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Calculating Means in Flow Cytometry

For flow cytometry data analysis, fluorescence and light scatter are measured using photomultiplier tubes that convert the emission spectra into a data point on the computer that is represented graphically. This is often presented on a histogram, dot plots, or density plots.

Remember: data analysis techniques cannot make up for bad data; before you start your experiment, check out our top tips for better flow cytometry data.


The arithmetic mean, geometric mean, and median

The mean is a measure of central tendency meaning it aims to identify a representative portion of a set of data. The arithmetic mean, or average, is the sum of the individual values of all of the observations in the distribution divided by the number of observations.

The formula to calculate the mean is (Σx/N)

Σx = sum of all individual values

N = number of individual values

Those who commonly perform flow cytometry will also be familiar with the geometric mean, which is the Nth root of the product of all the observed value.


What is the geometric mean in flow cytometry?

Many flow cytometry software programs compute geometric means. This is because a majority of flow cytometry data are displayed on logarithmic scales and stored in logarithmic form; therefore, the values that are worked with are logarithms of the observed values.

Taking the arithmetic mean using logarithms is far easier than converting back to linear values.  


What does mean fluorescence intensity tell us?

Mean fluorescence intensity (MFI) values can be used to describe the expression levels of your protein of interest; however, this attribute of MFIs is situational and is largely restricted to protein expression on activated cells.

More routinely, MFI values reveal the signal intensity for a given fluorochrome; very bright dyes tend to have high MFI values whereas dim dyes have low MFI values, reflecting the signal intensity of the fluorochrome.

MFI is calculated using the geometric mean, median, or mean index.

  • Geometric mean MFI: more affected by outliers and skews towards them whether they are high or low; can be useful for detecting proteins with weak expression1
  • Median MFI: set at 50%, where 50% of cells are above or below the midpoint; also susceptible to the influence of outliers1,2
  • Arithmetic mean MFI: the average of the fluorescence intensities; it tends to skew to the right or towards higher MFIs1


The trends for the geometric mean, median, or arithmetic values tend to be the same: the geometric mean MFIs are lowest, median MFIs are in the middle, and mean MFIs are the highest.1

In general, median MFI is probably the best parameter to describe signal intensity, as its value lies between the geometric mean and the arithmetic mean, apart from some exceptions e.g., for weak protein expression.1

What is the mean fluorescence intensity ratio (MFIR)

The MFI ratio (MFIR) is usually defined as the ratio between the fluorescence intensity or geometric mean of stained cells and unstained cells, or cells stained with a control.3,4

The use of the MFIR has been found to strongly predict overall survival and time to treatment for patients with chronic lymphocytic leukaemia (CLL). This was seen using the geometric MFI index; however, calculating the percentage of positive cells and the use of the arithmetic mean MFIR saw no significant difference.6


Measurement using MFIR reduces subjective error potentially present when assessing of ZAP-70 expression, a marker of CLL, by percentage of positive cells.5


Find out more: BD Quantibrite™ Beads can be used to quantify antibody binding capacity (ABC) in flow cytometry.

  1. Goetz C, Hammerbeck C, Bonnevier J. Flow Cytometry Basics for the Non-Expert. Springer International Publishing; 2018. doi:10.1007/978-3-319-98071-3
  2. Shapiro HM. Practical Flow Cytometry. 4th ed. John Wiley & Sons; 2003.
  3. Ruiz F, Wyss A, Rossel JB, et al. A single nucleotide polymorphism in the gene for GPR183 increases its surface expression on blood lymphocytes of patients with inflammatory bowel disease. Br J Pharmacol. 2021;178(16):3157-3175. doi:10.1111/bph.15395
  4. Frerou A, Lesouhaitier M, Gregoire M, et al. Venoarterial extracorporeal membrane oxygenation induces early immune alterations. Crit Care. 2021;25(1):9. doi:10.1186/s13054-020-03444-x
  5. SMOLEJ L, VROBLOVA V, MOTYCKOVA M, et al. Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells. Neoplasma. 2011;58(2):140-145. doi:10.4149/neo_2011_02_140
  6. Wu YJ, Wang H, Liang JH, et al. Using the geometric mean fluorescence intensity index method to measure ZAP-70 expression in patients with chronic lymphocytic leukemia. Onco Targets Ther. Published online February 2016:797. doi:10.2147/OTT.S94613



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