[Histonet] Causes of false positive Congo Red

[Greg Dobbin]

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> 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> 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
>> = = =
>> ------------------------------
>> *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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>