Re: immunostaining, method validation

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From:Tony Henwood <>, Vicki L McKaughan <>
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Dear Vicki,

> We recently purchased a Ventana Nexes Immunostainer.  I have been told by our lab QA
> Director that I must do method validation/comparison studies for all the antibodies that we do.  She wants me to do 5 cases per antibody both with the Ventana and our
> old method which was basically manual.  If anyone else has encountered this I would like some suggestions on how to go about this study.
> It could prove to be very time comsuming and  costly.  Does anyone know specifically if CLIA  requires this for Histology?  Thanks for your help.
You are probably aware of the pro's and con's of each antibody you 
use. What tissue elements each antibody may cross react with for 
instance. The following could be useful as a guide.

1.	Test antibody on several positive tumours, both well and poorly 
differentiated, tumours with a good deal of antigen and especially 
those with small amounts present.
2.	Test tissues known to cross react eg salivary gland for PSA, 
benign lung for CEA etc.
3.	Try an Optimum titre run with the two techniques using an antibody 
of your choice.

This will probably require 3 or 4 sections per antibody run and the 
above should at least be done when ever a new antibody or a change in 
the technique is considered. I remember the difference in dilution of 
the localisation antibody that was required when I changed from a PAP 
to an ABC technique (1/100 to 1/300 for S100 for example).

I hope this helps.
Regards,  Tony

Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital

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