A lot of effort has gone into demonstrating mast cells from the earliest days of histological technique in the mid to late 1800s because of the interest aroused in some early medical microscopists by the selective, metachromatic demonstration of their granules. They sought to understand why some dyes stained the granules so selectively, believing that it might lead to discovery of a "magic bullet" which could target individual cellular components in the treatment of disease. It is now known that mast cell granules are composed predominantly of heparin, a sulphated acid mucopolysaccharide and thus are strongly metachromatic. This is a common basis for the demonstration of mast cells. Strongly metachromatic, intracytoplasmic granules can be confidently presumed to identify them in the vast majority of cases, although there may be some variation from species to species. In general, any method for mucin which depends on metachromasia will likely demonstrate mast cell granules. It should also be pointed out that Romanowsky stains contain azure dyes, either added directly or from polychrome methylene blue, so they can be expected to stain mast cells.
The simple metachromatic staining method below is for acid mucopolysaccharides, but will demonstrate mast cell granules. Other metachromatic staining methods which use ethanol and a low pH are often recommended for mast cells granules to emphasise sulphated acid mucopolysaccharides, i.e. the heparin in the granules, since these often resist loss of metachromasia from dehydration well enough to still be coloured differently than by the orthochromatic colour of the dye. There are many such methods, that of Maximow being an example.
However, metachromasia is not the only means used for their demonstration. Heparin contains some strongly negatively charged groups and these may also stain with basic dyes, even when the dyes do not display metachromasia, such as with basic fuchsin in Gomori's aldehyde fuchsin. Similarly, dyes whose metachromatic ability has been removed by other materials in the staining solution or treatment applied to the sections after staining may still demonstrate the granules, such as Llewellyn's aldehyde toluidine blue. The primary means of attachment of metachromatic dyes to heparin is by bonding to an acid group of the heparin, and this may still take place even if the metachromatic capability has been removed. Thus it is that some methods, using the metachromatic dye toluidine blue for instance, stain mast cell granules a deep blue instead of the metachromatic red of other methods. Red dyes which stain mast cell granules yellow are rarely, if ever, used for the purpose, although they are successful. This is simply because a group of tiny yellow granules is not easy to see in a field of red nuclei. Allen's method below uses neutral red but destroys any metachromasia with ethanol allowing red granules to contrast with blue nuclei.
Less well known are Csaba's method, which claims to be able to differentiate between young and mature mast cells, and Leder's esterase stain, which is one of very few methods for enzymes which is claimed to be successful after routine paraffin processing. Neither of these two methods is popular, Leder's perhaps because it is almost always invariably assumed that enzymes may not be demonstrated following chemical fixation.
Histology laboratories most frequently receive tissue in a 10% formalin variant of some kind, and mast cells are usually adequately fixed in this for demonstration. Both mercuric chloride and ethanol have been recommended as giving superior preservation. Mercuric chloride, however, is now deprecated and should be avoided unless its use is unavoidable. Ethanol fixation gives poor quality morphological preservation and is not recommended for routine diagnostic use, although the modern minimalist fixation of a few hours in the 10% formalin variant often brings this about anyway during the dehydration step of paraffin processing.
So what is the most appropriate method to use? To some extent that depends on the species being targeted, since staining varies from species to species. In human tissues, any of the metachromatic methods will generally be found suitable. Gomori's aldehyde fuchsin is usually considered to be very reliable. It does, however, also stain elastic fibres. Aldehyde toluidine blue does not stain elastic fibres and demonstrates human mast cell granules in an intense deep blue which contrasts well with nuclear fast red stained nuclei, and is the method I usually recommend.
|Aldehyde toluidine blue|
|Hughesdon's metachromatic method|
|Leder's esterase stain|
|Maximow's metachromatic stain|
|Simple metachromatic method|
Last updated February 2016.