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

¹ The Futhan/EDTA tubes are produced by Terumo Europe and can be purchased from PerSeptive Biosystems, 500 Old Connecticut Path, Framingham, MA 01701. At present, the Futhan/EDTA tubes are approved only for research use in the United States.
^a Address correspondence to this author at: Department of Immunology/IMM-18, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Fax 619-784-8307; e-mail hugli{at}scripps.edu

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: Biotrak^TM 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.© 1999 American Association for Clinical Chemistry

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