Pretreatment of serum containing hemoglobin vesicles (oxygen carriers) to prevent their interference in laboratory tests

Hemoglobin vesicles (HbV, diameter: 251 ± 81 nm) are artificial oxygen (O₂) carriers encapsulating concentrated hemoglobin (Hb) solution with phospholipid bilayer membrane, and their O₂ transporting ability in vivo has been extensively studied. It is important to clarify the interference of the HbV suspension in clinical laboratory tests performed on serum and to establish a pretreatment method to avoid such an interference. The HbV suspension, acellular Hb solution ([Hb] = 10 g/dl) or saline, was mixed with a pooled human serum at various ratios up to 50 vol% ([Hb] = 5 g/dl), and the magnitude of the interference effect of HbV and Hb on 30 analytes was studied. The mixture of the HbV suspension and serum was ultracentrifuged (50000 g, 20 min) to remove the HbV particles as precipitate, and the supernatant was analyzed and compared with the saline control group. The HbV particles were also removed by centrifugation (2700 g, 30 min) in the presence of dextran (Mw 200 kDa). The HbV suspension showed considerable interference effects in most analytes. The majority of these effects was more serious than those of the acellular Hb solution. These findings are thought to be due to the light absorption of Hb in HbV and/or the light scattering generated in the suspension that interferes with the colorimetric and turbidimetric measurements. The components of HbV may also interfere with the chemical reactions of the studied assays. However, removal of the HbV from the supernatant diminished the interference in most of the assays: this is an advantage of HbV in comparison with acellular chemically modified Hb solutions.