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Category 'Data-Analysis'
Status: Unanswered
Views: 9196

FSC-A, FSC-H

Stephanie Foan (Author)

29 April 2012 15:15

Hi,

Can anyone offer a brief explanation of the difference between FSC-A and FSC-H? I'm reading a paper that's gating out anything that doesn't fall on the diagonal when they're plotted against each other, I just can't find a good explanation for why?

Thanks

Roberto

02 May 2012 08:30

Hello, 

 

The plot FSC-A vs. FSC-H is used to discriminate doublets from single cells. In that plot, everything that is out of the diagonal is considered as a doublet.

They are removed in order to have right results because sometimes they can lead into false percentages.

 

I hope it may help you.

Roberto 

Stephanie Foan (Author)

02 May 2012 12:09

Hi,

Thanks Roberto - I'm sorry I wasn't quite specific enough though! I gather that the plot is used to gate out doublets, I just literally couldn't find an answer to what the -A, -H mean and why you'd use such a plot to gate them out... my colleague tells me it might be area and height... if that's the case then I'm still trying to redefine in my head the lymphocytes I stain in terms of area and height!

Am grateful for your help!

Steph

Roberto

02 May 2012 14:45

Hi, 

 

Your colleage is right, -A is for Area and -H for Height. I will try to explain:

When two cells pass at the same time in the cytometer, the doublet has the same height that one of them but the area is 2x, corresponding to the 2 cells.

Due to that, the doublets are out of the diagonal: the height value is the same that a single cell but the area value is higher that it should be.

So, all the events out of the diagonal have more area tahn corresponding to a single cell, so, are doublets or artifacts.

I would be better with an image, but it is not possible :(

 

I hope it may help you

Roberto

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