[Histonet] Help With Hemo Fading

Thu Jul 2 22:04:51 CDT 2009

Hi All,

Mordanted hematoxylin is quite colorfast when carefully 'blued' after binding to acid moieties in the tissues.  If one analyzes the entire process of H&E dyeing, one can 'see' several steps in which the 'bluing' process can be 'undone' or 'temporized'.  Note that I have always used personally prepared Gum Damar in Xylene to mount my sections.  It has always behaved better than expected.

Please recall that all of the currently used histologic procedures originated using water glasses from the dining room or kitchen and chemicals that were generated by magic.  When I was learning the procedure using the now-antiquated Coplin jar, I was overwhelmed by the inability of distilled water to 'blue' hematoxylin after acid differentiation.  For we new histologists at the time, the only thing that really 'set' the blue color was running tap water for 5 minutes AFTER 5 minutes in 35% EtOH, 2% in NaHCO3.  NB, the same magic rinse was required to stabilize the magenta of the PAS stain.  In those days, there was the complaint that even some tap waters in other parts of the country left some lack of color fastness with mordanted hematoxylin (it must be remembered that the active component of the H&E stain is NOT hematoxylin (those who are surprised must go the library!!)

In any case, I had a colleague who noticed that the slides in his collection were fading slowly in the closet in which he stored them (stored pretty much as we all do).  It was suggested that he perform a test of the storage environment in the following way.

        Using an aquarium aerator placed in the closet to bubble air thru 1.5 liters of distilled water with an appropriate indicator in a two-liter Ehrlenmyer with a two-hole stopper, the outlet taking the bubbled air out of the closet via an appropriate length of tubing.  Modify the above aerator and water volume to insure that the water will not be significantly evaporated during the overnight process (temperature, volume, and pressure are the appropriate variables).

This test should be run for several to many nights and days (or weeks) to determine whether at any time the environment 'goes' acidic.  [[The upside is that the experiment/test is neither labor intensive nor expensive (no grant proposal is required), though if you can wangle some stimulus money, you must let me know, because they will want to be able to count me as one of those who was put to work - albeit briefly.  If you run the above test for several years, you may be able to count me as being employed every time you refer to this suggestion.  [Perhaps not ethical, but we must do anything to keep unemployment under 10% lest there be declared another economic crisis whose fix we know by now will be the expected printing of more money to fund more studies of more fish in a pond or bugs on a wall.]]

Back to point, if at any time one observed the water to go acid, then the location of the storage is a likely candidate.  

If you use a machine to stain your sections, as many to most do,  then perhaps another environmental characteristic (such as humidity) is causing either continuous or seasonal ethanol dilution and thus leaving too much water in the sections, even though they do not appear milky when mounted.  In such a case, one must expect faster diffusion of acidic materials into the preparation.  NB, I only consider those airborne substances that are hydrogen rich - they can be inorganic or organic - simply anything that gets sprayed, mixed, dumped in the sink - anything that continuously gets aerosoled in the lab or is blown in with the 'conditioned' air.

Another test you might perform involves sealing slide boxes in plastic bags - see your local molecular biologist or check the kitchen area at KMart, Penny's, or Walmart for systems that pull a vacuum around food and seal it by creating a heat seal.  [If still unfamiliar, call Mom OR Mom-in-law!].  You must have a control box to compare over time.  One assumes that NOTHING can get in or out of such a sealed container.

The ultimate downside is that one does not or cannot control a process that one trusts to be automatic and credible.  

Hematologists may take note:  At the age of 40 I was laid up with a pulmonary 'problem' that included a few days with 104-105 degree spikes, dry coughing, and visits from the Savior who I sent away screaming, "Hell No!  I won't go!" - and in the process scarring the b'Jesus out of my kids.  In any case after passing the hospitalization test and ahaving received, i.v., every known broad spectrum antibiotic in the pharmacy, I got my doctors to give me erythromysin i.v.("we don't give enythromysin orally or i.v. - it bites!), the temp went down, and I felt better.  [If legionella is sensitive to erythromysin, why did so many die from that bug in Philadelphia? (This will not be on the test, but it should be.)]  Thus, came the day when I was told that my blood differential was so messy that it had been recommended that I submit to a bone marrow exam to determine my proximity to death.  I quietly demanded to know how the differential was done.  The answer?  The Coulter counter!  I quietly agreed to a bone marrow exam AFTER a manual differential was performed.  Later, the hematologist only wanted to know how I knew.  I told her that while working in a path lab at night during college (I know I wasn't 'certified'!!!), I had compared myself to the 'Counter' and I was frankly more consistent.  I admitted that my test was not statistical, but I had real confidence in the capacity of the eye and little in the photometer looking thru glass.

Machines may be necessary to do the work, but they do not clean themselves and they do not cry out when the 95% ethanol has become 87%.  With Coplin jars, variables were easy to manage.  They were more difficult the more we tried to do multiples of specimens.  This should be obvious in spite of the fact that automation is practical and manual is not - even though sometimes necessary.

When I started in histology, I began at the beginning of the Paraplast revolution, yet there were still those who formulated their own paraffin for the part of the planet in which they worked.

Sorry for the late-night babble, but I have vented stored up compulsion.

Cheers,

Fred Monson

P.S.  

Those who are required to volunteer do NOT.  Those who require others to volunteer fail.

Every tax is a means by which a government forces a citizen to pay for the vacations of those who work for the government.

When the USA has a public health plan, every union will be forced to accept it by the employer, and all those who want something better will be required to pay for the full load.  Government plan - 'free', all others - cost extra.  That's competition?  Why won't the insurance companies want to give 'free' public health care to their employees?  Their own actuaries will show them the way.

Three of four wars prosecuted by the United States during the 20th century were begun under democratically elected Presidents.  

One was fought entirely by volunteers.  

During only two of the four have a majority of citizens, at one time or another, wished the U.S.A. to lose(???).  

Three cheers for the Draft!!!!!  

Beware the resolve of an elected President not to lose.

Frederick C. Monson, PhD
Technical Director, CMIRT
West Chester University
West Chester, PA, 19383
610-738-0437
http://cmirt.wcupa.edu

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Rene J Buesa
Sent: Thu 7/2/2009 3:27 PM
To: histonet <@t> lists.utsouthwestern.edu; srnty <@t> aol.com
Subject: Re: [Histonet] Help With Hemo Fading

The fading most probably is caused by acid in the permanent slide, probably because the sections were passed through the alcohols very quickly after the acid differentiation, or they stayed little time in tap water after differentiation or no bluing agent was used.
It is unlikely that the mounting medium is acidic, although that could also be the cause also. An acid environment over the cover slipped section is the most probable "culprit" for the henatoxylin fading.
Check the staining protocol.
René J.

--- On Thu, 7/2/09, srnty <@t> aol.com <srnty <@t> aol.com> wrote:

From: srnty <@t> aol.com <srnty <@t> aol.com>
Subject: [Histonet] Help With Hemo Fading
To: histonet <@t> lists.utsouthwestern.edu
Date: Thursday, July 2, 2009, 2:42 PM

Hello everyone,

?

We are having problems with short-term hematoxylin fading and loss of detail. The pathologist is freaking out! I've seen hemo fade over a long period of time but not in a matter of a few months. Slides from one year ago are really bad.

?

I've been out of the business for a number of years and in the interim much has changed including reagents. These are GI tract biopsies processed by microwave. 

?

Any thoughts at all?

?

Thanks! Marg
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet