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@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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