Flow cytometry immunophenotyping of healthy platelets and hospitalized patients with suspected platelet dysfunction: Challenges for establishing a cutoff value

Daiane Keller Cecconello; Fabiane Spagnol; Ana Paula Alegretti; Diogo André Pilger; Mariela Granero Farias

doi:10.1016/j.htct.2023.07.002

Flow cytometry immunophenotyping of healthy platelets and hospitalized patients with suspected platelet dysfunction: Challenges for establishing a cutoff value

Hematol Transfus Cell Ther. 2023 Aug 28:S2531-1379(23)00156-6. doi: 10.1016/j.htct.2023.07.002. Online ahead of print.

Authors

Daiane Keller Cecconello¹, Fabiane Spagnol², Ana Paula Alegretti², Diogo André Pilger³, Mariela Granero Farias²

Affiliations

¹ Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil. Electronic address: daia_cecconello@hotmail.com.
² Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.
³ Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

PMID: 37652805
DOI: 10.1016/j.htct.2023.07.002

Abstract

Introduction and objective: Flow Cytometry (FC) is one of the techniques, which allows the identification and characterization of platelets. The detection of absent or reduced expression of the glycoproteins is the main objective of this technique. Abnormalities of glycoproteins lead to hemorrhagic syndromes. Among the main diseases, the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) stand out. We aimed to show a FC-based platelet assessment test for diagnostic use, which measures the expression of markers in normal patients, and evaluate these markers in patients with platelet disorders.

Methods: We examined a control group of 41 healthy adults to establish reference values and assess the variability of the relative expression of platelet markers and subsequently compared these findings to those of 30 patients with suspected platelet dysfunctions. We determined the mean fluorescent intensity (MFI) of the expressed parameters by FC using CD41, CD42a, CD42b and CD61 and SSC/FSC platelet-gated cells.

Results: We determined our baseline panel of markers and compared them to suspected platelet dysfunctions. Patients with suspected BSS presented increased levels of the MFI for the GPIIIa (CD61) and GPIIb (CD41). They showed significantly reduced levels of the GPIb (CD42b) and GPIX (CD42a). Patients with suspected GT showed normal expression of the GPIX (CD42a), increased expression of the GPIb (CD42b) and reduced levels of the GPIIIa (CD61). In this case, with reduced levels of only one marker, the GPIIb (CD41), values showed normal expression.

Conclusions: We describe the FC assay to support the diagnosis of different platelet disorders. Our study made it possible to implement a technique that brought benefits to care.

Keywords: Bernard-Soulier syndrome; Flow cytometry; Glanzmann thrombasthenia; Glycoproteins; Platelets.