Abstract
In the last two decades, different markers and diagnostic assays have been developed to overcome limitations of urinary cytology and improve the timely detection of urothelial carcinoma (UC). Among ancillary tests that can be used on cytological preparations, namely cell-based tests UroVysion® Fluorescence in situ Hybridization (U-FISH; Abbott Laboratories, Abbott Park, IL, USA) and ImmunoCyt/UCyt+® (uCyt; Diagnocure Inc, Quebec, Canada) have been approved by the U.S. Food and Drug Administration (FDA) for diagnosis of UC in patients with hematuria and/or monitoring for tumor recurrence in patients previously diagnosed with UC. The pre-analytical procedures, technique, and evaluation of U-FISH including imaging and automation are described, followed by discussion on the performance of the assay. U-FISH is used for the determination of neoplasia after an interpretation of atypical urinary cytology or finding residual neoplastic cells after intravesical bacillus Calmette-Guerin (BCG) treatment. This seems to be the most important indication. uCyt is another promising diagnostic assay; however, further validation studies are needed.
Most commonly used liquid- or non-cytology-based urine tests are the BTA and the NMP22 (Bladder Check). Both were approved by the FDA for detection of UC in symptomatic patients and for monitoring of patients with a history of UC.
No single ancillary test is being recommended as part of the routine evaluation in the Guidelines of the American Urological Association and the European Association of Urology at this time.The classification in The Paris System for Reporting Urinary Cytology lays the ground for prospective studies in search of cost-effective combinations of urine cytology with ancillary testing to improve diagnosis and clinical outcome of UC.
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Appendix
Appendix
UroVysion® Assay
The slide pretreatment with protease uncovers target DNA and is recommended in Pap-stained specimens. Decolorization is not mandatory since the stain is removed during further phases of FISH procedure. If using the archival slides, remove the coverslip and mounting medium in xylene. Place the slides in 1 % acid alcohol (HCL and 70 % alcohol) overnight or until decolorized. The U-FISH assay can subsequently be conducted either manually or automatically. The first step is denaturation of specimen DNA to expose single-strand target DNA. U-FISH probes should be prepared accordingly and applied to the selected area of slide. The area should be coverslipped and sealed immediately to ensure optimal conditions. Hybridization of probes to target DNA sequences follows under appropriate conditions. The procedure is finished with posthybridization washes to remove excessive probes. Slides should be dried in a dark area. The exact procedure of the FISH assay is described in the UroVysion kit datasheet. The procedure should be validated in each individual laboratory, together with positive and negative controls, to ensure optimal hybridization. Afterwards the specimen chosen for analysis is stained by DAPI solution. Slides are coverslipped and stored at −20 °C in the dark until analysis.
Automated Imaging Systems for UroVysion® FISH Analysis
The Duet TM System™ workstation integrates a microscope, CCD camera, motorized stage or slide-loader, computer, keyboard, mouse, joystick, monitor, and a dedicated software program. Up to 200 slides that have undergone the FISH procedure, can be loaded and run overnight for inspection the following day. This latter feature may be suitable for diagnostic laboratories that receive high volumes of abnormal or atypical urines. Similarly, the Ikoniscope oncoFISH Bladder Test System has an automated scanning microscope system coupled with an image analysis work station. It features automated slide loading and handling, low and high magnification scanning to identify cells of interest, and digital image acquisition [4]. The MetaSystems uses an automated fluorescent scanning microscope and analysis software with “tile-sampling” method [9].
The Bioview Duet System™ scans cells that are imaged at high resolution (under oil immersion) both in bright light illumination and in fluorescent illumination. Cells are classified by the system according to their morphological features, their staining on bright field (Giemsa or Papanicolaou stains, if target FISH is used), and according to the pattern of fluorescent signals. The automated microscope has micrometer-level precision in the X, Y, and Z axes which allows it to focus on cells and retain coordinate information for target cells. There are two modes of operation: (1) automatic scanning, which provides a gallery of all fields of view, and (2) manual scanning, which provides interactive control allowing the user to select the fields of view using either bright field or fluorescent illumination.
Similar to manual scoring, the automated system scans the FISH slides by locating and scoring the nuclei exhibiting abnormalities such as enlargement, irregular borders, and patchy DAPI staining. As identification of abnormal or malignant cells based solely on aberrant morphology may be misleading, the Bioview System™ classifies cells both by morphology on the DAPI fluorescence, as well as by superimposed FISH signals. Cells are ranked based on a combination of nuclear features, including size, shape, DAPI intensity, and DAPI standard deviation inside the nucleus.
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Bubendorf, L. et al. (2016). Ancillary Studies in Urinary Cytology. In: Rosenthal, D., Wojcik, E., Kurtycz, D. (eds) The Paris System for Reporting Urinary Cytology. Springer, Cham. https://doi.org/10.1007/978-3-319-22864-8_9
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