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Histotechnology and Diagnostic Cytology

FORMALIN PIGMENT

Formalin pigment has been a nuisance to histotechnologists ever since formalin was first introduced as a fixative in 1893 (J.Histotechn 6(4):181-184). This artifact usually occurs in blood containing tissues (eg spleen) regardless of whether the fixative is buffered or not. FAA (formalin-alcohol, acetic acid) is becoming more popular as a fixative because of its enhancement of nuclear staining but as a result of its use the inevitable formalin pigmentation is rife.

Winsome Garvey (J.Histotechnol. 13(3):163-165, 1991) has suggested three methods for its removal:

1. Treat hydrated sections in 8% ethanoic picric acid for 20 minutes followed by thorough washing to remove the yellow colouration from the section. Dipping the slides in saturated lithium carbonate will often hasten the decolourisation process. Failure to fully remove the picric acid may result in overoxidation and premature discarding of the haematoxylin solution.
2. Treat hydrated sections with 10% ammoniacal ethanol (10ml 28% ammonium hydroxide in 90ml 95% ethanol) for 10 minutes followed by thorough washing. Incomplete washing can impair eosin and phloxine staining. Tseung (Am.J.Med.Techn. 49(6):435-436) recommend 30 minutes treatment in a solution of 30ml acetone and 9ml 28% ammonium hydroxide.
3. Acidified Phenol/ethanol (APE). Place sections in a solution of: Phenol 5ml, HCl (Concent) 5ml, Ethanol (Absolute) 90ml.

Pigment removal should take 5 minutes, though an hour may be required for heavily pigmented cases.

Garvey prefers APE since phenol is a quicker than picric acid and with the addition of hydrochloric acid, enhancement of nuclear staining occurs. The risk of overoxidation of the haematoxylin solution is eliminated and there is an improved uptake of acid dyes.

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