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Peptide-conjugated glass slides for selective capture and purification of diagnostic cells: Applications in urine cytology
 
Danuta B. Wronska, Magdalena Krajewska, Natalia Lygina, Juhua C. Morrison, Dalia Juzumiene, W. David Culp, Shrikumar A. Nair, Martyn Darby, and Christopher M. Hofmann
Affinergy, LLC, Research Triangle Park, NC
BioTechniques, Vol. 57, No. 2, August 2014, pp. 63–71
Full Text (PDF)
Abstract

Obtaining a clear view of the cells of interest in diagnostic cytology can be challenging when specimens are contaminated with blood or other obscuring cells. In this study, we present a powerful technique for the selective capture of diagnostic epithelial cells directly on a microscope slide, highlighting its applications in urine cytology and immunocytochemistry (ICC). Using phage-display biopanning, we identified and synthesized a series of peptides that bind with high affinity to urothelial cells but not blood cells. We developed methods for conjugating the peptides to glass slides, and we used these slides to selectively capture both normal and cancerous epithelial cells from urine contaminated with blood cells. Unlike non-selective microscope slides, the peptide-conjugated slides selectively retained the cells of interest, recovering up to 75% of urothelial cells, while up to 98% of blood cells were washed away. The slides are compatible with Papanicolaou and hematoxylin and eosin (H&E) staining for cytology preparations, as well as ICC for detecting membrane-associated and nuclear cancer markers. We successfully detected the expression of carcinoembryonic antigen and survivin, two commonly measured bladder cancer markers. In addition to bladder cancer diagnostics, this technology has broad applications for increasing the quality of sample preparations in slide-based diagnostic testing.

Nearly 75,000 new cases of bladder cancer will be diagnosed in the United States in 2014 (1). Although the majority of urothelial carcinomas are low-grade, the disease has a high rate of recurrence (70%) and a 10%–30% rate of progression to high-grade lesions. Thus, bladder cancer requires lifelong surveillance (2, 3), making it one of the most costly per patient of all cancers. The total estimated annual expenditure for bladder cancer diagnosis, treatment, and surveillance in the United States is $3.7B, with total costs from diagnosis to death of $96,000–$187,000 per patient (3, 4).

Following diagnosis and treatment, bladder cancer patients are monitored with urine cytology and cystoscopy every three months for two years, with decreasing frequency but life-long surveillance thereafter (2, 5). In addition to cytology, ancillary diagnostic techniques such as the immunocytochemistry (ICC)-based test ImmunoCyt and the fluorescent in situ hybridization (FISH) test UroVysion are also used (6). In all cases, the primary cells of interest are epithelial cells that have been shed into the urine (7). However, as hematuria is the most common presenting symptom for bladder cancer, cytological interpretation can be more difficult if red and white blood cells (RBCs and WBCs) obscure the urinary epithelial cells (8-10). Similarly, ImmunoCyt and UroVysion have lower specificity and sensitivity, respectively, when sample preparations contain obscuring RBCs or WBCs (6, 11-14).

METHOD SUMMARY

We developed a new technique for the selective capture of both normal and cancerous epithelial cells directly on a microscope slide. Unlike non-selective microscope slides, our slides retain only the cells of interest while obscuring or unwanted cells are washed away. This technology has broad application in diagnostic cytology and slide-based immunocytochemistry (ICC), where contaminating cells often obscure the epithelial cells of interest.

Prior to analysis, urine of ten undergoes significant processing to remove RBCs, WBCs, and proteinaceous debris. Sample processing typically involves lysis of RBCs, followed by Cytospin or ThinPrep to deposit the cells on a microscope slide. With Cytospin, depositing the appropriate number of cells on the slide can be challenging; both too few and too many cells can limit the interpretation of cytology, ICC, and FISH. In these cases, the sample must be diluted or concentrated and prepared again, thus increasing time and cost (3). ThinPrep has the advantage of being less labor intensive and reducing the number of WBCs and amount of proteinaceous debris on the slide (8-10)(15, 16). However, ThinPrep reduces the morphological detail of stained cells and increases the occurrence of artifacts introduced by air-drying (9). As a result, slides prepared by ThinPrep have an increased frequency of atypical, inconclusive results (8-10). For urine specimens that remain atypical or bloody following processing, improved methods of slide preparation could ultimately increase the accuracy of clinical diagnoses for these patients (17).

In this study, we present a method for the selective capture of epithelial cells directly on microscope slides. Specifically, we have identified a series of peptides that bind with high affinity to epithelial cells but not blood cells, and we have developed methods for covalently attaching the peptides to glass slides. These peptide-conjugated slides can be used to selectively capture epithelial cells from a mixed population of cells, providing a new method for preparing urine specimens for analysis by cytology and ICC.

Materials and methods

J82 cells (HTB-1) and T24 cells (HTB-4) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Both the T24 and J82 cell lines display abnormal karyotypes and are derived from high grade bladder cancer patients with transitional cell carcinoma. Human whole blood was obtained from ZenBio (Research Triangle Park, NC). Specific supplies included streptavidin microbeads and LS columns (Miltenyi Biotec, Auburn, CA), NHS-derivatized glass slides (75.6 mm × 25.0 mm) and deactivation solution (MicroSurfaces, Inc, Englewood, NJ), ProPlate Trays (Electron Microscopy Sciences, Hatfield, PA), CellTracker dyes (Life Technologies, Grand Island, NY), N-α-Fmoc amino acids (Novabiochem, Merck KGaA, Darmstadt, Germany), positively charged glass slides (IMEB, Inc, San Marcos, CA), anti-CEA antibody (Abcam, Cambridge, MA), and anti-survivin antibody (Cell Signaling Technology, Danvers, MA).

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