title: Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular microchimerism in murine tissues.
abstract: Cellular microchimerism is defined as the presence of small populations of cells from one individual in another genetically distinct individual. The pivotal role of cellular microchimerism in a variety of immune settings is increasingly recognized, e.g. in context of pregnancy, transplantation and cancer. However, the detection of chimeric cells is overshadowed by technical limitations. This study aimed to overcome these limitations by testing the sensitivity and detection limit of a molecular biology approach (quantitative polymerase chain reaction, qPCR) and a cellular approach (flow cytometry) in order to identify experimentally induced cellular microchimerism in mice. Leukocytes isolated from lymph nodes or spleens of transgenic enhanced green fluorescent protein (eGFP) and CD45.1 mice respectively were used as targets to be detected as microchimeric cells among wild type (wt) or haploidentical cells. The detection limit of microchimeric cells by flow cytometry was 0.05% or lower for the respective eGFP(+) or CD45.1(+) cell subsets, which equals 48 cells or fewer per 1×10(5) wt cells. The detection limit of CD45.1(+) and CD45.2(+) cells among haploidentical CD45.1(+)2(+) cells by flow cytometry was 48 cells (0.05%) and 198 cells (0.2%), respectively. Using qPCR, a detection limit of 198 eGFP(+) cells per 1×10(5) wt cells, respective 0.2%, could be achieved. We here introduce two technical approaches to reliably detect low number of chimeric cells at a low detection limit and high sensitivity in transgenic mouse systems.
authors.affiliation: Laboratory for Experimental Feto-maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg - Eppendorf, Hamburg, Germany. Electronic address: k.thiele@uke.de.
authors.forename: Kristin
authors.initials: K
authors.lastname: Thiele
authors.affiliation: Institute of Medical Genetics, University of Rostock, Rostock, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Holzmann
authors.affiliation: Laboratory for Experimental Feto-maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
authors.forename: Maria Emilia
authors.initials: ME
authors.lastname: Solano
authors.affiliation: III. Department of Internal Medicine, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
authors.forename: Gunther
authors.initials: G
authors.lastname: Zahner
authors.affiliation: Laboratory for Experimental Feto-maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg - Eppendorf, Hamburg, Germany. Electronic address: p.arck@uke.de.
authors.forename: Petra Clara
authors.initials: PC
authors.lastname: Arck
mesh_terms: D000818:Animals; D046528:Chimerism; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007962:Leukocytes / Q000276:immunology*; D057230:Limit of Detection; D008198:Lymph Nodes / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013154:Spleen / Q000166:cytology*
chemical_list: D049452:Green Fluorescent Proteins
keywords: CD45.1; Flow cytometry; Microchimerism; Sensitivity; eGFP; qPCR