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!

Remember when multiplex analysis first emerged?  It changed the way we analyzed data from tedious one-analyte assays to a multiple-analyte analyses in a single run.  Using platforms such as Luminex’s bead based xMAP platform we are able to analyze hundreds of analytes at a time with great sensitivity. However the cost of multiplexing platforms was prohibitive for many labs. With the release of the new lower-cost MAGPIX instrument, which uses Luminex’s MagPlex® magnetic beads, multiplexing has become affordable for many more labs.

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.

Think you have what it takes to be a MasterPlex Guru? Take our quick 10-question quiz and see how where you fall on the MasterPlex IQ spectrum. (Answers are available at the end of the quiz)

We recently had a customer that was interested in MasterPlex QT because his current analysis software for his Bio-Plex instrument was reporting a lot of “OOR < ” or out of range concentration values (below the lower asymptote in this case) for points on the lower end of curve. This is what you would normally expect to see for values that fall below the minimum asymptote BUT the software did not have the capability to use weighting in calculating the lower asymptote which can greatly affect points on the lower part of the curve.
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The turnout at this year’s event was fairly successful given the alert of the swine flu that is going around. Our speaker, Ali Vahedi of Aviir, Inc., gave a very nice presentation on his evaluation of the AssayCheX beads and the MasterPlex AssayCheX plugin. Not surprisingly, his findings stirred a lot of attention from the crowd. He summed it up pretty nicely by saying that he ‘felt like Darwin presenting the theory of evolution for the first time.’ Curious to know what the buzz is all about? Read the AssayCheX white paper to find out.

Luminex multiplex expression data analysis has been taken to a whole new level in this major upgrade that features a new dashboard for managing your housekeeping genes and a makeover of the entire user interface.  Other improvements include:

• 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.

An online social community for nearly every niche exists on the internet.  MiraiBio understands this so now, there is one for the Luminex Community, MasterPlex Community, and DNASIS Community.

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!

MiraiBio has heard your voice and we realize that not all customers require a perpetual license when there is only the need to use our Luminex analysis software a couple of times a year.

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.

We have 3 improvements to report for DNASIS SmartNote this week, all having to do with more efficient sequence importing and exporting:

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”

Better communication improves any relationship, so we’ve started this blog to enhance communication with our customers. We’re going to use this blog to discuss what we’re doing, how we’re doing it and why. Our goal is to offer useful information, help our customers know us better and get to know them better, talk openly about what we’re about and build trust.

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!