Fig 1.
Outline of the filtration device and its components.
The device is composed of a filtration unit (A, individual components; B, assembled device; C, after filtration) and a water-tight storage cassette (D, individual components; E, assembled cassette).
Fig 2.
Capture of tumor cells from fluid by filtration.
PBS (100 ml) containing between 1,000 and 5 × 106 639V cells was processed using a device mounted with an 8-μm pore size polycarbonate membrane filter. DNA was extracted from the filters and tested for mutant FGFR3 (p.R248C) molecules using ddPCR. DNA from normal peripheral blood lymphocytes was used as a control for wild type FGFR3. A, ddPCR fluorescence amplitude plot. Vertical lines represent manually set cutoff settings. The results shown are from one of two independent experiments. B, Bar chart showing the number of recovered cells (calculated on the basis of mutant FGFR3 molecules) relative to number of input cells. Total counts of mutant molecules were calculated on the basis of three independent ddPCR tests, each measuring mutant molecules in 4% of the total DNA sample. Data of triplicate measurements (each on 4% of total DNA) from one experiment are represented as means ± SD. Percentages above bars represent the number of recovered cells relative to the number of input cells.
Fig 3.
Filtration-based enrichment of tumor cells on a background of normal lymphocytes.
Between 1,000 and 50,000 639V cells were spiked into 107 normal lymphocytes in 100 ml of PBS, and the suspension was processed using the filtration device. DNA was extracted from the filters and tested for mutant (p.R248C; mut) and wild type (WT) FGFR3 using ddPCR. A, ddPCR fluorescence amplitude plots of FGFR3 R248C-FAM probe fluorescence signal (blue, upper panel) and FGFR3 WT-HEX probe fluorescence signal (green, lower panel). Vertical lines represent manually set cutoff settings. The results shown are from one of two independent experiments. B, Bar chart showing the number of recovered cells (calculated on the basis of mutant and WT FGFR3 molecules) relative to number of input tumor cells. Total counts of FGFR3 molecules were calculated on the basis of three independent ddPCR tests, each using 4% of total DNA as template, and are represented as means ± SD. Percentages above the bars represent the number of recovered cells relative to the number of input cells.
Table 1.
Demographic characteristics and molecular markers in urinary cells from patients with transitional cell bladder tumor in TURBT specimens.
Table 2.
Demographic characteristics and molecular markers in urinary cells from patients with benign bladder histology.
Fig 4.
Enrichment of bladder tumor cells from urine.
Urine samples from patients with bladder tumors were divided into two fractions and processed by device filtration and sedimentation, respectively. Equimolar amounts of DNA from filters and sediments were tested for FGFR3 mutations using ddPCR.
Table 3.
Fractions of mutant (Mut) and wild type (WT) FGFR3 in urinary cells from paired samples processed by device filtration and sedimentation, respectively.