[Histonet] long answer RE: GMS Question

Fri May 1 16:13:29 CDT 2015

I totally agree and if I might add:

Pneumocystis jiroveci are difficult to see after PAS staining because of the strong mucin background staining. GMS is also more advantageous because it stains old and non-viable fungal elements more efficiently than PAS (Henwood, A. F., Prasad, L., & Bourke, V. M. (2013). The application of heated detergent dewaxing and rehydration to techniques for the demonstration of fungi: a comparison to routine xylene-alcohol dewaxing. Journal of Histotechnology, 36(2), 45-50). 

Also be aware of pseudo-fungi (eg Russell bodies), things that look like fungi are PAS positive but GMS negative. Pseudo-fungi will give a positive reaction with a GMS stain using periodic acid instead of chromic acid

Regards
Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

________________________________________
From: Gayle Callis [gayle.callis at bresnan.net]
Sent: Saturday, 2 May 2015 2:50 AM
To: 'Paula Lucas'; histonet at lists.utsouthwestern.edu
Subject: [Histonet] long answer RE:  GMS Question

Hi Paula,

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 Frieda 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)

-----Original Message-----
From: Paula Lucas [mailto:plucas at biopath.org]
Sent: Friday, May 01, 2015 8:17 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] GMS Question

Hello,

I think I already know the answer but I'm not sure why so if someone can
help me understand the theory behind it, I would greatly appreciate it.

Currently, we use the Richard Allen kit for the GMS stain and it uses
Periodic Acid as the 1st step.

We use a control tissue from a case we had that was positive for fungus and
it's a fungus ball from the Rt Maxillary.

We ran a test for fungus on a different and current case of the same tissue
(different patient): Rt Maxillary sinus.

The control tissue did work, but the patient's tissue did not, so the doctor
ordered a PAS for fungus and this clearly showed the fungal elements nicely.

My question is why would the control and patient tissue have different
results when they are both fungus balls from the same specimen source?

Thanks in advance,

Paula

Lab Manager

Bio-Path Medical Group

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