Modified hemoglobin preparations may potentially cause hypersensitivity and anaphylactic reactions, antibody-antigen reactions and other problems. Unfortunately response in animal safety studies may not reflect the same response in human. The next best test before clinical trial in human may be the use of human plasma in-vitro. This paper presents an in-vitro procedure based on complement activation (C3a) of human plasma. The procedure involves collecting heparinised blood and separating the plasma and freezing the heparinised plasma at -70-degrees-C until use. Each 100 lambda of control or test samples is added to 400 lambda of this plasma. This is incubated at 37-degrees-C, 60 rpm for 1 hour, then added to EDTA saline to stop the reaction and stored at -70-degrees-C until analysed by standard radioimmunoassay for C3a. (C3 measurement is not sensitivity enough). Using the screening test procedure described above, C3a levels (ng/ml) in plasma were: control, 1,980 +/- 280; Zymosan, 20,000; Hemoglobin preparation A, 2,227 +/- 617; Hemoglobin preparation B, 4,967 +/- 153; A 75% + B 25%, 3,967 +/- 270; A 50% + B 50%, 4,553 +/- 517; A 25% + B 75%, 4,920 +/- 430. Hemoglobin preparation A did not cause significant increases in C3a complement activation. Hemoglobin preparation B caused significant increase in C3a complement activation. Serial dilution of Hemoglobin preparation B in Hemoglobin preparation A continued to cause the same degree of C3a complement activation. This is not due to C3 exhaustion because Zymosan resulted in C3a of greater than 20,000ng/ml. This appears to show that this screening test can detect complement activation even at low concentrations of the hemoglobin preparation. Factors which cause complement activation may include endotoxin, toluene extractable factors like lipids, and organic solvents.
