[Histonet] Sulfated Alcian Blue

[Tony Henwood]

Peggy,

Have you tried the alcian blue tetrakis (methylpyridinium) chloride
(Sigma, Cat. No.A4045, 90% dye content)?

I would be interested to know whether this stains the amyloid better.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lee Wenk
Sent: Wednesday, 4 March 2009 7:35 AM
To: Nathanial nauss; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Sulfated Alcian Blue


Sulfonate Alcian Blue (SAB) is not always as specific as you would like.

Also, if you are using the newer alcian blue dye, it seems to have a 
different formulation than the old stuff that I have. The new alcian
blue 
will not dissolve in alcohol, so the newer dye doesn't demonstrate the 
amyloid, while my old dye does.

Old deposits of amyloid do not have beta pleats at regular intervals. 
Therefore, the Congo red dye (CR) will still bind, but will not line up
one 
right after the other | | | | |, but will be more random \  _  |  /.
When 
the CR dye are parallel to each other, they will show the apple green 
birefringence with the polarizing microscope. When the CR dye is
randomly 
arranged, there will be no apple green birefringence.

The other time this happens is with very overfixed amyloid, such as
months 
in NBF. Too many cross-links with NBF, so the CR dye can't bind right,
so 
they are not in parallel. We also had this happen once when the autopsy 
tissues were fixed in B5 (mercury fixative, long time ago). The resident

knew amyloid was an immunological problem, so put through tissue fixed
in 
B5. Congo red did not birefringe. So we went back to the NBF stock
bucket, 
submitted new tissue, and they all were wonderfully green birefringent.

The other problem with the SAB is that, if this is old amyloid, and the
beta 
pleats are messed up, SAB depends somewhat on the beta pleats.
Therefore, 
the green will be much paler in older amyloid than with newer amyoid.
(The 
green is due to to the blue of alcian blue staining the amyloid, and the

yellow of picric acid in the van Gieson also staining the amyloid, so
blue 
and yellow make green.)

We just had a case of non-birefringing green Congo red at our hospital a

couple of months ago, where it definitely looked by amyloid on the H&E,
but 
there was no birefringence on the patient's CR (control was great - all
3 
times that we repeated the procedure). Ot wasn't a fixation problem, or
a 
staining problem, but probably an "old amyloid" problem. Our resident
Dr. 
Tom Fennel just gave a talk on it at our state histology's winter
seminar 
Jan. 31, 2009. Here's what we did (all three):

1. View the Congo Red stained slides with a fluorescence microscope,
such as 
in microbiology auramine-rhodamine stain for AFB. When hit with green
light, 
the CR stained amyloid will fluoresce orange.
2. Use Crystal Violet or Methyl violet staining for amyloid. These
depend 
upon the carboxyl  ions of the amyloid for binding, not the beta pleats.
The 
amyloid should be violet, with the background blue/purple. However, it 
doesn't always demonstrate AA amyloid (some are low in surface carboxyl 
ions). So not always as sensitive as CR.
3. Use Thioflavin T or Thioflavin S for amyloid. Staining is (maybe)
with 
the P component of amyloid, not the beta pleats. Use a fluorescence 
microsope, using blue light (FITC filters), and the amyloid will
fluoresce 
yellow. However, other things also fluoresce yellow, such as fibrinoid 
material, JG granules, sometimes elastin, etc. So not as specific as CR.

By knowing your histology and knowing where in the tissue you are
thinking 
that there are amyloid deposits, the above three alternatives are a nice

addition, for when Congo red is not demonstrating the apple green 
birefringence.

Since in our recent case, 3 out of 4 stains demonstrated amyloid (CR 
fluorescent, Crystal violet and TFT were all positive, while CR 
birefringence was negative), it was diagnosed as amyloid, with, I think,
a 
note that older deposits of amyloid sometimes demonstrate no
birefringence 
with CR.

If this person needs help with find staining procedures for CV/MV or 
TFT/TFS, could someone email them? I'm on vacation, and don't have
access to 
my home or work computer.

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


----- Original Message ----- 
From: "Nathanial nauss" <nsnauss <@t> bellsouth.net>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, February 27, 2009 10:42 AM
Subject: [Histonet] Sulfated Alcian Blue


I need some help, has any one used the sulfated alcian blue to stain for

amyloid. We have a case that looks like it should be positive but it is
not 
staining with the Congo Red. Any help would be great.
Nathaniel
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