Table 1.
Patient’s characteristics.
Fig 1.
Flowchart of the CTC detection method using an automated cytology-based, microfluidic platform.
(A) Macroscopic view and scanning electron microscope image of the 3D metal (nickel) filter. (B) Overview of the disposable CTC filtration device containing a 3D filter inside. (C–E) Overview of sequential processes of CTC detection including filtration, CTC transfer to a glass slide, and staining. (C) Appearance of the multi (four)-channel automated CTC enrichment apparatus with a monitor panel. (D) Transfer of tumor cells (COLM-5) from the 3D filter to a glass slide by brief centrifugation. (E) Resultant transferred tumor cells on a glass slide after centrifugation visualized by Pap staining (right column). Inset: enlarged view of Pap-stained tumor cells. Bar = 20 μm.
Fig 2.
Comparison of the flow rate and cell morphology between manual and automated filtration apparatuses.
(A) Effect of flow rate on the morphology of COLM-5 tumor cells transferred to glass slides and stained with Pap. (B) Changes in the percentage of tumor cells with healthy morphology transferred to glass slides with increasing flow rate. P<0.01 (2 ml/min vs 5, 10 ml/min). NS: not significant. Bars = standard deviation (SD). (C) Changes in parameters such as pressure, flow rate, and velocity at each measurement point with a decreasing aperture ratio. Comparison of the manual device with a syringe pump and the automated apparatus with a pressure control system. A = automated, M = manual. (D) Overview of the manual-type microfluidic device using a syringe pump as a control to compare with the automated device. Three measurement points are shown. Schematic representations of each parameter are also shown in the right lower. Gray circles = leukocytes, Black circle = CTC.
Fig 3.
Identification of CTCs in peripheral blood (PB) and draining venous blood (DVB) from CRC patients.
(A) Representative CTCs in DVB and PB from the same patient detected by pan-cytokeratin ICC and Pap staining with separate specimens. (B) Representative CTCs in DVB and PB stained by combined Pap and pan-cytokeratin ICC on the same specimen. De-stained Pap slide used for subsequent ICC. (C) Representative CTCs in DVB and PB stained by combined Pap and triple IF (Alexa Fluor 488-cytokeratin/Alexa Fluor 568-CD45/Hoechst 33342) on the same specimen. Bar = 10 μm.
Fig 4.
Differentiation of CTCs from circulating non-neoplastic cells in DVB and PB from CRC patients.
(A) Leukocyte cluster stained for Pap/CD45 and a macrophage stained for Pap/CD68. (B) Circulating megakaryocytes in PB stained for Pap/CD61, showing multinucleated giant cell morphology. (C) Circulating endothelial cells in DVB stained for Pap/CD34, showing cell cluster formation. Bar = 10 μm.
Fig 5.
Collection of DVB and numbers of CTCs in DVB and PB from CRC patients.
(A) DVB was obtained by puncture of the main trunk of the mesenteric vein of the resected colon. Enlarged view of mesenteric vein (right). (B) Variations in DVB volumes collected. (C) Numbers of CTCs in PB from CRC patients (n = 26) and healthy volunteers (n = 14). p<0.05. (D) Numbers of CTCs in PB and DVB from individual CRC patients (n = 26). p<0.01.
Fig 6.
Numbers of CTCs in PB and DVB from CRC patients in relation to the tumor stage (I–IV).
(A) Representative CTCs stained by Pap and cytokeratin ICC from patients with stage I (upper) and stage II–IV CRC (lower). Bar = 10 μm. (B) Numbers of CTCs in PB and DVB from CRC patients according to stage. p<0.05 (Stage I vs Stage II–IV) for DVB. NS (= not significant) for PB. Bars = standard deviation (SD).