Appendix II J. Flow Cytometry
Flow cytometry consists of a multiparametric analysis of optical properties of individual particles in a fluidic system.
Cells or particles in suspension are individually distributed into a linear array (stream), which flows through a detection device. Solid tissues have to be reduced to a single-cell suspension to be analysed.
The spectrum of parameters measurable by flow cytometry includes volume and morphological complexity of cells or cell-like structures, cell pigments, DNA content, RNA content, proteins, cell surface markers, intracellular markers, enzymatic activity, pH, membrane and fluidity.
It is possible to collect 2 morphological parameters plus 1 or more fluorescence signals per single cell. The multiparametric analysis allows the definition of cell populations by their phenotype.
Apparatus
Focusing, magnifying, and choice of light source are optimised to allow the automatic detection and measurement of morphological differences and staining patterns. Flow cytofluorimetric analysis meets the following criteria:
Flow cytometry is characterised by the automated quantification of set parameters for a high number of single cells during each analysis session. For example, 100 000 particles or more (practically unlimited) are analysed one after the other, typically in about 1 min. The detection limit is as low as 100 fluorescent molecules per cell.
A flow cytometer apparatus has 5 main components:
fluidic system and Flow Cell
The single cell is exposed to the light source and detected in the flow cell. The fluidic system carries the suspended cells individually from the sample tube to the laser intercept point. To achieve this, the sample stream is drawn out to a very thin fluid thread by a sheath fluid in the flow cell (hydrodynamic focusing). The light beam is focused in an elliptical shape, by 2 confocal lenses, into the flow cell channel through which the cells pass. The flow rate must be constant during routine cell surface marker analysis and must ensure a suitable distance between the cells to allow counting.
Light Sources
Commonly used light sources are:
Signal Detection
When a particle passes across the light beam, it scatters some of the light in all directions. Fluorescent dyes, when added to the particle, give off their own light (fluorescence), which is also radiated in all directions. 2 types of signals may thereby be generated:
The instrument′s light detectors collect some of this scattered and fluorescent light and produce electronic signals proportional to the amount of light collected.
Scatter
2 parameters of light scattering are measured:
Forward scatter correlates with the cell volume while side scatter is influenced by parameters such as the shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness, and correlates with the morphological complexity of the cell, so that the higher the SS intensity, the higher the cell complexity. As a function of the morphological characteristics of cells, scatter signals will always be generated during a flow analysis; they are defined as intrinsic parameters.
Fluorescence
Depending on the type and number of light sources, when a cell passes through the sensing area, it will emit fluorescent light. Fluorescence signals are generated from fluorescent dyes naturally present in the cells (for example, co-enzymes, chlorophyll, seaweed pigments) and/or from fluorescent probes taken up by the cells when stained for the analysis of specific characteristics (for example, fluorescent antibodies, nucleic acid dyes, pH probes, calcium probes, fluorescent proteins). Nowadays, there is a large number and a wide range of different types of fluorescent probes available. The optical filters must be adapted to the fluorochromes used and changed if necessary. Each fluorescent probe is characterised by its excitation spectrum and its emission spectrum. They are chosen depending on the nature of the excitation source and the detection system, and according to the specific purpose of the analysis.
Signal Management and Analogue to Digital Conversion
Scatter and fluorescence signals emitted by cells when passing across the laser beam are sorted and addressed to their detectors using optical filters. The detectors are transducers (photomultiplier tubes (PMTs)) that convert light signals radiated from the cells into voltage pulses.
The process of counting each pulse in the appropriate channel is known as Analogue to Digital Conversion (ADC). The process is finally shown as a frequency histogram.
Amplification
Voltage pulses need to be amplified for optimal visualisation. The amplification process accentuates the differences between cell signals, and consequently increases the resolution among cell populations of different characteristics (for example, the differentiation of viable from non-viable cells, or non-specific fluorescence from antigen-specific fluorescence after staining with a fluorescent monoclonal antibody).
There are 2 methods of amplification: linear or logarithmic; the choice between the 2 types is made for every single signal according to the morphological characteristics of the cells and the staining reagents used (for example, fluorescent monoclonal antibodies, nucleic acid dyes).
Linear amplification, which enhances the differences among strong pulses, is used with those parameters that generate high intensity signals, for example:
Logarithmic amplification, in contrast, is for weak pulses and parameters or analysis conditions that may generate both weak and strong pulses, for example:
Compensation of fluorescence signals
Each fluorescent dye has an absorption wavelength spectrum and a higher emission wavelength spectrum. When using 2 or more fluorescent probes simultaneously for staining cells (for example, 4-antigen immunophenotyping), the fluorochromes emission spectra may overlap. As a consequence, each fluorescence detector will sense its own specific fluorescent light and a variable quantity of light emitted by the other fluorescent probes. This results in signal over-evaluation and poor separation of the cell populations.
The solution is in the use of an electronic matrix that allows the selective subtraction of the interfering signals from each fluorescence signal after detector sensing (fluorescence compensation).
Fluorescence compensation requires the use of fluorescence calibrators, preferably positive cell samples stained with the fluorochromes of interest, combined in a manner equivalent to that for the antibody used for the analysis.
Signal Plotting and Display
After amplification and compensation, the signals are plotted in 2 or 3 dimensions. Histograms show the signal intensities versus the cell counts for a given parameter. Cytograms, in which each dot represents a cell, result from the combination of 2 signal intensities (dual-parameter dot plots). The type and number of plots and signal combinations are chosen on the basis of the specimens and dyes used. When analysing acquired data, the flow cytometry software can also generate other kinds of graphs (such as overlays, surface plots, tomograms, contour plots, density plots, overlay plots). Statistical data such as mean fluorescent intensities (and their shifts in time or their dependence on cell function) can also be used.
Data Analysis
Different kinds of cell populations may be present inside the cell suspensions to be analysed, some of which are unwanted (such as dead cells, debris or macro-aggregates), or simply not relevant for the analysis (for example, granulocytes when studying lymphocytes). This depends on the cell sample type (whole blood, bone marrow, cell cultures, biological fluids, cell suspensions from solid tissues) and on the handling procedures (for example, staining methods, lysis, fixation, cryopreservation, thawing, paraffin-embedded tissue preparation).
As a consequence, not all the signals generated during a flow cytometry analysis belong to the cells to be studied. 2 strategies are adopted to exclude unwanted and irrelevant cell signals.
The 1st is used during data acquisition. It is a noise threshold, applied to 1 (or more) significant parameter(s), set to acquire only the cells with signal intensities higher than the pre-defined discrimination value for that parameter. Due to its characteristics of a strong signal with a low grade of interference, forward scatter is the parameter most often used as discriminator.
The 2nd, applied during data analysis, consists of the use of gating regions to restrict the analysis only to signals from those populations that satisfy given morphological and expression profile characteristics. 2 types of logical gating are commonly used. The 1st is the morphological gate. The cell populations are identified using their morphological signals (FS and SS). A region gate is drawn around the population of interest (for example, lymphocytes, viable cells) then the fluorescence plots are gated into the selected region. The 2nd is the fluorescence-based gate. The cell population of interest is identified on the basis of the expression intensity of an antigen or a dye, then a gate region is drawn around it. Afterwards the fluorescence plots are gated into the selected region.
The analysis software allows the creation of multiple gate regions, using a sequential logic order. This feature is especially useful when studying rare cell populations or for sorting purposes.
CONTROLS
Internal control
The system’s optical alignment must be validated before analysis using adapted fluorospheres and the optimum fluidic stability is checked. The data obtained are reported and allow the periodical review of control values against the mean performance value. A positive control is highly desirable to prove that the test antibody is functional and to allow the proper setting of the flow cytometer. The positive control must include samples known to be positive for the marker of interest.
External control
To ensure reliability in the data obtained or to check inter-laboratory reproducibility, participation in a proficiency testing study is recommended.