# Accurate determination of the positive population when control and test tube are highly overlapping

## mahmood bozorgmehr (Author)

Dear friends

When analysing the percentage of posive cells stained for one marker, there are some situations where the histogram of the positively stained cells highly overlapes that of the negative-control cells (e.g. isotype tube).

According to "current protocols in immunology-2011" (page: 5.2.2.-2nd paragraph) in such cases we are supposed to find the chanel number corresponding to the intersection of the test and control histograms and then calculate the difference between their positive areas after this channel.

However, in some artiles I see that the authors simply find the last channel number of the control (isotype control) histogram and consider this channel as the point after which the test histogram would be positive.

It it obvious that especially in cases where control and test histograms have a high overlap, the methods we select for analysis (intersection or end channel neumber of control) would highly affect the percentage of posivtive cells and results in high differences.

I would really like to know whether the first method is reliable. Although many articles follow the first method, there are numerous publications that choose the 2nd method, which I suppose is wrong.

Thanks