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.

A 6-year-old spayed female Scottish Fold cat presented with lethargy and anorexia. A complete blood cell count indicated severe anemia and mild thrombocytopenia. Examination of peripheral blood smears revealed marked changes in the erythroid lineage, including the presence of basophilic stippling and Howell-Jolly bodies as well as an increase in nucleated erythrocytes, polychromatophils, ovalocytes, and schistocytes. Additionally, some erythrocytes contained a ring or figure-eight shaped structure known as a Cabot ring, which were especially observed in polychromatophilic erythrocytes. Hemolytic diseases (Mycoplasma infection and IMHA) were diagnostically excluded, and the cat was treated through prednisolone administration, whole blood transfusion, and administration of vitamins (K2 and B12); however, the anemia progressively worsened. Cabot rings were observed until Day 22 and subsequently disappeared as the number of nucleated RBCs increased, and the erythrocyte lineage shifted to immature population. On Day 42, peripheral blood examination revealed further left shifting and appearance of many rubriblasts. The patient died at home on Day 43. Necropsy revealed neoplastic cells infiltrating the bone marrow and other organs, which were immunopositive to CD71 which is an erythroid lineage marker. In humans, Cabot rings have been observed in megaloblastic anemia, lead poisoning, myelodysplastic syndrome, and myelofibrosis; further, they are thought to be related to stressed bone marrow and dyserythropoiesis. This is the first case report of a cat with Cabot rings, which are suggestive of defects in erythroid lineage production.

BACKGROUND: Immune checkpoints are regulators of the immune system response that allow self-tolerance. Molecules such as Programmed Cell Death Protein 1 (PD-1) and its Ligand (PD-L1) participate in the immune checkpoint by signaling co-inhibition of lymphocyte responses. In cancers, PD-L1 expression is associated with the immune evasion mechanism, which favors tumor growth. The use of anti-PD-1/PD-L1 drugs is already well described in solid tumors, but still not fully understood in hematologic malignancies. Myelodysplastic neoplasms (MDSs) are heterogeneous bone marrow disorders with an increased risk of progression to Acute Myeloid Leukemia (AML). The MDS affects hematopoietic stem cells and its pathogenesis is linked to genetic and epigenetic defects, in addition to immune dysregulation. The influence of the PD-L1 on the MDS remains unknown.
METHODS: In this study, we evaluated the mRNA expression of the PD-L1 in 53 patients with MDS, classified according to the WHO 2016 Classification.
RESULTS: Patients with dyserythropoiesis presented significantly higher PD-L1 expression than patients without dyserythropoiesis (p= 0.050). Patients classified as having MDS with an excess of blasts 2 (MDS-EB2) presented a significant upregulation in the mRNA expression of the PD-L1 compared to the MDS with an excess of blasts 1 (MDS-EB1) (p= 0.050). Furthermore, we detected three patients with very high levels of PD-L1 expression, being statistically classified as outliers.
CONCLUSIONS: We suggested that the high expression of the PD-L1 is associated with a worse prognosis in the MDS and functional studies are necessary to evaluate the possible use of anti-PD-L1 therapies for high-risk MDS, such as the MDS-EBs.

One of the major pathophysiologies of malaria is the development of anemia. Although hemolysis and splenic clearance are well described as causes of malarial anemia, abnormal erythropoiesis has been observed in malaria patients and may contribute significantly to anemia. The interaction between inadequate erythropoiesis and Plasmodium parasite infection, which partly occurs in the bone marrow, has been poorly investigated to date. However, recent findings may provide new insights. This review outlines clinical and experimental studies describing different aspects of ineffective erythropoiesis and dyserythropoiesis observed in malaria patients and in animal or in vitro models. We also highlight the various human and parasite factors leading to erythropoiesis disorders and discuss the impact that Plasmodium parasites may have on the suppression of erythropoiesis.

1.5-year-old yellow-collared macaw (Primolius auricollis) was presented as a referral case for chronic breathing difficulties and coelomic distension. The bird was in poor body condition, and coelomic distension and green-colored urates were noted during the physical examination. Radiographic images revealed a large coelomic space-occupying soft-tissue lesion that was ultrasonographically confirmed to be hepatomegaly; the liver had a heterogeneous echogenic pattern. An ultrasound-guided fine needle aspirate of the liver was performed. The cytological results revealed immature hematopoietic cells with signs of dyserythropoiesis and were consistent with extramedullary hematopoiesis (EMH). The plasma biochemistry panel revealed a marked increase in aspartate aminotransferase and bile acids, consistent with severe hepatic disease. Following the results of the diagnostic tests, chemotherapy was initiated using hydroxyurea. Two weeks after the initial presentation and treatment, the bird died and a full postmortem examination was performed. Macroscopic examination confirmed severe hepatomegaly and severe splenomegaly. Histopathological examination of tissue samples confirmed severe EMH in the liver and spleen, splenic and renal hemosiderosis, and acute pulmonary congestion. The bone marrow was normal. The final diagnosis was pathogenic idiopathic EMH, and this case was unusual in both its presentation and severity. Extramedullary hematopoiesis is usually related to myeloid proliferative disorder, chronic blood loss, hemolytic disease, or chronic inflammatory disease. Mycobacteriosis and parasitic infection have been reported to be associated with EMH in birds; however, the inflammatory patterns seen in those cases were lacking in this case. Myeloproliferative neoplasia also appears an unlikely disease condition in this case considering that histopathology found normal architecture in the studied bone marrow; however, bone marrow abnormalities in locations other than the one sampled could not be excluded. A short review of homeostatic and pathogenic hematopoiesis in birds is provided to support the likely diagnosis of idiopathic EMH.

The case of an 83-year old male patient is described. He presented to our hospital with a 2 week history of intermittent syncope and was diagnosed with atrial fibrillation. His CBC showed bicytopenia with anemia and neutropenia in context of a leucoerythroblastic blood film and blastemia. As the patient was previously diagnosed with stage IV diffuse large B cell lymphoma; treated with CHOP-R a bone marrow biopsy was requested to rule out acute leukemia. A cellular bone marrow aspirate showed presence of a myeloblast infiltrate (32.6%; confirmed by flow cytometry). Interestingly blast phagocytosis of terminally differentiated cells along with cannibalism of other blasts was identified. The diagnosis of therapy-related acute myeloid leukemia was reached and a palliative consultation was requested. To our knowledge, this is the first report of blast cytophagocytosis and cannibalism associated with a therapy-related acute myeloid leukemia with a complex karyotype.

Diamond−Blackfan anemia (DBA) is one of the inherited bone marrow failure syndromes marked by erythroid hypoplasia. Underlying variants in ribosomal protein (RP) genes account for 80% of cases, thereby classifying DBA as a ribosomopathy. In addition to RP genes, extremely rare variants in non-RP genes, including GATA1, the master transcription factor in erythropoiesis, have been reported in recent years in patients with a DBA-like phenotype. Subsequently, a pivotal role for GATA-1 in DBA pathophysiology was established by studies showing the impaired translation of GATA1 mRNA downstream of the RP haploinsufficiency. Here, we report on a patient from the Dutch DBA registry, in which we found a novel hemizygous variant in GATA1 (c.220+2T>C), and an Iranian patient with a previously reported variant in the initiation codon of GATA1 (c.2T>C). Although clinical features were concordant with DBA, the bone marrow morphology in both patients was not typical for DBA, showing moderate erythropoietic activity with signs of dyserythropoiesis and dysmegakaryopoiesis. This motivated us to re-evaluate the clinical characteristics of previously reported cases, which resulted in the comprehensive characterization of 18 patients with an inherited GATA-1 defect in exon 2 that is presented in this case-series. In addition, we re-investigated the bone marrow aspirate of one of the previously published cases. Altogether, our observations suggest that DBA caused by GATA1 defects is characterized by distinct phenotypic characteristics, including dyserythropoiesis and dysmegakaryopoiesis, and therefore represents a distinct phenotype within the DBA disease spectrum, which might need specific clinical management.

BACKGROUND: Two Labrador retriever littermates were identified based on incidentally noted marked microcytosis and inappropriate metarubricytosis. Muscle atrophy was noted and associated with distinctive pathological findings in biopsy samples from 1 dog studied. The disorder represents a rare clinical entity of suspected congenital dyserythropoiesis and polymyopathy. Clinicopathologic changes were similar to a previously reported syndrome of congenital dyserythropoiesis, congenital polymyopathy, and cardiac disease in 3 related English Springer Spaniel (ESS) dogs, but the dogs reported here did not have apparent cardiac disease.
METHODS: Bone marrow aspiration, electromyography, muscle biopsies, and an echocardiogram were performed on dog 1. Results supported dyserythropoiesis and congenital polymyopathy similar to reports in ESS dogs, but did not identify obvious cardiac disease.
CONCLUSIONS: The clinicopathologic changes of dyserythropoiesis and polymyopathy provide an easily recognizable phenotype for what appears to be a low morbidity syndrome. Early recognition may decrease unnecessary testing or euthanasia.

Hematopoiesis occurs in the bone marrow, producing a complete spectrum of blood cells to maintain homeostasis. In addition to light microscopy, chromosome analysis, and polymerase chain reaction, flow cytometry is a feasible and fast method for quantitatively analyzing hematological diseases. However, because sufficient specific cell markers are scarce, dyserythropoietic diseases are challenging to identify through flow cytometry.
Bone marrow samples from C57BL/B6 mice and one healthy donor were analyzed using traditional two-marker (CD71 and glycophorin A) flow cytometry analysis. After cell sorting, the gene expressions of membrane proteins in early and late erythropoiesis precursors and in nonerythroid cells were characterized using microarray analysis.
Among characterized gene candidates, aquaporin 0 (AQP0) expressed as a surface protein in early- and late-stage erythropoiesis precursors and was not expressed on nonerythroid cells. With the help of AQP0 staining, we could define up to five stages of erythropoiesis in both mouse and human bone marrow using flow cytometry. In addition, because patients with dyserythropoiesis generally exhibited a reduced population of APQ0high cells relative to healthy participants, the analysis results also suggested that the levels of APQ0high cells in early erythropoiesis serve as a novel biomarker that distinguishes normal from dysregulated erythropoiesis.
AQP0 was successfully demonstrated to be a marker of erythroid differentiation. The expression levels of AQP0 are downregulated in patients with dyserythropoiesis, indicating a critical role of AQP0 in erythropoiesis. Accordingly, the level of AQP0high in early erythroid precursor cells may serve as a reference parameter for diagnosing diseases associated with dyserythropoiesis.

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.
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.
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).
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.