Multi Wavelength Cell Scoring: Why is there cytoplasmic intensity for negative cells?
#1
This question comes up occasionally for the Multi Wavelength Cell Scoring (MWCS) application module:
If a cell scores negative for cytoplasmic staining, how can it have a cytoplasmic pixel intensity value great than zero?

There are two reasons that you might see a cytoplasmic intensity value > 0 for a negative cell.

1) If you have multiple channels that you are measuring (e.g. nuclei in DAPI channel, two stains of interest in FITC and Texas Red), and if a cell as found as positive in one channel (FITC) but negative in the other channel (Texas Red), it will still report out the Texas Red intensity in the cytoplasmic area defined by the FITC channel.

   

2) This can happen even if you are only measuring on two wavelengths (e.g. nuclei in DAPI channel, stain of interest in FITC): The algorithm can define a cytoplasmic area based on the minimum/maximum width and intensity above background settings, but then call a cell as negative because it does not pass the minimum stained area criteria.  Assuming that the minimum stained area was defined appropriately for the cell type, this seems to happen mostly when there are clumps/clusters of cells and a small amount of the cytoplasmic area for a positive cell was assigned to a negative cell.

          
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