[Histonet] Causes of false positive Congo Red

[John Kiernan]

Greg, your method is incompletely described in your Histonet post, but it looks quite different from the "traditional" Highman's procedure (Arch. Path. 41:559-562). What method were they using "at another
lab" to get correct red amyloid that is green (dichroic, not fluorescent) with crossed polars?
John Kiernan
https://www.schulich.uwo.ca/anatomy//people/faculty/emeriti/kiernan_john.html
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________________________________
From: Greg Dobbin via Histonet <histonet at lists.utsouthwestern.edu>
Sent: June 19, 2024 8:53 AM
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Causes of false positive Congo Red

Hello experts,
*Some background:*
I know that Congo Red can bind nonspecifically to non-amyloid components
such as collagen and elastin under certain conditions (eg Carnoys fixative,
insufficient differentiation, insufficient alkalinity, etc). However,
everything I have been able to read on the topic suggests that
over-staining is "easily" differentiated from true amyloid staining by
using polarizing light microscopy. That is, true amyloid produces apple
green fluorescence while non-amyloid components produce silver/grey color.

*My question:*
I want to know if anyone has encountered false positive staining that *is
apple green* in color? We had a few bone marrow core biopsies that stained
bright green but were later found to be negative when stained at another
lab. We subsequently threw out all of our working solutions and made up
everything fresh and repeated the previous (false positive) specimens and
they were indeed negative in our lab as well.

*In order to prevent this from happening again, I need to attempt to
understand what may have caused this to happen in the first place. *

This is where the vast collective knowledge of this group comes in. :-)
Can anyone offer some insight as to possible causes?

*Our Congo Red method:*


Deparaffinize sections and bring them to water.

Stain in Hematoxylin for 1 minute

Add 0.5ml of 1% Sodium Hydroxide to 50 ml of stock alkaline salt solution.

Wash slides in running water

Place in *working* alkaline salt solution from step 2 for 20 minutes

Add 0.5 ml of 1% Sodium Hydroxide to stock Congo red solution.

Start to filter *working* Congo red solution when 15 mins are left in step 6

Place sections in the *working* Congo red from step #8 for 20 minutes.

Dehydrate the slides one at a time in 3 changes of absolute ethanol, 6 dips
each.

Dip the slide 10 times in a coplin of xylene.

Continue dehydrating the other slides.

Coverslip the slides.

*Greg Dobbin*
1205 Pleasant Grove Rd
Route 220
York,  PE      C0A 1P0
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