In this section, we will:

 

 

We now have all the essential data in our Data Grid; however, we still need to determine if the patient serum tested is a 'positive', 'negative', or 'weak' cross-match to the donor cells.

If you are starting the tutorial series from this step please open the HLA Custom Data_Completed.fey found in the FCS Express Sample Data folder. This layout has completed all of the previous steps in the tutorial.

To begin, we must first create custom tokens that normalize the 'sample' median fluorescence values based on a 'negative' value. We will use the median MESF FITC values from the B- and T-cells of the PBS NEGATIVE CONTROL as the 'negative' value and subtract this value from the median MESF FITC value of the samples. We will begin by inserting the Custom Tokens window.

The Custom Tokens window appears on the layout. We will now dock the window to the right side of the layout. For more information on docking, please see the Docking tutorial.

The Custom Tokens window is now docked on the right side of the layout. Now, we will create the custom tokens that represent the MESF channel shift of the samples relative to the PBS NEGATIVE CONTROL sample. For more information on creating custom tokens, refer to the Custom Token Tutorial.

 

 

We will now copy the appropriate median token value in the Data Grid into the custom token definition. Refer to Figure T15.25 for the following steps.

The Custom Data editor window appears on top with the statistic token definition in the editor window.

 

 

Please refer to Figure T15.26 for the following steps.

 

 

We have now created the Custom Token definition for the B-cell median MESF shift of the PBS NEGATIVE CONTROL sample, called 'BShift PBS'. By definition, this shift should equal zero. We will now create the Custom Token for B-cell median shift of the AB NEGATIVE HUMAN SERUM sample, calling it 'BShift NegSerum', by copying and pasting the definition of the 'BShift PBS' Custom Token. We will then drag and drop the 'Bcell Median' statistic token (of the AB NEGATIVE SERUM sample) from the Data Grid into the appropriate place in the Custom Token definition of 'BShift NegSerum'.

 

 

 

The 'BShift NegSerum' Custom Token is now defined as the B-cell median of the AB NEGATIVE HUMAN SERUM – the B-cell median of the PBS NEGATIVE CONTROL, as shown in Figure T15.27.

To define the rest of the B shift Custom Tokens, repeat Steps 16 through 28 using the appropriate B-cell median tokens from the Data Grid for the POSITIVE HUMAN SERUM and PATIENT SERUM samples. Use the Custom Token names 'BShift PosSerum' and 'BShift Patient', respectively.

Again, repeat Steps 16 through 28 to define the T shift Custom Tokens using the appropriate T-cell median tokens from the Data Grid for all the samples. Remember to subtract the T-cell median of the PBS NEGATIVE CONTROL from all the samples and substitute 'T' for 'B' in the Custom Token names.

When you are done, the Custom Tokens window should look similar to Figure T15.28, which shows the definition for the 'TShift Patient' Custom Token.

 

 

We will now add the B and T Shift Custom Tokens to the Data Grid, so we need to add two columns to the Data Grid. Refer to Figure T15.29 for the following steps.

 

 

A New Column Name dialog appears, similar to previously shown Figure T15.8.

A new column labeled 'BShift' appears on the Data Grid. Using steps 29 through 32, insert a new column labeled 'TShift' to the right of the newly created 'BShift' column. When you are done the Data Grid should appear similar to Figure T15.30.

 

 

We will now drag and drop the Custom Tokens into the cell of the new BShift and TShift columns. Please refer to Figure T15.31 for the following steps.

 

 

The Data Grid updates to reflect the insertion of the 'BShift PBS' Custom Token value, which is shown as '0.00' in Figure T15.31. This makes sense because the B-cell median MESF fluorescence value of the PBS NEGATIVE CONTROL sample is being used to define the negative. Continue to fill in the Data Grid by dragging and dropping each Custom Token into the appropriate cell of the BShift or TShift columns. When you are done, the Data Grid should look like Figure T15.32.

 

 

We can now close the docked Custom Tokens window.

 

 

 

Next, we will create and use token classifiers in the data grid.