Immuno-analysis of microparticles: probing at the limits of detection

Abstract

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.

Figure 1. Endothelial MP quantitation and characterisation by FCM.

Culture supernatants were collected from control or TNF activated (100 ng/ml overnight) hCMEC/D3 cells and labelled with either annexin V-FITC or lactaherin-FITC in combination with either CD54-PE or CD105-PE. (a) The total numbers of MP per 10³ cells are shown with dark grey bars representing the levels of PS + MP, light grey bars representing double + MP and white bars indicating the numbers of Ag + MP. Annexin V is shown on the left hand side of each graph vs lactadherin on the right, CD54 is on top and CD105 below (mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.0001). (b) The numbers of annexin V and lactadherin positive MP per 10³ cells were compared between the 6 replicate wells analysed over 3 individual experiments (n = 18). Spearman’s test shows a significant correlation between the two PS labels (r = 0.96; p < 0.0001). (c) Mean fluorescence intensity (MFI) data for each individual label were pooled (n = 36) and differences between resting (NS) and TNF MP were assessed (mean ± SE; ***p < 0.0001). (d) Median fluorescence intensity for arbitrary size gates (a–e) (smallest to largest) for each individual label (mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001).

Figure 2. Quantitative EM shows no influence of vesiculation agonist on MP size.

(a) Representative transmission electron micrograph of purified, negatively stained MP collected with the JEOL 1400 TEM at 20 000 X Mag. (b) Minimum vs maximum Feret diameters (μm) for all MP analysed (n = 1690) with Image J software, illustrating overall endothelial MP size distribution. A significant correlation was confirmed between the two parameters (Spearman’s r = 0.96; p < 0.0001). (c) No significant differences in the distribution (Median, 25–75% percentile and range) of maximum Feret diameters (μm) between resting (NS) and TNF MP were observed (Mann Whitney test; p = 0.38).

Figure 3. A direct correlation between MP size and marker expression.

(a) Immuno-gold labelled MP grids were imaged at 60 000X Mag with the JEOL 1400 TEM. All images are to the same scale where scale bars are measured in micron. Closed arrow-heads indicate gold positive MP whilst open arrow-heads indicate gold-negative MP. (b) Spearman’s correlation shows a significant relationship between the size of MP, measured as the maximum Feret diameter (μm), and the number of gold particles bound per MP for the annexin V label (r = 0.68; p < 0.0001). (c) The median size, measured as the maximum Feret diameter in μm, was compared between gold-positive and gold-negative MP for each of the markers of interest (Mann Whitney test; ***p < 0.0001).

Figure 4. Comparative dual labelled FCM and immuno-EM show distinct differences in the percentage of Ag+ MP.

(a) Purified MP samples dual immuno-labelled with either annexin V or lactadherin (10 nm gold, closed arrowheads) followed by anti-CD54 or CD105 Abs (15 nm gold, open arrowheads) were negatively labelled and imaged at 100 000 X Mag on the JEOL 1400 TEM. (b,c) The percentages of PS+ (dark grey), double + (light grey) and Ag + (white) were determined from the total numbers of MP analysed for each marker combination and for both the FCM (n = 3) and immuno-EM (n = 1) experiments. CD54 data is shown on the top row whilst CD105 is below for both annexin V (b) and lactadherin (c) labels.

Figure 5. Flow cytometry setup (methods) (a–d) Definition of the MP gate on FSC vs SSC cytograms.

(a) Fluorescent beads were used to set the protocol and allow evaluation of its stability. (b) PBS analysis demonstrates the purity of the machine where all the events detected correspond to the electronic noise of the instrument. (c) Analysis of a cell culture supernatant shows that the majority of the events fall with the MP gate. (d) Arbitrary gates (a–e) divide the MP gate to assess the relationship between MP size and Ag labelling. (e,f) examples of excluding swarm detection. (e) Number of MP positive for Annexin V detected in a serial dilution of the culture supernatant. (f) The time of Flight (TOF) parameter within a positive MP population where the left gate represent the single events and the right gate indicates the coincident events. (g,h) Examples of labelling combinations. CD105 vs annexin V and lactadherin on TNF-stimulated cells (g, left and right respectively). CD54 vs annexin V staining on resting vs TNF-stimulated cells (h, left and right respectively). MP were counted in the upper left quadrant for Ag only (Ag+), upper right for double labelled (double), lower right of PS marker only (PS+) and lower left for negative (Neg).