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


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