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Semiautomation of preparation of fixed paraffin-embedded tissue for DNA analysis
C Cohen1, R A Santoianni, R J Tickman
1Department of Pathology and Laboratory Medicine, Emory University College of Medicine, Atlanta, Georgia 30322.
Abstract:
The usual manual preparation of single-cell suspensions from fixed paraffin-embedded tissue sections for flow cytometric (FCM) DNA ploidy analysis is a time-consuming, labor-intensive technique that requires 70 minutes to deparaffinize and rehydrate 50 microns sections as the initial step. Manual deparaffinization was compared with two semiautomated methods using an automatic slide stainer with either a 70-minute or 35-minute schedule. Samples from 6 normal tissues and 21 tumors (13 diploid and 8 aneuploid) were prepared using all three methods and analyzed by FCM. The mean cell counts in all samples were over 10(6). The DNA indices for the three samples prepared from a given tissue showed no significant differences. Using the 70-minute automation schedule, no aneuploid peaks were lost, and the ratio of G0G1 normal cells to aneuploid tumor cells was maintained. The automation of deparaffinization can thus provide a significant reduction in the labor need to produce single-cell suspensions for FCM; it can be especially helpful when handling large numbers of tumors. At the same time, the automated procedure decreases the exposure to hazardous chemicals and lowers the chance of losing tissue.
Insights
Automating tissue deparaffinization for flow cytometry (FCM) significantly reduces labor and chemical exposure. This method effectively prepares single-cell suspensions for DNA ploidy analysis without losing critical tumor data.
Area of Science:
- Biotechnology
- Oncology
- Cell Biology
Background:
- Manual preparation of single-cell suspensions for flow cytometric DNA ploidy analysis is labor-intensive.
- The process involves a lengthy deparaffinization and rehydration step for paraffin-embedded tissues.
Purpose of the Study:
- To compare manual deparaffinization with semiautomated methods for preparing single-cell suspensions.
- To evaluate the efficiency and accuracy of automated deparaffinization for flow cytometry (FCM) DNA ploidy analysis.
Main Methods:
- Manual deparaffinization was compared against two semiautomated schedules (70-minute and 35-minute) using an automatic slide stainer.
- Single-cell suspensions were prepared from normal tissues and tumors (diploid and aneuploid).
- Samples were analyzed by FCM for DNA ploidy.
Main Results:
- Automated deparaffinization yielded comparable DNA indices to manual methods across all samples.
- No aneuploid peaks were lost using the 70-minute automated schedule.
- The ratio of normal to tumor cells was maintained, indicating accurate sample preparation.
Conclusions:
- Semiautomated deparaffinization significantly reduces labor requirements for single-cell suspension preparation for FCM.
- Automated methods decrease hazardous chemical exposure and minimize tissue loss.
- This automation is particularly beneficial for high-throughput tumor analysis.