Flow cytometry DNA Analysis in the Clinical Management of Cancer

 

           Flow cytometric DNA analysis has the potential to significantly enhance the clinical care of tumour patients in the future. The majority of these research have looked at aneuploidy and % S phase as indicators of disease recurrence and survival. These two DNA analysis parameters have been correlated with traditional diagnostic parameters for the tumour being studied, such as cytology (e.g., lung and uterine), state of differentiation (e.g., prostate), pathological tumour grade or stage (e.g., bladder, prostate, and lymphoma), number of involved lymph nodes (e.g., breast cancer), and others, in several excellent studies. This topic was given its own volume in the Annals of the New York Academy of Science (Vol. 468, 1986). "Generalizations cannot be made when using flow cytometric DNA analysis to clinical decision making," as Merkel et al. (1987) pointed out.

An aberrant DNA histogram's prognostic significance for an individual patient must be determined using the relevant data set for that tumour type." The nonclinical reader should consult McNicol's outstanding review to put flow cytometry in its right context within the traditional diagnostic environment, which includes immunohistochemical and ultrastructural studies as well as clinical and biochemical results (1987).

        Fig:Rat prostatic carcinoma aneuploidy increases with age. 

Flow Cytometry Controls

 

You may or may not want to utilise isotype controls, but if you do, make sure you have other suitable controls in place, which we shall discuss later. To identify what is particular, isotype controls for surface staining have been created. The proper isotype control is an antibody raised against an irrelevant antigen from the same antibody subclass, using the same conjugated fluorophore, purchased from the same source as your test antibody, and utilised at the same dose as your test antibody. They serve to guarantee that the staining observed is due to specific antibody binding rather than an artefact, to rule out non-specific binding to Fc receptors, and to remove non-specific antibody or fluorophore (such as PE) binding to cellular components. 

Intracellular staining can be more difficult than cell surface staining, and isotype controls may not be acceptable in this situation. In order to correctly calculate your positive population, it is a good idea to have different controls. Unstained samples, negative samples stained with antibody that does not express the antigen of interest, and known positive samples are all examples. Compensation values for multicolor panels must be calculated using single stained compensation controls. Antibody binding beads labelled with your planned multicolor panel can also be used to calculate compensation values. Finally, FMO controls may be used to assess fluorescence spread, gating limits, and prevent decreased sensitivity.

Flow cytometry gating

 Flow cytometry is a technique for detecting and quantifying the physical and chemical properties of a population of cells or particles. 

A sample comprising cells or particles is suspended in a fluid and injected into a flow cytometer device in this procedure.I

n flow cytometry, the term "gating" refers to the process of choosing a certain cell type.

Gating is a term used frequently in flow cytometry to describe the process of picking a specific section of data to study. On histograms, linear gates are employed, however on dot plots, polygons, rectangles, or circles can be used to gate.Polygon gates (freeform gates), histogram gates, quadrants, and boxes are the four primary types of gates. Each of them can be used to select certain cell populations. We can pick CD62L+ (right gate) and CD62L populations using the histogram gates in Fig. 1d, for example (left gate).These gating mechanisms can then be used to identify cell populations that can be further investigated. Quadrant gates are also commonly employed to divide protein expression into four areas: Q1, Q2, Q3, and Q4.Quadrants are used to gate CD4 and CD8 expression in mouse thymii (Fig. 1b–c).Box gates, like histogram gates, can be used to select a specific cell type; in this case, CD8+ T cells are selected (Fig. 1a, bottom panel). Gating is likewise hierarchical, with each gate building on the one before it.A typical gating hierarchy that would be employed in mouse thymii stained with CD4 and CD8 is shown below (Fig. 1b, c). The live gate, P1, is an upstream gate, whereas the quadrant gates Q1-Q4 are downstream gates, meaning they are after P1. As a result, quadrant gates 1–4 are considered to be P1's dependents.

Typical gating hierarchy.

P1 (live gate)

–– Q1 (CD8+CD4−)

–– Q2 (CD8+CD4+)

–– Q3 (CD8−CD4+)

–– Q4 (CD8−CD4−)

Fig. 1 Interpreting dot plots. Histograms, dot plots, and density plots are shown on various

lymphocyte populations in human and mouse. Cells were harvested, stained with the antibodies indicated below, acquired on a BD LSRFortessa™ and analyzed using FlowJo® software. (a) hCD8 A405 expression is shown in human PBMCs using both a histogram (top panel) and dot plot using SSC-A (bottom panel). (b, c) Mouse thymocytes were stained for mCD4 and mCD8 and are shown using a pseudocolor dot plot (top panel, b), monochromatic dot plot (bottom panel, b), and density plot (c). (d) Human PBMCs were stained for hCD62L, and gates were set on the negative and positive populations