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Chapter 17 : Polychromatic Flow Cytometry

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Polychromatic Flow Cytometry, Page 1 of 2

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Abstract:

For nearly half a century, flow cytometry has been a tool used by biologists to study features of individual cells in a rapid and unbiased manner. The measurement of cellular features has relied upon intrinsic properties of the cells which can be examined by laser-light scattering or by the addition of extrinsic fluorescent probes, such as dyes or fluorochrome-conjugated antibodies that can make cellular features quantifiable. The early flow cytometers relied on only one or two lasers, or a mercury arc bulb, for excitation light, and generally measured only one fluorescent parameter. In the late 1970s, Stohr (1) and Dean and Pinkel (2) described dual-laser excitation for flow cytometry, which permitted the simultaneous use of two spectrally distinct fluorochromes, signaling the genesis of polychromatic flow cytometry. At roughly the same time, Loken and colleagues proposed a method to overcome the difficulties caused by the spectral overlap of fluorochromes excited off a single laser—a method we now refer to as “compensation” (3). These developments laid the foundation for future high-polychromatic flow cytometry, as the bases for both multi-laser excitations as well as exciting multiple fluorochromes off each laser had now been described.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Figures

Image of FIGURE 1
FIGURE 1

Schematics of the main components of a flow cytometer. A single-cell suspension goes through a tightly focused stream into the flow cell, where it encounters the different lasers. Insert A shows the position of different components of hydrodynamic focusing using a coaxial stream. Insert B shows the different components of acoustic focusing, where a capillary line of particles can be created using acoustic-radiation pressure. The signals emitted using the blue-laser excitation can measure physical characteristics of cells in addition to fluorescence measurements via transformation of fluorescence into voltage (see insert C). Each fluorescence channel measured is assigned to a PMT.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 2
FIGURE 2

Schematics of the voltage pulse and time delay. Area, height, and width are the signals recorded for each single particle (text in red), and these measurements are influenced by the threshold and window-gate extension that are set up by the user (text in green). The voltage pulse generated by the red laser will need to be retrospectively assigned to the same particle-voltage pulse from the blue laser (time delay).

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 3
FIGURE 3

Example of dot plots generated by CST and QC Levy-Jennings. On a daily basis, eight peak beads are run on the cytometer after the CST performance check. (A) After doublet exclusion the fluorescence intensity is measured for each of the eight peaks in each detector. (B) Median fluorescence intensity of each PMT for each laser is generated by CST software (example of 1 Violet PMT V585). (C) Robust CV, robust SD of MFI of Violet PMT V585. Blue line represents the PMT voltage, the rCV is in yellow, rSD is in green, and the baseline PMT is the red line showing intersection of rCV with rSD. (D) Levy-Jennings plots, which display the performance characteristics of an instrument over time, are useful for assessing instrument variations. Here a Levy-Jennings plot of the MFI of bead standards at a constant voltage is shown for four PMTs across several months, with the performance of each detector shown in a different color.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 4
FIGURE 4

Illustration of Ficoll-Hypaque and whole blood as sample to stain. (A) Illustration of Ficoll-Hypaque. In noncoagulated blood after centrifugation in a Ficoll gradient, the plasma is on the top layer, followed by a layer of mononuclear cells, and then by a pellet of granulocytes and red blood cells at the bottom. (B) Illustration of red blood cell lysis. (C) A FSC versus SSC dot plot of the two types of samples: left, ammonium chloride-lysed sample; right, sample after a Ficoll gradient.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 5
FIGURE 5

Summary tables of the most common fluorochromes used in flow cytometry. The left table shows fluorochromes and cytosolic dyes while the right table shows dyes to stain nucleic acids.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 6
FIGURE 6

Illustration of the stain index. (A) Theoretical stain index (SI) representation. When testing different reagents, the effective brightness is measured using the stain index. SI = /, where is the difference between the median fluorescence intensity of the positive and negative peaks of fluorescence, and is the spread of the negative peak. (B) SI for CD3 BV510. (C) SI for CD27 BV650.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 7
FIGURE 7

Illustration of titration of monoclonal antibody. An FCS file represented independently (A) or concatenated (B). (C) Determination of the separating concentration by graphing the ratio of the positive to negative peaks against the dilution. The histogram represented in red (A), or the dot plot highlighted by the red square (B) or red arrow (C), all mark the selected concentration for the given antibody.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Image of FIGURE 8
FIGURE 8

Tubes (staining) of CLIP panel organized by lineage. In black are markers present in all stainings of one lineage, and in blue are markers specific to one staining. *, Lineage mix = CD3+CD19+CD20+CD56+CD16+CD14.

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
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Tables

Generic image for table
TABLE 1

List of the most abundant families of antigens and number of members with assigned CDs

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17
Generic image for table
TABLE 2

Summary of types of dyes used in flow cytometry

Citation: Biancotto A, McCoy J. 2016. Polychromatic Flow Cytometry, p 149-167. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch17

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