[Histonet] RE: Question about fixation terminology

Sarah Dysart sdysart <@t> mirnarx.com
Thu Oct 3 15:28:44 CDT 2013 

http://www.ihcworld.com/_protocols/general_ICC/fixation.htm

This article specifically addresses the differences.  You are right about the cross linking, but alcohol and acetone are still considered fixatives.

Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jones, Lynne
Sent: Thursday, October 03, 2013 2:33 PM
To: histonet <@t> lists.utsouthwestern.edu
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

>

>

>

>

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>Histonet mailing list

>Histonet <@t> lists.utsouthwestern.edu<mailto:Histonet <@t> lists.utsouthwestern.edu>

>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

>


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