Formalin Pigment

Formalin pigment is a brown, granular, doubly refractile deposit seen both intracellularly and extracellularly in tissues which have been fixed with a simple formalin solution, such as formal-saline. It is also known as acid formaldehyde hematin, as it is formed from hemoglobin by the action of formaldehyde at acid pH. The hematin being referred to in this context is the derivative of hemoglobin and not the oxidation product of hematoxylin, called hematein.

As it is formed from hemoglobin it is more likely to be encountered with bloody specimens, and simple formalin solutions can produce it in quite short periods. It also tends to form in tissues stored for extended periods and in which the formalin solution is not changed regularly.

Formation of this artifact can be largely avoided by using a fixative such as 10% neutral buffered formalin. Formalin pigment is produced in an acid pH, so buffering the fixative to neutrality or slight alkalinity effectively stops it from forming. This is not absolute, however, and it is still sometimes encountered in tissues fixed with 10% NBF if the tissues are very bloody or kept for months in fixative.

In small amounts the pigment does not interfere with slide examination, but large amounts can be distracting. It may be removed from sections prior to staining quite easily by immersing in ethanolic picric acid, a method given below. It may also be removed with alkalis such as 1% sodium hydroxide in absolute ethanol, but these very frequently cause sections to detach from slides and should be avoided.

 

Removing formalin pigment

1. Bring sections to water via xylene and ethanol.
2. Place into saturated picric acid in absolute ethanol for 1 hour.
3. Optionally, treat with saturated aqueous lithium carbonate to remove picric acid discolouration.
4. Wash well with water.
5. Continue with the staining method.

 

Note

1. Most formalin pigment will be removed fairly rapidly, and the time given should be adequate.
2. Heavy deposits may take longer. Sections may be left overnight if necessary.
3. Treatment with lithium carbonate is only required if picric acid discolouration interferes with staining.

 

Reference

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.