Quantitative in situ analysis of FoxP3 + T regulatory cells on transplant tissue using laser scanning cytometry

Hidenori Takahashi, Phillip Ruiz, Camillo Ricordi, Victor Delacruz, Atsushi Miki, Atsuyoshi Mita, Ryosuke Misawa, Scott Barker, George W. Burke, Andreas G. Tzakis, Hirohito Ichii

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)113-125
Number of pages13
JournalCell transplantation
Volume21
Issue number1
DOIs
StatePublished - 2012

Keywords

  • Foxp3
  • iCys
  • Laser scanning cytometry (LSC)
  • Quantification
  • T regulatory cells (Tregs)
  • Transplant biopsy

ASJC Scopus subject areas

  • Cell Biology
  • Transplantation
  • Biomedical Engineering

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