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

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.