[Histonet] Thioflavine S for Amyloid

[Lee & Peggy Wenk]

Congo red is the gold standard for amyloid staining. It is the most 
sensitive of the amyloid stain, at about 97%. However, sometimes the Congo 
red will not stain the amyloid protein, such as when the amyloid is a large, 
very old deposit. In that case, more and more amyloid is being crammed into 
the same space, and the beta pleats become warped. For Congo red staining to 
work, the beta pleats must be consistently at a certain distance apart (7 um 
apart, if I remember correctly). Congo red is a linear dye, with 2 SO3- 
groups, one at each end. So it binds to the amyloid protein pleats, one dye 
right after another  | | | | |, and that's why it will polarize. But if the 
beta pleats are not lining up | \ \ | \ / / | | \ /, then the Congo red, may 
not be able to bind, as the binding sites on the amyloid are not the correct 
distance apart. And it the Congo red can bind, it is not lining up | | | | 
|, but is binding in all directions, similar to the warped beta pleats. 
Therefore it will not birefringe/polarize. Overfixation in formalin will do 
the same thing, as there will be too many formalin cross-links, warping the 
beta pleats.

Therefore, when it is suspected that the person really has amyloid, but the 
Congo red isn't working (remember, it's 97% sensitive, which means it's not 
demonstrating amyloid 3% of the time), it's good to have back ups, which we 
have used, and have been able to demonstrate amyloid when the Congo red 
doesn't work. Since the alternatives aren't as specific or sensitive for 
amyloid as Congo red, when we have to go to our backups, we tend to do all 
three, on the theory that even though they aren't as sensitive or specific, 
since they are all staining a different aspect of amyloid, and if all three 
are showing positivity, then it most be amyloid.

Congo red, viewed with fluoresence microscope. Use the auramine-rhodamine 
AFB filters (hit it with 540 green light), and the Congo red-amyloid will 
fluoresce orange.

Crystal violet or Methyl violet - polychromatic dyes that bind to carboxyl 
ions on amyloid. So one dye component stains the amyloid a violet color, 
while the other dye components stain the background a blue-purple. Need to 
use aqueous mounting media, so not a permanent stain. Not as sensitive 
(about 70%) or specific as Congo red. Will stain mucin. AL Amyloid also 
tends to have a lower concentration of surface carboxyl ions, so tends to 
have negative staining with the CV or MV stains.

Thioflavin T (TFT) and Thioflaving S (TFS) are fluorochromes, so need a 
fluorescence miscroscope, blue light excitation at 490, similar to FITC, and 
these dyes will fluoresce yellow. These dyes appear to stain the P component 
on amyloid, which is a pentagonal shaped polysaccharide protein, which is a 
normal protein in our blood (alpha-globin), but for some reason attaches 
itself to amyloid. It's a fast stain, easy to do, very sensitive for 
amyloid. However, you do need a FITC fluoresence microscope, and since it's 
an aqueous mount, it's not permanent. It's not specific for amyloid, as 
other components will be stained and fluoresce yellow, or will autofluoresce 
yellow (such as elastin fibers, dense connective tissue, lipofuchsin, a lot 
of other granules). I've only used TFT, so I can't say if TFS is any better.

So TFS is good for a back up, but I would continue with Congo red, or the 
Amyloid red from Anatech, to be the primary amyloid stain.

Peggy A. Wenk, HTL(ASCP)SLS
Beaumont Health Systems
Royal Oak, MI 48073

Opinions expressed are mine, and do not reflect on my place of employment.

-----Original Message----- 
From: Mitchell Jean A
Sent: Friday, April 12, 2013 2:20 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Thioflavine S for Amyloid

Would appreciated some feedback/input from labs using Thioflavine S staining 
protocol for amyloid screening.  Any advantages/disadvantages to this 
procedure vs Congo Red?

Thanks much!!

Jean Mitchell, BS HT (ASCP)
University of Wisconsin Hospital & Clinics
Neuromuscular Laboratory
600 Highland Avenue
Madison, WI  53792-5132



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