[Histonet] Re: H&E staining question

Wed Jul 3 20:48:01 CDT 2013

The lab I work in now did GMA for all Bone marrows as well, of course with B5 fixation.  Some of the haematologists still miss the old days.  In those days everything revolved around quality of work.  Now we have to balance practicality, cost, OH&S and time to get a compromised method.

regards
Tony

>>> Patsy Ruegg <pruegghm <@t> hotmail.com> 7/4/2013 10:36 am >>>
I did a lot of GMA work on bone marrows and lymph nodes back in the late 70's and early 80's, we had a special H&E developed by my hematopathologist that was Hematoxylin (Harris before Gills came out) eosin and azure we called it HEA I just ran across the protocol a few days ago i should post it for old time sake, it was brilliant for 2 micron sections of hematopoetic tissue, hematologist are the best morphologist in my opinion and demanded thin high quality histology sections.  The nuclear detail with HEA is phenomenal.  A little off topic we did not use an oxidizer.  

> Date: Wed, 3 Jul 2013 17:06:25 -0700
> From: akemiat3377 <@t> yahoo.com 
> To: akemiat3377 <@t> yahoo.com; Tony_Reilly <@t> health.qld.gov.au; TJohnson <@t> gnf.org; linda.prasad <@t> health.nsw.gov.au 
> Subject: Re: [Histonet] Re: H&E staining question
> CC: histonet <@t> lists.utsouthwestern.edu 
> 
> Oh by the way, the reason I did a Harris hematoxylin and eosin/phloxine on GMA (plastics) was because our pathologists at OHSU hated Gills and were used to seeing Harris hematoxylin on all of our regular surgical and autopsy slides. We only did GMA on kidney, liver, some GI and BM core bx's. 
> 
> I was just a plain ole histologist that wanted to please her pathologists!  Anything can be accomplished if you are creative and innovative!
>  
> Akemi Allison BS, HT(ASCP)HTL
> Director
> Phoenix Lab Consulting
> E-Mail: akemiat3377 <@t> yahoo.com 
> 
> 
> 
> 
> ________________________________
> From: Akemi Allison <akemiat3377 <@t> yahoo.com>
> To: Tony Reilly <Tony_Reilly <@t> health.qld.gov.au>; Teri Johnson <TJohnson <@t> gnf.org>; Linda Prasad (SCHN) <linda.prasad <@t> health.nsw.gov.au> 
> Cc: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
> Sent: Wednesday, July 3, 2013 4:54 PM
> Subject: Re: [Histonet] Re: H&E staining question
> 
> 
> Tony: 
> I actually was going to suggest this.  You beat me to it!  I used to do GMA on 1.5 microns sections and did a Harris hematoxylin and eosin/phloxine routinely (by the way, this was not recommended back in 1980. Gills hematoxylin was the norm!). 
> 
> To get the results I wanted, I would routinely oxidize the slides in 1% periodic acid and rince in running tap water before staining in hematoxylin.  the staining was intensified and was beautiful!  I came up with this because my PAS's with hematoxylin counterstains looked better than routine H&E's.  I figuired it was worth doing the GMA's routinely using 1% periodic acid since we didn't do that many. Funny how we stumble on our modifications!  
> 
> Science never sleeps!!
>  
> Akemi Allison BS, HT(ASCP)HTL
> Director
> Phoenix Lab Consulting
> E-Mail: akemiat3377 <@t> yahoo.com 
> 
> 
> 
> 
> ________________________________
> From: Tony Reilly <Tony_Reilly <@t> health.qld.gov.au>
> To: Teri Johnson <TJohnson <@t> gnf.org>; Linda Prasad (SCHN) <linda.prasad <@t> health.nsw.gov.au> 
> Cc: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
> Sent: Wednesday, July 3, 2013 4:06 PM
> Subject: [Histonet] Re: H&E staining question
> 
> 
> Hi Linda
> 
> For a little more information (not an article) go to:
> http://www.ihcworld.com/royellis/problems/problem21.htm 
> 
> regards
> Tony
> 
> 
> 
> 
> Tony Reilly  B.App.Sc. , M.Sc.
> Chief Scientist, Anatomical Pathology
> Pathology Queensland-PA Laboratory 
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> 
> >>> Teri Johnson <TJohnson <@t> gnf.org> 7/4/2013 12:37 am >>>
> Hi Linda,
> 
> There is no journal article, merely an observation on my part many years ago.
> 
> Teri
> 
> On Jul 2, 2013, at 10:05 PM, "Linda Prasad (SCHN)" <linda.prasad <@t> health.nsw.gov.au> wrote:
> 
> > Hi Teri,
> > Oxidising the slides in periodic acid prior to H&E improves the HE. Would really appreciate if you could please send me the reference for that article. Thank you Teri.
> > 
> > 
> > Linda Prasad, MSc, BSc
> > 
> > Hospital Scientist | Histopathology
> > t: 02 9845 3316 | f: 02 9845 3318 | e: linda.prasad <@t> health.nsw.gov.au | w: www.schn.health.nsw.gov.au 
> > m: 0425 314 267
> > 
> > 
> > 
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> > ________________________________________
> > From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of Teri Johnson [TJohnson <@t> gnf.org] 
> > Sent: Wednesday, 3 July 2013 3:24 AM
> > To: histonet <@t> lists.utsouthwestern.edu 
> > Subject: [Histonet] Re: H&E staining question
> > 
> > Hi Brett,
> > 
> > I agree with the others, the storage in 70% ETOH is likely not the cause of the washed out appearance of the staining.
> > 
> > *       Did the lab that did the H&E staining run a daily control? How did that look? Perhaps the tap water they are using went a little funny? Perhaps the depar xylene needs refreshing? The hematoxylin is used up? If all the other H&Es done that day look good, then look further.
> > 
> > *       Look at the fixative, it is possible to get bad batches of that. It is also possible to cram a lot of tissue into a small space resulting in inadequate fixation. Or perhaps it was bloody and not changed to fresh fixative? Was one sample fixed at room temperature and the other fixed at 4 degrees C? Was the person doing the necropsy the same among animal batches?
> > 
> > *       Two things might help get better hematoxylin staining - I recall a journal article about using antigen retrieval pretreatment to improve H&E staining on samples that had been stored in fixative for a prolonged period of time. That might help.  Years ago,  I also noticed that the hematoxylin counterstained PAS slides looked better than our H&Es, so we put a bucket of 0.5% periodic acid as an oxidation step before hematoxylin. It needs to be rinsed well so there is no periodic acid carryover (that'll kill your hematoxylin!), but that might improve your staining.
> > 
> > If you get this figured out, please let us know the cause and fix.
> > 
> > Teri Johnson
> > Manager, Histology
> > GNF - San Diego, CA
> > 858-332-4752
> > 
> > 
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