Background/Purpose: Deregulation and activation of the classical complement system is known to be associated with systemic lupus erythematosus (SLE). As such, several investigators have proposed that the determination of C4d fragment, the activation product of C4 in its soluble or cell-bound form to erythrocytes (EC4d), or B-lymphocytes (BC4d) could help diagnose SLE. However, one of the challenges in determining complement activation is the stability of these products post-phlebotomy. Our objective was to quantify the effects of time and temperature on soluble and cell bound C4d.

Methods: Venous blood was collected in EDTA-containing tubes from 12 subjects with SLE (67% females, mean age 39 years) and 15 normal healthy volunteers (NHV) (48% females, mean age 38 years). All subjects enrolled provided informed consent. Following venipuncture, the blood was equally distributed into polypropylene tubes and stored at either 4°C or ambient room temperature (22°C) for up to two days post phlebotomy. These conditions mimic the transportation condition of a typical specimen. Starting on the day of collection (day 0), and the following two days, plasma was isolated from the stored whole blood. Soluble plasma C4d concentration was determined using immunoassays (MicroVue C4d EIA, Quidel). EC4d and BC4d were determined from stored whole blood using flow cytometry and reported as net mean fluorescence intensity (MFI). The relationship between the change in the markers from baseline as a function of time and temperature was estimated with linear mixed-effect models.

Results: Soluble C4d (baseline levels 0.8±0.3 µg/ml) was significantly affected by both time (increase of 1.1 ± 0.1 µg/ml per day) and storage conditions (increase of 0.7 ± 0.1 µg/ml at ambient temperature versus 4°C) (Table 1). After two days storage at ambient temperature, soluble C4d levels increased by more than 2 fold compared to baseline plasma concentrations. By comparison, EC4d and BC4d levels were not significantly impacted by time and storage at ambient temperature. EC4d and BC4d levels were 15.9 and 62.5 net MFI after two days storage at room temperature, respectively, by comparison to 15.8 and 65.5 net MFI at day 0. In this small population of subjects, elevated CBCAPS (EC4d>14 net MFI or BC4d>60 net MFI) were 73% sensitive for SLE and 100% specific (vs. NHV).

Conclusion: Our data suggest soluble C4d is unstable during transportation and delivery of specimen to the clinical laboratory. However, cell-bound C4d is a stable measure of complement activation and could be used as complement activation biomarker in multi-centered studies.