Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation.
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 degrees 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 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees 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.


CONCLUSIONS
The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.
Pfeifer
P H
PH
The Futhan/EDTA tubes are produced by Terumo Europe and can be purchased from PerSeptive Biosystems, 500 Old Connecticut Path, Framingham, MA 01701, USA.
Kawahara
M S
MS
Hugli
T E
TE
D000328:Adult; D000925:Anticoagulants; D001550:Benzamidines; D001800:Blood Specimen Collection; D015926:Complement C3a / Q000032:analysis*; D015934:Complement C4a / Q000032:analysis*; D004492:Edetic Acid; D005260:Female; D006146:Guanidines; D006801:Humans; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D011863:Radioimmunoassay
D000925:Anticoagulants; D001550:Benzamidines; D006146:Guanidines; D015926:Complement C3a; D015934:Complement C4a; D004492:Edetic Acid; C032855:nafamostat