3.0 FIBRIN

3.1 MARTIUS YELLOW - BRILLIANT CRYSTAL SCARLET BLUE (MSB) TECHNIQUE - FOR FIBRIN
(Lendrum 1962)
Fibrin is an insoluble fibrillar protein formed by polymerisation of fibrinogen, which is one of the plasma proteins and is seen in tissues where there has been tissue damage.

PRINCIPLE
Martius yellow, a small molecule dye, together with phosphotungstic acid in alcoholic solution stains red cells. Early fibrin deposits may be coloured, but the phosphotungstic acid blocks the staining of muscle, collagen and connective tissue fibres. Brilliant crystal scarlet, a medium sized molecule, stains muscle and mature fibrin. Phosphotungstic acid removes any red stain from the collagen. The large molecule dye aniline blue stains the collagen and old fibrin.

REFERENCES
p 101 Cook H C, Manual of Histological Demonstration Techniques. p 134 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2ndEd.

SPECIMEN
Standard paraffin section - coated slide may be used to aid adhesion Fixation -10% neutral buffered formalin Post fixation in Bouin's fluid enhances staining

CONTROL
Use control positive for fibrin eg. inflammatory appendix due to peritonitis.

REAGENTS

(A) WEIGERT'S IRONHAEMATOXYLIN
(see Van Gieson's Technique 2.3)

(B) MARTIUS YELLOW SOLUTION
95 % alcohol 100.mL
Phosphotungstic acid 2.0 g
Martius yellow C.I. 10315 (Acid yellow 24) 0.5 g

(C) 1% BRILLIANT CRYSTAL SCARLET
C.I.16250 in 2.5 % ACETIC ACID (Synonym: Crystal Ponceau Acid Red 44)

(D) 1% PHOSPHOTUNGSTIC ACID
(Synonym: Dodecatungstophosphoric Acid)

(E) 1% ANILINE (SOLUBLE) BLUE C.I. 42755 IN 1% ACETIC ACID
OR 0.5% METHYL BLUE (ACID BLUE 93) C.I. 42780 IN 1% ACETIC ACID

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Post-fix in Bouin's fluid at 60øC for 1 hour.
3. Wash in water
4. Stain with Weigert's Haematoxylin - 15-30 mins.
5. Wash in water
6. Stain in Martius Yellow in a coplin jar for 2-3 mins and rinse in distilled water.
7. Stain with Brilliant Crystal Scarlet for 10mins and rinse in distilled water.
8. Treat with Phosphotungstic acid for 5 mins until no red remains in the collagen and rinse in distilled water.
9. Stain with Aniline Blue for 3-5 mins (control microscopically)
10. Rinse in water, dehydrate, clear and mount.

RESULTS
Fibrin, Red (early fibrin may colour yellow and very old fibrin blue)
Red blood cells Yellow
Collagen Blue
Nuclei Blue/black
Muscle Red (paler than fibrin)
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3.2 LENDRUM'S PICRO-MALLORY METHOD FOR FIBRIN
(Lendrum 1947)
Fibrin is present in tissue where there has been tissue damage and is one of the plasma proteins.

PRINCIPLE
This stain is very similar in principle to the trichrome techniques where small molecule dyes are used to stain less porous tissue followed by larger molecule dyes to stain looser textured structures such as collagen.

REFERENCES
p 99 Cook H C, Manual of Histological Demonstration Techniques.

SPECIMEN
Standard paraffin section - coated slide may be used to enhance adhesion Fixation -10% neutral buffered formalin

CONTROL
Fibrin positive control eg inflamed appendix (peritonitis)

REAGENTS
(A) WEIGERT'S IRON HAEMATOXYLIN (see Van Gieson's Technique) 2.4 (
B) PICRO - ORANGE
Dissolve 0.2g of Orange G [CI 16230] in 100 mL of 80% alcohol saturated with picric acid.
(C) ACID FUCHSIN (CI 42685)
1% Acid Fuchsin in 3% trichloroacetic acid.
(D) 1% PHOSPHOMOLYBDIC ACID
(E) 2% ANILINE (SOLUBLE) BLUE C.I. 42755 IN 2% ACETIC ACID

PROCEDURE
1. Deparaffinize and hydrate to distilled water
2. Post-fix in Bouin's fluid at 60øC for 1 hour (to enhance staining).
3. Wash in water to remove yellow colour.
4. Stain with Weigert's Haematoxylin - 15-30 mins.
5. Wash in water - 10 mins
6. Rinse in 95% alcohol
7. Stain in Picro-orange for 2 mins
8. Wash in water 5 - 10 secs.
9. Stain with acid fuchsin for 5 mins, then rinse in water.
10. Dip sections in equal parts Picro-Orange and 80% alcohol for a few seconds.
11. Differentiate in 1% Phosphomolybdic acid until colours are clear (5-10 mins), then rinse in water.
12. Stain with soluble Aniline Blue for 2 - 10 mins, then rinse in water.
13. Dehydrate, clear and mount.

RESULTS
Fibrin Clear red
Red blood cells Orange/yellow
Collagen Blue
Nuclei Blue/black
Muscle Paler red

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