Optimization of detection method of monocyte-platelet complexes in whole blood
Methods: The study included ten healthy volunteers. Peripheral venous blood was sampled by direct venipuncture to a 3.8% sodium citrate containing tube. Tubes were gently inverted to ensure mixing of whole blood with anticoagulant. Thirty microliters of whole blood (either fixed or unfixed) were immediately immunolabeled with 20 microliters of fluorescent monoclonal antibody mix (CD45-eFluor450, CD14-PerCP Cy5.5 and CD41a-APC, containing 2% of mouse IgG to prevent non-specific binding) for 20 minutes in order to identify monocyte-platelet complexes. Sample was diluted with 2 ml of phosphate buffer saline (PBS) or 2 ml of 0.5% formaldehyde and subjected to flow cytometry. Monocyte-platelet complexes were detected by simultaneous expression of all three markers. The MPC positive gate was defined based on fluorescence minus one control (FMO-IgG), including IgG1-APC (Figure 1).
Figure 1. Flow cytometric analysis of monocyte-platelet complexes in mildly fixated whole blood.
Monocytes were labeled with CD45-eFluor450, CD14-PerCP Cy5.5 and platelets with CD41a-APC. Monocytes were gated based on their high expression of CD14 and presence of CD45 antigen. Minimum of 2 500 events was collected in the monocyte gate (A). Fluorescence minus one control, containing IgG1-APC isotype antibody was used to define double positive monocyte-platelet complexes based on APC histogram(C) and dot plot (D) to set a marker excluding for non-specific binding and autofluorescence. Dot plot containing the pre-defined gate of double-positive CD14+CD41a+ then used to determine the percentage of monocytes positive fir CD41a (B).
The flow cytometric data was acquired at medium to high flow rate using CD45 threshold set on 200 to avoid collection of unwanted erythrocyte events.
Different sample processing protocols were utilized to identify the method that minimizes the in vitro cell activation and preserves most of the complexes, formed in vivo:
1. Whole blood samples were stained immediately (within 2 minutes) after collection for 20 minutes at room temperature and then fixed.
2. One hundred microliters of whole blood was immediately fixed with equal volume of cold formaldehyde solution resulting in different final concentrations (0.25-4%) before immunolabeling for 20 minutes .
2. Erythrocyte lysis was performed either after (using eBioscience RBC lysis solution) or simultaneously with cell fixation (using BD lyse reagent).
3. Whole blood sample pre-fixed with 0.25% formaldehyde was then stained and finally fixed with 2 ml of 0.5% formaldehyde.
4. The stability of MPC was assessed for the timeline of 24 hours at 4˚C.
Results and discussion: The time from blood collection to the fixation of the sample is a critical factor which increases the number of MPC by in vitro cell activation. Staining of the samples for 20 minutes immediately after blood collection results in 35.4±3.5% MPC, and each 30 minute delay prior blood processing adds 20-25% of MPC. On the other hand, when samples are fixed immediately after blood collection, MPA content remains stable for a longer time. The concentration of the fixative also affects the MPA concentration. Higher formaldehyde concentrations (1-4%) change neutrophil and monocyte scatter properties and result in clumping of dead cells at 4%. Immunolabeling in high concentrations of formaldehyde (1-2%), although permanently stabilizes the cell membrane, reduces the fluorescence of the most fluorochromes.
Lower concentrations of methanol-free formaldehyde (0.5-0.25%) do not interfere with immunostaining and are able to temporarily stabilize the amount of MPC without reducing the level of fluorescence. The percentage of monocytes, complexed with platelets in mildly fixated whole blood of healthy donors is 6,5±2,2%.
Lysis of red blood cells leads to further activation of platelets and monocytes and increases complex formation, and therefore it is preferable to avoid it. The RBC lysis without fixation requires washing steps, which markedly affects the amount of MPC. Simultaneous lyse-no-wash protocol using commercially available reagents, containing 1% of formaldehyde bears the drawbacks of the high concentration fixation discussed above.
The samples fixed in 0.5% formaldehyde remain stable MPC content for 24 hours at 4˚C.
Conclusions: The amount of monocyte-platelet complexes is an early marker of immunoactivation and atherothrombosis (Furman et al. 2001, Harding et al., 2007). However, the high sensitivity of the method, requires thorough optimization of the parameters of sample preparation and analysis, to obtain stable and accurate results in order to exclude in vitro platelet activation.
In this study we have developed a method of processing and analysis of monocyte-platelet complexes in whole blood, which minimizes the effects of in vitro activation of platelets and monocytes, allowing to quickly and effectively enumerate the percentage of MPC.