Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools

Abstract

Background and Objectives Microparticles (MPs) are small phospholipid vesicles of less than 1 μ m, shed in blood flow by various cell types. These MPs are involved in several biological processes and diseases. MPs have also been detected in blood products; however, their role in transfused patients is unknown. The purpose of this study was to characterize those MPs in blood bank conditions. Materials and MethodsQualitative and quantitative experiments using flow cytometry or proteomic techniques were performed on MPs derived from erythrocytes concentrates. In order to count MPs, they were either isolated by various centrifugation procedures or counted directly in erythrocyte concentrates.Results A 20-fold increase after 50 days of storage at 4 ° C was observed (from 3370 ± 1180 MPs/ μ l at day 5 to 64 850 ± 37 800 MPs/ μ l at day 50). Proteomic analysis revealed changes of protein expression comparing MPs to erythrocyte membranes. Finally, the expression of Rh blood group antigens was shown on MPs generated during erythrocyte storage.Conclusions Our work provides evidence that storage of red blood cell is associated with the generation of MPs characterized by particular proteomic profiles. These results contribute to fundamental knowledge of transfused blood products.

Measurement of microvesicle levels in human blood using flow cytometry

“…A 1-h delay resulted in about a 50 2 100% increase in platelet and annexin V1 microvesicle counts with longer delays associated with larger increases in microvesicle counts. Storage of red cell concentrates lead to an exponential increase in red cell microvesicles, with a 20-fold increase from 6 to 50 days of storage (17). Vigorous agitation for 1 2 2 h lead to 200 2 900% increases in platelet and/or annexin V1 microvesicles (11,14).…”

“…To prepare platelet free plasma the sample is typically spun twice at 2,000g for 15 min, a total of 60,000g-min of sedimentation, 60 times more than the preparation of PRP. This additional centrifugation of whole blood or platelet rich plasma removes cells and platelets but also reduces red cell, platelet, and annexin V1 microvesicles to a variable extent from sample to sample (16,17,20). Figure 1 shows total light scatter particles and platelet-derived (CD411) microvesicles in platelet rich plasma versus platelet microvesicles in platelet free plasma.…”

Erythrocyte-Derived Microvesicles Amplify Systemic Inflammation by Thrombin-Dependent Activation of Complement

“…Erythrocyte-derived microvesicles increase significantly in number during storage 15,16 and therefore large amounts are given to patients at the time of transfusion. Microvesicle release is a coordinate and active process coined ectocytosis to account for the fact that these vesicles (ectosomes) bud directly from the cell membrane.…”

Red Blood Cell Microparticles: Clinical Relevance

“…After dilution and antibody staining, samples were analyzed by flow cytometry. In this semi-quantitative study, an increase in the number of EMPs during storage from approximately 3.3 ± 1.2 × 10 9 /l on day 6 to approximately 20 ± 10 × 10 9 /l after 35 days of storage was observed [12]. We also tested the ability of EMPs to support coagulation: EMPs were isolated from 42-45 days stored erythrocyte concentrates by centrifugation; then, an increasing amount of EMPs was added to standard plasma and the prothrombin time (PT, Quick time) was measured.…”