Methods to Examine Complement Activation by HIT Immune Complexes

Current assays for measuring complement activation by immune complexes (ICs) are largely indirect, non-specific, and employ non-physiologic conditions. To address these drawbacks, we developed methods for measuring the complement activating effects of ICs that cause heparin induced thrombocytopenia (HIT), a blood clotting disorder. These assays rely on the formation of in situ ICs in undiluted healthy donor plasma and/or whole blood as a physiologic source of complement. Once formed, complement-bound ICs in plasma or whole blood are detected using immunocapture and/or flow-based assays. While these methods are designed to study the complement activating properties of HIT ICs, the immunocapture and flow-based assays can be readily extended to other ICs of known composition.