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.