[Histonet] Causes of false positive Congo Red

[John Kiernan]

Dear Greg,

This is the same as on p.126 in my copy of Carson's 2nd edition (1997) and also in the 11th and last (2008) edn of Churukian's "Manual of the Special Stains Laboratory". The only stock solution that can be expected to change with time is the Stock Congo red solution, because solutions of dyes with large anions are unstable in the presence of inorganic salts. Churukian (p.195) said it could be kept for 2 months.

The correct staining you got with newly made solutions suggests that your earlier stock Congo red stock solution was too old. Evidently you solved the problem yourself!

In their "Troubleshooting Histology Stains"  book, Horobin & Bancroft (1998, p.45-47) stressed the need for a freshly made dye solution. They also suggested ignoring "pink background" and checking that it's not dichroic. They also listed various Congo-positive and dichroic materials that aren't amyloid. An unidentified yellow compound is often present in Congo red and it may sometimes cause generalized yellow background staining.

John Kiernan
https://www.schulich.uwo.ca/anatomy//people/faculty/emeriti/kiernan_john.html
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________________________________
From: Greg Dobbin <greg.dobbin at gmail.com>
Sent: June 20, 2024 8:44 AM
To: John Kiernan <jkiernan at uwo.ca>; histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Causes of false positive Congo Red

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Hi John,

I must apologize again. We used to use the method from Carson's book. We now make up the reagents as follows:


Stock alkaline salt solution

Sodium chloride............. 2g

Distilled Water............... 20mL

Stir until the salt is dissolved, then with continuous stirring on a magnetic stirrer add

80mL of 100% denatured ethanol.

Some salt may precipitate out after the ethanol is added.



Working alkaline salt solution

Stock alkaline stock solution...50 ml

1% Sodium Hydroxide…………0.5ml

Filter and use within 15 minutes


Stock Congo red solution

Congo red................................ 0.1g

Stock alkaline salt solution........ 50mL

Stir well with the magnetic stirrer and let stand overnight or for a minimum of 3 hours if the slides need to be ready the same day that the order was placed.



Working Congo red (Congo red)

Stock Congo red...................... 50ml

Sodium hydroxide 1%............... 0.5ml

Filter and use within 15 minutes.

On Thu, Jun 20, 2024 at 9:31 AM Greg Dobbin <greg.dobbin at gmail.com<mailto:greg.dobbin at gmail.com>> wrote:
Good day John,
Very nice to hear from you again! I have been consulting your textbook in my investigations!
Sorry about the brevity of the description of our method. I felt like my post was already too long
and it might scare off some would-be contributors! :-) And yes, I incorrectly referred to the dichroic green as "fluorescent"-thank you.

Our method follows the Puchtler method described on pages 132-3 in Frieda Carson's "Self-Instructional" textbook (1990) as does
the hospital that repeated our false-positive Congo Reds. Note, once we re-made our reagents, our results returned to accurate staining.
Greg

On Thu, Jun 20, 2024 at 2:49 AM John Kiernan <jkiernan at uwo.ca<mailto:jkiernan at uwo.ca>> wrote:
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<mailto:histonet at lists.utsouthwestern.edu>>
Sent: June 19, 2024 8:53 AM
To: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu> <histonet at lists.utsouthwestern.edu<mailto: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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