[Histonet] Prussian Blue Reaction

Mon Jun 7 18:24:41 CDT 2021

The following might be useful:

Iron Histochemistry - A Review

It is convenient to divide iron-containing complexes in human tissues into two categories: those in which the iron is loosely bound to proteins and easily released by mild acid treatment (eg hemosiderin) and those in which the iron is more strongly bound (masked iron) and cannot be released by mild acid hydrolysis (eg haemoglobin) (1).

Iron in the body is stored in the forms of hemosiderin (ferric hydroxide polymer) or ferritin (a ferrous iron-protein complex) (1). Iron in tissues occurs mainly in the ferric state (2,3).

The reactions that have been used for the detection of iron in tissues include (2-5):

1.	The Quincke reaction using ammonium sulphide
2.	The Perls reaction, using ferrocyanide, for ferric and the Turnbull Blue reaction, using ferricyanide for ferrous iron.
3.	Coloured lakes, eg haematoxylin (Mallory's Method)
4.	Coloured precipitates with organic chemicals not classified as dyes (eg bathophenanthroline). 

Ferric iron may be converted into ferrous iron by ammonium sulphide (Quinke's reaction) and the ferrous sulphide thus formed can then be demonstrated using the Turnbull blue reaction (3,5).

Some iron-containing compounds (hemoglobin, malaria pigment, formalin pigment) do not react with the Perl's method because the iron is present in bound form. These compounds can be unmasked using hydrogen peroxide and then demonstrated using the Perl's reaction (1).

Interestingly, it is possible to remove excess iron pigment from tissue sections. Iron can be removed by (5):

.	15 min in 1% sodium dithionite in 0.1M acetate-HCl  buffer (pH 4.5)
.	3 hours in 2.4N HCl
.	30 min in 3.7N H2SO4
.	15 min in 5% Oxalic acid

Heavily pigmented tissues may need to have these times extended (5).

References

1.	Barka, T., Anderson, P.J., (1963) "Histochemistry: Theory, practice and bibliography" Harper & Row Publishers Inc, New York, p172-174.
2.	Davenport, H.A., (1961) "Histological and Histochemical Technics" W.B. Saunders Co., Philadelphia, 280-284.
3.	Gabe, M., (1976) "Histological Techniques" Masson, Paris, p311-317.
4.	Lynch, M.J., Raphael, S., etal "Medical Laboratory Technology and Clinical Pathology" 2nd Ed, W.B Saunder Co, Philadelphia, p1135-1136.
5.	Morton, D., (1978) "A comparison of iron histochemical methods for use on glycol methacrylate embedded tissues" Stain Tech 53(4):217-223.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: Mac Donald, Jennifer via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, 8 June 2021 6:41 AM
To: Jay Lundgren <jaylundgren at gmail.com>
Cc: histonet at lists.utsouthwestern.edu; John Kiernan <jkiernan at uwo.ca>
Subject: Re: [Histonet] Prussian Blue Reaction

Thank you.

From: Jay Lundgren <jaylundgren at gmail.com>
Sent: Monday, June 7, 2021 1:35 PM
To: Mac Donald, Jennifer <jmacdonald at mtsac.edu>
Cc: John Kiernan <jkiernan at uwo.ca>; Gudrun Lang <gu.lang at gmx.at>; histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] Prussian Blue Reaction

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Supposed to be insoluble.  Try potassium permanganate followed by oxalaic acid.  But book says insoluble.

On Monday, June 7, 2021, Mac Donald, Jennifer via Histonet <histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>> wrote:
The instrument malfunction and it was overstained.

From: John Kiernan <jkiernan at uwo.ca<mailto:jkiernan at uwo.ca>>
Sent: Monday, June 7, 2021 9:50 AM
To: Gudrun Lang <gu.lang at gmx.at<mailto:gu.lang at gmx.at>>; Mac Donald, Jennifer <jmacdonald at mtsac.edu<mailto:jmacdonald at mtsac.edu>>
Cc: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Prussian Blue Reaction

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Overstained? Doesn't that mean the tissue contains a lot of iron and you are seeing where it is - which was the reason for doing Prussian blue histochemistry. Gudrun Lang correctly says that mineral acids won't remove it. Oxalic acid is said to dissolve Prussian blue (? by chelation); I've never tried this. If it works, you will no longer see where the iron is. To see features other than the distribution of iron, why not just stain another section from the block with a general-purpose stain like Giemsa or H&E?
John Kiernan
London, Canada
= = =
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From: Mac Donald, Jennifer via Histonet <histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu><mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>>>
Sent: June 7, 2021 12:47 AM
To: Gudrun Lang <gu.lang at gmx.at<mailto:gu.lang at gmx.at><mailto:gu.lang at gmx.at<mailto:gu.lang at gmx.at>>>
Cc: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu><mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>> <histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu><mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>>>
Subject: Re: [Histonet] Prussian Blue Reaction

The tissue was overstained and the blue was interfering with interpretation

-----Original Message-----
From: Gudrun Lang <gu.lang at gmx.at<mailto:gu.lang at gmx.at><mailto:gu.lang at gmx.at<mailto:gu.lang at gmx.at>>>
Sent: Sunday, June 6, 2021 2:18 AM
To: Mac Donald, Jennifer <jmacdonald at mtsac.edu<mailto:jmacdonald at mtsac.edu><mailto:jmacdonald at mtsac.edu<mailto:jmacdonald at mtsac.edu>>>
Cc: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu><mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>>
Subject: AW: [Histonet] Prussian Blue Reaction

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Hi Jennifer,
Why do you want to reduce the staining?

I ask, because the impact of hydrochloric acid on the tissue may influence the following results anyway.
I think, that the prussian blue pigment cannot be removed in an easy way. It is resistent to solvents and mineral acids.
https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.epsilonpigments.com%2Finorganic-pigment%2Fprussian-blue%2FPrussian-Blue&data=04%7C01%7Cjmacdonald%40mtsac.edu%7C0fbc82a2b13749a4222608d928cbfe52%7Ccc4d4bf20a9e4240aedea7d1d688f935%7C0%7C0%7C637585679205067185%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000&sdata=KjvijcfrVPGZKGsAn6qX5rMKtulHpmsAzqHEkwz%2B96Y%3D&reserved=0<https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.epsilonpigments.com%2Finorganic-pigment%2Fprussian-blue%2FPrussian-Blue&data=04%7C01%7Cjmacdonald%40mtsac.edu%7C684bae0e8884489b44cb08d929f3cae3%7Ccc4d4bf20a9e4240aedea7d1d688f935%7C0%7C0%7C637586952354386054%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=d22NsJ2m3eoU6bFZ3mvyb6rIaFr%2BcMu4tuS40b1hcsk%3D&reserved=0><https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.epsilonpigments.com%2Finorganic-pigment%2Fprussian-blue%2FPrussian-Blue&data=04%7C01%7Cjmacdonald%40mtsac.edu%7C2eeb915058594b1d14b508d929d455c4%7Ccc4d4bf20a9e4240aedea7d1d688f935%7C0%7C0%7C637586814671423758%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=xKy3v4qvO4spk0zrRxXWZfcsHWxbQC%2FIQ1FbpMB390Y%3D&reserved=0<https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.epsilonpigments.com%2Finorganic-pigment%2Fprussian-blue%2FPrussian-Blue&data=04%7C01%7Cjmacdonald%40mtsac.edu%7C684bae0e8884489b44cb08d929f3cae3%7Ccc4d4bf20a9e4240aedea7d1d688f935%7C0%7C0%7C637586952354386054%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=d22NsJ2m3eoU6bFZ3mvyb6rIaFr%2BcMu4tuS40b1hcsk%3D&reserved=0>>
-for-Solvent-Based-Inks.html

On the other hand, if the blue colour doesn't interfere with your following staining, you can try to simple make a "double stain".

Regards
Gudrun

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Von: Mac Donald, Jennifer via Histonet
[mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>]
Gesendet: Sonntag, 6. Juni 2021 06:34
An: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu><mailto:histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>>
Betreff: [Histonet] Prussian Blue Reaction

Does anyone know of a way to remove/reduce the Prussian blue reaction?
Thanks,
Jennifer

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