Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, “SYTO-high”) corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, “SYTO-low”) corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.


Introduction
Flow cytometry can quantify markers of platelet reactivity through the measurements of the expression of platelet surface antigens using specific fluorescence-coupled antibodies [1][2][3]. Many aspects of platelet physiology can be evaluated simultaneously by measuring several surface markers via using a combination of different antibodies and agonists [4]. Flow cytometry can be performed in patients regardless of platelet count, as platelets are analyzed individually [5,6]. Furthermore, flow cytometry only requires small sample volumes and can be performed on whole blood or platelet-rich-plasma [4,7]. However, flow cytometry requires experienced laboratory personnel and advanced equipment, and is currently not standardized across laboratories [8].
Immature platelets are newly produced platelets that are more reactive than mature platelets [9][10][11]. Compared with mature platelets, immature platelets contain larger amounts of RNA derived from megakaryocytes providing the ability to produce proteins that contribute to platelet reactivity [12,13]. RNA degenerates in a time-dependent manner [14]. Strong evidence suggests a positive association between immature platelets and the risk of cardiovascular events in patients with ischemic heart disease [15][16][17][18]. Hence, exploring and characterizing immature platelets is of great clinical interest.
The most common method to quantify immature platelets is immature platelet count (IPC) or the immature platelet fraction of the total platelet pool (IPF). These parameters are most often measured with a fully automated assay using monochromatic automated flow cytometry (e.g., Sysmex ® ). This method uses a predefined algorithm to classify immature platelets on the basis of manufacturer-decided cut-offs for forward-scattered (FSC) light (corresponding to cell size), side-scattered (SSC) light (corresponding to granularity), and the staining of nucleic acids using fluorescent dyes [19]. However, this method does not allow for the further investigation of the reactivity of immature platelets.
The cell-permeable SYTO-13 fluorescent dye exhibits bright green fluorescence upon binding to nucleic acids, including RNA. The amount of SYTO-13 correlates with the quantity of RNA within the platelet and with the number of immature platelets [20]. Hence, SYTO-13 staining using flow cytometry provides a method for subdividing a platelet population into immature and mature platelets, enabling the detailed investigation of immature platelets [20]. In addition, SYTO-13 staining may be advantageous compared with other nucleic acid dyes (e.g., thiazole orange (TO)), as SYTO-13 staining is stable over time and shows better correlation with immature platelets measured with automatic assays compared with TO [20]. Therefore, although more demanding and time-consuming, manual flow cytometry using SYTO-13 staining is a promising method for subdividing immature platelets from mature platelets and subsequently investigating the reactivity of both platelet populations.
The international standardization of advanced laboratory methodology between laboratories is very important. The present protocol describes in detail the experimental design and workflow to examine platelet reactivity and platelet maturity using whole blood flow cytometry.

Flow Cytometer
All analyses were performed on a CytoFLEX S (B75442) flow cytometer (Beckman Coulter, Miami, FL, USA) equipped with a violet laser (405 nm), a blue laser (488 nm), a yellow-green laser (561 nm), and a red laser (638 nm), and with 11 detectors.

Blood Sampling
See Section 6.1.  • CRITICAL STEP: HEPES buffer, antibodies, and PE isotype control should reach room temperature before further processing.

Preparations of Working Dilutions of Agonists
10. Agonists were collected from the freezer.

11.
CRITICAL STEP: keep agonists in the freezer until immediately before use, thaw at room temperature for 5 min.

26.
CRITICAL STEP: antibodies and PE isotype control must not be exposed to direct light for a prolonged period. 27. A total of 5 µL whole blood was added to each test tube at 20 s intervals. 28. Each sample was mixed slightly by gently shaking the tube.

29.
CRITICAL STEP: incubate for exactly 10 min in darkness. 30. After incubation, 2 mL PBS-0.2% PFA was added to each test tube for fixation with 20 s intervals. See Section 6.4. 31.
CRITICAL STEP: after adding fixative, each test tubes must be vortexed immediately for 8-10 s. 32. After fixation, the test tubes must rest in darkness for 1 h.

Preparing the Analysis
33. The waste bottle was emptied and refilled with sheath fluid container. 34. CytoFLEX and the associated computer were started. 35. CytExpert software was started. 36. The system startup program was run: see Section 6.6. 37. The daily quality control program was run: see Section 6.7. 38. A new experiment was created from the template: see Section 6.8.

46.
CRITICAL STEP: the placement of the test wells in CytExpert software must match the added wells on the plate. Test Tube 2 containing the antibody cocktail, HEPES buffer, and whole blood (see Section 4.2). 48. P-selectin expression < 15% was considered to be acceptable (no evidence of preactivation). 49. In the case of P-selectin expression > 15%, the results should be evaluated with caution.

Identifying Single Platelets
50. Single platelets were identified for further analysis using the gating strategy as shown in Figure 1. Minor adjustments are to be expected between test tubes; see Section 6.9.

Data Quality
47. Preactivation was evaluated on the basis of P-selectin expression in Test Tube 2 containing the antibody cocktail, HEPES buffer, and whole blood (see Section 4.2). 48. P-selectin expression < 15% was considered to be acceptable (no evidence of preactivation). 49. In the case of P-selectin expression > 15%, the results should be evaluated with caution.

Identifying Single Platelets
50. Single platelets were identified for further analysis using the gating strategy as shown in Figure 1. Minor adjustments are to be expected between test tubes; see Section 6.9.

Defining Gates and Measurements
51. The expression of activation-dependent platelet surface markers (antifibrinogen, CD63, and P-selectin) was evaluated via the percentage of positive platelets and with median fluorescence intensity (MFI) corresponding to the amount of the expression of a surface marker. 52. Test Tube 1 (negative sample) was used to set the positive gates, as shown in Figure  2A. The positive gates were set to include 1-2% events for antifibrinogen and CD63, 0.1-0.2% events for P-selectin, and 0.02-0.04% events for SYTO-13 on the negative control. Gates must be applied to all tubes, although minor adjustment can be expected between test tubes. Preactivation was evaluated through the positive expression of P-selectin in Test Tube 2 containing the antibody cocktail, whole blood, and

Defining Gates and Measurements
51. The expression of activation-dependent platelet surface markers (antifibrinogen, CD63, and P-selectin) was evaluated via the percentage of positive platelets and with median fluorescence intensity (MFI) corresponding to the amount of the expression of a surface marker. 52. Test Tube 1 (negative sample) was used to set the positive gates, as shown in Figure 2A.
The positive gates were set to include 1-2% events for antifibrinogen and CD63, 0.1-0.2% events for P-selectin, and 0.02-0.04% events for SYTO-13 on the negative control. Gates must be applied to all tubes, although minor adjustment can be expected between test tubes. Preactivation was evaluated through the positive expression of P-selectin in Test Tube 2 containing the antibody cocktail, whole blood, and HEPES buffer instead of an agonist, as shown in Figure 2B. Furthermore, using the HEPES sample, all single platelets were subdivided into quintiles according to SYTO MFI. SYTO-high platelets occupied the first quintile (20% of platelets with the highest SYTO-13 expression) and SYTO-low platelets occupied the fifth quintile (20% of the platelets with the lowest SYTO-13 expression), as shown in Figure 2C. In Test Tubes 3-6, containing agonists, the MFI and percentage of positive platelets of activationdependent platelet surface markers of all single platelet were evaluated as shown in Figure 2D, SYTO-high platelets in Figure 2E, and SYTO-low platelets in Figure 2F.
SYTO-13 expression) and SYTO-low platelets occupied the fifth quintile (20% platelets with the lowest SYTO-13 expression), as shown in Figure 2C. In Test 3-6, containing agonists, the MFI and percentage of positive platelets of activ dependent platelet surface markers of all single platelet were evaluated as sho Figure 2D, SYTO-high platelets in Figure 2E, and SYTO-low platelets in Figure

Results
53. Table 1 shows the flow cytometric analysis of platelet reactivity in 20 healthy viduals investigated using the current protocol. The expression of antifibrinog dicates platelet-to-platelet aggregation, as this process is mediated through fi gen binding [21]. The expression of CD63 indicates the release of dense granule taining high concentrations of ADP that increase platelet reactivity [1]. The e sion of P-selectin indicates the release of α-granules containing proteins particip in platelet reactivity, cell adhesion, and coagulation [1].   Table 1 shows the flow cytometric analysis of platelet reactivity in 20 healthy individuals investigated using the current protocol. The expression of antifibrinogen indicates platelet-to-platelet aggregation, as this process is mediated through fibrinogen binding [21]. The expression of CD63 indicates the release of dense granules containing high concentrations of ADP that increase platelet reactivity [1]. The expression of P-selectin indicates the release of α-granules containing proteins participating in platelet reactivity, cell adhesion, and coagulation [1]. 54. Table 2 shows the platelet reactivity of SYTO-high platelets (immature platelets) and SYTO-low platelets (mature platelets) in 20 healthy individuals investigated via flow cytometry using the described protocol. SYTO-high platelets were statistically significantly larger (higher FSC) than the SYTO-13-low platelets. Furthermore, SYTO-high platelets had overall statistically significantly increased platelet reactivity compared with the SYTO-low platelets.  All values are presented as median and interquartile range. Differences were analyzed using the Mann-Whitney test. Test significance was two-tailed with a probability value of p < 0.05. All significant p-values are highlighted with bold. Abbreviations: MFI: median fluorescence intensity, FSC: forward scatter, Antifib: antifibrinogen, P-sel: P-selectin, COL: collagen-related peptide, ADP: adenosine diphosphate, TRAP: thrombin-receptor-activating peptide, AA, arachidonic acid. Figure 3 shows platelet reactivity in 20 healthy individuals in all single platelets, subdivided into SYTO-high platelets (immature platelets) and SYTO-low platelets (mature platelets). Overall, SYTO-high platelets had the highest platelet reactivity, and SYTO-low platelets had the lowest platelet reactivity.

Summary and Conclusions
We provided a protocol to investigate how platelet reactivity is related to platelet maturity in whole blood. Platelet reactivity was measured via the expression of antifibrinogen, CD63, and P-selectin. Furthermore, all single platelets were subdivided on the basis of SYTO-13 labelling into 20% SYTO-high platelets (immature platelets) and 20% SYTOlow platelets (mature platelets) corresponding to the RNA content within the platelets. Simultaneously, the platelet reactivity of each platelet subpopulation was measured.
This protocol requires experienced laboratory personnel and advanced equipment. Currently, the method has not been standardized across laboratories. These factors may limit implementation into routine clinical practice. However, the method provides a valuable research tool to analyze platelet reactivity and its relation to platelet maturity.

Blood Sampling
Whole blood was collected in tubes anticoagulated with 3.2% sodium citrate (Terumo Europe, Leuven, Belgium) using a large needle (21-gauge) with a minimum of stasis. Samples were then allowed to rest for 1 h before any sample preparation and fixation as previously described [5].

HEPES Buffer
HEPES buffer was prepared by mixing the following: 4.003 g NaCl, 0.101 g KCl, 0.103 g MgCl 2 , 0.504 g glucose, 2.383 g HEPES, and 0.5 g BSA. Then, sterile water was added until a total volume of 500 mL. Lastly, the pH of the HEPES buffer was adjusted to 7.4, and it was stored at -20 • C until use.

Mixture of PBS-0.2% PFA Fixation
One tablet of PBS was diluted in 200 mL demineralized water. Then, 240 µL 37% PFA was added to 44.4 mL PBS. The fixation buffer was then ready for use.

Flow Cytometer Settings
All analyses were performed with the corresponding filter configurations for the fluorescence detectors, as shown in Table 3. An unstained sample was used to adjust the gain for all fluorochromes to maximize the separation of positive and negative events. Settings were confirmed by matching the isotypic controls. The sampling rate was set to slow to minimize platelet activation. For each test well, a minimum of 10,000-15,000 platelets or a total sample time of 600 sec was assessed.

System Startup Program
Before analyzing the samples, different startup programs were run using the incorporated programs according to manufacturer's instructions.

Daily Quality Control Program
The quality control of fluorescence and particle size, and the alignment of the optical and fluidic systems were standardized and controlled with QC beads (CytoFLEX Daily QC Fluorophores, REF B53230, Beckman Coulter, Miami, FL, USA) daily according to the manufacturer's instructions.

Experiment Creation
• "New Experiment from Template" was selected on the Start page. • "Browse" was selected to save the experiment in the preferred location. • "Template" was selected to choose the specific template (a template must be created before analysis).

Identification of Platelets
Platelets were identified via forward-scatter (FSC) and sideward-scatter (SSC) characteristics, and using specific platelet marker CD42b. Additionally, CD45 (a specific leucocyte marker) was used to exclude single leucocytes and platelet-leucocyte aggregates, thereby ensuring a pure platelet population. Lastly, by employing a FSC peak and FSC intensity plot, microparticles and platelet-platelet aggregates were excluded. Hence, single platelets were included for further analysis. This identification strategy was employed in all further experiments. The volume and concentration of CD42b and CD45 were selected to ensure the optimal separation of negative and positive events.

Stability of SYTO-13 Dye
The stability of SYTO-13 staining in whole blood was investigated for a total of 3 h comparing incubation times of 10 and 30 min.  The analysis started by running an unstained sample, analyzed in 6 min, followed by the analysis of a stained sample for 4 min.

•
The unstained sample was used to set the gate to subdivide platelets into SYTO-13positive and SYTO-13 negative.

•
Steps 6 and 7 were repeated for a total of 3 h.

•
The MFI and percentage of positive platelets were stable after 10 min of incubation followed by 60 min of resting. On the basis of these findings, we decided to use these time intervals in our future analyses.

Titration of Antibodies
To find the optimal concentration of SYTO-13, antibody, and activation-dependent

Titration of Antibodies
To find the optimal concentration of SYTO-13, antibody, and activation-dependent surface markers, titration experiments were performed using whole blood resting for 60

Titration of Antibodies
To find the optimal concentration of SYTO-13, antibody, and activation-dependent surface markers, titration experiments were performed using whole blood resting for 60 min prior to sample preparation, incubation time of 10 min followed by fixation, and 60 min of resting in the dark. Furthermore, the selected concentrations were controlled with matching isotypic controls with similar concentrations showing minimal nonspecific binding.

•
A final SYTO-13 concentration of 19.23 µM was selected. • A final P-selectin concentration of 0.10 µg/mL was selected. • A final CD63 concentration of 7.69 µg/mL was selected. • A final antifibrinogen concentration of 0.013 µg/mL was selected.
We experienced considerable lot-to-lot variation in antibodies. Antibody titration is, therefore, crucial when changing lot numbers to achieve the same percentage of positive platelets and MFI values.

Stability of Activation Dependent Surface Markers
We examined the stability of the activation-dependent surface markers to investigate if the expression of surface markers was stable over a period of 60 min after fixation (selected as the expression of SYTO-13 that was stable after 60 min of resting), as shown in Table 4. Overall, our results show no significant difference in the expression of activation-dependent surface markers between the two time points. In the final protocol, we decided to include a 60 min rest period after incubation, as both SYTO-13 and the activation-dependent surface markers were stable after 60 min. Table 4. Comparing the expression of activation-dependent platelet surface analyzed directly after fixation and analyzed after 60 min of resting after fixation.  Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.