Platelets and von Willebrand factor

Platelet light microscopy and platelet indices

Automated and manual platelet counting are covered in Chapter 74 . Alternatively, the proportion of platelets to red blood cells on a Giemsa-Wright stained blood film can be used along with the red blood cells counted on an automated hematology analyzer to calculate the platelet count. ^, Besides a platelet count, an automated hematology analyzer can provide other indices such as mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), platelet large cell ratio (P-LCR), and immature platelet fraction (IPF). MPV, PDW, and IPF increase with peripheral platelet destruction because young platelets released from the bone marrow are larger and can be a helpful indication of bone marrow response. The MPV is an important laboratory variable since it can help to categorize different inherited platelet disorders (IPDs). For instance, small platelets (microcytic platelets) are characteristic of Wiskott–Aldrich syndrome (WAS) ; conversely, large platelets (macrocytosis) are characteristic of Bernard-Soulier syndrome (BSS) and MYH9 mutation-associated platelet diseases. Under the light microscope, a platelet should be less than one-third the size of a red blood cell and contain purple cytoplasmic granules, which are alpha granules ( Fig. 80.1 A). An absence of these purple granules is characteristic for gray platelet syndrome (GPS) (see Fig. 80.1 B). White cells should also be examined since the presence of Döhle body-like pale blue cytoplasmic inclusions in neutrophils, in conjunction with large platelets, is characteristic for May-Hegglin anomaly or other MYH9 -mutation associated platelet disorders (see Fig. 80.1 C).

Platelet structure by platelet transmission electron microscopy

Platelet transmission electron microscopy (PTEM) is a valuable tool for the laboratory diagnosis of various hereditary platelet disorders since it was first used to visualize fibrin-platelet clot formation in the 1950s. ^, PTEM employs two main methods to visualize platelet ultrastructure, whole mount (WM) TEM and thin section (TS) TEM. ^, WMTEM is a quick and simple way to examine platelet electron opaque inclusions and dense granules (DG), also known as delta granules, by laying platelet-rich plasma (PRP) on a TEM grid. The high calcium content in DG blocks the electron beam of TEM and creates a sharp dark shadow ( Fig. 80.2 ). WMTEM is considered the gold standard test for diagnosing DG deficiencies in Hermansky-Pudlak syndrome (HPS), combined alpha-delta platelet storage pool deficiency, ^, Paris-Trousseus-Jacobsen syndrome, WAS, thrombocytopenia with absent radii syndrome (TAR), Chediak-Higashi syndrome, and other platelet DG deficiencies. TSTEM is a preferred method to visualize platelet alpha granules, other organelles, and inclusions ( Fig. 80.3 ). Distinct and sometimes pathognomonic ultrastructural abnormalities are found in GPS with virtually absent alpha granules, ^, White platelet syndrome, Medich giant platelet disorder, X-linked GATA-1 macrothrombocytopenia, ^{, ,} and the recently described York platelet syndrome. ^,

Bleeding time

To assess the platelet functions as summarized in Table 80.1 , over the past century, many iterations of the bleeding time were employed. ^, The test is performed on the volar aspect (the surface of the arm on the same side as the palm) of the forearm. A blood pressure cuff is applied at 40 mm Hg to provide uniform intravascular pressure at the site of the incision. A small incision (1 cm long and 1 mm deep) is made using a disposable template that provides a uniform incision from test to test. The result is influenced by the direction of incision, with a shorter time obtained if the incision is parallel to the sides of the forearm compared with a perpendicular incision. Blood at the site of injury is gently blotted with a filter paper every 30 seconds until no blood is detectable on the paper (time required, 4 to 8 minutes). Because the test lacks sensitivity and specificity for diagnosing bleeding diatheses and exhibits high intertechnologist imprecision, it is no longer routinely used.

Platelet function analyzer (PFA-100 and PFA-200)

The PFA-100 analyzer ( Fig. 80.4 ) is a global test of platelet and VWF function under high shear flow. ^, The analyzer utilizes whole blood specimens collected in a 3.2% buffered citrate tube (109 mmol/L trisodium citrate). When platelets pass through a small perforation in a membrane with embedded activators (collagen/epinephrine and collagen/adenosine diphosphate [ADP]), they are activated and interact with VWF. The time required for adhering and aggregating platelets to close the perforation is measured, known as the closure time. Prolonged collagen/epinephrine closure time (PFA-CEPI) and normal collagen/ADP closure time (PFA–CADP) are likely related to aspirin effect ( Table 80.2 ). Prolonged PFA–CEPI and–CADP can be seen in inherited or acquired platelet disorders and von Willebrand disease (VWD; see Table 80.2 ). PFA-100 is not affected by coagulation factor deficiencies such as hemophilia A or B. Although PFA-100 is simple to operate, it has several limitations. The closure times can be affected by anemia or thrombocytopenia, and it is insensitive to some platelet disorders such as storage-pool deficiency and the antiplatelet effect of thienopyridine drugs. To overcome the latter, a new INNOVANCE PFA-200 System (Siemens Healthcare Diagnostics, Deerfield, IL) was developed and has shown promising results, though its laboratory performance remains to be independently evaluated in both clinical and laboratory studies.

Platelet aggregation tests

The original platelet aggregation method and the first aggregometer were described by Dr. Gustav Born in 1962, which laid the foundation for understanding platelet biology and clinical laboratory testing for platelet dysfunction. After this invention, various platelet aggregation tests using an array of agonists ( Table 80.3 ) were established. Platelet aggregation can be detected by either light transmission or electrical impedance methods. Both methods have become preferred platelet function tests. Of the two methods, light transmission aggregometry (LTA) is more commonly used. It employs PRP and platelet-poor plasma (PPP) harvested from whole blood collected into 109 mmol/L trisodium citrate. Since the platelet count can affect results, PRP is then adjusted to approximately 250 × 10 ⁹ /L to assure reliable results. After adjusting the maximum light transmission by PPP, the patient’s PRP is placed in the testing chamber in the light path and warmed to 37 °C with continuous stirring and continuous measurement of light transmittance. After an agonist is added, increased transmission of light is recorded over time as the platelets aggregate (see Fig. 80.5 ). The tracing of platelet aggregation with epinephrine or ADP usually shows two waves. Right before the first wave of aggregation, there is an initial increase of turbidity immediately after the addition of an agonist. This is caused by an initial platelet shape change. During the first wave of aggregation, platelet granule and cytoplasmic contents are released. The released ADP and thromboxane A2 (TXA2) cause further activation of platelets, which triggers the second wave of aggregation. Sometimes, the first wave of aggregation is reversible when a low dose agonist (e.g., 2µM ADP) is used, and the secondary wave does not occur. Other agonists, like collagen and arachidonic acid, do not demonstrate separate primary and secondary waves because aggregation and secretion are coinciding. The maximum percentage of aggregation is frequently used as the final result to assess platelet function. An alternative method was developed using anticoagulated whole blood. In this case, an electric probe is placed in the specimen, and an alternating current is passed through the blood. Upon activation, platelets are attracted to the electrodes. As more platelets attach to the electrodes, the flow of current is impeded and plotted over time. Although LTA remains the “gold standard” for platelet aggregation, the impedance method has several advantages, such as no preanalytical sample preparation and a smaller sample size requirement. The patterns of responses to different agonists can be used to diagnose platelet disorders ( Table 80.4 ).

Platelet secretion studies

Upon initial activation, platelets secrete a wide variety of mediators into the microenvironment. These mediators are stored in the platelet dense and alpha granules, as summarized in Table 80.5 . DG secretion was initially evaluated by exploiting the ability of platelets to take up radioactively labeled serotonin and then release of the label following platelet activation. This serotonin release assay (SRA) is very labor-intensive and requires the use of radioisotopes. An alternative method is adenosine triphosphate (ATP) release using a lumi-aggregometer, in which luciferin and luciferase are added to the patient specimen before an agonist is added. Conversion of luciferin to its product by luciferase is dependent on ATP, the only source of which is released from the platelet DG. In the conversion of luciferin to its product, photons are emitted and are detected by a fluorometer in a quantity that correlates with the ATP released. This method allows for the simultaneous assessment of aggregation and secretion in the same reaction by using either LTA or impedance aggregometry.

Platelet support of plasma coagulation

Platelets contribute to the coagulation cascade by providing phospholipids, such as phosphatidylserine, which is required by the coagulation factors’ activation. They also secret coagulation factors V, XIII, VIII, and VWF from alpha granules to facilitate local clot formation. Platelet support of plasma coagulation can be evaluated by measuring the amount of residual prothrombin in a serum specimen after coagulation in the test tube is complete. However, this test is not routinely performed in clinical laboratories.

Platelet contractile function

Platelet contractile function can be evaluated by measuring clot retraction. Upon activation, platelets form filopodia, slender projections from the surface of the platelets that attach to fibrin(ogen) strands via their surface receptors glycoprotein (GP) IIb/IIIa. Platelet actin tethers to membrane receptors and organizes with cytoplasmic myosin within these filipodia, forming a rudimentary muscle that contracts at the conclusion of the hemostatic process. This has been described as the in vivo suture, drawing the edges of the injury closer together as healing is initiated. This clot retraction is dependent on the contractile proteins in platelets and can be qualitatively measured in a test tube. Clot retraction is abnormal in Glazmann thrombasthenia and some acquired disorders, most notably with monoclonal gammopathy; nevertheless, platelet contractile function is not routinely tested in clinical laboratories.

Platelet receptors and function using flow cytometry

Flow cytometry is a useful adjunct in evaluating platelet function in selected situations. Flow cytometry can be used to evaluate the platelet surface antigen expression and activation state. Platelet surface antigens include the VWF binding receptor glycoprotein (GP) complex Ibα/β-IX-V (CD42a–d), fibrinogen receptor GPIIb/IIIa (CD41/CD61), and collagen binding receptors GPIa (CD49b) and GPVI. GP surface expressions can be measured with the use of fluorescent-conjugated GP-specific antibodies. The fluorescent intensities correlate with GP expressions. Flow cytometry can be used to measure platelet surface glycoprotein deficiencies in BSS, Glanzmann thrombasthenia (GT), and collagen receptor deficiencies ( Table 80.6 ).

Flow cytometry can be used to evaluate platelet function by measuring activation markers upon platelet activation. The most commonly used platelet activation markers are P-selectin (CD62P), activated GPIIb/IIIa complex, and lysosomal-associated membrane protein (CD63). CD62P is located on the alpha granule membrane, while CD63 is on the membrane of DG and lysosomes. Both CD62P and CD63 translocate to the surface of plasma membrane after platelet activation with ensued granule release. Decreased expression of CD62P and CD63 upon platelet activation can be seen in AG or DG deficiencies, respectively. PAC-1 is a mouse monoclonal antibody that detects the neoepitope of GPIIb/IIIa conformation changes upon platelet activation. PAC-1 binding on the platelet surface is indicative of platelet activation. A lack of PAC-1 binding by activated platelets suggests GT or other platelet disorders. Currently, the flow cytometric method of platelet function testing remains largely a research tool and has not been widely used in clinical laboratories.

Molecular genetic testing

Genotyping has a confirmatory role in the diagnosis of IPD. Molecular genetic analysis by Sanger sequencing was first employed in the early 1990s to identify the causal mutations in the genes coding for GPIIb/IIIa and GPIbα/β−IX-V in GT and BSS, respectively. With the advent of next-generation sequencing (NGS), several novel platelet disorder-associated gene mutations have been identified, for example, NBEAL2 , GFI1B and VPS33b for GPS, ACTN1 for autosomal dominant thrombocytopenia, RBM8A for thrombocytopenia absent radii, STIM1 for York platelet syndrome, and ANKRD26, ETV6, GATA-1, GATA-2 and RUNX1 for thrombocytopenia with a predisposition to hematologic malignancy. For further discussion on these molecular techniques, refer to Chapter 64 . Although NGS-based approaches may potentially revolutionize the diagnosis of IPD, preliminary clinical and laboratory phenotypic characterization remain crucial and indispensable. Due to the growing list of heritable thrombocytopenias and their disease associations, a new, evolving classification has been proposed. ^,

Evaluation of antiplatelet medication

Antiplatelet medications are commonly used in patients with cardiovascular conditions (see Table 80.7 ). Evaluation of the effects of antiplatelet medication has two main clinical indications. First, it is crucial to know whether the medication is effectively inhibiting the platelet functions as intended for the patient to receive the desired reduced risk of thrombosis. Second, many patients who are taking antiplatelet medications need emergency or elective procedures and may be at increased risk for hemorrhage during those procedures. Increased risk of hemorrhage in patients taking antiplatelet medications has been demonstrated but is widely variable among patients. Unfortunately, whether laboratory tests of the medication effects on platelet function indeed can accurately predict hemorrhagic or thrombotic outcome is still controversial. Several techniques are used to measure the effects that medications have on platelet function. They include the PFA-100 and -200 analyzers, Verify-Now, PlateletWorks, Multiplate, Thromboelastography (TEG), Platelet Mapping, and platelet aggregation assays. Since some of these tests are still being clinically validated and are beyond the general scope of this chapter, only selected methods are discussed.