Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
Some mesotheliomas contain hyaluronic acid and can be stained with alcian blue or colloidal iron. The removal of this staining with hyaluronidase can be of value in distinguishing between carcinomas and mesotheliomas. Chondroitin sulphates A and C are digested, as well as hyaluronic acid, with this method. Following treatment with hyaluronidase, the sections are stained with Colloidal iron followed by Curtis stain. Loss of staining, when compared to a control, duplicate section establishes the presence of one or more of the three hyaluronidase labile mucins.
By matching the ionic strength and/or the pH of the solvent, solutions of the copper phthalocyanins, (eg Alcian blue), are used to react with, and thus demonstrate, the ionisable moiety of acidic proteoglycans. The moiety in question can be:
sulphated residue
carboxylated residue
hyaluronic residue
chondroitin residue
This method utilises a solvent pH of 2.5.
Specific enzymatic hydrolysis of residues eliminates their visualisation, in this case, hyaluronic residues.
Technical Points
1. A known positive control section must be used to ensure correct differentiation has been achieved.
2. Step 6 - Red background staining results if the neutral red stain is applied directly from tap water.
Method
1. Bring two test and two control sections to distilled water
2. Incubate one test and one control section in hyaluronidase at 37°C 1 hr
3. Wash well in running tap water 10 mins
4. Rinse in distilled water
5. Stain all sections in Alcian blue stain 30 mins
6. Wash well in running tap water 5 mins
7. Rinse in distilled water
8. Counterstain with filtered neutral red stain 1 min
9. Rinse in distilled water
10. Blot dry
11. Dehydrate well in absolute alcohol, clear and mount.
Results
Digested slides: XE "Acid mucin,:45-"
Areas containing hyaluronic acid, hyaluronosulphate or chondroitin sulphates A and C.Unstained
Other acid mucins....................................Blue
Nuclei..................................................Red
Undigested slides:
Hyaluronic acid, hyaluronosulphate and chondroitin sulphates A and C.......Blue
Other acid mucins...................................................................Blue
Nuclei.................................................................................Red
Muscle................................................................................Yellow
Reagent Formulae
1. Hyaluronidase solution
40 mg hyaluronidase (H-3506, Sigma), dissolved in 40.0 mls normal saline
2. Alcian blue stain pH 2.5 –see method for Alcian Blue
3. Neutral red – see method for Gram stain
References
1 Lillie, R.D. 1977. H.J.Conn's Biological Stains, 9th edition. Williams and Wilkins.
2 Zugibe, F.T. 1970. Diagnostic Histochemistry. The C.V.Mosby Co, p84-87.
3 Pearce AGE,(1953),Histochemistry Theoretical and Applied, 1st ed., Churchill.