A disadvantage of assessment of immunocytochemical staining using conventional microscopy is that it is very difficult to quantify the intensity of the immunostaining.

Although it is possible to estimate the proportion of cells that are immunostained in a population by performing manual cell counts, the accuracy of the estimate is dependant on the total number of cells that are counted and the proportion of positive cells.

These limitations are overcome in flow cytofluorimetric analysis. In this technique, the measurement of the size and refractive index of individual cells in a fluid stream is deteremined.

These cells intersect a laser beam and reflected light is collected by a photomultiplier tube set at right angles, hence "side scatter." Refracted light around the cell is proportional to size and is collected incident to the laser beam, hence "forward scatter."

If the cells are incubated with fluorescent proteins, usually an antibody against a specific cell surface antigen this can be excited by the high energy laser and the Stokes-shift emitted light is collected.

Up to three wavelengths can normally be detected, allowing cellular discrimination according to five parameters. The collected light from each cell is converted into electronic analogue data which is processed by the computer and can be presented as coordinates on a dot plot or as a historgram on screen.

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