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Polyacids & Displacement

in Trichrome Staining

Displacement is the term used to describe the phenomenon of one charged group being able to replace another similarly charged group in chemical reactions. In practical histological terms, it refers to the ability to replace a dye already attached to tissue groups by another dye which is similarly charged. In other words, within the context of trichrome staining, using one acid dye to replace another on tissue amino groups.

Polyacids are high molecular weight compounds such as tungstophosphoric (phosphotungstic) acid and molybdophosphoric (phosphomolybdic) acid. Most trichrome methods use these in some way or other. There are exceptions, however. The commonest method, van Gieson’s stain, does not use a polyacid at all. The strongly acidic picric acid provides what acidity is required. There is also at least one variation of Lendrum’s Picro-Mallory stain which uses trichloracetic acid rather than one of the acids above. It should be noted that trichloracetic acid differs from the other two in that it does not contain any metal atoms.

Their use is integral to trichrome staining methods, although there is some disagreement as to how they function. The most usual explanation is that they function as if they were very high molecular weight acid dyes. On this basis, they would displace other acid dyes already attached to tissues. Due to the high molecular weight, and the inferred large molecule size, the displacement would first involve the most easily accessible structures such as collagen. While other tissues could be involved, a longer time would be needed for those structures to be affected. This approach envisages that the dye attached to the collagen is removed and replaced by the polyacid. When the next dye is applied it, in turn, replaces the polyacid. This approach results in a sharper contrast between the cytoplasmic staining dye and the collagen staining dye.

The alternate explanation is similar, but the dye applied following the polyacid is considered to link to the tungsten or molybdenum in the tissue rather than to replace it. In this respect it is thought to be similar to, but not the same as, mordant dyeing. This explanation is based on the observation that sections stained with trichrome methods using polyacids can be shown to have tungsten or molybdenum atoms still present in those structures after they have been stained with the subsequent dye.

The first explanation, however, explains why the use of chemicals such as trichloracetic acid can differentiate acid dyes by replacing them. Since this chemical does not have metal atoms it could not participate in a metal-dye complex such as envisioned with the tungsten or molybdenum of the polyacids. It should also be noted that in a mass action approach reactions are not permitted to go to completion, so some residual metal atoms from the polyacids should be expected.

Personally, I prefer the first explanation but do not exclude the second.