, Christopher VandenBussche2 and Syed A. Hoda3
CBL Path, Rye Brook, NY, USA
Department of Pathology, Johns Hopkins University Department of Pathology, Baltimore, MD, USA
New York Presbyterian Hospital Weill Cornell Medical College, New York, NY, USA
In 2016, approximately 76,000 new cases of bladder carcinoma will be diagnosed in the United States. Of these, around 58,000 will be in men and 18,000 in women. Approximately 16,000 bladder cancer-related deaths will occur in 2016. About 50 % of bladder cancer cases are diagnosed while it is noninvasive, and around 35 % are diagnosed when it has invaded into deeper layers of the bladder, but the disease is still confined to the organ (American Cancer Society, Cancer Facts and Figures, 2016).
More than 90 % of the bladder cancers are urothelial in origin. The examination of urine cytology is an efficient method to diagnose primary and recurrent carcinoma of urinary tract, i.e., bladder, urethra, ureters, and renal pelves. A urinary sample can be collected as voided specimen, through urinary catheter, or by cystoscope using instrumental lavage (washings) or brushings.
Urine cytology specimens comprise the largest proportion of non-gynecological specimens in most cytology laboratories and are increasingly being processed as liquid-based preparations (LBP). In this chapter, cytology and differential diagnosis of nonneoplastic and neoplastic lesions of the lower urinary tract collected via different methods are illustrated. The utility of ancillary tests such as immunocytochemistry is also addressed.
The Paris System (TPS) for Reporting Urinary Cytology
The Paris System (TPS) is a standardized international reporting system for urinary cytology specimens. The system was conceived by participants of two urine cytology symposia in 2013 at the 18th International Congress of Cytology (ICC) held in Paris. TPS guidelines are based on consensus, global participation, and evidence-based practice. The primary emphasis of TPS is the detection of high-grade urothelial carcinoma (HGUC), with the understanding that urinary cytology has low sensitivity, specificity, and reproducibility for the detection of low-grade urothelial neoplasms (LGUN). The reporting system is sponsored by the American Society of Cytopathology (ASC) and the International Academy of Cytology (IAC). TPS diagnostic categories include negative for HGUC (NHGUC); atypical urothelial cells (AUC); suspicious for HGUC (SHGUC), HGUC, LGUN; and other malignancies, both primary and secondary. Many of these categories utilize strict cytomorphological criteria and in particular focus on nuclear-to-cytoplasmic ratio, hyperchromasia, nuclear border irregularity, and chromatin quality (Figs. 3.1–3.24).
NHGUC: The negative category implies that no cytomorphological features of HGUC are identified in the specimen. If a specimen does not meet the criteria of AUC, milder atypical changes can be classified into this category. NHGUC includes cases in which LGUN cannot be excluded. If there is a cause for atypia without the presence of any features of HGUC (i.e., urolithiasis, treatment-related changes, viral cytopathic, or other reactive changes), this category is indicated.
AUC: In order to be classified as AUC, the cells should be non-degenerated, non-superficial cells with a nuclear-to-cytoplasmic ratio of at least 0.5 and at least one of the following: mild to moderate hyperchromasia, nuclear border irregularities, and/or irregular clumpy chromatin. The number of AUC is not a criterion, but evidence suggests the number correlates with risk.
SHGUC: This category is used for more severe atypia that quantitatively falls short of a definitive diagnosis of HGUC – 5–10 HGUC cells. The cells should be non-degenerated, non-superficial cells with a nuclear-to-cytoplasmic ratio of at least 0.5 and at least moderate hyperchromasia. In addition, the suspicious cells should have either markedly irregular nuclear borders or irregular clumpy chromatin.
HGUC: These cells may be from carcinoma in situ (CIS) or papillary HGUC. The requirements are nuclear-to-cytoplasmic ratio of 0.7 or above, moderate-to-severe hyperchromasia, irregular nuclear borders, and coarse, clumpy chromatin.
LGUN: A combined cytological term for low-grade papillary urothelial neoplasms include urothelial papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade papillary urothelial carcinoma (LGUC), and flat, low-grade intraurothelial neoplasia. This diagnosis technically falls under the category of NHGUC, but may be used if clinical and cystoscopy findings correlate with a papillary lesion and no features of HGUC are identified.
Other malignancies, both primary and secondary: Used for non-urothelial malignancies, both primary and secondary – non-urothelial carcinoma including squamous cell, adeno-, and small cell carcinoma and non-epithelial malignancies including lymphoma, melanoma, sarcoma, and metastatic tumors to lower urinary tract.
Indication, Collection, and Laboratory Processing of Cytological Samples
The principal indications for the use of cytology in disorders of urinary tract (bladder, urethra, ureters, and renal pelves) are as follows:
The diagnosis of HGUC: The cytological techniques are of limited value in the identification of LGUC.
The routine follow-up and monitoring of patients with a history of bladder cancer, for the early detection of recurrences. The current standard of care consists of cystoscopy and urine cytology evaluation every 3–6 months for the first year and at reduced intervals subsequently.
Methods of Specimen Collection
The principal methods of specimen collection are:
Catheterized urine (CU)
Direct sampling techniques
Bladder washings, barbotage, or lavage
Collection by retrograde catheterization of ureters
Direct brushings of ureters and renal pelves
The method of specimen collection and processing depends on the clinical circumstances and goal of the examination.
This is the easiest method of cytological investigation of the urinary tract. A small volume of the collected specimen is preserved in liquid-based preservative medium, or if received fresh, it can be refrigerated for 24 h until processing.
The specimens are collected via a urinary catheter and preserved and processed as described for voided urine specimens.
Direct Sampling Techniques
Bladder Washings (Barbotage or Lavage)
This technique may be utilized during cystoscopic examination to obtain well-preserved cells from high-risk patients. It is the specimen of choice for DNA ploidy analysis. Bladder washings have significantly better diagnostic yields.
Retrograde Catheterization of Ureters or Renal Pelves
This procedure is used to establish the nature of a radiologically detected space-occupying lesion of ureter or renal pelvis, including a calculus, blood clot, neoplasms, or inflammatory masses. In the ureters, other causes include a stricture or extraneous pressure.
The Direct Brushing Procedure
Brushing is performed through a ureteral catheter to investigate space-occupying lesions in the ureters or renal pelves.
Ileal Conduit Urine
After cystectomy for HGUC, an artificial bladder is often constructed from a segment of the small intestine, usually the ileum. The status of these organs must be monitored after treatment due to the propensity of urothelial tumors to recur.
Laboratory Processing of Urinary Specimens
Several techniques are available for processing urinary specimens for microscopy including LBP, cytocentrifugation, and membrane filtration. The two LBP, ThinPrep (TP, Hologic, Bedford, MA) and SurePath (SP, BD Diagnostics, Burlington, NC), are illustrated in Chap. 1.
Normal urothelial histology. Normal urothelial lining in distended bladder. There are approximately seven layers of urothelial cells. The surface is lined by larger superficial cells with abundant cytoplasm (“umbrella cells”). Capillary vessels are present immediately beneath the epithelium in the lamina propria (H&E)
Normal urothelial cells in voided urine. (a) Superficial (umbrella) cells: normal voided urine may contain relatively few urothelial cells. These cells are of various types, shapes, and sizes and comprise superficial (umbrella) cells and a few cells from the intermediate and deeper urothelial cell layers. Umbrella cells are the predominant cell type in voided urine and are 20–30 μm in diameter, with one flat plane. They are somewhat similar in size to cervicovaginal intermediate squamous cells, but may be larger. Umbrella cells may be mono-, bi-, or multinucleated. The nuclei are central and round with regular finely granular chromatin and occasional small nucleoli (SP). (b) Intermediate cells originating from the deeper layers of the urothelium: resemble parabasal squamous cells from lower genital tract in size and configuration. These cells are often spherical or round with scanty basophilic cytoplasm and spherical bland nucleus. They desquamate singly or in clusters (TP). (c) Multinucleated umbrella cells are rare in voided urine, except after diagnostic and therapeutic procedures. They are normally seen in specimens from ureter or renal pelves. Giant umbrella cells may be mistaken as neoplastic (TP)
Catheterized urine. (a) The cytological features of catheterized urine (CU) are usually similar to voided samples, except for higher cellularity, more sheets, clusters and urothelial tissue fragments, and background inflammation. Some umbrella cells show reactive features with prominent single and multiple nucleoli. The nuclei however are regular and pale (TP). Catheterized urine is considered instrumented specimen and therefore may have artifacts not seen in voided urine, particularly the presence of increased urothelial tissue fragments which may be at first worrisome for low-grade urothelial neoplasia (LGUN). Kannan and Bose  found that evenness of fragment borders and the presence of cytoplasmic collars were distinguishing features of instrument artifact. (b) Since urothelial tissue fragments have some three-dimensionality, inflammatory cells and other background cells may appear on a different plane of focus in SP. This does not occur in TP, as fragments seen in those preparations are flat (SP)
Washing specimens in the absence of disease. (a) Bladder washing specimens are usually highly cellular and often include a larger number of urothelial tissue fragments and single urothelial cells. In fact, a specimen that is sparsely cellular may indicate an inadequate sample. Prather et al.  determined that a cutoff of 20 well-visualized, well-preserved urothelial cells per ten high-power fields increased the positive predictive value of bladder wash (TP). (b) Deeper urothelial cells are more frequent and may form tightly packed three-dimensional clusters in a “papillary” configuration with relatively smooth borders. In these instances, the cells are often more elongated with regular, oval nuclei, bland chromatin, and small nucleoli. Umbrella cells usually outline these groups. These large clusters are commonly mistaken as representing a papillary tumor, particularly, if the cytologist is unaware of how the specimen was procured (SP). Washings and brushings from upper urinary tract are richly cellular and generally appear similar to bladder wash. Large sheets of urothelial cells may be forcibly removed from surface of ureters or renal pelves resulting in complete denudation of ureteral surface
Ileal conduit urine. (a) Ileal conduit urine (ICU) used in monitoring patients after cystectomy for bladder cancer normally contains a rich population of poorly preserved intestinal cells. The intestinal cells may have vacuolated or granular degenerative cytoplasm and fragmented and/or pyknotic nuclei. Background mucin and Paneth cells may sometimes be seen. Wolinska and Melamed  studied ICU and were able to trace the transition from intestinal epithelial cells in clusters to degenerating rounded cells that resemble histiocytes (TP). In less degenerated samples, well-preserved columnar mucus-producing cells with peripheral and round dark nuclei are noted. (b) In this specimen, numerous degenerative cells distract from rare malignant cells in the field; this patient had a recurrence of HGUC (SP). (c) In histological specimens taken from a benign ileal conduit, progressive shortening and ultimately flattening of villi can be seen, as well as an increased number of goblet cells (H&E)
Squamous cells. Urine samples are often contaminated by squamous cells from anogenital region, trigone, or urethra, though this contamination may be limited by procuring a sample through catheterization or instrumentation. Superficial squamous cells are characterized by a small, pyknotic nucleus and abundant cytoplasm, and intermediate cells have a slightly larger, vesicular nucleus. Smaller parabasal cells may be seen, which have an increased nuclear-to-cytoplasmic ratio and share some cytomorphological overlap with urothelial cells. Note cluster of deeper urothelial cells. Pear-shaped Trichomonas vaginalis organisms indicate contamination from the lower gynecologic tract. Occasionally atypical squamous cells (ASC) may be seen, such as koilocytes; in these instances, they should be noted and clinical correlation should be suggested. It can be difficult to isolate the origin of ASC, as they could represent any lesion from condyloma to HGUC with squamous differentiation in voided urine (see Fig. 3.23a, b) (TP). Owens and Ali  followed 32 patients with ASC in the urine; 8 patients had bladder carcinoma with squamous differentiation, 2 had cervical carcinoma, and 22 (69 %) had benign follow-up
Acute and chronic cystitis. Mixed inflammatory cells comprise of polymorphonuclear leukocytes, lymphocytes, and eosinophils. Desquamated benign urothelial cells can be seen. These findings are usually nonspecific and require clinical correlation. If necrotic debris is observed, it should raise awareness for tumor in which only necrotic cells are present. In some instances, repeated infections may cause cystitis follicularis, which may result in the presence of dendritic cells with prominent nuclear chromatin and prominent nucleoli, lymphoid aggregates, reactive urothelial cell atypia, and glandular cells, indicating glandular metaplasia . Melamed-Wolinska bodies (dense, round eosinophilic inclusions) can be observed in degenerating urothelial cells. Multiple inclusions of varying sizes may be present and are thought to represent degenerative lysosomal aggregates (TP)
Crystals. Rarely, true uric acid crystals can have a variety of shapes and sizes and are typically yellow or colorless. Possible shapes include rhomboids, hexagonal plates, needles, and rosettes. They typically form under acidic conditions and are not necessarily a sign of an underlying pathological process (TP). Calcium carbonate crystals, which usually appear as large yellow-brown or colorless spheroids with radial striations, are also commonly seen in urinary specimens. They can also be seen as smaller crystals with round, ovoid, or dumbbell shapes. Triple phosphate crystals (struvite) appear as “coffin lids” and form in alkaline urine. They are classically associated with Proteus mirabilis infection. For the most part, crystals are simply distracting noncellular element seen in urine specimens. Wright and Halford  found that TP of urine cytology had the same numbers of casts and debris as cytospin preparations, but the amount of crystals and red blood cells (RBCs) were reduced in TP