[Histonet] Causes of false positive Congo Red

[Greg Dobbin]

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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