Consensus protocol for platelet desialylation (β-galactose exposure) quantification using lectins by flow cytometry: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

IF 5.5 2区医学 Q1 HEMATOLOGY

Journal of Thrombosis and Haemostasis Pub Date : 2025-03-24 DOI:10.1016/j.jtha.2025.03.017

Alexandre Kauskot, Sofia Ramstrom, Thomas Nipoti, Dianne E van der Wal

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引用次数: 0

Abstract

Introduction: Platelets contain many heterogeneous carbohydrates (glycans): often capped by sialic acid. The removal of sialic acid (desialylation) is important for platelet function and clearance, leading to novel diagnostic markers. Platelet desialylation can be easily measured using inexpensive, user-friendly lectins, and flow cytometry.

Aims: Here, the Platelet Physiology Scientific and Standardization Committee (SSC) of the International Society for Thrombosis and Haemostasis (ISTH) carried out a survey to assess current methods used for platelet desialylation. Based on the survey results, a consensus protocol was drafted and tested.

Methods: A survey/questionnaire was posted on the ISTH Platelet Physiology SSC pages. Washed platelets and diluted apheresis platelets were diluted to 50 and 200x10⁶/mL, ± CaCl₂. Platelets were stained with a concentration range of either β-galactose binding fluoresceine-conjugated lectins Ricinus communis agglutinin 1(RCA-1) or Erythrina cristagalli lectin (ECL) respectively. As positive controls, different recombinant sialidases were tested.

Results: Results of the survey (n=20) showed that flow cytometry and RCA-1 are mostly used to assess platelet desialylation. Calcium did not significantly influence lectin binding and optimal binding was achieved with ECL and RCA-1 at 2 and 5 μg/mL, respectively. The specificity of lectins varied, particularly after sialidase treatment, compared to cold-stored platelets. These findings contribute to standardization of desialylation measurements, particularly in patient samples.

Conclusions: Our findings demonstrate that flow cytometry using RCA-1 and ECL is a robust method for quantifying platelet desialylation. The proposed standardised protocol addresses key pre-analytical variables, enabling reproducible and accurate analysis of platelet glycosylation.

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来源期刊

Journal of Thrombosis and Haemostasis 医学-外周血管病

CiteScore

24.30

自引率

3.80%

发文量

321

审稿时长

1 months

期刊介绍： The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community. Types of Publications: The journal publishes a variety of content, including: Original research reports State-of-the-art reviews Brief reports Case reports Invited commentaries on publications in the Journal Forum articles Correspondence Announcements Scope of Contributions: Editors invite contributions from both fundamental and clinical domains. These include: Basic manuscripts on blood coagulation and fibrinolysis Studies on proteins and reactions related to thrombosis and haemostasis Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.