Optimizing Workflows and Processing of Cytologic Samples for Comprehensive Analysis by Next-Generation Sequencing:...

The value and suitability of cytology specimens for molecular diagnosis has been demonstrated by numerous studies. In practice, however, the success rates vary widely across institutions depending on the disease setting, institutional practices of acquisition, handling/processing, and testing methodologies. As the number of clinically relevant biomarkers continues to increase, more laboratories are turning to next-generation sequencing platforms for testing. Although amplicon-based next-generation sequencing assays, interrogating a limited genomic territory, can be performed with minimal input material, broader-based next-generation sequencing assays have higher DNA input requirements that may not be met if the small tissue samples are not acquired and handled appropriately. We briefly describe some of the process changes we have instituted in our laboratories when handling cytologic material to maximize the tissue available for broad hybrid-capture-based next-generation sequencing assays. Among the key changes established were the consolidation and preservation of previously discarded supernatant material in cytologic samples, the introduction of mineral oil for deparaffinization of cell blocks, and adjustments in the molecular laboratory process and bioinformatics pipelines. We emphasize that even minimal changes can have broad implications for test performance, highlighting the importance of a cohesive group-based approach among clinical, cytopathology, surgical pathology, molecular, and bioinformatics teams.