Functional validation of a modified platelet desialylation test for immune and hereditary thrombocytopenias

BACKGROUND: Thrombocytopenia arises from heterogeneous inherited and acquired disorders, and identifying the underlying platelet clearance mechanisms remains challenging. Platelet desialylation, characterised by loss of sialic acid and consequent exposure of terminal β-galactose residues recognised by the Ashwell-Morell receptor, represents an alternative pathway of hepatic platelet clearance. This study aimed to validate a modified platelet desialylation test (PDT) and assess its applicability in multitransfused, alloimmunized, and hereditary thrombocytopenia patients as a functional in vitro assay to detect plasma-induced platelet desialylation and explore its potential as a complementary biomarker of platelet clearance mechanisms. METHODS: PDT performance was evaluated using plasma from 20 healthy donor pools, 10 antibody-positive refractory patients, and 15 individuals with hereditary thrombocytopenias by FITC-RCA-I flow cytometry. Analytical validation included assessment of reproducibility, incubation time, lectin concentration, and ratio-based result interpretation. RESULTS: β-Lactose showed significantly lower expression mean fluorescence intensity (MFI) than both untreated reference control and neuraminidase-treated conditions (p < 0.00001), whereas patient plasma exhibited higher fluorescence than the reference control (p < 0.0001). A 1 h incubation using 0.3 μL per reaction RCA-I-FITC stock solution (5 mg active conjugate/mL) provided stable and reproducible discrimination. In inherited thrombocytopenias, 66.7% of patients were PDT-positive despite HLA/HPA antibodies being detected in only 13.3%. PDT-positive samples showed increased desialylation-associated MFI (p = 0.002) with a strong correlation between MFI and ratio (ρ = 0.877). The assay required approximately 4 h and cost US$3.62 per test. CONCLUSION: The PDT is a practical, reproducible, and cost-effective assay capable of detecting physiologic, enzymatic, and immune-mediated platelet desialylation, providing a complementary biomarker for investigating thrombocytopenic disorders.