ANTERIOR PITUITARY
Structure and function
The anterior lobe (or adenohypophysis) is the glandular portion, composed of nests and cords of cells within an interlacing network of small capillaries. The cells of the anterior pituitary produce growth hormone (GH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), melanocyte stimulating hormone (MSH), b -lipotropin (b -LPH), follicle stimulating hormone (FSH), luteinising or interstitial cell stimulating hormone (LH or ICSH) and thyroid stimulating hormone (TSH). Secretion is regulated by 'releasing' and 'inhibiting' factors produced in the hypothalamus and transported to the anterior lobe via the hypophyseal portal veins. A negative feedback system controls the secretion of these factors³⁷.

Specimen preparation
Pituitary tissue must be fixed promptly to prevent the diffusion and/or loss of hormones, a problem that occurs when interruption to the blood supply is prolonged or the gland is traumatised during removal³⁸. Post mortem specimens should be collected within 4 hours of death³⁹. Fixation in 10% neutral buffered formalin preserves morphological detail and retains hormones in situ, but for specific staining techniques other fixatives are recommended^40,41,42.

The gland should be sectioned in the horizontal plane for optimal examination of the medial 'mucoid' wedge and lateral 'acidophil' wings of the anterior lobe. This enables evaluation of the distribution and relative frequency of the different cell types³⁸.

Staining
Three cell types (acidophils, basophils and chromophobes) can be identified in thin haematoxylin and eosin stained sections as their respective secretory granules have differing affinities for acidic and basic dyes^1,43 (Table 1) although some simple special stains will give improved cellular differentiation (Table 2). Other techniques allow basophils to be separated into two subtypes (Table 3), and when this information is combined with morphological criteria and the pattern of cell distribution, three or more subtypes can be identified. Unfortunately, some of these methods are complex and require experience to obtain good quality, consistent staining results. Those which use a strong oxidising agent (especially performic acid) can also disrupt tissue and result in section loss⁴². Acidophils can be divided into two subtypes by selective staining of prolactin producing cells⁴⁸ using either Herlant's erythrocin stain41 or Brooks' carmoisine technique⁴⁴. The orange G-acid fuchsin-light green (OFG) method of Slidders⁴⁰ is a suitable routine stain for the differentiation of acidophils, basophils and chromophobes, and the architectural pattern of the gland is well demonstrated with a reticulin stain⁴³. A control section of normal pituitary should always be stained in parallel with the test case to allow comparison with normal morphology and cell distribution³⁸.

Immunocytochemical techniques using specific antisera to pituitary hormones have enabled the identification of five distinct cell types in the anterior lobe^48,49 (Table 4). Studies comparing conventional dye techniques with immunocytochemical localisation of pituitary hormones show that the conventional methods do not reliably differentiate the various cell types present^47,48,50,51. Some anterior pituitary cells contain more than one hormone and chromophobes, previously thought to be resting acidophils or basophils, or transitory cells, may contain hormones. In addition, cells containing the same hormone can stain variably with dye techniques³⁸. Such discrepancies can be attributed to the staining of substances other than hormones⁵¹ or may reflect different stages in cell maturation or the cell secretory cycle⁴⁷.

Ultrastructural features of the different cell types and their granules^11,43 are not sufficiently distinct to allow definitive cell identification1 but are of value when used in conjunction with immunocytochemistry¹¹. It should be noted that there is significant inter-species variation in the structure of the pituitary gland and information from animal studies cannot be extrapolated to humans⁴⁸.

Pathology
Variations in the number, size and distribution of the different cell types in the anterior pituitary are seen in a number of conditions. During pregnancy and lactation the gland may increase in size by up to one third due to a true hyperplasia of prolactin secreting cells. Changes in other cell types occur in some endocrinopathies, following surgical removal of pituitary hormone target organs, and in conjunction with certain drug therapies. The gland also shows age related changes^43,48. The most significant lesions seen in the pituitary gland are benign adenomas^37,38. Current classifications identify ten types of pituitary adenoma based on immunocytochemical demonstration of specific hormone production and tumour cell ultrastructure^48,49. Tumour cells may be non-secretory or produce one or more hormones: chromophobic adenomas (the most common type) are able to produce any of the anterior pituitary hormones; acidophilic adenomas produce PRL and/or GH; and basophilic adenomas produce ACTH, b -LPH and/or endorphins⁴⁸. TSH, LH or FSH secreting adenomas are uncommon³⁷.

Orange G - Acid Fuchsin - Light Green⁴⁰
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in mercury-containing fixatives or 10% neutral buffered formalin. A known positive control (pituitary) should be included.

REAGENT PREPARATION
1 Orange G solution
G (CI 16230) 0.5 g
Absolute ethanol 95 ml
Distilled water 5 ml
Phosphotungstic acid 2 g

2 Acid fuchsin solution
Acid Fuchsin (CI 42685) 0.5 g
Distilled water 99.5 ml
Glacial acetic acid 0.5 ml

3 1% phosphotungstic acid
Phosphotungstic acid 1 g
Distilled water 100 ml

4 Light green solution
Light Green (CI 42095) 1.5 g
Distilled water 98 ml
Glacial acetic acid 2 ml

5 Celestine blue/alum haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Remove mercury pigment with iodine/thiosulphate.
3 Stain nuclei with celestine blue/alum haematoxylin.
4 Rinse in 95% ethanol.
5 Stain sections with orange G solution for 2 minutes.
6 Rinse in distilled water.
7 Stain sections with acid fuchsin solution for 2-5 minutes. Staining is progressive and should be continued until the basophils, but not the background, are strongly stained.
8 Rinse in water.
9 Treat sections with 1% phosphotungstic acid for 5 minutes.
10 Rinse in water.
11 Stain sections with light green solution for 1-2 minutes.
12 Rinse in water.
13 Dehydrate, clear and mount.

RESULTS
Nuclei - blue/black
Acidophils - orange/yellow
Basophils - magenta red
Chromophobes - pale grey/green
Erythrocytes - ;yellow
Stroma - green

TECHNICAL NOTES
1 If tissue has been fixed in formalin, staining can be enhanced by mordanting sections in picro-mercuric-alcohol (a saturated solution of picric acid in absolute alcohol containing 3% mercuric chloride) overnight. This solution is particularly toxic and care is required when preparing or handling it. Subsequently, mercury pigment must be removed with iodine/thiosulphate and the sections washed well in water to remove picric acid staining. Helly's or Bouin's fluids can also be used.
2 Iodine/thiosulphate treatment is recommended, even when mercury-containing solutions have not been used, to enhance staining.

POSTERIOR PITUITARY
The posterior lobe (or neurohypophysis) is the neural portion containing pituicytes (specialised supportive cells similar to the neuroglial cells of the central nervous system) and unmyelinated nerve axons that originate from neurosecretory cells located in the hypothalamus. The posterior lobe stores and secretes two hormones, oxytocin and vasopressin (anti-diuretic hormone). These are produced by the neuronal cell bodies in the hypothalamus, transported to the posterior lobe along the axons by carrier proteins (neurophysins) and stored as neurosecretory granules (Herring bodies) at the nerve terminals1.

The nerve axons can be demonstrated using silver impregnation techniques. Neurosecretory substances stain blue to purple with Alcian Blue or Aldehyde Thionin in the following anterior pituitary staining methods: permanganate-aldehyde thionin-periodic acid-Schiff-orange G (PM-AT-PAS-OG)⁴⁶, bromine-alcian blue-orange G-acid fuchsin-light green (Br-AB-OFG)⁸ or; Bargmann's modification of Gomori's chrome haematoxylin⁴⁵. Immunocytochemical techniques using specific antisera can demonstrate the hormones secreted (oxytocin, vasopressin), the neural origin of the tissue (S100 protein, neurone specific enolase) and the glial nature of pituicytes (glial fibrillary acidic protein).

Disorders of the posterior pituitary are very rare and usually result in vasopressin deficiency or inappropriate release of vasopressin³⁷.

Between the two main lobes lies the intermediate lobe (a poorly defined region in humans) that contains cystic structures lined by epithelium and filled with colloidal material (remnants of the embryonal Rathke's pouch)1. In other species (amphibians) the cells of the intermediate lobe secrete MSH but in humans the function of these cells is uncertain⁵².