Minimizing delays in DNA retrieval: The “freezer method” for glass coverslip removal. Letter to the editor regarding comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas
Bibliographic record
Abstract
Removing the glass coverslips for retrieving DNA from archival slides can be time-consuming, as pointed out by Khode et al.1 Traditionally, xylene has been used as the solvent for glass coverslip removal, which can take several days and thereby impose delays in molecular assays. We want to bring their attention to a method that applies "freezing" to old stained slides that can overcome this problem. The method was described for slides requiring restaining or destaining,2 but we have been using it in projects that involved molecular analysis, shortening the time required for getting the slides ready for DNA extraction. According to the "freezer method,"2 the slide should be placed flat in the freezer as the initial step. Although the original recommendation described a maximum exposure of 10 minutes to one hour, in our experience slides can rest in the freezer (at a temperature of −20°C) for only 1 to 2 minutes. After removing the slide from the freezer and wearing eye protection, immediately place the tip of a scalpel blade under one corner edge of the coverslip, lift up the coverslip, and remove it. The slide should be still frozen/cold when using the blade, otherwise the technique will not work. If the coverslip fails to lift off, return the slide to the freezer for an additional minute. If the coverslip still does not lift off during that period of time, it may indicate that the mounting medium was applied too recently. After the coverslip has been removed, allow the now uncoverslipped slide to return to room temperature before soaking it in xylene for 1 or 2 minutes until the remaining mounting media has been removed. The slide can be then sent for manual or laser capture microdissection for collecting cells for DNA extraction. Similar approaches have described how to remove coverslips from Araldite-mounted preparations using an ice block on the surface of the coverslip and liquid nitrogen.3 Apparently the basic principle consists of the differences in freezing between the glass slide and the mounting media. Archival stained smears and cytospin preparations are a precious source of DNA for molecular analysis and in some cases might be the only resource for detecting molecular abnormalities such as mutations in epidermal growth factor receptor (EGFR), BRAF, and KRAS genes to direct targeted therapies.4, 5 We anticipate the broad use of archival slides in studies involving cases with exhausted histological material or those with only cytological specimens as a source for DNA. The "freezer method" for coverslip removal can facilitate the preparation of these samples. The authors made no disclosures. Gilda da Cunha Santos, MD, PhD, FRCPC, FIAC1,2 Monica Schroder, MLT2 Julie Baoqian Zhu, MSc, MLT, SCT(ASCP)2 Mauro Ajaj Saieg, MD, PhD3 William R. Geddie, MD, FRCPC1,2 Scott L. Boerner, MD, FRCPC1,2 Scott McDonald, BSc, MLT2 1Department of Laboratory Medicine andPathobiologyUniversity of TorontoToronto, Ontario, Canada 2Laboratory Medicine ProgramUniversity Health NetworkToronto, Ontario, Canada 3Department of PathologySanta Casa Medical School Sao PauloSao Paulo, Brazil
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.009 | 0.027 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.002 | 0.004 |
| Scholarly communication | 0.003 | 0.003 |
| Open science | 0.005 | 0.001 |
| Research integrity | 0.016 | 0.023 |
| Insufficient payload (model declined to judge) | 0.003 | 0.004 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".