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!

I stumbled upon an interesting article this week in the Journal of Food Protection titled “Microbead-Based Immunoassay Simultaneous Detection of Shiga Toxins and Isolation of E. coli O157 in Foods”. It shows how the implementation of the xMAP technology has improved the sensitivity of current technology, specifically in detecting food-borne pathogens and toxic markers.  With improved sensitivity and specificity, the article also states that when using the xMAP technology, you can develop rapid and reliable results.

How can I get even more value out of the xMAP technology?

Hitachi Solutions America, Ltd. has engineered analysis software called MasterPlex QT to ultimately save you time through its ease-of-use interface while providing versatility of analysis tools and customization of reports.  MasterPlex QT allows MagPlex and/or MAGPIX users to save time by simply selecting the Best Fit curve-fitting button to create the optimum curve fit for your data. This feature automatically selects the optimal model equation and weighting algorithm with different parameters to optimize for: Root Mean Square Error (RMSE), R-Square, or lowest Standard Deviation of % Recovery.

MasterPlex QT’s optimized model equations present accurate and precise curve-fitting from one experiment to the next, offering reliable and reproducible results.

Reporting has not been this easy to do as MasterPlex QT has highly flexible graphing capabilities and a Custom Report Generator, allowing you to create a report that fits your current report templates.

This one-two combination of xMAP technology and the power of MasterPlex QT can help you achieve more consistent results in less time.

For more tips on optimizing xMAP data analysis, refer to our “Top 10 Tips Luminex/Bio-Plex Data Analysis.”

Reference:

Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods. Clotilde LM, Bernard C 4th, Hartman GL, Lau DK, Carter JM. J Food Prot. 2011 Mar; 74(3):373-9.

Sample A = 20 MFI – 10 MFI (background) = 10 MFI (50% reduction)
Sample B = 5000 MFI – 10 MFI (background) = 4990 MFI (0.2% reduction)

As can clearly be seen, slight MFI changes on the lower end of the curve can dramatically affect MFI values on the lower end where it barely makes a dent with points on the higher end of the curve. To make things worse, if Sample A were on the really flat part of the curve, this issue will be magnified as really small changes in MFI greatly affect the concentration values.

An MFI change on the lower part of the curve has greater weight

A change in MFI has greater weight on the lower end of the curve because values here have smaller variance. This is a natural situation that arises in almost all fields, including chemical- and immuno-assays, in which the variance of a dependent variable varies across the data. Simply using the 4PL or 5PL model equations without weighting will assume equal variance across the entire curve which is certainly not optimal.

Heteroscedasticity - Nonconstant Variance in Immunoassay Data

Ok…back to my story on the true case. We first wanted to compare apples to apples so we imported the data into MasterPlex QT and used the 5PL model equation with no weighting. The results were very similar where both analysis software had calculated the minimum asymptote to roughly about 8. This means that any sample with an MFI lower than 8 is considered out of range (for the lower part of the curve). Both analysis software reported out of range results since most of the samples were on the lower part of the curve. Not good.

We then applied 1/Y weighting to our 5PL model equation and the minimum asymptote was lowered to about -4. I know what you are thinking right about now; how it is possible to have a negative MFI value for the minimum asymptote? This is something that is certainly possible with background subtraction on background wells with higher MFI values than the actual samples. Anyway, with weighting applied, we were able to recover 95% of the out of range values =) It is also worthy to note that the majority of these points were interpolated rather than extrapolated so they were in fact within standard range. This is not to say to adding weighting will always push the minimum asymptote lower. What can be said with confidence is that the lower asymptote is more accurate with weighting than without weighting because it takes into account the natural heteroscedastic nature of bioassay data. In this particular case, having weighting was able to bring a large majority of the unknown samples into calculable range.

For more details regarding heteroscedasticity, please review point #2 of our 10 Tips for Luminex/Bio-Plex Data Analysis post.

You may also be interesting in reading our previous blog post on the 5PL nonlinear regression model equation.

This is sort of non-related but I just wanted to add that according to our most recent survey results, users who switch to MasterPlex QT from their old analysis software, save (on average) 83 minutes per analysis!

• Data Reporting – A highly flexible data reporting interface will allow you to create your own reports and export them in many useful formats such as CSV, Excel, or PDF.
• Professional Charts – MasterPlex EX will include a new powerful charting engine for you to create and customize your own charts.

In addition to the online community members, MiraiBio’s own employees will be browsing around as well joining in on all the conversations.  So whether you were just curious about something or need help troubleshooting, feel free to drop by and start a converation!

We are now happy to introduce the new Term Licensing options for our MasterPlex QT and GT Luminex data analysis software.  Term Licensing durations include the 3, 4, 5, 6, 7, 8, 12, or 24 month options.  The software license expires at the end of the purchased term and also includes all major upgrades released during the term.

For those that do not need to run analyses 24/7, this may be the cost-efficient choice for you.  To find out more details, please visit the MasterPlex QT product page or the MasterPlex GT product page at the MiraiBio Store.

1. Accession number recognition – DNASIS SmartNote now recognizes accession numbers in sequences you import (or will ask you to enter one if you paste in a raw sequence). This gives you better control over the naming of your sequences, so you can keep better track of which results belong to which sequence.

2. Import multiple sequences at once – If you’d like to import several sequences into DNASIS SmartNote and you know their accession numbers, you can now enter them all (separated by commas). To try it out, click on “Get Sequences” under DNASIS SmartNote’s “Sequences” tab, then select “Search for sequences from NCBI Entrez” or “Search for SNPs from NCBI DB”

3. Export raw results from xTAG Software tool – For those who are using DNASIS SmartNote’s new tools for designing tagged, gene-specific oligos (GSO) for their Luminex assays using Universal Array beads, the xTAG tool can now export results as comma-separated values (CSV), so you can transfer the data to Microsoft Excel or other spreadsheet programs. The output fields are: “Sequence Name”, “Bead Region” and “Oligo Sequence”

We’ll be discussing Luminex instruments, assays, bioinformatics software, and anything else that helps biologists work more efficiently, particularly if it has to do with software.

Please let us know if you have an idea, topic or something you’d like us to write about.

Let’s start the conversation!