KRAS mutation analysis using FNA residual tissues via the “water-burst” sample preparation method. (A) Workflow for sample collection and DNA preparation. After submitting patient specimens obtained via FNA for cytopathological diagnosis, residual specimens and the needle washing solution were collected, centrifuged, and separated into a pellet and supernatant. DNA was prepared by the “water-burst” method, in which the precipitate was centrifuged and suspended in water, and dPCR analysis was then performed. The supernatant was directly subjected to the dPCR reaction. These methods do not require DNA purification, and it takes about 2.5 h to obtain genetic information after the collection of a sample. Purified DNA was also subjected to the assay as control (see Table 2). (B) DPCR plot of the KRAS G12/G13 mutation assay in the collected tissues were resuspended using water (left large panels). The plot graph shows the pattern of detection of KRAS tissues obtained via centrifugation from FNA residual tissues or needle rinsed fluids. The threshold (solid pink line) was manually set to extend to an amplitude of 2,000 or 1,000 (FAM mutant or HEX wild-type probe) above the maximum background intensity value. The asterisk indicates the results for the KRAS Q61 mutation assay for patient 9 (right small panels; see Table 2).