[Histonet] Trichrome staining and fibrin

Wed Jun 14 20:02:13 CDT 2006

Terry,

I figured that one urban myth deserved another!

Your comment on the leap of faith with most empirically derived tinctorial 
stains is certainly very true.
But then, Histopathology is full of such leaps. e.g. simple descriptive 
terms like acidophilia, basophilia etc., which are of course dependent on 
technique.
Another example is the infamous classification "Malignant Fibrous 
Histiocytoma" very few of which, it now turns out, have anything to do with 
Histiocytes.
The magnitude of that leap of faith is always greatly reduced by using a 
standardized, reproducible, tinctorial stain with appropriate controls.
If validation of  these results by confirmation with biochemical analysis, 
histochemistry, immunohistochemistry or EM etc. follows,
then, if it walks like a duck and quacks like a duck, chances are it's a 
............!
However, when definitive identification of a fibrinoid deposit is necessary, 
we have always confirmed the presence of fibrin by IHC.

Bryan

----- Original Message ----- 
From: "Marshall Terry Dr,Consultant Histopathologist" 
<Terry.Marshall <@t> rothgen.nhs.uk>
To: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>; "Timo Väisänen" 
<timo.vaisanen <@t> oulu.fi>; <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, June 14, 2006 8:39 AM
Subject: RE: [Histonet] Trichrome staining and fibrin

Well, that's telling me:-)

All these years, the success I've had with primary mercury fixation (I had 
the original article once), and lack of success with anything other was 
entirely due to something else, the techs washing too much in one instance 
and little enough in another.
Who would have thought it.
However it may explain my observation that "At times it works and at times 
not."

Actually, the only time you can be confident that you are looking at fibrin, 
is when it is in strands - easily recognised in H&E.
To call anything else fibrin on any tinctorial stain is something akin to a 
leap of faith.

But - I'll get you back re. your "bees can't fly" thing.

Take this/that:   :-)

The "science has proved that bees can't fly" urban myth originated in a 1934 
book by entomologist Antoine Magnan, who discussed a mathematical equation 
by Andre Sainte-Lague, an engineer. The equation proved that the maximum 
lift for an aircraft's wings could not be achieved at equivalent speeds of a 
bee. I.e., an airplane the size of a bee, moving as slowly as a bee, could 
not fly. Although this did not mean a bee can't fly (which after all does 
not have stationary wings like the posited teensy aircraft), nevertheless 
the idea that Magnan's book said bees oughtn't be able to fly began to 
spread.

Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path
 Consultant Pathologist
 Rotherham General Hospital
 South Yorkshire
 England
        terry.marshall <@t> rothgen.nhs.uk

-----Original Message-----
From: Bryan Hewlett [mailto:bhewlett <@t> cogeco.ca]
Sent: 13 June 2006 17:14
To: Marshall Terry Dr, Consultant Histopathologist; Timo Väisänen;
histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome staining and fibrin

Terry,

Actually NOT!
True, Lendrum's original papers(1962) recommended fixation in picro-mercuric
alcohol for 3 weeks (a tad unnecessary that!).
However, both Prof. Lendrum and Bill Slidders also successfully used
formaldehyde fixed material that was treated in Bouin immediately prior to
staining.
The key to all of the Masson Type trichromes on formaldehyde-fixed tissue,
is to use a pretreatment in picric acid.
This re-aligns the reactive side chains on the proteins, so that there is a
predominance of basic amino groups and hence maximal binding of anionic
dyes.
Mercury fixation is simply not necessary! Nor is Zinc substitution. (see
Troubleshooting Histology stains: Horobin and Bancroft 1998, page 129)
The lack of consistency, in obtaining good red colour, is due variability in
the staining procedure (the post dye water rinses), NOT the fixation.
Once this is addressed, the inconsistency goes away.
That is true for the original Masson, Picro-Mallory (all variants), MSB and
Masson 44/41.
Achieving consistent good red colour with formaldehyde fixed material is
easy, if the staining mechanisms are understood and those unnecessary water
rinses removed.
Been doing it for over 40 years!

Bryan
(Engineers can prove that the bumble bee simply cannot fly. However, the
bumble bee doesn't know that and flies anyway)

----- Original Message ----- 
From: "Marshall Terry Dr, Consultant Histopathologist"
<Terry.Marshall <@t> rothgen.nhs.uk>
To: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>; "Timo Väisänen"
<timo.vaisanen <@t> oulu.fi>; <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, June 13, 2006 11:27 AM
Subject: RE: [Histonet] Trichrome staining and fibrin

Bryan,

You know full well that MSB required initial staining in mercury. I know
that zinc is an adequate replacement.
Attempting to get a consistent good red colour with formalin fixed material
is futile, as is post-fixation.
At times it works and at times not.

IMO, Masson is useless for fibrin.

Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path
 Consultant Pathologist
 Rotherham General Hospital
 South Yorkshire
 England
        terry.marshall <@t> rothgen.nhs.uk

-----Original Message-----
From: Bryan Hewlett [mailto:bhewlett <@t> cogeco.ca]
Sent: 13 June 2006 15:52
To: Timo Väisänen; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome staining and fibrin

Timo,

Try the Lendrum MSB trichrome variant, it was designed to demonstrate
fibrin.
I will send the method via a separate e-mail.

Bryan

----- Original Message ----- 
From: "Timo Väisänen" <timo.vaisanen <@t> oulu.fi>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, June 13, 2006 10:33 AM
Subject: [Histonet] Trichrome staining and fibrin

> Hello all,
>
> I have struggled with getting Masson Trichrome (Goldner) staining to work
> with
> kidney biopsies. The problem is that our pathologist does not get positive
> fibrin staining to glomeruli in diseased kidneys. Those areas of the
> glomeruli
> that should contain fibrin, at least according to the pathologist, are
> green
> (Lichtgrun) and not red, as they should be, whatever I do. I have tried to
> enhance the staining with Bouin's fixative (+56C) pretreatment but it did
> not
> change the overall situation. However, the red stain was more intense in
> skin
> samples containing fibrin (formalin fixed). I have also tried another
> Trichrome
> staining with Crocein Scarlet 7B without any luck. In our lab kidney
> biopsies
> are routinely fixed with alcoholic Bouin's. As far as I know it should be
> compatible with the Masson protocol we use. Any ideas? Any good protocols
> that
> I could try?
>
> Thank's in advance,
>
> Timo
>
>
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