title: Four-color flow cytometric analysis of peripheral blood donor cell chimerism.
abstract: Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.
authors.affiliation: Department of Surgery, Thomas E. Starzl Tranplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Metes
authors.forename: Alison
authors.initials: A
authors.lastname: Logar
authors.forename: William A
authors.initials: WA
authors.lastname: Rudert
authors.forename: Adriana
authors.initials: A
authors.lastname: Zeevi
authors.forename: Jennifer
authors.initials: J
authors.lastname: Woodward
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Demetris
authors.forename: Kareem
authors.initials: K
authors.lastname: Abu-Elmagd
authors.forename: Bijan
authors.initials: B
authors.lastname: Eghtesad
authors.forename: Ron
authors.initials: R
authors.lastname: Shapiro
authors.forename: John J
authors.initials: JJ
authors.lastname: Fung
authors.forename: Massimo
authors.initials: M
authors.lastname: Trucco
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Starzl
authors.forename: Noriko
authors.initials: N
authors.lastname: Murase
mesh_terms: D000911:Antibodies, Monoclonal; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D006680:HLA Antigens / Q000276:immunology; D006650:Histocompatibility Testing; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D007962:Leukocytes / Q000276:immunology*; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction; D018183:Transplantation Chimera; D014181:Transplantation Immunology
chemical_list: D000911:Antibodies, Monoclonal; D006680:HLA Antigens