Characterization of Bernard-Soulier syndrome, Glanzmann's thrombasthenia using immunomagnetic cell separation technique

Abstract

To characterize human platelet membrane glycoprotein (GP), we developed an immunomagnetic cell isolation method using monoclonal antibodies (MoAbs) that specifically bind to Ib and IIb-IIIa complexes of GP. When added to plateletrich plasma (PRP), anti-GPIb MoAb inhibited ristocetin-induced platelet aggregation, and anti-GPIIb-IIIa MoAb inhibited ADP- and collagen-induced platelet aggregation. Each MoAb was incubated with monosized polystyrene beads (4.5μm in diameter) coated with secondary antibody and then with normal PRP for 30 minutes. More than 90% of the platelets bound to the beads and were eliminated from PRP. However, the binding between the platelets and the beads coated with each MoAb was inhibited by pretreatment of PRP with the same type of MoAb.Platelets from patients with Bernard Soulier syndrome (BSS) and Glanzmann's thrombasthenia (GT) were added to the beads coated with each MoAb. BSS platelets with defective GPIb and GT platelets with defective GPIIb-IIIa did not bind to the beads coated with anti-GPIb MoAb and those coated with anti-GPIIb-IIIa MoAb, respectively.Our immunomagnetic isolation method using anti-GP MoAbs is very simple and useful for detecting abnormalities such as GP deficiency and especially diagnosing BSS and GT.