An Automated High-throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood

Overview

Journal Anal Chem

Specialty Chemistry

Date 2013 Feb 8

PMID 23387387

Citations 24

Authors

Mengxia Zhao

Perry G Schiro

Jason S Kuo

Karen M Koehler

Daniel E Sabath

Viorica Popov

Qinghua Feng

Daniel T Chiu

Affiliations

Soon will be listed here.

Abstract

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

Citing Articles

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References

Riethdorf S, Muller V, Zhang L, Rau T, Loibl S, Komor M . Detection and HER2 expression of circulating tumor cells: prospective monitoring in breast cancer patients treated in the neoadjuvant GeparQuattro trial. Clin Cancer Res. 2010; 16(9):2634-45. DOI: 10.1158/1078-0432.CCR-09-2042. View

Nagrath S, Sequist L, Maheswaran S, Bell D, Irimia D, Ulkus L . Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007; 450(7173):1235-9. PMC: 3090667. DOI: 10.1038/nature06385. View

Powell A, Talasaz A, Zhang H, Coram M, Reddy A, Deng G . Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One. 2012; 7(5):e33788. PMC: 3346739. DOI: 10.1371/journal.pone.0033788. View

Jeffries G, Lorenz R, Chiu D . Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation. Anal Chem. 2010; 82(23):9948-54. PMC: 2995829. DOI: 10.1021/ac102173m. View

Kahn H, Presta A, Yang L, Blondal J, Trudeau M, Lickley L . Enumeration of circulating tumor cells in the blood of breast cancer patients after filtration enrichment: correlation with disease stage. Breast Cancer Res Treat. 2004; 86(3):237-47. DOI: 10.1023/B:BREA.0000036897.92513.72. View

Gross H, Verwer B, Houck D, Hoffman R, Recktenwald D . Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7). Proc Natl Acad Sci U S A. 1995; 92(2):537-41. PMC: 42776. DOI: 10.1073/pnas.92.2.537. View

Sieuwerts A, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M . Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst. 2009; 101(1):61-6. PMC: 2639293. DOI: 10.1093/jnci/djn419. View

Mego M, Mani S, Cristofanilli M . Molecular mechanisms of metastasis in breast cancer--clinical applications. Nat Rev Clin Oncol. 2010; 7(12):693-701. DOI: 10.1038/nrclinonc.2010.171. View

Daugherty P, Iverson B, Georgiou G . Flow cytometric screening of cell-based libraries. J Immunol Methods. 2000; 243(1-2):211-27. DOI: 10.1016/s0022-1759(00)00236-2. View

10.

Chaffer C, Weinberg R . A perspective on cancer cell metastasis. Science. 2011; 331(6024):1559-64. DOI: 10.1126/science.1203543. View

11.

Goda K, Ayazi A, Gossett D, Sadasivam J, Lonappan C, Sollier E . High-throughput single-microparticle imaging flow analyzer. Proc Natl Acad Sci U S A. 2012; 109(29):11630-5. PMC: 3406874. DOI: 10.1073/pnas.1204718109. View

12.

Nakagawa T, Martinez S, Goto Y, Koyanagi K, Kitago M, Shingai T . Detection of circulating tumor cells in early-stage breast cancer metastasis to axillary lymph nodes. Clin Cancer Res. 2007; 13(14):4105-10. DOI: 10.1158/1078-0432.CCR-07-0419. View

13.

Schiro P, Kuyper C, Chiu D . Continuous-flow single-molecule CE with high detection efficiency. Electrophoresis. 2007; 28(14):2430-8. DOI: 10.1002/elps.200600730. View

14.

de Bono J, Ashworth A . Translating cancer research into targeted therapeutics. Nature. 2010; 467(7315):543-9. DOI: 10.1038/nature09339. View

15.

Zheng S, Lin H, Liu J, Balic M, Datar R, Cote R . Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. J Chromatogr A. 2007; 1162(2):154-61. DOI: 10.1016/j.chroma.2007.05.064. View

16.

Alunni-Fabbroni M, Sandri M . Circulating tumour cells in clinical practice: Methods of detection and possible characterization. Methods. 2010; 50(4):289-97. DOI: 10.1016/j.ymeth.2010.01.027. View

17.

Allard W, Matera J, Miller M, Repollet M, Connelly M, Rao C . Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004; 10(20):6897-904. DOI: 10.1158/1078-0432.CCR-04-0378. View

18.

Bottcher S, Ritgen M, Pott C, Bruggemann M, Raff T, Stilgenbauer S . Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation. Leukemia. 2004; 18(10):1637-45. DOI: 10.1038/sj.leu.2403478. View

19.

Balic M, Dandachi N, Hofmann G, Samonigg H, Loibner H, Obwaller A . Comparison of two methods for enumerating circulating tumor cells in carcinoma patients. Cytometry B Clin Cytom. 2005; 68(1):25-30. DOI: 10.1002/cyto.b.20065. View

20.

Dharmasiri U, Witek M, Adams A, Soper S . Microsystems for the capture of low-abundance cells. Annu Rev Anal Chem (Palo Alto Calif). 2010; 3:409-31. DOI: 10.1146/annurev.anchem.111808.073610. View