Quantitative and three-dimensional analysis of human Langerhans cells in epidermal sheets and vertical skin sections

We used confocal laser scanning microscopy to analyze and compare Langerhans cells (LCs) in normal skin of six subjects. Acetone-fixed epidermal sheets and 25-microns vertical skin sections were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies directed against HLA-DR. An individual threshold setting algorithm compensating for the differences in background fluorescence was applied to identify specific fluorescence. No statistically significant difference was found in the relative volume of epidermal HLA-DR reactivity between epidermal sheets (14 +/- 5%) and vertical skin sections (13 +/- 6%) or in the number of dendrites per HLA-DR+ LCs (7.8 +/- 3.1 and 5.9 +/- 3.1, n = 58, respectively). However, statistically significant higher background intensity was found in vertical sections than in epidermal sheets. Three-dimensional (3D) reconstructions of HLA-DR+ LCs revealed a concentration of HLA-DR to one or a few intracellular vesicles in 42 of 58 analyzed LCs in epidermal sheets and in 18 of 58 analyzed LCs in vertical sections. Direct contact between dendrites from different LCs was not found. The results indicate that both skin forms are suitable for quantitative studies. Owing to less background intensity and larger tissue volume, detailed 3D analysis of LCs is preferably performed on epidermal sheets rather than on vertical sections.