Editorial: Harnessing the Power of Multicolor Flow Cytometry

2012, Volume 22, Number 3


William G. Finn, M.D.

In this issue of the Warde Report, Dr. John Carey, scientific director of flow cytometry at Warde Medical Laboratory, provides details of the changes involved in our recent deployment of 6-color flow cytometry. This enhancement is likely to bring greater value—both economic and medical—to Warde laboratory clients and co-tenants. The expansion of polychromatic flow cytometry platforms does more than simply reduce the number of assays (“tubes”) necessary to run a given analysis. It also optimizes the ability to isolate specific cell types, both in reactive and neoplastic diseases, that are defined by specific simultaneous combinations of multiple markers rather than by hierarchical lists of expressed antigens.

Global patterns of marker expression, including expected sequences of marker acquisition based on higher dimensional analysis, are more important than simply the list of markers expressed by a given cell type. In polychromatic flow cytometry, each measured parameter is analogous to a spatial coordinate, and the aggregate of many parameters forms in essence a high dimensional “shape.” 1 The familiar histograms of a flow cytometric analysis are effectively 2-dimensional projections of a higher dimensional space. Just as we gain more information by walking through a 3-dimensional room than we do by examining a 2-dimensional snapshot of that room, we gain more information about cell populations when we can characterize them based on aggregate phenotypes of numerous markers, analyzable from many different vantage points in a higher dimensional analysis.

For many years, flow cytometry has been a valuable tool in the detection of abnormal cell populations, often in very low numbers, in the background of normal cellular consituents. This function can be enhanced when panels are designed around not only the likely characteristics of the abnormal cells being targeted, but also around the normal cellular constituents of a given sample. For instance, panels capable of discerning the maturation patterns of normal B-lymphocyte precursors (hematogones) in bone marrow may more effectively enable the interpreter to detect abnormal B-lymphoblasts in acute lymphoblastic leukemia. 2 Likewise, the ability to isolate normal germinal center B-lymphocytes in a lymph node sample may allow for a more sensitive detection of partial lymph node involvement by lymphoma.3 Similar approaches allow distinction of normal from leukemic myeloblasts, 4 and so on. These types of scenarios are better enabled by more available fluorescence channels (colors) that allow a fuller immunophenotyping of normal maturation patterns based on aggregate marker combination patterns.

Polychromatic flow cytometry (10-color and beyond) has been used in the research setting for some time, but the diagnostic utility of polychromatic flow cytometry is likely to expand as clinical laboratories gain more experience with higher dimensional analysis in clinicalgrade instruments. For instance, the use of flow cytometry in the diagnosis of myelodysplastic syndromes — now somewhat controversial 5 — is likely to become more mainstream as more laboratories deploy higher dimensional analyses allowing for standardization of myeloid maturation patterns in healthy and diseased bone marrow.

In the era of genomics and personalized medicine, the continued development of more and more “colors” in diagnostic flow cytometry is resulting in the transition of this technology toward an iterative, cytomic discovery tool that will allow us to keep pace with advances in diagnostic classification and therapeutic monitoring.


  1. Finn WG: Beyond gating: capturing the power of flow cytometry. Am J Clin Pathol 2009;131:313-314.
  2. McKenna RW, Washington LT, Aquino DB, et al: Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry. Blood 2001;98:2498-2507.
  3. Reichard KK, McKenna RW, Kroft SH: Comparative analysis of light chain expression expression in germinal center cells and mantle cells of reactive lymphoid tissues. Am J Clin Pathol 2003;119:130- 136.
  4. Harrington A, Olteanu H, Kroft S: The specificity of immunophenotypic alterations in blasts in nonacute myeloid disorders. Am J Clin Pathol 2010;134:749-761.
  5. Wells DA, Ogata K: On flow cytometry in myelodysplastic syndromes, with caveats. Leuk Res 2008;32:209-210.