseases such as AML and B cell acute lymphocytic leukemia 24. Therefore, CD123 is considered to be a unique marker of leukemic transformation 25. MDS could be described as a preleukemia status; thus, we doubt whether some kinds of malignant stem cells may be similar to leukemic stem cell. They also exist in the BM of MDS patients, which are exactly the malignant clone of MDS, and contribute toward transformation into AML 26. We found increased CD34-positive cell expression in MDS and leukemic transformation. Our results are in agreement with a recent report that indicated an increase in BM CD34+ cells as detected by FCM in MDS patients. Cytopenia in the early stages of MDS may reflect a balance between the capability of cell production and degree of cell apoptosis. Furthermore, a decrease in the apoptosis of MDS clonal cells, especially CD34+ cells, may be one of the mechanisms leading to disease progression 27. Combined therapy with granulocyte colony stimulating factor and erythropoietin improves cytopenia in a considerable proportion of MDS patients and this clinical effect is reported to be associated with a decrease in the apoptosis of BM cells 28. A recent paper has reported that a molecule, p38 mitogen-activated protein kinase, which is involved in regulating apoptosis and controlling the cell cycle, was overactivated in the early stages of MDS, but not in advanced stages 29. In our study, we observed increased expression of CD13, CD33, CD56, CD16, and CD15. Although the increased CD33 expression did not correlate with the BM blast count, it did correlate with a higher risk of disease progression. On myeloid cells, the CD33/CD16 ratio of immature to mature myeloid cells was estimated in order to distinguish MDS patients with myeloid dysplasia from MDS patients without myeloid involvement 18,30. Also, increased expression of CD13 was observed in their marrow mononuclear cells, suggesting a maturation shift results were obtained using unfractionated BM. Some studies have reported a much higher CD13 expression in high-risk groups than low-risk groups with BM blast count although the blasts are frequently positive for CD13 31. Moreover, we found increased intensity of CD56 expression in MDS patients. Thus, these features of nonblast granulocytes in leukemic patients were suspected not to be clonal changes related to leukemia but rather, reactive changes or changes because of immaturity. Nevertheless, it was reported recently that CD56 expression on monocytes was the only discriminating marker between chronic myelomonocytic leukemia and MDS 20,32. CD56 expression is frequently seen in regenerating BM after chemotherapy or stem cell transplant, on granulocytes and monocytes during G-CSF primed stem cell collections and during infections 27. We found an increase in the expression of CD105 and CD117 in the MDS and leukemic group compared with the non clonal disorder group. Hooman et al. 33 reported that analysis of an increased expression of CD105and CD117 in the MDS and leukemic group may thus also aid the assessment of erythroid dysplasia in MDS. Moreover, the CD105 level was significantly associated with the degree of erythroid dysplasia assessed by morphology 34.
| Conclusion|| |
We conclude that an accurate FCM evaluation of marrow dysplasia was carried out in this study with a panel of multiple MoAbs. Flow cytometric immunophenotypic evaluation may be valuable in difficult cases in which morphology and cytogenetics are nondiagnostic but clinical suspicion of MDS is high because it is easy to gate and the results are easy to interpret. Gating the granulocytic region is a relatively easy method for MDS immunophenotyping. Among the parameters studied, SSC, CD10, CD123, and CD56 were the most useful for differentiating MDS from nonclonal disorders, whereas immunophenotypic changes in MDS appear to be useful for differentiating MDS from nonclonal disorders.
| Acknowledgements|| |
The authors thank Dr M. Yacoubi, assistant professor, Department of Histopathology, KFU, and Dr Maha Ameen, professor, Department of Histopathology, for their active participation in the pathological study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]