Multicolor immunophenotyping of tissue sections by laser scanning cytometry (LSC)

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Abstract
In lymphatic organs the quantitative analysis of the spatial distribution of leukocytes would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen. Analysis of them in solid tissues is difficult to perform but would yield important data in a variety of clinical and experimental settings. We have developed an automated analysis method for LSC suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for DNA and up to three antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (Argon Laser). Due to the heterogeneity in cell density measurements are repeatedly performed at different threshold levels (low threshold: regions of low cellular density, germinal centers; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to determine data from single- (FITC), up to triple-staining (FITC/PE-Cy5/APC). Percentage and cellular density of cell-subsets is quantified in different structural regions of the specimen. Comparison with manual analysis of identical specimens showed very good correlation. With LSC a semi-automated operator-independent and immunophenotyping of lymphatic tissues with simultaneously up to four antibodies is possible. This technique should yield new insight into processes during diseases and should help to quantify the success of therapeutic interventions.

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