The work presented here demonstrates a novel technique for epithelial cell capture from urine samples for slide-based analysis. In particular, we demonstrated a slide configuration that can capture and isolate scarce urothelial cells found in human urine, even in the presence of contaminating blood cells. Importantly, the slides are compatible with Papanicolaou staining for urine cytology and can be used with ICC for detecting cancer marker expression. Thus, our peptide-conjugated slide technology represents a powerful new technique for preparing a purified cell population for diagnostic testing.
D.B.W., W.D.C., N.L., M.K., D.J., and J.C.M. conceived and developed the experiments presented here and analyzed the resulting data. M.K., M.D., and S.A.N. established protocols for peptide conjugation to glass. M.K. conjugated the peptides to the slides and performed peptide density quantification. N.L. and D.J. per formed cell capture assays. J.C.M. and N.L. performed cytology staining and ICC experiments. W.D.C. performed phage display and isolated the epithelial cell binding peptides. S.A.N. was responsible for peptide design and manufacturing. D.B.W. provided leadership to the project. C.M.H. wrote and revised the manuscript, and all authors read and approved the text.
We acknowledge William Siesser for his role in developing the initial ideas for peptide-conjugated glass, Jonathan White, Yuchen Chen, and Phil Hamilton for synthesis and purification of the cell binding peptides, Wesley Storm for his help editing the manuscript, and Bruce Lamb for his guidance as head of the R&D group at Affinergy.
All authors were employees of Affinergy at the time this work was completed. The peptides and methods described here are contained in an unpublished patent application filed by Affinergy. There are no marketed products to declare.
Correspondence Address correspondence to Martyn Darby, Affinergy, LLC, Research Triangle Park, NC. E-mail: [email protected]">[email protected]References
1.) Siegel, R., J. Ma, Z. Zou, and A. Jemal. 2014. Cancer statistics, 2014. CA Cancer J. Clin. 64:9-29.
2.) Canfield, S.E., C.P. Dinney, and M.J. Droller. 2003. Surveillance and management of recurrence for upper tract transitional cell carcinoma. Urol. Clin. North Am. 30:791-802.
3.) Kipp, B.R., M.B. Campion, E. Coffman, A. Smith, J.D. Tomisek, G.G. Browne, J.R. Panella, R. Desai. 2006. An evaluation of ThinPrep UroCyte filters for the preparation of slides for fluorescence in situ hybridization. Diagn. Cytopathol. 34:479-484.
4.) Botteman, M.F., C.L. Pashos, A. Redaelli, B. Laskin, and R. Hauser. 2003. The health economics of bladder cancer: a comprehensive review of the published literature. Pharmacoeconomics 21:1315-1330.
5.) Lotan, Y., and C.G. Roehrborn. 2003. Sensitivity and specificity of commonly available bladder tumor markers versus cytology: results of a comprehensive literature review and meta-analyses. Urology discussion 118 61:109-118.
6.) Mitra, A.P., and R.J. Cote. 2010. Molecular screening for bladder cancer: progress and potential. Nat Rev Urol. 7:11-20.
7.) Yoder, B.J., M. Skacel, R. Hedgepeth, D. Babineau, J.C. Ulchaker, L.S. Liou, J.A. Brainard, C.V. Biscwotti. 2007. Reflex UroVysion testing of bladder cancer surveillance patients with equivocal or negative urine cytology: a prospective study with focus on the natural history of anticipatory positive findings. Am. J. Clin. Pathol. 127:295-301.
8.) Wright, R.G., and J.A. Halford. 2001. Evaluation of thin-layer methods in urine cytology. Cytopathology 12:306-313.
9.) Nassar, H., R. Ali-Fehmi, and S. Madan. 2003. Use of ThinPrep monolayer technique and cytospin preparation in urine cytology: a comparative analysis. Diagn. Cytopathol. 28:115-118.
10.) Voss, J.S., B.R. Kipp, A.K. Krueger, A.C. Clayton, K.C. Halling, R.J. Karnes, M.R. Henry, and T.J. Sebo. 2008. Changes in specimen preparation method may impact urine cytologic evaluation. Am. J. Clin. Pathol. 130:428-433.
11.) Caraway, N.P., A. Khanna, R.L. Fernandez, L. Payne, R.L. Bassett, H.Z. Zhang, A. Kamat, and R.L. Katz. 2010. Fluorescence in situ hybridization for detecting urothelial carcinoma: a clinicopathologic study. Cancer Cytopathol. 118:259-268.
12.) Sullivan, P.S., J.B. Chan, M.R. Levin, and J. Rao. 2010. Urine cytology and adjunct markers for detection and surveillance of bladder cancer. Am J Transl Res. 2:412-440.
13.) Mitra, A.P. 2010.Urine cytologic analysis: special techniques for bladder cancer detection. Dako, Carpinteria, CA.
14.) Têtu, B. 2009. Diagnosis of urothelial carcinoma from urine. Mod. Pathol. 22:S53-S59.
15.) Elsheikh, T.M., J.L. Kirkpatrick, and H.H. Wu. 2006. Comparison of ThinPrep and cytospin preparations in the evaluation of exfoliative cytology specimens. Cancer 108:144-149.
16.) Luthra, U.K., P. Dey, J. George, M.A. Abdulla, A.A. Shaheen, Z.A. Sheikh, and S.S. George. 1999. Comparison of ThinPrep and conventional preparations: urine cytology evaluation. Diagn. Cytopathol. 21:364-366.
17.) Raab, S.S., D.M. Grzybicki, C.M. Vrbin, and K.R. Geisinger. 2007. Urine cytology discrepancies frequency, causes, and outcomes. Am. J. Clin. Pathol. 127:946-953.
18.) Siegel, D.L.. Cell-surface selection and analysis of monoclonal antibodies from phage libraries.
19.) Fields, G.B., and R.L. Noble. 1990. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161-214.
20.) Wang, C.-J., Z.-G. Zhou, A. Holmqvist, H. Zhang, Y. Li, G. Adell, and X.-F. Sun. 2009. Survivin expression quantified by Image Pro-Plus compared with visual assessment. Appl. Immunohistochem. Mol. Morphol. 17:530-535.
21.) Raab, S.S., C.H. Stone, E.M. Wojcik, K.R. Geisinger, L. Dahmoush, F.U. Garcia, D.M. Grzybicki, J.E. Janosky. 2006. Use of a new method in reaching consensus on the cause of cytologic-histologic correlation discrepancy. Am. J. Clin. Pathol. 126:836-842.
23.) Halling, K.C., W. King, I.A. Sokolova, R.G. Meyer, H.M. Burkhardt, A.C. Halling, J.C. Cheville, T.J. Sebo. 2000. A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J. Urol. 164:1768-1775.
24.) Comploj, E., C. Mian, A. Ambrosini-Spaltro, C. Dechet, S. Palermo, E. Trenti, M. Lodde, W. Horninger. 2013. uCyt+/ImmunoCyt and cytology in the detection of urothelial carcinoma. Cancer cytopathology 121:392-397.
25.) Têtu, B., R. Tiguert, F. Harel, and Y. Fradet. 2005. ImmunoCyt/uCyt+ improves the sensitivity of urine cytology in patients followed for urothelial carcinoma. Mod. Pathol. 18:83-89.
26.) Rödel, F., T. Sprenger, B. Kaina, T. Liersch, C. Rodel, S. Fulda, and S. Hehlgans. 2012. Survivin as a prognostic/predictive marker and molecular target in cancer therapy. Curr. Med. Chem. 19:3679-3688.