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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Cytometry A. 2012 Nov 2;83(2):242–250. doi: 10.1002/cyto.a.22222

Calcium-phosphate microprecipitates mimic microparticles when examined with flow cytometry

Michael C Larson 1,2,*, Maia R Luthi 3, Neil Hogg 1, Cheryl A Hillery 2,4
PMCID: PMC3615643  NIHMSID: NIHMS444850  PMID: 23125136

Abstract

There are increased levels of circulating microparticles in several disease states. Flow cytometry is a common method to examine microparticles, but their small size necessitates the use of markers to specifically distinguish microparticles from artifact. Annexin V, which binds phosphatidylserine, is a commonly used marker for microparticle detection. Annexin V requires millimolar calcium ion for optimum binding. Ca++ can precipitate with phosphate in phosphate-buffered saline (PBS).

Calcium-phosphate microprecipitates were formed by titrating Ca++ into PBS and examined using flow cytometry. Calcium-phosphate microprecipitates were compared with microparticles derived from aged donor blood units.

Microprecipitates were approximately 0.7–0.9 μm in diameter compared to standard beads of known size. The microprecipitates disappeared with the addition of Ca++ chelator. When we added fluorescently-labeled antibodies to microprecipitates, the median fluorescent signal increased with increasing Ca++ concentration regardless of specificity of the antibody. When repeated with a biological sample, there was an apparent increase in the fluorescent signal that returned to baseline after Ca++ chelation.

The flow cytometry signal of calcium-phosphate microprecipitates overlaps with the microparticle signal. Since Ca++ is essential for annexin V binding, it is essential to avoid artifacts from calcium-phosphate microprecipitates when using any buffer or biological fluid containing phosphate. This also highlights the potential utility of flow cytometry for the analysis of crystals in biological fluids.

Keywords: Microparticles, Calcium, Phosphate, Annexin V, Microvesicles

Introduction

Circulating microparticles (MP) are small (<1 μm in diameter) membrane fragments shed from cells following a host of physiologic and pathophysiologic stimuli. MPs are normally found in small numbers (relative to other circulating cells) in the vascular space, where they participate in complex intercellular signaling pathways (1). The number of circulating microparticles (MPs) greatly increases in numerous disease states, including cardiovascular disorders (1, 2), infectious diseases (3), cancer (4), and hemolysis (5, 6). MPs are currently under intense study, with pathologic, prognostic, or even protective functions being reported (1–6). For example, red blood cells (RBC) may shed MPs as a protective means to dispose of irreversibly-altered hemoglobin or bound immunoglobulin, preventing otherwise healthy RBCs from premature degradation (7). However, RBC MPs also house potentially harmful macromolecules, like hemoglobin that can scavenge nitric oxide (8), or pro-coagulant (9) or immune-modulating phospholipids (10). More functions of, and even ways to modulate these transient bioeffectors will undoubtedly be uncovered in the near future.

Flow cytometry is the preferred method for examining MPs. However, caution is required, as the small size of MPs relative to the wavelength of interrogating light results in considerable artifact (11,12). Thus, markers are required to identify flow cytometry events as true MPs (11–13). In particular, fluorescently-labeled annexin V, which binds to phosphatidylserine (PS), a phospholipid normally on the inner cellular leaflet of the plasma membrane but which is externalized on MPs, has been used for this purpose (14). As millimolar calcium ion concentrations are required for annexin V-PS binding (15), many studies of MPs have either 1) isolated MPs from biological fluids, followed by re-suspension in phosphate-buffered solutions and addition of a calcium salt (16–20); or 2) added calcium salt directly to plasma of patients with impaired phosphate excretion (and elevated plasma phosphate) (21). As calcium-phosphate has limited solubility, it is possible that calcium-phosphate precipitation can occur in phosphate-containing fluids when Ca++ is added (22,23).

The purpose of this study was to examine if the formation of calcium-phosphate microprecipitates interferes with flow cytometry determination of microparticles. Calcium-phosphate microprecipitates were compared with microparticles obtained from expired donor blood, a robust source of red cell-derived microparticles. Our results indicate that calcium-phosphate microprecipitates and microparticles overlap in light scatter (and therefore, size); and that fluorescently-labeled antibodies may bind non-specifically to microprecipitates. Thus, experiments examining MPs should be carefully designed 1) to avoid microprecipitate formation or 2) to confirm the cellular basis of the MP by a secondary method.

Materials and Methods

All buffers were sterile-filtered using 0.20 μm syringe filters (Corning, Tewksbury, MA, USA) and either kept on ice or at room temperature for 30+ minutes prior to mixing and examination with flow cytometry. Dulbecco’s phosphate buffered saline (containing no Ca++ but 9.5 mM phosphate) at pH 7.3–7.4 (PBS) and Hank’s balanced salt solution at pH 7.4 (which contains 0.77 mM phosphate and 1.3 mM CaCl2, HBSS) were from Invitrogen (Life Technologies, Grand Island, NY, USA). Normal saline (0.9% w/v) adjusted to pH 5–7.4 (NS) and Calcium chloride were from Sigma-Aldrich (St. Louis, MO, USA) or Fischer Scientific (Pittsburgh, PA, USA) when protocols were independently repeated by a second researcher. Tris-HCl for Tris-buffered saline (50 mM Tris with 150 mM NaCl) at pH 7.4–7.6 (TBS) was also from Fischer. CaCl2 was mixed in purified water at 100 mM final stock concentration. Expired (>42 day-old) leukocyte- and platelet-reduced donor blood units (N=4) in adenine-citrate-dextrose and Solution 1 were obtained from the BloodCenter of Wisconsin (Milwaukee, WI, USA) as a known source of MPs (6). Platelet-rich plasma was obtained from a healthy, consented donor in accordance with IRB-approved protocols. Platelet-rich plasma was isolated by centrifugation of citrate-anticoagulated whole blood at 200× g for 10 minutes, followed by 1500× g for 15 minutes at room temperature to avoid cold-induced platelet activation. Potassium EDTA (Fischer) was diluted 6% w/v in purified water, and was added to achieve 0.1% final concentration at the peak of the Ca++-PBS titration (5 mM CaCl2). (This concentration of EDTA is used clinically to anticoagulate whole blood.) Beads with known nominal diameters were from Sigma (0.55 μm), Solulink (San Diego, CA, USA) (0.8 μm), Pierce (1.0 μm), New England Biolabs (Ipswich, MA, USA) (2.0 μm), and Bang’s Laboratories (Fishers, IN, USA) (3.0 μm). PE- labeled mouse anti-human PECAM-1 (CD31) and rat anti-mouse IgG1 isotype control antibodies were from BD Pharmingen (San Jose, CA, USA) and used at 0.1μg/ml. Annexin V-PE was also from BD Pharmingen, and used at 1:100 (per manufacturer’s recommendation). APC-labeled anti-mouse IgG1 isotype from eBioscience (San Diego, CA, USA) was used at 1:200. Streptavidin Cy3 was used at 1:50 per the manufacturer (Invitrogen). Lactadherin-FITC (Haematologic Technologies, Essex Junction, VT, USA) was used per manufacturer’s instructions as a Ca++-independent PS-probe to confirm PS staining on biological samples.

Flow cytometry was initially performed on an Accuri C6 and subsequently on a LSRII for confirmation and comparison (BDIS,); the Accuri C6 was chosen initially, as its sheath fluid is distilled water, while the LSRII sheath fluid is a proprietary phosphate-buffered solution. Experiments titrating Ca++ into the various buffers (Figure 2A) were carried out independently by two researchers. The Accuri C6 was run at the default “slow” flow rate of 11 μl/min, with FSC-H and SSC-H threshold at 10000. The threshold was empirically determined to provide an acceptable minimal level of background (a maximum 100 events/minute on sterile-filtered distilled water), as a smaller threshold resulted in increased noise (data not shown). (The background “noise” can be seen in Figure 2 as roughly 1–2 events per microliter in sterile-filtered saline. The events/μl rate reported by the cytometer was checked against beads with a known concentration, and was consistently on the same order of magnitude [data not shown].) For experiments using side scatter to approximate size, no SSC-H threshold was applied. All samples were vortexed immediately prior to flow cytometry examination. Samples were acquired in at least 3 separate triplicates for 30 seconds or 10000 events (at minimum); 50,000 events (at minimum) were recorded of samples containing RBCs. Flow cytometry analysis was primarily done using C-Flow Plus (BD Biosciences). Singlet populations were gated based on the smallest forward scatter width by height (Figure 1F). A standard curve for estimating the size based on the side scatter area of singlet microprecipitates was established by a linear fit of the various beads, as the forward scatter at these small sizes is not accurate for estimating size (24). This was repeated on the LSRII; however, the sample intake of the LSRII may contaminate samples with phosphate from the sheath fluid drip during normal operation. The settings of the LSRII were as follows: FSC voltage: 550; SSC voltage: 355; FSC threshold: 1500, which resulted in a background of <1 event/sec on sterile-filtered water; slow flow rate. Samples were collected for 30 seconds or 15,000 events. Flow cytometry analysis from the LSRII was done using Cyflogic (CyFlo, Ltd., Turky, Finland).

Figure 2. Flow cytometry of calcium-phosphate microprecipitates and microparticles.

Figure 2

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Representative light scatter images and <1 μm gate of A) sterile-filtered normal saline (NS) and B) sterile-filtered PBS with identical increases in calcium, normalized to volume. A, far right) flow cytometry examination of stock CaCl2. B, far right) Addition of EDTA to 5 mM CaCl2 in PBS. C) Quantification of the total number of flow cytometry events, normalized to volume. There was a significant increase in of the total number of flow cytometry events even at 1 mM CaCl2 relative to control. The addition of EDTA significantly reduced the total (ungated) number of flow cytometry events per μl. D) For qualitative comparison, expired donor blood (as a known source of RBC-derived MPs); platelet-rich plasma; and 0.55, 2 and 3 μm beads are shown. N=3–5. *, p<0.05; **, p≤0.005 compared to 0mM control; #, P <0.01 compared to 5 mM CaCl2 alone.

Figure 1. Microparticle (MP) threshold gating.

Figure 1

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Forward scatter height (FSC-H) vs. width of A) ultrapure water with no threshold. B) The same water after the empirically-determined minimum FSC-H threshold was applied. Beads with 0.55 μm nominal diameter C) without or D) with the threshold. E) Twice sterile-filtered (0.20 μm filter) 0.55 μm beads show the minimum threshold to be between 0.55 and 0.20 μm. F) The upper limit of what was considered a MP was established using FSC of 1 μm beads. All samples were collected for 30 seconds or 10000 events using otherwise identical settings.

For microscopic examination of the microprecipitates, PBS with increasing concentrations of Ca++ was examined directly under the microscope. Additionally, aged blood was washed 3 times with sterile normal saline (Sigma). Washed RBCs were then diluted 1:500– 1:1000 into sterile-filtered PBS with 0, 2.5, or 5mM CaCl2 added, and the solution was placed on glass slides. Cells along with microprecipitates were immediately examined with microscopy. Brightfield images were acquired on a Nikon Eclipse using a 40x objective with 200ms exposure using NIS-Elements software (Nikon, Melville, NY, USA). Image adjustment and analysis was performed using ImageJ (US NIH, http://imagej.nih.gov/ij/, Bethesda, MD, USA). When performing image analysis, particles with less than 10 or more than 10,000 pixels were excluded (this arbitrary range encompassed both the precipitates and RBCs).

As a way to overcome the potential for precipitate formation, EDTA was used in the presence of annexin V with increasing Ca++. This was compared against Lactadherin, which is another PS-binding protein that does not require Ca++. RBC MPs in the supernatant of aged blood were isolated by gentle centrifugation (325× g for 10–12 minutes to remove RBCs). The MP-containing supernatant was then diluted 1:100 in sterile-filtered PBS. Annexin V at 1:100 with or without 0.1% EDTA was added to label RBC MPs. Ca++ was titrated into the solutions as they were examined with flow cytometry.

Statistical analysis was done using Student’s 2-tailed t-test when comparing 2 groups or ANOVA for comparison of 2 or more groups with differing amounts of CaCl2. For data displayed on a logarithmic scale, data were first log-transformed prior to applying the t-test. An alpha of 0.05 was used to determine significance. Data are presented as mean ± standard error of mean unless explicitly stated.

Results

Microparticle (MP) gating

MPs are challenging to study using flow cytometry (11–13). To ensure correct threshold and gate settings, we used a series of steps to empirically define a correct MP gate. Figure 1A shows the forward scatter signal height by width for distilled and autoclaved water with no threshold on the Accuri C6. We applied a minimum threshold resulting in 1–2 events per μl as an acceptable background (Figure 1B). Figure 1C shows beads with a 0.55 μm nominal diameter without the threshold. The beads are clearly seen with the threshold applied (Figure 1D), and a few residual beads can be seen after repeated filtration of the beads with a 0.20 μm pore-diameter filter (Figure 1E). This ensures the minimum size range of the threshold is below the International Society of Thrombosis and Hemostasis (ISTH) recommendation of 0.5 to 0.9 μm for platelet-derived MPs (25). The upper limit of what we considered a MP was established by the FSC-H of 1 μm beads (Figure 1F). The minimum threshold was maintained in subsequent experiments; the upper limit was re-established in each set of experiments using the same beads when a MP-gate was needed.

Calcium-phosphate microprecipitates and MPs light-scatter signal

Shown in Figure 2A and 2B are representative dot plots using the 1 μm forward-scatter gate (representing the upper limit of MPs) based on the FSC-H intensity of commercial beads (as shown in Figure 1). All dot plots display data from 5 μl of sample or 10,000 events from different samples or conditions.

There was no change in the number of detected events after the addition of CaCl2 to sterile-filtered normal saline (Figure 2A). However, there was a concentration-dependent increase in flow cytometry events after the addition of Ca++ to PBS. This increase in total events in PBS is striking, being ~200-fold more at 2.5 and ~7500-fold more at 5 mM CaCl2 added than the 0 mM CaCl2 control. These flow cytometry events essentially disappeared with the addition of EDTA to chelate Ca++ (Figure 2B, 2C). Figure 2D compares RBCs and RBC-derived MPs from an expired donor unit, platelet rich plasma, and various commercial beads. Comparison of figure 2B and 2D shows clear overlap between the light scatter of microprecipitates and microparticles derived from biological sources.

Buffer, instrument, time and temperature effects on microprecipitate formation

CaCl2 was also titrated into other commonly used buffers to assess the potential of calcium-dependent microprecipitates. Hank’s balanced salt solution (HBSS) and Tris-buffered saline (TBS) were used along with normal saline (NS) as a negative control. Detection was performed using two different cytometers to examine machine-specific effects (Figures 3A and 3B).

Figure 3. Buffer, instrument & time factors in the formation of microprecipitates.

Figure 3

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Ca++titration in various buffers, including normal saline (NS), Tris-buffered saline (TBS), Hank’s balanced salt solution (HBSS), PBS and EDTA added using A) an Accuri C6 (water sheath); or B) an LSRII (PBS sheath). Singlet beads of various sizes (0.55, 0.8, 1, 2 and 3 μm) were identified and gated based on the least FSC-H to width ratio (see Figure 1F). The SSC-A was then used to determine a linear-fitted standard curve. The standard curves from the C) Accuri C6 and D) LSRII, along with the average SSC-A of the precipitate singlets are shown. Based on the standard curves, the microprecipitate singlets formed in these conditions measured between 0.64 and 0.97 μm in size. E) Representative 5-minute time-course of microprecipitate formation and dissolution with EDTA. F) The rate of microprecipitate formation (averaged over 5-second intervals for 30 seconds) is shown. No gating was used. N=3–12. *, p<0.05; **, p<0.01; ***p<0.001 compared to 0 mM CaCl2 control; #, p<0.001 compared to 5 mM CaCl2 alone.

As shown in Figure 3A generated using the Accuri C6 cytometer, there was minimal increase in the number of events as a function of Ca++ concentration in TBS and HBSS (from a minimum of 4 to a maximum of 15 events/μl, indicating a 3 to 4-fold increase of baseline). Titration into PBS gave similar results to Figure 2C. With the addition of the chelator, EDTA, to the 5 mM CaCl2 solutions, there was no difference in the number of events in TBS and HBSS; however, as before, there was a marked drop in the number of total events per μl of calcium-containing PBS (p<0.001).

Figure 3B shows similar experiments using the LSRII. Again, there is a Ca++ concentration-dependent increase in the number of events. However, substantially more events were observed at lower Ca++ concentrations, and microprecipitates were also observed in non-phosphate buffers (for example, there was a 10-fold increase above baseline in the number of events in HBSS with only 1 mM CaCl2 added; adding 2.5 mM CaCl2 resulted in a 100-fold increase). We observed that the LSRII intake drips phosphate-containing sheath fluid into the sample when positioning the sample; consequently there was an unknown amount of phosphate in all samples detected using the LSRII. This result highlights the fact that instrument factors can also be responsible for microprecipitate formation, and not simply buffer choice.

While forward scatter is used to determine cellular size, it does not accurately represent size of the sub-micron particles (24); however, side-scatter has been used for this purpose (26). To estimate the size of the microprecipitates based on side scatter, a linear standard curve was established using multiple types of beads with known nominal diameters. Multiplets were excluded based on their disproportionate height to width (see Figure 1F).

There was no difference between the size of singlets from 2.5 mM and 5 mM CaCl2 conditions tested in either the Accuri C6 or the LSRII (p>0.2). The estimated diameter of the microprecipitates using the Accuri C6 was 0.76 ± 0.12 μm (Figure 3C) compared to the 0.84 ± 0.13 μm using the LSRII (Figure 3D). From the standard curves, the apparent diameter of these microprecipitate singlets (an estimated 0.64–0.97 μm) fell into the commonly accepted microparticle range between 0.1 and 1 μm, and the 0.5 to 0.9 μm platelet-MP window recommended by the ISTH (25).

The time required for microprecipitate formation was examined at room temperature. The time from which CaCl2 was added to PBS until the appearance of microprecipitates generally was less than the sample handling and cytometer lag time. Figure 3E shows a representative real-time experiment. In this series, data were collected at 30 seconds intervals, following the addition of 2.5 mM CaCl2 and immediate replacement of the sample into the cytometer. This process was repeated again with an additional 2.5 mM CaCl2 and then the chelator. Figure 3F shows the precipitate formation rate for different Ca++ concentrations in PBS. The formation and EDTA-dependent dissolution of microprecipitates occurs relatively quickly.

Calcium-phosphate solubility is temperature-dependent (22). To assess this temperature dependence, experiments were carried out keeping all tubes and buffers on ice or at room temperature. In the solutions containing phosphate (PBS and HBSS), there was no significant difference in the mean total events/μl between the two temperatures across the range of CaCl2 added (data not shown).

Microprecipitate interaction with fluorescently-labeled antibodies

Since antibodies can interact non-specifically via charged residues (27), the fluorescent signal of the calcium-phosphate precipitates were examined in the presence or absence of fluorescently-labeled antibodies (Figure 4).

Figure 4. Microprecipitate interaction with fluorescently-labeled antibodies.

Figure 4

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Representative tracing of the fluorescent signal of the microprecipitates in A) the absence or B) presence of fluorescently-labeled antibodies. C) Increasing the calcium concentration significantly increased the median fluorescence intensity (MFI) at 2.5 and 5 mM CaCl2 in PBS relative to antibodies in PBS alone. D) Annexin V-PE, mouse anti-human isotype control IgG1-APC, mouse anti-human CD31 (PECAM-1)-PE, and streptavidin-Cy3 were also tested in PBS with increasing calcium, and the MFI reported. No gating was employed. N=3–4. *, p<0.05; **, p≤0.005 relative to control; #, p<0.05 relative to 2.5 mM CaCl2; †, two-way ANOVA p<0.05.

In the absence of fluorescently-labeled antibodies, there was no significant alteration in the median fluorescent signal with increasing Ca++ (Figure 4A). In contrast, when anti-human IgG1 PE-labeled antibodies were added, there was a Ca++-dependent increase in the average median fluorescent intensity (MFI) of the microprecipitates (Figure 4B, 4C).

The above procedure was repeated using different fluorophore-labeled probes, including annexin V, APC-labeled anti-IgG1, PE-labeled anti-CD31 (PECAM-1), and streptavidin Cy3 (Figure 4D). This phenomenon may be protein-specific, since there was a significant increase in MFI signal with the addition of 5 mM CaCl2 using annexin V, anti-IgG1, and anti-PECAM-1, but not streptavidin. FITC-labeled streptavidin yielded similar results as Cy3-labeled (data not shown).

The MFI at 0 mM CaCl2, was largely variable, since the events were essentially background. Thus, comparisons were carried out across the different buffers and calcium concentrations using a two-way ANOVA. There was a significant difference between the MFI of microprecipitates with annexin V and the isotype antibody (p<0.05).

Microprecipitates in microparticle-containing biological fluids

RBCs naturally shed MPs as they age or are damaged; the concentration of MPs in donor blood accumulates over time (6). Thus, to examine the potential overlap between microprecipitates and a known source of MPs, donor blood aged beyond expiration (42 days) was diluted in PBS and examined with flow cytometry.

As shown in Figure 5A and C, there was an increase in MP-gated events normalized to RBCs with the addition of 5 mM CaCl2, similar to the order-of-magnitude increase in buffer-only experiments (refer to Figure 2).

Figure 5. Microprecipitates mimic microparticles in stored blood.

Figure 5

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A) Representative light scatter dot plot of 1:1000 aged donor blood:PBS with various CaCl2 added. B) Quantification shows a significant increase in MP-gated events with the addition of 5mM CaCl2. C) Corresponding PECAM-1 by FSC-H. D) Quantification of PECAM-1+ MP-gated events, normalized to volume. The addition of EDTA after 5mM CaCl2 resulted in an overall 21 ± 22% change in MP-gated events from baseline (trending toward significant decrease from 5 mM CaCl2 [p=0.08]). Light microscopy was employed to visualize the formation of microprecipitates, and the size of the precipitates were measured. Washed RBCs in PBS with E) 0, F) 2.5 and G) 5 mM CaCl2 are shown under 40x objective magnification in the upper panel; bar=5μm, and birefringent microprecipitates are highlighted with arrows. The lower panels are the results of automatic particle/cell identification. H) Histogram showing the results of automatic particle analysis, indicating no difference in the size of microprecipitates (geometric mean area of 1.67 ± 0.51 μm2 excluding particles that were clearly RBCs). N=3–7. *, p<0.05 compared to 0 mM CaCl2; #, p<0.05 compared to 5 mM CaCl2 alone.

In the presence of PE-labeled anti-human PECAM-1 (CD31) antibodies, there was also an accompanying increase in the number of PECAM-1+ events at 5 mM CaCl2 despite the WBC- and platelet-reduced nature of the biological fluid (Figure 6B and D). This was analogous to the increase seen in buffer-only experiments (refer to Figure 5).

Figure 6. Annexin V and EDTA co-incubation.

Figure 6

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A) Representative dot plots of aged donor RBC MPs isolated into PBS (i) with 5 mM CaCl2 (ii) and 0.1% EDTA (iii). B) With the addition of 5mM CaCl2, there was an increase in the total number of events per μl. There was a significant increase in the MFI following the addition of EDTA. C) For confirmation that the MPs were truly PS+, lactadherin (a more sensitive, calcium-independent PS-probe) was used in Ca++-free PBS. N=4. *, p<0.05; ***, p<0.001 compared to 0 mM control and 5 mM CaCl2 and EDTA.

With the addition of EDTA to chelate Ca++, the concentration of total and PECAM-1 positive MP-gated events again decreased to near baseline, similar to the result seen in buffer-only experiments (refer to Figure 2).

Microscopy was used to confirm the findings, and particle analysis was used to automatically determine the area of the microprecipitates. Representative images of RBCs in PBS with 0, 2.5, and 5 mM CaCl2 added are shown in the upper portion of Figures 5E, 5F, and 5G, respectively. An automatic particle identification algorithm was used, and the particle outlines are shown directly below. The microprecipitates are identifiable as birefringent, sub-cellular structures. Using a particle analysis algorithm, the area of the microprecipitates was determined.

Analysis of the calcium-phosphate microprecipitates revealed that while there was a clear difference in numbers, the size of a microprecipitates was similar between those formed either in 2.5 or 5 mM CaCl2 in PBS (p=0.93, Figure 5H). The geometric mean area of the microprecipitates was 1.67 ± 0.51 μm2 in the conditions tested. Assuming a grossly spherical shape, the diameter as measured via this method is 0.53 ± 0.16 μm, overlapping with the range of that suggested by flow cytometry, as well as the range commonly used to define a microparticle.

Annexin V-PS binding in the presence of EDTA

Annexin V has a high affinity for PS/Ca++ as revealed by a dissociation constant (Kd) of 7 nM (28) or lower (29); whereas EDTA is a relatively weak Ca++ chelator, with an approximate Kd of 50 nM (30). Thus, to overcome the potential for precipitate formation, annexin V binding to PS and calcium was examined in the presence of EDTA.

Annexin V was added at 1:100 to a 1:1000 dilution of RBC MPs in PBS. The events per μl significantly increase with the addition of 5 mM CaCl2; however, there was an inconsistent change in the annexin V MFI signal. With the addition of 0.1% EDTA, the number of events per μl significantly decreased, returning to baseline. There was also a significant increase in annexin V MFI compared to baseline. For comparison and assurance of surface PS on the MPs, a more sensitive and calcium-independent PS probe, lactadherin (31), was also used in identical Ca++-free conditions (Figure 6C).

Discussion

The results of this study suggest that, when analyzing MPs with flow cytometry using annexin V (and hence, adding Ca++ as an essential co-factor), one must consider microprecipitates in phosphate-containing samples or buffers. Such precipitates closely mimicked MPs in light scatter; and bound non-specifically to fluorescently-labeled proteins, potentially leading to false-positive cytometry events.

When applied to a biological system with MPs, the addition of Ca++ appeared to cause the formation of PECAM-1 antigen-positive MPs. However, this population was essentially abolished after the subsequent addition of a Ca++ chelator. Additionally, the differences in affinity for Ca++ between annexin V and the relatively-weak Ca++-chelator EDTA were used to differentiate MP from microprecipitate. This represents one way to differentiate MPs from microprecipitate. An alternative way to distinguish MP from artifact is the complementary use of microscopy to confirm flow cytometry findings. Microscopy provides qualitative data to compliment quantitative findings obtained with flow cytometry.

While the use of annexin V staining is routinely used for cell-surface PS staining, it is not entirely sensitive (31,32) nor specific (33) for surface-exposed PS as its binding requires Ca++, multiple PS molecules, and the lipid headgroups around the PS molecules not to interfere with the electrostatic PS-Ca++ bridge (29). On physiologic membranes, the primary phospholipid surrounding PS that enables annexin V binding is phosphatidylethanolamine (PE) (31). Additionally, concerns have been raised about the addition of Ca++ to the effects of down-stream assays (34,35). This report highlights the utility of calcium-independent phospholipid probes, such as lactadherin for specifically targeting PS (36) or duramycin for specifically targeting PE (37), for the detection of MPs.

Interestingly, the size of the microprecipitates did not differ between 2.5 and 5 mM CaCl2 in PBS. The size of a crystal is determined by numerous factors. Solute concentration is a vital factor, but the interplay of concentration, nucleation rate, and growth rate determine the ultimate size of a crystal (22). In direct visualization, it can be seen that the average singlet microprecipitate size was similar between the 2.5 and 5 millimolar; but the precipitates were much more numerous in the 5 millimolar calcium chloride/PBS solution.

There are numerous reports of increasing amounts of circulating MPs in patients with end-stage renal disease (16,17,21,38). The contribution of artifactual vs. actual microprecipitates in such studies need to be carefully addressed, since calcium-phosphate crystals have been implicated in vascular pathologies (23,39). Patients with end-stage renal disease typically have altered mineral homeostasis (i.e. hyperphosphatemia and hypercalcemia). The results of this study additionally point out the potential utility of flow cytometry for microcrystal analysis, as distinguishing MPs from microprecipitates can easily be done with the addition of a chelating agent. This provides an exciting alternative approach for elucidating the effects of microcrystals in human diseases.

In summary, when designing experiments involving MPs, researchers need to carefully consider factors increasing the potential for microprecipitate formation—including reagents, buffers, instruments, and endogenous crystals in the samples of interest.

Acknowledgments

Research support:

The research presented in this report was done with funding support from U54 HL090503, P01-HL44612, and T32GM080202

Footnotes

No conflict of interest declared.

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