RE: [Histonet] Iron stain on frozen sections

From:Tony Henwood


Following is an excerpt from a paper I published a year or so ago:

Henwood, A.F., (2002) "Microwave Perl's Stain for urgent frozen sections"
Aust J Med Sc 23(2):68-69).

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


Anthony F. Henwood
Laboratory Manager,
The Children's Hospital at Westmead,
Locked Bag 4001,
Westmead, 2145, AUSTRALIA 


A rapid Perls' Stain for hemosiderin using a microwave oven is described. It
takes one minute to perform and is particularly suitable for urgent frozen
section diagnosis. 

Urgent frozen section diagnosis is usually restricted to the interpretation
of haematoxylin and eosin stained sections (1). With the increased
utilisation of microwave modifications of routine special stains, it is
possible to perform a range of such stains on frozen sections (1). To be
applicable to urgent frozen section diagnosis, a special stain needs to be
rapid, preferably taking 1-2 minutes to perform, easy to prepare, using
stock solutions with few preparation steps and produce results equal to
those obtained on paraffin sections.

The following microwave Perls' technique for hemosiderin takes about one
minute to perform.

Frozen sections can be fixed in Shoobridge's fixative (10ml concentrated
formalin in 30ml absolute alcohol) (2) or 95% alcohol. Kennedy and Forbes
(1) have also successfully used Wolman's fixative (5% acetic acid in
absolute ethanol).

1. Rinse fixed frozen sections in water and then place in a coplin jar
containing 20m1 each of 2% Potassium Ferrocyanide and 2% Hydrochloric acid.
Place in the microwave oven and microwave for 20 seconds at 650 watts.
Slides can be left in this solution for up to 3 minutes. Remove slides if
the solution begins to turn cloudy.

2. Rinse slides in water.

3. Counterstain using agitation, with eosin (as routinely used for the
frozen section HE) for 5-7 seconds.

Other counterstains such as Nuclear Fast Red (3) or 0.25% basic fuchsin (4)
can also be used, though eosin is readily available at the frozen section
site and works quite well.

Since the frozen block is usually fixed and processed to paraffin, sections
of this block should also be stained with the routine Perls' as a quality
control procedure.


Iron in the body is stored in the forms of hemosiderin (ferric hydroxide
polymer) or ferritin (a ferrous iron-protein complex) (5). Iron in tissues
occurs mainly in the ferric state (6,7). 

Microscopically, hemosiderin appears similar to other yellow to brown
pigments, such as melanin and fine carbon dust. Macrophages can contain any
of these pigments. Heavily pigmented macrophages, often masking the cell
nucleus, can be confused with malignant melanoma. The identification of the
pigment as being hemosiderin, especially at the time of frozen section, is
diagnostically useful.

The rapid Perls' stain described above uses the same solution as used by
Schaffner (4). This solution is easier to prepare than others reported in
the literature (3). The stain takes approximately one minute to perform and
gives results equivalent to those obtained in paraffin sections. It is
especially applicable to frozen section diagnosis.


1. Kennedy, A., Foulis, A.K. (1989) "Use of microwave oven improves
morphological and staining of cryostat sections", J.Clin.Pathol. 42:101-105.

2. Shoobridge, M.P.K., (1978) "Improving frozen sections by wet fixation",
(Abstract) Pathology 10:195.

3. Brian, N.T., (1983) "Rapid Metallic Histological staining using the
microwave oven", J.Histotechnol. 6(3): 125-129.

4. Schaffner, R., (1986) "The Perls' Iron staining procedure for use in the
microwave oven using a temperature probe", J.Histotechnol. 9(2): 107-108.

5. Barka, T., Anderson, P.J., (1963) "Histochemistry: Theory, practice and
bibliography" Harper & Row Publishers Inc, New York, p172-174.

6. Davenport, H.A., (1961) "Histological and Histochemical Technics" W.B.
Saunders Co., Philadelphia, 280-284.

7. Gabe, M., (1976) "Histological Techniques" Masson, Paris, p311-317.

-----Original Message-----
From: Connolly, Brett M []
Sent: Friday, 19 March 2004 6:03 AM
Subject: [Histonet] Iron stain on frozen sections

Along with the bone marrow thread.....

Has anyone had experience with iron stains on frozen sections?  What
fixative? Procedure?


Brett M. Connolly, Ph.D.
Merck & Co., Inc.
MRL, Imaging Research
PO Box 4
West Point, PA 19486
PH 215-652-2501
fax. 215-652-2075

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