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

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.

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.

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.

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.