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Ancillary Studies in Urinary Cytology

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The Paris System for Reporting Urinary Cytology

Abstract

Though cytology and cystoscopy are complementary, together they fail to detect a significant number of patients with UC. In the last two decades, many different assays, or ancillary tests, have been developed to overcome the limitations of urinary cytology and improve the timely detection of UC. No ancillary test has of yet demonstrated sufficient sensitivity/specificity to be useful as a stand-alone screen for UC. The ancillary tests that have recognized utility include UroVysion® Fluorescence in situ Hybridization (U-FISH) (Abbott Laboratories, Abbott Park, IL, USA); BTA™ (Polymedco Inc., Cortlandt Manor, NY, USA); and the NMP22™ (Bladder Check) test (Alere Inc., Waltham, MA, USA). Several other tests for tumor-associated antigens and PCR-based tests for mutations have been proposed, and within the past several years, next-generation sequencing has begun to be applied to detect mutations or epigenetic changes of UC. The most promising ancillary tests are described in this chapter.

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Authors and Affiliations

  1. University Hospital Basel, Basel, Switzerland

    Lukas Bubendorf

  2. MD Anderson Cancer Center, Houston, TX, USA

    Nancy P. Caraway

  3. Department of Pathology and Cell Biology, University of Massachusetts Memorial Medical Center, Worcester, MA, USA

    Andrew H. Fischer

  4. Department of Pathology, Chaim Sheba Hospital, University of Tel Aviv, Tel Aviv, Israel

    Ruth L. Katz

  5. Medical Faculty, Porto University, Porto, Portugal

    Fernando Schmitt

  6. University of Ljubljana, Ljubljana, Slovenia

    Margareta Strojan Fležar

  7. Princess Margaret Cancer Center, University Health Network, Toronto, ON, Canada

    Theodorus H. Van der Kwast

  8. Department of Pathology, Medipath & American Hospital of Paris, Paris, France

    Philippe Vielh

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  1. Lukas Bubendorf
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  2. Nancy P. Caraway
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  3. Andrew H. Fischer
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  4. Ruth L. Katz
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  5. Fernando Schmitt
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  6. Margareta Strojan Fležar
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  7. Theodorus H. Van der Kwast
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  8. Philippe Vielh
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Correspondence to Lukas Bubendorf .

Editor information

Editors and Affiliations

  1. Department of Pathology, Loyola University Medical Center, Maywood, IL, USA

    Eva M. Wojcik

  2. Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Wisconsin State Laboratory of Hygiene, Madison, WI, USA

    Daniel F.I. Kurtycz

  3. Department of Pathology/Cytopathology, The Johns Hopkins University, Baltimore, MD, USA

    Dorothy L. Rosenthal

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 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-stranded target DNA. U-FISH probes should be prepared accordingly and applied to the selected area of the 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 post-hybridization 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 [10]. The MetaSystems uses an automated fluorescent scanning microscope and analysis software with “tile-sampling” method [16].

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, according to 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 and 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. (2022). Ancillary Studies in Urinary Cytology. In: Wojcik, E.M., Kurtycz, D.F., Rosenthal, D.L. (eds) The Paris System for Reporting Urinary Cytology. Springer, Cham. https://doi.org/10.1007/978-3-030-88686-8_9

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