[Histonet] Partially muddy slides

histotech <@t> imagesbyhopper.com histotech <@t> imagesbyhopper.com
Tue Nov 16 11:49:57 CST 2010

Regarding incomplete deparaffinizing:  I did give that some thought, but
wouldn't that affect ALL the slides and not just  hit or miss?  It's
primarily the small biopsies that are being affected, and I would think
those would clear the paraffin much easier than the big specimens?

Just as a troubleshooting step, I will increase the deparaffinzing times in
the stainer.

Jeff, I wondered if the collecting nurse/doctor was possibly placing the
tissue in saline prior to formalin?  A cautery effect... Would that show up
as muddy or more burned/desiccated?

Please keep the ideas coming!


-----Original Message-----
From: Victor Tobias [mailto:victor <@t> pathology.washington.edu] 
Sent: Tuesday, November 16, 2010 12:14 PM
To: histotech <@t> imagesbyhopper.com
Subject: Re: [Histonet] Partially muddy slides

What about incomplete paraffin removal? Without seeing the slides it is 
hard to tell.

Victor Tobias
Clinical Applications Analyst
University of Washington Medical Center
Dept of Pathology Room BB220
1959 NE Pacific
Seattle, WA 98195
victor <@t> pathology.washington.edu
206-598-7659 Fax =================================================
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On 11/16/2010 9:09 AM, histotech <@t> imagesbyhopper.com wrote:
> Hi Histonetters!
> We are having an issue in our lab with some inconsistent staining.  
> It's an odd situation.  One small biopsy can stain reasonably well 
> over most of the tissue, but then one area of it is muddy.  This does 
> not affect all the slides in the same staining rack, nor does it 
> affect all the small biopsies that were processed in the same 
> processing cycle.  Troubleshooting this has been difficult, at best. 
> It's sort of hit or miss on when/where the muddy-ness appears.
> Here are my thoughts:
> ** I don't know if this is a processing issue, a staining issue or 
> perhaps even a collection issue.
> ** If it affected all small biopsies equally, I would be tempted to 
> question the processing/fixation.  But it doesn't affect across the 
> board.
> **  If all the staining was muddy, I would look to the stainer, but again,
> even within the same slide, we can have clear and muddy areas.
> **  The stainer is a Leica XL (about 3 years old),  hooked up to tap water
> for the washes.
> **  We are using a regressive staining method with Fisher brand Protocol
> Harris hematoxylin and Protocol Eosin Y.  We use acid alcohol and ammonia
> water for differentiating and bluing.
> **  The xylene we use is recycled, but verified for purity.
> **  The alcohols are recycled, but the last alcohol prior to the
> xylene/coverslipping is "pure", not recycled (for just in case).
> Suggestions on what could be causing this issue would be greatly 
> appreciated.
> Michelle
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