Identification and distribution of chimeric cells in epidermal and dermal compartments of the skin in patients with GvHD. Presence of chimeric keratinocytes, is characterized by CD45 negativity, AE1/AE3 positivity, and X and Y markers (arrows), in the superficial layers of epidermis in a patient in whom a biopsy was taken late in the course of GvHD (day 60) (A), and in the suprabasal layer of epidermis in a patient in whom a biopsy was taken earlier in the course of GvHD (day 35) (B). Presence of chimeric endothelial cells is characterized by CD31 positivity, CD45 negativity, and X and Y markers (arrows) in microvessels of the superficial dermis in all patients with GvHD (C), and in larger vessels of the deep dermis in only one patient with GvHD (D). All scale bars represent 10 μm. Formalin-fixed, paraffin-embedded skin biopsy specimens were cut into 5-μm thick sections. Sister following sections were used for combined immunohistochemistry, FISH, immunofluorescent study and for TUNEL assay. An indirect immunoperoxydase procedure was performed with monoclonal mouse anti–human CD45 antibody (clone PD7/26 and 2B11; Dako, Carpinteria, CA) and secondary biotinylated antibody (Nexes; Ventana, Tucson, AZ). For FISH, the CEP X (SpectrumGreen)/Y (SpectrumOrange) DNA probe (Vysis, Downers Grove, IL) kit was used. Immunofluorescent labeling was carried out with monoclonal mouse anti–human cytokeratin (clone AE1/AE3; Boehringer Mannheim, Indianapolis, IN) and anti–human CD31 (clone JC70A; Dako) as primary antibodies, and AMCA-conjugated horse anti–mouse IgG antibody (Vector Laboratories, Burlingame, CA) as secondary antibody. Bright-field and fluorescent images were obtained by a Provis AX 70 microscope (Olympus, Rungis, France), with wide-field eyepiece number 26.5, providing a field size of 0.34 mm² , through a UPlan FI 40×/0.75 NA objective, and were captured with a ColorView III digital camera (Olympus) using analySIS software version 3.2 (Soft Imaging System).