Flow cytometry

8.5 Flow cytometry

Fluorescence is the emission of photons by molecules that have absorbed light. Electrons move from a ground to an excited state, and on return to the ground state, a photon is emitted, of lower energy than the excitation light, which is represented by an increase in wavelength. The excita- tion of a fluorescent material, e.g. fluorescein by blue light, leads to an emission of green light, but the reverse is not possible. The shift in wavelength is an important and useful feature, since it is possible to excite several fluorescent dyes (used to label proteins, etc.) simultaneously by a single excitation wavelength, in say, the ‘blue’ region, yet by choosing dyes with different emission wavelengths. It enables separate information to be retrieved from the emissions from each dye, using selective filters for different wavelengths (see Fig. 8.5).

In flow cytometry, cells are channelled into a thin liquid stream through the passage of a narrow beam of light, these days normally a blue (488 nm) laser beam. As individual cells pass through the laser beam photomultipliers situated around the plane of the laser, two types of information may be derived: (i) scattering of the excitation (488 nm) laser beam; and (ii) photons

8.5FLOW CYTOMETRY

Pressure difference between two buffer inlets

PMT 3 Cells in buffer

PMT 2

PMT 1

Sheath buffer Photodiode

Light beam split by Forward-angle

dichroic mirrors collecting lens

nm

90 collecting lens

lon ger

with obscuration bar

Obscuration bar Beam

and

dump

Beam-shaping lenses Liquid carrier stream

Laser

nm only

To waste

Forward-angle light scatter

90 light scatter

Carrier stream Sheath fluid

Incident laser beam

Cell

C H A P T E R 8: Lymphocyte structure C H A P T E R 8: Lymphocyte structure

There are two types of light scattering:

1 forward scatter (Fsc), the degree of which is related to cell size; and

2 side scatter (Ssc) which is related to granularity. These scatter characteristics may be utilized to display and define the cell type being investigated in a mixed population of cells. Such cells can be selected or gated to specifically examine the fluorescence on the surface of the chosen population.

The flow cytometer can analyse the distribution of antigen expression on mixed cell pheno- types to provide discrete information on each population (see Fig. 8.8). Information from up to three fluorescent dyes can be monitored in selected cell populations, usually green-, orange- and red-emitting dyes.

In flow cytometry, generally directly labelled monoclonal antibodies to a particular antigen are used (but this is a relatively insensitive method of labelling). Sensitive photomultipliers are used to detect the light emitted by the entire cell. It allows the simultaneous analysis of three cellu- lar molecules using different labels. Under these circumstances the three labelled antibodies can

be incubated simultaneously with the cell. The green-emitting label is fluorescein or some close derivative. The orange-red is phycoerythrin, although in common use it is described as red. The third colour is more difficult and examples are PerCP (Becton-Dickinson) and Badshaw’s Tri- colour. Some of these stains are relatively simple to couple in the laboratory, e.g. fluorescein, others are not. Usually the development of dyes for flow cytometry is designed around an assumption that the most commonly used laser for excitation is the blue 488 nm.

Fig. 8.5 (opposite) The continuous-flow cytometer. Cells, in dilute suspension, are injected into the centre of a plastic nozzle through which a stream of sheath fluid flows continuously. As the cell and sheath buffer inlets are at different pressures, the concentric buffer streams emerging from the nozzle run at different rates and therefore do not mix; the cells are thus constrained at the centre of the carrier stream. Under stable conditions, each cell should follow virtually the same path.

Laser light, usually from an argon ion laser, is passed through a set of shaping lenses to produce a beam with an elliptical cross-section, which is aimed at the buffer stream falling to waste. The buffer stream acts as a vertical cylindrical lens and disperses a small proportion of the laser light in the same horizontal plane as the beam. However, the majority of the light continues forward through the buffer stream and is absorbed harmlessly by the cylindrical beam dump. The optical system is aligned so that, provided the laser beam does not hit a cell, no light signal is recorded by the instrument; the horizontally dispersed light is absorbed by obscuration bars placed across the front face of the collecting lenses in the forward angle and at 90° to the incident beam.

Any cell interacting with the laser beam acts as a spherical lens and disperses the light in all directions out of the horizontal plane (shown in the inset). Light is collected by detector systems placed in the direction of travel of the beam (forward-angle light scatter) and at right angles to the direction of travel (90° light scatter). The cell is labelled with one or more fluorochromes. The emitted light that is shifted to a longer wavelength, is resolved from the original exciting wavelength of the laser (for example, 488 nm for the laser and fluorochrome combinations most frequently used) by a combination of dichroic mirrors and long- and short- pass filters. The filters direct the light to a series of photomultiplier tubes (PMT 2 and PMT 3 in the figure). PMT 1 is being used in this case for the detection of the 90° light scatter signal. The integral of the electrical impulse thus generated is digitized and stored or analysed in a computer.

Although the laser beam can be of sufficient intensity to burn a hole in a piece of card, the high thermal capacity of water protects the cells from harm. Viable cells can be sorted using this instrument.

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1 or 10% transmission

FITC and PE

neutral density

stained cell

filter

488 nm

Forward-angle light scatter detector

Laser

495 nm long-pass dichroic mirror

90 light scatter PMT

Interference filter

515 nm

Absorbance filter 560 nm short-pass

dichroic mirror Phycoerythrin PMT

550 nm short-pass 570 nm long-pass filter

filter

Fluorescein PMT

Fig. 8.6 Lens and mirror combinations used to resolve the signals emitted by stained cells. Laser light is monochromatic, stable and very powerful. Thus even subtle shifts of wavelength and minor changes of direction caused by refraction through a single cell can be detected by judicious selection of filters and dichroic mirrors. In principle, light near the original exciting wavelength is used to measure forward-angle light scatter (FALS) (using a photodiode placed in the forward angle) and 90° light scatter (a weaker signal so it is detected using a photomultiplier). Thereafter, light of the exciting wavelength is stripped off using a pair of 515-nm filters which pass only longer (emitted) wavelengths. The dichroic mirror placed at an angle of 45° to the light beam, and after the stripping filters, directs long wavelength light to the photomultiplier set to detect red light (phycoerythrin PMT). Shorter wavelengths are not interrupted in their passage to the photomultiplier set to detect green light (fluorescein PMT). As the mirror does not split the wavelength perfectly, it is ‘backed- up’ by long- and short-pass absorbance filters, as shown in the diagram. The final traces of ‘breakthrough’ between the red and green channels can be removed by electronic processing of the photomultiplier signals.

Fig. 8.7 (opposite) Multiparameter cytometry. (a) and (b) show an isometric (three-dimensional) and planar (two-dimensional) projection of the same data. Whole blood was stained with fluorescein- and phycoerythrin- conjugated antibodies, the erythrocytes lysed by osmotic shock and the resulting cell suspension washed by centrifugation. (a) In this histogram, forward-angle light scatter (FALS) is plotted along the y- (rear) axis; 90° light scatter (90°-CLS) along the x- (left) axis and frequency on the z- (vertical axis). Although the relative numbers of cells in the different populations may be easily appreciated from this type of display, the nature of the populations (in terms of the parameters being measured) is more easily appreciated from a planar view, as in (b). (b) An approximation to the relative abundance of the different populations is achieved in the two-dimensional display by different pixel densities, representing three selected ‘levels’ in the frequency data. The four populations of cells marked in the figure have the following characteristics and identity:

1 low FALS and 90°-CLS ared-cell ghosts and platelets; 2 medium FALS and low 90°-CLS alymphocytes; 3 high FALS and low 90°-CLS (also low abundance) amonocytes; 4 high FALS and high 90°-CLS agranulocytes.

Electronic gates were set around the ‘lymphocyte’ population which was then analysed for the presence of T- and B-lymphocyte markers. Even though other cells are present, both stained and unstained, they are now ‘gated out’ from further analysis.

Histograms (c) and (d) show three- and two-dimensional plots of fluorescent emissions of the cells examined in (a) and (b). (c) It is possible to discern three distinct populations: one massive population in the centre of the distribution (1), and more minor populations on the x- (2) and y- (3) axes. The x-axis corresponds to the red fluorescence associated with an antibody against a T-cell marker; whereas the y-axis corresponds to the green fluorescence associated with an antibody against a B-cell marker. The three cell populations, more easily seen in (d), have the following characteristics and identities:

1 high red and high green fluorescence athis is a population of B lymphocytes (see below) expressing a T-cell marker;

2 high red, low green athis is the staining pattern of the normal blood T-lymphocyte population; 3 low red, high green athis is the staining pattern of the normal B-lymphocyte population in the blood;

however, in this patient, it is a very minor population. These cells are derived from a patient with B-cell chronic lymphocytic leukaemia and so contain a monoclonally expanded population of aberrant B lymphocytes. The residual normal B lymphocytes aon the y-axis in (c)ahave been virtually replaced by leukaemic cells, whereas the T-lymphocyte population is virtually unchanged. This type of qualitative and quantitative analysis can provide crucial data for haematological diagnosis of disease.

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(a) Forward-angle light scatter (b) 90 light scatter

(c) Control (d) Positive immunofluorescence

27.05 16.07 CV CV 83.39 23.62

T cells 1P256 negative

T cells 1P256 positive

Channel 14 to 255 Integral 538 Peak 37 at 18% In interval 5.32

Channel 14 to 255 Integral 10037 Peak 289 at 71% In interval 98.02

Fig. 8.8 Flow cytometry parameters. The data in the figure were derived from the analysis of the T-cell line CEM, in each case analysing 10 000 events. Forward-angle light scatter, 90° light scatter and fluorescent data are plotted as frequency histograms using an ascending logarithmic horizontal scale divided into 256 channels. (a) Forward-angle light scatter (FALS). The intensity of the FALS signal is directly proportional to the volume of the cell being measured. The data have a clear bimodal distribution. There is a minor population of particles with a very low FALS signal (extreme left of distribution), probably due to cell debris and inorganic particles (described in flow cytometry jargon by a variety of epithets of Anglo-Saxon derivation), and a major population with a peak value just midway along the axis athese are the viable CEM cells. Although the standard polystyrene beads used to set up the instrument would have given a FALS distribution with a very low coefficient of variation, living cells aeven from a cloned cell lineashow marked variation in cell size. It is possible to examine a proportion of the above population only by setting an electronic ‘gate’. In this instance the lower gate would be set to the right of the minor population, and the upper gate to the extreme right of the major population. It is then possible to confine the measurement of a second parameter purely to those cells falling within the preset FALS values: they are ‘gated in’.

(b) 90° light scatter (90°-CLS). This signal is proportional to the volume of the cell, but is also affected by other parameters such as granularity, surface topography, etc. Although in this instance, where we are using a tumour-cell line with a homogeneous cytoplasm, this parameter yields little additional information over FALS alone, its true value may be appreciated with reference to Fig. 8.7(a,b).

Histograms (c) and (d) show the relative fluorescent intensity of cell populations stained with a directly conjugated irrelevant monoclonal antibody (c) or a pan-T monoclonal antibody (d). In each case, we are examining the fluorescence associated with the FALS ‘gated-in’ population alone. (c) The majority of the signals given by the cells in this population could not be detected above the electronic ‘noise’ of the instrument. In order to set the fluorescent intensity limits for this negative control, it is convenient to set the lower cursor (depicted as vertical line) to a channel number which excludes (to the left) about 95% of the population in the example shown. Around 5.3% of the cells fall in the interval between channels 14 and 255 (to the right of the cursor). (d) This population of cells has been analysed with the same instrument settings as in (c), and 98% of the 10 000 cells analysed gave a fluorescent signal falling in the channel interval defined in (c). They are the specifically stained population. The computing software used on this instrument can use much more precise definitions of the positive and negative populations and carry out a ‘channel-by-channel’ analysis to obtain full information from the data.

In examples (c) and (d) we have used electronic cursors to define the fluorescent population only for analysis and integration. It is possible, however, to define a ‘gated-in’ population, either alone or with reference to the FALS gates already set from histogram (a). One may then examine a third parameter in relation to cells of a certain size (perhaps lymphocytes rather than platelets) and with a certain fluorescent attribute (for example, pan-T-positive lymphocytes in a population of peripheral blood mononuclear cells). The ability to measure using correlated parameters on a per cell basis provides these instruments with exquisite analytical and preparative capabilities.

The emergence of flow cytometry as an important research and diagnostic tool is the result of three major developments: (i) the generation of monoclonal antibodies to a variety of cell antigens, used for the cell phenotyping; (ii) characterization and analysis of the functional significance of cell subsets and cell receptors; and (iii) the development of instrumentation which is relatively cheap, reliable and simple to use.

Typically, cells may be analysed at a rate of several hundred/second, but flow rates an order of magnitude higher are within the capabilities of the instruments.

MATERIALS Cells to be investigated Labelled antibody 2% paraformaldehyde in phosphate-buffered saline (PBS)

METHOD

1 Centrifuge 2 × 10 5 cells at 250 g for 5 min at 4°C. Discard the supernatant and agitate the pelleted cells.

2 Add 10 µl of each labelled antibody and incubate at 4°C for 30 min.

3 Centrifuge the mixture of cells and antibody at 250 g for 5 min at 4°C. Discard the supernatant and agitate the pelleted cells.

4 Fix cells with 2% paraformaldehyde in PBS.

5 Cells may be stored in the dark at 4°C for up to 1 week.

Technical applications Clinical uses: immunophenotyping, cellular deficiencies, tumour-cell detection and tumour

progression, cell kinetics, chromosomal analysis, and the analysis of ploidy. Research interest: analysis of receptor distribution, cell surface and intracellular molecules (see

Section 4.5.1 for methods on permeabilization of cells in preparation for flow cytometry), changes in expression, functional analysis of receptor behaviour.