Before we see how the concepts outlined in previous chapters can be used we should consider the reasons why they might have to be used. Some of the sources of variation in making a measurement with a ruler were discussed in Section 2.2. It is very important to appreciate where sources of variation in flow cytometry might be encountered, because variation gives rise to distributed data from the population being analysed. Flow cytometers are somewhat complex, and sources of variation can enter at every step in making a quantitative recording. We have already seen that the measurements we make are indirect (Section 2.2) in that there is conversion of light energy into electrons. But the process starts even further back down the line. Initially, the sample is prepared and stained; next it flows through the laser focus from which the fluorescence is elicited; this is then collected and focused onto the photomultiplier; converted to a voltage as already described; amplified and subjected to signal processing, including analogue-to-digital conversion; and finally stored electronically. These are just the physical processes, all of which can introduce variation, but there are also biological factors to be included. Some of these factors are more important than others, but each will be considered.

Preparation and staining

There are usually many steps in the preparation and staining processes. In solid tissues a disaggregation step is required to produce a single cell suspension, and this frequently involves enzymatic treatment.