[Histonet] why do we use xylene? was - Peloris Processor and isopropanol

[Barbara Bublava]

Dear Histonetters, Hallo Gudrun,

In 1968 Romeis (german book about Microscopic Techiniques) named
Isopropanol as an Intermedium when used at 56°C.

In a later edition ( I dont have it by hand right now) he once more
mentions the potential of Isopropanol.
Searching Histonet archives or Google also gives some results

One protocol suggested isopropanol with 50°C at the last
change and then going directly to paraffin. I did try that and neither
me nor my pathologist could see differences to usual xylene processing
(on autopsy material; HE, Trichrome; Chloroacetate Esterase, Prussian Blue).

For me it sounds too good to be true.  I am afraid of missing something
important because IF it is that easy - why do we all use hazardous
xylene or expensive and smelly substitutes on our processors?

Maybe someone out there can tell us why isopropanol isn´t the
intermedium of choice?

Barbara Bublava
Dep. of Forensic Medicine
Vienna, Austria

Gudrun Lang wrote:
> Yes, I've been told, that it works. But what is the histochemical background? 
> Proper fixed tissue would'nt suffer too much from high temperatures, I think. What is about too thick, underfixed tissue? Wouldn't it become hard and tough, difficult to cut? What about shrinkage?
> What about collagen-rich tissue? I don't want to produce glue.
> Is the whole process sensitiv to the duration in the high temp.? What is the recommandation for the maximum duration in 80 degrees?
> What are the critical points?
> 
> Many questions, I know. But I don't like black boxes. 
> 
> Gudrun Lang
>  
> Biomed. Analytikerin
> Histolabor
> Akh Linz
> Krankenhausstr. 9
> 4020 Linz
> +43(0)732/7806-6754
> 
> -----Ursprüngliche Nachricht-----
> Von: Phil McArdle [mailto:pmcardle <@t> ebsciences.com] 
> Gesendet: Montag, 02. April 2007 20:30
> An: gu.lang <@t> gmx.at
> Cc: histonet <@t> lists.utsouthwestern.edu
> Betreff: Re: AW: [Histonet] Peloris Processor and isopropanol
> 
> I'm with a laboratory microwave vendor:
> 
> Never mind 74˚ C - some microwave protocols call for temperatures as 
> high as 82˚ or 84˚ C (!) in order to exceed the boiling point of 
> isopropanol, facilitating its replacement with paraffin. Naturally, this 
> caused some concern and resistance in the anatomic pathology community. 
> However, look at your own question about IHC: when you consider antigen 
> retrieval protocols call for 100˚C, even 84˚ pales by comparison. Of 
> course, there are many reasons you want to minimize the amount of time 
> spent at high temperatures, but there is no problem with the majority of 
> tissue types for the relatively short timeframes involved in microwave 
> processing.
> 
> Also, by the paraffin step, the tissue has already been fixed, 
> dehydrated, and defatted; while it is in a sense still "tissue," it's 
> radically different from the tissue as received. You certainly don’t 
> want to burn or "cook" anything, but 82°C isn’t really extreme in this 
> context. (I'd be at least as concerned about how a given paraffin and 
> additives hold up at high temperatures, so it's always a good idea to 
> check with the manufacturer.) There also is the issue of vacuum; since 
> the purpose of high temperature is to get above the boiling point of 
> isopropanol, and vacuum causes BP depression, vacuum should allow you to 
> lower the temperature to something you're more comfortable with.
> 
> More information may be found at
> 
> http://www.ebsciences.com/pdf/EBS_MW_COMPANION.pdf
> 
> Very best regards,
> 
> Phil McArdle
>