Quantitative analysis of cell distributions in 3 dimensions. A BM specimen from a patient with SAA was fluorescently labeled with antibodies to CD34, CD8, BODIPY 493/503 (adipocytes), and DAPI (nuclei) (A,E). Original confocal images are presented in top panels (A) CD34⁺ cells (red)/adipocytes (green) and (E) CD8⁺ cells (white)/blood vessels (red). Bottom panels display segmented images of (B) CD34⁺ cells (red)/adipocytes (green) and (F) CD8⁺ cells (blue)/blood vessels (red), computationally extracted from the original fluorescence images, to measure the distance to adipose cells (C) and blood vessels (G), respectively. Clustering of CD34⁺ cells (D) and of CD8⁺ cells (H) was analyzed by comparing experimental data (white columns in panel D) and (black columns in panel H) with a Monte Carlo simulation (hatched columns in panels D and H) of randomly distributed cells of same density in a similar volume. Representative images and results from one patient are presented. The analysis was repeated independently 2 to 4 times for 4 different specimens.