[Histonet] Question about fixation terminology
joelle weaver
joelleweaver <@t> hotmail.com
Thu Oct 3 15:21:11 CDT 2013
Surely I will find those that disagree with this post, however what I was "classically" trained about fixation categories generally falls within the information below...which I took the liberty of reposting here since it is pretty clear & straightforward from Leica. http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives/
I hope this will help.
" Traditionally fixing agents were termed “coagulant” or “non-coagulant” based on their effect on soluble proteins in solution. 7 Coagulant fixatives were said to result in a permeable meshwork of protein strands whereas non-coagulant fixatives which are additive in nature, formed extensive cross-links producing a less permeable gel. These terms are still encountered in modern histological literature but a more systematic approach has recently been taken to classification. 2 There are two major mechanisms which are important in fixation of proteins and protein complexes: denaturation, and addition and cross-link formation". Denaturation: " Most commonly this effect is induced by dehydrants such as the alcohols or acetone. These reagents remove and replace free water in cells and tissues and cause a change in the tertiary structure of proteins by destabilizing hydrophobic bonding. Hydrophobic areas, frequently found on the inside of protein molecules, are released from the repulsion of water and become free to occupy a greater area. hydrophilic areas of protein water molecules are loosely bound by hydrogen bonds and removal of water also destabilizes these bonds. The changes produced in the conformation of the protein molecules cause a change in the solubility of the protein, rendering water soluble proteins insoluble, a change that is largely irreversible if the protein is returned to an aqueous environment." 2, 7 Addition and cross-link formation: " The non-coagulant fixing agents chemically react with proteins and other cell and tissue components, becoming bound to them by addition and forming inter-molecular and intra-molecular cross-links. Because these agents are reactive compounds they bind to a variety of chemical groups in tissues, often affecting the charge at the site of attachment. This can have an effect on the subsequent staining characteristics of a particular protein as well as altering its molecular conformation and thus its solubility" . For further and more in depth discussion of formalin fixation ( and the article I am usually referred to in order to make corrections to my own chemistry in publications) is 1985!! Fox, et al. "Formaldehyde Fixation", Journal of Histochemistry and Cytochemistry, vol. 33, No. 8. pp. 845-855. pdf available at http://www.gofindpdf.com/readpdf/formaldehyde-fixation.html Joelle Weaver MAOM, HTL (ASCP) QIHC
> From: jonesly <@t> mir.wustl.edu
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Thu, 3 Oct 2013 19:32:51 +0000
> CC:
> Subject: [Histonet] Question about fixation terminology
>
> Hello -
> Our research group does a fair amount of autoradiography with frozen sections and we sometimes perform IHC or routine stains. I am not a histologist (nor do we have one in our current group), so I assumed that the correct answer was alcoholic formalin, because the other options were either inappropriate or not fixatives.
>
> I was taught (by an old-school cell biologist) that both alcohol and acetone act as dehydrating agents when used on frozen sections, and are not true fixatives, because they don't cross-link proteins. "Real" fixatives don't just preserve against decay, they also modify the tissue (i.e., cross-linking or denaturing proteins). I am pretty sure I was taught that acetone does not "fix" tissue, and that "fixing" tissue to a slide is an imprecise/slang term derived from "affix". (This was at least a decade ago, so my memory could be faulty.)
>
> How is acetone a fixative? I thought it just replaced water, and preserved the structure of frozen sections by drying them out. (Please be kind - I'm not a histologist. I've sat at the cryostat sectioning mouse brains, and I know when we use it, but I don't understand what the acetone actually does.)
>
> How do you describe the difference between preserving tissue by drying (with acetone) and cross-linking the proteins (with alcoholic formalin)?
>
> I would really appreciate clarification from the guru's on HistoNet. I no longer spend much time in the lab, but I edit our manuscripts, so try make sure we use the correct terms in describing how the work is done. (Some reviewers get picky about terminology.)
>
> Thanks,
>
> Lynne Jones
>
> Lab Manager
> Radiochemistry Research Group
> Radiological Chemistry and Imaging Lab
> Washington University School of Medicine
> St. Louis, MO
>
>
>
>
>
>
> Message: 2
>
> Date: Thu, 3 Oct 2013 10:21:42 -0400
>
> From: "Lee & Peggy Wenk" <lpwenk <@t> sbcglobal.net<mailto:lpwenk <@t> sbcglobal.net>>
>
> Subject: Re: [Histonet] HT HistoDeck question...
>
> To: "Watson, Linda" <Linda.Watson <@t> bms.com<mailto:Linda.Watson <@t> bms.com>>, "Stephenson, Sheryl"
>
> <SStephenson <@t> lifecell.com<mailto:SStephenson <@t> lifecell.com>>, <histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>>
>
> Message-ID: <335B9C2DCE414D1CB831DCCB07DEDC94 <@t> HP2010>
>
> Content-Type: text/plain; format=flowed; charset="iso-8859-1";
>
> reply-type=original
>
>
>
> I agree, there is probably more than one correct answer to this question,
>
> depending upon whether you are planning on doing stains for lipids, IHC,
>
> immunofluoresence or muscle enzymes.
>
>
>
> But I don't think (A) full strength 37-40% formaldehyde solution would ever
>
> be the correct answer. Unless you put a gauze in the bottom on the coplin
>
> jar, pour a little conc. formaldehyde on the gauze, put a lid on the coplin
>
> jar, and fix the section in formaldehyde vapors. But the question does say
>
> SOLUTION, not VAPOR. So I still think A is wrong. And most likely full
>
> strength acetic acid (C) is wrong - would eat the tissue off the slide.
>
>
>
> That leaves cold acetone (B) , which is good for some antibodies and some
>
> enzymes, or alcoholic formalin (D) which might be OK, but most of the time
>
> things either like alcohol and hate formalin, or they like formalin and
>
> don't like alcohol. So I would think most FS that we want to fix would not
>
> particularly like alcoholic formalin.
>
>
>
> And none of the solutions listed are good for lipids.
>
>
>
> So, given the question (with incomplete information) and the choices of
>
> answers, I would still side with (B) cold acetone.
>
>
>
> Now - a little aside - for the questions on the ASCP HT and HTL exams - if
>
> it is a new question, the people on the HT/HTL exam committee would be
>
> looking at it intensely before it goes on the exam for the first time. If
>
> the committee people are having problems answering it (like we are here),
>
> the question would be reworked until all the issues are resolved (such as
>
> putting in "for lymph node IHC" into the question). If it makes it past the
>
> committee, and the stats from the exam show that many people are having
>
> problems answering it, the question is pulled from the exam and is not used
>
> in the person's score. The question is then sent back to the HT/HTL exam
>
> committee, to try to figure out why examinees were having problems, and the
>
> question reworked again.
>
>
>
> As someone who has written exam questions at the school for 20+ years, I can
>
> tell you that it really is hard to write exam questions. I think I've
>
> covered everything in the question so that it is straight forward, and then
>
> half the students read something into the question that I never thought of,
>
> or come up with a written answer that I never considered. So I either have
>
> to throw out the question or give the point to the student, depending upon
>
> what's going on. And then mark the question for a re-write next year. And
>
> that doesn't include me marking the wrong answer on my master sheet. It
>
> happens!
>
>
>
> Peggy A. Wenk, HTL(ASCP)SLS
>
>
>
> -----Original Message-----
>
> From: Watson, Linda
>
> Sent: Thursday, October 03, 2013 9:40 AM
>
> To: Lee & Peggy Wenk ; Stephenson, Sheryl ;
>
> histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
>
> Subject: RE: [Histonet] HT HistoDeck question...
>
>
>
> For frozen cut sections, would the fixation also depend on what you plan on
>
> doing with it. For example, H&E, Special Stain or IHC? Please correct if I
>
> am wrong. I think that is a trick question!!!
>
>
>
> >-----Original Message-----
>
> >From: histonet-bounces <@t> lists.utsouthwestern.edu<mailto:histonet-bounces <@t> lists.utsouthwestern.edu> [mailto:histonet-
>
> >bounces <@t> lists.utsouthwestern.edu<mailto:bounces <@t> lists.utsouthwestern.edu>] On Behalf Of Lee & Peggy Wenk
>
> >Sent: Thursday, October 03, 2013 8:16 AM
>
> >To: Stephenson, Sheryl; histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
>
> >Subject: Re: [Histonet] HT HistoDeck question...
>
> >
>
> >Personally, I think it's "a" is a wrong answer, and that you are correct
>
> >that "b" is a better answer. My students and I have found a couple of
>
> >other
>
> >questions that we thought had the wrong answer indicated in the study
>
> >set.
>
> >
>
> >Peggy A. Wenk, HTL(ASCP)SLS
>
> >-----Original Message-----
>
> >From: Stephenson, Sheryl
>
> >Sent: Thursday, October 03, 2013 7:21 AM
>
> >To: histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
>
> >Subject: [Histonet] HT HistoDeck question...
>
> >
>
> >Hi,
>
> >Please clarify why this answer to the HistoDeck study question is a)
>
> >and
>
> >not b).
>
> >
>
> >Here is the question:
>
> >
>
> > 'Frozen section slides cut from fresh, unfixed tissue specimens are
>
> >optimally fixed in which of the following solutions?
>
> >a) 37%-40% formaldehyde
>
> >b) Cold acetone
>
> >c) Acetic acid alcohol
>
> >d) Alcoholic formalin
>
> >
>
> >Thanks,
>
> >
>
> >
>
> >Sheryl Stephenson | Histology Technician
>
> >
>
> >
>
> >
>
> >
>
> >_______________________________________________
>
> >Histonet mailing list
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> >Histonet <@t> lists.utsouthwestern.edu<mailto:Histonet <@t> lists.utsouthwestern.edu>
>
> >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> >
>
>
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