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Glossary

3-dimensional analysis or 3-D plots or Histograms – Data display for flow cytometric data. Terms “histogram” and “plot” can be used for 3 parameter (multivariate) graphs. Also known as Multivariate Plots.

Absorbance filter – Dyed or colored glass. Works by absorption of unwanted wavelengths. Can fluoresce. Inexpensive—not often used alone.

ADC – Instrument compartment that converts the electrical signal (stream of electrons) into numbers. Also known as Analog-Digital Conversion.

Amplification – Increase of the signal detected to adjust signal intensity.

Analog-Digital Conversion – Instrument compartment that converts the electrical signal (stream of electrons) into numbers. Also known as ADC.

Analyzers – (When used in terms of flow cytometry) Instruments used to measure properties of particles (whole cells, nuclei, chromosomes, diatoms, plankton, bacteria, viruses) by moving these particles through a detection chamber.

Aneuploid – Sample from whole cells or nuclei containing more (or less) DNA than a normal cell. Proper term is “DNA Aneuploid”. Has a DI less than 2.0 or greater than 2.0

Aneuploid Fraction – % of the total cycling population that is aneuploid

Antibody affinity – Measurement of how strongly an Antibody attaches to only one antigen or epitope.

Antibody Conjugation – Molecular attachment of a fluorochrome to an antibody.

Antibody specificity – Measurement of how selective an Antibody is. How well an antibody differentiates one antigen or epitope from another antigen or epitope.

Antigen – Molecule on a cell we are trying to identify or collect.

Autofluorescence – Fluorescence associated WITH the Cell, caused usually by components and chemicals within the cell structure itself.

Back Gating – Gating on populations in one histogram or plot and then examining other plots to see where that population falls. Useful for example is determining where a particular fluorescent population falls within the light scatter gates. Some software does not require grating. These packages show the population in the other parameters by coloring related events identified by a region in a single histogram.

B.A.D (Background, Aggregates and Debris) – Fraction of the histogram from the first G0G1 to the last G2M calculated to contain background, aggregates and debris. Measures the amount of background, aggregates and debris within the cycling part of the histogram. If aggregates are gated rather than modeled, B.A.D. in invalid and can NOT be used.

Band pass filters – Transmit wavelengths in a narrow range around a specified wavelength indicated in the specifications of the filter.

Boolean or Complex gates – Gates set by adding or subtracting populations – utilizes the ‘and’, ‘not’ and ‘or’ (Boolean) functions when defining the population of choice.

Bivariate Plots or Histograms – Data display for flow cytometric data. Terms “histogram” and “plot” can be used for two parameter (bivariate) graphs.

Bleed over Fluorescence – Also known as Cross over or Spill over fluorescence, or See “Spectral overlap”.

Capped surface staining or Capping – Antigens when attached to antibodies migrate to one end of the cell and can be internalized by the cell.

CDs –See Cluster Designations. Also known as Clusters of Differentiation.

Channels – 1) Bins or Columns where the events are assigned based on their fluorescence or light scatter, which collect the measurements varying in intensity.

2) the ‘Parameters’ themselves; such as the “Green Channel”

Cluster Designations – Terms designated by the International Workshop on Human Leukocyte Differentiation Antigens to name antigens found on the surface of immunological cells, used to identify a cell type or subset. Also known as CDs or Clusters of Differentiation.

Clusters of Differentiation– See Cluster Designations. Also known as CDs.

Coincidence Detection – Process for the detection of two or more ‘events or incidences’ occurring within the same proximity during sorting.

Compensation – Removal of undesired signal cross over from a channel designated for another fluorophore. This process should not be referred to as Subtraction. (Histogram subtraction is a means of obtaining information about the difference between two histograms, eg Sample histogram –Test histogram.)

Complex or Boolean gates – Gates set by adding or subtracting populations – utilizes the ‘and’, ‘not’ and ‘or’ (Boolean) functions when defining the population of choice.

Confocal pinhole alignment – Alignment using the same number of pinholes as the number of light sources.

Conjugation – (When used with Antibody) molecular attachment of a fluorochrome to an antibody.

Contour Plot – Data analysis plot, valuable to show the change in the number of events at a location on the plot. As the number increases the color changes, the contours get closer together. Similar to a topographical map.

Cross over Fluorescence – Also known as Bleed over or Spill over fluorescence, See “Spectral overlap”.

CV or Coefficient of Variation – Statistic that is the standard deviation / mean. Indicates how concise the population is / how well the procedure was executed.

Cytometer – Apparatus used to measure cells.

Cytometry – Measurement of physical/chemical characteristics of cells or other biological particles.

Density Plot or pseudo-color Plot – Data analysis plot, valuable to show the change in the number of events at a location on the plot. As the number increases the color changes, usually from cooler blues to more intense reds and oranges.

DI – see DNA Index.

Dichroic filters – Filters that pass one signal and reflect another – useful when you need both signals. Also known as Dichroic mirrors.

Dichroic mirrors – Filters that pass one signal and reflect another – useful when you need both signals. Also known as Dichroic filters.

Diploid – Sample from whole cells or nuclei containing a normal complement of DNA. Proper term is “DNA Diploid”. Has a DI of 1.0.

DNA Index – Measurement indicating the amount of shift from one G0G1 peak to another G0G1 peak. Normally this is the ratio of the Aneuploid G0G1 peak / Diploid G0G1 DNA peak. Also known as DI.

Doublet discrimination – Discrimination between single cells and cells clumped together (aggregates) utilizing the width of the pulse and time of flight, width of the pulse and height of pulse or width of the pulse and area of pulse. When histogram dot plots are display for these parameter combinations, doublets are suppose to fall off the primary axis of population. This technique does not differentiate between aggregates and elongated cells so some normal and tumor cells may be identified incorrectly. Technique is also subject to variations in gates set by user so reproducibility may be an issue.

Enrichment – Sorting with high recovery first to ensure the collection of all events prior to a sort for purity.

Epitope – Single site on an antigen.

Extrinsic fluorescence – Fluorescence added to the Cell.

FACS – Registered trademark of Becton Dickinson, stands for Fluorescence activated cell sorting.

Flow Cytometer – Apparatus used to measure the properties of single cell suspensions as they flow through the instrument.

Flow Cytometry – Measures cells in fluid suspension as they pass one by one through the measurement apparatus called a Flow Cytometer.

Flow cytometry standard (FCS 2.0 or 3.0) File format – format used to save flow cytometry data. Includes identifying information about the sample in the “header” and measurement information for each cell or event analyzed. The header is in text format while the measurement information is in binary format.

Flow cell – Apparatus where the sheath and sample meet, also known as the flow chamber. Can be one of several types; jet-in-air, quartz cuvette, or a hybrid of the two.

Flow chamber – Apparatus where the sheath and sample meet, also known as the flow cell. Can be one of several types; jet-in-air, quartz cuvette, or a hybrid of the two.

Fluor – Fluorescent substance used in biological staining to produce fluorescence in a specimen. Also known as Fluorochrome, Fluorescence probes, or Fluorophore.

Fluorescence – Excitation light energy is absorbed by fluorescent molecule, the molecule transitions to an excited state and as it returns to unexcited ground-state, a specific wavelength of light is emitted.

Fluorescence Intensity Statistics – Statistics used in measurement of the fluorescence. Include Arithmetic and Geometric Mean, median, peak channel or mode.

Fluorescence Minus One Controls – Controls used to help establish a cutoff for “negativity” in multiply stained samples. Cells are stained with all the reagents except one. Also known as FMO controls.

Fluorescence probes – Also known as Fluorochrome or Fluorophore. See “Fluor”.

Fluorochrome – Also known as Fluorescence probes or Fluorophore. See “Fluor”.

Fluorophore – Also known as Fluorochrome or Fluorescence probes. See “Fluor”.

Fluorophore Excitation / Absorbance – Light absorbed by the fluorochrome.

Fluorophore Emission / Fluorescence – Light given off or emitted by the fluorochrome.

FMO Controls – Controls used to differentiate positive from negative. Cells are stained with all the reagents except one. Also known as Fluorescence minus one controls.

Gates – Areas designated around populations in order to identify events of interest..

Hierarchical gates – Organize gating information as “tree”- much like a family tree. For example; Lymphocytes are gated and from that gate, T or B cells, and from those gates, subsets of T and B cells – three generations, parent, child and grandchild.

Hydrodynamic focus – Focusing of a sample injected into a stream of sheath fluid as it passes through a small (70-200 µm) orifice, so that the sample fluid flows in a central core that does not mix with the sheath fluid. This flowing together is termed Laminar flow.

Hypo-Diploid – Sample from whole cells or nuclei containing a cell cycle with less than normal complement of DNA. Proper term is “DNA Hypo-Diploid”. Has a DI less than 2.0.

Immunophenotyping – Classification of cells by evaluating combinations of antigen expression.

Intercept – Point at which the laser intersects the stream, illuminating the cells – where they emit their fluorescence and scatter the light. Also known as Intersection point, Laser intersection point, Interrogation point.

Interference filters – Mirrors – constructed from multiple deposited dielectric coatings (very thin metal layers) sandwiched together. Unwanted wavelengths eliminated by internal interference.

Interrogation point – Point at which the laser intersects the stream, illuminating the cells – where they emit their fluorescence and scatter the light. Also known as Intersection point, Laser intersection point, Intercept.

Intersection point – Point at which the laser intersects the stream, illuminating the cells – where they emit their fluorescence and scatter the light. Also known as Interrogation point, Laser intersection point, Intercept.

Intrinsic fluorescence – Fluorescence that comes WITH the Cell, including autofluorescence.

Laminar flow – Sheath and Sample fluids stream together through the flow cell without mixing

Laser intersection point – Point at which the laser intersects the stream, illuminating the cells – where they emit their fluorescence and scatter the light. Also known as Intersection point, Interrogation point, Intercept.

Light scatter – Light deflected by any object as it passes through the laser beam.

Linear – Data display where one increment indicates a linear change in the data. Useful where there is a one to one ratio of fluorescence to cell characteristic, such as in DNA analysis.

Linearity factor – Typically 2.0. Amount of DNA in G2/M is 2x the amount in the G0/G1 cells, so the G2/M peak is located twice as far out on a linear scale than the G0/G1 peak.

Logarithmic – Data display where one increment indicates a 10 fold change in the data. Log scaling is valuable for immunofluorescence since there is a need to display a wide dynamic range of fluorescence when evaluating surface markers found on cells.

Long pass filters – Filters that transmit wavelengths above a certain wavelength indicated on the specifications of the filter

Markers– Lines drawn around populations in order to distinguish them from other populations and to collect data about that population. Also known as Regions.

Modeling – Analysis of cell cycle components, calculates the % of each phase of the cell cycle.

Monoclonal – Antibody made to one (mono) epitope on the antigen.

Multivariate Plots or Histograms – Data display for flow cytometric data. Terms “histogram” and “plot” can be used for 3 parameter (multivariate) graphs. Also known as 3-dimensional analysis or 3-D plots.

Parameter – Each measurement from each detector. Include such measurements as light scatter and fluorescence area, height, and peak.

Peak fluorescence – Measurement of the fluorescence emitted from a fluor at the highest amount or number of events present.

Piezo Electric Crystal – Apparatus that vibrates up to 200,000 times per second to break up the stream into droplets for sorting.

Polyclonal – Antibody made to many (poly) epitopes on the antigen.

Population Statistics – Percentage of population in a sample that is positive for an antigen or the Percentage of population that is positive for an antigen within a certain gate or region.

Preamplification – Process in the electronics to strengthen signals so that they can travel from remote detectors to central electronics.

Probes – Substances (as DNA in genetic research) used to obtain specific information for diagnostic or experimental purposes (may or may not be fluorescent).

Pseudo-color Plot or Density Plot – Data analysis plot, valuable to show the change in the number of events at a location on the plot. As the number increases the color changes, usually from cooler blues to more intense reds and oranges.

Pulse Area – Area under the electronic pulse. Also known as integral.

Pulse height – Height of the electronic pulse.

Pulse width – Width of the electronic pulse. Also known as time of flight.

Purity – Recovery of the sample with the least contamination from unwanted cells or particles.

Quantum efficiency – (When used with Antibodies) the amount of excitation necessary to give off the largest number of photons. Generally the higher the quantum efficiency, the better.

Recovery – Highest number of desired cells or particles recovered, regardless of contamination from other, unwanted cells or particles.

Regions – See Markers.

Resonance Energy Transfer – Process by which a fluorescent molecule can be excited and transfer this energy to a nearby other fluorescent molecule which then emits. Used in Tandem dyes.

Sheath flow rate – Volume of sheath that passes through the cytometer in a given period of time.

Short pass filter – Filter that transmits wavelengths below a certain wavelength indicated on the specifications of the filter and reflect light above.

Single pinhole alignment – Alignment using one pinhole for all light sources.

Sample flow rate – Rate at which the sample flows. Variable and regulated by the difference in pressure between sample and sheath.

Sorter – (When used in terms of flow cytometry) Apparatus that analyzes and separates or sorts particles passing through.

Specificity – (When used with Antibody ) measurement of how well an Antibody attaches to only one antigen or epitope without contamination from other antigens or epitopes.

Spectral overlap – Light emitted from one fluorochrome can be measured by several detectors. Also can be known as Cross over, Bleed over, or Spill over Fluorescence.

Spill over Fluorescence – Also known as Cross over, Bleed over, or See “Spectral overlap”.

Stoichiometry – Relationship of an antibody and a biological variable such as a fluorochrome. For instance, a 1 to 1 fluorochrome to antibody (F:P ratio).

Stokes Shift – Energy difference between the lowest energy peak of absorbance and the highest energy of emission

Subtraction– A means of obtaining information about the difference between two histograms, eg Sample histogram –Test histogram.. This process should not be referred to as Compensation but often is.

Tandem dyes – Dyes which utilize random energy transfer by coupling two fluorophores together in an “intimate association”, close enough so that the emission of the first will excite the second. Allows for more fluorochromes to be measured from a single excitation source.

Tetraploid – Sample from whole cells or nuclei containing a cell cycle with twice the normal complement of DNA. Proper term is “DNA Tetraploid”. Has a DI of 2.0

Thresholding – Instrument setting to eliminate unwanted events from the analysis, such as debris or RBC’s. Usually set on size so that only events of a size larger than the setting will be counted. NOTE: In DNA analysis, a fluorescence threshold is usually used to keep small fluorescent events from being excluded from the analysis. Also, use care in setting the threshold in sorting. The instrument will be blind to small particles which fall below the threshold.

Time of flight – Width of the electronic pulse. Also known as pulse width.

Titration – Dilution of antibodies to determine the appropriate staining concentration in order to saturate all antigens on cell and minimize non-specific binding.

Univariate Plots or Histograms – Data display for flow cytometric data. Terms “histogram” and “plot” can be used for single parameter (Univariate) graphs.