Comparison of strategies to detect and quantitate uniquely marked cells in intra- and inter-species hemopoietic chimeras.
Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopoietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA-based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse-to-mouse) and xenogeneic (mouse-to-human, monkey, sheep) backgrounds. DNA-based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10(-4). The sensitivity of immunofluorescence-linked flow cytometric-based detection of nucleated leukocytes approached 10(-3), whereas flow cytometric-based detection of fixed human erythrocytes was feasible at cell frequencies of 10(-5). Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.
Biomedical Sciences Division, Lawrence Livermore National Laboratory, California 94550.
M G
MG
Pallavicini
R G
RG
Langlois
M
M
Reitsma
M
M
Gonzalgo
D
D
Sudar
T
T
Montoya
H U
HU
Weier
S
S
Haendel
D000818:Animals; D015415:Biomarkers; D016026:Bone Marrow Transplantation; D002678:Chimera; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D004912:Erythrocytes / Q000737:chemistry; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006021:Glycophorins / Q000032:analysis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008253:Macaca mulatta; D051379:Mice; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012756:Sheep; D013045:Species Specificity; D014183:Transplantation, Heterologous
D015415:Biomarkers; D015342:DNA Probes; D006021:Glycophorins; D004247:DNA