MAST CELLS
By Robyn Siebert and John Stirling

Structure and function
Mast cells are normally present in small numbers in the connective tissue of all organs, but particularly in the dermal layer of skin (around blood vessels and nerves), and are identified by their cytoplasmic granules. Mast cells have been considered the tissue equivalent of the circulating basophil but, while there is evidence that they arise from common precursor cell in the bone marrow, there is no evidence that mature basophils are able to differentiate into mast cells67. The two cell types are readily distinguished by their morphology on light microscopy67,68 and the presence of chloroacetate esterase activity in mast cells69.

Mast cells play an important role in immunity, with specific involvement in type I (anaphylactic) hypersensitivity reactions. When IgE antibodies are raised against a particular allergen they can bind to mast cell surface Fc receptors. Subsequent exposure to the allergen triggers mast cell degranulation and the release of chemical mediators, such as histamine and heparin, into the surrounding tissues5. Mast cells are also involved in delayed hypersensitivity, cytotoxicity, immunoregulation and inflammation70.

The content of mast cell granules varies between species and in some pathological conditions. Human mast cells contain histamine, heparin and various proteins, and although serotonin is not normally present, it has been demonstrated in mast cells in the stroma of carcinoid tumours and in mastocytosis68.

A subpopulation of mast cells has been identified at mucosal surfaces, such as in the gastrointestinal and respiratory tracts, in rats and humans. These mucosal mast cells show some structural and functional differences from connective tissue mast cells68,71 and require special fixation conditions or modified staining protocols for their demonstration70,72-75.

Pathology
Increased numbers of mast cells are found in many pathological conditions. Mast cell hyperplasia in the skin (mastocytosis) manifests with skin lesions and may present with symptoms of urticaria and flushing due to the chemical mediators released during mast cell degranulation. Children may develop single mastocytomas or the multiple cutaneous lesions of urticaria pigmentosa. In adults, multiple organ involvement can occur (notably affecting bone, liver, spleen and lymph nodes) even without apparent skin lesions (systemic mastocytosis). Lesions of the bone may be localised or widespread, osteoclastic or osteoblastic76. Increased mast cell numbers are also seen in some inflammatory bowel diseases (ulcerative colitis, Crohn's disease) and in parasitic infections68. Cutaneous neurofibromas, benign and malignant breast lesions, and some soft tissue tumours also show high numbers of mast cells.

Specimen preparation
Fixation must be rapid to avoid cytoplasmic degranulation and deterioration of granule contents. Neutral buffered formalin (10%) preserves morphology and enables demonstration of connective tissue mast cell granules using a variety of staining techniques. Special fixation is required for the demonstration of mucosal mast cells as aldehydes reversibly block cationic dye-binding sites74. Carnoy's fluid (minimum fixation time: two hours) or isotonic-formol-acetic acid (1.5% formalin, 0.5% glacial acetic acid - minimum fixation time: two days) are suitable73. Alternatively, mucosal mast cells can be demonstrated in aldehyde fixed tissue by increasing the staining time74.

Staining
Mast cells are not readily identified in haematoxylin and eosin stained sections (the granules are refractile and do not stain) but are well demonstrated by a number of special staining methods. The most common are metachromatic dye techniques and the demonstration of chloroacetate esterase activity.

Metachromatic dye techniques
Metachromatic dyes, such as Toluidine Blue and Azure A, demonstrate the strongly sulphated acid mucopolysaccharide (heparin) content of mast cell granules. The acidified toluidine blue method of Churukian and Schenk77 is simple, rapid and reliable. Sections are oxidised with permanganate, decolourised and then stained in an acidified solution of Toluidine Blue. The low pH (3.2) of the solution minimises background and enhances nuclear staining (both nuclear and background staining can be eliminated by lowering the pH to 0.568).

Acidified toluidine blue77
SPECIMEN PREPARATION
Cut 3 to 5 m thick paraffin sections from tissue fixed in 10% neutral buffered formalin. Tissue known to contain mast cells (neurofibroma, skin) is used as a control.

REAGENT PREPARATION
1 0.5% aqueous potassium permanganate
Potassium permanganate 0.5 g
Distilled water 100 ml

2 2% aqueous potassium metabisulphite
Potassium metabisulphite 2 g
Distilled water 100 ml

3 Acidified toluidine blue solution (pH 3.2)
Distilled water 99.75 ml
Glacial acetic acid 0.25 ml
Toluidine Blue (CI 52040) 0.02 g

METHOD
1 Dewax and rehydrate sections.
2 Transfer sections to potassium permanganate solution for 2 minutes.
3 Rinse in distilled water.
4 Transfer sections to potassium metabisulphite solution for 1 minute (or until sections appear white).
5 Wash in tap water for 3 minutes.
6 Rinse in distilled water.
7 Place in acidified toluidine blue solution for 5 minutes.
8 Rinse in distilled water.
9 Dehydrate rapidly, clear and mount.

RESULTS
Mast cell granules and other strongly sulphated acid mucopolysaccharides - purple
Nuclei - blue

TECHNICAL NOTE
Mast cells and eosinophils can be demonstrated in the same section by staining with Congo Red before acidified toluidine blue78 and this technique can be modified for undecalcified bone sections79.

A prolonged staining protocol74 can be used to demonstrate mucosal mast cells in aldehyde fixed tissue, connective tissue mast cells after prolonged aldehyde fixation or the presence of low numbers of mast cells in highly cellular tissue (such as lymph node). Sections are treated with a 0.5% solution of Toluidine Blue in 0.5 mol/l HCl (pH 0.5) for 5-7 days, filtering the solution on alternate days. The mucosal mast cell granules stain dark blue against a clean background. An eosin counterstain (1% eosin in 70% ethanol for 20 seconds) will improve contrast and allow tissue orientation. Eosinophils are demonstrated after counterstaining with eosin at pH 10.

Chloroacetate esterase activity
Mast cell granules contain proteases, including esterases that rapidly hydrolyse the a -chloroacyl esters of a -naphthol and naphthol AS80. Chloroacetate esterase activity is demonstrated by a simultaneous capture technique using the substrate naphthol AS-D chloroacetate and diazonium salts such as pararosaniline, Fast Blue RR or Fast Garnet GBC81,82,83. Mast cell granules are demonstrated after a short incubation time, before other tissue staining becomes apparent. Leucocytes of the myeloid series are also demonstrated. Pararosaniline is preferred as it forms an insoluble red/pink reaction product and sections can be mounted in synthetic resin. Fast Blue RR forms a vivid blue reaction product and Fast Garnet GBC a red product, but both are soluble in organic solvents and require an aqueous mountant.

Chloroacetate esterase82,84
SPECIMEN PREPARATION
Cut 3 to 5 m thick paraffin sections from tissue fixed in 10% neutral buffered formalin. Do not use acid-containing fixatives such as Zenker's or Bouin's. Fix smears/imprints for 30 minutes in a solution of 9 parts methanol, 1 part formalin. Wash in tap water and air dry. Tissue known to contain mast cells (or kidney - tubule lining cells; liver - hepatocytes, Kupffer cells) is used as a control.

REAGENT PREPARATION
1 4% pararosaniline in 2 mol/l HCl
Pararosaniline (CI 42500) 0.4 g
Distilled water 8.4 g
Concentrated hydrochloric acid 1.6 ml
Dissolve the pararosaniline in the water. Add the acid slowly.

2 4% aqueous sodium nitrite
Sodium nitrite 0.4 g
Distilled water 10 ml

3 0.07 mol/l phosphate buffer pH 6.5
a) Disodium hydrogen orthophosphate (anhydrous) 9.465 g/l
b) Sodium dihydrogen orthophosphate 10.452 g/l
Mix 30 ml of solution a) and 70 ml of solution b).

4 Substrate solution
Naphthol AS-D chloroacetate 0.01 g
(Store below 0C)
N-dimethylformamide 1 ml

5 Mayer's haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Mix 0.1 ml pararosaniline (solution 1), with 0.1 ml of sodium nitrite (solution 2). Leave for 30-60 seconds.
3 Add 30 ml of buffer (solution 3). Check that the solution is at pH 6.3.
4 Add substrate (solution 4), mix and filter.
5 Immediately place sections in the solution and incubate at room temperature for 30 minutes.
6 Check microscopically; if the reaction is incomplete, refilter the solution and reincubate sections for further 15-30 minutes.
7 Wash slides in water for 5 minutes.
8 Counterstain in Mayer's haematoxylin for 5 minutes.
9 Wash in water.
10 Dehydrate, clear and mount.

RESULTS
Esterase activity - red/pink
Nuclei - blue

TECHNICAL NOTES
1 Sections should not be treated with iodine/thiosulphate to remove mercuric pigment as this may reduce the intensity of staining.
2 Do not overheat sections when drying as this destroys enzyme activity.
3 Solutions 1 and 2 keep for at least a month but the reaction solution must be made up fresh each time.
4 If the solution turns red when the buffer is added (step 3) the reaction of pararosaniline with the nitrite was incomplete and a fresh solution should be prepared.

A combination of Human Platelet Factor 4 (HPF 4) which binds to heparin and an anti-HPF 4 antibody can be used to specifically demonstrate mast cell granules in formalin-fixed, paraffin-embedded tissue sections by immunocytochemical techniques85. An antiserum directed against histamine has also been used in an indirect immunofluorescent technique on frozen sections. Positive immunostaining in mast cells was only produced in tissue fixed in 4% carbodiimide in O.1 mol/l phosphate buffer (pH 7.4)86. Lectins (000) have been used in studies on the heterogeneity of mast cells87.

References
Safety Data