Platelet Activation

Platelet Activation, Staining, and Analysis

Scope

This procedure describes the preparation of fresh or fixed, unstimulated or in vitro activated platelets from unlysed whole blood; staining of platelets; and setting up the FACS brand flow cytometer prior to acquisition of platelet data. When performing assays to detect in vivo platelet activation or analyzing in vitro platelet activation, it is critical that artifactual stimulation of the platelets be minimized during blood collection and sample handling. Even with careful preparation, low levels of inadvertent platelet activation can be difficult to avoid. Therefore, nominally unstimulated platelets should always be analyzed in parallel with in vitro stimulated samples to measure specific effects. The investigator has several options when performing platelet assays by flow cytometry. Anticoagulant, blood collection, fixation, agonist, and gating/analysis strategies are independent variables; the only limiting factor is the antibody. Some guidelines are outlined here.

Reagents and Equipment Required

1. ACD VACUTAINER blood collection tube (BD Cat. No. 4816).
2. Falcon disposable 12 x 75-mm capped polystyrene test tubes (BD Cat. No. 2058) or equivalent.
3. Micropipettor with tips (BD Electronic Pipette, BD Cat. No. 343246) or equivalent.
4. Adenosine diphosphate (ADP), 2 x 10–4 M (Bio/Data, Inc, Phila, PA, Cat. No. 101312). Prepare according to manufacturer’s instructions. Store at 2° to 8°C.
5. 1% paraformaldehyde solution prepared in PBS with 0.1% sodium azide. Store at 2° to 8°C in amber glass for up to 1 week.

   WARNING: Formaldehyde is harmful by inhalation, in contact with skin, and if swallowed. It is irritating to eyes and skin. Exposure can cause cancer. Possible risks of irreversible effects. Can cause sensitization by skin contact. Keep locked up and out of the reach of children. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing and gloves. If swallowed, seek medical advice immediately and show the container or label. Dispose of according to federal, state, and local regulations.

   WARNING: Sodium azide is harmful if swallowed. Keep out of reach of children. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing. If swallowed, seek medical advice immediately and show this container or label. Contact with acids liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.

6. Centrifuge
7. Wash buffer: phosphate-buffered saline (PBS) with 0.1% sodium azide (Dulbecco’s PBS without calcium, magnesium, or phenol red, pH 7.2 ± 0.2).1 Filter the PBS through a 0.2-μm filter prior to use; store at 2° to 8°C.
8. Vortex mixer
9. Staining medium: PBS with 0.1% sodium azide and 2% fetal bovine serum (FBS). Store at 2° to 8°C.
10. BD fluorochrome-conjugated monoclonal antibodies to human platelet antigens. Refer to the appropriate reagent package insert for more information.
11. Arg-Gly-Asp-Ser (RGDS) (Sigma Cat. No. A9041). Prepare 10 mg/mL in PBS. Use RGDS with conjugated PAC-1 antibody
    NOTE: Some commercial preparations can include lipopolysaccharide (LPS), a potent platelet activator.
12. FACS brand flow cytometer. Refer to the appropriate instrument user’s guide for detailed information.
13. CaliBRITE beads. Refer to the BD CaliBRITE Beads package insert for detailed information.
14. FACSComp software for instrument setup and CellQuest software for acquisition and analysis. Refer to the appropriate software user’s guide for detailed information.

Procedure

Blood Collection

WARNING:All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing and gloves.

Our labs have successfully used ACD, sodium citrate, and EDTA anticoagulants. Other anticoagulants can also work, however. Heparin anticoagulant activates platelets and is not recommended for measuring in vivo platelet activation.

NOTE: PAC-1 binding to the fibrinogen receptor is pH and Ca ++ sensitive. 2 PAC-1 will not bind to EDTA-treated blood and PAC-1 binding is typically higher in sodium citrate than in ACD.

The following protocol is designed to minimize artifactual activation of platelets during blood draw. 3

1. Label collection tubes and organize them conveniently in a tube rack.
2. Collect ~2 mL of blood aseptically by venipuncture into any type VACUTAINER tube using a 20-gauge needle. Discard this blood because it contains activated platelets. Release tourniquet and collect blood into a labeled ACD VACUTAINER tube. This blood will be used for staining.
3. Perform activation and staining or fixation of platelets within 10 minutes of blood collection. 3,4

Whole Blood Activation

This procedure is one example of a variety of methods that can be used to activate platelets. Our research labs have successfully used ADP, epinephrine, phorbol 12-myristate 13-acetate (PMA), thrombin, and thrombin-receptor agonist peptide (TRAP).

1. Within 10 minutes of blood collection, pipette 50 μL of ADP solution into a 12 x 75-mm test tube.
2. Add 0.45 mL of whole blood. Gently swirl to mix.
3. Incubate at room temperature (20° to 25°C) for 2 minutes.
4. Stain or fix immediately.

Fixation

Fixation of the blood with paraformaldehyde prior to staining inhibits spontaneous platelet activation. For clinical testing, fixing platelets can make the assay more manageable. Fixation has an effect on activation-dependent platelet antibodies. PAC-1 will not bind to paraformaldehyde-fixed platelets and CD62P binding is decreased. If fixation is not desired or possible, proceed to the next section, Direct Immunofluorescence Staining, and use fresh whole blood.

1. Within 10 minutes of blood collection, pipette 100 μL of unstimulated or activated whole blood into a 12 x 75-mm test tube containing 1 mL of cold (2° to 8°C) 1% paraformaldehyde solution.
   

   NOTE: 100 μL of whole blood yields enough fixed blood for 20 tests. If additional fixed blood is required, prepare the appropriate number of tubes. Do not increase the volume of the blood or paraformaldehyde in the tube as this will increase the possibility of platelet aggregation.

2. Fix platelets at 2° to 8°C for a minimum of 2 hours. Fixed platelets are stable up to 5 days. Store at 2° to 8°C.
3. Before staining, centrifuge the fixed blood at 1200 x g for 5 minutes at room temperature (20° to 25°C).
4. Remove the supernatant, and add 1 mL of room-temperature wash buffer.
5. Resuspend the pellet by vortexing thoroughly. Centrifuge at 1200 x g for 5 minutes at room temperature.
6. Remove the supernatant and resuspend the pellet in 1 mL of room-temperature staining medium.

Direct Immunofluorescence Staining

Single- or multicolor staining can be used in the assay. With multicolor staining, one antibody conjugate can be used to threshold data acquisition to analyze only those blood cells that bind an activation-independent, platelet-specific antibody, 4,5 for example, CD61 or CD42a. Another antibody conjugated to a different fluorochrome can be used to simultaneously assess the binding of platelet-associated, activation-dependent antibodies, for example, CD62P or PAC-1. The combination of CD61, CD62P, and PAC-1 reagents represents a three-color assay that reports two aspects of platelet activation. 3 The following is a multicolor staining procedure.

1. Label 12 x 75-mm tubes.
2. Add appropriate volume of activation–independent, platelet-specific antibody to the appropriately labeled test tube.
3. Add appropriate volume of isotype control or platelet activation–dependent monoclonal antibody to the appropriately labeled test tubes. Test and control antibody concentrations should be matched. Consult the monoclonal antibody data sheet for each test antibody to determine the amount of control (in μg) to use.
   NOTE: To demonstrate specific PAC-1 binding, include one tube with 10 μL RGDS solution in the staining mixture. RGDS peptide competitively inhibits PAC-1 binding. Refer to the PAC-1 data sheet.
4. Using a fresh micropipettor tip each time, carefully add to the bottom of each tube either:
   

    

   • 5 μL of unstimulated or activated fresh whole blood within 10 minutes of blood collection, or
   • 50 μL of unstimulated or activated fixed whole blood suspension within 5 days of fixation

    

   
   Exercise care to prevent blood from running down the side of the tube. If blood remains on the side of the tube, it might not be stained with the reagent. If this occurs, discard and try again.
5. Gently swirl the tubes to mix.
6. Incubate for 15 to 20 minutes at room temperature in the dark.
7. Add 1 mL of cold (2° to 8°C) 1% paraformaldehyde solution to each tube and vortex. Store stained and fixed cells at 2° to 8°C in the dark for at least 30 minutes, but not more than 24 hours. 3 Analyze samples on a flow cytometer.

Data Acquisition and Analysis

Acquisition and analysis can be performed on scatter gating (Figure 1 through Figure 4) or fluorescence gating (Figure 5 through Figure 8). Scatter gating (gating on forward scatter [FSC] and side scatter [SSC]) can be difficult when the platelet count is low or when there is aggregation in the sample. In both normal and disease states, and especially when activated, platelets and red blood cells can have overlapping light scatter signatures. For scatter gating, exclude debris and background noise by setting the appropriate FSC threshold.

Fluorescence gating (gating on FL3 and SSC) can be done on the activation-independent platelet marker, and then the light-scatter profile of the positive population can be independently analyzed. Venous blood typically demonstrates three subpopulations of particles (Figure 6). The majority of the particles consist of single intact platelets. A second population, typically representing 5% of all particles, exhibits greater light scatter than single platelets and represents platelets associated with large white blood cells (WBCs). 3,6 A third population, representing 5% to 15% of the particles whose light scatter is lower than single intact platelets, includes platelet-derived microparticles with an average diameter of 0.1 μm.5 For fluorescence gating, exclude debris and background noise by setting the appropriate FL3 threshold.

The FACS brand flow cytometer should be calibrated using FACSComp software with CaliBRITE beads. The lyse/no-wash setup can be used for platelets. However, since platelets are much smaller than leucocytes, the events are not optimally displayed along each axis. The following procedure uses fluorescence gating and a setup that optimally displays platelet events.

1. Select logarithmic amplification for FSC and SSC gains.
2. Set the flow rate to low to minimize coincident events.
3. Trigger acquisition on positive events using an activation-independent platelet-specific antibody conjugated to an FL3 conjugate (Figure 5).
4. Use platelets stained with an isotype control or unstimulated platelets stained with activation-dependent monoclonal antibodies to adjust FL1 and FL2 PMT voltages. FL1/FL2 baseline signals should be depicted squarely in the first decade in an FL1 vs FL2 dot plot. For PAC-1, use resting platelets stained with PAC-1 and RGDS (Figure 7).
5. Use activated platelets stained with an activation-independent, platelet-specific antibody conjugate in FL3 and a mix of activation- dependent antibody and isotype control to adjust compensation. For example, use platelets stained with PAC-1 FITC/Mouse IgG1 PE/CD61 PerCP to adjust compensation of FITC from FL2. To adjust compensation of PE from FL1 when using PAC-1 FITC, stain with PAC-1 FITC/CD62P PE/CD61 PerCP in the presence of RGDS to inhibit PAC-1 binding.
6. Acquire 5,000 to 10,000 activation-independent platelet events.
7. Display the total platelet population, including those associated with WBCs, or any light-scatter gated subpopulation as twocolor dot plots and statistically analyze (Figure 8).

References

1. Jackson AL, Warner NL. Preparation, staining, and analysis by flow cytometry of peripheral blood leukocytes. In: Rose NR, Friedman H, Fahey JL, eds. Manual of Clinical Laboratory Immunology. 3rd ed. Washington, DC: American Society for Microbiology; 1986:226-235.
2. Shattil SJ, Motulsky HJ, Insel PA, Flaherty L, Brass LF. Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine. Blood. 1986;68:1224-1231.
3. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood. 1987;70:307-315.
4. Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thrombosis and Haemostasis. 1991;65:467-473.
5. Abrams CS, Ellison N, Budzinski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood. 1990;75:128-138.
6. Jennings LK, Ashmun RA, Wang WF, Dockter ME. Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann’s thrombasthenia in whole blood by flow cytometry. Blood. 1986;68:173-179.