Simple methods to determine and compare the sensitivity of flow cytometers

Knowledge of the sensitivity of a flow cytometer is important both in choosing an instrument for a particular application and in determining whether the resolution obtained on real samples is limited by instrumental factors or sample variability. The coefficient of variation (CV) obtained for measurement is given by CV2 = CVS2 + CVI2 + 1/n + K(delta VB2)/n2, where CVS is the variability due to the sample, CVI that due to variations in illumination of the sample particles, n the number of photoelectrons emitted from the photomultiplier photo cathode per sample particle (which is the signal intensity), delta VB2 the mean square background noise voltage (usually due to background illumination), and K a constant. Measurement of the CV of light flashes from a light emitting diode as a function of their intensity allows development of standard curves which can be used to directly determine the degree to which the resolution obtained on actual samples is limited by signal intensity and background light. The use of standard particles allows relative calibration of the curves for different instruments and which also ranks their sensitivities.