[Histonet] RE: air drying special stain slides rather than

Wed Sep 12 10:13:21 CDT 2012

EWayne (et al):
So, there you have it!
He (or she) who still uses xylene in the histology lab is just because he (or she) has decided to do so!
At this moment what you describe is standard procedure for several private labs in the US and the US Virgin Islands, Canada, Russia and Spain.
Besides dewaxing with dishwasher soap and air drying before cover-slipping, you can also eliminate xylene from tissue processing by just following the instructions outlinedin the articles I sent. you.
Try to contact as many colleagues as you can and "spread the word": xylene is out of our lives, as long as we want to.
Thank you for the information
René J.

________________________________
From: E. Wayne Johnson <ewj <@t> pigsqq.org>
To: Rene J Buesa <rjbuesa <@t> yahoo.com> 
Cc: "Mayer,Toysha N" <TNMayer <@t> mdanderson.org>; "'histonet <@t> lists.utsouthwestern.edu'" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Wednesday, September 12, 2012 9:49 AM
Subject: Re: [Histonet] RE: air drying special stain slides rather than

Dishwashing machines are not at all common in China even in Beijing so 
we could not find dishwasher detergent nearby.   But we took a bus and subway
ride toward the city center to Zhongguancun (the computer district) and found a Carrefour's
(Jia Le Fu 家乐福) that had one brand of powdered detergent "Finish" (Reckett Benckiser).
Finish was formerly called "Electrasol".  Actually I was a bit afraid of "Finish".  If I had known
it was the same thing as the familiar Electrasol, I would not have had any concerns.

Anyway, we took the Finish powder back to the lab where we had a covered water bath waiting at 90C.
I mixed 40 grams in with 2L of tap water and we followed the protocol Rene' sent with some test
tissues from some pigs we examined a few days ago (lung, liver, heart, spleen, kidney, gut).
We heated the detergent solution on an induction stove and poured in into some square glass jars in the water bath.
The procedure took the paraffin right off.  We did an H&E and dried the slide in the 60C oven after
a water wash to clean up after Eosin.  Ver-r-ry nice result.

Jane tried the technique then by herself with 3 more slides including one slide with some honking big pieces of pig cerebellum.
The sections all stayed put on the slides.  Sometimes we can lose most of the cerebellum in processing, so
we think it is a good demonstration that section loss is not going to be much of a problem.
The stain was a Harris hematoxylin regressed with 1% HCl.  We blued some with
tap water and some with Scott's.  I sort of prefer the plain tap water bluing.
Our Eosin is an Eosin/Biebrich Scarlet (Acid Red 66)/Orange G that we make up.  

We usually use a treatment in alcoholic Picric Acid to get rid of formalin pigment but we omitted that step today and the
slides turned out well.  Indeed these pigs were a field necropsy and the formalin was simple 10% unbuffered formalin in
plain local farm tap water, but there were only some traces of formalin pigment.  We are wondering if perhaps the detergent is 
taking out some of the formalin pigment for us.

We got a few white paraffin spots on one slide but even that would not be an issue in reading or photographing the slide.
Jane thinks she can tweak the procedure to eliminate the paraffin spots.
Jane's opinion on the procedure?  She will be bottling up all of the xylenes and alcohols and storing them away first thing tomorrow
morning.

This fixes a big problem for us because the histolab is on the first floor along with some offices.  We do our work under 
a fume hood and we are careful, but we  had an incident where students left containers of xylene uncapped outside the hood
overnight in hot weather vaporizing a large amount of xylene into the hallway.  Not cool.

We moved the tissue processor and autostainer to a remoter spot on the 4th floor 
but the water quality issues there made our autostainer a problem.  We can now bring our autostainer back and set it
up for special and routine stains.  The procedure with detergent from beginning to end
is significantly shorter than the xylene alcohols stain alcohols xylene procedure and we will dramatically reduce
our consumption and waste output of xylene and our consumption of ethanol.  Very cool.

Thanks

EW Johnson
Enruikang Ag Tech
Beijing

On 9/12/2012 3:13 AM, Rene J Buesa wrote: 
EWayne:
>All mounting media contains a so called "solvent". Permount contains toluene (not as nasty as xylene) and will "penetrate" the section provided it is absolutely dehydrated (in an oven in this case).
>You just have to finish the HC (or IHC) protocol and pop-in the slides in the oven.
>Balsam of Canada (the resin) is dissolved in xylene (always) so the "penetration" is also assured.
>Under separate cover I am sending you my articles.
>Try this method, you will love it!
>René J.
>
>
>From: E. Wayne Johnson mailto:ewj <@t> pigsqq.org
>To: Rene J Buesa mailto:rjbuesa <@t> yahoo.com 
>Cc: "Mayer,Toysha N" mailto:TNMayer <@t> mdanderson.org; mailto:'histonet <@t> lists.utsouthwestern.edu' mailto:histonet <@t> lists.utsouthwestern.edu 
>Sent: Tuesday, September 11, 2012 1:14 PM
>Subject: Re: [Histonet] RE: air drying special stain slides rather than
>
>I am convinced to give it a try because I also have trouble will the 
>loss of some stains in dehydration.
>I was concerned that the slides would not clear well after oven 
>dehydration.  I will see how it
>works for me.
>
>I can see clearly how going from counterstain to oven will save much 
>hassle with xylene and alcohols as well as
>not washing out some special stains.  I have tried some of the isopropyl 
>alcohol and acetone dehydration called for
>in some of the stain procedures and it would be great if the slides 
>could just be popped into the oven.
>
>What mounting medium are you using?  Does it matter?  I am a bit worried 
>about penetration of the mountant
>into the tissue section if there is no xylene in the tissue.  Will 
>neutral balsam still work ok?
>
>Rene:  if you have a link to the paper you talked about on eliminating 
>xylene, I am interested.  Xylene is becoming
>more and more of an issue and a pain for us.
>
>EWJohnson
>Enruikang Ag Tech
>Beijing.
>
>
>On 9/12/2012 12:01 AM, Rene J Buesa wrote:
>> Toysha:
>> Perhaps you have not oven dried stained slides before, and that explains some of your comments, like:
>> 1- if the stained slides are completely dried, the "miscibility" you point out is not an issues, because there is nothing to mix with;
>> 2- if you dehydrate → clear the stained sections that will take about 15 minutes per group of up to 25 slides, or even more depending on the protocol used in your automated stainer, but if your group of slides in their rack are placed in an oven at 60ºC for 5 minutes it will just that, 5 minutes reducing the usual TAT for each staining procedure;
>> 3- any oven can accommodate more than 100 stained slides in their racks and the TAT is shortened by oven drying, no matter how many slides you are working with;
>> 4- I really do not know where you can find that "extreme heat" can affect the tissue sections. All tissue sections are fixed → processed → dried (usually at the same 60ºC before staining) → stained and an additional step at 60ºC to dry before cover-slipping is just that, an additional step at 60ºC
>> 5- The so called "Lean" technologies do not refer to staining only, they have to do with the whole work-flow and an additional drying step at 60ºC cannot affect in a negative way to the work-flow
>> 6- after staining you will oven dry the sections.
>> I think you should try the method instead.
>> René J.
>>
>>
>> ________________________________
>> From: "Mayer,Toysha N"<TNMayer <@t> mdanderson.org>
>> To: "'histonet <@t> lists.utsouthwestern.edu'"<histonet <@t> lists.utsouthwestern.edu>
>> Sent: Tuesday, September 11, 2012 11:41 AM
>> Subject: [Histonet] RE: air drying special stain slides rather than
>>
>>
>> Ooh, great question for my students next semester. 
>> Your answer is the counterstain, some counterstains may require dehydration after rinsing, or some may not. Adjusting the times of the counterstain is not the issue as much as  the solvent of the counterstain.
>>  
>> Rene, while I do acknowledge that the xylene may/will cause hazards, we must think of the miscibility of the clearant and the dehydrant, as well as the amount of time involved.  The amount of time involved to blot and air dry the slides will affect the TAT for the specimen.  5 min may be ok if you have a small amount of slides, but with a larger number of slides, it will be considerably more than 5.  Also Lean methodologies would not apply in that case. With automation, the extreme heat involved with a stain dryer may affect the tissue on the slide.
>>
>> There are some stains that can be blotted, cleared and coverslipped, but using the alcohol to remove excess water and counter stain is better in my opinion.
>>
>>
>> Toysha N. Mayer, MBA, HT (ASCP)
>> Instructor, Education Coordinator
>> Program in Histotechnology
>> School of Health Professions
>> MD Anderson Cancer Center
>> (713) 563-3481
>> tnmayer <@t> mdanderson.org
>>
>>
>>
>>
>> Message: 16
>> Date: Tue, 11 Sep 2012 10:32:08 -0400
>> From: "Diana McCaig"<dmccaig <@t> ckha.on.ca>
>> Subject: [Histonet] air drying special stain slides rather than
>>      dehydrate    and clear
>> To:<histonet <@t> lists.utsouthwestern.edu>
>> Message-ID:
>>      <DCFD9E6A390E294AAF3A2561CD32E5C417A90529 <@t> ckhamail1.ckha.on.ca>
>> Content-Type: text/plain;    charset="us-ascii"
>>
>> I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip.
>>
>>
>>
>> Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping.  Reason given is that the counterstain gets washed out.  Wouldn't adjusting the times be a better resolution.
>>
>>
>>
>> I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process.
>>
>>
>>
>> Diana
>>
>>
>>
>> ------------------------------
>>
>> Message: 17
>> Date: Tue, 11 Sep 2012 07:52:05 -0700 (PDT)
>> From: Rene J Buesa<rjbuesa <@t> yahoo.com>
>> Subject: Re: [Histonet] air drying special stain slides rather than
>>      dehydrate    and clear
>> To: Diana McCaig<dmccaig <@t> ckha.on.ca>,
>>      "histonet <@t> lists.utsouthwestern.edu"
>>      <histonet <@t> lists.utsouthwestern.edu>
>> Message-ID:
>>      <1347375125.72189.YahooMailNeo <@t> web121405.mail.ne1.yahoo.com>
>> Content-Type: text/plain; charset=iso-8859-1
>>
>> Diana:
>> The most simple answer to your question is: "Because that is the way it has been done for more than 150 years".
>> The second question would be: "Is it necessary?" and the short answer to this question is: NO!!!
>> As a matter of fact, one of the steps I have developed to totally eliminate xylene from the histology lab refers to the "clearing" of stained sections, not only "special stains" (the so called HC and IHC) but the routine as well (the H&E).
>> Now, the "secret" to a successful drying of the stained slides is NOT to let them air dry because that will take not only too much time, but you can never be sure if the section is completely dry and if you add the mounting medium to a not completely dried section, you will have transparency problems.
>> The correct way of doing that is by drying the stained sections during 5 minutes at 60?C in an oven.
>> Under separate cover I am sending you something I published about your question and other aspects of how to completely eliminate xylene from ALL steps in the histology laboratory.
>> Ren? J.
>>
>>
>> ________________________________
>> From: Diana McCaig<dmccaig <@t> ckha.on.ca>
>> To: histonet <@t> lists.utsouthwestern.edu
>> Sent: Tuesday, September 11, 2012 10:32 AM
>> Subject: [Histonet] air drying special stain slides rather than dehydrate and clear
>>
>> I was hoping to get information on why special stains are dehydrated, cleared and mounted vs allowing them to be blotted dry, air dried then coverslip.
>>
>>
>>
>> Every procedure I have ever encountered always indicates to dehydrate and clear but I have heard where some labs are blotting the slides , allowing to air dry (probably not set standard time) and dipped in xylene prior to cover slipping.? Reason given is that the counterstain gets washed out.? Wouldn't adjusting the times be a better resolution.
>>
>>
>>
>> I understand residual water could be present and cause long term issues on storage but wanted some other opinions on this process.
>>
>>
>>
>> Diana
>>
>>
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