[Histonet] IHC mystery

[Truscott, Tom]

Hi Cindy, I don't use the Dako system or any of the antibodies you mention, but this kind of sounds like an antibody diluent problem, where either what you dissolve the antibody in doesn't spread out evenly on the slide or the antibody doesn't dissolve well in the diluents you use. In an automated system, it just could be that the rinse cycle in between reagents is incomplete. I have seen something like this happen where oily or waxy blotches contaminated the slide before the run . Good luck, Tom T 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cindy Pyse
Sent: Friday, August 09, 2013 9:51 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC mystery

Happy Friday Histonetters

 

I have a mystery. First a little background. We stain our immuno’s slides on a Dako 48 with the PT modules for pretreatment. We have not changed any protocols or antibodies since we optimized and validated the instruments. The majority of our antibodies are Dako but we do use other vendors, our detection is the Dako Flex. The pretreatment used is either high or low Dako pretreatment solution with the Her-2 and p16 using there FDA approved pretreatment. The her-2 and p16 slides are deparaffinized with xylene and rehydrated to water as the FDA protocol requires. All other antibodies are placed directly into the preheated pretreatment solution. All controls are precut and placed on Dako slides and prebaked for 30 minutes in a 58⁰ oven for 30 minutes. We cut our patient tissue and place them on the bottom of the slides and bake them in the same oven for 45 minutes. 

 

Recently we have noticed that some of the ER and PR (breast tissue) is not staining completely, with an occasional Her-2 giving us the same staining problem. Sometime it is part of the section not staining at all, almost like a hydrophobic effect with part of the patient tissue not staining past an almost perfect line. Sometimes it seems like the parts of the patient tissue within the section is lifted off, but when you look at the slide the tissue is there it just does not stain. All of our controls stain perfectly. All other antibodies are not effected.

 

Some of the things we have tried. Leaving the slides longer in the oven. Since it is breast tissue and the pretreatment has a surfactant in it. We have checked the level of the slides on the instruments. Since I do have two instruments, we have plotted the slides to see if it is a certain rack or instrument. We have checked if the pretreatment modules are running correctly since we have three. I have three tech running the slides. I am having an engineer coming in to look at both instruments.

 

There is no common denominator I can find. I have talked to my TRS from Dako and their Tech Service. No one can come up with the cause of the problem. I have been doing histology for over 30 years and immuno’s for over 20 years and I have never had a problem that I couldn’t figure out the cause. This time I need expert help. Does anyone have ideas? I am willing to trying anything to solve this problem. 

 

Please help!

Cindy

 

Cindy Pyse CLT, HT(ASCP)

Laboratory Manager

X-Cell Laboratories of WNY

20 Northpointe Parkway Ste 100

Amherst, NY 14228

716-250-9235 Ext. 232

cpyse <@t> x-celllab.com

 

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