The utility of a single tube 10-color flow cytometry for quantitative and qualitative analysis in myelodysplastic syndrome- a pilot study
Introduction
Myelodysplastic syndrome (MDS) is a clonal myeloid disorder that results in ineffective hematopoiesis and ≥1 cytopenia. The diagnosis of MDS in clinically suspected patients is that of exclusion based on peripheral blood and bone marrow morphology, blast count, and cytogenetics. However, low-grade MDS poses a diagnostic challenge, often due to absent morphological or cytogenetic criteria [1]. Only 30–40 % of MDS show cytogenetic abnormality [[2], [3], [4]]. Multiparametric flow cytometry (FCM) offers a more objective and robust diagnostic tool that aids in the diagnosing MDS particularly, the low-grade MDS without ring sideroblasts [[5], [6], [7]]. This has been documented in recent studies and recommendations [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]]. Also, it is a fast technique to analyze cell lineages, abnormal co-expression, and maturation asynchrony [19]. The prognostic value of FCM in MDS is proven in some studies [20,21].
Two types of analytical approaches are used either separately or combined in the flow cytometric analysis of MDS. The qualitative approach is similar to the morphological assessment of dysplasia in maturing cells of the bone marrow (BM) myeloid lineages. The maturation patterns of erythroid, granulocytes, and monocytes assessed using relevant markers, and any significant ‘deviation from normal’ suggest MDS [22]. Another approach, the pattern-based approach, is a subjective methodology that requires adequate experience and knowledge of the normal and abnormal maturation patterns [22]. Comprehensive reviews of maturation patterns in MDS and non-MDS are in the literature, and most patterns differentiate abnormal from normal [16,17,[23], [24], [25]]. However, this method uses an exhaustive antibody panels and lacks objectivity and established reference ranges. Despite international recommendations from the working group on FCM in MDS, these methods are hardly available worldwide at most platforms, mainly due to cost constraints, lack of expertise, and standardization.
To make FCM more objective, Ogata et al. have expressed aberrations on blast progenitor compartment by mean fluorescence intensities (MFI) or percentage of gated myeloblasts. [11,26] Chopra et al. analyzed both the qualitative and quantitative flow cytometric parameters of hematopoietic cells in patients with suspected MDS [13]. Based on these parameters, a score could be applied to exclude non-MDS from low-grade MDS cases [13]. Most of the studies on the quantitative approach have used 2 or 3 tubes. A single tube evaluating multiple markers will cut the cost and conserve time in processing for flow cytometric evaluation of MDS [[27], [28], [29], [30]]. This study aims to evaluate if a single 10-color screening flow cytometry tube can be used to differentiate MDS from the cause of secondary dyspoiesis. Reproducibility of the parameters suggested by Ogata et al., albeit in a single 10-color tube, is also evaluated.
Analysis of maturation patterns on multi-axial dot plots could be help screen abnormal patterns visually. Though subjective, we tried to assess the patterns in proven-MDS and the control group to look for any differences.
Section snippets
Study plan
After approval from the Institute Ethics Committee and obtaining written informed consent, 56 consecutive patients with cytopenia of an unidentifiable cause requiring bone marrow examination were included, in this prospective observational study, from September 2016 to May 2018. Patients with haematolymphoid malignancy (acute leukemia or lymphoma), dry tap on bone marrow aspiration, and those treated for MDS were excluded from this study.
Bone marrow aspirates from 56 consecutive patients with a
Statistical analysis
Statistical analysis was performed using R 3.5.1 statistical software (2018). The student's t-test was used to differentiate between parametric data sets. Kruskal Wallis Rank sum test was used to compare desired parameters amongst different patient groups with the control group. The expression of FCM parameters on control samples and non-MDS samples was used to calculate reference ranges.
The reference ranges (RR) / cut-off for the parameters analyzed by FCM were determined based on receiver
Patient characteristics
There were 23 patients of untreated proven-MDS, including high-grade (n = 10) and low-grade (n = 13) MDS cases. These included MDS-excess blasts (EB)1 (n = 7) and MDS-EB2 (n = 3), MDS- single lineage dysplasia (SLD) (n = 4), MDS-multilineage dysplasia (MLD) (n = 6), MDS-multilineage dysplasia with ring sideroblasts (MLD-RS) (n = 1), and MDS-unclassified (U)(n = 2) based on their cytogenetics report. The median age of the 23 proven MDS patients was 47 years (range 24–68 years). The male: female
Insight and comparison with previous literature
It challenging to distinguish low-grade MDS from non-MDS in clinically suspected MDS patients for a hematologist, even in the light of morphology and cytogenetics. The abnormalities of blast compartment cannot be appreciated on morphology unless they expand to ≥5% or an Auer rod is visualized. A complementary methodology that can distinguish reactive changes in the bone marrow from abnormal findings based on analysis of blast progenitor compartment could enhance the diagnostic power of FCM in
Conclusion
With a limited number of antibodies and a single tube that can effectively distinguish MDS from non-MDS, this study highlights that this tube can be applied in clinical practice to diagnose MDS in resource-constrained and busy laboratories. A negative FCM analysis will exclude myelodysplastic syndrome and prompt further exploration of secondary causes in cases of idiopathic cytopenia of unknown significance. Larger multi-centric studies to validate the usefulness of quantitative FCM
Author contribution
RC, JS, and PD have contributed to data collection, analysis and writing of manuscript; CS has applied statistics; AC, MP, and HPP have supervised the work, contributed to manuscript draft development including conception of the study, interpretation and review.
Funding source
None to disclose.
Funding
None.
Data availability statement
For original data please contact [email protected]
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgement
None to disclose.
References (44)
- et al.
Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: consensus statements and report from a working conference
Leuk. Res.
(2007) - et al.
Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome
Blood
(2001) - et al.
Update on developments in the diagnosis and prognostic evaluation of patients with myelodysplastic syndromes (MDS): consensus statements and report from an expert workshop
Leuk. Res.
(2012) - et al.
Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation
Blood
(2003) - et al.
Detection of hematopoietic maturation abnormalities by flow cytometry in myelodysplastic syndromes and its utility for the differential diagnosis with non-clonal disorders
Leuk. Res.
(2007) - et al.
Flow cytometric analysis of myelomonocytic cells by a pattern recognition approach is sensitive and specific in diagnosing myelodysplastic syndrome and related marrow diseases: emphasis on a global evaluation and recognition of diagnostic pitfalls
Leuk. Res.
(2008) - et al.
Revising flow cytometric mini-panel for diagnosing low-grade myelodysplastic syndromes: introducing a parameter quantifying CD33 expression on CD34+ cells
Leuk. Res.
(2018) - et al.
The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia
Blood
(2016) - et al.
Multiparameter flow cytometry is instrumental to distinguish myelodysplastic syndromes from non-neoplastic cytopenias
Eur. J. Cancer
(2016) - et al.
Flow cytometry diagnosis in myelodysplastic syndrome: current practice in Latin America and comparison with other regions of the world
Leuk. Res.
(2019)
Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of myelodysplastic syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts
Haematologica
Identification of novel cytogenetic markers with prognostic significance in a series of 968 patients with primary myelodysplastic syndromes
Haematologica
Cytogenetic features in myelodysplastic syndromes
Ann. Hematol.
Incidence, characterization and prognostic significance of chromosomal abnormalities in 640 patients with primary myelodysplastic syndromes. Grupo Cooperativo Español de Citogenética Hematológica
Br. J. Haematol.
Minimal diagnostic criteria for myelodysplastic syndromes and separation from ICUS and IDUS: update and open questions
Eur. J. Clin. Invest.
Proposed minimal diagnostic criteria for myelodysplastic syndromes (MDS) and potential pre-MDS conditions
Oncotarget
Diagnostic application and clinical significance of FCM progress scoring system based on immunophenotyping in CD34+ blasts in myelodysplastic syndromes
Cytometry B Clin. Cytom.
Establishment and validation of an updated diagnostic FCM scoring system based on pooled immunophenotyping in CD34+ blasts and its clinical significance for myelodysplastic syndromes
PLoS One
Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study
Haematologica
Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome
Leukemia
Flow cytometry in myelodysplastic syndrome: analysis of diagnostic utility using maturation pattern-based and quantitative approaches
Ann. Hematol.
Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group
Leukemia
Cited by (3)
High Expression of CD300A Predicts Poor Survival in Acute Myeloid Leukemia
2023, Acta HaematologicaDiagnostic and Prognostic Values of Integrated Flow Cytometric Score in Myelodysplastic Syndrome
2023, Chinese General Practice
- 1
These authors have contributed equally to the manuscript.