Related Experiment Videos
Effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented lesions
1Department of Pathology, University of Sydney, Concord Hospital, Sydney, Australia.
Objective:
To investigate the effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented melanocytic lesions.
Study Design:
Twenty banal compound nevi with various grades of pigmentation were bleached by 0.5% and 1% KMnO4 for 0 to 20 minutes and by 10% H2O2 for 24 hours prior to Feulgen staining. DNA microdensitometry was performed by video image analysis to measure the integrated optical density (IOD) in nuclei from nevomelanocytes, lymphocytes and spinous keratinocytes. The DNA index of nevomelanocytes was calculated using spinous keratinocytes as the diploid controls.
Results:
There were significant decreases in IOD (P < .05) in the nuclei of nevomelanocytes, lymphocytes and spinous keratinocytes after treatment with 1% KMnO4 for 5 and 10 minutes, but no significant changes were detected after treatment with 0.5% KMnO4 for 5 and 10 minutes. Severe tissue damage was observed in the Feulgen-stained slides treated with 1% KMnO4 for 15 and 20 minutes and with 10% H2O2 for 24 hours. There was no significant change in DNA index in any bleached sets measured.
Conclusion:
KMnO4 can affect Feulgen-DNA content if used in high concentrations or for long periods of incubation. The DNA index, which is derived from internal controls, is not affected by the bleach procedure.
Insights
Melanin bleaching agents like potassium permanganate (KMnO4) can alter Feulgen-DNA measurements in pigmented lesions. However, the DNA index, using internal controls, remains unaffected by these bleaching procedures.
Area of Science:
- Histopathology
- Molecular Biology
- Dermatopathology
Background:
- Feulgen-DNA microdensitometry is a method used to quantify DNA content in cells.
- Pigmented melanocytic lesions pose challenges for accurate DNA analysis due to melanin interference.
- Melanin bleaching techniques are explored to improve DNA quantification in these lesions.
Purpose of the Study:
- To evaluate the impact of melanin bleaching on Feulgen-DNA microdensitometry.
- To determine if bleaching affects DNA content measurements in nevomelanocytes and surrounding cells.
- To assess the reliability of the DNA index after bleaching procedures.
Main Methods:
- Twenty compound nevi were treated with varying concentrations and durations of potassium permanganate (KMnO4) and hydrogen peroxide (H2O2).
- Feulgen staining was performed, followed by DNA microdensitometry using video image analysis to measure integrated optical density (IOD).
- The DNA index was calculated using spinous keratinocytes as diploid controls.
Main Results:
- High concentrations (1% KMnO4) or prolonged incubation significantly decreased IOD in nevomelanocytes, lymphocytes, and keratinocytes.
- Lower concentrations (0.5% KMnO4) or shorter durations did not significantly alter IOD.
- Severe tissue damage occurred with higher concentrations/longer times; however, the DNA index remained unchanged across all bleached samples.
Conclusions:
- Potassium permanganate can interfere with Feulgen-DNA content measurements at higher concentrations or longer exposure times.
- The DNA index, derived from internal controls, is a robust parameter unaffected by the tested melanin bleaching protocols.
- Careful consideration of bleaching agent concentration and duration is necessary to avoid artifacts in DNA microdensitometry.