Comparison of fresh/frozen isolation method in DNA preparation of meningiomas for flow cytometry.

ABSTRACT

The level of G2/M-phase can represent tumor grading to discriminate benign from atypical meningiomas using flow cytometric analysis. In this study, we compare two tumor DNA prepared methods using fresh and frozen samples for flow cytometric analysis. The specimens were obtained from tumoral tissues of 28 microsurgically resected meningiomas as approved by the institutional review board. Single-cell suspensions were prepared from fresh and frozen tumor tissues using fresh and frozen isolation methods. The coefficient of variation (CV) of DNA and the level of G2/M-phase were assessed by flow cytometric analysis. For fresh samples prepared using the fresh isolation method, atypical meningiomas had significantly higher G2/M-phase levels than those of benign meningiomas. In contrast, for fresh samples prepared using the frozen isolation method, the levels of G2/M-phase in benign and atypical meningiomas were severely interfered. Benign meningiomas could not be discriminated from atypical meningiomas based on the level of G2/M-phase. Additionally, frozen samples prepared using the frozen isolation method had significantly higher values of G2/M-phase in benign and atypical meningioma than those of fresh samples using fresh isolation method. CV was used to estimate the quality of DNA fixation. The diploid G0/G1 peak determined from fresh samples obtained using the fresh isolation method had a smaller CV than those for frozen samples obtained using the frozen isolation method. DNA prepared from fresh samples obtained using fresh isolation method is more suitable for discriminating benign from atypical meningiomas using flow cytometric analysis.