Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular microchimerism in murine tissues

TY - JOUR

T1 - Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular microchimerism in murine tissues

AU - Thiele, Kristin

AU - Holzmann, Carsten

AU - Solano, Maria Emilia

AU - Zahner, Gunther

AU - Arck, Petra Clara

N1 - Copyright © 2014 Elsevier B.V. All rights reserved.

PY - 2014/4/1

Y1 - 2014/4/1

N2 - 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.

AB - 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.

U2 - 10.1016/j.jim.2014.03.009

DO - 10.1016/j.jim.2014.03.009

M3 - SCORING: Journal article

C2 - 24657636

VL - 406

SP - 74

EP - 82

JO - J IMMUNOL METHODS

JF - J IMMUNOL METHODS

SN - 0022-1759

ER -