Higher levels of specific cell-derived MPs are associated with various diseases, including thrombosis (platelet MPs) (26), congestive heart failure (endothelial MPs) (27), breast cancer patients (leukocyte MPs) (28), and women with preeclampsia (29). This suggests the number of total MPs and/or a subset of cell-specific MPs might be used to predict or monitor such pathologic conditions.
When labeling cells with antibodies (Abs) [i.e., CD3 (30), CD4 (31)], DNA dyes [i.e., Hoechst 33342 (32), propidium iodide (17), 7-aminoactinomycin-D (7-AAD) (30)] or lipid membrane dyes/probes [i.e., annexin V (33), CFSE (34), PKH26 (35)], or Cell Tracker CM-DiI (lipophilic carbocyanine dye containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (36) for flow cytometry, a known amount of cells is used so that the experiment can be reproduced. Although it might seem appropriate to label MPs with Abs or annexin V and then analyze/quantitate MPs, this erroneous procedure is common practice among researchers who use flow cytometry to analyze MPs. Given the intervariation in centrifugation protocols between laboratories (Table 1) and variation in the number of MPs between genders, within the same genders (menstruating vs. nonmenstruating women (37) and pregnant vs. nonpregnant women (10, 29) and between physiological and pathological conditions (vasculitis (38), diabetic retinopathy (39), and cancer (40), an accurate quantitation of total and/or subsets of MPs are required for optimal and reproducible staining of MPs (i.e., Abs and fluorescent dyes). In our hands, MPs are first quantitated using a known concentration of fluorescent beads and then a known amount of MPs are fluorescently labeled, rather than adding Abs/membrane fluorescent dyes to a known volume of plasma or supernatant with an unknown amount of MPs. For example, apoptotic MPs released from HTR-8/SVneo cells (trophoblast cell line that expresses the angiotensin II receptor type 1 (AT1) were first quantitated using a fluorescent bead-count assay (19). Then known amounts of apoptotic MPs were titrated, labeled with two different concentrations of the anti-AT1 Abs and analyzed by flow cytometry. Figure 2A shows a 1.6-fold increase in AT1 mean fluorescence intensity (MFI) between 0.1 × 106 MPs and 0.5 × 106 MPs labeled with AT1 Abs, respectively, suggesting that the optimal number of MPs labeled by anti-AT1 Abs is 0.1 × 106 MPs. However, it appears that labeling low amounts of MPs (0.05–0.1 × 106 MPs) with anti-AT1 Abs is associated with higher variability (MFI) compared with labeling higher amounts of MPs between 0.25–1.0 × 106 MPs, suggesting that the lower limit of detection for the number of MPs is around 0.25–0.5 × 106 MPs. Although it seems counterintuitive for an Ab to bind fewer antigens (Ag) at a high Ab to Ag ratio, this could be explained by Ab saturation. For example, bivalent Abs have high avidity at low concentration and low avidity at high concentrations. When the Ag binding sites become more saturated due to the increase in Ab concentration, the Ab undergoes monovalent binding, which has less avidity for the Ag and therefore dissociates more rapidly. This suggests at low MP numbers (0.05–0.1 × 106 MPs), the Ag binding sites become more saturated, resulting in higher frequency of low avidity binding Abs as shown in Figure 2B (66.6 ± 4.1% and 77.9 ± 1.8%, respectively). Furthermore, at higher MP numbers (0.25–0.5 × 106 MPs) there are more Ag binding sites to allow for higher avidity binding Abs as also shown in Figure 2B (84.7 ± 1.9% and 92.5 ± 1.2%). In addition to Ab saturation, the amount of MPs used is necessary for reproducibility of an experiment. For example, if the number of MPs used in Figure 2 was not determined, then one might conclude that 618 ± 137 μl is the optimal volume to label MPs in future experiments. However, Figure 3 clearly shows that the concentration of plasma MPs (MPs per μl) can vary between individuals. Therefore, if the amount of plasma used for labeling MPs with anti-AT1 Abs was determined solely by the volume shown in Figure 2 (618 ± 137 μl) rather than an the optimal number of MPs (0.25–0.5 × 105 MPs), then suboptimal amounts of MPs (6, 13, and 24 × 105 MPs) would be labeled with AT1 Abs rather than the optimal number of MPs.
Two general methods used to enumerate circulating MPs are the fluorescent bead count-based and flow rate-based method. The bead count-based method allows for quantitation of MPs based on the addition of known quantities of standardized fluorescent beads to the samples. The method is suitable for reproducible and accurate measurements of MP numbers (21). However, because Trucount™ beads are added to each sample, this method has the potential of becoming labor intensive and expensive depending on the number of samples analyzed. By contrast, the flow rate method is less expensive. The method uses a derived calibration factor to convert cytometer events into absolute counts (41). Although this technique requires the use of Trucount™ beads to calibrate the machine only before and after samples, it also can become cumbersome and impractical when biological samples with different viscosities are used because calibrations are needed before and after each different biological sample. Therefore the flow rate method is more beneficial for clinical flow cytometry that routinely analyze a single biological sample such as blood or plasma.
Alternatively, we use a less expensive fluorescent bead count-based assay termed single bead-enhanced cytofluorimetry to quantitate the number of MPs (19) in Figures 1 and 2. This procedure uses Flow-Check™ Fluorospheres (10-μm average diameter), does not require calibration of flow rates or specialized flow cytometric analytic software and can be applied to all flow cytometers. Furthermore, 10 μm sized beads are easily differentiated from the MPs and background “events” when analyzed by light scatter and allows for a separate gate to count beads based on fluorescence (Fig. 4). We initially applied this strategy to determine the total number of MPs released into the supernatant in our in vitro model culture system for analysis of apoptotic MPs derived from JEG-3 cells, an extravillous trophoblastic cell line, undergoing various methods of cell death: chemically induced hypoxia and heat stress (necrosis like)-induced cell death (17). Furthermore, to accurately compare the amount of DNA and lipid membranes present in apoptotic and necrotic MPs (Table 2), the number of MPs was quantitated using this fluorescent bead-count method and equal numbers of MPs were labeled with DNA and membrane lipid dyes. We also applied this fluorescent bead-count method to quantitate the number of in vivo MPs in maternal and nonmaternal plasma prior to labeling the plasma MPs with control isotype, anti-human leukocyte antigen-G (HLA-G), placental alkaline phosphatase (PLAP) Abs and counter-staining with a DNA dye, PicoGreen (10) (Table 2).