Direct effects of circulating blood microparticles on fibrin formation and structure were studied. Clots made from platelet-free plasma and from microparticle-depleted plasma obtained by filtration was studied in parallel, including clots from the microparticle-depleted plasma replenished with phospholipids. Fibrin formation was induced by exogenous thrombin without Ca2+ to prevent formation of endogenous thrombin and exclude indirect kinetic effects of microparticles related to thrombin generation. In the presence of natural microparticles or exogenous phospholipids the maximal turbidity of fibrin clots was significantly smaller, indicating structural distinctions from the clots formed in the absence of microparticles. Scanning electron microscopy and confocal microscopy showed that clots formed from platelet-free plasma, i. e. in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contained 0.1-0.5-μm-large CD61-positive granules associated with fibrin fibers that were identical to the particles found on the surface of filters used for microparticle removal. The results show that platelet-derived microparticles bind to fibrin and affect its structure. The revealed interactions of cell-derived microparticles with fibrin highlight a previously unknown role of microparticles in hemostasis and thrombosis as constituents and modulators of a fibrin clot structure.

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.

To determine parameters of predictive value in CML, a retrospective clinico-pathological study was performed. This included laboratory data and (pretreatment) bone marrow biopsies of 120 patients with a monotherapy by busulfan (BU) and 50 patients with interferon-alpha 2b (IFN) treatment. Median survival in the BU group was 39 months and in the IFN-treated patients 65 months. Morphological features (CD61-positive megakaryocytes, argyrophilic fibres, pseudo-Gaucher cells) were evaluated by morphometry. Additionally, we measured the incidence of apoptosis (in situ end-labelling technique) and the expression of the proliferating cell nuclear antigen (PCNA). The ratio between the proliferative and apoptotic cell fraction was coined leukaemia turnover index (LTI). In order to estimate the impact of clinical and various morphological as well as dynamic features of prognostic significance, a multivariate analysis was carried out using the classification and regression tree approach (CART). Discrimination of single disease parameters revealed that fibrosis remained the most significant variable for survival in both therapeutic groups. Indicators of myeloid metaplasia such as occurrence of erythro-normoblasts and/or splenomegaly were important clinical parameters for prognosis. Inclusion of morphological as well as dynamic disease features in risk classification resulted in a substantial improvement of prognostic efficiency compared to other predictive scores which could be demonstrated by means of ROC-analysis.

OBJECTIVE

Bone marrow histopathology reveals a striking heterogeneity at diagnosis of Philadelphia chromosome positive (Ph1+) chronic myelogenous leukaemia (CML). Based on semiquantitative evaluations of the number of megakaryocytes and the content of fibres, various histological subtypes have been postulated. However, little information exists on whether these groups represent stable categories of the different classification systems and whether therapeutic regimes exert any influence on the putative shift of histological patterns.

RESULTS

A retrospective clinicopathological study was performed on 396 bone marrow biopsies derived from 173 patients. There were at least two representative trephines taken at diagnosis and at median intervals of 16 months. Processing of the specimens involved immunostaining with CD61 (megakaryopoiesis) and Ret40f (erythropoiesis) and Gomori's silver impregnation technique. Based on morphometric analysis and in accordance with the general appearance of bone marrow histology three different histological subtypes were distinguished. These consisted of a granulocytic (51 patients), a predominantly megakaryocytic (73 patients) and a myelofibrotic pattern (49 patients). Follow-up biopsies revealed that a significant transition of histological groups occurred and that, independently of treatment modalities, the myelofibrotic category was associated with an unfavourable prognosis. Of the 124 patients without myelofibrosis at onset, 42% later transformed into the myelofibrotic subtype. However, these patients showed no prevalence of either a pre-existing granulocytic or megakaryocytic growth. Myelofibrotic changes were significantly associated with interferon (IFN) and busulfan (BU) therapy. On the other hand, a transition of a myelofibrotic into a nonfibrotic subtype was detectable in 17 of the 49 patients under study and related to hydroxyurea (HU) treatment.

CONCLUSIONS

Histological classification systems of bone marrow features in CML do not represent stable patterns, but may be significantly altered by therapy, in particular IFN and HU.

The purpose of this study was to evaluate the in vivo viability of human platelets cryopreserved at -80 degrees C by using SCID mouse model and flow cytometry. The fresh human platelets were frozen with 5% DMSO at -80 degrees C for 10 days, thawed, and centrifuged for concentration. A 100 ml aliquot of concentrated platelets was injected into the SCID mouse tail vein by using a 1 ml insulin-syringe fitted with a 29-gauge ultra-fine needle. The whole blood was collected into heparinized capillary tube at 0.5, 2, 4, 6, 12, and 24 hours after infusion via a tail vein and was labelled with CD61-PE. Then the human platelets in mouse whole blood were detected by flow cytometry. The 30 minute time point was used as 100% to calculate the survival time of human platelets. The results showed that the survival time of cryopreserved human platelets were more significantly decreased than that of fresh platelets in SCID mice. Survival rates at 4 hours after transfusion of fresh platelets and cryopreserved platelets in SCID mice were 79.5% +/- 9.1% (n = 8) and 40.6% +/- 6.6% (n = 8) respectively, and a T(1/2) estimated were 7 hours for fresh platelets, but 2.5 hours for the cryopreserved. In conclusion, platelets survival time in SCID mice was shortened after frozen with DMSO at -80 degrees C.

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34+ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.

In chronic myeloid leukemia (CML), it has been assumed that the number of CD34(+) progenitor cells (PGCs) provides useful diagnostic and prognostic information regarding the evolution of accelerated phase and blastic crisis. However, until now no information is available about changes of this peculiar precursor cell population during therapy or possible associations with the other bone marrow constituents. For this reason, a retrospective clinicopathological study was performed on 83 patients with CML including 209 sequential bone marrow biopsies (intervals ranging between 6 and 143 months) and immunohistological staining of CD34(+) cells (QBEND10), megakaryocyte precursors (CD61), and erythropoiesis (Ret 40f). According to treatment modalities, three different groups of patients could be distinguished that received either monotherapy by interferon-alpha2b (IFN-alpha2b) or hydroxyurea (HU) and a combination of both. In comparison with a control group, morphometry revealed a significant increase in the quantity of CD34(+) PGCs per hematopoiesis (cellularity) in the CML bone marrow before treatment. Independently of treatment modalities and presentation of clinical findings nonresponding patients were generally characterized by a higher amount of progenitors in the initial biopsy specimens. Furthermore, calculation of the CD34(+) cell growth index showed a significant and rapid progression in nonresponding patients and in those developing an accelerated or blastic phase during therapy. This feature was prominently expressed following IFN treatment and related to a failing regeneration of nucleated erythroid precursors. In patients with a myelofibrotic bone marrow at onset no differences in the number of CD34(+) PGCs were recognizable in the pretreatment biopsies. This finding contrasted a significant and gradual change in progenitor cell frequency under treatment and evolving myelofibrosis. Opposed to HU therapy, the latter feature was explicitly detectable in the IFN group. In conclusion, the incidence of CD34(+) PGCs in the CML bone marrow reflects therapeutic efficacy. By demonstrating a significant relationship between fiber content and quantity of CD34(+) cells during treatment, experimental findings concerning the complex functional interactions between the fibrous stroma compartment and progenitor cell differentiation and proliferation are elucidated.

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL-3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.

BACKGROUND

Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts.

OBJECTIVE

To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation METHODS: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR.

RESULTS

At a concentration of 40 µg/ml, the leaf extracts of Justicia adhatoda for 72 h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels.

CONCLUSIONS

Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.

Primary myelofibrosis (PMF), per WHO criteria, is a clonal myeloproliferative neoplasm that usually presents with a proliferation of granulocytic and megakaryocytic lineages with an associated fibrous deposition and extramedullary hematopoiesis. The bone marrow histologic findings of this disorder are typically characterized by the presence of myeloid metaplasia with an associated reactive fibrosis, angiogenesis, and osteosclerosis. However, marked myelofibrosis is not solely confined to PMF and may also be associated with other conditions including but not limited to acute megakaryoblastic leukemias (FAB AML-M7). Here, we describe a rare case of a non-megakaryoblastic acute myeloid leukemia with marked myelofibrosis with osteosclerosis and an isolated trisomy 19. A 19-year-old male presented with severe bone pain of one week duration with a complete blood cell count and peripheral smear showing a mild anemia and occasional circulating blasts. A follow up computed tomography (CT) scan showed diffuse osteosclerosis with no evidence of hepatosplenomegaly or lymphadenopathy. Subsequently, the bone marrow biopsy showed markedly sclerotic bony trabeculae and a hypercellular marrow with marked fibrosis and intervening sheets of immature myeloid cells consistent with myeloblasts with monocytic differentiation. Importantly, these myeloblasts were negative for megakaryocytic markers (CD61 and vWF), erythroid markers (hemoglobin and E-cadherin), and lymphoid markers (CD3, CD19, and TdT). Metaphase cytogenetics showed an isolated triosomy 19 with no JAK2 V617F mutation. The patient was treated with induction chemotherapy followed by allogenic hematopoietic stem cell transplantation which subsequently resulted in a rapid resolution of bone marrow fibrosis, suggesting graft-anti-fibrosis effect. This is a rare case of a non-megakaryoblastic acute myeloid leukemia with myelofibrosis and osteosclerosis with trisomy 19 that may provide insights into the prognosis and therapeutic options of future cases.

An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation (IAC - AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61- positive EVs and CD9- positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g. exomeres and exosomes) for further analysis. Field-emission scanning electron microscope elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in ng/mL range using hydrophilic interaction liquid chromatography - tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37-80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84-6.6%.

Detection of atypical megakaryocytes in bone marrow biopsies, especially in cases of myelodysplastic syndromes (MDS), chronic myeloproliferative disorders (CMPD) and acute leukemias, is facilitated by staining for markers such as Ulex europaeus agglutinin (UEA)-J, CD31, CD61 and von Willebrand factor (VWF), the latter being considered the most sensitive. Recently, LAT (linker for activation of T cells), a molecule involved in T-cell activation and platelet aggregation, was found to be expressed by megakaryocytes and platelets in tissue sections. We compared VWF and LAT immunoreactivity on megakaryocytes in 64 bone marrow biopsies from 12 normal controls (NC), and from patients with MDS (n=18), CMPD (n=21) and acute megakaryocytic leukemia (AML-M7, n=13). Immunostaining was performed on paraffin sections with polyclonal antibodies against VWF and LAT. Immunoreactivity was evaluated by counting positive megakaryocytes in 10 high-power fields, and values were compared using Student's t test for paired data. Both VWF and LAT predominantly stained the cytoplasm of megakaryocytes, although LAT was also recognizable on the cell membrane. In most biopsies, the immunoreactivity of the two antibodies was quite similar. No significant differences were noticed between the mean values of VWF+ and LAT+ megakaryocytes. However, in 22 cases (5 NC; 5 MDS; 6 CMPD; 6 AML-M7), the number of LAT+ megakaryocytes was at least 30% higher than VWF+cells, while in 3 cases opposite findings were found. In 3 AML-M7 cases, anti-LAT antibodies stained numerous megakaryocytes, but anti-VWF staining was practically negative; in another 5 AML-M7 cases, anti-LAT labeling was much stronger than anti-VWF staining. LAT represents a useful immunohistochemical marker for megakaryocytes in normal and pathological conditions. It seems to be expressed by megakaryocytes more than VWF in most cases and, particularly, in conditions associated with poorly differentiated megakaryocytes, such as acute megakaryocytic leukemias. The use of LAT staining should be recommended in association with other megakaryocyte markers in the study of bone marrow biopsies in cases of hematopoietic disorders.

Hemoperfusion (HP) is one of the important treatment modalities in extracorporeal therapy for patients with acute intoxication. Its use has declined during the past 20 years despite its efficacy, because of its side effects, especially an increased risk of bleeding. Mechanisms of hemostasis impairment have not been clearly elucidated and studies demonstrating the mechanism are lacking. It is not clear which step of the hemostatic process is impaired during HP, and whether it leads to an increased risk of bleeding. We performed both in vivo and in vitro studies to elucidate the mechanism of impairment in the hemostatic process. In patients with acute pesticide intoxication who underwent HP, the platelet count decreased rapidly during the first 30 minutes from 242.4 ± 57.7 × 10³/μL to 184.8 ± 49.6 × 10³/μL, then gradually decreased even lower to 145.4 ± 61.2 × 10³/μL over time (p < 0.001). As markers of platelet activation, platelet distribution width increased continuously during HP from 41.98 ± 9.28% to 47.69 ± 11.18% (p < 0.05), however, mean platelet volume did not show significant change. In scanning electron microscopy, activated platelets adhered to modified charcoal were observed, and delayed closure time after HP in PFA-100 test suggested platelet dysfunction occurred during HP. To confirm these conflicting results, changes of glycoprotein expression on the platelet surface were evaluated when platelets were exposed to modified charcoal in vitro. Platelet expression of CD61, fibrinogen receptor, significantly decreased from 95.2 ± 0.9% to 73.9 ± 1.6%, while those expressing CD42b, von Willebrand factor receptor, did not show significant change. However, platelet expression of CD49b, collagen receptor, significantly increased from 24.6 ± 0.7% to 51.9 ± 2.3%. Thrombin-antithrombin complex, a marker for thrombin generation, appeared to decrease, however, it was not statistically significant. Fibrin degradation products and d-dimers, markers for fibrinolysis, increased significantly during HP. Taken together, our data suggests that hemoperfusion leads to impairment of platelet aggregation with incomplete platelet activation, which was associated with reduced thrombin generation, accompanied by increased fibrinolysis.

BACKGROUND

No diagnostic tests reliably distinguish primary immune-mediated thrombocytopenia (pIMT) from other causes of thrombocytopenia.

OBJECTIVE

The purpose of the study was to evaluate diagnostic sensitivity and specificity using modified direct and indirect platelet-associated immunoglobulin (PAIg) assays and reticulated platelets (RP) by flow cytometry for the classification of thrombocytopenic dogs and differentiating pIMT.

METHODS

Platelets were isolated from plasma samples of thrombocytopenic dogs and nonthrombocytopenic healthy and ill dogs. For direct PAIg, they were analyzed by flow cytometry after incubation with anti-human amylase fluorescein isothiocyanate (FITC, negative control), anti-canine IgG-FITC, anti-canine IgM-FITC, and anti-human CD61-conjugated fluorochrome (AF647). For indirect PAIg, platelets from normothrombocytic dogs were incubated with thrombocytopenic dog plasma and analyzed similar to direct PAIg. RP percentages were determined based on forward light scatter vs thiazole orange fluorescence.

RESULTS

Seventy-five thrombocytopenic dogs, 16 nonthrombocytopenic ill dogs, and 24 healthy dogs were evaluated. Diagnostic sensitivity and specificity utilizing direct IgG was 29.4% and 75.9%, respectively; when combining direct/indirect assays (IgG/IgM), it was 76.5% and 65.5%, respectively, for distinguishing pIMT. For RP, no significant difference between pIMT and sIMT was noted. RP>> 8% with positive PAIg had a sensitivity of 94% and specificity of 27.6% for distinguishing pIMT. There was a significant difference in platelet concentration and CD61% staining between control and pIMT.

CONCLUSIONS

The combined modified assays resulted in fair diagnostic sensitivity and specificity for the diagnosis of pIMT. The modification of the immunoglobulin assays improved diagnostic accuracy; however, a single panel to accurately classify thrombocytopenia remains elusive.

OBJECTIVE

To detect whether preeclampsia influences neonatal intrahepatic hematopoiesis, given that an activation of fetal neutrophils and monocytes during the course of this disorder occurs.

METHODS

We examined liver samples from 10 neonates of hypertensive/preeclamptic women at 27 to 28 weeks of gestation delivered by a cessarian section. All neonates were placed in incubators but they all died within 24 hours due to immaturity. The control group comprised 10 fetuses of the same gestational age, after voluntary abortion due to a neural defect. Specific antibodies against CD34, glycophorin C, hemoglobins A and F, myeloperoxidase, CD61, CD68, terminal desoxynucleotidyl transferase and the pax-5/B-cell specific activator protein, were used in each sample.

RESULTS

Neonates from hypertensive/preeclamptic women, in comparison with controls, showed: a statistically significant reduction of erythropoiesis by 25% (p=0.015); a statistically significant increase of granulopoiesis (p=0.019); a statistically significant increase in the expression of CD68 positive cells of the monocytic lineage (p=0.017); a statistically significant increase in the expression of CD34 progenitor/stem positive cells (p=0.021). No statistically significant differences were observed in both examined groups, concerning megakaryopoiesis and B lymphopoiesis.

CONCLUSIONS

Preeclampsia of pregnancy has an impact on neonatal intrahepatic hematopoiesis by increasing granulopoiesis, reducing erythropoiesis and triggering endothelial and stem cell activation. We suggest that these findings reflect a state of persistent inflammation and a loss of red blood cell production possibly contributing to the neonatal morbidity related to this disorder.

Objective: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients.

Methods: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF⁺MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations.

Results: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF⁺MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF⁺MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF⁺MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF⁺MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05).

Conclusion: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF⁺MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

题目: ET患者血浆MP水平测定及其与JAK2V617F突变关系的研究.

目的: 检测原发性血小板增多症（ET）患者血浆中的微颗粒（MP）水平，并分析其与JAK2V617F突变的关系.

方法: 抽取56例ET患者及28例正常人空腹静脉血，枸橼酸钠抗凝(1∶9)，以藻红蛋白标记的特异性荧光抗体CD235a-PE、CD61-PE、CD142-PE和CD62E-PE 分别标记红细胞微颗粒（RMP）、血小板微颗粒（PMP）、组织因子微颗粒（TF⁺MP）及内皮细胞微颗粒（EMP），FCM检测4种MP绝对数。试剂盒抽提基因组DNA，PCR扩增目标DNA，琼脂糖凝胶电泳筛选26例JAK2V617F突变标本.

结果: ET组的RMP（157.2±304.9/μl vs 21.3±18.4/μl）、PMP（1378.9±2454/μl vs 113.8±97.1/μl）、TF⁺MP（123±354.6/μl vs 21±15.7/μl）、EMP（1434.7±2601.9/μl vs 291.7±297.7/μl）高于正常对照组（P<0.05）；8例血栓组的RMP（258.2±327.7/μl vs 55.6±63.8/μl）、PMP（1853±1874.7/μl vs 444.7±712/μl）、TF⁺MP（120.4±112.5/μl vs 60.3±128.3/μl）、EMP（1637.1±1563.5/μl vs 629.4±1000.2/μl）高于25例非血栓组（P<0.05）；17例脾肿大组的RMP（306.8±491.1/μl vs 59.3±51/μl）、PMP（2944.8±3767.6/μl vs 334.8±420.2/μl）、TF⁺MP（162.9±166.8/μl vs 31.0±28.7/μl）、EMP（3031.8±4048.8/μl vs 701.5±729.2/μl）高于11例非脾肿大组（P<0.05）；26例JAK2V617F突变组的TF⁺MP高于未突变组（154.7±516.3/μl vs 100.5±126.6/μl）（P<0.05），另外3种MP在这2组间无明显区别（P>0.05）.

结论: ET组、血栓组、脾肿大组4种MP水平均高于对照组；JAK2V617F突变组患者TF⁺MP数高于未突变组，另3种MP水平在突变与未突变组之间无明显区别.

BACKGROUND

Preeclampsia (PE) is associated with platelet activation, which may be involved in its pathogenesis promoting coagulation and mediating inflammation. We investigated whether the platelet activation status together with the frequency of platelet-leukocyte aggregates/PLA and monocyte tissue factor/TF expression could be used as laboratorial biomarkers for PE diagnosis and prognosis.

METHODS

Ninety-seven women were evaluated including severe PE/sPE (N=15), mild PE/mPE (N=20), normotensive pregnant/NP (N=31) and non-pregnant women/nonP (N=31). Platelet markers were analyzed by flow cytometry.

RESULTS

Platelet counts and CD41a expression by platelets were lower in NP and sPE vs nonP. The expression of CD61 was lower during pregnancy. Altered balance of platelet marker expression was also observed in NP and sPE vs nonP. No significant differences in the PLA and TF expression by monocytes were observed among the groups. There are several correlations between platelet activation markers, especially in sPE, which suggest a relevant role of the hemostatic/immunological cross-talk in this disease.

CONCLUSIONS

PE is not associated with increased platelet activation markers. It cannot rule out a role of platelet activation in the PE pathophysiology. Despite those correlations, we did not find a putative laboratorial biomarker that could be useful by itself for PE diagnosis and prognosis.

Peripheral acute leukemia (PAL) is extremely rare. We report on the first case of acute megakaryocytic leukemia presenting with PAL and complex chromosomal abnormalities. At diagnosis, the patient had 10.5-21% marrow blasts and 55-60% peripheral blasts which expressed CD42b and CD61. Two related clones with the karyotype of 48,X,-X,+3,+9,del(9)(q12q31) x 2,+17,i(17)(q10)/ 49,idem, +18 were revealed by conventional cytogenetics, but no AML-1/ETO fusion transcript was detected by RT-PCR assay. The significance of this case is discussed concisely.

To evaluate treatment-related changes of the reticulin stain-measured fibrosis in Ph(1+)-CML, a clinicopathological study was performed on sequential trephine biopsies of the bone marrow following either interferon (IFN) or busulfan (BU) monotherapy. Using the monoclonal antibody CD61 for the identification of megakaryopoiesis and Gomori's silver impregnation method, number of megakaryocytes and density of argyrophilic (reticulin and collagen) fibers were determined by morphometry. We studied specimens from 26 patients with IFN-alpha 2b (including nine patients with additional IFN gamma) therapy and from 23 patients who had received BU. In both groups, repeated bone marrow biopsies (total 125) revealed a significant increase in the fiber content, as well as in the number of megakaryocytes during treatment. To assess the dynamics of myelofibrosis more precisely, computation of differences in the degree of fiber density between the first and last examination was carried out. Regarding the considerable variations in the biopsy intervals, a so-called myelofibrosis progression index (MPI) was calculated. Following this rationale, we were able to demonstrate that, in comparison to the BU-group, speed of progression of bone marrow fibrosis was significantly increased in CML patients treated with IFN. Preliminary statistical analysis indicated a relationship between myelofibrosis on admission, which was always associated with increased growth of megakaryocytes, and the MPI with survival. Even when these parameters were regarded, prognosis was significantly more favorable in the IFN-treated patients. The failure of IFN and BU to inhibit the evolution of myelofibrosis may be related to several conversely acting pathomechanisms. Among others, the inability of both therapeutic agents to reduce the number of megakaryocytes more effectively should be taken into consideration.

The immunosuppressive agents administered to maintain the remission of idiopathic nephrotic syndrome (INS) may have a deleterious effect on several cell types. The aim of this study was to analyze platelet activation and reactivity in children with INS treated with cyclosporin A (CyA). The study groups comprised 16 children with remission of INS induced by CyA and 16 children with glucocorticosteroid-induced remission 8 weeks from the onset of INS relapse. Fifteen healthy children served as controls. Surface expression of CD61, CD62P, and CD42b on resting and thrombin-stimulated platelets was analyzed with flow cytometry. No differences between groups were found in CD61, CD62P, and CD42b surface expression, but markers of the coagulation cascade and fibrinolysis or endothelial injury (F1+2 prothrombin fragments, tissue plasminogen activator inhibitor 1) were elevated in patients treated with CyA compared with children on steroids and healthy controls. No correlations between markers of platelet function and CyA concentration were found. We postulate that CyA administration in nephrotic patients causes an activation of thrombinogenesis but does not influence platelet activation and reactivity in INS.

The two acute myelomonocytic leukemia sister cell lines MOLM-17 and MOLM-18 and the Epstein-Barr-virus positive non-malignant B-lymphoblastoid cell lines (B-LCLs) B422 and B423 were established from the bone marrow sample of a 60-year-old Japanese male in the advanced leukemic phase of refractory anemia with excess of blasts, a subtype of myelodysplastic syndromes (MDS). MOLM-17/-18 are proliferatively responsive to the growth factors present in the culture supernatant of the 5637 cell line. The B-LCLs are constitutively growth factor-independent. MOLM-17 and B422 were established at eight months after the initial diagnosis, while MOLM-18 and B423 were derived from a sample one month later. Immunophenotyping of the first leukemia sample revealed a mixed lineage leukemia immunophenotype with positivity for terminal deoxynucleotidyl transferase (TdT), CD13 and CD19; the second sample revealed solely myeloid characteristics with positivity for CD13, CD41 and CD61, whereas TdT was negative. MOLM-17/-18 showed immunomarker profiles typical of the myelomonocytic lineage. The karyotype analysis of MOLM-17/-18 revealed various non-random numerical and structural abnormalities including del(5)(q?), -7, der(11)add(11)(p11.2)add(11)(q23), add(17)(p11.2), add(18)(p11.2), -20, -22 as common aberrations. Treatment with tumor necrosis factor-alpha induced pronounced cellular differentiation of both cell lines into macrophage-like cells. The overall profile of MOLM-17/-18 based on their extensive immunological, cytogenetic and functional characterization suggests that these cell lines together with the paired B-LCLs B422 and B423 may represent scientifically significant in vitro models which could facilitate investigations into the pathobiology of MDS.

Elastin-like polypeptide (ELP) coatings have been shown to have non-thrombogenic properties both in vitro and in vivo. In this work, we expand our understanding of this phenomenon by investigating the interaction of these coatings with leukocytes. Citrated whole blood was exposed to a shear rate of 300 s^-1 for 2 hours at 37 °C on ELP1- and ELP4-coated polyethylene terephthalate (Mylar™) surfaces in a cone and plate device. Scanning electron microscopy and flow cytometry were used to measure leukocyte activation and platelet-leukocyte aggregation in response to the ELP1 and ELP4 coatings on the surface and in the bulk, respectively. Surface analysis showed little leukocyte activity on the surface of uncoated positive controls. Both the tissue factor (TF) expression (indicative of leukocyte activation) and CD61 expression (indicative of platelet-leukocyte aggregates), in the bulk were decreased by 40% and 20%, respectively, with the ELP coating of Mylar™, while a two- to three-fold increase in CD11b upregulation (indicative of leukocyte activation) for ELP1 and ELP4 was determined. Two of three bulk markers indicated that ELP-coated Mylar™ decreased the leukocyte response compared to the uncoated Mylar™, while the third, CD11b, indicated an increase in leukocyte response to the ELP coatings.

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit(-)Tie2(-)CD41(+) Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41(+)CD42b(+)CD61(+) platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future.

CD62p expression was an important monitoring parameter for preserved platelets quality. To setup an optimized flow cytometry assay for preserved platelets based on the CD62p expression on platelets, the platelet samples were collected, 0.1 mmol/L persantine and 1.1 mmol/L EDTA were added into the modified TB S used to replace PBS dilution; the methodological evaluation were carried out. Results showed that 0.1 mmol/L persantine and 1.1 mmol/L EDTA achieved to prevent platelets activation during the test procedure. The favorable negative or positive samples were prepared for check of fluorescence antibody's quality to ensure the validity of results. CD61 was used to identify platelets for assay and improve veracity of assay. The special injector was also replaced by special big syringe needle for blood collection to reduce in vitro artifacts, and the prepared sample can be steady-going for 48 hours at 4 degrees C after fixed by 1% paraformaldehyde. It is concluded that this flow cytometry assay for CD62p positive platelets is simple and efficient.