[Histonet] Iron pigment removal procedure

Tony Henwood AnthonyH <@t> chw.edu.au
Mon Jan 7 16:19:08 CST 2008

This was a review I did for our journal a few years back. It might be

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
*	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).


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


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Poteete,
Jacquie A.
Sent: Tuesday, 8 January 2008 3:28 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Iron pigment removal procedure

Does anyone have a procedure for the removal of iron pigment for FFPE
tissue prior to IHC staining?  We have had no luck using the reference
material we have available, so any help would be very much appreciated.

Jacquie Poteete MT(ASCP)QIHC
Lead Technologist, IHC Laboratory
Saint Francis Hospital
Tulsa, OK
japoteete <@t> saintfrancis.com
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Histonet <@t> lists.utsouthwestern.edu

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