[Histonet] Microwave processing

[Bryan Hewlett]

Rene, and John,

The answer for use of this instrument for routine IHC is to make sure that 
fixation PRECEDES processing, they are not one and the same!
If your routine fixation is NBF, then prefix in NBF before rapid processing.
True, some of the speed advantages are lost, but rapid processing can still 
be used to good effect in the workflow.
Any change in the routine fixative used can, and does, have dramatic effects 
on the antigen to be demonstrated by IHC.

The effects on antigens such as CK's, vimentin etc. can be positive, as Rene 
indicates no HIER is required.
On the other hand, the effects on ER, HER2 and many other surface proteins 
can be devastating!
There is a reason that the fixation protocol for HER2, EGFr and other 
surface markers calls for fixation in NBF,
i.e. many surface proteins are known to be stripped away by alcohol 
fixation!!
FISH for HER2 is not a problem with alcohol fixation, except that the 
proteolysis step will have to be removed or greatly curtailed.
This means you will no longer be following the approved methodology!!!

IMHO, to not parallel process and compare/validate all results would 
constitute a gross dereliction of duty!!!


Bryan

----- Original Message ----- 
From: "Rene J Buesa" <rjbuesa <@t> yahoo.com>
To: "John PJ Coleman" <jcolclefa <@t> aol.com>; 
<histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, February 22, 2006 6:06 PM
Subject: Re: [Histonet] Microwave processing


> John:
>  I know this instrument and the technology is safe. You will not need to 
> do HIER for IHC since the tissue will be fixed in an alcohol based 
> fixatives and I don't think that you will need to run parallel runs.Maybe 
> you will have to dilute your antibodies a little more than for formalin + 
> HIER.
>  The turn around time is 120 cassettes per hour, and with 4 of these 
> instruments (which I personally think is a "bit too much") what you will 
> have to do is to rethink your whole staff scheduling.
>  Try to get a book titled:
>  Microwaves for the Art of Microscopy by L.P Kok and M.E.Boon, Coulomb 
> Press Leyden 2003 (you can get it at http://www.coulomb.nl
>  This instrument is ideal for a large volume reference lab like yours.
>  Hope this will help you.
>  René J.
>
> John PJ Coleman <jcolclefa <@t> aol.com> wrote:
>  I am the Senior tech of a large hospital corporation. My
> administration has just won funding for 4 Sakura Microwave rapid
> processing units. We run FISH her 2 on formalin fixed paraffin embedded
> tissue as per FDA protocol. As I tech, I am not in favor of tossing
> routine processing wholesale in favor of a completely new technology
> without thorough testing and parallel processing. Also, we are a
> regional reference lab for IHC and have a panel of 115 antibodies, all
> optimized for formalin fixed paraffin embedded tissue. We run an
> average of 350 IHC slides a day, max 580 per day and would have to
> re-optimize these to use in the new formalin free world while keeping
> our FFPE procedures in parallel for our reference lab work. Much like
> running 2 labs. If anyone has any insight, or if anyone currently uses
> these instruments for routine and/or IHC, feel free to call or email,
> and I'll check the postings on this string. We are also taking
> invitations to come out and see these things in use real time.
>
> John PJ Coleman-757 335-2159
> http://members.cox.net/captainmadman/
> http://journals.aol.com/jcolclefa/Waytoomuchboringcraptosayusually/
>
>
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