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Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular
Kristin Thiele1, Carsten Holzmann2, Maria Emilia Solano1
1Laboratory for Experimental Feto-maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
Insights
Researchers developed sensitive methods to detect cellular microchimerism, the presence of cells from one individual in another. These techniques, flow cytometry and quantitative polymerase chain reaction (qPCR), overcome technical limitations in identifying these rare cells.
Area of Science:
- Immunology
- Cell Biology
- Genetics
Background:
- Cellular microchimerism involves cells from one individual residing in another.
- Its role in pregnancy, transplantation, and cancer is increasingly recognized.
- Technical limitations hinder the detection of microchimeric cells.
Purpose of the Study:
- To overcome technical limitations in detecting cellular microchimerism.
- To test the sensitivity and detection limits of flow cytometry and quantitative polymerase chain reaction (qPCR).
- To identify experimentally induced cellular microchimerism in mouse models.
Main Methods:
- Used leukocytes from transgenic enhanced green fluorescent protein (eGFP) and CD45.1 mice.
- Detected target cells as microchimeric cells among wild type or haploidentical cells.
- Employed flow cytometry and qPCR for detection and sensitivity analysis.
Main Results:
- Flow cytometry detected microchimeric cells at 0.05% or lower (≤48 cells per 1×10^5 cells).
- Detection limits for specific cell subsets using flow cytometry were 48 cells (0.05%) and 198 cells (0.2%).
- qPCR achieved a detection limit of 198 eGFP+ cells per 1×10^5 cells (0.2%).
Conclusions:
- Introduced two reliable technical approaches for detecting low numbers of chimeric cells.
- Demonstrated high sensitivity and low detection limits for both flow cytometry and qPCR.
- These methods overcome existing limitations in identifying cellular microchimerism in experimental systems.
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.
