Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2615-2615
Author(s):  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Erythroid Cell ◽  
Multiparameter Flow Cytometry ◽  
Cell Populations ◽  
Aberrant Expression ◽  
Hla Dr ◽  
Cd16 Expression

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.

Detection of Dysplastic Features by Multiparameter Flow Cytometry in Myelodysplastic Syndromes in Relation to Cytomorphology and Cytogenetics.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2461-2461
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Erythroid Cell ◽  
Multiparameter Flow Cytometry ◽  
Cell Populations ◽  
Erythroid Cells ◽  
Monocytic Cell ◽  
Aberrant Expression ◽  
Cell Lineages ◽  
Cd16 Expression

Abstract Dysplastic features can be detected in different cell lineages in myelodysplastic syndromes (MDS) by multiparameter flow cytometry (MFC). The aim of the present study has been the assessment of the flow cytometric detection of dysplastic features previously published to occur in MDS in relation to findings in cytomorphology (CM) and cytogenetics (CG). We analyzed 307 bone marrow samples from patients with suspected (n=130) or proven (n=177) MDS by MFC, CM, and CG in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 3.5%) and from 0% to 23% (median, 3%; r=0.271, p<0.0001). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 5), 1 for monocytes (0 to 5), and 0 for erythroid cells (0 to 2); median total number=3, range 0–11. The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (13.7%), CD15 (10.4%) and CD64 (10.4%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (67.1%), aberrant coexpression of CD56 (32.9%), aberrant pattern of CD13/CD16 expression (31.6%), aberrant pattern of CD11b/CD16 expression (24.1%), and reduced expression of CD33 (13.4%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (42.7%) and of CD16 (18.9%). The most frequent dysplastic features observed in the erythroid cell populations included a lack of CD71 expression (15.0%) and an aberrantly homogeneous expression of CD71 (9.1%). As compared to cases with no indication of MDS by CM (=non-MDS) cases with MDS according to CM were significantly associated with a reduced side-scatter signal in granulocytes (ratio granulocytes:lymphocytes 6.53±1.27 vs. 7.44±1.17, p<0.0001) as well as a higher number of dysplastic features in granulocytes (1.98±1.09 vs. 1.00±1.31, p<0.0001), monocytes (0.81±0.84 vs. 0.35±0.63, p<0.0001), and erythroid cells (0.33±0.47 vs. 0.20±0.40, p=0.061). Particularly, an aberrant expression of CD56 in monocytes occurred more frequently in 33 cases with CMML as compared to non-MDS cases (84.8% vs. 15.7%, p<0.0001). In cases with possible MDS according to CM the differences to non-MDS cases were less pronounced (reduced side-scatter signal in granulocytes 7.77±1.47 vs. 7.44±1.17, n.s., dysplastic features in granulocytes 1.90±1.21 vs. 1.00±1.31, p=0.003, monocytes 0.50±0.63 vs. 0.35±0.63, n.s., and erythroid cells 0.30±0.47 vs. 0.20±0.40, n.s.). In cases with aberrant cytogenetics (n=80, excluding those with –Y as sole aberration) dysplastic features by MFC occurred more frequently as compared to cases with normal karyotypes (reduced side-scatter signal in granulocytes 6.35±1.18 vs. 7.09±1.38, p<0.0001, dysplastic features in granulocytes 2.05±0.95 vs. 1.68±1.31, p=0.021, monocytes 0.80±0.89 vs. 0.71±0.81, n.s., and erythroid cells 0.38±0.49 vs. 0.31±0.48, n.s.). A total of more than two dysplastic features in blasts, granulocytes, and monocytes and/or a blast count >5% occurred in 68.4%, 63.3%, and 35.3% of cases with MDS, possible MDS, and non-MDS according to CM (p=0.0005). Interestingly, in some cases aberrant antigen expression has been observed in cell lineages not rated dysplastic by CM. This evaluation suggests that MFC may be used to identify dysplastic features in patients with suspected MDS. Sensitivity and specificity varies between MDS subtypes and should be clearly defined in future studies.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

Multiparameter Flow Cytometry Identifies Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes: Correlation with Cytomorphology and Cytogenetics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2376-2376
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Median Number ◽  
Multiparameter Flow Cytometry ◽  
Strong Correlations ◽  
Trisomy 8 ◽  
Prognostic Information ◽  
Accurate Identification ◽  
Hla Dr

Abstract Myelodysplastic syndromes (MDS) are diagnosed and classified based on cytomorphology (CM) and cytogenetics. The accurate identification of dysmyelopoiesis and quantification of bone marrow blasts requires a high degree experience in CM. Multiparameter flow cytometry (MFC) has been shown capable of identifying dysplastic features in all lineages and to reveal prognostic information in MDS. We analyzed 23 bone marrow samples from patients with untreated MDS by MFC, CM, and cytogenetics in parallel. In addition, four normal bone marrow samples and one bone marrow sample with hereditary sideroachrestic anemia (HSA) were analyzed by MFC for comparison. Diagnoses included RA (n=4), RARS (n=1), RAEB-1 (n=7), RAEB-2 (n=6), 5q- syndrome (n=2), and CMML (n=2). CM revealed dysgranulopoiesis and dyserythropoiesis in 16 and 14 cases, respectively, and blasts counts ranging from 3% to 20% (median, 9%). Karyotype aberrations included 5q- (n=3); trisomy 8 (n=2); del(11q) (n=2); t(3;11) (n=1); loss of chromosome X (n=1); and trisomy X, trisomy 8, and 5q- (n=1); while n=13 cases had normal karyotypes. Blast counts as determined by MFC ranged from 1.9% to 14.8% (median, 4.7%; correlation with CM: r=0.466, p=0.029). The median number of aberrant features detected by MFC were 4 for blasts (range, 2 to 9), 3 for granulocytes (1 to 8), 3 for monocytes (0 to 7), and 0 for erythrocytes (0 to 2). The presence of dysgranulopoiesis (CM) correlated with hypogranulation (MFC; 50% vs. 17%, p>0.05) and expression of HLA-DR (38% vs. 17%, p>0.05) and CD7 (13% vs. 0%, p>0.05) in granulocytes as well as with the lack of CD16 expression (63% vs. 0%, p=0.035), the expression of CD2 (25% vs. 0%, p>0.05), and the dim expression of CD66 (63% vs. 40%, p>0.05) on monocytes. Combining these parameters led to strong correlations between dysgranulopoiesis (CM) and at least one dysplastic feature by MFC for granulocytes (75% vs. 17%, p=0.023) and monocytes (81% vs. 33%, p=0.054). Combining the granulocytic and the monocytic MFC scores revealed dysplastic features in 100% cases with dysgranulopoiesis (CM) and in 50% without (p=0.013). No clear-cut correlations were found between dyserythropoiesis (CM) and dysplastic features in erythroid cells as detected by MFC. The most striking MFC features of cases with 5q- syndrome included CD2-positivity (100% vs. 6%, p=0.003) and CD34-positivity (100% vs 28%, p=0.042) in monocytes as well as hypogranulation (100% vs. 32%, p=0.055) and HLA-DR-positivity (100% vs 21%, p=0.023) in granulocytes. Dysplastic features that were detected by MFC were related to each other. Thus, in blasts the expression of CD56 was strongly related to the expression of CD64 (p=0.0003) and the dim expression of CD45 (p=0.0003). In addition, the expression of CD11b was associated with the expression of CD15 (p=0.001). In granulocytes, the lack of CD33 was associated with a lack of CD64 expression (p=0.0004). In monocytes, the dim expression of CD45 was associated with a lack of CD14 expression (p=0.0005). The case with HSA displayed an aberrant pattern of CD71 expression. These data suggest that MFC is capable of reproducibly detecting dysplastic features in different compartments of the bone marrow. Strong correlations between CM and cytogenetic finding and MFC are detectable. Future studies will define the role of MFC in optimizing the diagnosis of MDS and the prognostication in patients with MDS.

scholarly journals Activation of JAK/STAT Signaling in Megakaryocytes Is Necessary and Sufficient for Myeloproliferation In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 949-949
Author(s):  
Q. Jeremy Wen ◽  
Brittany Woods ◽  
Qiong Yang ◽  
Chiu Sophia ◽  
Gu Lillu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Diphtheria Toxin ◽  
Bone Marrow Cells ◽  
Erythroid Cell ◽  
Spleen Weight ◽  
Wild Type ◽  
Myeloid Progenitor

Abstract Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPN). It is has been long known that abnormal megakaryocytes secrete elevated levels of cytokines such as TGFβ, resulting in pathologies including bone marrow fibrosis. Two recent studies showed that megakaryocytes regulate the quiescence of HSCs, raising the possibility that megakaryocytes may promote the MPNs by influencing the biology of non-malignant HSCs. However, the mechanism by which megakaryocytes regulate the initiation and progression of MPNs is largely unknown. To study the role of megakaryocytes in the MPNs, we analyzed the phenotype of PF4-Cre/Jak2V617F mice in which Jak2 is expressed in the megakaryocyte lineage from the endogenous locus, in contrast to previous studies, which used transgenic models. Selective activation of Jak2V617F was confirmed by allele-specific qPCR. CD41+ cells were positive for mutant Jak2, whereas sorted stem/progenitor cells and erythroid cells were Jak2 wild-type. Furthermore, flow cytometry showed that Stat5 activation was present in megakaryocytes, but not in erythroid or myeloid cells. Activation of JAK-STAT signaling caused an expansion of megakaryocytes in the bone marrow and spleen and a modest increase in the platelet count. Surprisingly, PF4-Cre/Jak2V617F mice also displayed a robust expansion of TER119(low)/CD71(high) and TER119(high)/CD71(high) red cells in the spleen, increased hematocrit and splenomegaly. Histological examination of the spleen revealed expansion of the erythroid lineage coupled with disrupted splenic architecture and fibrosis. This PV-like phenotype was fully penetrant and comparable to that of Vav-Cre/Jak2V617F mice, which express mutant Jak2 in all hematopoietic lineages. Profiling of hematopoietic progenitors by flow cytometry demonstrated that myeloid progenitor populations were expanded and skewed toward the erythroid-megakaryocyte lineage with a significant increase in Pre Meg-E, Pre CFU-E and MKPs in the PF4Cre/Jak2V617F mice. In addition, LSK cells were increased in both the bone marrow and spleen. Cytokine profiling of the plasma revealed increased levels of several cytokines, including Il-6, which is known to be upregulated in human JAK2 mutant PV megakaryocytes. Significant increases in Cxcl1, Cxcl2, and Ccl11 were also detected. Real-time qPCR analysis confirmed increased expression of these cytokines/chemokines in Jak2V617F-mutant CD41+ cells. Furthermore, IL6 treatment increased EPO-dependent colony formation of wild type LSKs and MEPs, and also enhanced expression of the erythroid cell markers CD71 and Ter119. To further explore the role of megakaryocytes in the MPNs, we used a strategy in which expression of the diphtheria toxin receptor (DTR) sensitizes cells to diphtheria toxin (DT). We transduced c-Kit+ cells from PF4-Cre/iDTR+/- mice with MPLW515L and transplanted the cells to irradiated mice. As expected, both iDTR+/- and PF4-Cre/iDTR+/- mice developed a PMF-like phenotype, including leukocytosis, thrombocytosis, splenomegaly and myelofibrosis (Fig 1). Treatment of these animals with DT caused significant reductions in megakaryocytes in the bone marrow and spleen as well as a decrease in the platelet count of PF4-Cre/iDTR+/- mice. Of note, DT also significantly reduced the white count and spleen weight, while restoring splenic architecture. PF4Cre/iDTR+/- mice also showed significant reduction of c-Kit+ myeloid progenitor cells. Therefore, depletion of megakaryocytes significantly attenuated the disease phenotype of MPLW515L induced MPN in vivo. Together, these two model systems reveal that JAK2 activation in megakaryocytes is sufficient and necessary for MPNs and support the development of megakaryocyte differentiation therapy in the disease. Moreover our data resonate with studies in MPN patients in which a JAK2V617F low allele burden in the setting of full-blown, clinical MPN. figure 1 Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. figure 1. Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. Disclosures Levine: Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Analysis of the Immunophenotypic Features of Blast Cells, Lymphocytes and Granulocytes by Multiparametric Flow Cytometry in Bone Marrow (BM) Cells of Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4843-4843
Author(s):  
Xin Du ◽  
Jing Huang ◽  
Maohua Zhou ◽  
SuXia Geng ◽  
Jianyu Weng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Flow Cytometric Analysis ◽  
Control Group ◽  
Consistent Finding ◽  
Multiparametric Flow Cytometry ◽  
Hla Dr ◽  
Number Of Patients ◽  
Blast Cells

Abstract Abstract 4843 Background The myelodysplastic syndromes (MDS) are a group of clonal heterogeneous bone marrow disorders characterized by peripheral cytopenias, ineffective hematopoiesis, and unilineage or multilineage dysplasia. Multiparametric flow cytometry is increasingly being used as an adjunct to the establishing of MDS. While many antigens have been described to be aberrantly expressed in MDS the findings are generally heterogeneous and there is no consistent finding that would be present in all cases with MDS. Aim To investigate the immunophenotypic features of MDS and non-MDS patients and the characteristic of subtypes of MDS. Methods BM samples were collected from 22 MDS patients including 3 RA (2 male, 1 female, median age 57), 3 RAS (2 male, 1 female, median age 72), 12 RAEB (6 male, 6 female, median age 67.5), 4 MDS-AML (2 male, 2 female, median age 69.5) and 20 non-MDS (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies and using the conventional and secondary gating strategies to analysis the immunophenotypic features of BM cells. Results This study showed that the proportion of blast cells increased significantly than non-MDS group (P=0.002). As the disease progressing, the percentage of blast cells became higher and significantly difference compared to the non-MDS group (P=0.226, P=0.464, P=0.001 and P=0.000, respectively). The expressions of CD34+ and CD7+ on blast cells were significantly difference between MDS and non-MDS groups (P=0.005, and P=0.002, respectively). Compared with the subtypes of MDS and non-MDS group, the expressions of CD34+ and CD7+ on blast cells became high gradually (P=0.534, P=0.487, P=0.009, P=0.004 and P=0.294, P=0.166, P=0.002, P=0.001) and the high percentage of blast cells and high expression levels of CD34+ and CD7+ might indicate poor prognosis. The expression of CD7+ on lymphocytes was similar with CD34+ and CD7+ on blast cells, but the expressions of CD19+ and CD56+ on lymphocytes were no significantly difference (P=0.076, P=0.252, respectively). The expressions of antigens on granulocytes showed that the expressions of CD15+CD11b+, CD10+ and HLA-DR were significantly difference between MDS and non-MDS groups(P=0.000, P=0.009 and P=0.007, respectively), meanwhile, as the disease progressing, the expression rates of CD15+CD11b+, CD10+ and HLA-DR in subtypes of MDS increased gradually and the survival time of these patients who had over-expression of these antigens was shorter than control group(P=0.002). However, the expressions of CD33+, CD13+, CD56+ and CD15+CD11b- were significantly difference between subtypes of MDS and non-MDS group (P=0.059, P=0.588, P=0.063 and P=0.207, respectively). Conclusions Our results showed that using multiparametric flow cytometry to analyze the immunophenotypic features of BM cells could provide clinically useful information for the diagnosis, classification and prognosis of MDS patients. Particularly, the percentage of blast cells, the expression of CD34+ and CD7+ on blast cells, the expression of CD7+ on lymphocytes and the expression of CD15+CD11b+, CD10+ and HLA-DR on granulocytes may provide the much more useful information. However, further studies including larger number of patients with a longer follow-up are necessary to confirm these results. Disclosures No relevant conflicts of interest to declare.

Multiparameter Flow Cytometry in Patients with Suspected Myelodysplastic Syndromes Adds Significant Prognostic Information: A Study on 1,013 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1722-1722
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Continuous Variable ◽  
The Other ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Information ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Cox Analysis

Abstract Abstract 1722 Backgroundn: Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal (SSC) in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Prognostic Impact of Multiparameter Flow Cytometry in Patients Analyzed for Suspected MDS.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2806-2806
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Expression Pattern ◽  
Multivariable Analysis ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Impact ◽  
Cd16 Expression ◽  
Equity Ownership ◽  
Cox Analysis ◽  
Bone Marrow Blasts

Abstract Abstract 2806 Background: Multiparameter flow cytometry (MFC) is increasingly used to evaluate patients with suspected myelodysplastic syndromes (MDS). The prognostic impact of distinct MFC findings has been controversial yet. Aims: Assess the respective impact of different antigen expression aberrancies on overall survival (OS) in suspected MDS in relation to established prognostic parameters. Methods: We studied 804 patients (pts) who were analyzed for suspected MDS by cytomorphology (CM), cytogenetics and MFC in parallel (f/m 463/341; median age 70 yrs, range 2–89). Pts had been included in a previous study evaluating the diagnostic role of MFC (Kern et al., Cancer 2010). Data on OS was available in all pts (median OS 6.2 yrs, median follow-up 3.2 yrs). CM revealed MDS in 493 (61.3%) pts; 170 (21.1%) pts had evidence of dysplasia which was not sufficient to diagnose MDS by CM; in 141 (17.5%) pts MDS was excluded by CM. Karyotypes were favorable / intermediate / unfavorable according to IPSS in 684 (85.1%) / 89 (11.1%) / 31 (3.9%) pts. MFC was performed following recent ELN Working Group recommendations (Westers et al., Leukemia 2012) in myeloid progenitor cells (MPC), granulocytes, monocytes and erythroid cells. MFC parameters included, each compared to normal bone marrow, increased or decreased expression of antigens, expression of normally not expressed antigens, aberrant expression pattern of pairs of antigens as well as cross-lineage expression of lymphatic antigens. Results: Considering all 804 pts, i.e. regardless of confirmation of MDS by CM, the following MFC parameters were associated with OS: MPC >5% (p<0.001, hazard ratio (HR) 2.5), expression of CD5 (p<0.001, HR 4.1), CD56 (p=0.043, HR 2.0), CD7 (p=0.015, HR 2.2) in MPC; reduced side-scatter signal (p<0.001, HR 1.9), aberrant CD13/CD16 expression pattern (p=0.007, HR 1.4), aberrant CD11b/CD16 expression pattern (p=0.003, HR 1.6), expression of CD56 (p<0.001, HR 2.1), reduced expression of CD33 (p=0.018, HR 1.6) in granulocytes; expression of CD56 (p<0.001, HR 1.6) in monocytes; reduced expression of CD71 (p<0.001, HR 2.1) in erythroid cells. A score was devised calculating for each pt the sum of all HRs for the respective parameters found positive. Pts were separated into 4 groups: group 1 (n=263 pts) had a score of 0; group 2 (n=259) had a score >0 and below the median (2.135); group 3 (n=149) had a score above the median and below the 75th percentile (4.961); group 4 had a score above the 75thpercentile. Significant differences in OS were observed: OS at 4 years in groups 1, 2, 3, and 4 amounted to 82.4%, 67.1%, 54.7%, and 36.2%, respectively (p=0.001 for 1 vs 2, p=0.022 for 2 vs 3, p=0.003 for 3 vs 4, p<0.001 for all other comparisons). Cox analysis of the MFC score revealed a significant association with OS (p<0.001, HR 1.4 per group). Other parameters univariably related to OS were: diagnosis of MDS by CM (p<0.001, HR 2.2), percentage of bone marrow blasts by CM (p<0.001, HR 1.9 per 10% increment), cytogenetic group according to IPSS (p<0.001, HR 5.1 per group), WBC count (p<0.001, HR 1.2 per 10 G/L increment), hemoglobin level (p<0.001, HR 0.8 per g/L), platelet count (p<0.001, HR 1.4 per 100 G/L increment), and age (p<0.001, HR 1.5 per decade). Multivariable Cox analysis including diagnostic markers as covariates revealed an independent impact on OS for all of them: MFC score (p<0.001, HR 1.3), diagnosis of MDS by CM (p=0.003, HR 1.4), bone marrow blasts by CM (p=0.015, HR 1.4), and cytogenetics according to IPSS (p<0.001, HR 2.9). The addition of age or of peripheral blood counts as covariates into the multivariable analysis still indicated an independent impact of the MFC score on OS. Limiting the multivariable analysis to cases with MDS proven by CM still resulted in an independent impact of the MFC score on OS (p=0.001, HR 1.2). Interestingly, if only cases were considered with signs of dysplasia by CM, which are not sufficient to diagnose MDS, multivariable analysis also revealed an independent impact of the MFC score on OS (p=0.016, HR 1.3). Conclusions: The present data indicates that MDS-related findings by MFC provide prognostic information not just in pts with MDS proven by CM but also in a comprehensive cohort of pts being diagnosed for suspected MDS. Furthermore, even in pts with evidence of dysplasia not sufficient to diagnose MDS by CM a prognostic impact of MFC was demonstrated. This data thus suggests to integrate MFC into the diagnostic work-up of pts with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Multicolour Flow Cytometric Finetuning of the Classification of Myelodysplastic Syndromes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2614-2614
Author(s):  
Arjan A. van de Loosdrecht ◽  
Theresia M. Westers ◽  
Guus Westra ◽  
Angelica Draeger ◽  
Vincent H. Van der Velden ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Surface Expression ◽  
New Drugs ◽  
Refractory Anemia ◽  
Aberrant Expression ◽  
Clinical Behaviour ◽  
Flow Cytometric ◽  
Hla Dr

Abstract The WHO classification of myeloid disorders contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). The considerable differences in clinical behaviour of pure refractory anemia (RA) versus refractory cytopenia with multilineage dysplasia (RCMD) with or without ringsideroblasts are of importance in the management of patients with MDS. Flow cytometry may add additional diagnostic criteria to adequately discriminate RA from RCMD (+/− ringsideroblasts; (RS)) and may contribute in identifying Idiopathic Cytopenia of Undetermined Significance. We developed a 4-colour flow-cytometric procedure that comprises all differentiation stages of granulocytic, monocytic and erythroid lineages, instrumental for the recognition of various subpopulations within all three lineages in normal bone marrow samples. In 43 evaluable patients with MDS (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1 and 2), aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. Flow-cytometry identified aberrancies in granulopoiesis and monocytopoiesis in 93% and 74% of the cases, respectively. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia was detected, whereas in 59% abnormal surface expression of CD14, CD36 and CD33 indicating aberrant differentiation of monocytes. We defined aberrant myeloid blasts by a leukaemia associated phenotype (LAP) according to the definitions used in acute myeloid leukaemia. In 47% of the patients a LAP was detectable by demonstrating co-expression of CD5, CD7, CD19 and CD56 on CD34+ myeloid blasts. In all patients diagnosed as RA/RARS and MDS-U (n=12) according to WHO criteria, additional flow aberrancies were identified including a leukaemia associated phenotype of myeloid blasts in 41% of the cases. Only in 3 out of 28 cases with RCMD/RCMD-RS no erythroid aberrancies were detectable by flow-cytometry. In 9 normal control BM samples, no flow-cytometric abnormalities were present. It is concluded that flow-cytometry in MDS identifies aberrancies in the granulocytic and monocytic lineages and may classify patients with multi-lineage aberrancies not otherwise determined by cytology (WHO). Flow-cytometry may discriminate pure RA or MDS-U from RCMD. Since new drugs are emerging in low-risk MDS, the value of flow-cytometry might be of importance to further refine the classification in MDS. The exact role of these aberrant differentiation patterns on IPSS, clinical behaviour, impact on treatment decisions and as tool in disease monitoring have to be determined in future prospective studies.

P-ERK1/2 Is a Predictive Factor of Response to ESA Treatment in Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 294-294
Author(s):  
Emilie Frisan ◽  
Patrycja Pawlikowska ◽  
Cécile Pierre-Eugène ◽  
Valérie Bardet ◽  
Laure Gibault ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Conflicts Of Interest ◽  
Marrow Cell ◽  
Wilcoxon Test ◽  
Response To Treatment ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Bone Marrow Biopsies

Abstract Abstract 294 Endogenous serum erythropoietin (sEPO) less than 500UI/L and a transfusion requirement lower than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents (ESA) in low/int-1 myelodysplastic syndromes (MDS). However, the highest response rate hardly reaches 60% suggesting that other factors may influence the response. To investigate the biological signature of response to ESA, we enrolled 100 low/int-1 MDS patients in a prospective study of erythropoiesis at diagnosis before they were treated with ESA. According to the IWG 2006 criteria, 43 patients were non-responders. These patients had significantly higher serum EPO level, higher number of transfusion per month, and lower number of bone marrow-deriving BFU-E and CFU-E than responders. Analysis of CD34+-deriving erythroid progenitors by in vitro liquid culture, demonstrated that all MDS patients (n=54) had an increased apoptosis and a delayed expression of erythroid marker, glycophorin A (GPA). A collapse of EPO-induced DNA synthesis was observed in non-responders, while EPO-dependent erythroid cell differentiation and survival to Fas-induced apoptosis was equivalent in the two groups. Thus, non-responders exhibited an early and isolated default in EPO-induced cell proliferation, suggesting a defect in EPO-R signaling. Immunofluorescence to p-ERK1/2 before and after EPO-R stimulation in immature erythroblasts was negative in 6/8 non-responders, and positive in all 11 responders. Immunohistochemistry to p-ERK1/2 on bone marrow biopsies in 5 non-responders was negative and positive in immature cells in 4 responders. By flow cytometry, p-ERK1/2 expression in the CD71+/GPA− bone marrow cell fraction corresponding to immature erythroblasts (n=30) was significantly lower in non-responders (n=16) than in responders (n=14; Wilcoxon-test: p<0.0001). Receiver operator curve (ROC) analysis of the flow cytometry test demonstrated a good predictive value for the response to ESA with a 0.96 area under the curve (AUC) [95%CI: 0.89 – 1.00]. ROC were also constructed for BFU-E number, serum EPO level, and number of transfusion per month and the AUC were computed. p-ERK1/2 was equivalent to BFU-E and superior to serum EPO level or number of transfusion in predicting the response to ESA. Although requiring validation in a larger cohort, these results suggest that p-ERK1/2 is a ready tool available for the prediction of response to ESA in MDS patients. Disclosures: No relevant conflicts of interest to declare.

Identification by flow cytometry of the prostaglandin-producing cell populations of term human decidua

1991 ◽  
Vol 131 (2) ◽  
pp. 327-334 ◽  
Author(s):  
E. R. Norwitz ◽  
P. M. Starkey ◽  
A. López Bernal ◽  
A. C. Turnbull
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Human Leukocyte ◽  
Cell Types ◽  
Cell Populations ◽  
Decidual Cell ◽  
Leukocyte Antigen ◽  
Hla Dr ◽  
Pure Cell

ABSTRACT Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0·25% bovine serum albumin at 37 °C. PGF2α and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2α (fmol/106 cells per 3 h) exceeded that of PGE2 at 273 (108–322) 322) versus 97 (38–127) respectively (median (range)). The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2α and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2α and PGE2 production rates in human term decidua in vitro. Journal of Endocrinology (1991) 131, 327–334

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