Mast Cells - RCPAQAP

Aim of special stain:: Demonstrate mast cells in tissue sections
Diagnostic applications:: Demonstrate the presence of mast cells in mast cell tumours
Material supplied for surveys:: Composite block containing tumour and normal skin tissue cut at 3µm
Recommended control:: Skin with neurofibroma and leiomyoma of the uterus

1. Azure A for Mast cells staining method

Reagents – commercial source unknown, unless specified:

1. Stock Azure A
0.2% aqueous solution of azure A (Dyes – commercially available)

2. Working Azure A
a) Absolute ethanol	15 mL
b) Distilled water	30 mL
b) Stock azure A	5 mL
Make up fresh just prior to use

3. Uranyl Nitrate
0.5% aqueous solution uranyl nitrate.
CAUTION: Toxic, radioactive. Wear gloves and prepare under hood. Solid store in fridge.

Method:

De-wax sections to water.
Stain in working azure A in a coplin jar for 10 minutes.
Wash in water.
Differentiate in uranyl nitrate until there is a good colour contrast, 10 seconds.
Wash in water.
Blot sections and allow to air dry.
Clear in xylene and mount.

Results:

Mast cells granules – Pink
Background – Blue

2. Toluidine blue for Mast cells staining method

Reagents – commercial source unknown, unless specified:

1. 0.5% Toluidine blue, pH 0.5
a) Toluidine blue	0.5 g
b) 0.5 N HCL	100 mL
i) Conc HCL	42 mL or 10.5 mL
ii) Distilled water	958 mL or 240 mL

Method:

Dewax to water.
Stain in toluidine blue solution in a Coplin jar for 20-30 minutes.
Rinse in water and check staining microscopically.
Air dry.
Clear in xylene and mount in a neutral mounting medium.

Results:

Nuclei – blue green
Mast cells – deep violet
Cytoplasm – light blue
Erthrocytes – green to Yellow

3. CSABA’s Mast cell staining method

Reagents – commercial source unknown, unless specified:

1. Sodium acetate stock
Anhydrous Sodium Acetate 82.4g (powder form) or 136.09g (crystal form)
Make up to 1000 mL with distilled water

2. 1N HCL
a) HCL (specific gravity 1.19)	83.5 mL
b) Distilled water	916.5 mL

Preparation of Buffer- pH 1.42
a) 1N HCL	24 mL
b) Sodium acetate	20 mL
c) Distilled water	56 mL

Preparation of Stain
a) Alcian blue	0.36 g
b) Safranin	0.18 g
c) Ferric ammonium	0.48 g
d) Buffer pH 1.42	100 mL

Method:

Bring sections to water.
Stain in Alcian blue-safranin for 10-20 minutes.
Rinse in water.
Dehydrate in tertiary butyl alcohol, clear in xylol, and mount in synthetic resin.

Results:

Young mast cells – Blue
Mature mast cells – Red
Background – Pale blue

4. Giemsa for Mast cells staining method

Reagents – commercial source unknown, unless specified:

1. Giemsa solution (changed every week)
a) Giemsa	40 mL
b) Distilled water	360 mL

2. 1% Glacial Acetic Acid (changed every week)
a) Concentrated Glacial Acetic Acid	4 mL
b) Distilled water	400 mL

Method:

Deparaffinise and hydrate to water.
Giemsa solution for 10 minutes.
Wash in running water for 2 minutes.
5 dips in Glacial Acetic Acid.
Rapidly wash in running water for 2 minutes.
Air dry, clear and mount.

Results:

Mast cells granules – purple
Red blood cells and background – pink
Micro-organisms, fungi, parasites – Purplish-blue

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

2017 RCPAQAP Technical General Survey 3.
Grizzle. William and Bancroft John D. “The dispersed neuroendocrine system, Cytoplasmic granules, and other Organelles” in Theory and practice of Histological Techniques, 6^th Edition. Churchill Livingstone 2011:16:304.
Mathilde Leclere, Michel Desnoyers, Guy Beauchamp, and Jean-Pierre Lavoie. Comparison of Four Staining Methods for Detection of Mast Cells in Equine Bronchoalveolar Lavage Fluid. The Journal of Veterinary Internal Medicine; 2006; 20:377–381.