Vol. 19 • Issue 5 • Page 34
Platelet testing is a high-complexity test that requires skill to both perform and interpret results. It’s imperative that the laboratory standardizes methods to ensure consistent quality results.
The Clinical and Laboratory Standards Institute (CLSI) is an international, interdisciplinary, non-profit, standard-developing and education organization committed to developing documents classified as either standards, guidelines or reports to provide consensus opinion to promote good laboratory practices. One of its most recent projects is the publication of the approved guideline for Platelet Function Testing by Aggregometry (H58-A Vol. 28 No 31).
Specifically, the guideline discusses standard procedures for using light transmittance aggregation, whole blood impedance aggregometry and low- and high-shear technologies. It also covers topics regarding anticoagulation, specimen storage, temperature, reference intervals, validation and analysis, reporting and troubleshooting.1A brief overview of the document follows; laboratories performing these tests should obtain a copy of the document in its entirety.
Process of Platelet Aggregation
Before exploring the new guidelines, the process of platelet aggregation should be reviewed. Platelets are produced by megakaryocytes, or very large cells (50 to 100 æm in diameter) produced in the bone marrow. Their surface membranes form protoplatelet extensions from which platelets “bud off” and are emitted into the circulation, where they number approximately 200,000-400,000 per microliter of blood.
Platelets have no nucleus and are the first responders in the vascular response to injury. They contain dense and alpha granules. Dense granules contain the nucleotide adenosine diphosphate (ADP), which activates other platelets; alpha granules, which contain proteins and protein mediators, are involved in inflammatory processes.
Platelets possess an affinity for adherence, especially to injured vessel walls, where they release their granule contents, then aggregate. Platelets adhere to proteins such as von Willebrand factor (vWF), a large extracellular matrix protein produced by endothelial cells. The platelet glycoprotein Ib/IX/V binds to vWF.2Platelets are then engaged through receptors for collagen and glycoprotein VI, leading to intracellular signaling that in turn activates platelets. This is followed by adhesion through the glycoprotein IIb/IIIa receptor. The receptor is involved in a platelet-platelet interaction with a IIb/IIIa receptor on another platelet, which attracts further platelets and results in platelet-platelet adhesion, called platelet aggregation.2
Platelet Function Tests
The new guidelines note that optical platelet aggregation is the most common method of measuring platelet aggregation. This procedure is a high-complexity laboratory test that uses platelet rich plasma (PRP) to which an aggregating agent is added (e.g., ADP, epinephrine, thrombin or ðarachidonic acid). Infrared light transmission is used to measure changes in shape and aggregation. Changes in shape result in a decrease in transmittance and aggregation, leading to an increase in light transmittance.3
Whole blood platelet aggregation is typically a high-complexity laboratory test that measures impedance change in blood viscosity. It is accomplished by looking at impedance between two electrodes immersed in a sample of whole blood; whole blood aggregation modulates platelet behavior in vivo due to its interaction with WBC. Leukocytes release prostacyclin and platelet activating factor. Erythrocytes are able to bind prostacyclin and release ADP.3
Lumi-aggregation also provides a measurement of aggregation and release defects; it detects secretion and storage pool deficiency and provides direct unequivocal measurement and quantification of ATP release. Luciferin-luciferase is added to the sample that enables it to measure platelet release of granules.3Additionally, the platelet function analyzer measures platelet function under flow and high shear conditions. It is dependent on plasma vWF, glycoprotein platelet receptor and platelet activation and simulates high shear platelet function on a membrane, which is reactive with either a coating of collagen and epinephrine or collagen and ADP. Under these conditions, platelet adhesion, activation and aggregation occur and result in the formation of a platelet plug. This is measured as closure time or the time it takes to occlude the aperture.1
Of utmost importance is to know what drugs the patient may be taking while being tested for platelet function. Many over-the-counter, herb supplements/drugs inhibit platelet function, and their effect can last seven to 10 days, which is the half-life of a platelet. Patients also should avoid fatty meals prior to testing.
To confirm sufficient platelet count, blood should be drawn into either evacuated tubes or plastic syringes with 19-21 gauge needles and be mixed well. If a syringe is used, the needle should be removed before adding the blood to the tube, and the blood should be gently added down the side of the tube. Minimize prolonged use of a tourniquet.
The anticoagulant of choice is sodium citrate, with the anticoagulant-to-blood ratio of one part trisodium citrate to nine parts of blood with a hematocrit of 45%. EDTA and heparin should not be used.
To prevent platelet activation, samples should be hand-carried, upright at room temperature. Testing should be performed within four hours of collection.
Quality Control, Assurance
The new guidelines also note that reference intervals should be determined for each laboratory and method. Quality control should be performed within manufacturer’s recommendations. Reagents must be functional and produce predictable results; it’s also crucial to demonstrate how a new shipment of reagents performs with normal platelets. If proficiency testing is not available, the laboratory should determine an in-house proficiency program.
Sample Preparation, Testing
PRP and platelet-poor plasma (PPP) are needed and, according to recommendations, should be obtained by centrifugation at room temperature. The speed should be in relative centrifugal force (RCF), also known as g-force. This is calculated by knowing the rotation radius of a centrifuge and its rotation speeds. A swing-out bucket and several combinations of time/force should be used (100 g for 10 minutes).
Platelet count should be standardized to a specific concentration of platelets with autologous PPP or a physiologic buffer. Counts should be adjusted to between 200-350 x 109 L. Samples that are lipemic, hemolyzed or icteric may interfere with testing; keep those samples at room temperature and capped to maintain pH.
The suggested agonists and dose range are:
• ADP (Adenosine diphosphate): 0.5 to 10 umol/L
• Arachidonic acid: 0.5 -1.6 mmol/L
• Collagen 1-5 ug/mL
• Epinephrine: 0.5-10 umol/L
• Ristocetin: high dose: 0.8-1.5 mg/mL; low dose: < 0.6 mg/mL
Whole Blood Aggregometry
This sample requires no preparation but should be handled and mixed gently and tested within three hours of collection. The concentration of the platelet count determines the need for dilutions. All whole blood platelet counts between 100-1000 x 109 L should be diluted with equal parts of preservative-free saline. For ADP, however, the concentration must be greater than 225 x 109 L to be diluted (if less than that test the sample neat).
Platelet function is recorded as a change of impedance and measured in ohms over a minimum of six minutes. Agonists used and dose ranges suggested are:
• ADP (Adenosine diphosphate): 0.5 to 20 umol/L
• Arachidonic acid: 0.5 -1.0 mmol/L
• Collagen 1-5 ug/mL
• Epinephrine: not recommended, <50% of subjects respond.
• Ristocetin: high dose: 1.0 mg/mL; low dose: 0.25 mg/mL
Flow and High Shear Devices
The platelet function analyzer developed by Kratzner and Borm became the prototype for the platelet function analyzer (PFA-100) as it is known today. This analyzer simulates high shear (5000-6000s -1) platelet function within disposable test cartridges. Cartridges should be warmed to room temperature. The value of a patient sample is compared to the laboratory-established reference interval for each cartridge type.
Donna D. Castellone is clinical project manager, Siemens Healthcare Diagnostics, Tarrytown, NY, and an ADVANCE editorial advisory board member.
For a list of references, go to www.advanceweb.com/labmanager
Alternatives to Platelet Aggregation Studies
By Janet L. Endres, MT(ASCP)
latelet aggregation studies are extremely useful for the laboratory evaluation of platelet function. Their use, however, is restricted because testing must be performed within four hours of specimen collection. If platelet aggregation testing is not available locally, specialized reference laboratories can employ other methods to diagnose several rare disorders.
Such alternatives are available for three platelet disorders characterized by absence or dysfunction of receptors on the platelet surface that play a role in normal aggregation. Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder in which expression of an important platelet receptor for fibrinogen is abnormal. Platelet aggregation with all agonists except ristocetin is markedly abnormal. Reference laboratories can perform flow cytometry with monoclonal antibodies to confirm absence of the fibrinogen receptor and support a diagnosis of GT.
In Bernard-Soulier syndrome (BSS), another rare autosomal recessive disorder, the platelet surface receptor for von Willebrand factor is abnormal. Platelet aggregation with ristocetin is absent. Even when platelet aggregation studies are available, the large platelet size and low platelet count associated with BSS makes aggregation studies difficult. Specialized reference laboratories use flow cytometry to determine whether the receptor is missing; DNA analysis is used to sequence the genes required for normal expression of the receptor.
As with BSS, the defect for platelet-type von Willebrand disease (PT-VWD) is found in the platelet receptor for von Willebrand factor. The only abnormal finding in platelet aggregation studies is presence of aggregation with low-dose ristocetin. DNA sequencing can identify mutations known to cause PT-VWD.
Finally, specialized tests can establish a diagnosis of type 2B von Willebrand disease, a plasma-based defect that routine testing and platelet aggregations cannot differentiate from PT-VWD.
As well, specimens for the alternative assays may be stored and shipped. Whole blood up to two days old is suitable for the flow cytometry assays. Citrated plasma may be stored frozen at -70ø C or below for up to one year prior to the type 2B-VWD platelet-binding assay, while whole blood samples for DNA analysis are stable for weeks or more.
Janet L. Endres is technical specialist, Hemostasis Reference Laboratory, BloodCenter of Wisconsin.