Representative images for the identified CTCs in parallel study.

<p>(<b>A</b>) Images of single CTCs. The upper row: cells harvested by IsoFlux with beads around cells. The lower row: cells harvested by Parsortix without beads around. (<b>B</b>) A cluster of CTCs (yellow arrow), single CTC (white arrow) and surrounding lymphocytes (arrowhead) were observed in samples harvested by Parsortix. Signals for nucleus, cytokeratin and CD45 were presented separately and then merged together. A CTC is defined as a CK positive/CD45 negative, nucleated and morphologically intact cell.</p

The proportion of PC3 cells expressing epithelial and mesenchymal markers.

<p>The proportion of PC3 cells expressing epithelial and mesenchymal markers.</p

Number of CTCs harvested by different systems in Parallel CTC analysis of prostate cancer patient samples.

<p>-: No data.</p><p>Number of CTCs harvested by different systems in Parallel CTC analysis of prostate cancer patient samples.</p

Parsortix system overview and cassette design.

<p>(A) Overview of Parsortix system. (B) Overview of the clamp holding the cassette where the blood passes through. (C) A diagram of the disposable isolation cassette. (D) Isolation principle inside the cassette. Blood is forced along a series of channels and to flow through a 10 μm patented step which separates particles on the basis of size and compressibility.</p

Spiked cancer cell lines test for capture rates.

<p>(A) Mean capture rate for spiked PC3, DU145 and MCF-7 after isolation. (B) Three repeated measurements for the mean diameter of PC3, DU145, MCF-7 and normal human lymphocytes. (C-E) representative examples of (C) smaller lymphocytes in the process of passing through the gap and exiting Parsortix cassette to the waste, magnification 100X); (D) captured pre-labeled single spiked cell in the stepped gap, magnification 200X; and (E) captured pre-labeled cluster of spiked cells in the stepped gap, magnification 200X.</p

Immunostaining of EpCAM and Vimentin for cultured cells after different cell transfer and fixation methods.

<p>EpCAM was shown as green signals and Vimentin was shown as red signals. Exposure time and magnification of images were the same for each method. Based on the same fixation, EpCAM signals by direct transfer were stronger than those by Cytospin. In addition, the morphology of cells was flattened after cytospin. Based on the same transfer method, paraformaldehyde provided the brightest signals and methanol provided the weakest and uneven signals.</p

Optimization of cell transfer and fixation for immunofluorescence analysis using spiked samples.

<p>(A) Representative images to show a CellTracker Green pre-labeled PC3 cell with damaged morphology (green arrow) after Cytospin (lower panel), compared with a round pre-labeled PC3 cell (green arrow) in direct cell loading method (upper panel). (B) and (C) Using both (B) PBS-4% paraformaldehyde and (C) KCl-acetone cell transfer and fixation methods, CK expression (green signals) on PC3 cells (green arrows) and CD45 expression (red signals) on lymphocytes (red arrows) were clearly detected with strong and specific fluorescence signals. (D) Percentage of cells retained after immunofluorescence staining process for cells with different re-suspension solutions and fixations. Both 70% and pure acetone fixation in combination with KCl re-suspension of cells achieved a retention rate of more than 90%. (E) Harvest rates of spiked PC3 cells using different cell transfer and fixation methods. KCl-acetone had the highest rate, Cytospin had a lower but more stable rate and PBS-4% paraformaldehyde had the lowest and most unstable rate.</p

Cell retention rates for different transfer and fixation combination using 20 μL cell suspension.

<p>Cell retention rates for different transfer and fixation combination using 20 μL cell suspension.</p

Comparison of the number and purity of harvested CTCs among three platforms.

<p><b>(A</b>) Number of harvested CTCs of each patient by different system. (<b>B</b>) CTC purity of the harvested sample of each patient by Parsortix and IsoFlux.</p

Viability of prostate cancer cell lines after isolation.

<p>(A) Pre-labeled cancer cell lines (labeled green) were identified with fluorescence microscope and then viewed in bright light to determine whether they were dyed with Trypan blue. Cells with clear cytoplasm were considered viable. (B) Cancer cells were cultured after isolation. Spiked pre-labeled cells (indicated by arrow) in 24-well plate, which were relative bigger and brighter comparing surrounding lymphocytes, under bright light and fluorescence views of the same area for spiked cells (labeled green) were present. Cancer cells attached to the plate bottom and started to proliferate on Day 4., They had grown well to form obvious clusters of proliferating cells by Day 6.</p