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 colour. 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 colours. 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 polymerised 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 colours 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 depolymerises 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. This dye, even in the dry state, almost always contains some crystal violet, produced by loss of one of its methyl groups. To remove this the solution of methyl green must be washed by shaking with a quantity of chloroform to extract the crystal violet until the chloroform is only very palely tinted with crystal violet. The long term stability of a cleaned solution is not universally accepted, some workers think that the cleaned solution may eventually form more crystal violet over time. It is recommended that the volume of solution cleaned be what will be used within a reasonable period of time.
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 desoxyribonuclease 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 colour 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.
|Einarson's gallocyanin chrome alum|
|Compare phenolic MGP solutions|
|Trevan and Sharrock's MGP|
|Hitchcock & Ehrich's Malachite-Acridine red|
|Roque's Methyl green-thionin|
|Plasma cells||Nissl bodies|
Last updated March 2016.