A method for detecting variability arising from errors in sample processing of paraffin-embedded tissue for DNA content analysis.

Abstract

We present a method for controlling variability that may arise from inconsistencies in sample preparation for DNA content analysis of paraffin-embedded tissue. Human tonsil tissue obtained from routine surgical specimens was embedded in paraffin according to standard protocols. Fifty-micrometer sections were cut from the block and analyzed each day for 20 days to establish control ranges. One tonsil tissue section was processed in parallel with each run of clinical specimens. In this context, a run was defined as the simultaneous processing of 50-microns tissue sections for extraction of cell nuclei (dewaxing and rehydrating). If the tonsil G0/G1 peak coefficient of variation (CV) exceeded 2 SDs of the established mean, and optimum instrument performance and staining were verified, all samples prepared with the tonsil control were reprocessed. Instrument performance and staining were assessed by using the appropriate external controls. By using this rejection rule (12s), the frequency of sample reprocessing in our laboratory was approximately 6%. When the run was repeated and the tonsil control CV was within acceptable range, the G0/G1 peak CV of the corresponding clinical specimens improved 25% of the time. Because most investigators are willing to accept higher CVs for paraffin-embedded tissue than for fresh tissue, it is desirable to have a control to detect decreased peak resolution, resulting from errors in sample processing.