[Histonet] Passing onto PAS discussion RE: GMS question

Sat May 2 11:03:35 CDT 2015

Bob, 

Culling said exactly the same thing about not needing precision weighing for
periodic acid.     I discovered, by a huge error in PAS staining results,
that  pre-weighing 1 gm of PA into a clear sample vial and letting the
pre-weighed PA stand at RT should NOT be done.  The PA degraded to the point
of very poor staining.   I thought I was doing myself a favor to have faster
solution preparation, and the pre-weighing efficiency idea was abandoned.
We sometimes learn the hard way.   Daily freshness is the key and we liked
1% periodic acid  (or 0.5% per the original McManus and Mowry method).   

 Our lab used 1% PA for a standard PAS stain for 10 minutes (never used for
fungus) most of the time.  It is interesting to note how many variations of
the PAS stain exist in this world.    It's important to check what PAS
method variation works best for the component you are looking for as one
variation may not be the best for the tissue component you want to see.
If over oxidation is suspected, reduce oxidation time to 5 min (original
McManus/Mowry method) and/or reduce concentration of PA to 0.5% periodic
acid instead of 1% PA OR do both.     Good positive controls are mandatory
for any particular tissue component in mind.   

The only time we used a periodic acid/methenamine silver staining was for 2
micrometer renal biopsies aka Jones Basement membrane stain, and our
methenamine silver was kept fresh with a 6 month expiration date on in house
preparation.   Fresh methenamine silver stock solution is important too.

Chromic acid cannot be drain dumped and hopefully people will have hazmat
collection available.  If you have to collect DAB, silver solutions, then
chromic acid (chromium trioxide is the stock chemical) can be collected.
It isn't that difficult.       

Thanks for the supportive comment on GMS.  Your comments on Histoplasma sp.
staining were interesting as not many of us have the opportunity to work
with ancient tissues.       

Gayle Callis  

-----Original Message-----
From: Bob Richmond [mailto:rsrichmond at gmail.com] 
Sent: Saturday, May 02, 2015 7:30 AM
To: Histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] GMS question

Gayle Callis's discourse on GMS staining is must reading for anyone who does
the GMS stain. Freida Carson in 1999 showed the importance of using chromic
acid - I was distressed that she didn't cite this paper in the 4th edition
of her book.

The most rigorous test of the GMS stain is probably the dead histoplasma in
ancient fibrotic granulomas. I've gotten these things to turn up with GMS
more than once - a finding of potential clinical importance.

For control material, I don't think there's any substitute for infected
human (or mammalian) tissue, preferably though not necessarily with a
species identification of the fungus. I think either active histoplasmosis
or invasive aspergillosis might provide the best material. The big academic
centers that are still doing autopsies could be helpful in getting control
material for everyone.

Periodic acid should indeed be made up fresh with the dry chemical, but
precision weighing isn't required - I used to keep a small plastic measure
in the stock bottle so I could simply spoon out what I needed for the day.

I'm not sure what the rules are for disposing of chromic acid, but it's a
significant hazmat - toxic metal, strong acid, strong oxidant. I think you
can neutralize it and precipitate the chromium, but I'd have to look up the
method.

Bob Richmond
Samurai Pathologist
Maryville TN
******************************************
Gayle Callis wrote:

The same site of infection for your control and patient tissues does not
mean the fungus species was the same.  Now for addtional commentary.

The classic GMS uses chromic acid as the oxidizer, stronger than periodic
acid.   This is something to beware of since you had a false negative result
with the kit.     Periodic acid should be freshly made up from periodic acid
crystals just before use (Culling insisted on fresh periodic acid reagent)
then discarded after that day - something that is not going on with a kit
where all components are sent to you in ready to use liquid form.   I think
if you had used the classic GMS with chromic acid to start with, you would
have had the results seen with Periodic Acid/Schiffs.   Part of the problem
is the not all fungus species stain well either PAS or Periodic acid/GMS,
and even classic GMS.   All these factors can present a problem when trying
to diagnose fungal infections.    Lee Luna in AFIP manual, pointed out "it
has been found that the time of exposure to methenamine-silver nitrate
solution for complete development may vary according to type and/or strains
suspected."   He advised if Nocardia asteroides is suspected, then two
slides should be run:  one for 60 minutes and one for 90 min in the silver
solution.   Histotechs should be aware these possible problems.     Fungi
are difficult at best even when doing fungus cultures and treat.
Fortunately, there are antibodies for some fungi i.e. Aspergillus, Candida)
but you would probably need the specific fungus as a control for the
antibody being used.   Fungus IHC experts can weigh in on the latter topic.
I don't know what fungus species was in your  "fungus ball" control block,
but our clinical lab fungus ball control contained Aspergillus sp. from a
post mortem human lung.  With the researcher, he only had pure Aspergillus
sp. infecting the tissues.    It would be nice to know what species of
fungus is in your control tissue block????

The publication by Freida Carson along with Jerry Fredenburgh and John
Maxwell   "Inconsistent detection of Histoplasma capsulatum with periodic
acid in GMS fungus stain"  , J Histotechnology, 1999 is a profound statement
about what you just experienced.   Their tissue control i.e. Aspergillus sp.
stained adequately with periodic acid/GMS but the H. capsulatum fungus in
tissue did not stain.  They studied several different times, temperatures,
periodic acid concentrations and fungus species.  They had additional
controls containing fungi other than Aspergillus sp. for better sampling of
PA/GMS on
different  fungi.   Having different fungus species control blocks is a
luxury many labs do not enjoy.  The reason chromic acid is not in kits is
due to shipping, health and safety hazards, must be handled carefully and
collected for proper, safe disposal so it is easier to make up "GMS" kits
with periodic acid.    If you have to collect your silver solutions for safe
chemical disposal, then chromic acid shouldn't be a big problem to do the
same.

Also, since Periodic acid/Schiffs is popular and commonly used for fungus
staining,  PAS can also present false negative results or weak staining  due
to the weaker periodic acid oxidation, even when the periodic acid is made
in house, fresh for the day.   Chromic acid/Schiffs has been recommended by
people on Histonet to improve fungus staining over PAS.   PAS stain always
seem to work well with Candida sp. and Aspergillus sp. but classic GMS was
always better in my hands.   I only used classic GMS, prepared in house, and
controlled the development with a microscope to avoid under and over silver
staining of fungus.

I will be happy to send the Carson et al publication and scan the AFIP
manual pages with photos on fungus staining by Luna.   I apologize for long
discourse as chromic acid/GMS stain is one of my favorite special stains to
perform along with a long standing interest in fungus staining.

I hope this helps.

Gayle M. Callis
HTL/HT/MT(ASCP)
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