Melanin & Enterochromaffin
In most texts melanin would be considered as a pigment rather than as intracellular granules. However, it is a normal constituent of various cells and is obviously a cytological inclusion. Due to that, I am including it as a cytological granule. I have included enterochromaffin since both stain with the same methods.
Melanin is found in many places throughout human tissues, sometimes in quite heavy concentrations, dark brown eyes for instance, and it may obscure the cells enough that cellular detail is difficult to see. In that case it may be necessary to remove the melanin so that cellular morphology may be examined. In addition, it may be found in malignant melanomas, in which case it may be necessary to demonstrate its presence in order to determine whether the malignant cells have crossed a limiting membrane and are invading tissue, and also remove it so that the cells may be examined.
Fortunately, melanin is easily removed by oxidation with potassium permanganate, i.e. using a Mallory bleach. It may be done with, or without, sulphuric acid, but it is sometimes necessary to increase the concentration of potassium permanganate above the 1% or so used for the mild oxidation in many staining methods, although light deposits may be removed with the standard concentrations of a Mallory bleach. Very heavy melanin deposits may require higher concentrations of potassium permanganate for bleaching within a reasonable time, although exceeding 10% is not recommended. These higher concentrations are prone to causing detachment of sections from slides, so they should be thoroughly baked on with a properly applied adhesive. Invariably, eosin staining is poorer following bleaching, especially with higher concentrations of permanganate.
If it is necessary to show that the removed material was melanin, then an appropriate staining method should be applied to a second section as well. Any material bleached out that is stained in the second section may be presumed to be melanin. It is not always necessary to prove the removed material was melanin, and a pathologist may simply need melanin removed to examine the cells and nuclei carefully, without them being obscured.
There are two basic approaches to demonstrating melanin. Both depend on it being a reducing substance. The silver reduction methods are quite popular, and of these the Masson Fontana is in common use. The melanin is seen as black granules. There are many variations of these methods, differing primarily on how the silver solution is made and the temperature at which reduction takes place. Methenamine silver solutions may also be used without prior oxidation of the section. This usually takes longer for reduction than ammoniacal silver, but is less likely to loosen the sections from the slide. The second approach is Schmorls reaction, which is based on the reduction of ferrocyanide to ferricyanide and trapping of the ferricyanide with a ferric salt to produce prussian blue deposits. Melanin is then blue.
Both of these methods demonstrate other materials, such as enterochromaffin and lipofuscins. That is why a bleached section is often required to provide evidence that the deposit is melanin and not something else. Melanin is the most easily bleached of the materials that may be stained.
Enterochromaffin is found in some cells of the intestine. It is also known as "argentaffin" because it reduces silver solutions directly without any auxiliary reducing agent, as does melanin. Also, like melanin it stains blue with Schmorls ferrocyanide reduction. Usually it is quite clear that these granules are not melanin due to their location, but it may occasionally be required to demonstrate its presence positively, as in carcinoid tumours. To differentiate enterochromaffin from melanin, it is usual to use the diazonium salt, fast red salt B, which selectively stains it orange-red and leaves melanin unstained.
|Diazonium reaction (enterochromaffin)|
Last updated January 2019