Quantitative histology by multicolor slide‐based cytometry

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Abstract
Background In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. Methods We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed “multiple thresholding,” which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA‐fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single‐ (Ar) or dual‐laser excitation (Ar‐HeNe) in order to analyze single‐ (FITC) up to four‐color (FITC/PE/PECy5/APC) stained specimen. Results Percentage and cellular density of cell‐subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r2 = 0.96, P < 0.0001). With LSC, semiautomated operator‐independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. Conclusions We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.