Flow cytometry antibodies, kits, reagents

Chapter 3: Data Analysis

Two-Parameter or Bivariate Histograms

These are graphs that display two measurement parameters, one on the x-axis and one on the y-axis, and the cell count as a density (dot) plot or contour map. The parameters could be SSC, FSC, or fluorescence.

An example is the dual-color fluorescence histogram in Figure 17. Lymphocytes were stained with anti-CD3 in the FITC channel (x-axis) and anti–HLA-DR in the PE channel (y-axis). CD3 and HLA-DR are markers for T and B cells, respectively. See Figures 15 and 16 for additional examples of two-parameter histograms.

 Two-parameter (dual-color fluorescence) histogram

Figure. 17. Two-parameter (dual-color fluorescence) histogram. FITC, fluorescein isothiocyanate; log comp, logarithmic scale with compensation applied; PE, phycoerythrin.

In Figure 17, R2 encompasses the PE-labeled B cells — note their positive shift along the PE axis. R5 contains the FITC-labeled T cells (positively shifted along the FITC axis). The top right quadrant, R3, would show the cells that are stained for both antibody markers, in this case making them activated T cells. In this sample there are no activated T cells. R4 contains cells negative for both FITC and PE (no shift).

Currently, flow cytometry can be performed on samples labeled with ≥17 fluorescence markers simultaneously (Perfetto et al. 2004). Therefore, a single experiment can yield a large set of data for analysis using various two-parameter histograms.