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Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System.

Xu L, Mao X, Imrali A, Syed F, Mutsvangwa K, Berney D, Cathcart P, Hines J, Shamash J, Lu YJ - PLoS ONE (2015)

Bottom Line: The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02).Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively).Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China.

ABSTRACT
Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier "liquid biopsy" than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.

No MeSH data available.


Related in: MedlinePlus

Parsortix system overview and cassette design.(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.
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pone.0138032.g001: Parsortix system overview and cassette design.(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.

Mentions: The Parsortix system (Fig 1A and 1B) utilizes microfluidic based particle separation technology, with a disposable cassette (Fig 1C) containing a separation structure comprising a series of steps across which cells are forced to pass, leading them to a terminal gap of 10 μm. Most blood cells pass across the steps and through the terminal gap and cells whose size and rigidity prevent them from passing through the step structure (e.g. CTCs) are retained in the cassette (Fig 1D).


Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System.

Xu L, Mao X, Imrali A, Syed F, Mutsvangwa K, Berney D, Cathcart P, Hines J, Shamash J, Lu YJ - PLoS ONE (2015)

Parsortix system overview and cassette design.(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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4580600&req=5

pone.0138032.g001: Parsortix system overview and cassette design.(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.
Mentions: The Parsortix system (Fig 1A and 1B) utilizes microfluidic based particle separation technology, with a disposable cassette (Fig 1C) containing a separation structure comprising a series of steps across which cells are forced to pass, leading them to a terminal gap of 10 μm. Most blood cells pass across the steps and through the terminal gap and cells whose size and rigidity prevent them from passing through the step structure (e.g. CTCs) are retained in the cassette (Fig 1D).

Bottom Line: The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02).Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively).Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China.

ABSTRACT
Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier "liquid biopsy" than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.

No MeSH data available.


Related in: MedlinePlus