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

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).

Buffers and culture conditions

Printing buffer composition: Peptides were first dissolved in DMSO, followed by addition of 50 mM EDTA (pH 8.0) to a final DMSO content of 20% (DMSO was omitted for peptide EBP-0). Glycerol was then added to a final concentration of 7.3% (v/v). Slide wash buffer consisted of phosphate buffered saline (PBS) with 0.05% Tween 20 (v/v). Cell capture, slide blocking, and wash buffers were formulated as follows: PBS with 0.5% bovine serum albumin (BSA) (w/v) and 2 mM EDTA. ICC blocking buffer consisted of PBS with 2% BSA and 10% goat serum. T24 cells were cultured in McCoy's 5a Modified Medium, and J82 cells were cultured in Eagle's Minimum Essential Medium. Cultures were incubated at 37°C, 5% CO2 and supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.

Phage display

Cell-sur face biopanning of phage-display libraries was done on human urothelial cells lines using magnetically activated cell sorting as previously described (18). In order to identify urothelial cell binding peptides that do not bind WBCs, phage display was carried out in the presence of urothelial cells (T24 or J82) and WBCs (1:9 ratio). Urothelial cells were biotinylated and conjugated to streptavidin magnetic microbeads, and phage libraries were incubated with the mixture of cells. The cell-phage mixture was then added to an LS Column and placed in a magnetic field. After multiple washes to remove the WBC population and loosely bound phage, the column was removed from the magnetic field, and urothelial cells were eluted. Urothelial cell binding phage were amplified overnight in E. coli (XL1-Blue), followed by three additional rounds of biopanning. Using a fluorescently labeled anti-M13 antibody, phage pools from each round were analyzed by fluorescently activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA) to identify enriched cell binding pools. Selected pools were titrated on lawns of E. coli, and individual phage were amplified and screened by FACS. Phage that bound T24 and J82 cells without binding WBCs were selected for DNA sequencing by rolling circle amplification (Sequetech, Mountain View, CA), and peptide sequences were deduced from the resulting DNA sequences.

Peptide synthesis

Peptides (Table 1) were synthesized on a PTI Symphony synthesizer using standard Fmoc chemistry (19). To facilitate unidirectional attachment to NHS-derivatized glass slides, the peptide amino termini and any internal lysine residues were acetylated, and a flexible PEG linker and lysine residue were added to the C terminus. For measurements of peptide density on the slides, peptides were also synthesized with a trypsin-cleavable site near the C terminus. Following synthesis, simultaneous cleavage and side chain deprotection was achieved by treatment with a trifluoroacetic acid (TFA) cocktail. Crude peptide was precipitated with cold diethyl ether and purified by reverse-phase HPLC using a Vydac C18 silica column (10 µm, 120 Å, 250 × 22 mm) with a linear gradient of water/acetonitrile (0.1% TFA). Homogeneity of the purified peptides was evaluated by analytical RP-HPLC, and molecular mass was confirmed by MALDI-TOF-MS.

Table 1. 



Table 1.   (Click to enlarge)



Covalent attachment of peptides to glass slides

Peptides were attached to glass slides using N-hydroxysuccinimide (NHS) chemistry. To create discrete peptide-conjugated regions, the NHS-derivatized glass slides were mounted in a ProPlate tray with 4, 8, or 16 distinct wells (2.67 cm2, 1.13 cm2, or 0.28 cm2 per well, respectively), and peptides were dissolved in printing buffer (0 or 200 µM) and pipetted into each well (946, 396, or 100 µl, respectively). Slides were incubated for 2 h (30°C, 75% relative humidity) then washed 3 times (5 min each) with washing buffer. Unreacted NHS groups were hydrolyzed using deactivation solution.

Peptide density on slides

Peptide density on the slides was quantified by cleaving the peptide with trypsin (5 µg) in ammonium bicarbonate buffer (1.7 mL, 100 mM, pH 8.5). Slides were covered with the trypsin solution, sealed, and incubated for 5 h (37°C with shaking), after which the released peptide fragment was collected and dried in a SpeedVac. The pellet was suspended in water, and peptide was quantified by HPLC using a standard curve prepared from the trypsinized peptide fragment.

Selectivity of peptide-conjugated slides

WBCs (labeled with CellTracker Green) and urothelial cells (J82; labeled with CellTracker Red) were mixed (7:1, 8:1, or 72:1 ratio) and applied to peptide-conjugated slides. Slides without peptide were used as a control. After 15 min, unbound cells were removed by tipping the slide to allow the liquid to drain. Slides were then washed 3 times by immersing in wash buffer, then fixed in 4% paraformaldehyde. The number of each cell type bound was counted manually or by Image-Pro Plus 7.0 software (Media Cybernetics, Rockville, MD) using fluorescence microscopy (QIClick camera, QImaging, Surrey, BC, Canada) with a 10× objective (N = 5 fields per well; N = 2 wells). After determining the final ratio of WBC:J82 on the slides, WBC depletion was calculated as 1 - (final ratio/start ratio).

Sensitivity of peptide-conjugated slides

Peptide EBP-35 was conjugated to slides using a 16-well ProPlate tray (0.28 cm2). J82 cells labeled with CellTracker Red were applied to individual wells (384, 192, 96, or 48 cells; N = 4 wells each). After 15 min, unbound cells were washed away, and captured cells were fixed and counted manually by fluorescence microscopy. A no-peptide control was included for the lowest cell input.

Cytology preparations

For experiments with cancer cell lines, J82 urothelial cells (7.5 × 104 cells) and anti-coagulated whole human blood (100 ml) were spiked into fresh urine (20 µL). Cells were recovered by centrifugation (500 × g, 10 min), suspended in capture buffer, and either added directly to peptide-conjugated slides (EBP-35) and unmodified controls or fixed with 4% paraformaldehyde in suspension, centrifuged, suspended in capture buffer, and smeared on poly-L-lysine slides. After 15 min, peptide-conjugated slides and unmodified controls were rinsed in wash buffer and fixed, and all slides were stained with hematoxylin and eosin (H&E).

For endogenous cell capture experiments, a voided urine specimen (40 mL; second morning void) was obtained from a healthy female donor, and cells were isolated by centrifugation (500 × g, 10 min). The cell pellet was washed in distilled water, centrifuged, suspended in capture buffer, and smeared on poly- L-lysine slides or deposited on peptide-conjugated slides (15 min). The peptide-conjugated slides were washed 3 times in wash buffer, after which all slides were fixed in 4% paraformaldehyde, dried, and subjected to Papanicolaou or H&E staining. Using bright field microscopy, Papanicolaou stained slides were qualitatively observed (40× objective), while the number of small cells (basal and intermediate) and large cells (squamous and superficial) bound to H&E stained slides were manually counted (10× objective).

Immunocytochemistry (ICC)

A suspension of urothelial cells (J82) in capture buffer was applied to peptide-conjugated slides for 15 min, after which slides were washed 3 times in wash buffer to remove unbound cells. As controls, cells were smeared on positively charged slides or poly-L-lysine slides without rinsing. All slides were fixed in 4% paraformaldehyde, dried for 30 min at 37°C, and then washed with distilled water and PBS. For carcinoembryonic antigen (CEA) staining, slides were covered with blocking buffer (1 h at room temperature) then immunostained with rabbit IgG anti-CEA (1:100). For survivin staining, cells were permeabilized with Triton X-100 (0.5% in PBS) for 10 min prior to blocking, then labeled with rabbit monoclonal anti-survivin (1:100). Survivin-labeled cells were also labeled with red fluorescent phalloidin (1:400) to positively identify whole cells. All slides were incubated overnight at 4°C, washed in PBS, labeled with goat anti-rabbit Alexa Fluor 488 (30 min, 37°C), then washed 3 times in PBS. To visualize nuclei, CEA-labeled slides were counterstained with DAPI (200 ng/mL) for 2 min, followed by a final PBS wash. Slides were imaged by fluorescence microscopy (40× objective). For CEA-labeled slides, Image-Pro Plus 7.0 software was used to quantify the CEA expression area in each cell according to published methods (20). Briefly, a color standard was established such that negative controls (no CEA antibody) demonstrated no CEA-positive signal when the standard was applied. The color standard was applied to all slides, and average CEA expression area per cell was calculated by averaging across all analyzed cells for each slide type (8 images per slide, 40× objective).

Results and discussion

Isolation of urothelial cell binding peptides

Phage display was performed against T24 and J82 urothelial cells with 10 different phage libraries, representing over 20 billion peptide sequences. Cell-surface biopanning revealed a series of phage that bound to T24 and J82 urothelial cells but did not bind to WBCs or RBCs. Synthetic peptides based on six of these sequences were synthesized for the work presented here (see Table 1). Peptide purity, as determined by RP-HPLC, was ≥92%. The synthetic peptides have micromolar affinity for J82 and T24 urothelial cells, and they do not bind to blood cells or platelets (as determined by FACS; data not shown).

Conjugation of urothelial cell binding peptides to glass slides

The peptides in Table 1 were conjugated to NHS glass slides through the lysine epsilon amine, and the surface density of peptide on the slides was found to be 29.9 pmol/cm2 (N = 2). Hydrolyzing the NHS groups prior to conjugation eliminated peptide attachment to the slides, confirming covalent attachment. The reproducibility of peptide attachment was assessed by preparing slides on 4 separate days, with an average peptide density of 30.0 ± 4.0 pmol/cm2 (%CV 13.5). Although it was possible to obtain higher or lower peptide densities by varying the peptide input concentration, subsequent experiments revealed that higher densities did not increase cell capture, while lower densities led to decreased cell capture (data not shown). Thus, 200 µM was selected as the optimal peptide input concentration.

Selective capture of urothelial cancer cell lines

In a clinical setting, urine specimens may contain significant numbers of contaminating blood cells (17, 21). To demonstrate peptide selectivity for the urothelial cells of interest, moderate to heavy contamination was simulated by applying mixtures of WBCs and J82 cells (start ratio 7:1, 8:1, or 72:1) to the slides. The end ratio of WBC:J82 cells captured on the slides was calculated after washing away unbound cells. The observed WBC depletion was ≥81% for all peptide-conjugated slides, with depletion ≥95% for 3 of the peptides (Table 2). Meanwhile, sensitivity of urothelial cell capture was measured by applying low numbers of J82 cells (48–384 cells) to individual peptide-conjugated wells. The percent recovery increased with increasing input cell number, ranging 40%–75% within the clinically relevant range (Table 3).

Table 2. 



Table 2.   (Click to enlarge)



Table 3. 



Table 3.   (Click to enlarge)



To demonstrate that the peptide-conjugated slides enrich for urothelial cells from urine contaminated with whole blood, J82 cells and anti-coagulated whole blood were spiked into fresh urine. The cells were deposited on slides, fixed, and stained with H&E (Figure 1). Samples prepared on poly- L-lysine slides were covered predominantly with red blood cells. Attempts to wash away the obscuring blood cells prior to fixing also removed the J82 cells, rendering the slides inadequate for subsequent analysis. However, peptide-conjugated slides could be rinsed to wash away the RBCs and WBCs, leaving the J82 cells on the slide for evaluation. Thus, the peptide-conjugated slides selectively capture scarce urothelial cells out of mixed populations heavily dominated by obscuring blood cells, making them superior substrates for diagnostic sample preparations from patients presenting with gross hematuria.



Figure 1.  Peptide selectively captures J82 urothelial cells and removes obscuring cell types. (Click to enlarge)



Capture of endogenous urothelial cells

Endogenous cells were obtained by processing voided urine obtained from a healthy donor. Recovered cells were applied to peptide-conjugated slides and subjected to Papanicolaou staining. Small basal urothelial cells as well as larger intermediate and superficial cells were observed with clearly distinguished nuclei (Figure 2). To assess peptide specificity for capture of endogenous urine cells, slides were prepared with four distinct regions. While unmodified regions captured only 8 cells from 40 mL of voided urine, the peptide-conjugated regions captured 27–59 cells (Table 4). When comparing the types of cells captured, EBP-8 and EBP-35 captured 3.5-fold and 2.1-fold more diagnostic basal and intermediate cells, respectively, while EBP-37 captured 7-fold more superficial and squamous cells. In a research or clinical setting, slides with multiple peptide-conjugated spots would thus enable investigators to capture, purify, and analyze multiple cell types on one slide using a single processing step. Together, these experiments demonstrate that the peptide-conjugated slides maintain their specificity for normal and cancerous urothelial cells when processing human urine specimens. Furthermore, the slides are compatible with Papanicolaou staining, the most prevalent method for evaluating cells on clinical cytology slide preparations (22).



Figure 2.  Papanicolaou staining of endogenous urothelial cells captured from urine on peptide-conjugated slides. (Click to enlarge)



Table 4. 



Table 4.   (Click to enlarge)



Characterization of captured urothelial cells

In the hands of inexperienced cytopathologists, cytology can have a lower sensitivity than FISH (23). Meanwhile, ICC can have a higher sensitivity than cytology for low-grade cancers (24, 25). As a result, the more expensive ICC- and FISH-based tests are commonly used to aid in the evaluation of epithelial cells deposited on slides. Carcinoembryonic antigen (CEA) is a membrane-associated cancer marker used in the FDA-approved ImmunoCyt ICC test to monitor bladder cancer patients, while survivin is a nucleus- and/or cytoplasm-associated cancer marker expressed in transitional bladder carcinoma cells that is correlated with poor clinical outcomes (26). Detection of CEA and survivin in cancer tissue and circulating tumor cells has prognostic and predictive relevance and may serve to guide treatment options.

To confirm that peptide binding does not interfere with ICC analysis, we evaluated the expression of CEA and survivin on peptide-conjugated slides. J82 urothelial cells were captured on peptide-conjugated slides, and their CEA expression was compared with cells deposited on positively charged slides or poly-L-lysine slides, two slide types commonly used for cytology preparations and ICC (Figure 3). CEA expression area per cell on the positively charged and poly-L-lysine slides was 207.7 and 333.8 µm2, respectively. The average CEA expression area per cell on the peptide-conjugated slides fell within the same range (253.3–276.5 µm2) (Table 5). Meanwhile, sur vivin expression detected by ICC in the nuclei of captured cancer cells was similar on both the peptide-conjugated and poly- L-lysine slides (Figure 4). Together, these results indicate that the peptides do not interfere with slide-based ICC analysis. Finally, as most urine specimens are fixed prior to analysis, J82 cells were fixed in PreservCyt (Hologic, Bedford, MA) prior to capture on the slide. PreservCyt is the same fixative used with the ThinPrep system, and the capture and CEA marker expression of preserved cells on the peptide-conjugated slides was comparable to fresh cells (Figure 3E). Similarly, endogenous cells from urine fixed in PreservCyt were captured on peptide-conjugated slides in numbers comparable to fresh cells (data not shown).



Figure 3.  J82 urothelial cells captured on peptide-conjugated slides express the carcinoembryonic antigen cancer marker. (Click to enlarge)



Table 5. 



Table 5.   (Click to enlarge)





Figure 4.  J82 urothelial cells captured on peptide-conjugated slides express the survivin cancer marker. (Click to enlarge)



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.

Author contributions

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.


Acknowledgments

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.

Competing interests

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]

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