Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry.
Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.
Gonchoroff
N J
NJ
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905.
Ryan
J J
JJ
Kimlinger
T K
TK
Witzig
T E
TE
Greipp
P R
PR
Meyer
J S
JS
Katzmann
J A
JA
D001943:Breast Neoplasms / Q000032:analysis / Q000235:genetics*; D002277:Carcinoma / Q000032:analysis / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D010232:Paraffin; D011003:Ploidies; D013010:Sonication; D014465:Ultrasonics
D004273:DNA, Neoplasm; D010232:Paraffin; D004247:DNA