A Novel Whole Blood Real-Time Microparticles Detection Assay Using Lactadherin-Coupled Dynabeads.

Abstract

INTRODUCTION: The size and heterogeneity of microparticles (MP) make identification, isolation, and characterization a constant challenge. The current literature reveals a wide assortment of identification and isolation techniques, often resulting in inconsistent or conflicting data. Flow cytometry, while a suitable technique for characterizing MP, suffers from several limitations. Purifying MP requires centrifugation, thereby inducing microvesiculation through shear stress. The constraints of cytometer lasers limit detection of MP size. Annexin V has long been considered the gold standard for identifying phosphatidylserine(PS)-positive MP. However, it only binds PS efficiently in the presence of Ca2+, which may promote microvesiculation as well as aggregation of MP and other soluble proteins. Recent data suggest that lactadherin, a milk fat glycoprotein, binds PS independent of Ca2+ and with greater specificity. We developed a Dynal bead-based assay, which eliminates sample processing while increasing specificity in identifying PS+ and tissue factor-bearing (TF+) MP.METHODS: Lactadherin, isolated from bovine whole milk, were coupled to 4.5 μm Dynabeads. Lactadherin-coated beads (LB, 4 x105 beads/sample) were incubated with 1 mL freshly drawn, citrated whole blood from healthy adult volunteers (N=12) for 10 min at 25°C. LB were collected using a magnetic cell sorter, washed, and labeled with either a cell-specific PE-labeled antibody or FITC-labeled antibodies against TF. Polyclonal anti-TF antibody-coupled Dynabeads were also used to capture TF+ MP, whose origins were then identified by cellular markers. To verify system response, blood samples were stimulated with lipopolysaccharide (LPS) to produce monocytic TF+ MP. Antibody binding was detected and analyzed using an EPICS XL flow cytometer with Expo32 software.RESULTS: LB-bound MP (9% of LB) stained for key cellular markers. Monocyte-derived MP (CD11b) accounted for 12%, whereas platelet (CD41a), endothelial (CD144), and erythrocyte (CD235a)-derived MP accounted for 13%, 52%, and 22%, respectively, verifying that MP arise from 4 major cell types. When circulating levels of various cell types are considered, CD11b+ MP contribute the greatest proportion. LB-bound TF+ MP detected as mean fluorescence intensity (MFI) increased significantly after LPS stimulation (1 μg/mL for 6 h at 37°C) of whole blood (3.2 vs 21.6, p<0.05, Mann-Whitney). CD11b expression was also enhanced (4.9 vs 81.7, p<0.05).CONCLUSIONS: We have developed a novel assay for identifying plasma MP and their cellular origins. This new method offers several advantages over current protocols. Using freshly drawn blood eliminates sample manipulation, shear stress-induced cellular activation, and further microvesiculation. It reduces preparation time, permitting multiple sample analysis in a timely manner. Lactadherin capture of PS+MP allows for sample collection using sodium citrate or EDTA, while reducing calcium-induced non-specific binding. Magnetic sorting of LB removes soluble plasma proteins, thus reducing background noise on flow cytometry analysis. The size of the LB improves the flow cytometer's ability to distinguish signal from noise - greatly improving specificity. Our results demonstrate MP may be more efficiently and accurately isolated from whole blood in a time-and cost-effective manner. It shall prove an ideal tool in characterizing MP in multiple center clinical studies.