From a cohort of 220 adults with newly diagnosed acute myeloid leukemia (AML), 8 (3.6%) exhibited a rare variant of aberrant membrane phenotype. It was characterized with typical myeloid morphologic and cytochemical patterns and absence of myeloid associated antigens (CD13, CD33, CD14, glycophorin A, CD61). According to the French-American-British criteria, disease in 5 patients was classified as M1 and in 3 patients as M2. CD34, CD38, HLA-DR, and CD45 were strongly expressed in 4 of 5, 3 of 3, 8 of 8, and 3 of 3 analyzed cases, respectively. CD7 antigen was strongly expressed in 4 of 6 patients. Except for predominance of male sex and high frequency of CD7 antigen expression, no other remarkable clinical or biologic characteristics were noted. Detected variant of AML with the unusual membrane phenotype (CD34+, HLA-DR-positive, CD38+, CD45+, CD7+) might represent an example of extreme asynchrony in sequences of morphologic and immunologic maturation or abnormal epitope expression on leukemic cell membrane molecules CD13 and CD33. Although the clinical significance of this AML variant is unclear, the existence of such cases demonstrates the continued need for simultaneous cytochemical and immunologic studies in the evaluation of acute leukemias.

UNASSIGNED

Immunohistochemistry (IHC) staining of core biopsy sections often plays an essential role in the diagnosis of acute megakaryoblastic leukemia (AMKL). The goal of this study was to define the relative sensitivities of commonly used stains for markers of megakaryocytic differentiation.

UNASSIGNED

The sensitivities of IHC stains for CD42b, CD61, and von Willebrand factor (vWF) were compared in 32 cases of pediatric AMKL.

UNASSIGNED

The sensitivities of CD42b, CD61, and vWF were 90.6%, 78.1% and 62.5%, respectively. When CD42b and CD61 were used together, the combined sensitivity increased to 93.6%. There were no cases in which vWF was positive when both CD42b and CD61 were negative.

UNASSIGNED

CD42b can reliably be used as a solitary first-line marker for blasts of megakaryocytic lineage, whereas CD61 may be reserved for infrequent cases that are CD42b negative. There is no role for the routine use of vWF when CD42b and CD61 are available.

Acute myeloid leukaemia (AML) with multilineage dysplasia (MD) is one of the four main categories of AML in the World Health Organization (WHO) classification. The role of bone marrow trephine biopsy (BMTB) histology and immunohistochemistry in the diagnosis of AML-MD is currently unclear. BMTBs were studied in 11 cases of AML-MD and two cases of myelodysplasia that subsequently transformed to AML. Among them, six cases showed trilineage dysplasia and seven showed bilineage dysplasia. With respect to conforming to the WHO definition of AML, documentation of an increased proportion of immature myeloid cells was possible on morphology and counting of immature cells following immunostaining with CD34, CD117 or HLA-DR antibodies. Recognition and quantification of dysplastic features in the haemopoietic lineages was made easier by immunohistochemistry with antibodies to ret40f (glycophorin C), myeloperoxidase, CD61 and/or CD42b, CD34, CD117 and HLA-DR. Based on this relatively small series of cases we show the utility of BMTB and immunohistochemistry as an aid to the diagnosis of AML-MD. This has to be seen not just in light of its utility at diagnosis, but also the role the diagnostic BMTB would play for purposes of comparison when follow-up BMTBs are submitted in this group of patients.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.

Adhesion is one of the important biologic characteristics of leukemic cells. We previously reported a new megakaryocytic-erythroid cell line, JAS-R. In this study, JAS-R cells were segregated into two types by the differences of attachment to culture dishes. One type (designated as JAS-RAD cells) adhered to the substratum of the culture dishes, while the other (JAS-REN cells) grew as a single-cell suspension. Adhesion of JAS-RAD was inhibited by treatment with RGDS oligopeptide. Flow cytometric analysis revealed that JAS-RAD cells had high expression of CD41a and CD61 versus low CD235a expression, and JAS-REN showed low expression of CD41a, and CD61, and high CD235a. The two phenotypes were reciprocally exchangeable by selecting adherent or suspended cells from each type of culture. Microarray analysis and RT-PCR revealed that JAS-RAD cells expressed four major alpha-granule genes and JAS-REN cells expressed beta-globin. Interestingly, erythropoietin was only secreted by JAS-RAD cells. With regard to transcription factors, it was shown that GFI1, FLI1 and RUNX1 were strongly expressed in JAS-RAD cells while GATA1, FOG1 and NFE2 were equally expressed by both types. These findings indicate that adhesion via integrins is related to the phenotypic shift of JAS-R cells between megakaryocytic and erythroid lineages.

At present, various researches presented how subtypes of hematological malignancies are related to stages of the immune response, because the activated immune system represents a promising form in cancer treatment. This study explores the relationship between the adaptive immune system (T cells), and the coagulation system (platelets, platelet membrane glycoproteins, platelets derivate microparticles) which seems to play an important role in host immune defense of patients with acute myeloblastic leukemia (AML) or B cell lymphoma (BCL), 2 of the most common hematological malignancies subtypes.Blood samples (n = 114) obtained from patients with AML or BCL were analyzed for platelet membrane glycoproteins (CD42b, CD61), glycoprotein found on the surface of the T helper cells (CD4+), protein complex-specific antigen for T cells (CD3+), platelet-derived microparticles (CD61 PMP) biomarkers by flow cytometry, and hematological parameters were quantified by usual methods.In patients with AML, the means of the percentage of the expressions of the molecules on platelet surfaces (CD61 and CD42b, P < .01; paired T test) were lower as compared to both control subgroups. The expression of cytoplasmic granules content (CD61 PMP) had a significantly higher value in patients with AML reported to controlling subgroups (P < .01; paired T test), which is suggesting an intravascular activation of platelets.The platelet activation status was presented in patients with low stage BCL because CD61 and CD42b expressions were significantly higher than control subgroups, but the expression of CD 61 PMP had a significantly decreased value reported to control subgroups (all P < .01; paired T test). T helper/inducer lineage CD4+ and T lymphoid lineage CD3+ expressions presented significant differences between patients with AML or low stage BCL reported to control subgroups (all P < .01; paired T test).Platelet-lymphocyte interactions are involved in malignant disorders, and CD61, CD42b present on platelet membranes, as functionally active surface receptors mediate the adhesion of active platelets to lymphocytes, endothelial cells, and cancer cells.

Human osteoclasts, in contrast to mononuclear phagocytes, are known to express a well-defined restricted range of myeloid antigens. To determine whether these antigenic differences are present in other species, we examined the immunophenotype of chicken and rabbit osteoclasts, macrophages, macrophage polykaryons, and monocytes and compared them with similarly derived and cultured human cells. Human, rabbit, and avian osteoclasts reacted with monoclonal antibodies against human beta 1 integrins (CD29, CD49b, CD49d), beta 3 integrins (CD51, CD61), as well as human macrophage-associated antigen CD68. Avian osteoclasts also reacted for CD11a/18 and CD14 which are not present on human osteoclasts. Avian and mammalian monocytes, macrophages, and macrophage polykaryons expressed all the above antigens. Both avian and human macrophage polykaryons produced by culture of peritoneal macrophages reacted with anti-CD51 antibodies indicating that expression of the vitronectin receptor alone does not distinguish between these cells in vitro.

OBJECTIVE

Randomized clinical trials have shown that peripheral blood stem cell transplantations (PBSCT) with appropriate doses of CD34+ cells are associated with rapid, complete and sustained recovery of marrow functions. Nevertheless, in a minority af patients delayed platelet recovery may occur and it remains to be established whether analysis of transplanted CD34+ cell subsets may demonstrate correlation with this phenomenon. We studied a series of 80 consecutive transplanted patients with the aim of evaluating the effect of CD34+ stem cell numbers and, in a subgroup of 32 patients, the effect of the lineage specific subset numbers on time to platelet engraftment (i.e. time to platelet counts higher than 20x10(9)/L for two consecutive days without the need for platelet transfusions).

METHODS

Different clinical and paraclinical factors were examined in a multivariate analysis for effect on platelet engraftment in 80 patients.

RESULTS

The number of CD34+ cells/kg infused was the most important factor predicting the time to platelet engraftment. Patients receiving more than 10x10(6) CD34+ cells/kg had prompt platelet engraftment. The majority of the patients (78%) received fewer than 10x10(3) CD34+ cells/kg and 17/62 (27%) of these patients experienced delayed platelet engraftment. In 32 patients receiving fewer than 10x10(6) CD34+ cells/kg we focused on the content of different lineage specific CD34+ subsets in the PBSC products. The most significant correlation was recognized for CD34+/CD61+ megakaryocytic cell number and platelet engraftment. An inverse correlation between the CD34+/CD38Eth subset and platelet engraftment was found, indicating that a high number of CD34+/CD38Eth in the PBSC product might increase the risk for delayed engraftment. These results were further confirmed by the observation that patients who experienced platelet engraftment after day 20 had significantly more CD34+/CD38Eth cells/kg infused than patients with fast engraftment.

CONCLUSIONS

The number of total CD34+ cells/kg infused was the most important factor predicting time to platelet engraftment. CD34+ subset analysis in a subgroup of patients suggests that a high number of uncommitted progenitors may be associated with slower platelet recovery than transplantation with a higher fraction of more committed peripheral blood stem cells.

In diabetic patients, where the membrane lipid microviscosity of blood platelets is altered, the availability of platelet membrane receptors may change concomitantly. Platelet hypersensitivity in diabetic subjects was previously hypothesized to result from the nonenzymatic glycosylation-induced loss in platelet membrane fluidity. In our present study juvenile type 1 diabetic subjects were compared with their relevant controls with respect to thrombin-stimulated platelet activation in relation to glycation-induced impairments of platelet membrane dynamics. Our results indicate that: (a) the mean steady-state fluorescence polarization (p) of both 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilino-8-naphthalenesulphonate (ANS) in membranes from diabetic subjects were significantly greater than for control subjects, thus indicating reduced membrane lipid fluidity in diabetic platelets in various membrane regions; (b) the significantly higher [(3)H]NaBH(4) reduction, indicating the increased attachment of glucose to protein amino groups, was attributed to the proteins extracted from diabetic platelet membranes; (c) CD62-positive resting platelets were not significantly more abundant in diabetic patients; (d) basically, unaltered amounts of PADGEM-140 membrane antigen (CD62) copies were detected in resting diabetic platelets; (e) significantly higher numbers of membrane glycoprotein β(3) were found in diabetic platelets; (f) thrombin-induced elevations in the expression of CD61 (β(3)) and CD62 (PADGEM-140) occurred to much higher extent in platelets of diabetic patients, thus pointing to more profound activation of diabetic platelets by thrombin; (g) the total amounts of platelet membrane glycoprotein β(3) was significantly reduced in platelet lysates from diabetic subjects. We conclude that glycation-induced rigidization of platelet membranes might hypersensitize diabetic platelets to aggregating agents by rendering platelet membrane receptors more exposed to the external environment. Thus, thrombin may bind more efficiently to the exposed glycoprotein receptors (due to glycation) in diabetic platelets. Such excessive exposure and displacements toward the external environment might favour the accelerated shedding of some membrane proteins in diabetic platelets. We further suggest that their subsequent replacements would render platelet intrinsic storage pools exhausted and thus, might explain the diminished total amount of β(3) found in platelets of diabetic patients.

We report evaluation in rhesus monkeys of a flow cytometric procedure (MicroFlow) that has previously been shown to allow assessment of micronucleated reticulocytes (MN-RETs) in the peripheral blood of rats and dogs. Reticulocytes (RETs) were labeled with anti-CD71-fluorescein isothiocyanate, DNA was stained with propidium iodide using RNase treatment, and anti-CD61-phycoerythrin was used to reduce interference from platelets. Flow cytometric data were compared with microscopic scores of peripheral blood and bone marrow using standard acridine orange staining. A single iv administration of cyclophosphamide (CP, 5 mg/kg) induced an approximately 10-fold increase in blood MN-RET frequency, with the peak occurring 2 days after administration. After daily CP treatment to approximate a steady-state condition, the frequency of MN-RETs in peripheral blood was approximately 25% of that in bone marrow, indicating strong selection against MN-RETs. Nonetheless, CP-treated animals exhibited markedly elevated blood MN-RET values (2.45-3.99%, n = 3; compared to a mean baseline of 0.12%, n = 6). These measurements closely reflected the increased frequencies observed in the bone marrow compartment (Spearman correlation coefficient = 0.9856, n = 6). These data suggest that MN-RET measurements in blood are suitable for assessing chemical-induced chromosomal damage and can be readily integrated into routine toxicity tests, allowing genotoxicity data to be obtained as an integral part of toxicity evaluations. Microscopy-based scoring is challenging due to the low frequency of RETs and MN-RET in monkeys, but sufficient numbers of cells are easily scored with the flow cytometric procedure.

Pneumatic tube system (PTS) is an integral part of large medical facilities providing rapid interconnection between units within the hospital and often used to transport blood samples. The aim of our study was to compare a wide variety of hemostasis assays to identify assays sensitive to this transport method and diagnostic relevance of the alterations.

Routine coagulation and platelet tests (APTT, PT, TT, fibrinogen, light transmission aggregometry (LTA) with ADP, collagen, ristomycin and epinephrine), whole blood flow cytometry platelet function test (levels of CD42b, CD61, CD62P, PAC1, annexin V binding and mepacrine release) and global coagulation tests (thromboelastography (TEG), thrombin generation (TGT), thrombodynamics (TD), thrombodynamics-4D (TD-4D)) were determined in PTS- and manually transported samples of 10 healthy volunteers.

There were no significant differences between the values of APTT, PT, TT or fibrinogen between the samples transported by PTS or manually. The results for LTA demonstrated increase in the collagen-induced aggregation (84 ± 7% versus 73 ± 5%), while the response to epinephrine was decreased (58 ± 20% versus 72 ± 7.4%). Flow cytometry-based platelet function test showed a pre-activation of platelets by PTS-transportation while all integral assays of coagulation tested in the present study (TEG, TGT, TD, TD-4D) demonstrated a hypercoagulation shift.

Transportation by PTS caused significant shifts in parameters of functional and integral assays that exceeded parameter variation values and sometimes even were comparable to normal ranges. The results obtained in this study indicate that using of PTS for such assays may cause sufficient alterations of results and can lead to patient's mistreatment.

OBJECTIVE

To investigate whether activation of circulating platelets was present in the fetal and maternal circulation in cases with vascular disease in the fetal-umbilical-placental circulation as identified by umbilical artery Doppler study.

METHODS

We studied 20 mother-fetus pairs with an abnormal umbilical artery Doppler study indicating umbilical-placental pathology and 9 normal pregnancy pairs. All pregnancies in these two groups had elective cesarean delivery. We also studied 15 healthy nonpregnant women. Blood was collected at delivery, and flow cytometry was used to measure platelet activation. The platelet population was specified by the antiglycoprotein IIIa (CD61) antibody and activated platelets by the anti-P selectin (CD62) antibody. Platelet activation in response to thrombin (0.03 to 0.25 U/mL) was also assessed.

RESULTS

In the normal, healthy, nonpregnant women, there was no evidence of platelet activation in the fetal circulation (median, 0.63% of platelet population). Platelet activation was present in the fetal circulation in pregnancies with placental insufficiency (median, 4.57%) compared with normal pregnancies (median, 1.19%) (P =.034). The fetal platelets from pregnancies complicated by placental insufficiency also showed resistance to challenge with increasing thrombin concentration compared with normal fetal platelets (at 0.25 U/mL thrombin concentration, placental insufficiency pregnancy 69.82% and normal pregnancy 81.49%, P =.003). In the maternal circulation there were no differences in platelet activation (normal 4.89%, placental insufficiency 5.16%, P =.33) and sensitivity to thrombin challenge.

CONCLUSIONS

In the fetal circulation, the presence of Doppler-detected umbilical-placental vascular disease was associated with significantly enhanced fetal platelet activation and resistance to thrombin challenge. These changes were not noted in the maternal circulation. This provides further evidence of a primary vascular pathology in the fetal-placental circulation independent of disease in the uteroplacental circulation when the umbilical Doppler flow velocity waveform reveals a high resistance pattern.

The effect of imatinib on myeloproliferative disease in transgenic (Tg) mice expressing the P230 BCR/ABL transcript is unknown. To investigate this issue, we administered imatinib (30 mg/kg per day) orally to P230 BCR/ABL-expressing Tg mice for 30 days. Following imatinib administration, the enlarged spleen was significantly reduced to within the normal size range. Infiltrating megakaryocytes in the long-axis section of the spleen were also significantly reduced. However, the cellularity of the bone marrow was not affected. Fluorescence-activated cell-sorting analysis revealed that infiltrating mature granulocytes in the spleen were reduced in number. The numbers of infiltrating CD34, CD117, CD61, and CD11b populations were also reduced in immature populations of the spleen. Real-time quantitative polymerase chain reaction analysis of messenger RNA revealed a dramatic reduction in the p230 BCR/ABL transcript for CD34, CD117, CD61, and CD11b populations in both bone marrow cells and spleen cells. Western blotting and immunoprecipitation analysis also revealed a marked reduction in P230 BCR/ABL protein expression in both bone marrow cells and spleen cells. Thus, imatinib administration had the intriguing effect of replacing clones with high expression of p230 BCR/ABL complementary DNA with clones with very low expression. These data show that imatinib may still be capable of eliminating and eradicating clones with high p230 BCR/ABL expression and healing the disease phenotype in Tg mice. Pluripotent clones with very low p230 BCR/ABL expression still survive as immature CD34, CD117, CD61, and CD11b populations.

BACKGROUND

This study was conducted to assess different cellular microparticles (MPs) in thrombocytopenic human immunodeficiency virus type 1 and their significance as disease activity markers.

METHODS

Thirty-five thrombocytopenic human immunodeficiency diseases and 25 healthy controls with matched age and sex were selected. Viral load was quantitated by COBAS real-time polymerase reaction (PCR) assessment of absolute T-cell subsets CD4, CD8 as a disease progress marker. Platelet MPs, platelet-derived monocyte MPs (CD42a, CD61), erythrocyte MP (CD235a), monocytic MP (CD14), and platelet activity MPs (CD62P, PAC-1) were assessed by multicolor flow cytometry FACSCalibur, while platelet functions were assessed by platelet function analyzer (PFA-100). CD42a, CD61, and platelet activity index represented by PAC-1 and CD62.

RESULTS

P-selectin in HIV-infected patient samples were significantly greater (P < 0.001) than among controls. There was a negative correlation between the proportion of PAC-1 and CD62 P-selectin-positive MPs and levels of CD4(+) T-cell counts (r = -0.403, P = 0.016; r = -0.438, P = 0.008), respectively. There was a negative correlation between collagen-ADP and levels of CD4(+) T-cell counts (r = -0.368, P = 0.03). There was a significant high expression level of CD14 monocyte MPs in patients than controls (P < 0.0001), overexpression of CD235a (P < 0.0001), and no correlation between CD14 and CD4, whereas there was a significant negative correlation with CD235a (r = -0.394, P = 0.019). A linear regression analysis of CD4 as a disease progression marker with other variable indicators in HIV patients showed that CD235a could be the most sensitive predictor similar to CD4.

CONCLUSIONS

Different cellular MPs and platelets activated in HIV patients could have a role in thrombotic events in these patients.

BACKGROUND

Platelets play a crucial role in the pathogenesis of acute coronary syndromes (ACS). The efficacy of antiplatelet treatment is pivotal in the success of percutaneous coronary intervention (PCI) performed in patients with ACS.

OBJECTIVE

The aim of the study was to investigate the effects of clopidogrel with or without abciximab on the expression of platelet surface receptors and platelet function in patients with ST-segment elevation myocardial infarction (STEMI) undergoing PCI.

METHODS

Thirty patients with STEMI were included in the study. During acute primary coronary intervention, patients received aspirin (acetylsalicylic acid) and clopidogrel in a loading dose of 300mg. Clopidogrel was the only antiplatelet therapy used by nine patients (group B). Twenty-one patients (group A) received additional abciximab. Blood samples were collected and analyzed twice: before and up to 22 hours after administration of antiplatelet therapy. The platelet aggregation was established as primary platelet-related hemostasis (closure time [CT] assessed using the PFA100 system). The absolute number of platelet surface antigens as CD41a, CD42a, CD42b, CD61, and CD62P were determined by flow cytometry analysis.

RESULTS

The study revealed a statistically significant increase in CT induced by adenosine diphosphate and adrenaline (epinephrine) +130 seconds (p < 0.0001) and +94 seconds (p < 0.0001), respectively, in group A patients post-therapy. While in group B the parameters of CT did not change after treatment. In addition, the absolute number of CD41a antigens (glycoprotein [GP] IIb/IIIa) increased significantly after treatment in group A. No significant changes were observed after treatment in the expression of CD62P (P-selectin) antigens in either treatment group. There was a significant reduction in the percentage of CD62P-positive platelets in group B after antiplatelet therapy.

CONCLUSIONS

The absolute number of GP IIb/IIIa receptors increases and platelets are not activated up to 12 hours after cessation of abciximab therapy. Treatment of STEMI patients undergoing PCI with a loading dose of clopidogrel reduces the percentage of active platelets but does not influence the CT.

A morphometric analysis has been performed on bone marrow trephine biopsies following sequential double-immunostaining with monoclonal antibodies PC10 (anti-proliferating cell nuclear antigen--PCNA) and Y2/51-CD61 (anti-platelet glycoprotein IIIa) to evaluate endoreduplicative activity of megakaryopoiesis. In addition to a control group, patients included different subtypes of chronic myeloproliferative disorders (CMPDs) like chronic myeloid leukaemia (CML), polycythaemia vera (P. vera), primary thrombocythaemia (PTH) and finally primary (idiopathic) osteomyelofibrosis (OMF). In comparison with the normal bone marrow and also with P. vera and PTH a significant increase in PCNA-labelling (late G1 and S phases) of megakaryocytes was recognizable in OMF, contrasting with a striking reduction of this marker in CML. Particularly in advanced stages of OMF, secondary folate deficiency leading to a megaloblastoid appearance of erythroid precursors is a frequent finding. In pernicious anaemia previous cytokinetic studies have demonstrated an arrest in the S phase (DNA synthesis) of the cell cycle due to vitamin B12/folate (haematinic) deficiency. A similar pathomechanism may also be effective in OMF. Consequently, a block in the S phase of the cell cycle is assumed which is in keeping with the increased numbers of PC10-positive megakaryocytes. Significant correlations were calculable between megakaryocyte sizes and PCNA-staining capacity in the normal bone marrow and CMPDs. According to morphometry small-sized (hypoploid) megakaryocytes showed a prevalence of PCNA labelling. This finding is confirmative with a hypothesis on the dynamics of endoreduplicative activity of megakaryocytes, i.e. the prolongation of G1/G2 phases in larger (polyploid) elements. On the other hand, some of the giant polyploid megakaryocytes may cease endoreduplication and enter into G0 phase, which could partially explain the predominance of PCNA-negative large-sized cells of this lineage.

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocytosis. The destruction of autoantibody-sensitized platelets by FcγR-bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP-mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte-based phagocytosis assay, we investigated the impact of disease-associated autoantibody isotype in antibody-mediated phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients' serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a CMFDA(+) CD14(+) gate. IgG-coated platelets showed a significantly higher phagocytic index than IgM-coated platelets (mean 796 ± 157 versus 539 ± 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP.

Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24-72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4 degrees C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6- to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8- to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.

A serum-free culture method was used to study the growth of megakaryocytic progenitor cells (CFU-Meg) from patients with elevated platelet counts. The culture technique was combined with immunocytochemistry (APAAP, CD61) for the identification of CFU-Meg derived cells in cytopreparations of cells eluted from the culture dishes. Twenty-six patients with primary thrombocythaemia (14 untreated patients, UPT, 12 treated patients, TPT), 14 patients with reactive thrombocytosis (RT) and 9 normal individuals were studied. Unstimulated growth of CD61-positive cells was detected in 8/14 UPT, 8/12 TPT, 12/14 RT and 5/9 normal subjects (with mean CD61-positive cell counts of 75, 579, 236 and 7 per cytopreparation respectively). Cultures supplemented with interleukin 3 contained CD61-positive cells in 11/14 UPT, 7/12 TPT, 14/14 RT and 5/9 normal subjects (with mean CD61-positive cell counts of 157, 589, 250 and 7 per cytopreparation respectively). Thus, this serum-free culture technique combined with sensitive positive identification of CFU-Meg derived cells failed to discriminate between PT and RT. These results cast doubt on the usefulness of serum-free culture assays for the detection of unstimulated CFU-Meg growth in the differential diagnosis of patients with elevated platelet counts.

The in vitro biosynthesis of metallothionein (MT) was investigated in thrombocyte precursors (megakaryocytes) isolated from human cord blood. Biosynthesis and induction of MT in magnetic cell sorting-separated CD61(+) megakaryocytes was confirmed by immunohistochemical staining using monoclonal mouse anti-MT. The presence of MT was detected both in the nuclear and in the cytoplasmic area. Using RT-PCR, in vitro upregulation/induction of total MT transcripts was observed in CD61(+) cells at 48 h post-treatment with 100 micromol/L of zinc supplement. Seven isoform-specific mRNAs namely, MT-1A, MT-1B, MT-1E, MT-1G, MT-1H, MT-1X, and MT-2A were detected in the similar cell populations left untreated with zinc.

Three dogs of different breeds, ages, and genders were presented with pale mucous membranes, depression, anorexia, and splenomegaly. Observed were severe normocytic, nor-mochromic, nonregenerative anemia, thrombocytopenia, and leukopenia. Blood smears contained large, atypical cells with blue vacuolated cytoplasm, cytoplasmic blebs, round to oval central nuclei, and elevated numbers of cytoplasmic fragment resembling macroplatelets. Bi- and multinucleated atypical cells were found mainly in spleen, lymph nodes, and bone marrow. A final diagnosis of acute megakaryoblastic leukemia (AMegL) was made based on morphology and positivity to the megakaryocyte-derived cell-specific markers von Willebrand factor and CD61. In case nos. 1 and 2, no treatment was initiated, and the dogs died on days 4 and 3, respectively. Case no. 3 received supportive therapy with prednisone, and after a brief improvement the dog died spontaneously 35 days after initial presentation. Only 11 cases of AMegL have been reported in dogs, and the specific diagnostic criteria have not been well established. The presence of vacuolization, cytoplasmic blebs, central round nuclei, cytoplasmic fragments, and multinucleated cells in these three cases were considered useful to differentiate AMegL from other hematopoietic neoplasms.

The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used for platelet counting cannot discriminate platelets from other cellular particles and precipitates that cause similar signals. Visual (chamber counting) methods are still frequently used in routine laboratories to verify low automated platelet counts (< 50 x 10/l) despite obvious technical and statistical drawbacks. The following report shows how platelet counts can be measured by multiparameter flow cytometry with the help of reference particles (fluorescent latex beads) and platelet-specific antibodies i.e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-alpha (CD42b) and anti-GP IIIa (CD61). The linearity of this method was highly satisfactory and the observed imprecision was within acceptable limits. At a platelet concentration of 10 x 10(9)/l the coefficient of variation (CV, n = 10) ranged from 5.3% (PCV = 0.456) to 5.6% (PCV = 0.148). Accuracy was evaluated by comparing results to the ICSH-selected method for platelet counting. The correlation of both methods was significant (P < 0.005) and Passing-Bablok's linear regression analysis showed no systematic differences between the two methods. Comparisons of this new platelet counting technique were also performed with routine visual methods, automated blood analysers (Technicon H-1, Sysmex E-5000) and a different flow cytometric method using only forward and side light scatter properties of platelets for their discrimination. The linear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 x 10(9)/l. At lower platelet concentrations, our new platelet counting technique correlated significantly only with the visual and the forward/side scatter methods.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Short-term and saturated simulated dives followed by decompression with air, cause a decrease in platelet count and increased activation of fibrinolysis. The aim of this study was to determine whether short-term dives with trimix as a breathing mixture induce the activation of platelets, and/or fibrinolysis.

METHODS

30 male divers were subjected to short-term hyperbaric exposures to 0.7 MPa. Thirty divers used air and then the same divers used trimix as a breathing mixture.

RESULTS

The mean platelet count dropped significantly after decompression only in the group breathing air. The number of CD62P positive platelets and the amount of platelet-derived micro particles were statistically significant higher after decompression in both exposures. The number of CD61 positive platelets increased significantly only in the group breathing air. We observed a significant decrease of factor XII and fibrinogen concentrations after decompression only in the group breathing air. A significant increase in the concentration of plasminantiplasmin complex in both groups was detected.

CONCLUSIONS

Short-term hyperbaric exposure and decompression performed according to current safety standards activates platelets and the fibrinolytic system. Trimix protects divers from a reduction in the amount of platelets, fibrinogen and factor XII in the course of these exposures.