Luminex MasterPlex QT User Manual

Tutorial for MasterPlex QT® v2.5

http://www.MiraiBio.com © 2007 Hitachi Software Engineering America, Ltd. All Rights Reserved.

Table of Contents

Running MasterPlex QT using an existing template .............................................3

Marking and Grouping Wells...............................................................................13

Designating the Standard/Known Concentrations:..............................................21

Select the Standard Curve Model .......................................................................26

Utilizing the Virtual Plate Feature........................................................................30

Analyzing a single QuantigenePlex plate............................................................40

Other Features to Explore...................................................................................45

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Running MasterPlex QT using an existing template

The following exercise will show how to run MasterPlex QT v2.5 using sample data that
are included when the program is installed.

When you start MasterPlex QT v2.5, you will see the plate wizard dialog box:

Click the Next button:

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Select the Import a new plate option, and click Next:

Click on the button with the folder icon to navigate to the .csv file. Navigate to

C:\Program Files\HitachiSoft\MasterPlex QT 2.5\examples, and select the file titled
IL5.csv.

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Next, click the Open button shown above.

Click the Finish button.

Click on the Template Manager icon shown circled above to select the template for this
plate.

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Click on the
M5PlexReplicates
template, and click on
the Load button.

Please note how the wells are color-coded.

• Copper = the background wells.

• Blue = the standard wells.

• Green = the unknown wells.

• Yellow = the (optional) control wells.

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Also note the black borders that show how the wells are grouped. Please note that
MasterPlex QT treats standard wells in a single group differently than background wells,
unknown wells or control wells grouped together in a single group:

• When standard wells are placed in one group, MasterPlex QT will use data from

that group to create a standard curve for each analyte. If you have replicate
standard wells, MasterPlex QT will determine which wells are replicates by
comparing their standard/known concentration values. For replicate standards,
you can choose to calculate the standard curves by either using the average values
for the replicate wells, or by using the individual values.

• When background, unknown or control wells are grouped together, you can

instruct MasterPlex QT to treat each group as a set of replicate wells, and
calculate the mean, standard deviation and %CV for each group when you
calculate your unknown concentrations.

In the above picture, there is one group of standard wells, and there are seven different
standard wells in that group. In this example, there are no replicate standards. There are
three groups of unknown wells, each with 6 replicates, and one group of control wells,
also with six replicates.

Before we calculate the unknown concentrations, we should select the model equation we
wish to use. To assign the model equation, right-click with your mouse on one of the
wells in the standard group, and select Assign Model Equation, as shown below.

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For this example, we will select the Five Parameter Logistics curve model, without
weighting:

Click on Assign Model. The following dialog box will show up:

Click OK.

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Click Exit to leave the Model Equations dialog box. The following dialog box will then
appear. Select Absolute Quantification, and click OK.

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You will then see the following dialog box:

Now, from the drop-down pick-list, select Concentration/Fold Change.

You can now toggle through the list of analytes on the left and view the concentration
information for each analyte and each well.

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Note for the IL-10 analyte, some of the wells show a value <26.47. If we go to the drop­down pick-list, and select Standard/Independent Values, we see that the lowest known
standard concentration for this analyte is 26.47.

So, what does <26.47 mean here? For 4-parameter logistic and 5-parameter logistic
curve models, when the best-fit curve is generated based on the data, there is a lower
limit for the Median Fluorescent Intensity (MFI) values that the model can use to
calculate an estimated concentration for a sample. When a sample has an analyte whose
MFI value is below this lower limit, the model cannot be used to calculate the estimated
concentration. However, if the MFI value for an analyte is below the MFI value for the

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lowest point on the standard curve, we can infer that the concentration for that analyte is
less than the concentration of the lowest point on the standard curve for that analyte. So,
in the example above, we cannot use the model to calculate an estimated concentration,
but we can infer that the concentration is less than 26.47 pg/mL. There is a similar upper
limit on the 4-parameter logistic and 5-parameter logistic curve models, with an
analogous “>” nomenclature when the MFI value is above the upper limit of the curve
model, but we can infer the concentration is above the highest point on the standard curve
for that analyte.

For many users, it is enough to know that we can infer “the concentration is below the
lowest point on the standard curve” or “the concentration is above the highest point on
the standard curve” when we measure a data point outside the range of the model curve.
If you would like additional details on this, please go to the www.MiraiBio.com website.
The presentation The Calculations at the link shown below provides a much more in­depth look at these issues:

http://www.MiraiBio.com/online-demo/online-demos.html

Going back to the exercise, in the plate above, if you wanted to save your work at this
point, you would select File → Save.

Note: When we read in a .csv file from the Luminex machine into MasterPlex QT,
the .csv file does not get altered. When we save the work we’ve done in MasterPlex QT,
it gets saved as an .mlx project file, leaving the original .csv file unaltered.

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Marking and Grouping Wells

We’ll open the IL5.csv plate again, but this time, instead of using a template, we’ll mark
the wells and enter the standard concentrations manually.

Go ahead and close the plate we were working on in the above tutorial by clicking on the
X in the top right-hand corner of the plate window:

Now, click on the New icon to read in a .csv file produced by the Luminex machine
(Note: If you are using build 163 or above, the New icon will not be present. In that case,
please click on the Open icon):

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Navigate to C:\Program Files\HitachiSoft\MasterPlex QT 2.5\examples, and select the
file titled IL5.csv.

You will see the window shown below:

This is the same plate we worked with before. If we did not have a template for this plate,
we would need to mark the wells (as shown below).

To mark wells, you highlight them, then right-click, and select how you want to mark
them.

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We’ll mark A1 as background: Highlight the well, then right-click, and from the menu,
select Mark Wells → Absolute Quantification → Background.

The well will change color to copper (you may have to click on another well to change
the highlighting to see the copper color indicating a background well).

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Now we will highlight B1 to H1, and mark them as Standard wells:

Now right-click on the wells, and select Mark Wells → Absolute Quantification →
Standards.

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