Re: [Histonet] RE: Endogenous biotin ??

From:"Katri Tuomala"

Have you ruled out the endogenous biotin by repeating the same test slide 
with Biotin block?
We tackle with endogenous biotin almost daily. Many types of cells have it 
in varying amounts. The reason we are seeing it now so often is to do with 
various heat retrievals combined with short fixation times.
We hardly ever had this problem before HIER, formalin fixation masked most 
of it, if it was present. Now we often end up repeating cases with a polymer 
detection system, which does not use avidin. Unfortunately it is still too 
expensive for us to use routinely, but that maybe the only route to go in 
the future. But then, the polymers may have their own problems.
We are now trying integrating the biotin block into the primary antibody 
diluents and the detection system to eliminate the extra steps you need to 
In your case the Ventana retrieval must be somehow stronger , than the 
manual one, therefore revealing the biotin present.
Unfortunately one can never predict which case will have endogenous biotin. 
The amount varies in tissues
of all kinds and many tumors have an abundant amount, sometimes even too 
much to block with a biotin block.
This problem will not be easily solved. If somebody has some brilliant 
ideas, pass them my way too...


Katri Tuomala
Hamilton, Ontario, Canada
----- Original Message ----- 
From: "Malam Jacqueline" 
Sent: Wednesday, December 13, 2006 10:48 AM
Subject: [Histonet] RE: Endogenous biotin ??

>I am trying to help someone from another hospital who is having immuno
> problems with non-specific staining in some of their negative controls 
> when
> using a Ventana Benchmark. I also got the same staining using their slides
> on my Benchmark XT. It can appear in mucin-producing cells of e.g. gastric
> tissue, lymphoid tissues and rather spectacularly in Leydig cells in 
> testis,
> but it does not appear in the same type of tissues all the time nor does 
> it
> appear when they do manual antigen retrieval and use their Ventana Nexus.
> The staining pattern has the appearance of endogenous biotin and testis 
> does
> indeed contain biotin; but I would have thought that any endogenous biotin
> in the tissues would have shown up on the Nexus-stained slides which had
> manual antigen retrieval. I am wondering if there is some neuro-endocrine
> involvement rather than biotin. Ventana have been extremely helpful but 
> are
> also scratching their heads too! The only significant difference between 
> the
> two methods is the fact that manual antigen retrieval is performed at a 
> much
> higher temperature and for a shorter time than the Benchmark. Fixation,
> processing, reagents and equipment malfunction has supposedly been ruled
> out.
> Your help and comments would be much appreciated by us both.
> Jacqui
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