Platelet Aggregation

Overview of Platelet Aggregation Studies

Definition: Platelet aggregation studies are in vitro tests that measure the ability of platelets to aggregate (clump together) in response to various agonists
Purpose:
- Diagnosis of Platelet Function Disorders: Helps identify inherited or acquired disorders of platelet function
- Evaluation of Unexplained Bleeding: Used to investigate patients with a personal or family history of mucocutaneous bleeding
- Assessment of Antiplatelet Therapy: Used to monitor the effects of antiplatelet medications (e.g., aspirin, clopidogrel)
- Research: Used to study platelet physiology and pharmacology
Key Principle: Measures the change in light transmission through a platelet-rich plasma (PRP) sample as platelets aggregate
Agonists Used: Different agonists stimulate platelet aggregation via different pathways, allowing for the assessment of specific platelet functions:
- Adenosine Diphosphate (ADP)
- Collagen
- Epinephrine
- Arachidonic Acid (AA)
- Ristocetin
- Thrombin Receptor-Activating Peptide (TRAP)

Components of Platelet Aggregation Studies

Specimen Collection:
- Collection Tube: Sodium citrate (light blue top) tube with a 3.2% or 3.8% sodium citrate concentration. It is very important to use the correct sodium citrate concentration and to fill the collection tube all the way to the fill line.
- Blood-to-Anticoagulant Ratio: The correct ratio is critical for accurate coagulation testing:
  - 9:1 ratio of blood to anticoagulant
- Collection Technique: Atraumatic venipuncture to avoid platelet activation
- Transport and Storage: Transport the sample to the laboratory promptly (within 30 minutes).
  - Maintain the sample at room temperature (18-24°C)
  - Do not refrigerate or freeze the sample
- Patient Preparation:
  - Medication Restrictions: Patients should avoid taking aspirin or other antiplatelet medications for at least 7-10 days before testing
Reagents:
- Platelet Aggregating Agents (Agonists): ADP, collagen, epinephrine, arachidonic acid, ristocetin, thrombin receptor-activating peptide (TRAP)
- Platelet-Poor Plasma (PPP): Used as a blank to calibrate the aggregometer and as a reference for 100% light transmission
- Saline or Buffer: Used to dilute reagents and samples
- Quality Control Materials: Commercially available control materials or pooled normal plasma may be used to monitor the performance of the assay
Instrumentation:
- Platelet Aggregometer: A specialized instrument that measures platelet aggregation by monitoring changes in light transmission through a stirred suspension of platelet-rich plasma
- Components of an Aggregometer:
  - Light Source: Provides a beam of light that passes through the sample
  - Cuvette Holder: Holds the sample cuvette
  - Stirring Mechanism: Continuously stirs the sample to maintain a homogenous suspension of platelets
  - Detector: Measures the amount of light transmitted through the sample
  - Recording Device: Records the change in light transmission over time, generating an aggregation curve

Procedure for Performing Platelet Aggregation Studies

Prepare Platelet-Rich Plasma (PRP):
- Centrifuge the sodium citrate whole blood sample at a low speed (e.g., 150-200 g) for 10-20 minutes to obtain PRP
- Carefully remove the PRP layer without disturbing the RBC layer
Adjust Platelet Count:
- Adjust the platelet count in the PRP to a standardized level (typically 200-300 x 10^9/L) by adding autologous platelet-poor plasma (PPP)
- Accurate platelet count adjustment is critical for obtaining reliable and reproducible results
Prepare Reagents:
- Dilute the platelet aggregating agents (agonists) to the appropriate concentrations according to the manufacturer’s instructions
- Warm the reagents to room temperature
Run the Assay:
- Pipette a specific volume of PRP into a cuvette
- Place the cuvette in the aggregometer
- Allow the sample to equilibrate at 37°C for a specific period (e.g., 2-5 minutes)
- Add the agonist to the PRP
- Start the stirring mechanism and the recording device
- Monitor the change in light transmission over time

Interpreting Results

Aggregation Curve:
- The aggregometer records the change in light transmission over time, generating an aggregation curve
- The curve typically consists of two phases:
  - Primary Aggregation: Initial wave of aggregation, caused by the agonist-induced shape change and adhesion of single platelets
  - Secondary Aggregation: Sustained aggregation, caused by the release of mediators from platelet granules (e.g., ADP, thromboxane A2) and activation of additional platelets
Parameters Evaluated:
- Maximum Aggregation: The highest percentage of light transmission achieved during the aggregation response
- Slope of the Curve: The rate of aggregation
- Lag Phase: The time between the addition of the agonist and the start of aggregation
- Shape of the Curve: The presence or absence of a secondary wave of aggregation
Normal Aggregation Response:
- Normal aggregation with all agonists (ADP, collagen, epinephrine, arachidonic acid, ristocetin)
Abnormal Aggregation Responses:
- Absent or Reduced Aggregation: Indicates a platelet function defect
  - May be specific to certain agonists or may affect all agonists
- Normal Aggregation with Some Agonists, Abnormal with Others: Suggests a specific defect in a particular platelet activation pathway

Specific Patterns of Platelet Aggregation

Glanzmann Thrombasthenia (GT):
- Absent or severely reduced aggregation with ADP, collagen, epinephrine, and thrombin
- Normal aggregation with ristocetin (because vWF binding to GPIb/IX/V is intact)
Bernard-Soulier Syndrome (BSS):
- Normal aggregation with ADP, collagen, and epinephrine
- Absent aggregation with ristocetin (even with added normal plasma)
von Willebrand Disease (vWD):
- Decreased aggregation with ristocetin
- Other agonists may show normal or mildly reduced aggregation
Aspirin Effect:
- Normal aggregation with ADP and epinephrine
- Absent or reduced aggregation with collagen and arachidonic acid
P2Y12 Inhibitors (e.g., Clopidogrel, Prasugrel):
- Reduced aggregation with ADP
- Normal aggregation with other agonists

Factors Affecting Platelet Aggregation Results

Pre-Analytical Variables:
- Improper Collection Technique: Traumatic venipuncture can activate platelets and affect results
- Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube can alter the test results
- Delayed Testing: Platelet function can change over time, so the test should be performed within the specified timeframe (usually within 2-4 hours of collection)
- Storage Temperature: Improper storage temperatures can affect platelet function
Analytical Variables:
- Instrument Malfunction: Ensure proper calibration and maintenance of the aggregometer
- Reagent Problems: Use fresh, properly stored reagents and follow the manufacturer’s instructions
- Air Bubbles: Air bubbles in the sample can interfere with light transmission
- Inadequate Stirring: Proper stirring is essential for maintaining a homogenous suspension of platelets
Patient-Related Variables:
- Medications:
  - Aspirin and NSAIDs: Inhibit cyclooxygenase (COX) and impair thromboxane A2 production
  - P2Y12 Inhibitors: Clopidogrel, prasugrel, and ticagrelor block the ADP receptor on platelets
  - GPIIb/IIIa Inhibitors: Abciximab, eptifibatide, and tirofiban block the GPIIb/IIIa receptor
- Underlying Medical Conditions:
  - Myeloproliferative Neoplasms (MPNs): Platelets may have abnormal function
  - Uremia (Chronic Kidney Disease): Uremic toxins can impair platelet function
  - Liver Disease: Can affect platelet function
  - Certain Infections

Troubleshooting Erroneous Results

If the platelet aggregation results are unexpected or inconsistent with the patient’s clinical presentation:
- Check the sample for clots or air bubbles
- Verify that the hematocrit and platelet count are within the acceptable range for the instrument
- Repeat the test on a fresh sample
- Review the patient’s medication list and medical history
- Ensure that the reagents and controls are within their expiration dates and stored properly
- Consider other pre-analytical or analytical factors that may have affected the results

Reflex Testing

If platelet aggregation studies are abnormal, additional testing may be performed to:
- Identify specific platelet function defects
- Flow Cytometry: To measure platelet surface markers (e.g., GPIIb/IIIa, GPIb/IX/V)
- Platelet Secretion Assays: To measure the release of substances from platelet granules (e.g., ADP, serotonin)
- Electron Microscopy: To examine platelet ultrastructure

Key Terms

Platelet Aggregation: The clumping together of platelets
Agonist: A substance that stimulates platelet activation and aggregation
GPIIb/IIIa: A platelet glycoprotein complex that binds to fibrinogen
Ristocetin: An antibiotic that induces vWF-dependent platelet agglutination
Platelet Aggregometer: An instrument that measures platelet aggregation
Light Transmission: A measure of the amount of light that passes through a sample
Mucocutaneous Bleeding: Bleeding from the skin and mucous membranes

Platelet Function

Thromboelastography