[Histonet] IHC antibody optimizing & validating

[joelle weaver]

I think that there is some variability to the methodology in this, and also depends on the constraints of your staining platform or if doing manual IHC. Below are my current habits, working alone and performing multiple validations at the same time...others may have their own way of accomplishing. The big thing to me is to keep excellent records for CAP. 
 
My opinion is, that the number of slides or different protocols needed to optimize is the number of slides or trials it takes to get the desired target staining versus high background, non-specific staining, other variables - until you are satisfied you have  the desired staining of the specific tissue element, antigen, protein bacteria....etc;  that the antibody targets. So I am not sure there is a specific required number, but if you do one for each retrieval stringency in your platforms ( automated),  and then change the incubation times if you can,  and maybe the AB titer a couple of trials, that is at least 4-8 slides, maybe more or less depending on your system. Sometimes you can hit it right off working from the specification sheet, but sometimes not. Usually after a couple of tests though, you can see if you are getting there or which direction to proceed. 
 
For setting up  a completely new protocol with unfamiliar antibody, I use an expected positive, sometimes normal, sometimes tumor section ( depending on the antibody) same tissue at this point, that I know to be well fixed and processed,  and change one of the staining variables after each attempt( tissue stays constant) sticking pretty close to the specification sheet if IVD. If none of the usual tweaks work,  I try different tissue and go back into it again. I document the changes and the results each time so I don't lose track of things on my worksheets.If you are doing an IVD,  then the specification sheet is the way to go with how to start and follow the recommendations and tweak for your lab conditions- changing one variable at a time until you are satisfied with the result. 
 
Usually the optimization and best slide/protocol is determined in consultation with a pathologist. Once the stain is optimized, I do parallel runs of known positives  and negatives ( usually at least 10 cases for IVD ( which may be 25 slides or more if needed) and more for ASR antibodies). Until you have enough to establish the stain is working consistently across multiple tissue samples, best to stress the tissue type you expect to use the stain on in your testing and to use your own fixation, processing and slide handling that you will again do with patient tissues in your parallel/validation studies. 
 
The pathologist or medical director will review all the validation slides and either accept and sign off on the test,  or send you back to the drawing board. For ASR, I consult literature to start, but there isn't much in the specification sheets as far as exact of manufacturer's recommendations. So this relies more on experience and close  review of results from optimization slides and tweaking from there. For those, I personally do at least 25 cases. For predictive markers I do 20 negative (have tumor-but non amplified/-) and 20 positive ( tumor and +/amplified) -so at least forty slides/tissue samples minimum for predictive markers, then you do the correlation. Keep everything for CAP.




Joelle Weaver MAOM, HTL (ASCP) QIHC
 
> From: CDavis <@t> che-east.org
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Tue, 25 Mar 2014 09:51:07 -0400
> Subject: [Histonet] IHC antibody optimizing & validating
> 
> Will you help me? I understand we are to use the known positives controls that the manufactures' recommends in the package insert when optimizing the stains, but I need to know what is your general procedure for optimizing (how many different staining protocols do you test) and validating a new antibody (how many different or "known" positive and negative tissues do you test [predictive markers I understand are 20])?
> 
> Cassandra Davis
> CDavis <@t> che-east.org
> 302-575-8095
> 
> 
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