Elsevier

Leukemia Research

Volume 107, August 2021, 106651
Leukemia Research

The utility of a single tube 10-color flow cytometry for quantitative and qualitative analysis in myelodysplastic syndrome- a pilot study

https://doi.org/10.1016/j.leukres.2021.106651Get rights and content

Highlights

  • Single tube 10-color flow cytometry for myelodysplastic syndrome yielded reproducible sensitivity and specificity for Ogata score.

  • The sensitivity of quantitative analysis improved by addition of CD38 expression on the CD34+CD19- cluster.

  • Addition of aberrant marker expression on the CD34+CD19- cluster further improved the diagnostic power of the tube in this pilot study.

  • Qualitative analysis by radar-plots may help in visual screening for myelodysplastic syndrome in a single tube.

Abstract

Introduction

Assessment of myelodysplasia (MDS) by flow cytometry (FCM) includes elaborate panels, and interpretation is observer-dependent. This study evaluates single tube 10-color FCM in a test cohort of clinically suspected MDS patients.

Methods

We analyzed fifty-six bone marrow (BM) samples from clinically suspected MDS patients in a morphology-blinded manner along with controls using a 10-color single tube flow cytometry. We analyzed the reproducibility of Ogata score and modified FCM scores, additionally incorporating the proportion of CD15, CD11b, CD56, and CD38MFI on CD34+CD19-cluster for each patient. Patients were grouped as proven-MDS, suspected-MDS, and non-MDS groups based on morphology and cytogenetics. Optimized multi-axial radar-plots were also used to analyze maturation patterns in the granulocytic, monocytic, and blast progenitor compartments of proven-MDS cases and controls.

Results

Flow cytometric abnormalities ≥3 were present in proven-MDS (n = 23) with a sensitivity and specificity of 78 % and 94 %, respectively, as per Ogata score. The addition of CD38 MFI to the score yielded sensitivity and specificity of 82 % and 88 %, respectively. Additional analysis of aberrant expression of CD15, CD11b, and CD56 increased the diagnostic power of the FCM score. A qualitative analysis of data also showed differences in maturation patterns in proven-MDS compared to the control group.

Conclusion

Single tube 10-color FCM scoring, including Ogata score, modified-FCM scores, and radar plots pattern analysis, showed significant abnormalities in proven-MDS cases in this pilot study. Large databases, including FCM-scoring and pattern-based analysis for normal BM maturation, could be further validated and standardized for screening MDS.

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.

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    These authors have contributed equally to the manuscript.

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