Doublet's discrimination:

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Doublet exclusion or discrimination is the method to ensure you count single cells and exclude doublets from your analysis.

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 An event is defined as a pulse. Pulses occur as a cell pass through the laser beam spots, and this passage generates signal from the detectors. This signal is monitored and processed by the cytometer’s electronics, in the form of Area, height and width Figure-1 (Left).

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Doublets were the single particles passing so close together through this laser spot, that the instrument was incapable of distinguishing them as individual events or particles Figure-1 (right). 

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This can be critical in cell sorting, cell cycle and DNA analysis. If a doublet containing a fluorescence positive and negative cell passes through the laser (Figure 1), it will produce a positive pulse leading to false positives in both analysis and sorting experiments.

 

 

 

 

 

 

 

 

 

 

 

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Figure 1. Voltage pulses for single (left) and doublet (right) events.

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The differences between the doublet pulse and the single particle pulse: both the area and the width of the doublet pulse are larger than the single cell’s but the heights of the two pulses are very close, if not identical. 

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Doublets (Green) can be distinguished from single cells (Red) by plotting FSC height vs FCS area. Doublets have increased area whilst similar height to single cells. Figure 2 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Doublets discrimination

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