Structure and function
Paneth cells are part of the epithelial lining of the human small intestine but are present only in the base of the crypts of Lieberkühn. Although the precise function of Paneth cells is unclear, lysozyme has been described in the secretory granules, Golgi apparatus and rER53, and immunoglobulins (IgA and IgG) have been demonstrated in the cell cytoplasm⁵⁴. The presence of lysozyme (an enzyme with limited bactericidal activity that is enhanced in the presence of immunoglobulins and complement) combined with the phagocytic capability of Paneth cells, suggests they are involved in regulation of the microbial flora of the small intestine1. Cationic trypsin immunoreactivity has been demonstrated in human Paneth cells, indicating a possible role in secretion of digestive enzymes⁵⁵, and their content of heavy metals, particularly zinc, suggests a role in the elimination of metals⁵⁶.

Pathology
The response of Paneth cells in pathological conditions is variable. Cell numbers may be decreased due to non-specific injury in inflammatory conditions and increased in regions undergoing regeneration and repair. Reports of alterations in coeliac disease are inconsistent⁵⁶. Characteristic inclusion bodies are seen (by electron microscopy) in Paneth cells in acrodermatitis enteropathica, a rare autosomal recessive disease characterised by primary zinc deficiency. Following dietary zinc supplementation these inclusions disappear⁵⁷. Similar inclusions have been reported in neoplastic Paneth cells⁵⁸.

Metaplastic Paneth cells have been demonstrated in other parts of the gastrointestinal tract in Barrett's oesophagus, chronic gastritis, various inflammatory conditions of the large intestine and in association with some tumours. The presence of metaplastic Paneth cells is thought to represent a non-specific response to the disease state (their evolution and function in these sites is unknown)⁵⁹.

Specimen preparation
Tissue fixation must be prompt as Paneth cells degranulate rapidly during autolysis. The granules dissolve in fixatives containing acetic acid but are well preserved in 10% neutral buffered formalin or mercuric chloride-formalin (formol sublimate)^56,60.

Staining
Paneth cells are recognised in haematoxylin and eosin stained sections by their large, intensely eosinophilic apical secretory granules and their localisation in the base of the crypts of Lieberkühn. They can also be demonstrated using Lendrum's phloxine tartrazine⁶⁰, Masson's trichrome (granules stain red with both methods)⁶¹ and Mallory's phosphotungstic acid haematoxylin (granules stain blue/black)^59,62. The granules stain variably (weakly) with the PAS technique^61,62 and react histochemically for tryptophan, tyrosine and sulphydryl and disulphide groups⁶³.

Lendrum's phloxine tartrazine⁶⁰ is a simple and reliable technique. Nuclei are stained with haematoxylin, followed by cytoplasmic staining with phloxine and subsequent differentiation in a solution of tartrazine in cellosolve. As differentiation continues the red phloxine staining is progressively removed from tissue components that then stain with the yellow tartrazine dye. Paneth cell granules have a strong affinity for phloxine and therefore retain their red colour even after lengthy differentiation. Dye techniques are adequate for demonstrating Paneth cells in sections but immunocytochemical techniques are required to identify specific substances contained within Paneth cells (lysozyme, immunoglobulins, cationic trypsin)^53,54,55.

Lendrum's phloxine tartrazine (Lendrum 1947)⁶⁰
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral buffered formalin or mercuric chloride-formalin. Avoid fixatives containing acetic acid. Small intestine should be used as control tissue.

REAGENT PREPARATION
1 Phloxine solution
Phloxine B (CI 45410) 0.5 g
Calcium chloride 0.5 g
Distilled water 100 ml

2 Tartrazine in cellosolve⁶⁴
Tartrazine (CI 19140) 2.5 g
Cellosolve (ethylene glycol monoethyl ester) 100 ml
This is a saturated solution.

3 Celestine blue/alum haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Stain nuclei with celestine blue/alum haematoxylin.
3 Stain in phloxine solution for 30 minutes.
4 Rinse briefly in water.
5 Differentiate with tartrazine in cellosolve until only the granules stain intensely red (control microscopically).
6 Rinse briefly in water (see note 3).
7 Rinse in 95% ethanol.
8 Dehydrate, clear and mount.

RESULTS
Nuclei - blue
Paneth cell granules - red
Background - yellow

TECHNICAL NOTES
1 Although Lendrum considered the use of an iron haematoxylin unnecessary, it does provide more intense nuclear staining.
2 Calcium chloride added to the phloxine solution intensifies the stain and prolongs the shelf life for up to one year.
3 Lendrum recommended prolonged differentiation followed by thorough washing in water to remove virtually all the yellow tartrazine staining. This facilitates the demonstration of Paneth cells by increasing the contrast. If intense yellow staining is preferred only brief washing is required at step 6.
4 Other tissue components can be demonstrated with this technique by varying the extent of differentiation. These include muscle, fibrin, keratin, viral inclusion bodies, pancreatic B-cell granules and Russell bodies. Appropriate control sections must be used to monitor differentiation.