Trichrome Stains

Although the word trichrome actually means three colour, it no longer has that specific significance. The term is used now to describe those staining methods which use two or more acid dyes of contrasting colours to selectively colour different basic tissue components. Most commonly they are used to demonstrate collagen, often in contrast to smooth muscle, but may also be used to emphasise fibrin in contrast to erythrocytes. Other components can also be selectively demonstrated.

The most widely accepted basis for these methods is an application of mass action, coupled with observations on the physical properties of various tissue components and their impact on staining. At the outset it must be made clear that the methods control how ionised acid dyes react with ionised basic tissues. That is the fundamental principle on which they depend, and the explanation is only about how that fundamental reaction can be manipulated.

There are several factors involved in determining which acid dye attaches to which tissue component. Some of these factors involve the tissue and some the dye, and the more traditional Masson type trichromes also incorporate what are often referred to as polyacids. These factors are all active simultaneously, but in a given circumstance an individual factor may be more or less important. For this reason, the explanations for multi-step and one-step methods differ slightly, although they do depend essentially on the same process.

The factors are:–


Common factors
Following appropriate fixation and processing, sections within the range of about 3 - 8 microns are cut. The section thickness can have an effect on the results, but within that range is usually satisfactory. They are baked onto the slides, and when ready to stain they are brought to water in the usual manner.

Trichrome methods invariably use dyes in acid pH solvents, usually dilute aqueous acetic acid. Usually the concentration of acetic acid matches the concentration of dye (1% dye in 1% acid, 2% dye in 2% acid etc.). This is not necessary, but is common practice. The acid pH itself is necessary to maximise the amount of dye that will attach to tissue amino groups, i.e. it is an accentuator.

This low pH has an effect on nuclear staining with alum hematoxylin, which is either removed or appears red. This is overcome by using ferric mordanted hematoxylin, which resists removal by acids better than aluminum mordanted solutions. Probably the two commonest techniques are Weigert's hematoxylin and the celestine blue hemalum sequence.

These techniques usually stain cytoplasm with a red dye, which is often called the plasma stain. Collagen is usually stained with a green or blue dye which may be called the fibre stain. Small molecular weight yellow dyes are sometimes included to stain erythrocytes so they contrast well with the red staining components, which can include fibrin. For consistency, I shall refer to these as the erythrocyte stain.

See also an explanation of yellowsolve methods.


Multi-step methods
Multi-step techniques include methods such as the Masson's trichrome, which is used to differentiate between smooth muscle fibres and collagen, or to demonstrate a change in the amount of collagen present. Other methods, such as Lendrum's Picro-Mallory may be used to demonstrate fibrin in sharp contrast to erythrocytes and other tissues. In these methods the dyes are applied sequentially, and staining is optimised at each step.

To selectively stain erythrocytes, small molecular weight yellow acid dyes such as picric acid and martius yellow are dissolved in ethanol. A polyacid may also be included. The solution is applied for a period then the sections are washed in water, usually tap water, to remove excess yellow staining from tissues. The erythrocyte limiting membrane permits the entrance of water. This dilutes the ethanol increasing polarity, and results in the dye aggregate size increasing, thus trapping the dye within the erythrocytes. This step is optional, and many techniques do not incorporate it. In those cases erythrocytes stain the same colour as cytoplasm.

The second dye (the plasma stain) is then applied. This is usually red, and of intermediate molecular weight. It is applied for long enough to deeply stain all of the tissue, including cytoplasm, muscle and collagen. If erythrocytes have been pre-stained with yellow dyes, they resist staining with this dye for some time. Otherwise they are also stained with it.

The polyacid is then applied. This has a large molecule, and removes (differentiates) the plasma stain from tissues. The first structures affected are collagen and bone. When it has been applied for long enough, these tissue components macroscopically appear much paler than muscle fibres or cytoplasm.

Following the polyacid, the contrast dye (the fibre stain) is applied. This has a molecular weight larger than the plasma stain, but considerably less than the polyacid. The dye is applied for long enough to strongly stain collagen fibres without beginning to replace the red staining of other components.

The commonest staining pattern is:–

In some methods staining with the fibre stain is accentuated to increase the contrast of the target element, usually fibrin, causing muscle and cytoplasm to appear blue tinged. There are also methods with a colour pattern different than above, yellow collagen for instance, or dark blue fibrin.


One-step methods
One-step trichrome staining methods are those that combine all of the dyes and other reagents into a single solution, which is applied for a specified time. The various tissue components are thereby coloured differentially. These methods include van Gieson's and Gomori's methods.

One step methods are technique dependant. They work satisfactorily provided that everything is standardised. This includes fixation, processing and section thickness as well as the formuation of the staining solution and the length of time for which it is applied. While individual techniques may be more or less tolerant of minor changes, in general, changing any of the parameters will require that the staining procedure be restandardised for consistency of results. In other words, one step methods work fine provided you don't change the procedure at all.

By incorporating all of the reactants into a single solution the various factors interact simultaneously resulting in various tissue components staining with different dyes. The basis is the trend towards reaching equilibrium caused by the reaction products participating in a mass action type reaction. It is for this reason that one-step methods must be so standardised, as changing any one of the parameters may result in a different equilibrium.

In practice, we do not let equilibrium be reached. This nearly always favors the fibre stain and would generally be undesirable. Instead, the process is interrupted by removing the solution when the desired results are obtained. This time period is one of the standardised factors. Proper staining is obtained by interrupting the progress towards equilibrium at a specified, repeatable point.

One step methods can be adjusted by altering any of several factors. However, this is usually a matter of trial and error. Change only one factor at a time and keep careful records.

In general, it will be found that increasing the time applied will bias the staining in favour of the fibre stain. It may also increase stain intensity. Decreasing the time, however, will bias staining in favour of the plasma stain. Erythrocyte staining is usually less affected. It may also reduce stain intensity.

Increasing the concentration of a dye and keeping the time constant will bias the staining in favour of that dye, while diminishing staining from the other dyes. In the case of the erythrocyte dye, this will often result in yellow tinged or orange cytoplasm. If it is the plasma stain, staining will be more intense in the cytoplasm and may result in some red collagen. It may also intrude into the erythrocytes causing them to stain orange, or be inconsistently coloured. If the fibre stain is increased, it will intrude into cytoplasmic structures, but will not generally affect erythrocyte staining.

Decreasing the concentration of a dye while keeping the time constant will reduce staining by that dye and increase the affect from the other dyes. If the erythrocyte dye is reduced, the plasma stain will invariably stain the erythrocytes to some degree, even obscuring staining by the erythrocyte dye completely. If the plasma stain is reduced there may be some yellow colouring in some structures such as fibrin or muscle, and there may be some infiltration of the fibre stain into other structures such as cytoplasm and muscle. If reduced too much, a stain of yellow and blue with an occasional red blob results, which is particularly unattractive. Reducing the fibre stain diminishes the staining of collagen, often leaving it with a red tinge.

Altering the amount of other ingredients (type and amount of polyacid, type and concentration of solvent) will also have an impact. These should be carefully evaluated as they can be inconsistent.



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