The Luminex and Bio-Plex platforms output 2 major pieces of data from an acquisition: MFI and bead count.
Let’s focus on MFI which stands for Median Fluoresence Intensity and NOT Mean Fluorescense Intensity.
Why do we use median and not mean?
Simply put, the median statistic is less sensitive to outliers where there is an inherent carryover of about 0.9% from these instruments. That is one of the reasons why Luminex recommends at least a minimum bead count of 35 to get statistically significant data but 50-100 is strongly suggested.
Here is an oversimplified example of why median is preferred over mean. Let’s say we are running a 1-plex with just 2 samples: A high control (well A1) and a low control (well A2). We have set a minimum bead count of 11. You will see in a second why I chose this odd number (no pun intended).
When we push the Acquisition button on our imaginary instrument, it begins reading well A1 with our high control and reads the following fluorescence intensities of 11 individual beads:
7833, 7609, 8335, 6900, 7354, 7234, 8120, 7777 (jackpot!), 7158, 7982, 7083
To figure out the median value, let’s place these readings in numerical order and the middle reading will be our median value.
6900, 7083, 7158, 7234, 7354, 7609, 7833, 7777 (jackpot!), 7982, 8120, 8335
Since we have an odd number of beads, the median value is simply the middle number which in this case is 7609. If this were an even count, we would have to take the average of the middle 2 readings after they are placed in order but that would just be too much work for me to do for this example. 7609 is the MFI value that this instrument would report for this particular analyte in well A1.
After the instrument reaches the minimum bead count for well A1, it moves on to well A2. (The probe actually goes back down in well A1 and spits out some of the leftover sample plus some additional sheath fluid but there is still a good chance there will be left over beads from A1.) Let’s say our imaginary instrument reports the following fluorescent intensities for A2 (our low control):
8001, 209, 7315, 199, 189, 217, 207, 186, 188, 208, 7917
Let’s place these readings in numerical order to figure out the MFI:
186, 188, 189, 199, 207, 208, 209, 217, 7315, 7917, 8001
The MFI value for the same analyte (we’re only doing a 1-plex) in well A2 is 208. Note though, that we had 3 pretty high readings (7315, 7917, 8001) that were most likely carried over from well A1. 3 out of 11 or 28% carryover is insanely high but I just wanted to illustrate this point that even though we had 28% carryover, we still have a respectable median (or MFI) value of 208 =)
If we used the mean fluorescence intensity, our value would be 2258 which is just bananas!
So in conclusion, the median statistic is preferred over the mean because the median is less sensitive to outliers or beads that are carried over from a previous well.
Thanks for tuning in!
If you have any questions or would like an explanation on other aspects of the Luminex technology feel free to comment below and we’ll try our best to address them!