What are you digesting with?

The acetic acid rinses are NOT a digestion step. They are a set up for
placing the tissue in an acidic solution of colloidal iron (which is usually
about a 30% acetic acid solution (10 mL stock colloidal iron, 18 mL d.
water, 12 mL acetic acid)). (Analogy - It's like going into 70% alcohol
before going into eosin, since eosin is made up in alcohol.)

For the digestion step of the colloidal iron, what most people are trying to
digest out is hyaluronic acid, which would differentiate acid mucins rich in
hyaluronic acid (such as the umbilical cord, dermis of skin, and to a lesser
amount in cartilage, aorta and bone) with those acid mucins which do not
have hyaluronic acid.

So what you need to use is an enzyme that will digest out the hyaluronic
acid, which is usually testicular hyaluronidase. Then stain to see if it
digested out. We used to get hyaluronidase from pharmacy, if I remember
correctly, though I don't know why it came from there. It was a liquid in an
ampule. Cut 2 slides of the patient's tissue, and use 2 positive control
slides (like umbilical cord). On 1 patient and 1 control slide, put on the
hyaluronidase (we did 1 hour at room temp), and keep the other patient and
control slides in water. After one hour, rinse off the enzyme, place the
digested slides in the with undigested slides, and do your stain. (Just a
note - we always did alcian blue afterwards, instead of colloidal iron.)

Results:
Those acid mucins rich in hyaluronic acid that HAD been digested, will no
longer stain blue since they are now digested out.
Those acid mucins rich in hyaluronic acid that had NOT been digested, will
continue to stain blue since they are still in the tissue.
Those acid mucins WITHOUT hyaluronic acid will continue to stain blue,
regardless of whether they were digested or not, since they always stain
blue with alcian blue (or your colloidal iron) and cannot be digested out.
Those components that do not contain acid mucins will NOT stain at all with
colloidal iron (or alcian blue).

(An analogy - PAS for glycogen - you need the diastase to digest out the
glycogen BEFORE you stain with PAS. If it was glycogen, it was digested out,
and the cells did NOT stain with PAS. If it was glycogen and you did not
apply the diastase, it will continue to stain with PAS. If it was not
glycogen, but something like mucin, it would NOT be digested out, so would
continue to stain. If it was not mucin or glycogen, then it wouldn't stain
either with or without digestion.)

There is also a sialidase digestion, which would differentiate acid mucins
rich in sialic acid from other acid mucins (though I think some sialomucins
also resist sialidase digestion.). However, I've never done this stain, and
don't even know which mucins contain sialic acid that can be digested out.
Hope someone is able to help you with this procedure, if this is what you
are looking for.

Hope this helps your problem.

Peggy A. Wenk, HTL(ASCP)
William Beaumont Hospital
Royal Oak, MI 48073


----- Original Message -----
From: <WWmn916@aol.com>
To: <histonet@pathology.swmed.edu>
Sent: Thursday, April 19, 2001 7:14 PM
Subject: Colloidal Iron


>     Greetings to all.
>     Brief and to the point:  Are colloidal irons (with/without digestion
> also) known for inconsistencies?  If so, are there tips to learn and
> remember?  Basically, we digest when requested, use 12% glacial
acetic/3min.,
> Colloidal Fe/1hr., back to 12% glacial acetic/3min/3changes each, and
finally
> into 5% Potassium Ferrocyanide-Hydrocloric Acid solution for 20 min.  We
> changed controls from umbilical cord to combination of pos/neg skin
sections.
>  The problem as I understand it, is the digested slides are still staining
to
> much blue and with other elements staining also.  I rinse the with and
> without separately and thoroughly before joining them together for the
> remainder of the procedure.  The procedure we have on hand is from old
AFIP
> manual.  More current literature suggest a 2% Pot/Ferr-Hcl Acid solution.
> Would a 5% solution be what is making the with digestion stain more than
it
> should?  Thoughts and comments are definetely appreciated.
>
> Deb King
> Central Histology Facility
> Sacramento CA
>
>