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Introduction Light transmission aggregometry (LTA) is regarded as the gold standard in platelet function diagnostics. However, there is a relevant degree of interlaboratory variability in practical applications. Objective The aim of the present study was to develop a practicable laboratory comparison on LTA and to analyze differences and influencing factors in regard to standardization in five specialized hemostaseological centers. Methods The study was performed on 30 patients in total. Each center performed LTA on blood samples from six healthy volunteers (three men and three women) using the inductors collagen (Col), adenosine diphosphate (ADP), arachidonic acid (ARA), and ristocetin. The LTA was performed three times using different methods as follows: (1) International Society on Thrombosis and Haemostasis recommendations with identical reagents, (2) in-house protocols and the identical reagents; and (3) in-house protocols and in-house reagents. Results A total of 396 measurements of 30 probands were performed. Even after standardization of the protocol and using identical reagents, there were significant differences between the centers regarding the final and maximum aggregation (p = 0.002 and

Next-generation sequencing is increasingly applied during the diagnostic work-up of patients with bleeding diathesis and has facilitated the diagnosis of rare bleeding disorders such as inherited platelet function disorders. Mutations in RAS guanyl releasing protein 2 (RasGRP2), also known as calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), underlie a recently described platelet signal transduction abnormality. Here we present the case of a consanguineous family originating from Afghanistan with two siblings affected by recurrent severe mucocutaneous bleedings. Platelet function testing demonstrated a marked reduction of aggregation induced by collagen and adenosine diphosphate. Whole exome sequencing revealed a novel homozygous nonsense RASGRP2 mutation segregating with the bleeding disorder in the family.

Inherited platelet disorders (IPD) form a rare and heterogeneous disease entity that is present in about 8% of patients with non-acquired bleeding diathesis. Identification of the defective cellular pathway is an important criterion for stratifying the patient's individual risk profile and for choosing personalized therapeutic options. While costs of high-throughput sequencing technologies have rapidly declined over the last decade, molecular genetic diagnosis of bleeding and platelet disorders is getting more and more suitable within the diagnostic algorithms. In this study, we developed, verified, and evaluated a targeted, panel-based next-generation sequencing approach comprising 59 genes associated with IPD for a cohort of 38 patients with a history of recurrent bleeding episodes and functionally suspected, but so far genetically undefined IPD. DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS, RBM8A, FERMT3, P2YR12, and MYH9 served as controls during the validation process. In 40% of 35 patients analyzed, we were able to finally detect 15 variants, eight of which were novel, in 11 genes, ACTN1, AP3B1, GFI1B, HPS1, HPS4, HPS6, MPL, MYH9, TBXA2R, TPM4, and TUBB1, and classified them according to current guidelines. Apart from seven variants of uncertain significance in 11% of patients, nine variants were classified as likely pathogenic or pathogenic providing a molecular diagnosis for 26% of patients. This report also emphasizes on potentials and pitfalls of this tool and prospectively proposes its rational implementation within the diagnostic algorithms of IPD.