Complement function and activation in human serum and plasma collected in different blood collection tubes

Complement analysis necessitates strict control of pre-analytical blood handling, including time, temperature, and additives. Here, we compared complement function and activation status across five different serum preparations and two plasma preparations. Serum was collected from ten healthy volunteers using glass tubes without additives, tubes with a silica clot activator (with or without a gel separator), and tubes containing thrombin (with or without a gel separator). Plasma was collected in the presence of EDTA or the thrombin inhibitor lepirudin. Serum and plasma aliquots were snap-frozen in liquid nitrogen and stored at -80 °C. Complement functional analysis was performed using Wieslab and Hycult Biotech pathway-specific assays. Complement activation was determined by quantifying specific activation markers: C1s/C1-INH, MASP-1/C1-INH, C3bc, C3bBbP, and sC5b-9. All serum samples exhibited increased complement activation compared to EDTA and lepirudin plasma, with serum tubes containing thrombin and gel separators showing the highest levels of complement activation. However, normal complement function was observed in all serum preparations, indicating that the complement activation and consumption that occurred did not affect complement functional analysis. While all tested serum tubes provided accurate functional activity, the type of tube and the presence of additives like thrombin and gel separators significantly influenced the degree of complement activation. We recommend preparing functionally active serum either in glass tubes or in silica clot activator tubes, and avoiding gel separators. For complement activation studies, lepirudin plasma is preferable over serum due to its complement functional capacity, low level of complement activation, and lack of excessive hemostatic activation.