Appendix 2: Glossary

Analogue-to-digital converter. An electronic chip which changes analogue (continuously variable) signals to digital (discontinuous, binary) signals. In general, electronic circuitry (for example, an amplifier) uses analogue signals, computers work on digital signals.

Aneuploid. Cytogeneticists use the word to describe a cell with an abnormal number of chromosomes. In flow cytometry, aneuploid is used to describe a cell whose DNA content is abnormal.

Autofluorescence. The fluorescence from an unlabelled cell. Cells contain substances which are fluorescent. The amount of autofluorescence observed will depend on the type of cell and the wavelength of excitation. Autofluorescence can limit the sensitivity of immunofluorescence detection.

Back gating. Light scatter is often used to select a particular population of cells by setting a 'gate'  (see below) on a cytogram of side vs. forward light scatter. If the desired population is not clearly apparent on the cytogram of light scatter, a fluorescently-labelled antibody is used to pick out the cells; a gate is set for positive fluorescence and the light scatter of these cells displayed. A region can now be set on the light scatter of the gated cells and used to define these cells on an ungated cytogram of light scatter (see Chapter 4, Section 4.3.2).

Blocker bar. See Obscuration bar.

Break-off point. In a cell sorter, the point at which the stream breaks up to form a droplet (seeChapter 2, Section 2.7).

Coefficient of variation (CV). A dimensionless property which measures the spread of a population distribution (see Chapter 4, Section 4.4.1).

Colour compensation. A correction factor applied to allow for spectral overlap (see below).

Contour plot. A display of a cytogram in which the density of cells is defined by contours (similar to those used on a cartographic map).

Cytogram. A two dimensional histogram in which two cell parameters are correlated.

Deflection plates. In a cell sorter, two plates with a high voltage applied to them (typically 5000 v). The stream of droplets passes between the plates, charged droplets are deflected.

Dichroic mirror. An optical interference filter which reflects one colour and transmits another. These filters either transmit longer wavelengths and reflect shorter (long pass) or vice versa (short pass).

Diploid. Cytogeneticists use the word to describe a cell with the normal number of chromosomes. In flow cytometry, diploid is used to describe a cell whose DNA content is normal.

Discriminator. See Threshold

DNA index (DI). The DNA content of tumour cells in G1 of the cell cycle compared to that of normal cells, hence normal cells have a DI of 1.

Dot plot. A representation of a cytogram in which each cell is represented by a dot on the two dimensional graph.

Droplet delay. In a cell sorter, the time difference between a cell crossing the laser beam and a droplet, containing that cell, being formed (see Chapter 2, Section 2.7).

FCS format. Flow Cytometry Standard format for files of data. The standard is published by the International Society for Analytical Cytology in its journal, Cytometry. FCS 2.0 can be read by all third party software; FCS 3.0 is increasingly being used, particularly for 
multi-parameter digital data.

Flow karyotype. The pattern of chromosomes as revealed by analysis in a flow cytometer (seeChapter 10, Section 10.7).

Gate. A gate is selected by defining a region on a univariate histogram or a cytogram (bivariate histogram). Only cells falling within the gate can pass through to the next stage of analysis (seeChapter 4, Section 4.3). Gates are also used to select desired populations for cell sorting.

Interrogation point. The point in the sample stream at which the laser light is focused. At this point the cells are measured, or interrogated.

Isometric plot. A pseudo-three dimensional representation of a two dimensional cytogram in which cell number is shown on the third (vertical) axis.

Jet-in-air (JIA). See Stream-in-air.

Linear amplifier. An electronic amplifier whose output signal is proportional to the input signal. (See Logarithmic amplifier below).

Logarithmic amplifier. An electronic amplifier whose output signal is proportional to the logarithm of the input signal.

Obscuration bar. A bar positioned to in front of a collection lens to block laser light. In a forward direction, an obscuration bar is used block the primary laser beam after it has passed through the flow cell or sample stream. In 'stream-in-air' instruments, an obscuration bar may also placed in front of the lens which collects 90° scatter and fluorescent light.

Pulse shape analysis. When a cell passes through the laser beam, scattered or fluorescent light is detected and an electronic signal (pulse) is generated. Analysis of the shape of the pulse can give information about the size and shape of the cell (see Chapter 2, Section 2..6.2).

Sheath fluid. The fluid which passes through the flow cell. The sample is injected into the stream of sheath fluid.

Spectral overlap. The fluorescence emission spectrum from one fluorochrome may overlap the emission spectrum of another. For example, the emission spectra of fluorescein and phycoerythrin overlap and cannot be completed separated. (See also colour compensation).

Stream-in-air. This phrase refers to the arrangement of the flow cell in a cell sorter. The laser beam is focused in the stream after it has emerged from the flow cell. Sometimes called jet-in-air (JIA).

Tetraploid. Describes a cell with double the DNA content of a normal (diploid) cell.

Threshold. The electronic circuitry is set to accept information (that is, to 'trigger') only when the input signal exceeds a set level (the threshold). The threshold is usually set on light scatter. Too low a threshold allows the system to accept too much noise, at too high a level some small particles may be excluded. This is sometimes called the discriminator setting.