Platelet adhesion on an oxygenator membrane is associated with thrombocytopenia or thrombus formation during extracorporeal circulation. The authors evaluated protein adsorption and platelet adhesion on three oxygenator hollow fiber membranes fabricated with polypropylene, silicone, and double layer polyolefin. Adsorbed proteins were analyzed by bicinchoninic acid protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. Platelet adhesion was assessed with enzyme immunoassays using monoclonal antibodies directed against CD42b and CD61. After 3 hr of incubation at 37 degrees C in whole blood, the amount of adsorbed protein was the least on silicone and increased from silicone < double polyolefin < polypropylene. The adsorbed protein pattern was similar; however, silicone showed less adsorption for all protein bands, and the gamma chain of fibrinogen was not detected. In contrast, double polyolefin showed the highest fibrinogen adsorption. The optical density at a wavelength of 450 nm for CD42b was 1.47 +/- 0.35 in polypropylene, 1.16 +/- 0.38 in silicone, and 1.85 +/- 0.19 in double polyolefin (p < 0.01 vs silicone) and for CD61 0.98 +/- 0.39 in polypropylene, 0.91 +/- 0.22 in silicone, and 1.69 +/- 0.25 in double polyolefin (p < 0.01 vs silicone and polypropylene). These data suggest that silicone is advantageous for long term extracorporeal respiratory support in terms of less platelet adhesion and no plasma leakage through the pores.

OBJECTIVE

To investigate the role of expression of platelet membrane glycoprotein CD31, CD61 and CD62p in the pathogenesis of decompression sickness.

METHODS

Mice were randomly divided into decompression sickness group and normal control group. The animals in decompression sickness group were exposed to 600 kPa compressed air for 60 minute, then they were rapidly decompressed to normal pressure in one minute. At 60th minute after reducing to normal pressure, the expression of CD31, CD61 and CD62p on platelet membrane in mice was measured by flow cytometry.

RESULTS

The mean fluorescence intensity of CD31, CD61 and positive percentage of CD62p on platelet membrane [(18.64 +/- 1.01), (271.06 +/- 24.25), (4.48% +/- 0.43%) respectively] in decompression sickness group were significantly increased compared with normal control group [(16.89 +/- 1.69), (234.09 +/- 15.96), (3.00% +/- 0.66%) respectively] (P < 0.05, P < 0.01).

CONCLUSIONS

Inadequately rapid decompression may induce up regulation of platelet membrane glycoprotein CD31, CD61 and CD62p expression in mice, which may lead to thrombosis.

Umbilical cord blood (UCB) is now commonly used as a source of stem cells for hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, platelet engraftment occurs at a median of 71 days which is significantly prolonged compared to allogeneic bone marrow. The number of megakaryocyte (MK) precursors in stem cell harvests appears to correlate inversely with the time to platelet engraftment. In an effort to increase the number of platelet precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41(+)) and stem cells (CD34(+) and AC133(+) cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p < 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34(+), AC133(+), CD41(+), and CD61(+) cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.

BACKGROUND

Cardiogenic embolism (CE) in cats is a devastating condition primarily associated with hypertrophic cardiomyopathy (HCM). Hypercoagulability may pose a risk for thrombus formation; however, no single test can predict CE development. Platelet microparticles (PMPs) released from platelet membranes are associated with thrombosis in humans.

OBJECTIVE

The aims were to validate flow cytometric PMP quantification in cats analytically and, in a pilot study, evaluate the procoagulant annexin V (AnV) positive PMP concentration in healthy cats and cats with asymptomatic HCM.

METHODS

With CD61 as a platelet marker, CD61+ AnV+ PMPs (0.3-1.0 μm) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Analyses were performed in 6 healthy cats and 5 cats with asymptomatic HCM. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses were calculated.

RESULTS

PMP concentrations were quantified with acceptable intra-assay CV for WB (CD61+ /AnV- ; 2.4%, 0.2%-8.4% (median, range), CD61+ /AnV+ ; 3.8%, 0.1%-12.5%) and PPP (CD61+ /AnV- ; 5.0%, 0.7%-12.8%, CD61+ /AnV+ ; 7.4%, 0.5%-15.3%), and acceptable inter-assay CV for WB in 10/11 cats (CD61+ /AnV- ; 6.2%, 1.4%-13.3%, CD61+ /AnV+ ; 6.4%, 0.7%-17.2%), but unacceptable for PPP (CD61+ /AnV- ; 15.6%, 5.8%-42.7%, CD61+ /AnV+ ; 27.8%, 8.4%-77.1%). For WB PMP concentrations, the pilot data demonstrated no differences between healthy cats and cats with asymptomatic HCM (4/5 with left ventricular outflow obstruction) for either the CD61+ /AnV- or the CD61+ /AnV+ PMPs.

CONCLUSIONS

Only WB PMP concentrations could be quantified reliably in cats in a clinical setting. PMP concentrations did not differ between healthy and asymptomatic HCM cats in this pilot study.

Hematogones, which are normal precursors of B lymphocytes in the bone marrow, may be mistaken for blast cells on flow cytometry and histology when their numbers increase. We report such a case in a 16 months old male who was unsuccessfully treated for a pre-B cell ALL on the basis of flow cytometry of the bone marrow which showed a substantial population of CD19 and CD10 expressing 'blast' cells. A diagnosis of AML M7 was made on a subsequent bone marrow biopsy in which the blast cells expressed CD61 and Factor VIII, while they were negative for CD10 and CD20. Also present were a few CD10 and CD20 expressing small lymphoid cells, which were interpreted as hematogones. This report reiterates the problem of mistaking hematogones for 'blast' cells on flow cytometry, especially in the marrow of very young children where hematogones are often prominent.

OBJECTIVE

In previous studies, we found that platelet microparticles (PMPs) bind to cord blood (CB) CD34+ cells and transfer adhesion molecules to them, which enhances their engraftment. Before applying this phenomenon in actual transplants, we investigated the effect of PMPs on cryopreserved CD34+ cells in CB.

METHODS

We cryopreserved 18 CB units, then evaluated the binding of PMPs to CD34+ cells after thawing, by varying the expression of platelet characteristic antigens (CD41a, CD61, CD62P and CXCR4) on these cells. Adherence of the CD34+ cells, coated with freeze/thaw-induced PMPs, to endothelium and fibronectin was also studied, as were the effects of thrombin-induced PMPs from both fresh and preserved CB platelets.

RESULTS

PMPs induced by freezing and thawing adhered less well to CD34+ cells than did those from fresh CB, and cells coated with these PMPs had poor adherence. However, thrombin-induced PMPs from both fresh and preserved CB platelets bound equally well to cryopreserved CD34+ cells and improved their adhesion properties.

CONCLUSIONS

PMPs could be a useful tool for enhancing engraftment after CB transplantation.

BACKGROUND

The diagnosis of 22q11.2 deletion syndrome depends on a time-consuming and expensive method, fluorescence in situ hybridisation (FISH).

OBJECTIVE

We aimed to determine new parameters which can aid for in the diagnosis of 22q11.2 deletion syndrome.

METHODS

Twenty two patients with 22q11.2 or 10p13 deletion were evaluated retrospectively.

RESULTS

Facial-dysmorphism and mental-motor retardation were detected in 100% of patients. Mean platelet (PLT) counts were lower (224,980 versus 354,000, p = 0.001), mean PLT volume (MPV) (9.95 versus 7.07, p = 0.002), and MPV/PLTx105 ratios (5.36 versus 2.08, p < 0.001) were higher in patients with 22q11.2 deletion compared with the control group. Area under the receiver-operator characteristic (ROC) curve was 0.864, sensitivity was 84.6%, specificity was 90.9%, positive predictive value (PPV) was 91.7%, and negative predictive value (NPV) was 83.3% when MPV was 8.6. Area under ROC curve was 0.864, sensitivity was 76.9%, specificity was 90.1%, PPV was 90.1%, and NPV was 76.3% when PLT was 265,500. Area under ROC curve was 0.906, sensitivity was 84.6%, specificity was 100%, PPV was 100%, and NPV was 84.6% when MPV/PLTx105 was 3.3. Expression of PLT surface markers which were not in the GPIb-V-IX receptor complex (CD61, CD41a) increased as the surface area increased, but markers which were in a complex (CD42a, CD42b) did not change.

CONCLUSIONS

High MPV/PLT value can be a good predictor for the diagnosis of 22q11.2 deletion syndrome. We suggest that in patients with facial dysmorphism and retardation in neurodevelopmental milestones and if MPV≥8.6fl, MPV/PLTx105 ratio≥3.3 and PLT count ≤265,500/mm3, the patients should be tested by FISH analysis to confirm the 22q11.2 deletion. If there are no macrothrombocytes, the 10p13 deletion should be tested in suspected cases.

Extracellular vesicles (EVs) are a heterogeneous population of submicron-sized structures released during the activation, proliferation, or apoptosis of various types of cells. Due to their size, their role in cell-to-cell communication in cancer is currently being discussed. In blood, the most abundant population of EVs is platelet-derived EVs (PEVs). The aim of this study was to estimate the absolute number and the origin of EVs in the blood of healthy dogs and of dogs with various types of cancer. The EV absolute number and cellular origin were examined by flow cytometry technique. EVs were classified on the basis of surface annexin V expression (phosphatidylserine PS+) and co-expression of specific cellular markers (CD61, CD45, CD3, CD21). The number of PEVs was significantly higher in dogs with cancer (median: 409/µL, range: 42-2748/µL vs. median: 170/µL, range: 101-449/µL in controls). The numbers of EVs derived from leukocytes (control median: 86/µL, range: 40-240/µL; cancer median: 443/µL, range: 44-3 352/µL) and T cells (control median: 5/µL, range: 2-66/µL; cancer median: 108/µL, range: 3-1735/µL) were higher in dogs with neoplasia compared to healthy controls. The estimation of PEV and leukocyte-derived EV counts may provide a useful biological marker in dogs with cancer.

Micromegakaryocytes (microMKs) are considered the most reliable dysplastic feature for myelodysplastic syndrome (MDS), particularly refractory cytopenia of childhood (RCC); there is no minimal threshold for the diagnosis of RCC. Since most RCC patients present with thrombocytopenia, the presence of microMKs should raise concern for MDS/RCC. This study attempted to investigate the prevalence of microMKs and associated marrow fibrosis in patients with thrombocytopenia unrelated to MDS/RCC and the need for establishing a threshold for microMKs for the diagnosis of MDS/RCC.

Bone marrow biopsies of pediatric patients with thrombocytopenia unrelated to RCC were examined for microMKs and fibrosis by CD61 immunohistochemical and reticulin stains respectively.

Thirty eight patients (1-18 years old) were included: 33 immune thrombocytopenia (ITP), 3 chronic thrombocytopenia, and 2 inherited macrothrombocytopenia. Fourteen cases (37%) had microMKs; four cases showed increased marrow fibrosis associated with microMKs (two had ITP and two had macrothrombocytopenia). All patients are alive and none developed MDS (follow up: 3months to 4 years).

MicroMKs can be seen in pediatric patients with thrombocytopenia unrelated to RCC. Hence the mere presence of microMKs is insufficient for the diagnosis of RCC in the pediatric population, and a quantitative threshold needs to be established.

OBJECTIVE

To study the effect of human umbilical blood (UB) mesenchymal stem cells (MSC) on the CD34(+) cells transplantation in NOD/SCID Mice.

METHODS

Umbilical blood CD34(+) cells (3.5 x 10(5) cells) alone or combined with umbilical cord MSC cells were transplanted into NOD/SCID mice that had been irradiated with (137)Cs (3.0 Gy) before transplantation. Changes in peripheral blood cells within 6 post-transplantation weeks were detected. The mice were sacrificed 6 weeks after transplantation. The human hematopoietic cells (hCD45(+)) and multi-lineage engraftment cells (CD3/CD19, CD33, CD14, CD61, and CD235a) in NOD/SCID recipients bone marrow, spleen, and peripheral blood were analyzed by flow cytometry.

RESULTS

In the 3rd post-transplantation week, white blood cells (WBC), platelets (PLT), and red blood cells (RBC) began to increase in both two groups. In the 6th post-transplantation week, WBC and PLT counts in CD34(+) + MSC group reached peak levels and were significantly higher than CD34(+) alone group (P < 0.05), while RBC level was not significantly different between these two groups P>> 0.05). hCD45(+) cell levels in bone marrow and peripheral blood were (42.66 +/- 2.57) % and (4.74 +/- 1.02) % in CD34(+) + MSC group, which were significantly higher than those in CD34(+) alone group [(25.27 +/- 1.67) % and (1.19 +/- 0.54) %, respectively, P = 0.006]. Also in the 6th post-transplantation week, the proportions of CD19(+), CD33(+), CD14(+), CD61(+), and CD235a(+) in CD34(+) + MSC group were significantly higher than those in CD34(+) alone group (P < 0.05), while the proportion of CD3(+) T lymphocyte in CD34(+) + MSC group was significantly lower than that in CD34(+) alone group (P = 0.003). The amplification of CD19(+) B lymphocyte was significantly higher than other blood cell lineages (P < 0.05).

CONCLUSIONS

The co-transplantation of MSC cells and CD34(+) cells can promote hematopoietic stem cell transplantation and hematopoietic recovery in vivo.

The aim of this study was to assess platelet reactivity in patients after ischemic stroke and to investigate the influence of hyperlipidemia (HL) on platelet activity markers. A total of 41 patients after ischemic stroke were divided into the following 2 groups: patients with HL and patients with normolipidemia. Expression of CD42b on resting, thrombin-activated blood platelets, and fibrinogen level was assessed. The CD42b-positive platelets were analyzed using the flow cytometer, anti-CD61, and anti-CD42b monoclonal antibodies. The results confirmed increased platelet reactivity to thrombin in all patients after ischemic stroke manifested by significantly lower CD42b expression and percentage of CD42b(+) platelets after activation by thrombin. The influence of HL on the expression of CD42b on resting and thrombin-activated platelets was not found. However, increased level of fibrinogen but no influence of HL on fibrinogen concentration was observed in patients after ischemic stroke. Increased susceptibility to platelet agonists was found in patients after ischemic stroke in the convalescent phase.

Umbilical cord blood (CB) has been widely used for related and unrelated transplants in pediatric patients. We present the case of an adult with secondary AML who received an unrelated, one-antigen mismatched CB transplant due to the lack of a matched donor. The patient was a 26-year-old female (35 kg/bw) who had received an autologous bone marrow transplant for Hodgkin's disease in April 1994 and, 6 months later, developed secondary MDS (RAEB, 46, XX, -7, +mar), which slowly evolved into acute myelogenous leukemia. In May 1995, she was transplanted with a 165 ml CB unit containing a total of 1.6 x 10(9) nucleated cells, 11 x 10(6) CD34+ cells and 7.2 x 10(5) CFU-GM. GVHD prophylaxis consisted of standard CsA and methotrexate. Myeloid engraftment occurred on day +28 (PMN>> 500) and full donor chimerism was confirmed twice (on days +33 and +56) by means of cytogenetics and DNA microsatellite analysis. Erythroid and megakaryocytic engraftment was documented by immunohistochemical analysis of a bone marrow biopsy on day +40, showing the presence of erythroblastic islands and isolated CD61+ immature cells. The patient did not develop GVHD but died on day +56 from idiopathic interstitial pneumonia and multiorgan failure. To our knowledge, this is one of the first case reports of unrelated mismatched CB transplantation in an adult.

Until now, studies on mixed chimerism (MCh) after allogeneic bone marrow transplantation (BMT) have predominantly focused on the B- and T-lymphocyte population, but not on distinct myeloid cell lineages like nucleated erythroid precursors and megakaryocytes. To evaluate the lineage-restricted MCh more explicitly in 10 patients with chronic myelogenous leukemia (CML), a quantitative analysis was performed on bone marrow biopsies following a sex-mismatched host/donor constellation. Techniques included immunophenotyping (antiglycophorin C, CD61) for the identification of erythro- and megakaryopoiesis and a simultaneously conducted genotyping with x- and y-chromosome-specific DNA probes. Normal bone marrow and specimens taken before BMT served as controls. Contrasting a total gender-dependent sex-typing in the latter samples in the early and late posttransplant period (up to 586 days), 3-9% erythroid precursors and about 16% megakaryocytes revealed a host-type origin. This significantly higher number of host megakaryocytes is explained by their polyploidy generating an increased probability to detect positive signals at a certain section level of the corresponding biopsies. A striking conversion of MCh to a recipient cell type was found in leukemic relapse with a more than 90% host-derived erythroid and megakaryocytic cell population in 4 patients approximately 643 days after BMT.

One case of acute megakaryoblastic leukemia (AMKL) with trisomy 21, trisomy 14 and unmutated GATA1 gene in a phenotypically normal girl was reported. The patient experienced transient myelodysplasia before the onset of AMKL. The bone marrow blasts manifested typical morphology of megakaryoblast both by the May-Giemsa staining and under the electronic microscopy. Leukemic cells were positive for CD13, CD33, CD117, CD56, CD38, CD41 and CD61 in flow cytometry analysis. Cytogenetic study showed karyotype of 48, XX, +14, +21 in 40% metaphases. Known mutations of GATA1 gene in Down syndrome or acquired trisomy 21 were not detected in this case.

We report a case of acute myeloid leukemia which we have classified as acute megakaryoblastic leukemia because of intracytoplasmic expression of CD61. Light microscopy demonstrated agranular blasts which, by cytochemical staining, were negative for myeloperoxidase. Using flow cytometry, the blast cells were seen to be positive for HLA-DR, CD7, CD13, CD33, and CD34, thus revealing their myeloid origin. Immunophenotyping on fixed blood smears additionally showed positive reaction with the CD61 antibody, demonstrating the megakaryoblastic differentiation of the blasts. After permeabilization of the cell membrane, the intracytoplasmic CD61 expression was confirmed by flow cytometry. Cytogenetic analysis disclosed a del(7)(q21-22). Our findings suggest that cytoplasmic expression of CD61 may precede the cell-surface expression of this antigen and should therefore be investigated in all cases of acute leukemias with undifferentiated morphology and negative cytochemistry to set apart early FAB-M7 from FAB-M0.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on β3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA-1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies.

We conducted a retrospective study to assess the changes in bone marrow (BM) stromal antigenic profile and fibrosis in chronic myeloid leukemia (CML) under combined interferon-alpha (IFN) and Ara-c therapy. Bone marrow biopsies were taken before therapy and twice (at 4 and 15 months) during therapy in 10 CML patients and compared with non-CML samples. Collagen and reticulin fibrosis was assessed by histochemical methods and phenotypic changes were studied by immunohistochemistry (APAAP) with antibodies directed against endothelial cell antigens, cell adhesion molecules, and HLA-DR. It was found that: (1) BM endothelial cells in patient and in control specimens showed a specific pattern of antigen expression: high expression of FVIII and CD34 (except on sinusoids for the latter), variable expression of UEA I, and no expression of HLA-DR and E-selectin. (2) Compared to non-CML controls, CML specimens at diagnosis showed an increased reticulin fibrosis and a decreased expression of CD61 on megakaryocytes and of CD31 on vessels and hemopoietic cells. (3) Treatment did not influence BM fibrosis, the vascular content of the BM, or the expression of the antigens tested except an increase in the number of CD34+ sinusoids (5/10 patients), an increase in the number of HLA-DR+, and a decrease in the number of CD34+ hemopoietic cells (6/10). (4) On therapy, difficulty in aspiration and/or reduced BM fragment numbers were noted in 8 of 10 patients whose bone marrow was still normocellular or slightly hypercellular. In conclusion, CML samples at diagnosis showed increased fibrosis and decreased CD31 and CD61 expression compared to controls. During the period of observation, combined therapy did not modify BM fibrosis; however, an increase in CD34+ sinusoids and a decrease in CD34+ hemopoietic cells were noted.

Five hundred twenty patients with de novo non-lymphocytic leukemia (ANLL) were classified according to morphocytochemical FAB criteria and then immunophenotyped using a set of 20 monoclonal antibodies (MoAb) of VI series. It was demonstrated that immunophenotyping increased the proportion of properly classified leukemias from 87% after morphocytochemical evaluation up to 97.5%. A first line diagnostic set was proposed for ANLL consisting of MoAbs detecting the following cell differentiation antigens: CDw65 (VIM2)--as a screening marker for the whole ANLL group, CD14 (VIM12)--as an indicator characteristic for M4 and M5 FAB subtypes, glycophorin A (VIEG4)--helpful in identification of erythroleukemia, CD15 (VIMD5)--which has a prognostic significance and CD41 (VIPI1)--important for identification of megacarioblastic M7 subtype. MoAbs detecting CD11b, CD61 and Ia-Dr may be used as the second line reagents.

An immunohistochemical and morphometric study was performed on 363 trephine biopsies of the bone marrow derived from 127 patients with chronic myeloid leukemia at standardized end points before and after allogeneic bone marrow transplantation (BMT). The purpose of this investigation was to evaluate features of CD61+ megakaryopoiesis related to successful engraftment. Further, we tried to elucidate possible associations of this lineage, including precursor cells, with the platelet count and reticulin fibrosis during the pretransplant and, specifically, post-transplant periods. A significant correlation was recognizable between the quantity of CD61+ megakaryocytes and the platelet values before BMT and also after completed hematopoietic recovery. In the very early post-transplant period, which is associated with severe thrombocytopenia, patchy regeneration of disarranged hematopoiesis occurred, including dysplastic megakaryocytes. According to planimetric measurements after BMT, the atypical micromegakaryocytes characteristic for chronic myeloid leukemia disappeared, and the engrafted donor bone marrow revealed a prevalence of normal-size cells of this lineage. On the other hand, normalization of megakaryocyte size was absent in sequential examinations of the few patients with a leukemic relapse who had a predominance of atypical dwarf forms comparable with chronic myeloid leukemia. Before BMT occurred, reticulin fiber density was significantly correlated with the number of CD61+ megakaryocytes and its precursor cell population. In 34 patients with myelofibrosis that occurred after myelo-ablative therapy and BMT, an initial regression was followed by an insidious recurrence of fibers concentrated in the areas of regenerating hematopoiesis. This postgraft reappearance of reticulin fibrosis was significantly associated with the quantity of megakaryocytes. Regarding engraftment parameters, pretransplant presence of (reticulin) myelofibrosis exerted a distinctive impact because of a delayed hematopoietic reconstitution according to standard clinical criteria. In line with this finding, slowed engraftment was also significantly related with higher pretransplant megakaryocyte and platelet counts.

OBJECTIVE

To evaluate the characteristics of P-glycoprotein (P-gp) expression of acute non-lymphocytic leukemia (ANLL) at different status and the prognostic and biological features in ANLL at diagnosis.

METHODS

Monoclonal antibody UIC2 and indirect immunofluorescence assay by flow cytometry were used to determine P-gp expression of 169 patients with ANLL, including 152 previously untreated, 7 refractory and 10 at remission.

RESULTS

P-gp was expressed in 28.9% of the previously untreated ANLL cases and P-gp was lower than that in 71.4% of the refractory cases (P < 0.05). No P-gp expression was found in the patients at remission. For previously untreated AN-LL, P-gp was highly expressed in hybrid acute leukemia (66.7%) and acute monoblastic leukemia (47.4%). P-gp expression was highly associated with surface markers [cluster of differentiation (CD) 34, CD7, CD14, CD42b and CD61] and unfavorable cytogenetic abnormalities. About 23% of P-gp-ANLL obtained complete remission, which was significantly lower than that (76%) in P-gp-cases.

CONCLUSIONS

P-gp expression is higher in refractory ANLL cases than that in cases at diagnosis or at remission. P-gp is an index of poor prognosis in adults with ANLL. P-gp+ ANLL cases have unique clinical and biological characteristics.

OBJECTIVE

To study the function of ZNF300 in the megakaryocytes differentiation and proliferation.

METHODS

Public data analysis of ZNF300 expression and megakaryocyte culture were used to reveal the correlation of ZNF300 expression with leukemia and megakaryocyte differetniation; ZNF300 overexpression was mediated by lentiviral or retroviral infection, and the differentiation and proliferation of K562 cells and primary mouse bone marrow cells to magekaryocytes were measured by flow cytometry, MTT assay and colony-forming test; the ZNF300 subcellular localization was tested by separating cytosolic and nuclear extracts combined with Western blotting. The dual-luciferase assay and ChIP-qPCR were used to study ZNF300 target gene.

RESULTS

ZNF300 expression upregulation correlated with megakaryoyte differentiation; over-expression of ZNF300 promoted CD41 and CD61 expression, inhibited cell cycle progress, and could reduce colony-forming unit. The ZNF300 locolized in nuclear and regulated C-MYC expression.

CONCLUSIONS

ZNF300 promotes megakaryocyte differentiation.

Two subclones (K562-L and -H) were previously isolated from K562 human leukemic cells according to hemoglobin production: K562-L was expressed in less than 5% and K562-H in more than 90% of dianisidine positive cells. 12-O-Tetradecanoylphorbol-13- acetate (TPA) suppressed the expression of the erythrocytic (glycophorin A) and myelocytic (CD11b) antigens in K562-L, but increased the expression of these antigens in K562-H. TPA increased the megakaryocytic (CD61) antigens in both cells. These findings suggest that there are distinct TPA responsible factors in K562-L and -H on the expression of the erythrocytic and myelocytic antigens.