Fat embolization following major trauma is reported to be a quite common event, while the clinical fat embolism syndrome (FES) seems to be a much rarer event. Fat embolism occurs in 2 up to 23% of patients with isolated femoral shaft fractures. This complication appears to be related not only to the fracture, but also to the timing of stabilization. Sometimes it may be impossible to perform histochemical reactions on frozen sections to detect fat emboli thus confirming diagnosis or suspicion of FES. The finding of fibrinogen and platelets around the apparently empty spaces in the blood vessels has been proposed as an evidence for vital reaction due to either a vital cellular reaction or a flotation mechanism, thus supporting an intravital fat embolism. We report a fatal case due to fat embolism syndrome in a young man hospitalized for a right femoral neck fracture, treated with orthopaedic surgery and subjected to an intra-surgery transesophageal echocardiography that revealed embolization of numerous highly echogenic bodies. Four hours after the onset of clinical symptoms the man died from respiratory failure. The autopsy confirmed the clinical diagnosis of fat embolism syndrome. The histological examination revealed a large amount of fat globules in cerebral and pulmonary arteries and in glomerular capillaries, as well as fibrin and platelet deposition confirmed by the positive results by Sudan III staining for lipids and immunohistochemistry with anti-CD61 and anti-fibrinogen antibodies. The quantitative classification of fat embolism was grade 3 of Sevitt's classification or grade 4 of Fineschi's quantification, according to the current quantitative microscopic methods used for grading fat embolism in pulmonary tissue.

BACKGROUND

Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).

OBJECTIVE

The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.

METHODS

Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.

RESULTS

The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0-4.3% (0-12,095/microL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.

CONCLUSIONS

These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet surface antigens, and certain hemostatic parameters was examined in 15 humans and 10 swine. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days resulting in a three-fold increase of total serum CoQ10 level. We observed a decline in plasma fibronectin (-20.2%), thromboxane B2 (-20.6%), prostacyclin (-23.2%), and endothelin-1 (-17.9%) level. Significant inhibition of vitronectin receptor expression was observed consistently throughout ubiquinone treatment. Inhibition of the platelet vitronectin receptor is a direct evidence of a link between dietary CoQ10 intake, platelets, and hemostasis. These findings may contribute to the observed clinical benefits by a diminished incidence of thrombotic complications in such patients.

BACKGROUND

Thrombocytopenia is a common haematological abnormality and no simple diagnostic test is available to diagnose thrombocytopenia pathogenesis.

OBJECTIVE

To evaluate sensitivity and specificity of reticulated platelets (RP) as a diagnostic test for thrombocytopenia with increased thrombopoietic activity.

METHODS

Prospective observational study in thrombocytopenic patients.

METHODS

A direct, whole-blood, dual-labelling flow cytometric method was used. Direct, whole-blood double coverage was achieved using a monoclonal anti-glycoprotein (GP)-III antibody (CD61 PerCP) for platelet identification and thiazole orange (Retic-count) as platelet mARN stain.

RESULTS

RP were measured in 101 thrombocytopenic patients and 104 non-thrombocytopenic controls. The mean RP percentage in 60 thrombocytopenic patients with no increased thrombopoietic activity was 7.5% (CI for 95%: 5.2-9.7) and RP absolute number was 3.2 x 10(9)/l (CI for 95%: 2.1-4.3). The mean RP percentage in 41 thrombocytopenic patients with increased thrombopoietic activity was 30.3% (CI for 95%: 25.1-35.5) and RP absolute number was 6.2 (CI for 95%: 4.8-7.7). The RP percentage cut-off for a diagnosis of thrombocytopenia with increased thrombopoietic activity was 11% [sensitivity 93%, specificity 85%, positive predictive value (PPV) 83%, negative predictive value (NPV) 95%].

CONCLUSIONS

RP measurement by flow cytometry, directly from whole-blood, is a useful screening test to differentiate between thrombocytopenia with high or low thrombopoietic activity. A RP percentage in excess of 11%, has a high sensitivity and good specificity for a diagnosis of thrombocytopenia with increased thrombopoietic activity.

We wanted to test the hypothesis that NO generation by L-arginine (LA) infusion will be beneficial in increasing blood flow to all organs to counteract the process of global ischemia during cardiopulmonary bypass (CPB) and to reduce platelet emboli by platelet inhibition. The effect of LA infusion on NO formation, vasodilation, and reduction of thromboembolic burden in organs and tissues after CPB was quantified with In-111-labeled autologous platelets in two major groups: 180 minutes CPB (CPB) and 90 minutes CPB plus 90 minutes reperfusion (RP). Platelets labeled with In-111 tropolone (650-780 µCi) were administered 24 hours before CPB and LA infusion (bolus, 10 mg/kg and infusion at 2 mg/kg/min, 21 pigs for 180 minutes CPB) in 8 groups of 30 Yorkshire pigs (30-35 kg, 6 pigs; LA 2 mg/kg/min, 3 pigs; sham-thoracotomy control, 6 pigs; unoperated control, 6 pigs). Two groups of 9 pigs (control CPB, 6 pigs; LA 2 mg/kg/min, 3 pigs) underwent 90 minutes of CPB and 90 minutes of reperfusion. All pigs were heparinized (ACT>> 400 seconds); CPB was instituted with a roller pump, an oxygenator (OX: Bentley Univox, 1.8 m2), and an arterial filter (AF: 0.25 m2, Bentley) at a blood flow of 2.5-3.5 l/min. Radioactive thrombi in OX and AF and emboli in viscera, brain, and connective tissues were imaged with a gamma camera and were finally measured with an ion chamber and a gamma counter. The percent of injected platelets (mean +/- SD) in the organs and tissues of all pigs was calculated. Cerebral emboli were mapped in 25 regions of both hemispheres of pig brain. Flow cytometry with antibodies to CD61 (GPIIIa) and CD62P (GMP-140:control) of porcine platelets was carried out with blood samples taken before, during, and after CPB. Coronary bypass with LA infusion decreased the amount of adherent thrombi in OX and AF (p < 0.07). The embolic burden in brain and lung also decreased. Regional cerebral mapping of In-111 platelets showed reduced emboli in almost all regions, including the medulla, hip pocampus, and posterior cerebral cortex in both LA-treated groups. Flow cytometry of blood samples demonstrated the shift of equilibria from single platelet to platelet-aggregate-microparticle during CPB and steady-state level after the first 5-10 minutes of initiation of CPB. The L-arginine infusion reduced thrombi and emboli during CPB in the pig model.

OBJECTIVE

To analyze the immunophenotype of blast cells in patients with acute leukaemia after polycythemia vera, together with the most relevant clinical and haematological disease characteristics.

METHODS

The immunophenotype was analysed in nine patients by immunofluorescence flow cytometry using a panel of 15 monoclonal antibodies. The DNA content of blast cells was determined using Vindelov's technique.

RESULTS

The most relevant clinical and haematological disease characteristics included: the presence of enlarged spleen and liver by 56% and 67%, respectively; a moderate degree of leucocytosis with thrombocytopenia while haemoglobin was normal in 50% of patients. All patients received alkylating agents or hydroxyurea, or both. Interestingly, the chronic phase in patients receiving this latter drug was shorter. All cases showed a myeloid phenotype, four of them reactive only to early myeloid antigens (CD13/33); in the remaining cases the blast cells displayed granulomonocytic (CD14+, CD15+), erythroid (CD71 ), or megakaryocytic (CD61+, CD41+) markers. Coexpression of lymphoid related antigens (CD7, TdT, or CD19) was also detected. The morphological assessment of blast cells agreed with the immunophenotyping in five out of the nine cases. Blast cells from all six patients analysed displayed a diploid DNA content and the proportion of S-phase cells ranged from 0.4% to 4%.

CONCLUSIONS

These findings suggest a pluripotential stem cell with myeloid commitment as the target cell of acute leukaemia after polycythemia vera.

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.

Treatment of the human erythroleukemia cell line K562 with the galactose-binding lectin, jacalin, results in rapid and profound alterations in the morphology and biochemistry of the cells. Within minutes of lectin addition, the cells adhere to the plastic tissue culture surface, and within hours, the cells spread on the surface, acquiring a monocyte-like appearance. Jacalin treatment results in elevated expression of CD61 (integrin beta 3) and CD14, a monocyte-associated cell surface antigen. These results suggest that jacalin treatment of K562 cells triggers intracellular events that result in differentiation along the monocyte lineage.

Glanzmann's thrombasthenia (GT) was diagnosed in two patients who presented with bleeding manifestations accompanied by absent platelet aggregation, secondary to adenosine-5'-diphosphate, adrenaline, arachidonic acid and collagen. Flow cytometry analysis for GPIIb/IIIa expression was done using CD61 and CD41 markers in these patients and their family members including siblings. The patients were sub typed as Type I as he had absent glycoproteins (GP) IIb/IIIa. Family studies by flow cytometry showed reduced GPII/IIIa expression in both the parents and one sibling. However, western blot showed abnormal GPIIb protein in all the family members including siblings. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. Patients' DNA was analyzed for mutation in both the GPIIb and GPIIIa genes by conformational sensitive gel electrophoresis (CSGE), followed by sequencing. CSGE showed defect in exon 12 and the promoter region of GPIIb. By sequence, it was confirmed that both the mutations were homozygous one was c.1028T>C and the other one was M33320.1:g.951G>A. For one of these mutations, restriction fragment length polymorphism (RFLP) was developed to look for the same mutation in all the family members. RFLP was developed using a restriction enzyme (TspRI) against the patient's mutation, c.1028T>C in exon 12 of GPIIb gene. RFLP followed by gel electrophoresis revealed that the mutation was heterozygous in all the family members. The findings by RFLP were double confirmed by direct DNA sequencing of the exon 12 in all the family members. Thus, TspRI marker may be used as a RFLP marker to predict the carriers in GT families, if the patients' mutation status is identified.

Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.

OBJECTIVE

To evaluate platelet-neutrophil aggregate (PNA) formation and neutrophil shape as indicators of neutrophil activation in dogs with systemic inflammatory diseases and after blood sample incubation with various platelet and neutrophil agonists.

METHODS

20 dogs with systemic inflammatory response syndrome (SIRS) and 10 healthy Beagles.

METHODS

Neutrophils were isolated from blood samples directly after blood sample collection and after incubation of blood samples with phorbol myristate acetate, collagen, adenosine diphosphate, epinephrine, or various concentrations of lipopolysaccharide or arachidonic acid. CD61+ neutrophils as an indicator of PNA formation were evaluated, and neutrophil size and granularity were assessed via flow cytometry.

RESULTS

Dogs with SIRS had more PNA formation, larger neutrophil size, and less granularity relative to control dogs, but no differences were evident when these dogs were grouped by whether they had sepsis (n = 6) or disseminated intravascular coagulation (12). A significant increase in PNA formation occurred after neutrophil incubation with all agonists, and incubation with phorbol myristate acetate elicited the strongest response. Neutrophils increased in size and decreased in granularity after incubation with all agonists except epinephrine. Incubation with lipopolysaccharide or arachidonic acid resulted in a dose-dependent effect on PNA formation and neutrophil shape.

CONCLUSIONS

SIRS appeared to increase the degree of PNA formation and neutrophil shape change. Similar changes after neutrophil incubation with platelet agonists suggested that platelet activation has a role in PNA formation. Additional studies are necessary to determine the clinical importance and diagnostic value of PNA formation in dogs with SIRS and sepsis.

This study assesses the value of immunologic and ultrastructural methods in disclosing the lineage commitment of cells from acute leukemias (ALs). Two hundred and fifty-one ALs were characterized morphologically, cytochemically, and immunologically. Myeloperoxidase (MPO) positivity in>> 3% of blasts was regarded as evidence of the myeloid origin of leukemic cells, cytoplasmic CD22 (cCD22) expression was taken as an indication for B-lineage acute lymphoblastic leukemia (ALL), and CD3+ (membrane or cytoplasmic) cases were classified as T-ALL. Diagnosis of minimally differentiated acute myeloid leukemia (AML-M0) was made when blast cells had undifferentiated features by light microscopy, reacted with at least one of the antibodies to myeloid-specific antigens (CD13, CD33, MPO), and lacked CD19, cCD22, and c/mCD3. Megakaryoblastic differentiation was demonstrated by the expression of CD41 and/or CD61. Following these criteria, 209 cases were classified as acute myeloid leukemia (AML) and 39 as ALL. Expression of lymphoid antigens was detected in 45% of AML cases and 30% of ALLs showed myeloid antigens. One case was regarded as a true biphenotypic leukemia because of the combined expression of MPO and CD33 for the myeloid lineage, and cCD3, CD2, and CD5 for the T-cell lineage. Two cases lacked signs of myeloid or lymphoid differentiation and were studied by electron microscopy methods. One displayed platelet peroxidase (PPO) activity and was classified as a megakaryoblastic variant, one other reacted with anti-CD33 and was considered AML-M0. We conclude that light microscopy and standard immunologic methods can accurately demonstrate the lineage orientation in greater than 99% of ALs. Integration with ultrastructural analysis can define the cell nature of virtually all cases of AL.

A megakaryoblastic cell line (MKPL-1) was newly established from the bone marrow of an adult patient with acute megakaryoblastic leukemia. This cell line grew in single cell suspension with a doubling time of 30 h and consisted of large primitive blasts with persistent development of giant cells carrying multilobed nuclei. MKPL-1 cells were positive for platelet GPIIb/IIIa (CD41) and GPIIIa (CD61), and expressed OKM5 (CD36), MY7 (CD13), and MY9 (CD33) antigens in the absence of erythroid and lymphoid markers. The cytochemical and morphologic characteristics of MKPL-1 were also consistent with those of megakaryoblasts. The cells did not, however, express ultrastructural platelet peroxidase which is considered to be another marker of the megakaryocytic lineage. Cytogenetic analysis of MKPL-1 revealed a model chromosome number of 92 with abnormal chromosomes including those found in the patient's bone marrow cells. Furthermore, MKPL-1 cells were serially transplanted into nude mice for nine passages with production of lethal tumors and leukemic manifestation. Thus, our megakaryoblastic cell line which can be maintained both in vitro and in vivo would be useful for further studies of the biology of megakaryopoiesis and megakaryoblastic leukemia.

The megakaryoblastic leukemia cell line MOLM-16 was established at relapse from the peripheral blood of a 77-year-old Japanese woman with minimally differentiated acute myeloid leukemia (AML-M0). Immunophenotyping of the fresh leukemic cells revealed a myeloid/NK precursor phenotype being positive for CD7, CD13, CD33, CD34, and CD56. In addition, megakaryocyte-associated antigens CD41 and CD61 were found to be positive. The established cell line designated MOLM-16 was proliferatively responsive to the treatment with various cytokines including EPO, GM-CSF, IL-3, PIXY-321, and TPO. MOLM-16 revealed characteristics of the megakaryocytic lineage in terms of immunophenotyping being positive for CD9, CD31, CD36, CD41, CD61, CD62P, CD63, CD110, CD151, thrombospondin, von Willebrand factor (vWf), and fibrinogen. Electron microscopic analysis showed positivity for ultrastructural platelet peroxidase in the nuclear envelope. The karyotype analysis of MOLM-16 revealed various numerical and structural abnormalities including t(6;8)(q21;q24.3), t(9;18)(q13;q21) and marker chromosomes. The extensive immunological, cytogenetic and functional characterization of MOLM-16 suggests that this cell line may represent a scientifically significant in vitro model which could facilitate the evaluation of megakaryocytic differentiation.

Background: Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Methods: Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. Results: MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Conclusions: Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells.

Keywords: cancer biomarker; cell-free DNA; exosomes; extracellular vesicles.

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.

OBJECTIVE

Does formation of platelet-derived microparticles correspond to platelet activation?

METHODS

The study was performed in 51 preterm newborns, 25 girls and 26 boys. The control group consisted of 55 term newborns, 25 girls and 30 boys. Blood samples were collected from the umbilical artery. The percentage of platelet-derived microparticles and platelet count were determined using flow cytometric analysis based on the CD61-positive antigen.

RESULTS

The percentage of platelet-derived microparticles was higher in preterm newborns (5.46) in comparison to term newborns (4.22, p < 0.01). We found 4.61% of platelet-derived microparticles in preterm female newborns and 6.28% in preterm boys (p < 0.0070). The platelet count was 256 x 10(3) microl in girls and 238 x 10(3) microl in boys. Female healthy term newborns presented higher values of platelet-derived microparticles (4.4%) than male newborns (4.07%, p = 0.4725, table 1). The platelet count in girls was found to be 308 x 10(3) microl and in boys 270 x 10(3) microl.

CONCLUSIONS

Higher percentage of platelet-derived microparticles in preterm newborns may provide a compensatory mechanism for the hemostatic system.

BACKGROUND

Co-presentation between dengue and diabetes mellitus (DM) can be expected. The author hereby assesses the effect of glycosylation process in poor glycemic control cases on the course of dengue infection.

METHODS

The aim of this work is to assess the effect of glycosylation of CD61 on the pathogenesis of dengue hemorrhagic fever. The hypothetical bioinformatics approach is used in this research.

RESULTS

Significant change of similarity between CD61 and dengue NS1 after glycosylation can be detected.

CONCLUSIONS

These findings might imply that immunomimicking process should be more difficult to occur.

BACKGROUND

Megakaryocytes are classically identified by their cellular morphology and expression of platelet glycoproteins.

METHODS

In this study, the expression of GPIIIa (CD61) on hemopoietic cells was analyzed by dual-fluorescence flow cytometry.

RESULTS

All monocytic cells (CD14+) were shown to coexpress CD61. As the expression of platelet protein on these monocytic cells cannot be reduced by treating the cells with anticoagulant (ethlyenediaminetetraacetic acid [EDTA]), this observation is not simply due to platelet adhesion. When sorted CD61(lo)CD14+ cells were studied by light and electron microscopy, platelets or platelet fragments could not be detected on the cell surface. These cells were found to have typical monocytic morphology but no megakaryocytic characteristics.

CONCLUSIONS

This finding demonstrates that without careful definition, the quantitation of megakaryocytic cells will be inappropriately high. A clear and unambiguous criteria for the identification of megakaryocytic cells is described based on the high expression of platelet glycoprotein (e.g., CD61(hi) or CD41(hi)) but not the monocyte marker (CD14(neg)).

BACKGROUND

Recent evidence suggests that the activation of platelets and their interaction with circulating cells are important independent risk factors for atherosclerosis. In non-uremic patients with symptomatic peripheral vascular disease, a relationship between serum homocysteine (Hcy) levels and platelet activity had been reported. The purposes of this study were to evaluate of effects of dialysis modality on platelet activity in patients with end-stage renal failure and to investigate the relationship between platelet activity, Hcy, and left ventricular hypertrophy (LVH).

METHODS

In age and sex matched 19 healthy subjects, 20 hemodialysis (HD) patients, and 18 continuous ambulatory peritoneal dialysis (CAPD) patients, the expression of platelet surface receptors CD41, CD61, CD42a, and CD62P were investigated. CD62P expression was statistically significantly increased in HD patients compared with CAPD patients and controls (34.4 +/- 22.5%; 17.3 +/- 19.6%, 12.0 +/- 15.6%, respectively, p < 0.05), but not in CAPD patients compared with controls. There was a positive correlation between CD62 expression and duration of dialysis in HD patients (r = 0.498, p = 0.026). Mean plasma Hcy levels in dialysis patients were higher than reference levels. However, we could not find any relationship between CD62 expression, Hcy, and LVH in both groups (p>> 0.05).

CONCLUSIONS

Hemodialysis and peritoneal dialysis (PD) have a different impact on the expression of CD62: peritoneal dialysis seems to have a more favorable effect. It may be possible that the differences in biocompatibility between PD and HD potentially contribute to differences in CD62 expression.

The initiation of hemodialysis using cuprophane membranes is followed by a rapid fall in the circulating neutrophil count. This neutropenia is caused by a transient sequestration of neutrophils in the lung due to homotypic aggregation, largely in response to generation of C5a by contact of plasma with the dialyzer. The transient nature of hemodialysis neutropenia is due to desensitization of neutrophils to stimulation by C5a, thus demonstrating desensitization in vivo. To examine the in vivo effects on surface phenotype of continuous exposure of neutrophils to C5a over 3 h, the surface expression of 22 antigens was examined by flow cytometry in patients undergoing dialysis. Neutropenia was prominent at 15 min and absent at 60 and 180 min of dialysis. CD10, CD11b, CD11c, CD13, CD18, CD35, CD45, CD66acde, and CD66b were upregulated at 15 min and remained upregulated at 180 min. CD61 and CD63 increased slightly at 15 min and returned to baseline by 180 min. CD16 and CD62L were down regulated at 15 min and normalized by 180 min. CD15s, CDw17, CD32, and CD44 were slightly down regulated at 15 min and then returned to baseline by 180 min. CD11a, CD15, CD24, CD31, and CDw65 did not change during dialysis. This study demonstrates the changes in surface phenotype of neutrophils during prolonged in vivo exposure to C5a over 3 h, during which time neutrophils become desensitized to subsequent stimulation by similar concentrations of C5a but maintain responsiveness to other chemotactic stimuli.

BACKGROUND

Transient nature of adhesive interactions occurring during cell margination is mainly dependent on expression of selectins which are shed by activated cells. This shedding in the circulation may play an important role as anti-inflammatory mediator. Haemodialysis is also associated with P-selectin (CD62P)/sialyl-Lewis(x) (CD15s) interactions which mediate platelet-leukocyte coaggregation. We further investigated the mechanisms underlying leukocyte margination during haemodialysis.

METHODS

CD15s, CD11b and CD61 expression on circulating leukocytes from patients dialysed on synthetic membranes (modified polyacrylonitrile (SPAN), polysulphone (PS), and polyacrylonitrile (AN69) was assessed by cytofluorometry in a prospective crossover trial. We measured plasma levels C3a/C3a desArg, soluble CD62P, and CD62E molecules obtained from patients and healthy individuals.

RESULTS

Expression of CD11b and CD15s was upregulated on neutrophils from patients dialysed with SPAN and PS membranes during the dialysis session. A significant negative correlation was found between the expression of CD11b or CD15s molecules and neutrophil counts as well as between CD15s expression and monocyte counts during haemodialysis. As assessed by CD61 expression on leukocytes, we observed that platelets bound significantly onto both neutrophils and monocytes during dialysis with both membranes. A significant positive correlation was found between the expression of CD11b molecules and the percentage of CD61+ monocytes counts during SPAN and PS dialysis. We found a significant increase of soluble CD62P in plasma samples obtained from haemodialysed patients before the dialysis session as compared to the levels detected in plasma from healthy individuals.

CONCLUSIONS

This study documents a major role of CD15s, CD11b, CD61, CD62P molecules in the transient leukocytes activation and margination during haemodialysis on synthetic membranes despite their low complement-activating properties.

Patients with end-stage renal failure undergoing chronic hemodialysis manifest alterations in cell-mediated immune response despite adequate urea clearance. We conducted a clinical trial in 8 patients (6 male, 2 female; 57.6 +/- 19.0 years) to investigate cell subsets and adhesion molecules on mononuclear blood cells during hemodialysis in patients on either cuprophane (n = 60 or polysulfone (n = 2). Cells were analyzed from the arterial line before, at 5, 10, 15, 30, 90 and 240 min with a panel of 33 surface markers including antibodies towards CD3, CD4, TCR-alpha/beta, CD7, CD8, CD11a-c, CD14, CD18, CD19, CD25, CD28, CD29, CD38, CD44, CD49a-e, CD51/61, CD54, CD56, CD58, CD62, CD106, and HLA class II using single fluorescence analysis. In vivo results were compared with in vitro tests. Our results suggest a substantial difference in the expression of various surface markers and cell adhesion molecules (lymphocytes: CD4, CD25, CD28, CD51, CD54; monocytes: CD49, CD54, CD58, CD62E, CD62P) compared to healthy persons, and a distinct modulation during hemodialysis (lymphocytes: CD11, CD18, CD49, CD51, CD61; monocytes: CD29, CD49, CD51, CD54, CD61) which might affect the immune response beyond the single dialysis session.

OBJECTIVE

During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material.

METHODS

We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P.

RESULTS

Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes.

CONCLUSIONS

This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.