Authors: Wei Guo & Hong Wu
Human leukemia has been determined and classified with the help of flow cytometry for the past two decades. Past attempts to detect leukemia blasts relied on both forward and side scatter (FSC and SSC) based on cell size and granularity. However, this technique failed to show a clean separation of blasts from normal lineage cells. In 1993, Borowitz, et al developed flow cytometric analysis to distinguish human leukemia blasts from other normal lineage cells by using fluorescence-conjugated CD45 antibodies (1). On CD45-SSC plots, the blasts are distinctly located below granuocytes in human acute myeloid leukemia. This technical advance has significantly improved diagnosis and classification of human leukemia.
Animal models mimicking human leukemia help us make a deeper understanding of leukemia progression and develop more effective therapeutic interventions in human leukemia. Recently, two groups of researchers published Bethesda proposals for classification of nonlymphoid and lymphoid hematopoietic neoplasms in mice (2,3). Similar to the French-American-British (FAB) criteria for human leukemia, the proposed classification depends on idenfication of leukemia blasts and their lineage identities. However, lack of a simple and sensitive methodology to identify leukemia blasts prevents us from characterizing leukemia in animal models and conducting subsequent translational research. Here we adopt the CD45-SSC analysis and characterize blasts and lineage cell populations of WT and Pten null T cell leukemia mice on CD45-SSC plots (4). The blast population is distinct from normal lineage cells, but its location is different from that of human blasts (4). The method is potentially useful for other murine leukemia models.
BD FACScan, FACSCalibur or any other FACS analyzer with more than three-color capability.
It takes 3 hours to harvest cells, stain with antibodies, and run stained cell samples on a FACS analyzer.
Thank James Y. Chen for helpful comments.
Figure 1: Identification of leukemia blasts by CD45-SSC analysis
Bone marrow samples from both WT and leukemia mice were analyzed by CD45-SSC FACS analysis (4). An abnormal blast population was detected in the leukemia mouse and constituted more than 20% of the total leukocytes (left panel), satisfying the FAB criteria for human acute leukemia. The cells in the blast and neutrophil regions were sorted and stained with Giemsa-Wright solution(Fisher, right panel). The sorted blast cells were large, immature, and morphologically distinct from normal lineage cells but similar to human leukemic blasts (right panel).
Figure 2: Characterization of cell populations on CD45-SSC plots
The cell populations on CD45-SSC plots have been characterized for WT and Pten mutant mice at different disease stages: wild-type (WT), chronic phase of myeloproliferative disorder (CP), and T cell leukemia during blast crisis (BC). Note: The lineage cells determined with surface markers are highlighted in pink and their percentages are denoted on FACS plots.
Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation, Wei Guo, Joseph L. Lasky, Chun-Ju Chang, Sherly Mosessian, Xiaoman Lewis, Yun Xiao, Jennifer E. Yeh, James Y. Chen, M. Luisa Iruela-Arispe, Marileila Varella-Garcia, and Hong Wu, Nature 453 (7194) 529 - 533 22/05/2008 doi:10.1038/nature06933
Wei Guo & Hong Wu, University of California Los Angeles
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.165. Originally published online 6 August 2008.