Measurement error in compensation.

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Therefore, a properly compensated measurement of FITC fluorescence requires the measurement of the fluorescence in the FITC channel as well as a measurement in the PE channel. In our ideal world, every cell could be exactly compensated, since all of these fluorescence could be measured exactly; since the fractional spillover is the same for every cell, we would be able to compute the exact contribution of FITC into PE (and vice versa) and end up with "pure" measurements.

Indeed, in the real world, the instrument can do quite an excellent job of compensation. However, there is a relatively small error in the ability of the FACS to measure the fluorescence from any particular cell. And note, from the equations above, that the error in computing the true compensated fluorescence the error in the primary measurement PLUS the error in the other channel multiplied by the compensation percentage. For very small compensations, only a small error is carried over; the larger the compensation, the greater the increase in the error.

This increased error especially affects cells that are dim in the compensated channel: primarily, because the process becomes one of trying to measure a small value above a large background (from spillover). Here, even small measurement errors in the determination of the fluorescence in the other channel will be significant with respect to the true signal we are trying to measure.

The fallout of this error is a broadening of the distribution of the compensated fluorescence (see example below): the error in the final measurement of a fluorescence is the sum of the errors in measuring both fluorescences.

Figure 3: Errors introduced by compensation

In a world of error-free measurements, compensation would not increase the "width" (c.v.) of a population. This is because the amount of fluorescence in the PE channel that arises from the FITC CD3 could be exactly determined and exactly corrected. The width in the PE dimension of the compensated population would be the same as for the uncompensated sample (left panels). However, in the real world, the FITC measurement and PE measurements are made with some inaccuracy. Therefore, it is impossible to exactly determine the amount of signal in the PE channel that arises from the FITC CD3. Thus, the compensated sample will have a width in the PE dimension that is equal to the original width plus an amount related to the error of the measurement: i.e., it gets wider (right panels). Remember that a single compensated fluorescence value depends on (in this case) two measurements: thus, the error in the compensation value will be the sum of the errors in both measurements.

Bottom line: error in compensation computations. Because of the errors in measuring fluorescence, we cannot guarantee that every cell will be perfectly compensated--just like we couldn't guarantee that we could measure the exact fluorescence on every cell. However, we can guarantee that we can accurately compensate a large population of cells: i.e., on average, the cells will be properly compensated. Again, this is analogous to the fluorescence measurement: we can guarantee that we can accurately determine the average fluorescence for a large population of cells­p;even if statistical errors limit the accuracy of the individual cell measurements.

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