P-selectin is an adhesion molecule found in the alpha granules of platelets. Activation occurs in response to a range of inflammatory and thrombotic agents resulting in rapid up-regulation. Flow cytometry methods have recently been described for the analysis of platelet P-selectin expression in whole blood. While introducing these methods into our laboratory it was noted that expression could be stimulated, in vitro, in a number of ways. This study shows that red cell lysis, the anticoagulant K3 EDTA and the time elapse between blood collection and antibody labelling had statistically significant effects on P-selectin expression. Post-labelling fixation, with CellFIX, caused no significant effect. We conclude that blood for P-selectin analysis should be collected in sodium citrate and that red cell lysis and centrifugation should be avoided. When comparing samples, the time between collection and labelling should be standardized. The relatively high CV for the assay indicates that all samples should be labelled and analysed in duplicate with the mean level reported.
Background: The β2-integrin CD11b (Mac-1) plays a crucial role in the firm attachment of leucocytes to the endothelium during the inflammatory response. Objective: This study aimed to determine whether the increased incidence of infections witnessed in elderly individuals compared to their younger counterparts was associated with deficiencies in basal expression and/or upregulation of CD11b. Methods: Flow cytometry was used to measure CD11b expression, before and after in vitro tumour necrosis factor alpha (TNF-α) stimulation, on neutrophils, monocytes and lymphocytes from healthy volunteers aged less than 36 years and Senieur-approximated 70–85 and over 85 year olds. The TNF-α levels in serum were measured using a commercially available enzyme-linked immunoassay technique. Results: The basal expression of CD11b on monocytes and lymphocytes was highest in the 70–85-year-olds and lowest in the >85-year-olds. Following in vitro stimulation using low (10 IU) and high (100 IU) TNF-α concentrations, subjects >85 years consistently showed significantly lower increases in CD11b expression on each of the three cell types. The maximal increase in CD11b expression was in the 70–85-year age group for neutrophils and monocytes and in <36-year-olds for lymphocytes. Serum TNF-α was significantly higher in the elderly groups. Regression analysis showed a significant association between TNF-α and expression of CD11b on lymphocytes before and after TNF-α stimulation and for neutrophils before stimulation. Conclusions: The results of this study suggest that CD11b expression on leucocytes may not be consistent throughout life. Such age-related changes could compromise the inflammatory response, rendering individuals >85 years old more susceptible to infections. Alternatively, the lower levels of CD11b expression in this group may represent downregulation and protection against excess leucocyte activation within the vascular system and may, therefore, provide a mechanism for successful ageing.
Small differences in levels of certain haemostatic components may be clinically significant. It is important therefore to eliminate potential sources of confounding variability. This study investigated the effect of removing tourniquet pressure prior to sample collection on plasma fibrinogen levels, platelet P-selectin and monocyte tissue factor expression. Blood was collected from the right arm under maintained tourniquet pressure and from the left arm following the release of pressure once the vein was sufficiently inflated for insertion of a needle. Whole blood was labelled within one hour of venepuncture to allow analysis of platelet P-selectin and monocyte tissue factor by flow cytometry. Plasma fibrinogen levels were analysed in samples stored at -70 degrees C, for all individuals at the end of the study using a method based on the Clauss technique. Intra-individual variability for each of the components was assessed by collecting samples under tourniquet pressure from four individuals on the same day on three consecutive weeks. Intra-individual variations were greater than assay CVs for all three components. There were no significant differences between the two tourniquet methods of collection for fibrinogen, P-selectin or tissue factor. In conclusion, there is no reason not to use a tourniquet during collection of blood for analysis of plasma fibrinogen, platelet P-selectin or monocyte tissue factor.
Summary. Inhibiting platelet and endothelial nitric oxide production favours platelet adhesion and aggregation, and arterial vasoconstriction. This study investigated the effect of N G -nitro-l-arginine methyl ester (L-NAME), a stereospecific inhibitor of nitric oxide synthesis, on P-selectin expression on platelets, platelet-derived microparticles and platelet-leucocyte aggregates, and on soluble P-selectin levels. Twelve healthy male volunteers were infused intravenously with L-NAME and then with a 10% solution of either l-or d-arginine. Blood pressure responses were recorded and whole blood and serum collected at baseline and after each infusion. P-selectin expression was analysed in all samples by flow cytometry. Serum levels of soluble P-selectin were batch analysed using an enzyme-linked immunosorbent assay at the end of the study. P-selectin expression on platelets, platelet-derived microparticles and platelet-leucocyte aggregates did not vary significantly from baseline levels following the infusion of L-NAME or l-or d-arginine. However, endothelial nitric oxide synthase inhibition caused a marked elevation of arterial blood pressure (P < 0AE01) that was restored to pretreatment values by l-but not d-arginine. Serum levels of the soluble form decreased significantly (P ¼ 0AE001) following the infusion of l-and d-arginine compared with samples taken at baseline and following L-NAME infusion. In conclusion, inhibition of constitutive nitric oxide synthase in the endothelium and platelets produced significant increases in blood pressure but did not alter platelet membrane expression of P-selectin.
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