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To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelets

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ± 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% ± 4% versus 0%). No difference, however, was observed in the number of bone marrow megakaryocyte colony-forming units (ITP, 118 ± 93/105 bone marrow cells; versus controls, 128 ± 101/105 bone marrow cells; P = .7). To demonstrate that circulating antibodies might affect megakaryocytes, suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally, the plasma glycocalicin index, a parameter of platelet and megakaryocyte destruction, was increased in ITP (57 ± 70 versus 0.7 ± 0.2; P = .009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P = .02; r = 0.7). In conclusion, most ITP megakaryocytes show ultrastructural features of (para-) apoptosis, probably due to action of factors present in ITP plasma.

Abstract 2932 Several studies have shown that hematopoietic precursor cells in low-risk myelodysplastic syndromes (MDS) undergo premature cell death. The mode of cell death is cell type specific and might include apoptosis, necrosis, and/or autophagy. Studies in MDS erythroblasts have shown enhanced apoptosis and enhanced autophagy (Houwerzijl EJ et al. Leukemia 2009), while megakaryocytes undergo a caspase-3 independent nonapoptotic cell death (Houwerzijl EJ et al. Blood 2005). Increasing data suggest that in MDS the bone marrow microenvironment is also affected. Some stromal cells, including mesenchymal and circulating endothelial cells, can carry similar chromosomal abnormalities as the neoplastic hematopoietic clone (Della Porta MG et al. Leukemia 2008, Lopez-Villar O et al. Leukemia 2009). In addition, recent data show that hematopoietic and endothelial cells have a common precursor, the hemangioblast (Lancrin C et al. Nature 2009). These findings raise the question whether endothelial cells in MDS also die prematurely. To define in more detail the underlying cell death pathways in MDS endothelial cells and to quantify vascularisation, immunohistochemical staining and electron microscopic analysis were performed on bone marrow samples of patients with refractory anemia (RA, n=6) and RA with ringed sideroblasts (RARS, n=6), and healthy controls (n=4). According to the MDS International Prognostic Scoring System (IPSS) the patients were categorized as low risk (n=6) and intermediate risk-1 (n=6). Immunohistochemistry of the MDS bone marrow biopsies demonstrated increased bone marrow microvessel density (MVD) determined by FVIII staining. In both RA and RARS MVD was increased compared to normal (number of vessels: 4 ± 1.6 and 3.8 ± 1.1/powerfield respectively, versus 0.4 ± 0.5 in healthy controls, magnification × 400). The increased number of endothelial cells stained strongly positive for VEGF in both RA and RARS compared to normal bone marrow. The elevated VEGF expression of these cells might be related to a significantly enhanced expression of hypoxia inducible factor-2α (HIF-2α) compared to normal controls, which was especially found in RARS endothelial cells. In contrast, immunostaining for HIF-1α was negative in MDS patients as well as controls. To expand these results ultrastructural analysis was performed on hematons of a subgroup of these patients (n=10). Hematons are compact spherical particles which contain hematopoietic progenitor cells residing within a finely arborized stromal framework, including adipocytes, mesenchymal cells, resident macrophages and endothelial cells (Blazsek I et al. Blood 2000). Hematons can be isolated from the bone marrow aspirate light density fraction. The ultrastructural analysis revealed irregularly shaped endothelial cells without pericytes and degradation of the basal membrane. Cytoplasmic vacuolization was present in endothelial cells, especially in RARS patients. The majority of these vacuoles were double membraned, a main characteristic of autophagy. Mitochondria were normal. No features of apoptosis were found, which was confirmed by a negative immunostaining for caspase-3 and caspase-8. Microvessels were irregularly shaped and showed frequent sprouting. These results indicate that endothelial cells from low-risk MDS patients are ultrastructurally abnormal, showing features of autophagy, but no apoptosis. Autophagy in these cells may be a type of cell death or a cell-rescue reaction to nutrient- and/or growth factor depletion in the microenvironment. On the other hand, in agreement with previous studies, angiogenesis, in low-risk MDS is increased, and appears to be HIF-2α and not HIF-1α mediated. Together, these findings may suggest that endothelial cells in MDS, similar to hematopoietic cells, show a high rate of cellular proliferation, which coincides with an increased susceptibility for premature cell death, i.e. autophagy. The findings might contribute to knowledge of the defective make-up of the stem cell compartment in MDS which requires a strong interaction between hematopoietic stem cells and the microenvironment. Disclosures: No relevant conflicts of interest to declare.

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced programmed cell death (PCD) of committed erythroid cells in the bone marrow compartment. Especially, enhanced apoptosis has been described. However, nonapoptotic forms of PCD have been demonstrated in MDS megakaryocytes characterized by the absence of chromatin condensation and caspase-3 and -8 activation (Houwerzijl EJ et al. Blood2005;105:3472–3479). Recent studies have indicated that besides apoptosis, necrosis and autophagic cell death can be recognized as PCD, whereby cells are capable to switch between the different types of PCD dependent on their cellular context. To define in more detail the underlying cell death pathways in MDS erythroblasts, immunohistochemical staining and ultrastructural and immunolabeling analysis were performed on bone marrow samples of low-risk MDS patients and normal controls (n=4). Immunohistochemistry of MDS bone marrow biopsies (n=23) demonstrated no positive staining of the erythroblasts for active caspase -3 and -8. To confirm these results ultrastructural analysis and immunoelectron microscopy was performed on mononuclear cells (MNC) and hematons of a subgroup of these patients (n=9). Hematons are compact hematopoietic complexes in which hematopoietic cells, including erythroblasts, are embedded in their own microenvironment. The ultrastructural analysis revealed that only a small fraction of erythroid cells of the MNC and hematon fraction of both MDS patients and healthy controls demonstrated features of apoptosis (2 ± 2% vs 0%). However, 52 ± 16% of immature and mature MDS erythroblasts contained cytoplasmic vacuoles in contrast to normal erythroblasts, in which vacuoles were only shown in the matured stage (12 ± 3%). These vacuoles were partly double-membraned and stained positive for the lysosomal marker LAMP (lysosome associated membrane protein)-2, catalase and the mitochondrial inner membrane protein (immunogold staining) underscoring the presence of autophagy of mitochondria and other cytoplasmic components. Morphometric analysis confirmed that the vacuolar surface in the cytoplasm of MDS erythroblasts was increased compared to controls (P

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced cell death of committed erythroid cells in the bone marrow compartment. To define in more detail the underlying type of cell death in MDS, immunohistochemical staining and ultrastructural analysis were performed on MDS and normal bone marrow samples. Immunohistochemistry of MDS bone marrow biopsies (n=20) demonstrated no positive staining of the erythroid lineage for active caspase -3 or -8. Ultrastructural analysis was performed in low-risk MDS patients (Refractory anemia (RA; n=3); RA with ringed sideroblasts (RARS; n=4) on isolated bone marrow hematons, which are compact hematopoietic complexes containing adipocytes, mesenchymal and endothelial cells, and hematopoietic cells. In vitro culture assays have demonstrated that these hematons contain a high number of progenitor and primitive cells. Ultrastructural analysis of the hematons of the MDS patients as well as the healthy controls demonstrated no signs of apoptosis in erythroid cells. In contrast, abnormalities compatible with autophagy were present in >60% of the MDS cells. Especially, cytoplasmic vacuoles with double membranes were noticed in basophilic and polychromatic normoblasts. Morphometric analysis of the erythroid cells showed no differences between healthy controls and MDS patients with regard to cell volume, mitochondria content or nuclear surface, but a significant increase was shown for the ratio area of vacuoles/cytoplasm in the MDS basophilic and polychromatic normoblasts (healthy controls vs MDS: 0.003 vs 0.013 (p