11.1 ATTWOOD'S METHOD FOR AMNIOTIC FLUID EMBOLISM

PRINCIPLE
All tissues are stained with phloxine, then the red stain is differentiated out with tartrazine in cellosolve. This method can be used to demonstrate many structures according to the degree of differentiation.

REFERENCE
P232 Lee G Luna, Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd Ed.

SPECIMEN
10% neutral buffered formalin fixation. Standard paraffin sections CONTROL Known positive tissue.

REAGENTS

(A) 1% ALCIAN GREEN SOLUTION
Alcian green 1 g Distilled water 100 mL Glacial acetic acid 2 mL

(B) PHLOXINE-CALCIUM CHLORIDE SOLUTION
Phloxine 0.5 g Calcium chloride 0.5 g Distilled water 100 mL

(C) TARTRAZINE-CELLOSOLVE SOLUTION
(Store in flammables cabinet) Tartrazine (CI 19140) 2.5 g Cellosolve (irritant to skin) 100 mL (also called 2-ethoxyethanol or ethylene glycol monoethyl ether)

(D) 0.5% POTASSIUM PERMANGANATE SOLUTION
Potassium permanganate 0.5 g Distilled water 100 mL

(E) 0.5% OXALIC ACID SOLUTION
Oxalic acid 0.5 g Distilled water 100 mL or Dilute 1% oxalic acid solution from reticulin fibre stain (method 2.5) 50/50 with distilled water.

PROCEDURE
Use control slide.
1. Deparaffinise and hydrate to distilled water.
2. Alcian green solution for 2 minutes.
3. Rinse in distilled water.
4. Potassium permanganate solution for 2 minutes.
5. Rinse in distilled water.
6. Oxalic acid solution for 2 minutes.
7. Rinse in distilled water
8. Stain with Mayer's Haematoxylin 3 minutes (or alum haematoxylin in linear stainer).
9. Rinse and blue in lithium carbonate solution.
10. Phloxine solution for 30 minutes.
11. Rinse in distilled water, then in cellosolve.
12. Differentiate each slide with tartrazine-cellosolve solution, checking until the phloxine is removed from erythrocytes (approximately 45 seconds).
13. Dehydrate in cellosolve, clear in histolene, two changes each.
14. Mount with Permount or Safety-mount.

RESULTS
Nuclei Blue/black
Mucin, ground substance of cartilage, granules of certain mast cells Green
Foetal squames, squamous epithelium, fibrin Red
Erythrocytes, plasma collagen Yellow
Inclusion bodies Red
NOTE The permanganate-oxalic bleach is not strictly necessary but it assists in the subsequent differentiation of the phloxine and tends to remove any background staining by Alcian Green.
Any excess staining with tartrazine can be removed by rinsing in water until the desired intensity is obtained.

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11.2 MALLORY BODIES (ALCOHOLIC HYALINE)

Alcoholic hyaline is a protein substance occurring within the cytoplasm of liver cells in certain patients who have liver disease due to excess alcohol consumption. It appears as a perinuclear pale-staining, often greyish zone in the liver cell. Alcoholic hyaline stains with the following stains but appears to be most satisfactorily stained with Luxol Fast Blue (personal communication forensic pathologist Dr KAP Lee).
Refer to Method 7.2.

REFERENCE
HC Cook, Manual of Histological Demonstration Techniques p243

SPECIMEN
Routine formalin fixation. Standard paraffin section.

CONTROL
Use known positive section of liver.

RESULTS
Stain Alcoholic hyaline H & E Weakly eosinophilic
Luxol Fast Blue Blue PTAH Blue Congo Red Red (weak)

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