Improved dna content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion
✍ Scribed by James Albro; Kenneth D. Bauer; Charles L. Hitchcock; Dr. Carl T. Wittwer
- Book ID
- 102813654
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 638 KB
- Volume
- 14
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
An improved method for the enzymatic digestion of formalin-fixed, paraffin-embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin-digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin-digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average GdG, coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin-treated samples. The apparent mean S-phase (a combination of S-phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin-treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin-fixed, paraffin-embedded liver tissue.