title: Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue using laser scanning cytometry.
abstract: There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune cells remains challenging due to a lack of objective  methodology. Laser scanning cytometry (LSC) is an imaging-based methodology that performs quantitative measurements on fluorescently and/ or chromatically stained tissue or cellular specimens at a single-cell level. In this study, we have developed a novel objective method for analysis of  immune cells, including Foxp3(+) T regulatory cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy sections using iCys® Research Imaging Cytometer. The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The  CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using iCys and compared with the results from conventional flow cytometry analysis (FCM). Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio  analyzed by iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas, and heart transplant biopsy sections and provide an objective quantification of Tregs within the grafts.
authors.affiliation: Miami Transplant Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Takahashi
authors.forename: Phillip
authors.initials: P
authors.lastname: Ruiz
authors.forename: Camillo
authors.initials: C
authors.lastname: Ricordi
authors.forename: Victor
authors.initials: V
authors.lastname: Delacruz
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miki
authors.forename: Atsuyoshi
authors.initials: A
authors.lastname: Mita
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Misawa
authors.forename: Scott
authors.initials: S
authors.lastname: Barker
authors.forename: George W
authors.initials: GW
authors.lastname: Burke
authors.forename: Andreas G
authors.initials: AG
authors.lastname: Tzakis
authors.forename: Hirohito
authors.initials: H
authors.lastname: Ichii
mesh_terms: D016516:CD4-CD8 Ratio; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000032:analysis*; D006801:Humans; D016030:Kidney Transplantation / Q000276:immunology; D047569:Laser Scanning Cytometry; D016031:Liver Transplantation / Q000276:immunology; D014066:Palatine Tonsil / Q000276:immunology* / Q000601:surgery; D016035:Pancreas Transplantation / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014180:Transplantation; D014181:Transplantation Immunology
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors