Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis.
OBJECTIVE
To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis.


METHODS
For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous.


RESULTS
Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern.


CONCLUSIONS
Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.
Sato
T
T
Department of Medical Technology, School of Allied Medical Sciences, Hirosaki University, Japan.
Chiba
M
M
Kamiyama
T
T
Miura
T
T
Kanagawa
Y
Y
Nakano
K
K
Sato
T
T
Ishikawa
T
T
Nozaka
H
H
Kogawa
R
R
Yasujima
M
M
D000818:Animals; D001940:Breast / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology / Q000648:ultrastructure; D002277:Carcinoma / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure; D002467:Cell Nucleus / Q000235:genetics*; D003593:Cytoplasm; D004273:DNA, Neoplasm / Q000032:analysis*; D004847:Epithelial Cells / Q000473:pathology / Q000648:ultrastructure; D005260:Female; D018226:Fibroadenoma / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005629:Frozen Sections / Q000592:standards; D006801:Humans; D008099:Liver / Q000166:cytology / Q000648:ultrastructure; D008297:Male; D008853:Microscopy; D003557:Phyllodes Tumor / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure; D011003:Ploidies; D051381:Rats; D011916:Rats, Inbred F344; D013194:Staining and Labeling; D017154:Stromal Cells / Q000473:pathology / Q000648:ultrastructure; D016707:Tissue Fixation
D004273:DNA, Neoplasm