[Histonet] Fraser-Lendrum stain

[Bryan Llewellyn]

Lendrum, Fraser and others published several fibrin stains, but I presume
the method you want is the Picro-Mallory, since that is the most well known.
There are several variants of the method, all published by Lendrum.  Some
are more complicated than others.  I include two of them here.

With this trichrome stain full and complete fixation is absolutely
essential.  Minimalist formalin fixation and quick processing will give
poorly stained erythrocytes with red or orange tinges instead of the yellow
they should have.  Lendrum and coworkers specified about seven days fixation
in formal sublimate (9 parts saturated mercuric chloride and 1 part
concentrated formalin) followed by processing thoroughly, sectioning,
degreasing with trichlorethylene for 48 hours, then refixing in
picromercuric ethanol (ethanol saturated with picric acid and mercuric
chloride).  Adequate results can be obtained with thorough formalin fixation
(24 hours), thorough processing (overnight), sectioning, followed by
refixing in Bouin's fluid at 56C for an hour or so.  However, results are
inferior to those obtained by the full procedure.  It is also a method where
experience is required for optimal results.  I always found the shorter
variant quite satisfactory.


Long variant (Picro-Mallory IV, I think)

Yellow mordant
Saturated picric acid in 80% ethanol - 100 mL
Orange G - 0.2 g.
Lissamine yellow - 0.2 g

Stock differentiator
picric acid - 2.5 g.
95% ethanol - 100 mL
Phosphotungstic acid - 25 g.
Dissolve and filter

Red stain
Acid fuchsin or biebrich scarlet - 1% in 1% acetic acid
or
Acid fuchsin - 0.5% in 1% acetic acid - 80 mL
Lissamine fast red - 1% in 1% acetic acid - 20 mL

Red differentiator
Stock differentiator - 40 mL
95% ethanol - 20 mL
Distilled water - 40 mL

Blue stain
Soluble or methyl blue - 1% in 1% acetic acid

Blue differentiator
Stock differentiator - 10 mL
Distilled water - 90 mL

Method
1.  Bring sections to water through xylene and ethanols.  Remove mercury
pigment.
2.  Stain nuclei with an acid resistant nuclear stain (Weigert, Celestine
blue hemalum).
3.  Place in yellow mordant for 2-3 minutes.
4.  Wash in distilled water until only erythrocytes are yellow.
5.  Place in the red stain for 5-10 minutes.
6.  Rinse with 1% aqueous acetic acid.
7.  Differentiate with the red differentiator, microscopically, until fibrin
stands out clearly.
8.  Rinse well in distilled water.
9.  Place in the blue stain for 5 minutes.
10. Rinse briefly with 1% aqueous acetic acid.
11. Place in the blue differentiator for 1-2 minutes.
12. Rinse with 1% aqueous acid briefly.
13. Dehydrate with ethanol, clear with xylene and mount in resinous medium.

Results
Fibrin - clear red
Erythrocytes - yellow
Connective tissue - blue




Shorter variant (Picro-Mallory V)

Picro-orange
Orange G - 0.2 g
Picric acid saturated in 80% ethanol - 100 mL

Yellow differentiator
Picro-orange - 50 mL
80% ethanol - 50 mL

Red stain
Acid fuchsin - 1% in 1% aqueous acetic acid

Differentiator
1% phosphotungstic acid in distilled water

Blue stain
Soluble or methyl blue - 2% in 2% aqueous acetic acid

Method
1.  Bring sections to water through xylene and ethanols.  Remove mercury
pigment.
2.  Stain nuclei with an acid resistant nuclear stain (Weigert, Celestine
blue hemalum).
3.  Rinse with 95% ethanol
3.  Place in picro-orange for 2 minutes.
4.  Without rinsing, place in red stain for 5 minutes.
5.  Rinse with distilled water.
6.  Dip into yellow differentiator.
7.  Rinse with distilled water.
8.  Place into phosphotungstic acid solution for 5 minutes.
9.  Rinse with distilled water.
10. Stain in blue stain for 2 minutes.
11. Rinse well with 1% aqueous acetic acid.
12. Dehydrate with ethanol, clear with xylene and mount in a resinous
medium.

Results
Fibrin - clear red
Erythrocytes - orange
Collagen - blue
Fibrinoid - varies red to orange


Bryan Llewellyn



----- Original Message -----
From: "Jill Songer" <jtsonger <@t> vt.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, January 14, 2004 12:54 PM
Subject: [Histonet] Fraser-Lendrum stain


> Would anybody in Histoland mind sharing their protocol for this fibrin
> stain with me? Many thanks.
>
> Jill Songer HT (ASCP)
> Histopathology Lab
> Veterinary Medical Teaching Hospital
> Virginia Tech
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>