Comparative analysis of feature-based ML and CNN for binucleated erythroblast quantification in myelodysplastic syndrome patients using imaging flow cytometry data.

Myelodysplastic syndrome is primarily characterized by dysplasia in the bone marrow (BM), presenting a challenge in consistent morphology interpretation. Accurate diagnosis through traditional slide-based analysis is difficult, necessitating a standardized objective technique. Over the past two decades, imaging flow cytometry (IFC) has proven effective in combining image-based morphometric analyses with high-parameter phenotyping. We have previously demonstrated the effectiveness of combining IFC with a feature-based machine learning algorithm to accurately identify and quantify rare binucleated erythroblasts (BNEs) in dyserythropoietic BM cells. However, a feature-based workflow poses challenges requiring software-specific expertise. Here we employ a Convolutional Neural Network (CNN) algorithm for BNE identification and differentiation from doublets and cells with irregular nuclear morphology in IFC data. We demonstrate that this simplified AI workflow, coupled with a powerful CNN algorithm, achieves comparable BNE quantification accuracy to manual and feature-based analysis with substantial time savings, eliminating workflow complexity. This streamlined approach holds significant clinical value, enhancing IFC accessibility for routine diagnostic purposes.

The impact of erythroblast enucleation efficiency on the severity of anemia in patients with myelodysplastic syndrome.

Anemia is the most common manifestation in myelodysplastic syndrome (MDS) patients, but the cause of ineffective hematopoiesis is not fully understood. Enucleation is an important event in the maturation process of erythroblasts. According to a series of morphological phenotypes of the pathological development of MDS erythroblasts, we speculate that there may be enucleation disorders. To verify this hypothesis, we cultured MDS bone marrow CD34+ cells in vitro and induced erythroblast development. The results showed that erythroblast enucleation in MDS was significantly lower than that in the normal group, and the rate of enucleation was positively correlated with hemoglobin concentration. Risk stratification of MDS was performed to further analyze the differences in enucleation among the normal group, low-middle risk group and high-risk group. The results showed that the enucleation rate of the high risk group was higher than that of the low-middle risk group but still lower than that of the normal group. Moreover, the expression of pERK and pAKT in MDS erythroblasts in the high risk group was higher than that in the normal group, while the expression of pERK and pAKT in the low-middle risk group was lower than that in the normal group. Furthermore, the enucleation of MDS was positively correlated with the phosphorylation degree of ERK and AKT. In conclusion, this study reveals that the enucleation of erythroblasts is one of the possible causes of anemia in MDS. Video Abstract.

B lymphocytes transdifferentiate into immunosuppressive erythroblast-like cells.

Recent studies have demonstrated that a particular group of nucleated cells that exhibit erythroid markers (TER119 in mice and CD235a in humans) possess the ability to suppress the immune system and promote tumor growth. These cells are known as CD45+ erythroid progenitor cells (EPCs). According to our study, it appears that a subset of these CD45+ EPCs originate from B lymphocytes. Under conditions of hypoxia, mouse B lymphoma cells are capable of converting to erythroblast-like cells, which display phenotypes of CD45+TER119+ cells, including immunosuppressive effects on CD8 T cells. Furthermore, non-neoplastic B cells have similar differentiation abilities and exert the same immunosuppressive effect under anemia or tumor conditions in mice. Similar B cells exist in neonatal mice, which provides an explanation for the potential origin of immunosuppressive erythroid cells in newborns. Additionally, CD19+CD235a+ double-positive cells can be identified in the peripheral blood of patients with chronic lymphocytic leukemia. These findings indicate that some CD45+ EPCs are transdifferentiated from a selective population of CD19+ B lymphocytes in response to environmental stresses, highlighting the plasticity of B lymphocytes. We anticipate a potential therapeutic implication, in that targeting a specific set of B cells instead of erythroid cells should be expected to restore adaptive immunity and delay cancer progression.

Ablation of Tmcc2 Gene Impairs Erythropoiesis in Mice.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 (Tmcc2) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2-/- pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2-/- nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71+TER119+ erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

Erythroblastic synartesis associated with lymphoproliferative disorder: There can be more than meets the eye.

Erythroblastic synartesis is a rare cause of acquired dyserythropoiesis. Only 9 cases have been previously reported. We hereby report 3 cases of patients diagnosed with erythroblastic synartesis associated with monoclonal immunoglobulin and an overt malignant lymphoid disorder. A different B-cell clone may produce the monoclonal immunoglobulin, forming a biclonal disorder. In light of these data and literature review, treatment targeting the paraprotein seems to be efficient to control synartesis and correct anemia. In the case of monoclonal gammapathy associated with chronic lymphocytic leukemia, therapeutics should be adapted to control both chronic lymphocytic leukemia and monitored monoclonal immunoglobulin titer.

Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts.

Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of ß-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

Pleckstrin-2 is essential for erythropoiesis in ß-thalassemic mice, reducing apoptosis and enhancing enucleation.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as ß-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in ß-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in ß-thalassemia. We hypothesize that compensatory mechanisms are required in ß-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in ß-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during ß-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in ß-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in ß-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in ß-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in ß-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.

Nucleated red blood cells participate in myocardial regeneration in the toad Bufo Gargarizan Gargarizan.

Heart regeneration is negligible in humans and mammals but remarkable in some ectotherms. Humans and mammals lack nucleated red blood cells (NRBCs), while ectotherms have sufficient NRBCs. This study used Bufo gargarizan gargarizan, a Chinese toad subspecies, as a model animal to verify our hypothesis that NRBCs participate in myocardial regeneration. NRBC infiltration into myocardium was seen in the healthy toad hearts. Heart needle-injury was used as an enlarged model of physiological cardiomyocyte loss. It recovered quickly and scarlessly. NRBC infiltration increased during the recovery. Transwell assay was done to in vitro explore effects of myocardial injury on NRBCs. In the transwell system, NRBCs could infiltrate into cardiac pieces and could transdifferentiate toward cardiomyocytes. Heart apex cautery caused approximately 5% of the ventricle to be injured to varying degrees. In the mildly to moderately injured regions, NRBC infiltration increased and myocardial regeneration started soon after the inflammatory response; the severely damaged region underwent inflammation, scarring, and vascularity before NRBC infiltration and myocardial regeneration, and recovered scarlessly in four months. NRBCs were seen in the newly formed myocardium. Enzyme-linked immunosorbent assay and Western blotting showed that the levels of tumor necrosis factor-α, interleukin- 1ß, 6, and11, cardiotrophin-1, vascular endothelial growth factor, erythropoietin, matrix metalloproteinase- 2 and 9 in the serum and/or cardiac tissues fluctuated in different patterns during the cardiac injury-regeneration. Cardiotrophin-1 could induce toad NRBC transdifferentiation toward cardiomyocytes in vitro. Taken together, the results suggest that the NRBC is a cell source for cardiomyocyte renewal/regeneration in the toad; cardiomyocyte loss triggers a series of biological processes, facilitating NRBC infiltration and transition to cardiomyocytes. This finding may guide a new direction for improving human myocardial regeneration.

Exploring dyserythropoiesis in patients with myelodysplastic syndrome by imaging flow cytometry and machine-learning assisted morphometrics.

BACKGROUND: The hallmark of myelodysplastic syndrome (MDS) remains dysplasia in the bone marrow (BM). However, diagnosing MDS may be challenging and subject to inter-observer variability. Thus, there is an unmet need for novel objective, standardized and reproducible methods for evaluating dysplasia. Imaging flow cytometry (IFC) offers combined analyses of phenotypic and image-based morphometric parameters, for example, cell size and nuclearity. Hence, we hypothesized IFC to be a useful tool in MDS diagnostics. METHODS: Using a different-from-normal approach, we investigated dyserythropoiesis by quantifying morphometric features in a median of 5953 erythroblasts (range: 489-68,503) from 14 MDS patients, 11 healthy donors, 6 non-MDS controls with increased erythropoiesis, and 6 patients with cytopenia. RESULTS: First, we morphometrically confirmed normal erythroid maturation, as immunophenotypically defined erythroid precursors could be sequenced by significantly decreasing cell-, nuclear- and cytoplasm area. In MDS samples, we demonstrated cell size enlargement and increased fractions of macronormoblasts in late-stage erythroblasts (both p < .0001). Interestingly, cytopenic controls with high-risk mutational patterns displayed highly aberrant cell size morphometrics. Furthermore, assisted by machine learning algorithms, we reliably identified and enumerated true binucleated erythroblasts at a significantly higher frequency in two out of three erythroblast maturation stages in MDS patients compared to normal BM (both p = .0001). CONCLUSION: We demonstrate proof-of-concept results of the applicability of automated IFC-based techniques to study and quantify morphometric changes in dyserythropoietic BM cells. We propose that IFC holds great promise as a powerful and objective tool in the complex setting of MDS diagnostics with the potential for minimizing inter-observer variability.

PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome.

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2Δ/Δ) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1+/-Ezh2Δ/Δ) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1+/-Ezh2Δ/Δ mice. Cdkn2a, a major PRC2 target encoding p19Arf, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1+/-Ezh2Δ/Δ erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1+/-Ezh2Δ/Δ mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

Real-world diagnostic testing patterns for assessment of ring sideroblasts and SF3B1 mutations in patients with newly diagnosed lower-risk myelodysplastic syndromes.

INTRODUCTION: The presence of ring sideroblasts (RS) and mutation of the SF3B1 gene are diagnostic of lower-risk (LR) myelodysplastic syndromes (MDS) and are correlated with favorable outcomes. However, information on testing and reporting in community-based clinical settings is scarce. This study from the Connect® MDS/AML Disease Registry aimed to compare the frequency of RS and SF3B1 reporting for patients with LR-MDS, before and after publication of the 2016 World Health Organization (WHO) MDS classification criteria. METHODS: Ring sideroblasts assessment and molecular testing data were collected from patients with LR-MDS at enrollment in the Registry. Patients enrolled between December 2013 and the data cutoff of March 2020 were included in this analysis. RESULTS: Among 489 patients with LR-MDS, 434 (88.8%) underwent RS assessment; 190 were assessed prior to the 2016 WHO guidelines (Cohort A), and 244 after (Cohort B). In Cohort A, 87 (45.8%) patients had RS identified; 29 (33.3%) patients had RS < 15%, none of whom underwent molecular testing for SF3B1. In Cohort B, 96 (39.3%) patients had RS identified; 31 (32.3%) patients had < 15% RS, with 13 undergoing molecular testing of which 10 were assessed for SF3B1. CONCLUSIONS: In the Connect® MDS/AML Registry, only 32% of patients with <15% RS underwent SF3B1 testing after the publication of the WHO 2016 classification criteria. There was no change in RS assessment frequency before and after publication, despite the potential impact on diagnostic subtyping and therapy selection, suggesting an unmet need for education to increase testing rates for SF3B1 mutations.

The role of ORC4 in enucleation of Murine Erythroleukemia (MEL) cells is similar to that in oocyte polar body extrusion.

The Origin Replication Complex subunit 4 (ORC4) is one in six subunits of the Origin Replication Complexes (ORCs) which is essential for initiating licensing at DNA replication origins and recruiting adaptor molecules necessary for various cellular processes. Previously, we reported that ORC4 also plays a vital role in polar body extrusion (PBE) during oogenesis in which half the chromosomes are extruded from the oocyte. We hypothesized that ORC4 might play a broader role in chromatin elimination. We tested its role in enucleation during the development of erythrocytes. Murine erythroleukemia (MEL) cells can be propagated in culture indefinitely and can be induced to enucleate their DNA by treatment with Vacuolin-1, thereby mimicking normal erythrocyte enucleation. We found that ORC4 appeared around the nuclei of the MEL cells with Vacuolin-1 treatment, gradually increasing in thickness before enucleation. We then tested whether ORC4 was required for MEL enucleation by down regulating ORC4 with siRNA-ORC4 during Vacuolin-1 treatment and found that this prevented MEL enucleation. These data are consistent with the model that ORC4 is required for erythroblast enucleation just as it is for oocyte PBE. They suggest a new model in which ORC4 expression is a marker for the initiation to the enucleation pathway.

Role of Plasma Gelsolin Protein in the Final Stage of Erythropoiesis and in Correction of Erythroid Dysplasia In Vitro.

Gelsolin, an actin-remodeling protein, is involved in cell motility, cytoskeletal remodeling, and cytokinesis and is abnormally expressed in many cancers. Recently, human recombinant plasma gelsolin protein (pGSN) was reported to have important roles in cell cycle and maturation of primary erythroblasts. However, the role of human plasma gelsolin in late stage erythroblasts prior to enucleation and putative clinical relevance in patients with myelodysplastic syndrome (MDS) and hemato-oncologic diseases have not been reported. Polychromatic and orthochromatic erythroblasts differentiated from human cord blood CD34+ cells, and human bone marrow (BM) cells derived from patients with MDS, were cultured in serum-free medium containing pGSN. Effects of pGSN on mitochondria, erythroid dysplasia, and enucleation were assessed in cellular and transcriptional levels. With pGSN treatment, terminal maturation at the stage of poly- and ortho-chromatic erythroblasts was enhanced, with higher numbers of orthochromatic erythroblasts and enucleated red blood cells (RBCs). pGSN also significantly decreased dysplastic features of cell morphology. Moreover, we found that patients with MDS with multi-lineage dysplasia or with excess blasts-1 showed significantly decreased expression of gelsolin mRNA (GSN) in their peripheral blood. When BM erythroblasts of MDS patients were cultured with pGSN, levels of mRNA transcripts related to terminal erythropoiesis and enucleation were markedly increased, with significantly decreased erythroid dysplasia. Moreover, pGSN treatment enhanced mitochondrial transmembrane potential that is unregulated in MDS and cultured cells. Our findings demonstrate a key role for plasma gelsolin in erythropoiesis and in gelsolin-depleted MDS patients, and raises the possibility that pGSN administration may promote erythropoiesis in erythroid dysplasia.