Flow Cytometry In Myelodysplastic Syndromes Research Articles

FLOW CYTOMETRY IN MYELODYSPLASTIC SYNDROME- CORRELATION WITH CYTOMORPHOLOGY RESULTS

FLOW CYTOMETRY IN MYELODYSPLASTIC SYNDROME- CORRELATION WITH CYTOMORPHOLOGY RESULTS

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Flow cytometry in myelodysplastic syndromes: present and future

Flow cytometry in myelodysplastic syndromes: present and future

Issue Highlights — May 2021

Issue Highlights — May 2021

Clinical implication of minimal residue disease monitoring by WT1 gene detection and flow cytometry in myelodysplastic syndrome with allogeneic stem cell transplantation

目的探讨骨髓增生异常综合征（MDS）患者在接受异基因造血干细胞移植（allo-HSCT）后基于WT1基因及流式细胞术（FCM）监测微小残留病（MRD）的意义。方法分别采用实时定量聚合酶链反应（RQ-PCR）和8色FCM检测2011年2月至2015年10月于北京大学人民医院接受allo-HSCT的MDS患者移植前及移植后骨髓中WT1基因表达及异常免疫表型，并与临床结果进行相关分析。结果92例患者中，40例（48.2%）移植后WT1阳性，9例（10.8%）FCM阳性（FCM检测骨髓表型异常髓系幼稚细胞≥0.01%），27例（29.3%）患者MRDco阳性[连续2次WT1≥0.6%（间隔2周）或连续2次FCM阳性（间隔2周）或同一骨髓标本WT1≥0.6%且FCM阳性]。单因素分析显示移植后FCM阳性、MRDco阳性与复发相关（P<0.001，P=0.017）。FCM（+）、FCM（−）组移植后2年累积复发率（CIR）分别为66.7%、1.2%（P<0.001）；MRDco（+）、MRDco（−）组移植后2年CIR分别为23.0%、1.6%（P=0.004）。移植后WT1、FCM、MRDco预测复发的敏感性均为66.7%，特异性分别为59.0%、96.4%、74.7%。结论FCM和基于WT1/FCM的MRDco可作为MDS患者MRD监测的有效指标；基于MRDco标准进行干预可降低移植后复发率。

The Use of Flow Cytometry in Myelodysplastic Syndromes: A Review.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematopoietic stem cell diseases categorized by dysplasia in one or more hematopoietic cell lineages, as well as cytopenia and functional abnormalities in bone marrow cells. Several MDS classification methods have been proposed to categorize the disease and help professionals better plan in patients’ treatment. The World Health Organization classification, released in 2008 and revised in 2016, is the currently and the most used classification method worldwide. Recent advances in MDS molecular biology and innovations in flow cytometry have enabled the development of new parameters for MDS diagnosis and classification. Several groups have published flow cytometry scores and guidelines useful for the diagnosis and/or prognosis of MDS, which are mostly based on detecting immunophenotypic abnormalities in granulocyte, monocyte, and lymphoid lineages. Here, we review the current literature and discuss the main parameters that should be analyzed by flow cytometry with the aim of refining MDS diagnosis and prognosis. Furthermore, we discuss the critical role of flow cytometry and molecular biology in MDS diagnosis and prognosis, as well as the current challenges and future perspectives involving these techniques.

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes.

The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. The rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become applicable in clinical practice, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians. In this review, we discuss the most relevant progresses in detection of marrow dysplasia by FCM in MDS

Clinico-Pathological Spectrum and Role of Flow Cytometry in Myelodysplastic Syndrome: Experience of a Tertiary Care Centre in North India

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell disorder characterized by morphological dysplasia which is subjective. There are numerous morphological mimickers of dysplasia which need to be differentiated from MDS. Cytogenetic abnormalities form an important component of the existing risk stratification protocols though seen in 30-70% individuals. Flow cytometry (FCM) on the other hand provides an objective evidence of dysplasia even in the absence of appreciable dysplastic morphological features or non-informative cytogenetics.Clinico-pathological profiles of 112 consecutive patients diagnosed as MDS over 45 months were analyzed. FCM analysis with single tube 6 colour combination using monoclonal antibodies against CD13, CD11b, CD16, CD34, CD45 and CD56 was applied in 43 patients. The panel was also applied in 14 other cases with morphological dysplasia and 21 age matched controls. Six prominent features, namely i) hypogranulatrity based on reduced side scatter ii) side scatter peak channel values (SSC PCV) ratio of the granulocytes and lymphoid cells, myeloid maturation pattern based on iii) CD13/16, iv) CD13/11b, v) CD11b/16 and vi) blast percentage based on CD34/SSC dot plot amongst all nucleated cells were noted. A score of 1 was assigned to each abnormality. A score of ≥3 was considered as "positive for MDS", 2 as ’suspicious for MDS’ and ≤ 1 as "negative for MDS". Cytogenetic studies could be done in 65 patients using G banding and counting 20 metaphases.There were 69 males and 43 females (M: F=1.6:1), with a median age of 55.5 (range 3-87 years). Majority of these patients presented with pancytopenia 67.8% (76/112) followed by bicytopenia in 29.5% (33/112). As per the recent WHO classification 2016, the cases were classified as MDS-single lineage dysplasia (n=7, 6.25%), MDS-multilineage dysplasia (n=48, 42.86%), MDS excess blast 1 (n=24, 21.42%), MDS execss blast 2 (n=18, 16.07%), Hypoplastic MDS (n=14, 12.5%), and refractory cytopenia of childhood (n=1, 0.9%). Chromosomal abnormality were detected in 32/65 (49.2%) patients, where complex karyotype was the most common abnormality. Most frequent individual chromosomal abnormality observed was chromosome 7 (11/32; 34.3%) in the form of monosomy and interstitial deletions. IPSS -R prognostic risk stratification showed majority of the patients (39/65, 59.9%) belonging to the very high and high risk groups. On FCM analysis, an SSC PCV ratio was found to differ significantly among the three groups (p <0.05) and a cut off value of < 6.16 was found to be compatible with a diagnosis of MDS. The most common finding was abnormal maturation pattern of granulocytes as reflected by the CD13/16 (95.3%), CD13/11b (69.8%) and CD11b/16 (74.4%) dot plots. An FCM score >3 was found in 84.7% of MDS patients. Overall sensitivity, specificity, positive and negative predictive values of this panel was found to be 86%, 64.2%, 90% and 62.5% respectively.MDS appears to have a relatively distinct clinicopathological profile in the Indian subcontinent, with a lower median age of presentation and a higher proportion of high IPSS-R risk group. Limited panel FCM can fairly predict the diagnosis of MDS using the objective scoring system and thus should be incorporated as a part of the routine work up in patients presenting with persistent cytopenia with or without morphological evidence of dyspoiesis. [Display omitted] DisclosuresRahman:SGPGI, Lucknow , India: Employment, Research Funding. Gupta:SGPGI, Lucknow, India: Employment, Research Funding. Singh:SGPGI, Lucknow, India: Employment. Kumari:SGPGI,Lucknow, India: Employment. Yadav:SGPGI,Lucknow, India: Employment. Kumar:SGPGI,Lucknow, India: Employment. Gupta:SGPGI,Lucknow, India: Employment. Nityanand:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment, Research Funding.

Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes.

Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10.

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50–60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches.

Immunophenotyping of myelodysplastic syndromes by flow cytometry and its potential clinical application

Myelodysplastic syndromes (MDS) is a heterogeneous group of clone bone marrow disorders. The diagnosis of MDS can be challenging when dysplastic features are minimal and cytogenetic analysis shows no abnormalities. The high sensitivity of flow cytometry (FCM) in the detection of dysplasia in myeloid lineages makes it a valuable tool to distinguish possible clonal causes of cytopenia(s) from non-clonal causes. Due to the heterogeneity and absence of specific FCM markers for MDS, the immunophenotyping of MDS by FCM remains a big difficulty. Here we will focus on how to perform immunophenotyping of MDS by FCM and the potential diagnostic, prognostic and therapeutic value of FCM in MDS. (Chin J Lab Med, 2016, 39: 332-335) Key words: Myelodysplastic syndromes; Immunophenotyping; Flow cytometry

Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features.

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10-<20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia.

Myelodysplastic syndromes: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

P. Fenaux1, D. Haase2, G. F. Sanz3, V. Santini4 & C. Buske5 on behalf of the ESMO Guidelines Working Group* Service d’Hematologie Clinique, Groupe Francophone des Myelodysplasies (GFM), Hopital St Louis (Assistance Publique, Hopitaux de Paris) and Paris 7 University, Paris, France; Clinics of Hematology and Medical Oncology, University Medicine, Goettingen, Germany; Department of Haematology, Hospital Universitario y Politecnico La Fe, Valencia, Spain; Functional Unit of Haematology, AOU Careggi, University of Florence, Firenze, Italy; Comprehensive Cancer Center Ulm, Institute of Experimental Cancer Research, University Hospital, Ulm, Germany

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Cutting Edge: Flow Cytometry in Myelodysplastic Syndromes

Flow cytometry (FC) is recognized as one of the important tools in the diagnosis of myelodysplastic syndromes (MDS), especially when cytomorphology and cytogenetics are inconclusive. Minimal requirements to analyze dysplasia using FC have been proposed by the International Workshops from the European LeukemiaNet on Standardization of FC in MDS. These should enable a categorization of FC results in cytopenic patients as "normal," "suggestive of," or "indicative of" MDS. Notably, FC should be considered as part of an integrated diagnosis, rather than as a separate technique. This article highlights tools to implement FC in the diagnostic workup of MDS. Current advances in FC for diagnosis and prognosis of MDS are also introduced.

P-051 Flow cytometry in myelodysplastic syndromes: A report from an international consortium and the European LeukemiaNet Working Group

P-051 Flow cytometry in myelodysplastic syndromes: A report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry in myelodysplastic syndrome: analysis of diagnostic utility using maturation pattern-based and quantitative approaches

Flow cytometry (FCM) is being increasingly evaluated for the diagnosis of myelodysplastic syndrome (MDS). We employed multiple FCM approaches to assess MDS. Five-color FCM, morphology blind, was done on bone marrow aspirates of 57 suspected MDS and 31 normal controls. Maturation pattern, quantitative FCM for low-grade MDS that awards FCM score, and expression of selected antigens on erythroid cells and CD34(+) blasts were evaluated. FCM results were correlated with clinical and laboratory workup. Patients (n = 57) included proven MDS (n = 14), suspected MDS (n = 13), and non-MDS (n = 30). By pattern-based approach, all proven cases were FCM positive. In suspected MDS, 11 (84.61 %) were positive including morphology-negative cases, and two (15.38 %) were intermediate. In non-MDS cases, 27 of 30 (90 %) were FCM negative, 2 of 30 (6.67 %) intermediate, and 1 of 30 (3.33 %) a hematinic-responsive case, positive. Quantitative parameters that characterized MDS included FCM score of >3, percentage CD34(+) B cells, and expression of CD11b, CD15, and CD56 on myeloblasts. CD71 MFI on CD235a(+) erythroblasts and CD38 MFI on myeloblasts were significantly lower in MDS. The former was present in FCM-intermediate suspected MDS but not FCM-intermediate non-MDS cases. Used in the overall clinical context, both maturation pattern recognition and quantitative approaches, the latter for low-grade MDS, are sensitive methods of diagnosing MDS, including cases negative by morphology and cytogenetics, especially if combined with evaluation of selected antigens, CD71 on CD235a(+) cells and CD38 on CD34(+) cells. The value of FCM in morphology-negative cases needs better definition of specificity through more extensive evaluation of secondary dyspoiesis.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: Report from the Dutch Working Party on Flow Cytometry in MDS

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005–2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Flow cytometry immunophenotyping for the evaluation of bone marrow dysplasia

The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and determining individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly a need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. Rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become clinically applicable, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators and the results should be easily understood by clinicians. In this report, we reviewed the most relevant progresses in detection of marrow dysplasia by FCM in MDS as defined by WHO criteria.