Flow cytometric measurement of intracellular bilirubin in human peripheral blood mononuclear cells exposed to unconjugated bilirubin

Clin Biochem. 1992 Aug;25(4):277-83. doi: 10.1016/0009-9120(92)80033-d.


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.

MeSH terms

  • Adult
  • Antigens, CD / analysis
  • Antigens, Differentiation, Myelomonocytic / analysis
  • Bilirubin / blood*
  • Bilirubin / pharmacology
  • Cytoplasm / chemistry
  • Flow Cytometry*
  • Humans
  • Leukocytes, Mononuclear / chemistry*
  • Leukocytes, Mononuclear / immunology
  • Lipopolysaccharide Receptors
  • Middle Aged
  • Monocytes / chemistry*
  • Monocytes / immunology
  • Reproducibility of Results


  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Lipopolysaccharide Receptors
  • Bilirubin