**2.1. Patients**

α2β1 with collagen [1]. Extension of the platelet plug requires activation of αIIbβ3 through an "inside-out" signaling cascade which enables receptor cross-linking with fibrinogen and vWF

Primary hemostasis relies on both adequate function and number of platelets. Abnormalities in platelet function and/ or number may be acquired (liver disease, chronic kidney disease) or inherited (inherited platelet function disorders, IPFDs or inherited platelet number disorders, IPNDs). The group of inherited macrothrombocytopenias is included in the heterogeneous IPNDs and are characterized by large platelets, thrombocytopenia and bleeding tendencies in

**Figure 1.** A normal blood film and three blood films demonstrating macrothrombocytopenia associated with muta‐ tions in different genes (*MYH9, NBEAL2* and *GFI1B*, respectively). (A) A blood film with platelets of normal appear‐ ance (black arrows). (B) *MYH9*-related disorder with characteristic inclusion bodies in the neutrophils (small black arrow) and large platelets (red arrow). Normal-sized platelets are also seen (long black arrow). (C) Gray platelet syn‐ drome showing distinctive pale or gray platelets (black arrows). (D) *GFI1B*-related thrombocytopenia (c.880-881insC mutation) resulting in red cells with atypical shapes and sizes (red arrow) and thrombocytopenia with platelets that appear large with normal granulation (long black arrow) as well as hypogranular or gray (short black arrows).

Unfortunately, inherited macrothrombocytopenia is under-recognized with the presence of large platelets on blood film examination often leading to a misdiagnosis of immune throm‐ bocytopenic purpura (ITP), resulting in subsequent inappropriate treatment with steroids or in some cases removal of the spleen [4]. Diagnostic algorithms have traditionally been based around biological laboratory tests examining functional properties and activation pathways

affected individuals (Figure 1A, Figure 1B, Figure 1C and Figure 1D) [3].

and activation of "outside-in" signaling events [1, 2].

388 Next Generation Sequencing - Advances, Applications and Challenges

Diagnostic assessment of patients with uncharacterized thrombocytopenia was performed as part of a human research ethics committee approved study conducted in accordance with the Declaration of Helsinki.

Following informed written consent, 20 ml of blood was taken from an antecubital vein and collected into EDTA tubes. This blood was easily transported, in some cases, over 1,000 km between diagnostic sites in Australia.

A total of 95 patient DNA samples were analyzed. This included two internal controls for which DNA-based diagnosis had previously been established by Sanger sequencing.

32 male patients (mean age 37.4 years, range 18–92 years) and 44 female patients (mean age 38.7 years, range 18–79 years) were included in the NGS assay. The mean age of the cohort was 38.1 years (range 18–92 years). Sixteen de-identified DNA samples were received from referring institutions for which no additional laboratory data were available.

Phenotypic testing data were available for 59 (62.1%) individuals. This included platelet functional analysis (PFA) (*n* = 25, 26.0% of the cohort), light transmission aggregometry / whole blood impedance aggregometry (LTA/WBIA) (*n* = 39, 41.3% of the cohort), flow cytometry (*n* = 45, 47.8% of the cohort) and electron microscopy (*n* = 12, 13% of the cohort). These phenotypic test results suggested a diagnosis to a "pathway level", that is, a description to the level of the suspected defective biochemical pathway, in only 11 cases. Pathway orientated defects included, storage pool disorders (*n* = 3), platelet glycoprotein deficiency (*n* = 3), platelet signaling defects (*n* = 2), platelet secretion defects (*n* = 2) as well as α-granule disorder (*n* = 1).
