Why Perform Platelet Aggregation

Platelet aggregation, the process by which platelets adhere to other platelets at sites of vascular injury, has long been recognised to be critical for haemostatic plug formation and thrombosis. Haematology laboratories perform platelet aggregation testing to diagnose specific bleeding disorders when a patient sample gives abnormal results to routine coagulation assays.

The response of the patients’ platelets in relation to different agonists, and different agonist concentrations, will show if the platelets have any qualitative or quantitative defects linked to specific disease states.

Disease states such as Glanzmann’s Thrombasthenia, Bernard-Soulier Syndrome and Storage Pool Disorders are diagnosed through interpretation of the response of patients’ plasma to a panel of agonists that reproduce platelet aggregation in vitro.

Platelet aggregation is also used in the diagnosis and typing of von Willebrands Disease, the most common hereditary bleeding disorder, using different concentrations of Ristocetin to type the disease state.

This is subsequently performed after an abnormal result is obtained from a von Willebrands Activity assay (also called Ristocetin Cofactor Activity assay) screening test. The gold standard methodology is the Born method utilising a light transmission platelet aggregometer.

Interpretation of the aggregation response is determined by evaluating maximum aggregation, final aggregation, lag phase, primary slope, secondary slope and time to maximum aggregation values for each agonist tested.

Platelets are required to adhere to the subendothelium of a vascular injury in order to initiate and perpetuate platelet plug formation. There are three main stages in platelet plug formation:

• Initiation (Platelets adhere to subendothelium via Thrombin activation and the Collagen-von Willebrand Factor complex)
• Extension (The platelet plug is extended by the activation of additional platelets via the release of Thromboxane A2 and other agonists from the original monolayer of platelets)
• Perpetuation (Close contact between platelets in the haemostatic plug and the fibrin meshwork help to perpetuate and stabilise the platelet plug)

Platelets are produced by fragmentation of megakaryocytes with around 4000 platelets generated per cell. A normal circulating platelet count is between 150 – 400 x 10⁹ / L, however only two-thirds of total platelets are circulating with one-third stored in spleen. 10% of the platelet pool is replaced every 24 hours.

They are discoid in shape, around 2 - 4 microns in size (Red blood cells are 7.5 microns) and composed of anucleated membrane bound cytoplasm housing a complex structure of microtubules, microfilaments and organelles/granules containing lysosomes, hydrolytic enzymes, peroxisomes, catalase, dense bodies, ADP, Ca2+, Serotonin, alpha granules, Fibrinogen, vWF, Factor V, PDGF and PF-4.

Disease States

Bernard-Soulier Syndrome (BSS)

BSS is a relatively severe bleeding disorder caused by a genetic defect in GP Ib or GP IX, which is autosomal recessive inheritance. Bleeding time is prolonged with both Thrombocytopenia and giant platelets. Platelets in patients with BSS do not agglutinate in response to Ristocetin (GP Ib is unable to bind vWF).

von Willebrand Disease (vWD)

vWD is the most common hereditary coagulation abnormality described in humans, although it can also be acquired as a result of other medical conditions. It arises from a qualitative or quantitative deficiency of von Willebrand Factor (vWF), which is a multimeric protein that is required for platelet adhesion. vWD disease is characterized by a reduced or abnormal function of vWF and can be subdivided into different types and sub-types:              

• Type 1 – partial reduction in vWF
• Type 2 – abnormal form of vWF (4 sub types)                                      

• 2A – loss of high molecular weight multimers
• 2B – abnormally high affinity for platelets
• 2M – defective GP 1b binding site
• 2N – reduced affinity for Factor VIII              

• Type 3 – total lack of vWF

Glanzmann’s Thrombasthenia (GT)

GT is an autosomal recessive disease caused by lack of expression / qualitative defects in GP IIb / IIIa. This causes a severe impairment or lack of platelet aggregation by all agonists.

Storage Pool Disorder (SPD)

SPD is characterized by defects in the alpha or dense granules in platelets, particularly a lack of granular non-metabolic ADP. Patients with ADP deficient "Storage Pool Disease" present a prolonged bleeding time due to impaired aggregation response to fibrillar Collagen.

Aspirin Like Disorder or Aspirin Ingestion

Aspirin inhibits the release of ADP from platelet storage pool, negating activity and preventing any platelet diagnostic testing until the platelet pool recovers. Disease states like Reye’s syndrome can mimic the diagnostic results of patients who have ingested Aspirin

Agonists

Arachidonic Acid (AA)

AA is a substrate for synthesis of Thromboxane A2 (TxA2). Aspirin permanently inactivates COX 1 & 2 preventing Arachidonic Acid conversion to Thromboxane A2. This Aspirin effect is evident for the lifespan of the platelets 7 – 10 days. AA is the initial screening test for Aspirin ingestion.

Adenosine Diphosphate (ADP)

ADP causes platelets to swell and encourages platelet membranes to adhere to each other. Low concentrations of ADP cause only primary, incomplete, reversible aggregation. Higher concentrations create biphasic aggregation and irreversible secondary aggregation caused by TxA2 formation. High concentration ADP causes both phases to fuse into a single phase.

Collagen

Collagen binds directly to membrane protein Ia, initiating aggregation. The characteristic response is a prolonged lag phase as TxA2 formation and secretion of granule contents is required prior to aggregation.

Epinephrine

Epinephrine inhibits cAMP formation & stimulates TxA2 formation. TxA2 inhibiting drugs will not allow aggregation in response to Epinephrine.        

Ristocetin

Ristocetin facilitates binding of vWF to GP Ib / IX / V. It produces a dose response testing for sensitivity. A normal patient requires both functional vWF and normal GP Ib / IX / V. A higher than normal aggregation response is seen for vWD Type 2B.

Platelet Aggregation testing in the modern laboratory

Platelet agonists are available from Helena Biosciences in high stock concentrations to allow for high and low dose dilutions recommended by CLSI guideline H58-A Platelet Function Testing by Aggregometry; Approved Guideline. They are suitable for use with any platelet aggregometer, including the AggRAM Platelet Aggregometer which utilises the gold standard Born methodology (light transmission) and is also available from Helena Biosciences Europe:

• Cat.No.5366 ADP 

• Cat.No.5367 Epinephrine
• 
Cat.No.5364 Arachidonic Acid 

• Cat.No.5368 Collagen 

• Cat.No.5199 Ristocetin

• Cat.No.5370 Ristocetin Cofactor Assay

The AggRAM offers fully customisable platelet aggregation and ristocetin cofactor testing by combining a high performance analyser with powerful software that allows up to 21 patient results to be overlayed and compared simultaneously.

• 4 or 8 Channels – Fully flexible, multiple agonist configurations and concentrations
• Flexibility – Customise your assay test sequence, calibration, dilutions, volumes, run times, display parameters, input additional agonists and create new screens 

• Half-volume – Fully optimised half volume settings, including programmable stirrer speed 

• Powerful data handling – Automatic calculation of slope, max% aggregation, time to max aggregation, lag phase, secondary slope and area under the curve (research use only) with full manual edit options 

• Security – Operator log on with password level protection 

• Database – Extensive database for patient results, quality control and standards. Data retrieval with the safety of full automatic backup
• Interface – LIMS bi-directional host interface
• Quality control – Evaluation and graphical Levey- Jennings display of QC data based on assigned Westgard rules with integrated corrective action log

---

Company information

• About us
• News
• Events
• Contact us

---

---