Hi Carla,
I certainly may be corrected by others on this but I personally don't 
think you will obtain statistically valid data by grading intensity of the 
reaction between cells or fields or sections. There is just so much 
room for subjectivity and for that matter, variability between runs or 
perhaps even between slides within runs!

What you may want to try is calculating a ratio of positive to 
negative (perhaps pos. cells vs. neg. cells in a given area). For 
instance: place a grid with 50 or 100 intersects on the computer 
monitor, choose a microscopic field (in a random or systematic- 
random manner), and then at each intersect decide whether the 
reaction at that point is positive or negative. If at any particular 
intersect you have trouble to decide whether it is  pos or neg, have 
a system that does not permit subjectivity, such as: for such points 
where the call could go either way, look at the space imediately to 
the left of the y-axis and above the x-axis and then make your 
decision. Determination of the number of fields you need to sample 
in order to obtain statistically valid data is very important and a 
matter for a statistician to explain and/or determine for you (which I 
most definately am not) unless you happen to have that training 
yourself.

Another idea would be to have the image analysis software either 
measure the area of or count the number of pixels in a given area 
(field) that are "this red or redder". This involves choosing a 
relatively arbitrational threshold for the degree of redness that is to 
be measured or counted and applying the exact same setting to all 
other fields and sections in the study. My personal experience 
(using Bioquant NOVA) has been this sounds great in theory but 
was very difficult to pull off! The good old-fashioned counting was 
much more reliable and for that matter, easier to defend in 
presentation or publication. 

I am eager to hear other ideas or methods that might also work! 
Good luck.
Greg

To:             	Histonet@lists.utsouthwestern.edu
From:           	"Carla M Conway" 
Date sent:      	Thu, 9 Sep 2004 07:22:02 -0700
Copies to:      	Subject:        	[Histonet] quantitative graded scale for IHC 

> Hello,
> 
> I would like to devise a quantitative graded scale for IHC results which
> would provide more info than just +/- staining.
> I have a reference where  IHC staining intensity was rated (from pale pink
> to dark red) using alkaline phosphatase/Vector-Red, however we are using
> Envision+/AEC which seems to be an all or nothing (red or no red) chromogen
> with no gradation. We just received ImagePro image analysis software, so
> this may be another route to try.  Thanks in advance for any comments or
> suggestions you may have.
> 
> Sincerely,
> 
> Carla Conway
> Western Fisheries Research Center
> Seattle, WA
> 
> 
> 
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> Histonet@lists.utsouthwestern.edu
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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Happiness is a journey, not a destination.

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