Genes 2025, 16(6), 621; https://doi.org/10.3390/genes16060621 (registering DOI) - 23 May 2025
Abstract
Inherited platelet disorders (IPDs) are rare bleeding disorders characterized by impaired platelet function and/or reduced blood platelet count. Their diagnosis typically relies on complex laboratory methods, including flow cytometry, aggregometry, and molecular genetic analysis. In recent years, immunofluorescence microscopy has been established as
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Inherited platelet disorders (IPDs) are rare bleeding disorders characterized by impaired platelet function and/or reduced blood platelet count. Their diagnosis typically relies on complex laboratory methods, including flow cytometry, aggregometry, and molecular genetic analysis. In recent years, immunofluorescence microscopy has been established as an alternative diagnostic method for IPDs. Background/Objectives: This study aims to validate a quantitative approach enhancing reproducibility through automated image analysis for diagnosing IPDs using immunofluorescence microscopy, with Bernard–Soulier Syndrome (BSS) and Glanzmann thrombasthenia (GT) as model IPDs. Methods: Native blood smears from patients with suspected BSS or GT were stained using a standardized immunofluorescence protocol targeting platelet surface glycoproteins, granules, and cytoskeletal components. The slides were analyzed using an automated fluorescence microscope, and a rule-based subpopulation analysis was implemented to quantify fluorescence signals. The results were compared to those of a healthy control group, as well as data from flow cytometry and molecular genetic testing. Results: The automated analysis successfully differentiated BSS and GT patients from healthy controls based on distinct fluorescence signal patterns. In BSS samples, CD42b (GPIbα) expression was absent or severely reduced, while GT samples showed a deficiency of CD41/CD61 (GPIIb/IIIa). The platelet size distribution confirmed macrothrombocytopenia in BSS patients. Flow cytometry and molecular genetic testing corroborated these findings, supporting the diagnostic reliability of the automated immunofluorescence microscopy approach. Conclusions: This proof-of-principle study demonstrates that automated quantitative immunofluorescence microscopy is a viable alternative for diagnosing IPDs, offering a standardized, objective, and efficient method, particularly in settings where flow cytometry is not feasible.
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(This article belongs to the Section Human Genomics and Genetic Diseases)
