Comparison of two methods of mechanical disaggregation of scirrhous breast adenocarcinomas for DNA flow cytometric analysis of whole cells.
Mechanical release of single cell suspensions from solid tumors for DNA analysis by flow cytometry has been shown to be optimal for adenocarcinomas in general. In the breast, many adenocarcinomas are fibrotic or scirrhous, creating difficulty in separating the neoplastic cells from the stroma. The authors have conducted a parallel study in 20 consecutive cases of scirrhous adenocarcinomas of the breast using two mechanical methods of cell disaggregation by: (1) mincing tumor tissue in culture media (MINCE); and (2) scraping the surface of the solid tumor with a scalpel blade and rinsing the blade in culture media (SCRAPE). Both methods were compared with PI staining and quantitation of percent debris and coefficient of variation (CV), as an index of resolution of the G0/G1 tumor peak. Debris was quantitated using Cell-FIT (Becton Dickinson Immunocytometry Systems, San Jose, CA) and Multicycle (Phoenix Flow Systems, San Diego, CA) software programs. With Multicycle, in addition to debris and aggregate determination, an index that included background, aggregates and debris (%BAD) occurring between the boundaries of the tumor G1 and G2 region was evaluated. In the 4 DNA diploid and 16 DNA aneuploid tumors, there was no significant difference in histogram quality measured by CV or amount of debris, aggregates, and %BAD between MINCE and SCRAPE methods. The authors conclude that either method of mechanical disaggregation will produce DNA histograms of comparable quality and degree of resolution. However, the scrape method may be advantageous in the mechanical disaggregation in scirrhous tumors. The scrape method also may be useful in small tumors, where tissue preservation for histology is paramount, and there is insufficient material to separately submit for flow cytometry. Combination of both MINCE and SCRAPE may provide higher cell yields, than using only one of these dissociation techniques. In addition, DNA analysis methods using intact cells obtained with the SCRAPE method result in percent CV values of similar resolution to those reported for methods producing bare nuclear suspensions from fresh tumors.
Torres
F X
FX
Division of Surgical Pathology, Henry Ford Hospital, Detroit, Michigan 48202.
Mackowiak
P G
PG
Brown
R D
RD
Linden
M D
MD
Zarbo
R J
RJ
D002293:Adenocarcinoma, Scirrhous / Q000235:genetics / Q000473:pathology*; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D002449:Cell Aggregation; D002470:Cell Survival; D004273:DNA, Neoplasm / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005260:Female; D005434:Flow Cytometry; D006652:Histological Techniques; D006801:Humans; D011003:Ploidies
D004273:DNA, Neoplasm