A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

Abstract

Background and aim: Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer.

Methods: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (<1.0-µm), by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS) exposing MPs binding Lactadherin was determined.

Results: By using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men.

Conclusion: We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

Keywords: coincidence occurrence; endothelial cell-derived; erythrocyte-derived; extracellular vesicles; flow cytometry; lactadherin; monocyte-derived; platelet-derived; tissue-factor.

Figures

Fig. 1

Construction of MP gates using size-calibrated fluorescent beads ranging from 200-nm to 3.0-µm. (A) Selection of bead subsets using the FACSAria instrument with a set SSC-A threshold of 200, and (B) construction of a MP gate with an upper limit of approximately 1 µm. (C) Selection of bead subsets using the FC500 instrument. (D) Threshold of 1 set on the SSC-A parameter eliminated the smallest (0.2 µm) beads. (E) Construction of a MP gate with an upper limit of approximately 1 µm. Background noise from the bead mix is shown in the left gate of the scatter plots.

Fig. 2

Side-by-side determination of absolute events within the MP gate using FACSAria (left panel) and FC500 (right panel) flow cytometers. (A,B) absolute events in non-filtered plasma. (C,D) absolute events in 0.22-µm-filtered plasma. TruCount beads are shown in the upper right corner. (E,F) evaluation of background noise contribution from 0.22-µm-filtered PBS analyzed under similar conditions (flow rate and time period).

Fig. 3

Side-by-side determination of Lactadherin-FITC positive events using FACSAria (left panel) and FC500 (right panel) flow cytometers. (A,B) Lactadherin-FITC positive events in non-filtered plasma. (C,D) Lactadherin-FITC positive events in 0.22-µm-filtered plasma (filtered prior to staining). (E,F) negative control for Lactadherin binding (unstained non-filtered plasma).

Fig. 4

Side-by-side determination of (A,E) CD41 + PMPs and (C,G) CD142+ (tissue factor-positive) MPs using FACSAria (left panel) and FC500 (right panel) flow cytometers. Right columns correspond to the respective isotype controls.

Fig. 5

Test for coincidence detection. (A) Serial dilutions of 200-nm beads mixed with a fixed number of Megamix beads, using the 900-nm sized beads as reference. We collected events triggered on side scatter (SSC-A) with a threshold of 200. Expressed is the ratio of 200-nm beads vs. 900-nm beads at each dilution. R2=0.951, as determined by linear regression. (B–C) To support our findings we conducted serial dilutions of 900-nm beads (R2=0.995) and 200-nm beads (R2=0.955), respectively. By serial dilution of plasma samples we observed a proportionally drop of all events within (D) the MP gate (R2=0.991) and of (E) all PS-positive events within the MP gate (R2=0.987).

Fig. 6

Detection of microparticle (MP) subpopulations in human plasma. (A) Platelet-derived MPs (CD41 + PMPs). (B) Monocyte-derived MPs (CD14 + MMPs). (C) Endothelial-derived microparticles (CD31+ /CD42b− EMPs). (D) Erythrocyte-derived microparticles (CD235a + ErytMPs). (E) Total tissue factor-positive microparticles (CD142+(TF) MPs). Right columns correspond to the respective isotype controls.