Hematology was one of the first medical disciplines to benefit from the clinical applications of CMF. Some of these applications are now regularly used for the diagnosis or therapeutic follow-up of various diseases. These applications concern the functional study of healthy cells as well as the demonstration of the pathological character of the analyzed cells.

The combination of immunofluorescence and flow cytometry (FCM) has become an essential element in the study of biological systems, especially in the discrimination between cells of a heterogeneous population. Indeed, the use of monoclonal antibodies (mAbs), directed against specific components, allows to distinguish lymphocyte subpopulations. In recent years, the identification and characterization of lymphocyte surface antigens (Ag) has progressed very rapidly. However, there is no single mAb that can recognize a particular cell with certain functions. This limitation has led users of the immunofluorescence technique to move from single to multiple labeling using combinations of mAbs revealed by different fluorochromes.

Compared to the initial investigations performed under the microscope, FCM also provides quantification of the number of recognized sites at the individual cell level and the possibility of sorting these cells according to the intensity of their labeling for further functional studies. Finally, it can be combined with the analysis of other parameters (cell cycle, Calcium) and thus inform us on the functional state of the cells.

Figure 1 : Emission spectra of 3 fluorochromes used in immunofluorescence.(© Becton Dickinson, Spectra Viewer)

For multiple labeling several fluorochromes are used simultaneously.
In this case, it is necessary that:

-their excitation wavelengths match the light sources of the cytometer,

-their emission wavelengths are sufficiently distant so that their signals can be analyzed separately.

-the weakly expressed antigens are revealed by high yield fluorochromes and the strongly expressed antigens with low yield fluorochromes.

-in the case of co-expression on a cell, use fluorochromes with little or no overlapping spectra.

The major obstacle in multiparameter analyses is the significant overlap in the emission spectra of the fluorochromes used to reveal the mAbs.
The introduction of multiple lasers in cytometers has made it possible to overcome such interference by making possible the excitation of several fluorochromes with distinct excitation and emission spectra.
To facilitate the choice of fluorochromes, there are many tools proposed by private companies:
Becton Dickinson, Thermo Fischer, BioLegend, Chroma...

Spectral cytometry, newly arrived on the market, significantly improves the separation of the various fluorochromes. It also offers tools to characterize fluorochromes:
Cytek

Before starting any immunolabeling protocol it is important to verify the quality of the reagents used by testing them on cells known to present the antigen being analyzed.
The concentration at which the reagent is effective should be tested by labeling these cells with various concentrations of the reagent and plotting the labeling intensity as a function of this concentration (Figure 2):

Overlapping emission spectra of the various fluorochromes used in cytometry require the use of electronic fluorescence compensations to subtract the superposition of the two fluorescence signals (Figure 3).

Figure 3 : Fluorescence overlapping.

Thus, without fluorescence compensation, a cell population labeled in green fluorescence (FITC) but not labeled in orange fluorescence (PE) is positioned on the bisector of the biparametric histogram of the two fluorescences (Figure 4a).
The compensation system artificially subtracts the orange fluorescence that results from the overlapping of FITC fluorescence into the PE channel (Figure 4b).

Figure 4 : Effect of fluorescence compensation

Each compensation percentage will thus be determined by analyzing the cells simply labeled with the various dyes. The negative cells should be adjusted correctly (A) and then the median over y of the positive population over x (B) should be adjusted so that it is equal to the median over y of the negative cells (Figure 5).


Determination of the positivity threshold

The common practice for determining a threshold of positivity is to use isotypic 'tagging' and position a cursor at the foot of the negative cell peak. This practice is not recommended for multiple labeling. Indeed, when more than one fluorescence is present and the compensation is set, the negative value of the labeling may be greater than in an unlabeled tube. To overcome this problem, the FMO (fluorescences minus one) method should be used, which consists in marking the cells with all the markers present in the study except the one of interest (Figure 6).