Platelet Aggregation Studies in Normal People

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Introduction

Haemostasis is a process that prevents blood loss from the body after injury. It has three processes: primary haemostasis, secondary haemostasis and fibrinolysis . Primary haemostasis occurs immediately after blood vessel injury. The first step in primary haemostasis is adhesion whereby platelets adhere to suendotheial cells through the Von Willebrand factor (VWF). This adhesion leads to platelet shape change, activation, release of granules contents and aggregation to form platelet plug. The primary haemostasis stimulates secondary haemostasis via activate coagulation factors that eventually convert fibrinogen to fibrin and form platelet fibrin thrombus. The third process of haemostasis is fibrinolysis, which leads to wound healing through removing the thrombus . This review outlines the screening tests of primary haemostasis and focuses on the platelet aggregometry test.

Screening Tests for Primary Haemostasis

The first step to investigating primary haemostasis is the full blood count, which measures platelet number and size. This test is used to exclude any defect in primary haemostasis due to thrombocytopenia . Morphological assessment of platelets is important to distinguish shape abnormalities such as Bernard-Soulier syndrome and Gray platelet syndrome. However, bleeding time (BT) is considered the functional screening test for primary haemostasis. It is used to diagnose VWD and platelet dysfunction by measuring the time from the start of bleeding to when it stops after making a small cut in the forearm. BT can be affected by many important factors such as operator technique, patient age and gender. For these reasons, it is rarely used now in practice . The best studied of all the primary haemostasis screening tests is the PFA-100. During this process, anti-coagulated whole blood is put in a cartridge and then rapidly aspirated through a membrane coated with agonists such as collagen and epinephrine or collagen and adenosine diphosphate. These agonists stimulate platelet adhesion, activation and aggregation, which lead to occlusion and stop blood flow in the test cartridge. In the PFA-100 test, closure time (CT) is measured, which reflects the time from aspirating the sample in an agonist coated membrane to occluding the blood flow in the test cartridge. The PFA-100 test is very simple, quick and easy to perform. In addition, it is a sensitive primary haemostasis screening test that can identify platelet function defects and VWD . The most commonly used screening test for assessing the platelet function is platelet aggregometry. There are two types of platelet aggregometry: light transmission aggregometry (LTA) and whole blood aggregometry (WBA) .

Light Transmission Aggregometry

Light transmission aggregometry is the gold standard for testing platelet function. It measures the transmission of the light through a test tube that contains platelet-rich plasma (PRP). After adding a specific agonist such as adenosine diphosphate (ADP), arachidonic acid (AA), collagen and epinephrine, the platelets activate and aggregate, which leads to an increase in light transmission through the test tube. The light signals transfer to a computer, which records these signals as a specific curve (.

These agonists activate platelet aggregation by binding to their receptors on the platelet surface. ADP, which is considered a weak platelet agonist, is also present in the platelet granules. It binds to two G- protein-coupled purinergic receptors (P2Y1 and P2y12) on the platelet surface to stimulate platelet aggregation. Through the P2Y1 receptor, platelet shape change is induced by activating phospholipase C, and then the primary wave platelet aggregation is initiated through calcium mobilisation. Full platelet aggregation response to ADP occurs through P2Y12 by inhibiting adenyl cyclase and stabilising platelet aggregation .

Arachidonic acid converts to thrombaxane A2 (TxA2) by cyclooxgenase and thromboxane synthase. TxA2 leads to mobilising calcium from intacellular storage sites and stimulates the secretion of platelet granules contents, which activates platelet aggregation .

Collagen has two important receptors on the platelet surface: GPIa/IIa (alpha2 beta 1 integrin) and GPVI. Collagen stimulates platelet adhesion through binding to the GPIa/IIa receptor. However, binding of collagen to the GPVI receptor leads to TxA2 formation, which is important for platelet aggregation .

Epinephrine is considered a weak platelet agonist like ADP. It stimulates platelet aggregation through the alpha2 adrenergic receptor, which causes the inhibition of adenyl cyclase and releases calcium ions from the endoplasmic reticulum .

Standardisation of LTA

LTA is affected by many pre-analytical and analytical factors, and these must be carefully controlled. The most important step that affects the LTA result is the PRP preparation . To prepare PRP, the whole blood sample is centrifuged at a specific gravitational force, which leads to separating red blood cells and white blood cells from the platelet-rich plasma. The gravitational force should allow the removal of all red blood cells and white blood cells from the plasma without loss too many platelets. At a 300 gravitational force, which is considered the highest force of centrifugation, the mean platelet volume (MPV) and aggregation rate will be lower in PRP than at other gravitational forces. When PRP is prepared at a 150 gravitational force, the red blood cells count will be high compared to 200, 250 and 300 gravitational forces. In contrast, white blood cell count is not significantly different between these four gravitational forces . The methodological standardisation of light transmission aggregometry is necessary because it is highly variable in the practices .

Blood Sample Collection

The blood sample for LTA should be collected from a subject who has abstained from smoking for at least 30 minutes and caffeine for at least two hours. In addition, drugs that reversibly inhibit the platelet function such as non-steroidal anti-inflammatory drugs should be stopped for at least three days before taking a sample, while medications that are known to irreversibly inhibit the platelet function such as aspirin and thienopyridines should be stopped for at least 10 days before sampling .

The blood sample for LTA should be carefully collected with minimal or no venous stasis, using a large diameter needle of at least 21 gauge into plastic or siliconised tubes. After that, the blood should be drawn into a buffered anticoagulant, which helps pH to remain stable during the LTA process. It is important that the first 23 ml of the blood sample to be discarded or used for tests other than LTA .

Preparation of PRP and PPP

Before centrifugation, the blood sample should be kept at room temperature for rest. The best gravitational force to prepare PRP by centrifugation is at 200 or 250 for approximately 10 minutes at 21 C . PPP should be prepared by centrifuging the remaining blood, which PRP was removed from at a 1500 gravitational force for about 15 minutes .

Choice of Agonists

There are specific agonists such as adenosine diphosphate, arachidonic acid, collagen, ristocetin and epinephrine that should be used in LTA for screen the platelet function. These agonists should be firstly used at a low concentration and then the concentration can be increased if there are abnormal results with low concentration.

 

2 mM of adenosine diphosphate, 5 mM of epinephrine, 1 mM of arachidonic acid, 1.2 mg mL-1 of ristocetin and 2 mg mL-1 of collagen are considered the lowest concentrations of these agonists that should be used during LTA test .

Primary Haemostasis Disorders

Mucocutaneous bleeding is the main complaint of patients with primary haemostasis defects. This bleeding pattern appears as epistaxis in addition to petechiae, ecchymoses and small bruises in the skin .

Von Willebrands Disease

This is an autosomal dominant disease that is characterised by a defect or deficiency in the Von Willebrand factor (VWF). VWF is important in the first step of primary haemostasis, which enhances platelet adhesion through binding with a specific receptor on the platelet surface. The most important lab investigation to diagnose VWD is to measure the VWF in the plasma, which will be low in this disease. Mild thrombocytopenia may present with this disorder in addition to prolonged PFA-100 CT. The response of platelets to all agonists in the LTA test is normal. In addition, the platelets can agglutinate normally with ristocetin .

Bernard-Soulier Syndrome

It is a rare autosomal recessive defect in the glycoprotein Ib (GpIb) receptor, the receptor of Von Willebrand factor. Bernard-Soulier syndrome is characterised by thrombocytopenia, giant platelet, prolonged bleeding time and prolonged CT in PFA-100 test. The platelet aggregation in response to ADP, AA and collagen are normal. In this disease, the platelet cannot agglutinate with ristocetin .

 

Glanzmann Thrombasthenia

This is an autosomal recessive disorder characterised by a defect in the glycoprotein (GPIIb/IIIa) forming the integrin alpha 11b beta 3, which is important for the development of stable platelet aggregate. Thus, the platelets in Glanzmann Thrombasthenia disorder cannot bind to VWF, fibrinogen or fibronectin. The platelet count and shape appear normal in this disease. However, this disorder is associated with prolonged PFA-100 CT and the absence of the platelet response to agonists except shape change only in the LTA test. Platelet agglutination with ristocetin at 1.5 mg/ml is normal, while with 0.5 mg/ml, ristocetin is absent in this disease .

Storage Pool Disease

This is the most common inherited platelet function defect and is characterised by a defect in the number, content or release the platelet dense granules. PFA-100 CT may be prolonged or normal depending on the extent of the disorder. In the LTA test, the platelet response to ADP and collagen is decreased, which appears as a primary wave only with ADP. However, AA can lead to normal platelet aggregation in this disorder. Platelet agglutination with 1.5 mg/ml ristocetin is normal but absent with low concentration of ristocetin .

Conclusion

The diagnosis and investigation of the primary haemostasis disorders require some important steps before conducting laboratory tests. Complete patient history including medical, surgical, family and past history in addition to physical examination are essential to diagnosing primary haemostasis defects and excluding other differential diagnoses. Full blood count and morphological assessment of the platelet is the initial step of the laboratory investigation. In addition, bleeding time and PFA-100 are also important to diagnosing primary haemostasis disorder.

Platelet aggregometry is the most commonly test used to assess platelet function. There are two types of this test: light transmission aggregometry (LTA) and whole blood aggregometry (WBA). LTA measures the light transmission through test tube that contains platelet-rich plasma after adding agonists that stimulate platelet activation and aggregation. LTA is widely used in the practices to identify and diagnose the different defects in the primary haemostasis. Since LTA is affected by many pre-analytical and analytical variables, the methodological standardisation is necessary in this test

Materials and Methods

Whole sample donated by three healthy individuals were generously provided by our supervisor. The ADP agonist solutions with final concentrations of 10 uM, 5 uM , 2.5uM and 1.25 uM were prepared by appropriate dilution with normal saline (9 grams of NaCl +1000 mL of distilled water) as shown in table 1. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared where whole blood samples were centrifuged at 100 g for 12 min to obtain the PRP. The obtained PRP from the blood samples were carefully extracted to avoid mixing with RBC, then transferred to clean tubes. After obtaining PRP, the residual blood samples were recentrifuged at 1700 g for 15 min to obtain PPP, which were carefully extracted and transferred to other tubes.

Table 1:

Final concentration

ADD

Dilution

Make 60 uL

10 uM

100 uM

1 in 10

6 ul + 54 ul

5 uM

50 uM

1 in 20

3 ul + 57 ul

2.5 uM

25 uM

1 in 40

1.5 ul + 58.5 ul

1.25 uM

12.5 uM

1 in 80

1.5 ul + 118.5 ul

*ADP stock =1 mM = 1000 uM

 

 

Plate aggregation tests were performed in siliconized glass cuvettes using Chrono-log Lumi aggregometer (560CA, Chrono-Log Corp., Haverton, PA, USA) according to manufacturer’s instruction. Prior to aggregation runs, the aggregometer was calibrated with 500 uL of PPP to 90 percent transmission. Next, PRP samples diluted with respective donor’s own PPP samples were incubated at 37 oC prior to aggregation runs. Platelet counts were performed using Emerald analyser PACKS-4 according to the manufacturer’s instructions and adjusted to 250 × 109/ L.

 

 

DRAFT

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