Platelet Function Evaluation
The ability to assess the efficacy of an individual's platelet function is valuable for patient management to evaluate appropriate antiplatelet effects and also when emergency reversal is required for a hemorrhagic complication or need for emergency open surgery. Additionally, as described above, many individuals have either inherent resistance to some agents or are poor responders, meaning they are unprotected during procedures for which antiplatelets are recommended (eg, stenting). Below we discuss the range of assays available including platelet counting, platelet aggregometry, bleeding time, platelet function assays, and point-of-care assays.
Platelet Counting
This is the first-line test for platelet function which continues to be used by surgeons and interventionalists alike, with a goal count of ≥80–100 000/μL being the recommended cut-off to proceed with an intervention. The gold standard is a manual count via phase contrast microscopy, but the majority of centers employ automated cell counter methods, optical counting methods, or flow cytometry. However, the absolute platelet count does indicate functionality of the platelets. Platelet function can only be measured by formal functional assays.
Platelet Function by Platelet Count Ratio Method
An indirect measurement of platelet function can be employed with available automated cell counters by stimulating platelets within anticoagulated whole blood samples with agonists such as ADP or epinephrine, inducing aggregation. A ratio between the platelet count in the control sample and the activated sample is then calculated, which correlates well with platelet aggregometry. This technique has been commercialized as the ICHOR Point of Care Hematology Counter for the acute setting.
Platelet Aggregometry
Optical Aggregometry. LTA was developed in the 1960s and has become the gold standard to test platelet function. In this assay, blood is centrifuged slowly to obtain platelet-rich plasma that is continuously stirred while a light source is shone through the sample. Platelet agonists such as ADP are then added to induce aggregation which results in increased light transmission, given a decrease in the turbidity of the platelet-rich plasma. A series of platelet agonists are employed through a range of concentrations to assess shape changes and aggregation responses. The test is labor-intensive and requires technical expertise, and may not be easily obtainable at all centers. Additionally, this test underestimates the degree of GP IIb/IIIa inhibitors, is not sensitive enough to test aggregates of <100 platelets (micro-aggregates), and is not reliable at detecting pre-existing aggregates, which is important in patients with hyperfunctioning of platelets. Resistance to various antiplatelet agents is monitored by selection of the appropriate platelet aggregation agonist. For example, aspirin therapy is monitored with ADP, arachidonic acid, or collagen, clopidogrel is monitored by platelet responses to ADP, and IIb/IIIa inhibitors are measured by the platelet response to ADP and thrombin receptor agonists.
Whole Blood Aggregometry, Flow Cytometry, and Laser Platelet Aggregometry. Because of the limitations of classic aggregometry, whole blood aggregometry was developed. In this assay, whole blood is stirred between two platinum electrodes which then become platelet-covered with aggregates after the addition of agonists, thereby changing the impedance in the circuit. This method is superior to measuring antiplatelet therapy with classic aggregometry, but is still insensitive to micro-aggregates. Nevertheless, the method is in widespread use in Europe. In order to assess small micro-aggregates, flow cytometry can be employed with high accuracy, but the equipment is expensive and requires a specialized operator. Employing a laser to detect platelet aggregates as small as a few platelets via light scattering has gained attention because of its accuracy, but its adoption has been limited.
Bleeding Time
This was the first test for platelet function and studies natural hemostasis using a simple technique used at the bedside. It requires a skin incision, often on the forearm, to a depth to disrupt capillaries. It has been used to predict surgical bleeding as it is a good test of platelet function. It does not require any special equipment but it has been criticized as invasive, insensitive, irreproducible, and with a subjective endpoint.
Platelet Function Assays
In an effort to mimic in vivo physiology including platelet adhesion, activation, and aggregation, several platelet function assays have been developed. Many of them seek to mimic vessel wall damage and thus base their assays on shear-induced platelet activation. For example, the clot signature analyzer measures the ability of flowing non-anticoagulated blood to form hemostatic plugs via fibrin formation in a tube of punched holes. The thrombotic status analyzer test employs hemodynamic forces on blood drawn through a capillary tube to induce platelet activation and capillary tube occlusion. The more well-known platelet function analyzer (PFA) exposes platelets in whole blood to high shear stress within a capillary tube coated with collagen and ADP/epinephrine and monitors the drop in flow rate as the platelets form a hemostatic plug. The test is used as a screening tool to assess platelet abnormalities and is simple and rapid in execution, but is limited by the absolute platelet count, blood count, and von Willebrand factor levels. Thromboelastography (TEG) is another method of testing the efficiency of blood coagulation. It is mainly used in surgery and anesthesiology and has been established as a sensitive test for hemostatic function assessment in several clinical settings. Although few centers are capable of performing it, TEG can assess platelet function, clot strength, and fibrinolysis by triggering clot formation followed by computerized coagulation analysis. The TEG trace is analyzed for the reaction time, which represents the rate of initial fibrin formation, and for the maximal amplitude, which correlates with the absolute strength of the clot. The use of TEG in NI procedures is underutilized and rarely described in the literature.
Point-of-care Platelet Function Assays (ICHOR and VerifyNow)
The ICHOR Point of Care Hematology Counter employs a ratio to measure platelet functionality pre- and post-activation. The VerifyNow Assay, previously known as the Ultegra Rapid Platelet Function Assay, was developed based on the premise that, in order to achieve significant clinical efficacy with antiplatelet agents, ≥80% of platelet receptors need to be blocked. Thus, monitoring of receptor blockade is crucial to ensure optimal dosing for patient outcomes. This system allows for rapid measurements of aspirin, clopidogrel, and IIb/IIIa inhibitor function at the bedside using a whole blood sample. The assay is based upon fibrinogen-coated bead agglutination in response to the proportion of available GP IIb/IIIa receptors. For aspirin, the activator is arachidonic acid, for P2Y12 inhibitors it is ADP, and for IIb/IIIa inhibitors it is thrombin receptor activating peptides, all with results that are available in minutes. In 2014 the US Food and Drug Administration (FDA) recalled the VerifyNow IIb/IIIa test due to concerns in the reporting of erroneous platelet aggregation unit results.