Flow Cytometric Analysis Of Bone Marrow Research Articles

Blastic plasmacytoid dendritic cell neoplasm: A case report and literature review.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a clinically aggressive tumor, which frequently presents as cutaneous lesions and subsequently progresses to bone marrow (BM) involvement and leukemic dissemination. BPDCN is a rare entity that belongs in the same class as acute myeloid leukemia-associated precursor neoplasms, according to the 2008 World Health Organization classification. The present study reported the case of a 26-year-old female who presented with evident thrombocytopenia, leukocytosis and anemia, but without skin lesions. The results of peripheral blood, BM smear and BM biopsy examinations detected numerous blastic or abnormal cells. In addition, flow cytometric analysis of BM demonstrated the presence of plasmacytoid dendritic cell-neoplastic precursor cells (CD4+, CD56+, CD123+, CD304+ and human leukocyte antigen-DR+ phenotype).

Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice.Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size.Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. DisclosuresNo relevant conflicts of interest to declare.

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull mice

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

A NUP98-HOXD13 Fusion Gene Impairs B Lymphocyte Development and Function In a Mouse Model for Myelodysplastic Syndrome

AbstractAbstract 279Transgenic mice which express the fusion gene NUP98-HOXD13 (NHD13) have been shown to develop characteristic features of Myelodysplastic syndrome (MDS) including impaired hematopoietic differentiation and peripheral blood cytopenias in the presence of normocellular or hypercellular bone marrow (BM). It is evident that B-cells play a role in the progression of MDS by immune modulation or as direct targets of mutations resulting in ALL, or as cells that influence the BM microenvironment in which a neoplastic myeloid clone evolves. Choi and colleagues suggested a block in differentiation during the early development of B lymphocytes in the BM of NHD13 mice leading to lymphopenia consistent with the observation in some MDS patients. In this study, we sought to further delineate the role of NHD13 on B lymphocytes which escaped the initial differentiation block in the BM. We hypothesized that NHD13 impairs maturation and function of IgM+ B lymphocytes contributing to immunodeficiency. To study this, we performed blood smear examination, Complete Blood Counts (CBC), quantitative ELISA for antibody concentrations, and flow cytometric analysis of B cell fractions from the BM and spleen in 8–12 week-old transgenic and wild type (WT) mice. CBCs revealed significant lymphopenia and ELISA showed higher IgM concentrations (n=10, p<0.001), reduced levels of IgG1 (n=10, p<0.05) and IgE (n=10, p<0.01). The IgG2a, IgG2b, and IgG3 antibody levels were comparable to WT counterparts. Flow cytometric analysis of BM and splenic B cell fractions revealed reduced numbers of B cells in Hardy fractions D and F (n=10, p<0.01) indicative of impaired differentiation prior to these stages; splenic fractions in NHD13 mice were comparable to WT controls. Next, to assess the peripheral maturation and functional efficiency of B lymphocytes in the context of a comprehensive immune stimulation, a cohort of five WT and five preclinical transgenic mice were injected with 100 μ g dinitrophenol (DNP) followed by a booster dose on day 21. Mice were euthanized on day 28 and whole blood, spleen, lymphnodes and BM were harvested. CBC evaluation revealed significant lymphopenia in NHD13 mice (n=5, p<0.001). Quantitative ELISA for DNP specific antibodies showed comparable levels of serum IgM and significantly reduced levels of serum IgG1 (n=5, p<0.001), IgG2a (n=5, p<0.001), IgG2b (n=5, p<0.01), IgG3 (n=5, p<0.001) and IgE (n=5, p<0.01). Flow cytometric analysis of peripheral blood showed reduced numbers of B220+ IgM+ B cells (n=5, p<0.01), but comparable percentages of CD4+ and CD8+ T-cells. Detailed flow cytometric analysis of B-cell fractions in the BM and spleen of DNP-stimulated mice revealed a reduction in subpopulations of B lymphocytes. The earliest B cell lineage population, Pre-Pro B, was comparable to the WT controls. Hardy Pro B fraction B (n=5, p<0.001) and Pre B fractions E (n=5, p<0.01) and F (n=5, p<0.01) from BM of stimulated mice were significantly reduced in contrast to fractions C and C', which were higher (n=5, p<0.05 and p<0.001 respectively), indicative of cell growth arrest at these stages. Flow cytometry of splenic B-cell fractions from the DNP-stimulated mice showed significantly lower Transitional 1 (n=5, p<0.01), Follicular (n=5, p<0.05) and Marginal Zone (n=5, p<0.001) populations upon antigenic stimulation suggestive of defective clonal expansion of IgM+ cells even after escaping the block in the BM. Histopathology of the spleen revealed smaller lymphoid follicles with poorly developed mantle and marginal zone regions in the transgenic mice when compared to WT controls, consistent with the flow cytometric data. This study indicates that when NHD13 mice are immunologically challenged, B lymphocytes undergo impaired differentiation in the BM and maturation in the spleen, as well as reduced antibody class switching and subsequently lower antibody production. Analysis of B cell subsets during development and specific IgG/IgE antibody production, suggest that the NHD13 transgene might impair VDJ gene recombination and class switch recombination that are critical during these phases of B cell development. Disclosures:No relevant conflicts of interest to declare.

Human Cord Blood-Derived Endothelial Progenitor Cells Engraft Following In Utero Transplantation, Integrate into the Developing Cytoarchitecture and Contribute to Ongoing Vasculogenesis.

Endothelial progenitor cells (EPC), whether isolated from the bone marrow (BM), peripheral (PB), or cord blood (CB), represent a promising tool for the development of novel cell therapies. EPC have been shown to contribute to re-endothelialization and neovascularization of damaged tissue, and have been proposed to be some of the primary regulators of tissue regeneration in organs such as the liver. Many studies have looked at the role of EPC in vasculogenic processes, but very few, if any, have focused their efforts on determining the complete differentiative potential of EPC upon transplantation in an experimental model that permits the robust formation of donor-derived tissue-specific cells in the absence of selective pressure to drive differentiation towards a specific phenotype. To this end, CB-derived EPC were obtained as previously described (Ingram et al. Blood:104,2004), transduced with a retroviral vector expressing dsRed, and transplanted (Tx) into 55–60 days old fetal sheep recipients (n=8) at concentrations ranging from 0.5–1.5 × 106cells/fetus. Recipients were then evaluated at 85 days post-transplant for the presence of donor (human)-specific cell types using flow cytometry and confocal microscopy. Using these methods, we found that levels of EPC engraftment in liver, as detected by dsRed expression, correlated directly with the Tx cell dose. Furthermore co-localization of CD31 or vWF was found within the dsRed+ cells. In animals receiving lower cell doses, EPC engrafted throughout the liver at the overall level of 0.12±0.03%; this number doubled in animals that received 2.6 × 106cells. Importantly, there was a preferential distribution of EPC around the vessels, with the EPC comprising 10 to 25% of the cells located around the perivascular areas, with some contributing directly to the endothelial layer of these vessels. Furthermore, expression of Connexin-43 and 45 in engrafted EPC demonstrated that the EPC had not only engrafted, but had also functionally integrated into the developing blood vessels. In addition, co-expression of albumin and alpha-fetoprotein in some of the engrafted EPC suggests that some of these cells may also have contributed to cells with a hepatocyte-like phenotype. Flow cytometric analysis of BM and PB of the transplanted sheep demonstrated that EPC engrafted and proliferated in the BM, with cells expressing CD105 (6.2±2.2) and CD146 (0.6±0.1), and continued to circulate in the PB with cells positive for CD105 (1.4±0.4) and CD146 (0.9±0.2). Of interest is that a CD45 negative aminopeptidase N+ (APN/CD13) population was found in both BM (18±7) and PB (5.6±2). This is particularly interesting, since CD13/APN is a potent regulator of vascular endothelial morphogenesis during angiogenesis. In conclusion, CB derived EPC are able to engraft and proliferate in vivo, integrate into the developing cytoarchitecture, and establish a circulating EPC pool ensuring long-term contribution to ongoing vasculogenesis.

Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure.

Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colony-forming cells, and AA progenitors fail to proliferate in long-term assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34+ and CD33+ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34+ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34+ cell reduction. A decrease in BM CD33+ cells was a common finding in MDS patients, but reduction in CD34+ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purified CD34+ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c-kit receptor expression on CD34+ cells, we found that the mitotically active CD34+ cells bearing the c-kit antigen were reduced in AA. With clinical improvement, CD34+ and CD33+ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34+ cells from recovered patents showed improved plating efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)