Cytological preparations for molecular pathology

Many biomarkers have been investigated using molecular assays and study designs that are dependent on the availability of tissue/cells. As described by Knoepp and Roh, archival stained smears are a cost-effective means of storing cytological material for molecular analysis. We agree that good-quality DNA can be harvested from a variety of cytological preparations. However, we want to highlight some advantages of using FTA R cards, which were not mentioned in the commentary. Simple storage methods make FTA R cards an ideal solution for the long-term storage of cytological material and their minimal requirements for safe transportation facilitate the transfer of the material over long distances and allow for multiinstitutional collaborations. Archival slides always suffer from the potential for slide breakage and require safer packing and handling procedures. Furthermore, differences in staining protocols might affect subsequent assays to a greater or lesser extent. The authors also recommend the use of unstained smears. We are not aware of any studies concerning the quality of the DNA extracted after long-term storage of unstained slides, but we predict that environmental conditions (heat and humidity) may potentially have an impact on DNA extraction/integrity. Analysis of a stained cytospin preparation produced from the same cell suspension of the needle rinse stored on the card may obviate the concern expressed in their commentary that the composition of the cellular material stored on FTA R cards is unknown. DNA extracted from the cards was successfully amplified, yielding amplicons of 1.5 kilobases. Furthermore, a sufficient amount of DNA to perform multiplex mutation analysis was obtained from only two 3-mm punches of each card although, potentially, 27 punches could be obtained. Recently, we conducted a study that included archival Romanowsky-stained smears, unstained alcohol-fixed cytospin preparations, frozen tissue, paraffin tissue blocks, and FTA R cards. Using a multiplex high-throughput sequencing platform, the percentage of successful results was similar using FTA R cards and frozen tissue. Although the number of cases was small for the other types of preparations, the success rates for archival stained smears and cytospin preparations were considerably lower. As cytopathologists, we should develop institutional policies for the use of the cytological material available that are dictated by the purpose of the analysis, the resources available at the point of collection, the amount of material needed, and the number/variety of tests that can potentially be performed. Although the development of multiple algorithms/protocols will be an additional challenge, it will eventually translate to “personalized cytopathology” for personalized medicine.