Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia

Loos, Kevin; Al-Rifai, Rawya; Ohlenforst, Sandra; Klein, Claudia; Oldenburg, Johannes; Pavlova, Anna; Pezeshkpoor, Behnaz

doi:10.3390/genes16060621

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Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia

by

Kevin Loos

^†,

Rawya Al-Rifai

^†,

Sandra Ohlenforst

,

Claudia Klein

,

Johannes Oldenburg

,

Anna Pavlova

and

Behnaz Pezeshkpoor

^*

Institute of Experimental Hematology and Transfusion Medicine, University Clinic Bonn, 53127 Bonn, Germany

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Genes 2025, 16(6), 621; https://doi.org/10.3390/genes16060621

Submission received: 15 April 2025 / Revised: 18 May 2025 / Accepted: 19 May 2025 / Published: 23 May 2025

(This article belongs to the Section Human Genomics and Genetic Diseases)

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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 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.

Keywords: inherited platelet disorders; Bernard–Soulier syndrome; Glanzmann thrombasthenia; immunofluorescence microscopy; automated diagnosis; flow cytometry; thrombocytopathy

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MDPI and ACS Style

Loos, K.; Al-Rifai, R.; Ohlenforst, S.; Klein, C.; Oldenburg, J.; Pavlova, A.; Pezeshkpoor, B. Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia. Genes 2025, 16, 621. https://doi.org/10.3390/genes16060621

AMA Style

Loos K, Al-Rifai R, Ohlenforst S, Klein C, Oldenburg J, Pavlova A, Pezeshkpoor B. Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia. Genes. 2025; 16(6):621. https://doi.org/10.3390/genes16060621

Chicago/Turabian Style

Loos, Kevin, Rawya Al-Rifai, Sandra Ohlenforst, Claudia Klein, Johannes Oldenburg, Anna Pavlova, and Behnaz Pezeshkpoor. 2025. "Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia" Genes 16, no. 6: 621. https://doi.org/10.3390/genes16060621

APA Style

Loos, K., Al-Rifai, R., Ohlenforst, S., Klein, C., Oldenburg, J., Pavlova, A., & Pezeshkpoor, B. (2025). Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia. Genes, 16(6), 621. https://doi.org/10.3390/genes16060621

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Automated Quantitative Immunofluorescence Microscopy Approach for Diagnosis of Hereditary Thrombopathies: A Proof of Concept Using Bernard–Soulier Syndrome and Glanzmann Thrombasthenia

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