Table 5.

Stratification of tests in the diagnosis of inherited platelet disorders.

Value of Commonly Used Assays in the Diagnosis of IPD (0: Low; 1: Reasonable; 2: High)#	Basic Panel	Extended Panel (Confirmation/Characterization)
Complete blood counts and blood smear: 2 Bleeding time: 0 PFA-100: 1 Thromboelastography: 0 Platelet aggregation: 2 Lumiaggregometry: 2 Flow cytometry: 2 Electron microscopy: 2 Molecular analysis: 2	Complete blood counts (automatic) Blood smear (platelet count and morphology, inclusion bodies in neutrophils, abnormalities in other blood cells) Basic coagulation and VWF study Platelet aggregation: basic panel of agonists: PAR-1 peptide (TRAP), ADP, arachidonic acid, epinephrine, collagen, ristocetin. Flow cytometric analysis of major platelet adhesive glycoproteins (GP Ib/IX; GP IIb/IIIa; GP Ia/IIa, GPVI)	Immunostaining of platelet proteins in blood smears. Platelet secretion: ATP release by lumiaggregometry; HPLC; uptake/release of serotonin (HPLC/ radiolabelled); release of CD62/CD63 by flow cytometry; release of granular proteins (PF4. Thrombospondin-1, VWF, P-selectin,) by ELISA. Agonist-induced activation of αIIbβ3 by flow cytometry (Binding of PAC-1 or fibrinogen labeled with fluorochromes). Clot retraction assay. Platelet aggregation: basic panel of agonists: PAR-4, collagen related peptide [CRP], convulxin, PMA, U46619, A23188; aggregation inhibition test with PGE1 or PGI2. Extended panel of platelet receptor expression (ADP receptors, TxA2, thrombin; CD31) by flow cytometry with selective antibody or ligand binding assays. Procoagulant activity by cytometry (annexin V binding). Measurement of second messengers (Ca2 +, IP3, cAMP, TxA2) by cytometry or biochemical assays. Assessment of signaling cascades by measuring phosphorylation of specific proteins by western blot or flow cytometry (VASP test). Platelet adhesion and spreading on adhesive surfaces (static and/or low flow). Platelet morphology/ultrastructure (electron microscopy, immunofluorescence microscopy, confocal). Molecular analysis (first-line HTS with a panel of selected genes; negative cases in HTS-gene panel can be analyzed by complete whole exome sequencing [WES) and/or complete genome sequencing [WGS].

vWF: von Willebrand factor; HTS: high throughput sequencing; ADP: adenosine diphosphate; PF4: platelet factor 4; For more details see previous publications [2,130,131]; #A score of 0 means that the test add little for the diagnosis of IPD, while test scored 2 are considered as diagnostic of IPD by them self. Evaluation of blood smears with classical MGG staining is almost mandatory in the diagnosis process of IPD, as it would provide essential information in the number and morphology of platelets. Blood smear can also inform of other blood cell abnormalities that are typical of some IPDs, such as inclusion bodies in neuthrophils present in some patients with MYH9-RD. Recently, immunofluoresecence staining of platelet proteins has been shown as a helpful assay in the diagnosis of IPD [132,133]. Bleeding time is an old test measuring the time to cessation of bleeding after a standardized incision in the forearm of the subject [134]. This test was shonw to have great variability among laboratories and low sensitivity (30%) among patients with mucocutaneous bleeding history [135], and it is longer recommended by experts for the diagnosis of IPD [130]. The Platelet Function Analyzer (PFA-100^©, Siemens) is a point-of care that automatically measures the time until occlusion of blood flow through a small orifice (150 µm diameter) in a membrane located inside a disposable cartridge, as a result of platelet adhesion and aggregation induced simultaneously by a high flow rate (4000-6000 s-1) and by a mixture of collagen with ADP or with epinephrine in the membrane The tests is highly sensitive for screening of severe IPD or VWD, but PFA-100 results can be normal in moderate disorders. Therefore, PFA-100 testing should be considered optional in the evaluation of platelet disorders [136,137]. Light transmission aggregometry, originally developed by Born, is still the gold-standard in platelet function testing and in diagnosis of IPD [138,139]. This assay, essentially measures the change in light transmission across a sample of platelet-rich-sample as platelets aggregate in response to an agonist added to the sample. Specific platelet aggregation profiles are diagnostic of certain severe IPD such as BBS or GT (see main text above). Much international effort has been done to standardize this milestone test [140]. Platelet aggregation can be combined with measurement of ATP release in lumiagregometry assays [137]. Agregation can also be mesured in whole blood by point-of-care such as Multiplate or by means of adapted flow cytometry o microfluidic assays [137]. Flow cytometry has became a routine procedure in the diagnosis of many hematological diseases including IPD. By means of fluorochrome-labelled specific antibodies or ligands specific for platelet proteins (surface receptors, granule markers, signaling proteins, etc.), flow cytometers can discriminate platelet populations with different expression of these proteins [141]. Electron microscopy allow detailed examination of platelet ultrastructure and can, therefore be diagnostic of some IPD such a GPS, a severe congenital defect of α-granules [142]. A less technical demanding procedure is whole mount electron microscopy, which allows quantification of dense granules in a non-manipulated drop of platelet rich plasma. Whole mount can be diagnostic of severe α-granules defect such as HPS [99]. Lastly, genetic diagnosis, i.e., identification of the underlying molecular defect, provides the definitive confirmation of an IPD. Until two decades ago, Sanger sequencing of candidate genes was the almost unique molecular tool, but currently HTS of panels of predefined genes, or in some cases even the whole exome o genome, have been introduced in first line genetic diagnosis of IPD [2,125,126,143].