Possible Mechanism for in Vitro Complement Activation in Blood and Plasma Samples: Futhan/EDTA Controls in Vitro Complement Activation

Abstract

Background: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. Methods: BiotrakTM RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 °C. Results: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22–23 °C) for 1 h and in plasma held for 24 h at 4 °C or −70 °C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. Conclusion: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Complement Factor C5a in Acute Exacerbation of Chronic Obstructive Pulmonary Disease

“…This may introduce error to in vitro measurements of in vivo-generated cleavage products, such as C3a or C5a. Addition of FUT-175 to plasma samples at the time of sample collection provides additional protection from ex-vivo activation and, therefore, ensures more accurate measurements reflecting the circulating levels of complement activation [23].…”

Measuring Circulating Complement Activation Products in Myeloperoxidase– and Proteinase 3–Antineutrophil Cytoplasmic Antibody–Associated Vasculitis

“…Conversely, even when thawed at this temperature, plasma collected with EDTA remained stable for up to 60 minutes, except for measures of C4d . Other investigators have shown that cleavage of complement proteins occurs in vitro despite the use of EDTA in blood collection . Careful and systematic sample processing to avoid in vitro complement activation is therefore required for more accurate measurement.…”

“…Careful and systematic sample processing to avoid in vitro complement activation is therefore required for more accurate measurement. Futhan, a broad‐specificity protease inhibitor, blocks in vitro complement activation . It has been shown to inhibit the esterolytic activities of C1r, C1s, and factor B and hemolytic activities mediated by both the classical and alternative complement pathways .…”

Acute phase proteins are major clients for the chaperone action of α2-macroglobulin in human plasma

“…C3 is an inherently unstable protein that converts to C3b by spontaneous hydrolysis of the thioester bond (Pangburn et al 1981). Furthermore, complement activation may occur in vitro even in the presence of protease inhibitors generating further cleavage products from C3b, including iC3b, C3c and C3d (Pfeifer et al 1999;Momeni et al 2012). Following hydrolysis to form C3b and subsequent cleavage to yield the inhibited form iC3b, C3 undergoes major conformational changes including progressive unfolding, although the precise nature of these changes is debated (Janssen et al 2007;Ajees et al 2007).…”