Confirmatory Testing for Diagnosis of Platelet Disorders

Laboratory investigation of a suspected inherited platelet disorder (IPD) requires both standard and confirmatory platelet laboratory testing. The former includes platelet count, mean platelet volume and other indices by a hematology analyzer, peripheral blood smear review by light microscopy, and platelet functional analysis. The latter encompasses platelet flow cytometry, platelet transmission electron microscopy (PTEM), and molecular genetic testing. Although the standard laboratory testing is an important first step to investigate a suspected IPD, the confirmatory testing is usually required to render a definitive diagnosis. This chapter will focus on these three confirmatory tests.

Platelet Transmission Electron Microscopy

Platelets are the smallest enucleated cellular fragments in peripheral blood with a mean volume ranging from 7.2 to 11.5 fL. Their intracellular organelles are mostly invisible by light microscopy. The transmission electron microscopy (TEM) can yield magnifications up to 500,000× with a resolution of 0.1 nm, which is sufficient to visualize platelet subcellular structures. PTEM was first used to visualize fibrin-platelet clot formation in 1955, and it was later used to evaluate platelet ultrastructural abnormalities. PTEM generally employs two methods: thin section (TS) ( Fig. 142.1A and B ) and whole mount (WM) TEM ( Fig. 142.1C and D ). The former method can also be used to examine white blood cells.

Figure 142.1, Examples of platelet and white blood cell electron microscopy.

The TS-PTEM involves platelet fixation, embedding, and sectioning. Platelets from platelet-rich plasma (PRP) or white blood cells from buffy coat are pelleted by centrifugation and then fixed with glutaraldehyde. The fixed pellets are stained in 1% osmic acid and embedded in plastic blocks. TSs can be cut via an ultramicrotome and stained with uranyl acetate and lead citrate to enhance contrast. TS-PTEM visualizes ultrastructures and abnormal inclusions in both platelets and white blood cells. Platelets are composed of the plasma membrane, cytoplasm, cytoplasmic organelles, and cytoskeleton. The plasma membrane has complex invagination throughout the entire platelet, to form a network called surface-connected canalicular system (CS). Platelets contain two main types of cytoplasmic granules: α-granules (AG) and dense granules (or δ-granules, DG). AG is the primary storage site of platelet secretory proteins (e.g., coagulation factor V, proteoglycans, platelet factor 4, and von Willebrand factor). Under TS-PTEM, AG usually present as uniform-sized round granules with mildly electron-dense content and a single layer membrane. On platelet activation, AG fuse with their nearby CS and release their content into the canalicular lumen, whereas the DG migrate to and fuse directly with the plasma membrane. Platelet cytoskeleton is essential for maintaining the discoid shape of platelets ( Fig. 142.1A ) by forming a circumferential microtubule scaffold underneath the plasma membrane ( Fig. 142.1B ). The platelet submembrane cytoskeleton is composed of actin, which also anchors platelet transmembrane glycoproteins (GP). Actin molecules can polymerize and form actin filaments, and regulate platelet shape change and pseudopodia formation on activation. Nevertheless, actin filaments are invisible by standard TS-PTEM.

The WM-PTEM examines platelet electron-dense structures such as DG and abnormal opaque inclusions in rare IPD such as York platelet syndrome. WM-PTEM preparation is made by applying a drop of PRP directly on a carbon-stabilized formvar grid. The grid is air-dried and then directly examined under an electron microscope. Platelet cytoplasm is transparent to the electron beam, whereas DG and abnormal electron-dense objects are inherently opaque ( Fig. 142.1C ). Normal platelets contain about 1–8 dense bodies per platelet. WM-PTEM is considered the gold standard method for diagnosing platelet DG deficiencies, e.g., Hermansky–Pudlak syndrome (HPS), Paris-Trousseau–Jacobsen syndrome (PTJS), Wiskott–Aldrich syndrome (WAS), thrombocytopenia with absent radii (TAR) syndrome, Chediak–Higashi syndrome (CHS), and combined alpha–delta platelet storage pool deficiency.

Platelet Ultrastructural Defects

Shape and Size Abnormalities

The average size of platelets is about 2–4 μm in diameter. Resting platelets are discoid in shape with a smooth cell border ( Fig. 142.1A and B ). Activated platelets, such as suboptimal sample collection, transportation, and storage, show irregular shapes and pseudopodia formation ( Fig. 142.1E ). Giant platelets of Bernard–Soulier syndrome (BSS) and MYH9 mutation–related disorders (MYH9-RD) are often spheroid in shape. However, we need to be aware that round and enlarged platelets can be a laboratory artifact when ethylenediaminetetraacetic acid (EDTA) is used as the anticoagulant for whole-blood collection and storage. Conversely, platelets of WAS are microcytic with an average diameter less than 1.8 μm.

α-Granule Defects

Gray platelet syndrome (GPS) is a rare IPD caused by AG deficiency. The platelets are gray appearing by the Giemsa–Wright stain on a peripheral blood smear. GPS is a genetically heterogeneous entity with its pathogenicity attributable to mutations in at least three genes, NBEAL2 , GFI1b , and VPS33b . Clinically, patients with GPS show heterogeneity in bleeding symptoms and platelet function assessed by platelet aggregation analysis. Under TS-PTEM, platelets contain virtually no AG in NBEAL2 mutation–associated GPS ( Fig. 142.1G ), whereas partial AG deficiency and occasional giant AG are observed in GFI1B or VSP33b mutation–associated GPS ( Fig. 142.1H ). Recent studies showed that GFI1B -GPS also has a concurrent DG deficiency.

Abnormal α-Granule Ultrastructure

Giant irregular-shaped alpha granules ( Fig. 142.1F ) and combined AG and DG deficiencies are the hallmarks of PTJS, which is caused by chromosome 11q23 deletion where FLI-1 gene is located. Other clinical features include congenital heart defects, trigonocephaly, mental retardation, and multiple organ malfunctions. Recent studies demonstrated that similar PTEM ultrastructural abnormalities are also present in FLI-1 mutation–associated IPD and other rare IPDs. It should be noted that occasional large and fuse AG may be due to prolonged and suboptimal whole-blood storage.

Dense Granule Deficiency

Platelet dense granule defects are a heterogeneous group of diseases. Some diseases such as HPS, CHS, and PTJS have a virtually complete absence of DG. Other disorders may have mild to moderate deficiency. Platelet light transmission aggregation and PFA-100 tests have about 50% sensitivity of detecting a DG deficiency. ATP or serotonin release test may serve as a screening testing for DG deficiencies. However, because of the significant variability of ATP release in the healthy population, the ATP release test still has insufficient sensitivity and specificity for diagnosing DG deficiencies. Because of these limitations of the standard platelet functional testing, WM-PTEM remains the preferred method for assessing platelet DG deficiency.

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