[Histonet] Isopropyl Alcohol in the histology lab

Tue Aug 13 15:22:01 CDT 2013

Probably the same non-effect.Although both procedures are methodologically different both deal with epitope reactions. The difference is how you visualize your results.
René J.

________________________________
From: "Rathborne, Toni" <trathborne <@t> somerset-healthcare.com>
To: 'Rene J Buesa' <rjbuesa <@t> yahoo.com>; Teri Johnson <TJohnson <@t> gnf.org>; "'gu.lang <@t> gmx.at'" <gu.lang <@t> gmx.at> 
Cc: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Tuesday, August 13, 2013 2:48 PM
Subject: RE: [Histonet] Isopropyl Alcohol in the histology lab

Rene,
You stated that immunoreactivity is not weakened by the use of IPA. Do you know how the use of IPA might affect FISH results? 
Toni
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, August 13, 2013 1:38 PM
To: Teri Johnson; 'gu.lang <@t> gmx.at'
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Isopropyl Alcohol in the histology lab

Teri:
The automatic coverslipper will wok on oven dried stained sections. I used them on a Sakura film coverslipper and my lab was in Miami Beach, and you do not more humid than that!
Xylene is the one weakening the immunoreactivity the most but I have tested the IPA and the weakening does not exist although there are so many antibodies that some may weaken the issue is: will the weakening affect the diagnosis or just will produce a weaker reaction? That you would have to test further.
But the bottom line is tha xylene should be eliminated.
René  J.

________________________________
From: Teri Johnson <TJohnson <@t> gnf.org>
To: 'Rene J Buesa' <rjbuesa <@t> yahoo.com>; "'gu.lang <@t> gmx.at'" <gu.lang <@t> gmx.at> 
Cc: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Tuesday, August 13, 2013 12:19 PM
Subject: RE: [Histonet] Isopropyl Alcohol in the histology lab

Thank you Rene. You raise many good points.

I have entertained the idea of eliminating xylene and believe it can be done successfully. It’s on my list of possible safety-related upgrades to the lab. I would very much like to get an automatic coverslipper in the lab, but don’t think that could work in a xylene-free environment. It might work using a substitute, but when the humidity in the lab gets high (we are a coastal city), it can overwhelm the aliphatic hydrocarbon.

I respectfully disagree with your assertion that immune reactivity is not further affected by processing after fixation. It is well known that alcohol and xylene can cause loss of immunoreactivity of proteins post-fixation. I think Bill Grizzle among others did some experiments and came to this conclusion. We tend to use antibodies and develop detection protocols that can work despite those factors. What concerns me is if we change the alcohol, will that increase or decrease any immunoreactivity that might cause us to have to revalidate all our antibodies? My gut feeling is “no”, but if others out there have experience in which it did, I do hope they speak up.

We do not do any alcohol recycling. I have worked in labs where we recycled ethanol and it can work quite well provided you don’t try recycling xylene-contaminated alcohol. You’ll never get the xylene out of it; at best you can recycle it as xylene, but most of the reagent will just end up in the waste container. Best just to relegate it to waste in the first place.

As for why I am asking, the catalyst for this is our new fume hood. It goes into intermittent alarm mode when in contact with ethanol, but mostly small amounts of methanol. They recommend we use IPA. Because I know IPA has a long history of use in Histology processing, I think it could be a reasonable substitution without creating a lot of problems downstream. But, as always, I try to tread carefully before leaping.

Gudrun, I am planning on a simple exchange of reagent alcohol by isopropanol as usual. If we use it in our processor, I’d like to try to use it universally as much as possible. That’s why I asked about holding tissues in it, and using it in the stain line. The more exceptions we have in the lab (IPA on processor, SDA-3A on stainer, etc) the more potential for error when doing exchanges.

Thanks again,

Teri

From:Rene J Buesa [mailto:rjbuesa <@t> yahoo.com] 
Sent: Tuesday, August 13, 2013 8:41 AM
To: Teri Johnson; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Isopropyl Alcohol in the histology lab

Teri:
It is a wise decision to substitute EthOL with Isopropyl alcohol (IPA) and to your questions:
1- although it is not advisable to keep tissues for long periods of time in EthOL sometimes this is done. EthOL will sometimes make the tissue brittle mostly because its chemical properties but IPA dehydrates more gently and is a larger molecule that EthOL so, if you use to keep tissues in 70EthOL you will be able to leave them in 70IPA more safely.
2- with a different dehydrating quality you will probably need to adjust your times in the auto-stainer or in your hand staining sequence. Your best option though is to totally eliminate xylene and the alcohols in your staining sequence. How? Dewax sections with 2% aq. dishwasher soap at 90ºC → distilled water → staining protocol → dry your stained sections in an oven for 5 minutes at 60ºC → coverslip as usual, but I am sure this may seem "too drastic" for you but I can assure you it works. Ask me if you want to.
3- Immnuno reactivity depends on how the tissue is FIXED and not in how it is processed. Had processing have any impact there would be scores of different protocols for all those who dehydrate or clear in different reagents. The BondMax IHC stained was developed in conjunction with the Peloris instruments and this later instrument was able to use EthOL, later IPA and even it has a "dry-out" step before infiltration (which I am not very fond of).
4- IPA is a safe substitute to EthOL but you have to consider other aspects such as costs and availability. Some labs lave "alcohol licenses" allowing them to use "200 grade EthOL", other can use only denatured EthOL but IPA will be always "pure" (about 98-99%).
Recycling is also an issue: I never recycled EthOL because it takes much more time and energy than recycling xylene, which I used to recycle. You will have to determine if recycling IPA is worth the cost and time.
RenéJ.

From:Teri Johnson <TJohnson <@t> gnf.org>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, August 12, 2013 5:18 PM
Subject: [Histonet] Isopropyl Alcohol in the histology lab

Dear colleagues,

I am entertaining the notion of switching over to isopropyl alcohol (IPA) for our tissue processing and have a few questions. I know the xylene-free processing and microwave processing protocols use this universally, but at this time I am only interested in getting rid of the ethanol/methanol/isopropyl alcohol blend.

1 - Are there any known issues with holding tissues in 70% IPA rather than 70% ethanol/reagent alcohol?
2 - Is there any impact on the stain line if you use it for H&Es and hydration/dehydration of histochemically stained slides?
3 - Have any of you who use IPA found any impact on immunoreactivity when compared to using other dehydrants?
4 - Is there anything else I have overlooked?

We have pure ethanol available to us if needed for particular stain applications.

Best wishes, and thanks in advance.

Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752

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