Haga Y, Kay H D, Tempero M A, Zetterman R K
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
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.
将人外周血单核细胞与胆红素在牛白蛋白溶液中于37℃孵育。对经胆红素处理的细胞进行组织学分析，结果显示细胞质中有显著的棕色色素沉积。这些细胞在氯仿 - 甲醇溶液中的匀浆与溶解在相同溶液中的纯胆红素呈现相同的吸收光谱。当用488nm激发经胆红素处理的细胞时，通过流式细胞术在585nm处检测到显著的自发荧光，且呈剂量依赖性，这与从细胞中化学提取的胆红素量具有显著相关性（r = 0.963，n = 34，p < 0.001）。流式细胞术分析自发荧光的批内和批间变异性较小（均小于5%）。当用荧光素标记的抗CD14抗体对经胆红素处理的细胞进行染色时，CD14阳性细胞群体能够被分离，且不受细胞内胆红素产生的自发荧光干扰。这些结果表明，对经胆红素处理的细胞进行流式细胞术分析能够定量细胞内胆红素，并且胆红素不会干扰表面抗原的分析。
