Influence of time, temperature and coagulation on the measurement of C3, C3 split products and C4

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Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18°C–22°C) and 4°C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4°C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4°C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occuring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4°C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4°C marginal increases in C3d levels only were observed. These results suggests that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.