Relative intensity values are in arbitrary units and can be expressed as scale values or channel numbers. The scale values take into account scaling factors such as the gain setting for the parameter. Channel numbers ignore the scaling factors, and use an integer scale for relative intensity ranging from 0 to the maximum value defined by the data collection program (i.e. 255, 1023, 4095).
If you are using calibration to calibrate a fluorescence parameter to absolute units, then all statistics computed on the calibrated platform are given in terms of those absolute units.
Definition of statistics
- Mean—The arithmetic mean. For a normal distribution, the mean = median = mode.
- Median—The median is the relative intensity value below which 50% of the events are found; i.e., it is the 50th percentile. In general, the median is a more robust estimator of the central tendency of a population than the mean.
- Mode—The relative intensity value which is most frequently found for a given parameter. This is the same intensity value at which the highest point on a histogram is found.
- Geom. Mean—The geometric mean. Can be a more applicable metric for a log-normal distribution. It is always less than or equal to the arithmetic mean.
- StdDev—The Standard Deviation is a measure of the spread of the dataset. Lower values indicate the data points are closer to the mean and give higher confidence to the mean value.
- Robust Standard Deviation—68.26% of the events around the Median are used for this calculation, and an upper and lower range set. The robust standard deviation is equal to (upper range + lower range) /2. If the upper range is off scale, the robust standard deviation is equal to the lower range, and vice versa when the lower range is off scale and the robust standard deviation is the upper range. The robust standard deviation is not as skewed by outlying values as the Standard Deviation.
- CV—The Coefficient of Variation is a normalized Standard Deviation. CV = StdDev/Mean. In FlowJo, the CV statistic is displayed in percent (i.e. a CV of 0.15 is displayed as 15). 1/CV is a common way to define the Signal to Noise Ratio.
- Robust CV—Equals 100 * 1/2( Intensity[at 84.13 percentile] – Intensity [at 15.87 percentile] ) / Median. The robust CV is not as skewed by outlying values as the CV.
- %ile—Percentile. This is the relative intensity below which n% of the events are found, where n is the selected value. n=50 is equivalent to the median.
- Frequency—The percentage of events (cells) in this gate out of the total number of events in the sample.
- Freq. of Parent—The percentage of events (cells) in this gate out of the parent gate (one level up).
- Freq. of Grandparent—The percentage of events (cells) in this gate out of the gate two levels up.
- Count—The absolute number of events (cells) in a gate.
- Correlation—The Pearson Correlation number for two parameters. This statistic requires that two input parameters are selected.
Note: A common question is “Which statistic should I use?”. There are many reasons to choose a mean over a median over a mode, or vice versa. It really depends on how your cells are expressing the markers that you use. Also, when using the term “MFI”, it is a good idea to clearly define it in your context.
More on means, median, geometric means, and modes can be found in this DD article.
Total Fluorescence of a gate (integrated area of a histogram)