The Spillover Spreading Matrix (SSM) values have been demonstrated to reveal fluorescence spectrum interactions that can limit the sensitivity of a reagent in the presence of brightly-stained cells on a different color. The SSM can thus be a useful tool in instrument quality control to ensure consistent sensitivity and performance. In addition, the SSM is a key element for predicting multicolor immunofluorescence panels, which will aid in the optimization and development of new panels.

 

The Spillover Spreading Matrix page is automatically created by FCS Express when either the Compensation or the Unmixing Matrix is calculated and depicts a spreadsheet with the Spillover Spreading values for every pair of dyes included in the compensation/unmixing matrix respectively (Figure 9.60).  The SSM automatically has conditional formatting automatically applied when created.

 

 

Figure 9.59  The Spillover Spreading Matrix.

Figure 9.59  The Spillover Spreading Matrix.

 

 

If errors are encountered when calculating the Spillover Spreading Matrix, the spreadsheet containing SSM values will report the error accordingly. Hovering the mouse over a spreadsheet cell reporting the error, will display a hint with additional details on the source of the error. An example a hover over error hint is depicted in Figure 9.61 below. In the example below the error occurred because of a lack of stained events in one of the controls.

 

 

Figure 9.60 An example of Spillover Spreading Matrix with errors.

Figure 9.60 An example of Spillover Spreading Matrix with errors.

 

 

 

More details on the Spillover Spreading Matrix can be found in the following publication:

Richard Nguyen et all,  Quantifying Spillover Spreading for Comparing Instrument Performance and Aiding in Multicolor Panel Design. Cytometry A. 2013 March ; 83(3): 306-315. doi:10.1002/cyto.a.22251.