Abstract
Platelets play a pivotal role in normal hemostasis, and derangement of their function can lead to hemorrhage or thrombosis. While progress has been made in elucidating the molecular mechanisms leading to platelet adhesion, aggregation, shape change and secretion, clinically useful tests of platelet function have lagged. A number of dedicated platelet function instruments that are much simpler to use and are now utilized as point-of-care (POC) instruments have now become available. Some instruments have been incorporated into routine clinical use and can be utilized not only as general screening tests of platelet function but as monitors of antiplatelet therapy and to potentially assess both risk of bleeding and/or thrombosis. Some of the factors that differentiate these tests are sample volume requirements, the use of whole blood, the presence of shear, POC status, need for a technician and expense. The following is a review of some of the commonly used tests of platelet function, along with their advantages and disadvantages. The tests and pertinent instruments described are based on aggregation, shear stress platelet contribution to clot strength, flow cytometry and serum and urinary thromboxane metabolites.
1
Introduction
Platelets play a pivotal role in both normal hemostasis and pathological bleeding and thrombosis . Most platelet function tests have been utilized for the diagnosis and management of patients presenting with bleeding problems rather than thrombosis . However, platelets are now implicated in the development of atherothrombosis, the leading cause of mortality in the Western world . Platelet function tests are increasingly being used for monitoring the efficacy of antiplatelet drugs used to treat these conditions and/or to try to identify patients at risk of arterial disease. On the other hand, as increasing number of patients are being treated with antiplatelet drugs, with which there is an increased risk of bleeding, especially during trauma and surgical procedures. Platelet function tests are also being proposed as presurgical/perioperative tools to aid in the prediction of bleeding and to monitor the efficacy of various types of prohemostatic therapies. This, coupled with the development of new, simpler tests and point-of-care (POC) instruments, has resulted in the increasing tendency of platelet function testing to be performed away from specialized clinical or research laboratories, where the more traditional and complex tests are still performed .
2
History of platelet function testing
An accurate platelet count in blood is important to eliminate thrombocytopenia as a potential cause of bleeding before any platelet function testing is performed. Though platelets were identified more than 120 years ago as distinct corpuscles in the blood, the routine application of accurate platelet counting and blood smears to study platelet morphology was not widespread until the 1950s. Phase contrast microscopy was the first accurate method used to count platelets in lysed blood . A fully automated full blood count, including a platelet count, became available in the 1970s, and it became possible to measure other important parameters such as mean platelet volume, platelet distribution width and platelet–large cell ratio . With convergence of flow cytometric and aperture impedance principles, platelet counting can now be performed by either optical or immunological methods, which may be more accurate in some samples .
Platelet function testing began with the application of the in vivo bleeding time by Duke in 1910. Bleeding time, regarded as the most useful screening test of platelet function until the early 1990s , was further refined by the Ivy technique and the availability of commercial spring-loaded template disposable devices containing sterile blades (i.e., Simplate II; Organon Technika, Durham, NC). Because of its limitations and with the availability of less invasive screening tests, use of the bleeding time has rapidly declined .
2
History of platelet function testing
An accurate platelet count in blood is important to eliminate thrombocytopenia as a potential cause of bleeding before any platelet function testing is performed. Though platelets were identified more than 120 years ago as distinct corpuscles in the blood, the routine application of accurate platelet counting and blood smears to study platelet morphology was not widespread until the 1950s. Phase contrast microscopy was the first accurate method used to count platelets in lysed blood . A fully automated full blood count, including a platelet count, became available in the 1970s, and it became possible to measure other important parameters such as mean platelet volume, platelet distribution width and platelet–large cell ratio . With convergence of flow cytometric and aperture impedance principles, platelet counting can now be performed by either optical or immunological methods, which may be more accurate in some samples .
Platelet function testing began with the application of the in vivo bleeding time by Duke in 1910. Bleeding time, regarded as the most useful screening test of platelet function until the early 1990s , was further refined by the Ivy technique and the availability of commercial spring-loaded template disposable devices containing sterile blades (i.e., Simplate II; Organon Technika, Durham, NC). Because of its limitations and with the availability of less invasive screening tests, use of the bleeding time has rapidly declined .
3
Currently available tests
3.1
Based on aggregation
Light transmission platelet aggregometry (LTA) is based on the principle that as platelets aggregate in response to the addition of an exogenous platelet agonist, the sample becomes more translucent and more light will pass through it ( Table 1 ). The original aggregometer described in 1962 by Born consisted of an absorption meter, and platelet function was performed at room temperature. In the same year, O’Brien reported on aggregation studies using a photoelectric colorimeter run at three different temperatures. This methodology has progressed to an instrument designed specifically for measuring platelet aggregation with a light source, a stirrer for mixing the blood sample and a spectrophotometer attached to a chart recorder or computer. The instrument is standardized for each subject using platelet-rich plasma as the most opaque setting possible (0% aggregation) and autologous platelet-poor plasma as the maximal transparent situation (100% aggregation). As platelets aggregate in response to the addition of an exogenous platelet agonist, the sample becomes “clearer” and an increase in light transmission through the test sample is recorded. Platelet aggregation response is calculated by dividing the distance from baseline to the maximal aggregation achieved by the distance from baseline to the theoretical 100% aggregation. The platelet aggregation pattern is classically thought of in terms of a primary response to the addition of an exogenous agonist, such as ADP, followed by a secondary response to the release of adenine nucleotides that are stored within the dense granules of platelets. These responses are often referred to as the first and second “waves” of aggregation. This biphasic response can be masked if high concentrations of agonists are added. Light transmission platelet aggregometry is regarded as the gold standard of platelet function testing, and by adding a panel of agonists at a range of concentrations to stirred platelets, it is possible to obtain a large amount of information about many different aspects of platelet function and biochemistry .
| Name of test | Principle | Advantages | Disadvantages | Clinical applications |
|---|---|---|---|---|
| Bleeding time | In vivo cessation of blood flow | In vivo test, physiological POC | Insensitive, invasive, scarring, high CV | Screening |
| Blood smear | Microscopic analysis of blood cell on glass slide | Diagnostic | Artifacts can occur | Detection of abnormalities in platelet size, number and granules and leukocyte inclusion bodies |
| Clot retraction | Measures platelet interaction with fibrin | Simple | Nonspecific | Detection of abnormalities in αIIbβ3 and fibrinogen |
| Full blood count | Automated impedance and/or flow cytometry-based analysis of cells | Rapid, precise, provides platelet distribution and MPV | Less accuracy and precision | Abnormalities in platelet number, size and distribution; immature platelet fraction now available |
| LTA | Platelet-to-platelet aggregation in response to classic agonists | Gold standard | Time-consuming, PPP and PRP preparation | Diagnosis of a wide variety of acquired and inherited platelet defects |
| WBA | Monitors changes in impedance in response to classic agonists | Whole-blood test | Older instruments require electrodes to be cleaned and recycled | Diagnosis of a wide variety of acquired and inherited platelet defects |
| Combined aggregometry and luminescence | Combined WBA or LTA and nucleotide release | Monitors release reaction with secondary aggregation | Semiquantitative | Diagnosis of a wide variety of acquired and inherited platelet defects, diagnosis of storage and release defects |
| Laser platelet aggregometer | Monitoring of aggregation using a laser | Detection of microaggregates, sensitive | Little widespread experience | Detection of platelet hyperfunction |
| VerifyNow | Fully automated platelet aggregometer to measure antiplatelet therapy | Simple, POC, test cartridges for aspirin, P2Y12 and GP IIb–IIIa | Inflexible, cartridges can only be used for a single purpose | Monitoring antiplatelet therapy |
| Ichor Plateletworks | Platelet counting preactivation and postactivation | Rapid, simple, POC, small blood volume | Indirect test measuring count after aggregation | Monitoring antiplatelet drugs, prediction of bleeding |
| Platelet adhesion assay | Adhesion and aggregation to polymer beads | Rapid | Requires counter and minishaker | Detection of platelet dysfunction |
| Gorog Thrombosis Test | High shear-dependent platelet function and thrombolysis | Simple, global test, rapid, POC | Fresh nonanticoagulated blood required | Measurement of platelet function and thrombolysis |
| Impact cone and plate(let) analyzer | Quantification of high shear platelet adhesion/aggregation onto surface | Small blood volume, rapid, simple, research and clinical versions available, POC | Instrument not yet widely available | Detection of inherited and acquired defects in primary hemostasis, detection of platelet hyperfunction, monitoring antiplatelet therapy |
| PFA-100 | High-shear platelet adhesion and aggregation during formation of a platelet plug | Whole-blood test, high shear, small blood volumes, simple, rapid, POC | Inflexible, VWF, Hct-dependent, insensitive to clopidogrel | Detection of inherited and acquired defects in primary hemostasis, monitoring aspirin, monitoring DDAVP therapy |
| O’Brien filterometer | High-shear platelet function | Simple | Nonphysiological surface, requires blood counter | Detection of defects in primary hemostasis |
| Endogenous thrombin potential assay | Global thrombin generation | Measures total thrombin-generating capacity within whole blood, PPP and PRP preparation | Requires fluorescent plate reader | Detection of clotting defects, monitoring prohemostatic therapy, measuring platelet procoagulant activity |
| Hemostasis Analysis System | Platelet contractile force, clot elastic modulus and thrombin generation time | Rapid, simple, POC | Measures mainly clot properties | Prediction of bleeding or thrombosis, monitoring rFVIIa therapy |
| Hemostatus device | Platelet procoagulant activity | Simple, POC | Insensitive to aspirin and GP Ib function | Prediction of bleeding |
| Thromboelastography (TEG or ROTEM) | Monitoring of rate and quality of clot formation | Global whole-blood test, POC | Measures clot properties only, largely platelet independent unless platelet activators are used | Prediction of surgical bleeding, aid to blood product usage, monitoring rFVIIa therapy, platelet mapping system can be used to monitor antiplatelet therapy |
| Aspirin Works | Immunoassay of urinary 11-dehydrothromboxane B 2 | Measures stable thromboxane metabolite in urine | Indirect assay, not platelet-specific, renal function and COX-1 activity dependent | Monitoring aspirin therapy and identifying poor responders at increased risk of thrombosis |
| Serum TxB 2 | Immunoassay | Measures stable thromboxane metabolite in serum | Dependent upon COX-1 activity | Monitoring aspirin therapy, detection of thromboxane production defects |
| Soluble platelet release markers (e.g., PF4, βTG, sCD40L, sCD62P, GPV | Usually by ELISA | Relatively simple | Prone to artifact during blood collection and processing | Detection of in vivo platelet activation |
| Flow cytometry | Measurement of platelet GPs and activation markers by fluorescence | Whole-blood test, small blood volumes, wide variety of tests | Specialized operator, expensive, samples prone to artifact unless carefully prepared | Diagnosis of platelet GP defects, detection of platelet activation in vivo or in response to agonists, monitoring antiplatelet therapy |
Although still considered the most useful diagnostic and research tool, LTA is relatively nonphysiological because separated platelets are usually stirred under low shear conditions during the test and only form aggregates after the addition of agonists, conditions that do not accurately mimic platelet adhesion, activation and aggregation upon vessel wall damage. Conditions such as lipemia and hemolysis, which impair light transmittance, will significantly affect LTA results. Conventional LTA using a full panel of agonists requires both high blood volumes and significant expertise to perform the tests and to interpret the tracings.
Whole blood aggregometry (WBA) is based on the principle that, as platelets adhere to electrodes, the resistance between the electrodes will increase. Whole blood aggregometry provides a means to study platelet function within anticoagulated whole blood without any sample processing . The test measures the change in resistance or impedance between two electrodes as platelets adhere and aggregate in response to classic agonists. Whole blood aggregometry has many significant advantages, including the use of smaller sample volumes and the immediate analysis of samples without manipulation, loss of time or potential loss of platelet subpopulations or platelet activation during centrifugation.
Lumiaggregometers assess the release of adenine nucleotides from platelet storage granules concomitant with the extent of aggregation response. Lumiaggregation is a quantitative bioluminescent determination and is based on the conversion of ADP released from the platelet dense granules to ATP, which then reacts with luciferin and luciferase to generate adenyl-luciferon. Light is emitted when oxidation of adenyl-luciferin occurs. The light emitted is proportional to the ATP-generated nanomoles . Studies of platelet secretion take the investigation of platelet function one step further than simple aggregation testing and allow the assessment not only of platelet aggregation but also release. This is useful in the diagnosis of bleeding disorders, such as storage pool disease and release defects, especially in cases where the patients have clinical bleeding in the absence of the abnormal aggregation tracings usually associated with these disorders . Secretion studies that provide a quantitative determination of second-wave aggregation can also be useful in the investigation of platelet activation or inhibition.
Currently, there are several commercially available instruments for measuring platelet aggregation based on the principles of light transmittance, impedance, luminescence and some in combination. Commercial aggregometers are easier to use, with multichannel capability, simple automatic setting of 100% and 0% baselines, computer operation and storage of results. PAP-8E (Biodata, Horsham, PA; Fig. 1 ) is a fully computerized eight-channel LTA. Using this instrument, one can test eight samples simultaneously. AggRAM (Helena Laboratories, Beaumont, TX; Fig. 2 ) is also a fully computerized four-channel LTA, but in conjunction with another module, it can serve as an eight-channel instrument. Payton Scientific (Buffalo, NY; Fig. 3 ) has several models of LTAs. One of their models is a two-channel instrument capable of performing LTA and lumiaggregometry. Chrono-log’s (Havertown, PA; Fig. 4 ) Model 700 is a two-channel device; however, in conjunction with other module, it can serve as a four-channel device. This instrument is capable of performing LTA, WBA and lumiaggregometry. Whole blood aggregometry can be performed using either disposable or reusable electrodes. The combined measurement of WBA or LTA with ATP luminescence helps define the secondary aggregation response or release reaction for the rapid detection of storage/release disorders and defects in thromboxane A 2 (TxA 2 ) production. Another five-channel, computerized WBA instrument (Multiple Platelet Function Analyzer or Multiplate Dynobyte Medical, Munich, Germany; Fig. 5 ) has disposable cuvettes/electrodes. All these instruments can be used for diagnosis and monitoring of antiplatelet therapy.
