Although intracellular granules are frequently present in histological preparations, they are often difficult to identify with the H & E stain and may be required to be demonstrated more prominently. Most often this is to identify mast cells, tissue eosinophils and plasma cells, which may increase in numbers in various conditions, and Nissl bodies in brain cells, often as a means of estimating nerve cell distribution. Melanin is important due to its association with malignant melanomas, and enterochromaffin due to its association with carcinoid tumors.
The selective demonstration of other granules may only be required very infrequently for diagnostic purposes, if at all. Paneth cell granules, for example.
Eosinophils are a blood cell and are easily stained. The name eosinophil means “eosin loving“. This, no doubt, refers to their bright pink stained granules following eosin staining. Unfortunately, the term “eosinophil“, or “eosinophilic“, is also sometimes used to describe other tissue components which stain brightly with eosin. In other words, “eosinophil” is sometimes used as a proper noun, i.e. a name for a blood cell, when the word “polymorphonuclear” may or may not precede it, and at other times it is used as an adjective to describe the stained appearance of other tissue components. In the latter sense, it often has the same meaning as acidophil, meaning that the component stains brightly with acid dyes.
The polymorphonuclear eosinophil has cytoplasmic granules which stain intensely with acid dyes. These may be referred to as “eosinophil”, “eosinophilic”, “acidophil”, or “acidophilic”. The terms are largely interchangeable in practice and refer to the same cells. There are other cells which have acidophil (or eosinophil) granules, but they are usually clearly identified and the term “eosinophil”, when used alone as a name of a cell, almost invariably refers to the polymorphonuclear eosinophil, whether in blood or tissue. Selective methods for the demonstration of these cells often use eosin, but some common methods use other acid dyes, such as chromotrope 2R or sirius red F3B. In fact, many acid dyes could be used for their demonstration by the addition of a little base to the dye solution in order to suppress much of the background staining.
Eosinophils are usually quite clearly identified in a routine H&E stain especially if the procedure used has a water or ethanol wash after eosin counterstaining to increase contrast between the tissue elements. Eosinophils will usually appear as bright pink, refractile granules in the cell’s cytoplasm. Due to their visibility in the H&E stain their selective demonstration is only infrequently required in diagnostic laboratories, although it may be necessary more frequently in research settings.
Although Romanowsky stains on peripheral blood smears show eosinophil granules very well, they are less prominent on tissue sections and either Lendrum”s method or alkaline sirius red are more successful in making them prominent.
Mast Cell Staining
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 sulfated 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 emphasize sulfated acid mucopolysaccharides, i.e. the heparin in the granules, since these often resist loss of metachromasia from dehydration well enough to still be colored differently than by the orthochromatic color 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 fibers. Aldehyde toluidine blue does not stain elastic fibers and demonstrates human mast cell granules in an intense deep blue which contrasts well with nuclear fast red stained nuclei, and is the method usually recommended.
Melanin and Enterochromaffin Staining
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, it has been included as a cytological granule. Enterochromaffin has been included 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, sulfuric 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 tumors. 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.
Nissl Body Staining
Nerve cells, or neurons, in the brain contain some RNA. This often appears to be in stripes, so an older name for it was tigroid, named after tigers, who have stripes (tigroid meaning tiger like). It is now more likely to be referred to as Nissl substance or Nissl bodies, or simply as Nissl, as it is named after a physician with that surname.
Nissl bodies contain significant amounts of RNA and this can be used to demonstrate them, such as with Einarsen’s method. Most staining methods which are used for nucleic acids will be successful including the methods for plasma cells, whose selective demonstration also depends on the presence of large amounts of RNA in the cell cytoplasm. In fact, the Unna-Pappenheim type methods stain Nissl bodies quite well. Nevertheless, they are not the most popular for doing so, and methods have been devised which demonstrate them clearly in good contrast to the rest of the brain tissue. These methods are very often used to give an overall view of the neuron content and distribution in sections of brain and spinal cord, rather than because of an interest in the Nissl bodies themselves. Since Nissl is confined to neurons, staining Nissl is tantamount to staining neurons, and it is this which makes its demonstration useful.
The original fixation method recommended was alcoholic. It has been suggested that this may have given sharper staining results due to removal of some cellular lipid which obscured the staining. It is recommended that frozen sections should be treated with ethanol and xylene to remove some of the lipids before staining. Formalin fixed paraffin sections will have this done during processing, but further treatment may still improve staining.
Thin sections are not usually of any benefit for staining Nissl and it is usually recommended that sections be thicker than normal, about 10 μmetres or thicker for paraffin sections, to give a sufficient depth of color.
Most staining methods follow Nissl’s original example and use blue, basic dyes to overstain the Nissl bodies, then remove excess dye by differentiating in a fluid which dissolves it out of the section, usually ethanol (95%), often with some cajeput oil or acetic acid.. In a well stained preparation the Nissl bodies are darkly stained, outlining nerve cells clearly against a pale background. Nuclei of all cells, being predominantly nucleic acid, will also be stained. Most of these methods are satisfactory. For paraffin sections the cresyl violet method differentiated with 95% ethanol acidulated with acetic acid will generally be found to be convenient and easily controlled.
Paneth Cell Staining
Paneth cells are located at the base of the glands of the small intestine and contain numerous cytoplasmic, acidophilic granules. For that reason, they can be seen in an H&E stain well enough for identification, and their demonstration with selective staining methods is rarely requested. They are often used for staining practice by those learning histological techniques due to their easy availability and because their demonstration uses an early “yellowsolve” staining method: Lendrum’s phloxine-tartrazine. The alkaline sirius red method also demonstrates them well. It is obvious that the methods for their demonstration are very similar to those for eosinophils, both being cells containing strongly acidophil granules.
Plasma Cell Staining
Plasma cells produce antibodies and are rich in cytoplasmic RNA. Their selective demonstration is based on this feature. Both DNA and RNA have an affinity for basic dyes or basic acting lakes and this can be used for their demonstration. These cells sometimes need to be demonstrated to confirm an increase in their numbers in a malignancy, as with a case of multiple myeloma, for instance. It should be noted that RNA is also found in some other cells, the tigroid of nerve cells in brain is rich in RNA, for instance, and the methods for plasma cells work equally well for staining those as well.
Einarson’s gallocyanin-chrome alum is a standard technique which demonstrates both nucleic acids in the same color. Gallocyanin is one of the dyes recommended as a substitute for hematoxylin in H&E staining, but in this case the mordant is chromium rather than iron. Alum hematoxylins do stain both nucleic acids, but the differentiation is greater with gallocyanin-chrome alum.
The methyl green-pyronin methods (MGP), usually referred to collectively as Unna Pappenheim stains, are a group of methods which use two basic dyes (methyl green and pyronin) to stain the two nucleic acids in different colors. There are several variations which seek to be more or less selective in the identification of DNA from RNA. Usually, with these techniques, DNA is stained green-blue and the RNA is stained red. This appears to be due to the DNA being more highly polymerized than the RNA, so that each nucleic acid reacts differently to each of the basic dyes at mildly acid pH. The controlling phenomenon is that in an acid medium basic dyes are restricted in their ability to stain acid tissue components, in this case nucleic acids, but one nucleic acid-dye combination is affected more than the other. This is similar to a one-step trichrome method in which different acid dyes of contrasting colors have differing affinities for basic tissue components from an acid medium. The significant difference with the two basic dyes used in methyl green-pyronin staining is that the pH range involved is quite limited, hovering around 4.8 or so. Below this both nucleic acids stain with pyronin and above this the methyl green predominates. Early methods used phenol to provide the mildly acid conditions required, but later techniques employed buffer solutions.
The staining is further complicated in that anything which depolymerizes the DNA causes it to stain red with pyronin. Although 10% formalin variants are usually reasonably satisfactory, decalcification often degrades the DNA staining excessively and nuclei may be pink.
Some methods for methyl green-pyronin staining require purified methyl green. However, this dye almost always contains some crystal violet (produced by loss of one of its methyl groups), which must be removed prior to staining.
For a simple means of demonstrating plasma cells in routine sections, the phenolic methods will generally be found reasonably satisfactory as they are fairly simple to do. For more selective demonstration of nucleic acids, the method of Trevan and Sharrock or Kurnick should be employed, but in critical applications duplicate sections should be stained after ribonuclease extraction to remove the RNA as a control and possibly after deoxyribonuclease extraction as a second control, although this is generally not considered necessary since DNA may be specifically demonstrated with the Feulgen technique.
There is also a related method by Hitchcock and Ehrich, which is similar to methyl green-pyronin but which uses Malachite green and acridine red to obtain the same color contrast. These two dyes are structurally related to methyl green and pyronin respectively and function similarly. A further modification of the methyl green-pyron stain was suggested by Roque, who replaced pyronin with thionin. Being a basic dye as well, thionin stains RNA while the methyl green continues to stain DNA. Although of interest, neither this technique nor the malachite green-acridine red technique have garnered much support.
- Drury, R A, and Wallington, E A, (1967).
Carleton’s histological technique., Ed. 5., pp. 379.
Oxford University Press, London, England.