Introduction

Formalin is a commonly used fixative in histology and pathology laboratories due to its efficacy in preserving tissue morphology and cellular details. However, a well-known drawback of formalin fixation is the formation of formalin pigment, also known as acid formaldehyde hematin (AFH). This artefact can interfere with microscopic examination and lead to diagnostic challenges.

What is Formalin Pigment?

Formalin pigment, or acid formaldehyde hematin, is a dark brown to black granular artifact that can form in tissues fixed with formalin. It is particularly prevalent in blood-rich tissues and arises due to the interaction of formaldehyde with hemoglobin under acidic conditions. This pigment can obscure histological details, complicating the interpretation of tissue sections.

Causes of Formalin Pigment Formation

The formation of formalin pigment is primarily due to:

1. Acidic Conditions: Formalin is often slightly acidic, and this acidity can lead to the production of formalin pigment, especially in blood-rich tissues.
2. Prolonged Fixation: Extended fixation times in formalin can increase the likelihood of pigment formation.
3. Inadequate Buffering: Unbuffered or poorly buffered formalin exacerbates the problem by allowing the pH to drop, promoting pigment formation.

Methods for Removal of Formalin Pigment

While preventing formalin pigment is ideal, removal methods are crucial when the pigment has already formed. Several techniques can effectively remove formalin pigment from tissue sections:

1. Saturated Alcoholic Picric Acid Solution: This is one of the most effective methods. Tissue sections are treated with a saturated solution of picric acid in ethanol, which dissolves the pigment without harming the tissue.Procedure:
   • Deparaffinize and hydrate the tissue sections.
   • Immerse the sections in saturated alcoholic picric acid solution for 30 minutes to 1 hour.
   • Wash the sections thoroughly with ethanol and then water to remove any residual picric acid.
2. Ammonium Hydroxide and Hydrogen Peroxide: This method uses a combination of ammonium hydroxide and hydrogen peroxide to bleach the pigment.Procedure:
   • Prepare a solution of 1% ammonium hydroxide and 3% hydrogen peroxide.
   • Treat the deparaffinized and hydrated sections with this solution for 30 minutes.
   • Rinse thoroughly with water.
3. Sodium Thiosulfate: A solution of 2% sodium thiosulfate can also be used to remove formalin pigment. Procedure:
4. Deparaffinize and hydrate the sections.
5. Immerse the sections in 2% sodium thiosulfate for 30 minutes.
6. Rinse with distilled water.

Prevention of Formalin Pigment Formation

Preventing formalin pigment formation is always preferable to removal. Here are some strategies to minimize or prevent its occurrence:

1. Use Buffered Formalin: Ensure that formalin is properly buffered to a pH of 7.0-7.2. Buffered formalin reduces the likelihood of acid formaldehyde hematin formation.
2. Minimize Fixation Time: Avoid unnecessarily prolonged fixation times. Standard fixation times are usually sufficient to preserve tissue morphology without promoting pigment formation.
3. Adequate Fixative Volume: Use an adequate volume of fixative relative to the tissue volume (usually 10:1) to ensure effective fixation and reduce the risk of pigment formation.
4. Fresh Fixative Solutions: Use fresh fixative solutions whenever possible. Old or reused formalin can become acidic over time, increasing the risk of pigment formation.
5. Rapid Post-Fixation Processing: Process tissues promptly after fixation. Delays in processing can lead to changes in pH and increase the risk of pigment formation.

Conclusion

Formalin pigment formation is a common artifact in histological preparations, but it can be effectively managed and prevented. Understanding the conditions that promote its formation and employing proper fixation techniques are crucial steps. When pigment does occur, several removal methods can restore the clarity of tissue sections, ensuring accurate histopathological evaluation. By following best practices in fixation and processing, the impact of formalin pigment can be minimised, leading to better diagnostic outcomes.

TRY THIS MCQ

Which of the following is false regarding formalin pigment?

Correct answer is D:

Lugol’s iodine can be used to remove mercuric pigments

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