Platelet function testing refers to in vitro measurements of platelets’ response to activation in an attempt to quantify the degree of platelet aggregation. It has evolved significantly over the last decade from a laborious laboratory based process to rapid point of care commercially available kits. Despite this, there still remains a surprising lack of standardisation of platelet function testing. Table 11.1 summarises some of the currently available methods, along with their incumbent advantages and limitations.
Light transmission aggregometry
Light transmittance aggregometry (LTA) is currently the gold standard for assessing platelet activation as it is able to measure the functional ability of platelets to aggregate in response to known agonists such as ADP. Platelet aggregation uses the principle that the amount of light transmitted through the sample increases proportional to an increase in platelet aggregation. Lack of standardization, poor reproducibility and spontaneous platelet activation through sample preparation are a few limitations of standard light transmittance aggregometry.
Whole blood aggregometry
Impendence aggregometry, a newer technique that measures electrical resistance due to aggregation of stimulated platelets on two platinum electrodes, has been shown to have improved reproducibility and sensitivity. The increase in resistance between electrodes is used to determine the amount of platelet aggregation after stimulation by an agonist. Impedance aggregometry correlates well with LTA.
Verify Now is a cartridge based rapid point of care test that measures aggregation of platelets via GIIb/IIIa receptors in response to ADP. The extent of platelet aggregation measured by a technique based on light transmittance aggregometry, is expressed as platelet reactivity units (PRU), which is specific to the action of the P2Y12 platelet receptor. Simplicity of technique, reliability of results, lack of reliance on lab equipment and good correlation with established tests of platelet function are advantages of this test. All point of care tests are limited by low to moderate sensitivity and specificity. However, VerifyNow? P2Y12 assay has been shown to predict clinical adverse outcomes.
Flow cytometry enables identification of expression of substances on platelets and leukocytes, particularly formation of platelet-leukocyte aggregates. Fluorophores (substances that fluoresce when stimulated with energy) attached to antibodies which bind to substances of interest; emit light when excited by energy from lasers. These lasers transmit light at a specific frequency aimed at particles flowing past the laser in single file. The intensity of light emitted by the fluorophores allows quantification of the substance targeted by conjugated antibodies. However, accuracy and reproducibility of results vary from one protocol to the next. The ability to assess multiple markers relating to antiplatelet activity at the receptor is the main advantage of flow cytometry. The main disadvantages of this technique is the need for experienced operators, time consuming process for analysis with narrow sample preparation windows, lack of standardized lab protocols and access to lab facilities.