Platelet Laboratory Testing: Integration of Molecular Testing and Advanced Methods.

Clinical Next Generation Sequencing to Identify Novel Platelet Disorders

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders characterized by normal or reduced platelet counts, bleeding diatheses of varying severities, and the presence (syndromic) or absence (non-syndromic) of involvement of other organs. Due to the lack of highly specific platelet function tests and overlapping clinical and laboratory features, diagnosing the underlying cause of IPDs remains challenging. In recent years, genetic testing via next-generation sequencing (NGS) technologies to rapidly analyze multiple genes has gradually emerged as an important part of the laboratory investigation of patients with IPDs. A systemic clinical and laboratory testing approach and thorough phenotype and genotype correlation studies of both patients and their family members are crucial for accurate diagnoses of IPDs.

Diagnosis of hereditary platelet disorders in the era of next‐generation sequencing: “primum non nocere”

Introduction: Inherited platelet disorders (IPDs) are rare conditions characterized by altered platelet function (IPFDs) and/or reduced platelet counts (IPNDs). Diagnosing IPDs is challenging which may results in delays, misdiagnosis and unappropriated treatment. In low and middle-income countries data on these disorders are scarce. Here, we describe a cohort of IPD patients at a reference center in Brazil. Methods: A descriptive analysis was conducted on patients with suspected or diagnosed IPDs at the Thrombosis and Hemostasis outpatient clinic of the Hospital das Clinicas, University of São Paulo, Brazil. From 857 patients identified between 1998 and 2023, 60 met the eligibility criteria for suspected or confirmed IPDs. Patients with acquired causes of platelet dysfunction were excluded. Results: The cohort comprised 60 patients, 65% with IPFDs and 35%, IPNDs. Women were 75%, with a median age of 48 years. IPDs were suspected based on clinical history, family history, and laboratory tests. In the IPND group, 62% had a family history of thrombocytopenia. In the IPFD group, family history was positive in 51% of cases. Previous misdiagnosis included ITP (immune thrombocytopenia) and von Willebrand disease. The bleeding phenotype, assessed using the ISTH BAT (Bleeding Assessment Tool) score, showed a median score of 6, with IPNDs scoring lower than IPFDs. Conclusions: Identifying IPDs is essential for proper treatment and follow-up. This study emphasizes the need for careful assessment of the familial history, bleeding risk, platelet count, morphology and function in diagnosing IPDs, particularly in low resource settings without access to advanced genetic testing.

Background: Congenital bleeding disorders (CBD) are a group of coagulopathies with different clinical and laboratory features. The prevalence of these disorders in different parts of the world is variable. Iran as a country with a high rate of parental consanguinity has a high rate of CBDs. This study was to report the prevalence of these disorders in Kurdistan province, west of Iran.\n\nMethods and materials: This descriptive study was conducted on patients suspected of a congenital bleeding disorder referred to hemophilia center of this province for evaluation of underlying bleeding diathesis. Diagnosis and classification of disorders were made by routine and specific laboratory tests.\n\nResults: Out of 107 patients, 65.4% affected by common bleeding disorders (hemophilia A and B), 23.4% affected by rare bleeding disorders (RBDs) and 11.2% had inherited platelet disorders. Factor VII deficiency (64%) was the most common RBDs and 9 patients had von Willebrand disease. Out of three patients with inherited platelet disorders, two had Glanzmann thrombasthenia.\n\nConclusion: CBD pattern though has similar patterns with total pattern of the country, some of the inherited platelet disorders are more common in Kurdish province. Determination of prevalence and distribution of these disorders can improve health system planning and resource allocation.\n\nKeywords: Congenital bleeding disorders, Rare bleeding disorders, Common bleeding disorders, inherited platelet disorders

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders associated with normal or reduced platelet counts and bleeding diatheses of varying severities. The identification of the underlying cause of IPDs is clinically challenging due to the absence of a gold-standard platelet test, and is often based on a clinical presentation and normal values in other hematology assays. As a consequence, a DNA-based approach has a potentially important role in the investigation of these patients. Next-generation sequencing (NGS) technologies are allowing the rapid analysis of genes that have been previously implicated in IPDs or that are known to have a key role in platelet regulation, as well as novel genes that have not been previously implicated in platelet dysfunction. The potential limitations of NGS arise with the interpretation of the sheer volume of genetic information obtained from whole exome sequencing (WES) or whole genome sequencing (WGS) in order to identify function-disrupting variants. Following on from bioinformatic analysis, a number of candidate genetic variants usually remain, therefore adding to the difficulty of phenotype–genotype segregation verification. Linking genetic changes to an underlying bleeding disorder is an ongoing challenge and may not always be feasible due to the multifactorial nature of IPDs. Nevertheless, NGS will play a key role in our understanding of the mechanisms of platelet function and the genetics involved.

Inherited Platelet Disorders: Diagnosis and Management.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.

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.

Inherited platelet disorders (IPDs) are rare conditions with diverse underlying pathophysiology which should be suspected in patients presenting with mucocutaneous bleeding or hemorrhages upon hemostatic challenges, in the presence or not of thrombocytopenia. Identifying IPDs is critical for providing appropriate care, preventing misdiagnosis, and avoiding unnecessary interventions, such as splenectomy. Syndromic IPDs, which may be associated with severe complications like kidney failure, infection, and malignancies, underscore the importance of accurate diagnosis and tailored management.Diagnosing IPDs remains challenging, requiring a comprehensive approach that integrates clinical assessment, evaluation of the bleeding history using standardized tools, like the ISTH-BAT, and first-line laboratory tests, such as light transmission aggregometry and flow cytometry. Second-line and specialized tests, including transmission electron microscopy, genetic analysis, and biochemical studies, may provide further insight in complex cases. Technological advancements, including multicolor flow cytometry and microfluidic tools, may in perspective improve IPD diagnostics by providing high-throughput and precise laboratory assays. In particular, mass cytometry and multi-omics may contribute to unraveling IPD pathophysiology, identifying novel markers, and refining disease classification. The application of artificial intelligence shows potential for improving diagnostic accuracy through the automated analysis of platelet morphology and function, from flow cytometry and digital microscopy assays, and for improving the understanding of pathogenic mechanisms of IPD through the examination of big data.This review summarizes current IPD platelet function testing strategies, emphasizing the need for a structured, tiered approach and examining emerging technologies and AI applications that could revolutionize diagnostic workflows, leading to personalized care and to an expanded understanding of IPDs.

Inherited bleeding disorders encompass a diverse group of conditions caused by genetic defects affecting coagulation factors, fibrinogen, von Willebrand factor, or platelet function. Despite major advances in quantitative and functional laboratory assays, a substantial diagnostic gap remains, particularly in patients with mild or atypical bleeding phenotypes. Genetic testing has become an important tool to complement traditional phenotypic testing, allowing for precise molecular characterisation and improved classification across the spectrum of bleeding disorders. This review summarises the role of genetic testing for rare coagulation factor deficiencies, von Willebrand disease (VWD), fibrinogen defects, and inherited platelet disorders (IPDs). Studies using next-generation sequencing (NGS), whole-exome sequencing, and whole-genome sequencing have identified numerous pathogenic variants, clarified inheritance patterns and helped to explain variable clinical presentations. In rare coagulation factor deficiencies, specific variants and inheritance patterns contribute to baseline factor levels. In VWD, molecular testing refines subtype classification and differentiates overlapping disorders. In fibrinogen disorders, large-scale sequencing efforts have uncovered extensive genetic heterogeneity and expanded variant databases. In IPDs, genomic studies have identified novel disease genes and improved diagnostic yield. Future work will focus on combining genetic data with functional and clinical information to improve diagnosis and guide personalised treatment. As sequencing technologies and bioinformatic tools evolve, genetic testing will play an increasingly central role in bridging the diagnostic gap and guiding precision medicine in inherited bleeding disorders. Large-scale community genetic analyses will foster data sharing and collaboration, enhance variant interpretation, and accelerate the translation of genomic discoveries into real-world benefits for the bleeding disorders community.

Background/Objectives: Inherited platelet disorders (IPDs) are diverse conditions characterized by abnormalities in platelet count and function. Next-Generation Sequencing (NGS) shows promise as a diagnostic tool in the diagnosis of IPDs. This study aims to assess the clinical value and limitations of using a targeted NGS panel in diagnosing children with suspected IPDs. Methods: We conducted a retrospective study of 93 children evaluated for suspected IPDs. A targeted NGS panel of 14 IPD-associated genes (RUNX1, WAS, ADAMTS13, ANKRD26, CYCS, GATA1, GP1BA, GB1BB, GP9, ITGA2B, ITGB3, MASTL, MPL, MYH9) was performed. Results: Genetic variants were identified in 30 patients (32.3% of the cohort). A total of 37 variants, of which 15 (40.5%) were novel, were found across 11 of the 14 genes on the panel (all except MPL, CYCS, and RUNX1). Variants were most frequently found in ITGB3 (18.9% of variants), GP1BA (16.2%), and ADAMTS13 (16.2%) genes. The majority of variants (64.9%) were classified as variants of uncertain significance (VUS), followed by likely pathogenic (LP) (27%) and pathogenic (8.1%) variants. Most variants were in a heterozygous state (73%). Specific cases highlighted complex genetic scenarios, such as co-occurring variants, and the identification of pathogenic and LP variants in patients initially presenting with immune thrombocytopenia. Conclusions: NGS helps to identify genetic causes, assess risk, manage, and provide genetic counseling in the management of IPDs. However, the prevalence of VUS underscores the need for a multidisciplinary approach to evaluate NGS results accurately.

Inherited platelet disorders are a heterogeneous group of rare conditions characterized by impaired platelet production or function, often leading to significant bleeding complications. Accurate diagnosis is essential for effective management and improved patient outcomes. However, the complexity of these disorders, coupled with the reliance on sophisticated diagnostic technologies, presents substantial challenges, particularly in settings with limited resources. This narrative review examines the advancements and challenges in the molecular diagnosis of inherited platelet disorders, highlighting the stark contrast between advanced and resource-limited healthcare environments. In high-income regions, cutting-edge molecular techniques, such as next-generation sequencing (NGS), have transformed diagnostic precision by accurately identifying genetic mutations, enabling tailored treatment strategies and better prognostic insights. Nevertheless, genetic testing is not routinely employed as a first-line diagnostic tool, even in advanced centers, because conventional phenotypic and paraclinical methods, including coagulation assays, platelet function studies, flow cytometry, immunofluorescence, and electron microscopy, remain essential for establishing initial diagnoses. These tests often suffice in characterizing severe or classical phenotypes, with molecular analysis serving as a confirmatory or complementary approach when needed. Conversely, in resource-limited settings, access to advanced genetic platforms is restricted due to cost, infrastructure, and expertise barriers, leading to delayed or incomplete diagnoses and continued reliance on less specific clinical or laboratory evaluations. Bridging this divide requires context-appropriate strategies, such as cost-effective genetic testing platforms, strengthened international collaboration, and capacity-building initiatives. Bridging the diagnostic gap in inherited platelet disorders demands a global commitment to equitable healthcare delivery. By prioritizing affordable diagnostic solutions and fostering collaborative efforts, it is possible to ensure that all patients, irrespective of geographic or economic circumstances, benefit from timely and precise diagnoses. Such efforts promise to enhance treatment efficacy, reduce morbidity, and elevate the quality of life for individuals affected by these complex disorders.

Platelets play an important role regarding coagulation by contributing to thrombus formation by platelet adhesion, aggregation, and α-/δ-granule secretion. Inherited platelet disorders (IPDs) are a very heterogeneous group of disorders that are phenotypically and biochemically diverse. Platelet dysfunction (thrombocytopathy) can be accompanied by a reduction in the number of thrombocytes (thrombocytopenia). The extent of the bleeding tendency can vary greatly. Symptoms comprise mucocutaneous bleeding (petechiae, gastrointestinal bleeding and/or menorrhagia, epistaxis) and increased hematoma tendency. Life-threatening bleeding can occur after trauma or surgery. In the last years, next-generation sequencing had a great impact on unrevealing the underlying genetic cause of individual IPDs. Because IPDs are so diverse, a comprehensive analysis of platelet function and genetic testing is indispensable.

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