Computerized image analysis of distinct cell marker parameters of glial fibrillary acidic protein: intensity of immunofluorescence and topography in human glioma cultures

Abstract

Glial fibrillary acidic protein (GFAP) is a 49 kDa component of 8 nanometers glial intermediate filaments. It is a definitive marker of both neoplastic and non-neoplastic glial cells. Malignant gliomas are heterogeneous in their structure and their expression of GFAP. Subjective visual impression of glioma cell cultures in early passage after explantation suggested two types of heterogeneity among the cells: 1) Indirect anti-GFAP immunofluorescence (IF) signal intensity appeared to vary among cells. 2). Sizes and shapes of cells appeared to vary. While many cultures lost GFAP+ cells by passage 2, some retained sufficient cells for study. Computerized microdensitometry and image analysis precisely assessed signal intensity, size and shape of glial cells from these latter cultures in passage 2 of cell culture. A Zeiss ICM 405 epi-illuminated fluorescent microscope with photographic interface to a Bio-Quant computerized microdensitometer measured IF signal intensity for GFAP. Normalized to the most intense reactivity, the mean intensity of GFAP expression of 26 glioma cells was 66.2%. The range of 5.2% to 100% around this mean reflected a high degree of heterogeneity of expression of GFAP by these cells. The effects of quenching of the fluorescent signal by the excitation beam were minimized in the following manner: a) Rhodamine anti-GFAP was substituted for more labile fluoresceine. b) A fluorescent filter and lens combination was selected to provide a high signal to quench ratio. c) The signal acquisition time was minimized and fixed to one standard interval. The image analysis system employed the described optics and computer.(ABSTRACT TRUNCATED AT 250 WORDS)