[Histonet] Immunostaining Scale

From:"J. A. Kiernan"

Dear Sharon W, Jeff G  and all others,

If intensity of an immunostain means the darkness of
the end product, this does not indicate the quantity
or local concentration of the antigen unless several
stringent conditions are met. With amplifying methods
(PAP, ABC and the like) the intensity of the colour
of the final product can be lower at sites of high
antigen concentration than at at sites of low antigen
concentration. The reasons are well documented in
textbooks and peer-reviewed journals. The classic
is probably Bigbee et al 1977 J Histochem Cytochem
25:443-447, They simply explained at least one reason 
why darker brown doesn't always mean more antigen.
The dissociation between antigen concentration and 
intensity of staining is sometimes called the
"Bigbee effect." 

Quantitation of immunostaining intensity is a
worthless exercise if you do not have ways to
control all the intermediate steps of the technique.
Even a crude 1-4 scoring system is meaningless
without rigorous criteria for all the steps in 
the immunostaining and the enzyme histochemistry
that generates the visible result. 

Amplification immunohistochemistry is designed to
collect Yes or No answers. All "weak" or "background"
staining cries out for explanation. The notion of
a simple scale of intensity of immunostaining is 
worthless without standards fo the procedures and
the anticipated results. 

John A. Kiernan MB, ChB, PhD, DSc
Dept of Anat & Cell Biol, 
U.W.O., London, Canade.
On Thu, 13 Nov 2003, Jeff Gordon wrote:

> Date: Thu, 13 Nov 2003 14:11:36 -0600
> From: Jeff Gordon 
> To: Sharon E Willman , histonet@pathology.swmed.edu
> Subject: RE: [Histonet] (Histonet) Immunostaining Scale
> Sharon, there is a current scale for immunostaining intensity.  It is expressed as a fraction, with the numerator being a number from 1-4 describing the intensity of the primary stain and the denominator being a number from 1-4 describing the background intensity.  For example, an optimal stain would read 4/1, meaning it has a strong primary stain (4) and absolute minimal background (1), and the next best stain would be a 3/1, and so forth.  Usually any background that is over 1 is unacceptable, so even a 4/2, which would show good strong staining of the primary antibody but slightly higher than normal background is unacceptable.  This is the only scoring of immunostaining that I am aware of.  To show you a sample of our scoring of stains chart based on different pretreatments of antibodies, check this link:  http://www.cellmarque.com/2000/Pages/pretreatmentcomparison.html 
> I hope that this helps.
> Jeff Gordon
> Cell Marque Corp.
> 1-800-665-7284, Ext. 12
> -----Original Message-----
> From: Sharon E Willman [mailto:sharon.willman@bms.com]
> Sent: Thursday, November 13, 2003 1:23 PM
> To: histonet@pathology.swmed.edu
> Subject: [Histonet] (Histonet) Immunostaining Scale
> Hi,
> My Pathologist would like to know if there is a scale for
> determining intensity of immuno stains.  For example (1-5) and
> what is the criteria for determing this.
> Any help would be most appreciated.
> Thanks,
> Sharon

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