Rare Event Detection and Analysis in Flow Cytometry: Bone Marrow Mesenchymal Stem Cells, Breast Cancer Stem/Progenitor Cells in Malignant Effusions, and Pericytes in Disaggregated Adipose Tissue
One of the major strengths of Flow Cytometry is its ability to perform multiple measurements on single cells within a heterogeneous mixture. When the populations of interest are relatively rare, analytical methodology that is adequate for more prevalent populations is often overcome by sources of artifacts that become apparent only when large numbers of cells are acquired. This chapter presents three practical examples of rare event problems and gives detailed instructions for preparation of single cell suspensions from bone marrow, malignant effusions, and solid tissue. These examples include detection of mesenchymal stem cells in bone marrow, characterization of cycling/aneuploid cells in a breast cancer pleural effusion, and detection and subset analysis on adipose-derived pericytes. Standardization of the flow cytometer to decrease measurement variability and the use of integrally stained and immunoglobulin capture beads as spectral compensation standards are detailed. The chapter frames rare event detection as a signal-to-noise problem and provides practical methods to determine the lower limit of detection and the appropriate number of cells to acquire. Detailed staining protocols for implementation of the examples on a three-laser cytometer are provided, including methods for intracellular staining and the use of DAPI to quantify DNA content and identify events with ³2N DNA. Finally, detailed data analysis is performed for all three examples with emphasis on a three step procedure: (1) Removal of sources of interference; (2) Identification of populations of interest using hierarchical classifier parameters; and (3) Measurement of outcomes on classifier populations.
Key wordsRare-event analysis Flow cytometry Breast cancer Cancer stem cells Adult stem cells Adipose pericytes Bone marrow-derived mesenchymal stem cells
The authors would like to thank Melanie Pfeifer and E. Michael Meyer for their excellent technical assistance, Peter Nobes and David Roberts (Applied Cytometry Systems) for their valued collaboration in data analysis and Cindy Collins and Brad Calvin (Beckman Coulter) for providing instrumentation, software, and the support of their flow cytometry group. This work was supported by the following granting agencies: Department of Defense (BC032981 and BC044784), NIH NHLBI (Production Assistance for Cellular Therapy (PACT) under contract N01-HB-37165, R01-HL-085819), the Hillman Foundation, and the Glimmer of Hope Foundation.
- 1.Triebel, F., Robinson, W. A., Hayward, A. R., and Goube de Laforest, P. G. (1981) Existence of a pool of T-lymphocyte colony-forming cells (T-CFC) in human bone marrow and their place in the differentiation of the T-lymphocyte lineage. Blood 58, 911–5.Google Scholar
- 5.Böyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97, 77–89.Google Scholar
- 6.Turgeon, M. L. (2005) Clinical Hematology: Theory and Procedures. Lippincott Williams & Wilkins, Philadelphia, PA, p. 570.Google Scholar
- 7.Park, C. H., Kimler, B. F., and Smith, T. K. (1985) Comparison of the supravital DNA dyes Hoechst 33342 and DAPI for flow cytometry and clonogenicity studies of human leukemic marrow cells. Exp Hematol 13, 1039–43.Google Scholar
- 8.Hoffman, R. A. and Wood, J. C. (2007) Characterization of flow cytometer instrument sensitivity. Curr Protoc Cytom 1.20, 1–18.Google Scholar
- 10.Donnenberg, A. D. and Donnenberg, V. S. (2005) Phenotypic and functional measurements on circulating immune cells and their subsets, in Measuring Immunity: Basic Biology and Clinical Assessment (Lotze, M. T. and Thomson, A. W., eds.), Elsevier Academic Press, San Diego, CA, pp. 237–56.Google Scholar
- 15.Zimmerlin, L., Donnenberg, V. S., Pfeifer, M. E., Meyer, E. M., Peault, B., Rubin, J. P., and Donnenberg, A. D. (2010) Stromal vascular progenitors in adult human adipose tissue. Cytometry A 77, 22–30.Google Scholar