Automated Quantitative Immunofluorescence Microscopy for Standardized Diagnosis of Inherited Platelet Function Disorders

Introduction Inherited platelet disorders (IPDs) comprise a heterogeneous group of rare bleeding disorders caused by defects in platelet function and/or number. Their diagnosis requires a combination of specialized assays, such as light transmission aggregometry, flow cytometry, and molecular genetic testing. However, these approaches are often labor-intensive, require fresh samples, and are not standardized across laboratories. Immunofluorescence microscopy has recently emerged as an alternative method for identifying characteristic protein expression defects in IPDs. This study aimed to establish and validate an automated quantitative immunofluorescence microscopy (IFM) approach for standardized and reproducible diagnosis of hereditary thrombopathies, using Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) as proof-of-concept models. Method Peripheral blood smears from patients with suspected BSS or GT and from healthy controls were prepared using a standardized IFM protocol. Platelet glycoproteins (CD42b, CD41/CD61), granule markers, and cytoskeletal components were stained with specific fluorophore-conjugated antibodies. Images were acquired using an automated fluorescence microscope and analyzed using rule-based subpopulation algorithms to quantify fluorescence intensity and platelet morphology. Results were compared with those obtained by flow cytometry and molecular genetic analysis. Fig. 1 Workflow for automated immunofluorescence microscopy in the diagnosis of inherited platelet disorders. Results Automated quantitative analysis reliably distinguished BSS and GT samples from healthy controls based on fluorescence signal intensity and localization. BSS platelets demonstrated markedly reduced or absent CD42b (GPIbα) expression and characteristic macrothrombocytopenia, while GT platelets exhibited loss of CD41/CD61 (GPIIb/IIIa) surface expression. Quantitative image data showed strong correlation with flow cytometric and genetic findings, confirming diagnostic accuracy. The automated workflow minimized observer bias and improved reproducibility compared to manual microscopy [ 1 ]. Conclusion This study provides proof-of-concept that automated quantitative IFM is a robust, objective, and standardized diagnostic tool for IPDs. The approach enables accurate detection of glycoprotein deficiencies and platelet morphological alterations, offering a practical alternative to flow cytometry-particularly valuable for laboratories with limited access to advanced platelet function testing Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany