Flow cytometric measurement of intracellular bilirubin in human peripheral blood mononuclear cells exposed to unconjugated bilirubin.
Human peripheral blood mononuclear cells were incubated at 37 degrees C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p less than 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both less than 5%). When bilirubin-treated cells were stained with fluorescein-labeled anti-CD14 antibody, the CD14 positive cell population can be fractionated without interference of autofluorescence derived from intracellular bilirubin. These results suggest that flow cytometric analysis of bilirubin-treated cells can quantitate intracellular bilirubin, and that bilirubin does not interfere with analysis of surface antigens.
Haga
Y
Y
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
Kay
H D
HD
Tempero
M A
MA
Zetterman
R K
RK
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D001663:Bilirubin / Q000097:blood* / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry* / Q000276:immunology; D018950:Lipopolysaccharide Receptors; D008875:Middle Aged; D009000:Monocytes / Q000737:chemistry* / Q000276:immunology; D015203:Reproducibility of Results
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018950:Lipopolysaccharide Receptors; D001663:Bilirubin