John,

Although I haven't carried out a scientific study by any means, I have
noticed a trend towards darker staining when the IHC reagents and the wash
buffer are at room temp. as opposed to fresh out of the refridgerator.  I
use only room temp wash buffer on my routine IHC runs but always store my
HercepTest wash buffer in the fridge.  If I forget to take this wash buffer
out early to warm and perform staining using wash buffer at roughly 4
degrees C, staining intensity is drastically reduced.  These results are not
surprising nor are they inconsistent with the way one would expect any
reactions to be affected due to temperature variances.  DISCLAIMER: THESE
ARE NOT CONTROLLED STUDIES.  THESE ARE PERSONAL EXPERIENCES ONLY AND I AM
NEITHER BASHING NOR ENDORSING ANY PRODUCTS OUT THERE.  

Glen Dawson

-----Original Message-----
From: Auld, John [mailto:John.Auld@rfh.nthames.nhs.uk]
Sent: Wednesday, December 12, 2001 7:25 AM
To: 'HistoNet Server'
Subject: RE: Her-2/neu


	Glen

	I am interested in your comments about temperature of the reagents.
We did a little experiment on how temperature affected immunostaining. My
lab in not air 	conditioned and he temperature varies uite a bit, between 14
and 33 C. I stained some slides at 14, 24, and 33 to assess what if any
effect this had. I expected 	stronger staining more background at the
higher temp, BUT  to my surprise  ER, at low and high temperatures, stained
less cels and stained them weaker. These 	were not in line with a
mislabelling and a few were repeated to check.

	Does anyone have any suggestions or seen similar

	John Auld FIBMS MSc
	Biomedical Scientist 3
	Immunocytochemistry
	Department of Histopathology
	Royal Free Hospital
	Pond Street
	London NW3 2QG
	Tel 020 7794 0500 ext. 6516

	


> -----Original Message-----
> From:	HistoNet Server [SMTP:histonet@pathology.swmed.edu]
> Sent:	Wednesday, December 12, 2001 7:47 AM
> To:	HistoNet Server
> Subject:	Daily Digest
> 
> 
> 
> Date: 11 Dec 2001 15:27:55 -0600
> From: "Dawson, Glen" 
> Subject: RE: Her-2/neu
> 
> Cathy,
>  
> I am currently doing FISH correlations on a number of cases done both here
> and elsewhere in which the patient tissue was a 3+ on core biopsies but
> were
> negative on subsequent big blocks using the DAKO HercepTest kit.  I am
> also
> monitoring the results closely on all HercepTest cases that I perform.
> Preliminary results are leading me to believe that the temperature of both
> the Kit reagents as well as the HercepTest wash buffer are a much bigger
> factor than I had believed.  There is a standing order here (Medical
> College
> of Wisconsin & Froedtert Hospital) to no longer do this test on breast
> core
> bx's until this problem is resolved.  I'll let you know what I find out.
>  
> Glen A. Dawson  BS, HT & IHC (ASCP)
> Lead IHC Technologist
> Milwaukee, WI
> 
> - -----Original Message-----
> From: Cathy Thornton [mailto:cthornton@usiw.net]
> Sent: Tuesday, December 11, 2001 9:17 AM
> To: histonet
> Subject: Her-2/neu
> 
> 
> Is anyone using anything other than Herceptest for clinical Her-2/neu
> stains.  If so what antibody and detection and could you discuss scoring
> and
> reporting the results??  Also has anyone done FISH correlations with IHC
> results within your own laboratory?
>  
> Pat Zeitlow
> Boyce and Bynum Pathology Laboratories
> Columbia, Missouri
> 
> 
>