Confocal microscopy images of microparticles generated by HIT serum, thrombin, and calcium ionophore.

Platelets and microparticles were identified using a platelet-specific primary antibody (mAb anti-GPIIb/IIIa) and a fluorescent secondary antibody (TR-conjugated goat antimouse). (A) Control platelets were incubated with patient serum, which tested negative for HIT in the presence of 0.1 U/mL heparin, (B) heparin alone, and (C) HIT serum in the absence of heparin. (D) Platelets were incubated with HIT serum in the presence of 0.1 U/mL heparin. As a positive control, (E) platelets were also incubated with 1 U/mL thrombin or (F) 10 μm calcium ionophore A23187. Platelet reactions with HIT serum in the presence of heparin demonstrated numerous brightly stained particles surrounding homogeneously stained platelets. This “starry sky pattern” was also observed in platelet reactions with thrombin and calcium ionophore. In comparison, very few stained particles were observed in platelet reactions with control serum or heparin alone. The results of a representative experiment are shown.