Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion.
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 G0/G1 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.
Albro
J
J
Department of Pathology, University of Utah Medical School, Salt Lake City 84132.
Bauer
K D
KD
Hitchcock
C L
CL
Wittwer
C T
CT
D000782:Aneuploidy; D016477:Artifacts; D001344:Autopsy; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D004247:DNA / Q000032:analysis* / Q000235:genetics; D019286:Endopeptidase K; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000648:ultrastructure; D016612:Paraffin Embedding; D010434:Pepsin A / Q000494:pharmacology; D016196:S Phase; D012697:Serine Endopeptidases / Q000378:metabolism*
D005404:Fixatives; D005557:Formaldehyde; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D010434:Pepsin A