Argentaffin and Argyrophil Granules. Cells containing neurosecretory granules, arise from neuroectoderm and migrate to their final position in the body rather than differentiating in situ. In many sites, such as the adrenal medulla, pituitary and pancreatic islets, they become aggregated together to form a recognisable endocrine gland, but in other parts, particularly the G.I. tract, they are scattered throughout the mucosa, forming a diffuse system. Argentaffin cells containing 5 hydroxy-trytomine (5HT) and argyrophil cell containing 5- hydroxytrytophane are important in the investigation of carcinoid tumours These tumours are found in the appendix and ileum and are usually argentaffin positive and argyrophil negative and those of the stomach are argrophil positive. The argentaffin cells are potential reducers in that with formalin fixation beta carboline is formed from 5HT and will reduce silver nitrate. Argyrophil cells are not reducers and stain negatively with silver stains.

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9.1 ALKALINE DIAZO TECHNIQUE FOR ARGENTAFFIN CELL GRANULES.
(Gomori 1952)

PRINCIPLE
The rationale of this technique is that certain diazonium salts are converted to diazo dyes by 5HT.

REFERENCE
p 273 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin section of formalin fixed tissue.
Note: This technique is not successful on unfixed or alcohol fixed tissue.

CONTROL
Distal small intestine.

REAGENTS
(A) 1 % Fast red B (C.I 37125) 5 mL (Store at 4øC - diazonium salts deteriorate with time)
(B) Saturated Lithium Carbonate 2 mL Keep solutions at 4øC before mixing, then use immediately (allow 30 minutes for solutions to come to 4øC from room temperature).

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Transfer sections to incubating solution at 4øC for 1 minute
3. Wash well in tap water
4. Lightly stain nuclei in haematoxylin
5. Wash well in tap water
6. Dehydrate through graded alcohols to histoclear.
7. Mount in Safety Mount
8. Haematoxylin staining must be light so that positive staining of granules is not masked.

RESULTS
Argentaffin cell granules Orange
Nuclei Blue
Background Yellow

NOTES 1. Other diazonium salts may be used, eg Fast Garnet GBC 2. This technique is only successful when fresh fixed tissue is used; the method rarely works with necropsy material. It is perhaps unnecessary to stress that, by the nature of the reaction, which depends on amine-aldehyde concentration, the techniques cannot be applied to unfixed material, or to tissues fixed in a non-aldehyde solution.

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9.2 GRIMELIUS SILVER METHOD FOR ARGYROPHIL CELLS,
ESPECIALLY A (a2 ) CELLS OF THE PANCREAS
(Grimelius, 1968)

PRINCIPLE
This is a silver technique where the silver salt first attaches to the tissue component and is then reduced by an externally added reducing reagent.

REFERENCE
p 269 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard formalin fixed paraffin sections.

CONTROL
Pancreas or stomach.

REAGENTS
(A) ACETATE BUFFER pH 5.6
Stock A: 0.2M Acetic Acid 1.2 mL Glacial Acetic Acid in 100 mL distilled water
Stock B: 0.2M Sodium Acetate 1.64 g Sodium Acetate (anhydrous) (MW 82) or 2.72 g Sodium Acetate Trihydrate (MW 136) in 100 mL distilled water
Mix 10 mL Stock A with 90 mL Stock B.
(B) SILVER SOLUTION Acetate Buffer (pH 5.6) 10 mL Redistilled water 87 mL 1 % silver nitrate (aqueous), fresh 3 mL
(C) REDUCING SOLUTION Hydroquinone 1 g Sodium sulphite (cryst) 5 g Distilled water 100 mL Freshly prepared.

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Transfer sections to silver solution at 60øC for 3 hours
3. Drain silver solution from slides
4. Place sections in freshly prepared reducing solution at 45øC for 1 minute
5. Rinse sections in distilled water
6. Counterstain if required
7. Wash in tap water
8. Dehydrate through graded alcohols
9. Clear in Histoclear and mount in Safety Mount

NOTES
1. If a weak positive reaction is obtained, the results can be improved by double impregnation. After the reducing stage, the sections are treated with 5 % sodium thiosulphate for 2 minutes, rinsed in distilled water and placed in freshly prepared silver solution at room temperature for 10 minutes. This is followed by fresh reducing solution for 1 minute at 45øC.
2. Counterstains are not normally used.

RESULTS
Argyrophil islet cells (A or a2 cells) - Positive (brown-black)
B & D cells - Negative Many other argyrophil cells - Positive (brown-black)

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