Exploring Imaging Flow Cytometry As a Tool for Evaluating Dyserythropoiesis in Myelodysplastic Syndrome
Authors: Rosenberg Carina A , Bill Marie , Kerndrup Gitte Birk , Hauerslev Mathias , Hokland Peter , Ludvigsen Maja 
Published: October 2019

Abstract
Introduction
In myelodysplastic syndrome (MDS), multicolor flow cytometry (FC) offers a supplement to the morphological, cytogenetic and molecular evaluation of the hematopoietic lineages in the bone marrow (BM). In addition to providing a robust enumeration of immature myeloid cells, FC allows for identification of aberrant antigen expression patterns. While aberrant findings are well-defined in the myelo-monocytic lineages, the erythroid and megakaryocytic cell lineages are less well characterized by conventional FC. Imaging flow cytometry (IFC) is a novel methodology that bridge multicolor FC and high-throughput capacity with collection of multispectral imagery of single cells. This allows for the simultaneous evaluation, quantification and subsequent statistical analysis of both surface-, nuclear- and cytoplasmic immunophenotypic and morphometric characteristics on a practically unlimited number of cells in suspension. In this study, we hypothesized the IFC technique to be useful in discriminating dyserythropoietic from normal BM.
Methods
Mononuclear BM cells from 6 healthy donors and 2 MDS patients were thawed and stained with monoclonal antibodies against CD235a, CD45, CD71, CD117 and CD105. DRAQ5 was included for DNA staining, and Zombie Violet was included for viability. Data acquisition was performed on an ImageStream®XMarkII (Amnis/Merck), and subsequent analysis was done using the IDEAS software package. The median number of acquired cells was 130,000 for healthy BM (range: 70,548-300,000) and for MDS samples the equivalent number was 253,255 and 260,000, respectively. Nucleated erythroid cells (NECs) were identified based on the CD45lowDRAQ5highCD71+ immunophenotype and the coexpression of either CD235 and/or CD105. Next, proerythroblasts (ProE), basophilic erythroblasts (BasoE) and poly-orthochromatic erythroblasts (Poly_OrthoE) were distinguished based on the expression of CD117 and CD105 (Fig. 1, upper panel). Morphometric features were examined within function masks covering the cellular, nuclear and cytoplasmic area. From these masks we calculated cell area, nucleus area, nucleus elongatedness, nucleus circularity score, and nuclear location (delta centroid XY) (Fig. 1, lower panel). Data from healthy controls (Fig. 1A) served as biological reference for phenotypic and morphometric measurements of MDS samples.
Results & perspectives
In one MDS patient with single lineage dysplasia (MDS-SLD) (Fig. 1B), morphological dysplastic changes were seen in less than 10% of erythropoiesis, and manifested as nuclear lobulations, cytoplasmic granules and megaloblastic changes. We examined 6470 NECs by IFC and found an increased elongatedness and a decreased circularity score of the nuclei in the ProE population, reflecting irregular nuclear shape. Moreover, for the ProE and BasoE maturation stages, we detected increased cell size as well as enlarged nuclei of BasoE. This finding corresponded to the megaloblastic changes seen by morphology. Taken together, data from IFC confirmed the morphological findings in this MDS patient.
The second patient (Fig. 1C) had MDS with excess blasts (MDS-EB-1). Morphologic examination showed mild dyserythropoiesis in less than 10% of erythropoietic cells with signs of multinuclearity, nuclear lobulations, and ringed sideroblasts. Interestingly, IFC analysis of 25,240 NECs identified aberrant delta centroid XY corresponding to decentralization of the nucleus in the ProE population, as also seen in the MDS-SLD patient. As such, with the applied IFC features, we were not able to detect changes corresponding to the morphological findings, which could be due to the low percentage of dysplastic erythroid cells. However, the deviant morphometric parameters detected by IFC in this patient identified additional dysmorphometric features of interest.
To conclude, we provide proof-of-concept of a novel experimental approach to study and quantify morphometric changes in samples from MDS patients. Certainly, the technique needs to be explored in a larger patient material and we are currently extending our study cohort. Using a different-from-normal approach, our preliminary data was sufficient to demonstrate dysmorphometric changes in two MDS patients with only mild morphological dyserythropoiesis. Thus, we propose that IFC holds great promise as a powerful tool in the challenging setting of MDS diagnostics.

Disclosures
No relevant conflicts of interest to declare.