Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations.

The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases.

The ex vivo function and expression of function-associated antigens of peripheral blood neutrophils and monocytes.

The availability of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors (rhG-CSF and rhGM-CSF) has prompted many studies of the analysis of antigen expression and function of monocytes and neutrophils from patients receiving these factors as therapeutic agents. Preparatory procedures for leukocytes are known to alter antigen expression and so function. We therefore investigated the use of a novel procedure in which live leukocytes are analyzed by flow cytometry without isolation from blood. The expression levels of CD11b, CD13, CD14, CD16, and CD18 antigens and L-selectin (TQ1 and Leu-8 epitopes) on neutrophils and monocytes from 15 normal individuals were determined and compared with a previously used method in which the leukocytes were fixed and the erythrocytes lysed before analysis. Significant differences for the apparent expression of CD11b, CD18, and L-selectin were observed between the two methods. The reasons for this were investigated. Since the new method allowed analysis of live cells, we also investigated whether modulation of antigen expression could be determined following receptor agonist interaction. This was found to be easily achievable, and we advocate using the new procedure where possible for the ex vivo analysis of function and function-associated antigens on monocytes and neutrophils.

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10(-4) to 10(-5). One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups.

Comparative expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells in patients with chronic lymphocytic leukaemia (CLL)

The aim of this study was to investigate the expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells of 13 patients with chronic lymphocytic leukaemia (CLL). This was carried out using a series of monoclonal antibodies (MAbs) against polymorphic and monomorphic class I and class II antigens, as well as to the transferrin receptor and assessed by flow cytometry and direct and indirect immunofluorescence. The expression of these molecules was assessed as mean fluorescent intensity (MFI). The results showed that cells from all 13 individuals expressed monomorphic class I antigens. The number of cases expressing polymorphic HLA-Bw6, -Bw4, -B7, -B27 and -A2 class I antigens on CD5- B cells was 11 (85%), 6(46%), 2(15%), 1(8%), 3 (23%), respectively, which was consistent with the expected population frequency distributions of these antigens. For each of the class I antigens on CD5+ and CD5- B cells, the ratio of the MFI was greater than 1 in 12 of 13 cases. For the transferrin receptor (CD71), this ratio was also almost always greater than 1. These results indicate that, unlike solid tumours where the loss or abnormal expression of class I and II antigens is a frequent event, the expression of class I antigens in CLL patients seems to be normal. This indicates that the loss of these antigens cannot provide the leukaemic cells with a selective advantage to escape immunological detection.

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood.

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.

This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.

Expression of functional antigens on neutrophils. Effects of preparation.

We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.

How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures.

For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, although there is no consensus as to the best method. In this study, we have examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whole blood, bone marrow and leucophoresis samples were analysed either directly after labelling with a vital nuclear dye (LDS-751) and fluorochrome-conjugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FACS Lysing Solution). By comparison with the results obtained from viable leucocytes in unmanipulated samples, it was found that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associated with a vital nuclear dye (in this instance LDS-751) and the CD34+ cells are detected with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.

von Willebrand factor release and P-selectin expression is stimulated by thrombin and trypsin but not IL-1 in cultured human endothelial cells.

The stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation. We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.

The anti-leukaemic effects and the mechanism of sodium selenite.

The anti-proliferative effects of selenium were studied both in vivo and in vitro. At a selenium concentration of 0.6 micrograms/ml, cells from patients with ALL-L1, L2 and AML-M1, M3 and M5 were more sensitive than cells from patients with CML. Cells from patients with AML-M2, CLL and leukaemic lymphoma were least sensitive. Normal bone marrow or peripheral blood cells were not sensitive to selenium at this concentration. In the mouse leukaemia models (L797, L615, L7712), the sensitivity of leukaemic cells were: L797 (93% cytotoxicity) greater than L615 (49.7% cytotoxicity) greater than L7712 (4.4% cytotoxicity). Sodium selenite injected i.p. increased the longevity of L797-inoculated mice. Administration of 40 micrograms selenium daily for 7 days resulted in a significant increase in the longevity of mice inoculated with 10(5) L797 cells. However, no remarkable increase of the longevity was observed in either L615- or L7712-inoculated mice after treatment with sodium selenite for 7 days. Treatment of the HL-60 leukaemic cell line with selenium caused a dose- and time-related decrease in DNA, RNA and protein syntheses as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. The inhibitory effect of selenium on DNA synthesis was reversed when selenium was removed from the medium, demonstrating that selenium-induced inhibition of DNA synthesis was due to interference with DNA biosynthesis rather than DNA template damage. These results suggest that the anti-leukaemic effect of sodium selenite is associated with inhibition of DNA replication, transcription and translation.

Gamma interferon augments functional and phenotypic characteristics of vitamin D3-induced monocytoid differentiation in the U937 human leukaemic cell line.

We studied the effect of gamma interferon on 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced differentiation of the human leukaemic cell line U937. Both phenotypic and functional aspects of 1,25(OH)2D3-induced differentiation were significantly augmented by gamma interferon (IFN). Gamma interferon had little effect alone but increased butyrate esterase staining and expression of CD14 antigen and the 40 kD Fc receptor (FcRII) in response to 1,25(OH)2D3. Ability to phagocytose IgG-opsonized bacteria, and superoxide burst in response to both IgG-opsonized bacteria and phorbol ester, was greater after incubation with both IFN and 1,25(OH)2D3 than with either agent alone. The degree of functional activation of cells showed a positive correlation with FcRII expression. In addition, IgG-induced generation of superoxide by differentiated cells was considerably reduced by pre-incubation with the anti-Fc receptor antibody IV3. We conclude that gamma interferon augments 1,25(OH)2D3-induced differentiation and functional activation of the U937 cell line. Increased functional activation may, in part, be due to up-regulation of surface FcR11.

Flow cytometric analysis of different adhesion molecules expression on circulating CD14- and CD64- human dendritic cell precursors.

Blood dendritic cell precursors (DCps) are identified as mononuclear leukocytes expressing HLA-DR but lacking the characteristic antigens associated with T cells (CD3), NK cells (CD16 and CD56) and B cells (CD 19). Dendritic cell precursors are distinguished from monocytes by their lack of expression of CD64 rather than of CD14. This study investigated whether CD14- DCps differed from CD64-DCps, which were predominantly CD14+, in their expression of five well-characterised adhesion molecules. There were significantly fewer cells expressing CD11b, CD18 and CD29 in the CD64-DCp population compared with CD14- DCps, and this CD64- DCp subpopulation also had a lower expression of CD11b and CD18. Our results suggest that the two DC precursor subpopulations may differ from one another in their binding characteristics to blood vessel walls and to other leukocytes.

Correlation of GP53 and P-selectin expression in myeloproliferative disorders and normal controls.

Platelet degranulation occurs when platelets are activated. Alpha degranulation releases P-selectin whereas lysosomal degranulation releases GP53. A correlation between these two markers might therefore be expected. We studied the correlation between P-selectin and GP53 in 50 patients with myeloproliferative disorders (MPD), 35 normal controls and 105 disease controls (patients with inflammatory bowel disease [IBD, n = 52], rheumatoid arthritis [RA, n = 26] and coronary artery disease [CAD, n = 27]) by flow cytometry before and after stimulation with thrombin ex vivo. There was no significant correlation between percentage expression of P-selectin and GP53 in unstimulated samples in normal individuals; r = 0.13, P = 0.3, n = 34. Mild thrombin stimulation (10 mU/ml) led to both alpha and lysosomal degranulation with a strong correlation (r = 0.62, P < 0.001, n = 35). Disease controls (IBD, RA and CAD) showed similar trends. In patients with MPD, in contrast, a strong correlation between the expression of these platelet activation markers was demonstrable in unstimulated samples (r = 0.37, P = 0.007, n = 50). P-selection and GP53 expression in stimulated samples also correlated well. The data support the existence of different control pathways for the steady state expression of P-selection and GP53. Heterogeneous steady state responses of P-selectin and GP53 may be physiological and loss of this heterogeneity may be a hitherto unreported and pathologically important feature of MPD. This lack of correlation appears to be specific to MPD and is not simply a function of increased in vivo platelet activation.

Platelet surface activation antigen expression at baseline and during elective angioplasty in patients with mild to moderate coronary artery disease.

Platelet activation is an important pre-thrombotic event. The elucidation of its pathophysiology could contribute to a reduction in the mortality associated with coronary artery disease-the foremost cause of death in the UK. We examined the platelets of 27 patients with angiographically documented coronary artery disease. All patients had stable angina and were taking their regular medication-including aspirin. We demonstrated significantly increased expression of GP53 and activated GPIIb/IIIa on the platelet surface using a sensitive flow cytometric method of detection. Comparison was made with a control group of 35 patients. Seventeen of the patients had coronary angioplasty carried out. Serial studies of these patients demonstrate an immediate and sustained increase in platelet activation and this has important implications for prevention of restenosis after angioplasty.

Evaluation of the in vitro antiproliferative properties of four novel anthracyclines YM1, 3, 4 and 6 in human leukemia cell lines.

The antitumor activity of novel doxorubicin analogues YM1, YM3, YM4 and YM6 was evaluated against drug sensitive U937 monocytic leukemia and CCRF-CEM lymphoid leukemia cell lines, as well as drug resistant CEM/VLB100 lymphoid multidrug resistant leukemia cell line by a [3H]thymidine incorporation assay. Different antileukemic activities of these new anthracyclines were observed in our studies. These novel anthracyclines produced a dose- and time-dependent inhibition in all the leukemic cell lines tested, while YM1 and YM3 were more effective than YM4 and YM6 against all the leukemic cell lines. The antitumor activity of all these novel analogues was lower than that of doxorubicin or epidoxorubicin in drug sensitive leukemic cells. The relative resistance values (IC50 of resistant cell line/IC50 of sensitive parental cell line) of YM1, 3, 4 and 6 were 27, 7, 5 and 14 respectively. These were lower than the resistance values for ADM and EDR which were 45 and 40 respectively. YM3 had a similar antileukemic activity against the CEM/VLB100 drug resistant leukemic cell line to ADM or EDR with a lower relative resistance value and a slightly increased IC50 value. Our results suggest that YM3 may be used in high dose for the clinical treatment of leukemias with possible less cardiotoxicity as well as less drug resistance.

Structure-dependent antitumor activities of novel anthracyclines YM1, YM3, YM4 and YM6: drug transport properties and effects on biomacromolecule synthesis in drug sensitive and resistant leukemia cells.

The antitumor activities of four novel doxorubicin (DOX) analogues, YM1, YM3, YM4 and YM6 in relation to their structure and drug transport properties, have been investigated in U937 monocytic and CCRF-CEM lymphoid drug sensitive leukemia cell lines, as well as in CEM/VLB100, a drug resistant subline displaying high levels of P-glycoprotein. Treatment of all cell lines with YM1, 3, 4 and 6 produced a dose-dependent decrease in DNA, RNA and protein synthesis as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. YM1 was more effective than YM3, YM4 or YM6 against the drug sensitive cells. The antitumor effects of all these DOX-analogues on macromolecule synthesis in U937 and CCRF-CEM cells were lower than that of DOX and epirubicin (EDR). A rapid accumulation of the novel anthracyclines was found in all cell lines compared with DOX or EDR. However, the maximal accumulation of the DOX-analogues was lower than that of EDR. There is a greater efflux from CCRF-CEM sensitive cells and less from CEM/VLB100 resistant cells of the DOX-derivatives when compared with EDR and DOX. Drug-induced cytotoxicity significantly correlated (P < 0.05) with drug retention levels in CCRF-CEM and U937 drug sensitive cells as indicated by an inverse correlation curve between anthracycline retention and drug-induced IC50 value. It was demonstrated that an increased level of drug retained within the sensitive cells would therefore produce a more cytotoxic effect of the drug. However, no such correlation was observed in CEM/VLB100 resistant cells. YM3 was shown to have an increased antitumor activity against CEM/VLB100 resistant cells compared with DOX with a lower resistance factor. These results showed that the antitumor effects of four novel DOX-analogues, like DOX or EDR, were associated with inhibition of DNA replication, transcription and translation. The finding that resistant leukemic cells are more susceptible to the cytotoxic effect of YM3 than DOX warrants further investigation to identify the intrinsic mechanism of resistance.

Flow-cytometric analysis of lymphocytes, leukaemias and lymphomas.

Flow cytometry may be used for immunophenotypic and karyotypic analysis of cells. The aim of this review is to provide insight into how the different leukaemias and lymphomas may be diagnosed by flow cytometry.

Programmed cell death (apoptosis) in immunity and haematological neoplasia.

Programmed cell death, also commonly referred to as apoptosis, is a genetically controlled sequence of events, often requiring protein synthesis, which results in cell death. Although initially described 20 years ago, it is only recently that its role as a mechanism in controlling cell population size and content has been fully realised. In this review the biochemical events of apoptosis are discussed briefly, followed by a more detailed look at the role of programmed cell death in the control of haemopoiesis and the maintenance of a balanced functional immune system. The role of proto-oncogenes and suppressor genes known to regulate programmed cell death is covered, and also their influence on the induction and maintenance of neoplastic disease. Finally, the potential role of apoptosis in the resistance of haematological malignancies to chemotherapy is commented on.

Flow cytometric analysis of microparticle phenotype and their role in thrombin generation.

BACKGROUND: Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation. METHODS: Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared. RESULTS: There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P < 0.001). A significant decrease in the number of CD42+, CD45+, CD15+, CD142+, and Annexin V+ microparticles was also observed. The change in CD42b+ microparticles correlated highly with the change in Annexin V+ microparticles (r = 0.68). Whilst the change in ETP correlated best with the change in CD15+ microparticles (r = 0.45) and the change in time to peak correlated with the change in Annexin V binding (r = 0.52) (P < 0.01). CONCLUSION: The presence of micropartcles in plasma significantly affects thrombin generation.