Chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) are blood cancers that affect lymphocytes and can be diagnosed by flow cytometry. Flow cytometry is a laboratory technique that analyzes cell properties, including cell surface markers such as cluster of differentiation 19 (CD19).
The main objective of this study was to explore the correlation of the number of CD19-positive cells with other CD antigens in patients with CLL and ALL.
After receiving ethical approval (Approval No. 5S/401), blood was collected from participants who had been diagnosed by a physician. Then the collected blood was prepared for flow cytometry analysis according to the protocol by staining with fluorescent antibodies.
The results of the current study showed that sex and different age groups had no statistical influence on the number of CD19-positive cells in the patients evaluated. The generated models did not reveal an association with the number of CD19-positive cells in patients with CLL and ALL. In patients with CLL, the number of cells expressing CD5, CD20, CD23, and CD200 was significantly and positively related with the number of CD19-positive cells. In patients with ALL, the number of cells expressing CD79 and CD99 was significantly and positively correlated with the number of CD19-positive cells. This comparison study also found that in patients with CLL, the number of CD19-positive cells was significantly higher than the number of cells expressing CD20, CD23, and CD200. In patents with ALL, there was a significantly higher number of CD19-positive cells than CD34-positive and CD79-positive cells.
In patients with CLL, there was a strong positive correlation between the number of CD19-positive cells and the number of cells expressing CD5, CD20, CD23, and CD200. Additionally, in patients with ALL, there was a positive correlation of CD79 and CD99 with the number of CD19-positive cells.
© 2024 The Authors.

Summary Cell atlas projects have nominated recurrent transcriptional states as drivers of biological processes and disease, but their origins, regulation, and properties remain unclear. To enable complementary functional studies, we developed a scalable approach for recapitulating cell states in vitro using CRISPR activation (CRISPRa) Perturb-seq. Aided by a novel multiplexing method, we activated 1,836 transcription factors in two cell types. Measuring 21,958 perturbations showed that CRISPRa activated targets within physiological ranges, that epigenetic features predicted activatable genes, and that the protospacer seed region drove an off-target effect. Perturbations recapitulated in vivo fibroblast states, including universal and inflammatory states, and identified KLF4 and KLF5 as key regulators of the universal state. Inducing the universal state suppressed disease-associated states, highlighting its therapeutic potential. Our findings cement CRISPRa as a tool for perturbing differentiated cells and indicate that in vivo states can be elicited via perturbation, enabling studies of clinically relevant states ex vivo .

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.
© 2022. The Author(s).

Inflammatory cytokines perturb hematopoietic stem cell (HSC) homeostasis and modulate the fitness of neoplastic HSC clones in mouse models. However, the study of cytokines in human hematopoiesis is challenging due to the concerted activities of multiple cytokines across physiologic and pathologic processes. To overcome this limitation, we leveraged serial bone marrow samples from patients with CALR -mutated myeloproliferative neoplasms who were treated with recombinant interferon-alpha (IFNa). We interrogated baseline and IFNa-treated CD34 + stem and progenitor cells using single-cell multi-omics platforms that directly link, within the same cell, the mutation status, whole transcriptomes and immunophenotyping or chromatin accessibility. We identified a novel IFNa-induced inflammatory granulocytic progenitor defined by expression and activities of RFX2/3 and AP-1 transcription factors, with evidence supporting a direct differentiation from HSCs. On the other hand, IFNa also induced a significant B-lymphoid progenitor expansion and proliferation, associated with enhanced activities of PU.1 and its co- regulator TCF3, as well as decreased accessibility of megakaryocytic-erythroid transcription factor GATA1 binding sites in HSCs. In the neoplastic hematopoiesis, the lymphoid expansion was constrained by a preferential myeloid skewing of the mutated cells, linked with increased myeloid proliferation and enhanced CEBPA and GATA1 activities compared to wildtype cells. Further, IFNa caused a downregulation of the TNFa signaling pathway, with downregulation of NFKB and AP-1 transcription factors. Thus, IFNa simultaneously initiated both – pro-inflammatory and anti- inflammatory – cell states within the same hematopoiesis, and its phenotypic impact varied as a function of the underlying HSC state and mutation status.

Measurable (minimal) residual disease (MRD) in B-acute lymphoblastic leukemia (B-ALL), as assessed by flow cytometry, is an established prognostic factor used to adjust treatment in most pediatric therapeutic protocols. MRD in B-ALL has been standardized by the Children's Oncology Group in North America and more recently in a multicenter Foundation for the National Institutes of Health-funded study. This article outlines the reagents, instrument setup, and analysis protocols required for the reproducible detection of residual leukemic cells in patients following induction therapy for B-ALL. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Staining and flow cytometry for B-acute lymphoblastic leukemia (B-ALL) measurable residual disease detection Support Protocol: Specimen collection, handling, storage, and shipping Basic Protocol 2: Analysis and interpretation of data for B-ALL measurable residual disease detection Basic Protocol 3: Analysis of samples lacking sufficient CD19+ events.
© 2022 Wiley Periodicals LLC.