RE: Mast cells

From:Bryan Hewlett

Hi John,

As usual, you are right on the money!

You wrote;
>>If it doesn't work on dog tissues, the human and dog tryptases
may be too different as antigens despite their biochemical

I believe this to be the case. We have only performed IHC for mast cell
tryptase on one sample of canine skin(badly fixed at that), it was negative.

You then wrote;
>>Another consideration: THE FIXATIVE.
Rat and mouse mast cell granules are unusual in being
well preserved by aqueous formaldehyde. In many other
species the granules dissolve in aqueous fixatives (and
also in the extracellular fluid in vivo, after being
discharged from the cell). Human mast cell granules are
partly preserved by aqueous fixatives. Dogs have very
soluble heparin, and discharged granules are hard to
find. To preserve mast cells as stainable entities the
optimal fixative is non-aqueous: Carnoy, or an "alcoholic
Bouin," for example.

I have assumed, perhaps incorrectly, that the sample Richard is testing was
fixed in formaldehyde.
If Dog heparin is very soluble,that may be a problem with the suggested
cationic dye techniques.
Both human mast cell tryptase and histamine are very well preserved by this
fixative, so IHC reactivity should not be an issue.
Histamine, as detected by IHC, is well preserved in both formaldehyde and
Carnoy, with no pre-treatment being required.
Mast cell tryptase requires proteolytic pre-treatment following
formaldehyde, I trust that Richard will have followed the recommendation on
the data sheet.
If the tissue was fixed in a non-aqueous fluid, then NO proteolysis is

You also wrote;
>>Failure to find mast cells after fixation in an aqueous
mixture (except in rats & mice) is just what's expected.

This is certainly true of mucosal mast cells, since they contain lower
amounts(10%?) of heparin than connective tissue mast cells.
However, in Human material the long versions of Alcian blue and Tol blue
stains largely circumvent that problem. In fact, Enerbach suggests that the
heparin is not lost but 'masked' by formaldehyde fixation and proteolytic
pre-treatment unmasks the heparin.
A number of years ago, a pathology resident from Japan did a study with us
on the distribution of mucosal mast cells in normal human gut and in
patients with mastocytosis.. We utilized paired GI biopsies, one fixed in
NBF and one fixed in Carnoy, from each patient. We compared several staining
techniques including long tol blue and mast cell tryptase. In short,
comparing the cationic dyes methods, the highest density of mucosal mast
cells was found by the long Tol blue technique. This was true for both
fixatives. As expected the Carnoy fixed material gave higher absolute
numbers. Interestingly, the density of mast cells detected by IHC for
tryptase was higher than that detected by tol blue and was essentially equal
for both fixatives.
I believe that this was published in a Japanese surgical pathology journal.
Unfortunately, I do not have the reference but I can try to locate it if you
are interested.
We no longer use Carnoy fixation and cationic dyes for mast cells. NBF
fixation and IHC for tryptase are now our method of choice.


Bryan R. Hewlett
Technical Specialist
Anatomical Pathology
Hamilton Regional Laboratory Medicine Program
Ontario, Canada

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