Correlation between the cytology of urine sediment in fresh sample and smears stained by Papanicolaou and Giemsa methods.

BACKGROUND: Urine excreted by the body has a variable composition in different physiological and pathological conditions. The cells that come from the renal pelvis, ureters, bladder, and urethra are carried by the urine, and therefore, they can be observed in fresh samples and in smears with Giemsa and Papanicolaou stain. AIM: The aim of this study was to show that high correlation that exists between the cytological examination of fresh urine samples and smears stained with Papanicolaou and Giemsa methods.
MATERIALS AND METHODS: A total of 45 cases with no tumor of the urinary tract and 36 patients with lower urinary tract neoplasms were included in the study (20: Low-grade urothelial tumors; 16: High-grade urothelial tumors, squamous carcinomas, and adenocarcinomas). The sediments in the urine samples were observed in fresh specimen and in smears stained with Papanicolaou method.
RESULTS: The meticulous observation of fresh urinary sediments allowed identification of diverse cellular types associated with varied pathologies.
CONCLUSIONS: The cytological examination of urinary samples in fresh smears, and its later diagnostic confirmation with the Papanicolaou stain is important not only as a diagnostic procedure of tumoral or non-tumoral pathologies, but also as a method for the 'screening' of pre-cancerous lesions or carcinoma in situ, especially in high-risk populations.

Introduction

The renal calyces, the renal pelvis, the ureters, the bladder, and a part of the urethra are lined by the urothelium. The most superficial cells of the urothelium are big and frequently multinucleate and receive the name of ‘umbrella’ cells. The underlying cells have a predominantly pyriform shape (sometimes with an elongated end, especially those arising from the renal pelvis) and the deepest cells are small, with a high nuclear:cytoplasmic (N/C) ratio [Figure 1].[12]

Figure 1

Cytology of the urinary tract. (a) Normal urothelial cells: A group of deep cells with high N/C ratio (thin arrow); umbrella cell (thick arrow) (b) Cells with elongated cytoplasm arising from the renal pelvis (arrow) (c) A big multinucleated umbrella cell showing microvacuolated cytoplasm (voided urine) (d) Umbrella cell obtained from bladder washing, similar to that showed in Set C, but with better nuclear details (Pap, ×400)

About 80% of the normal bladders present with buds that extend into the lamina propria, called Brunn's nests, which are lined by columnar epithelium. If the Brunn's nests develop to become large with distended cysts (cystitis cystica), many columnar cells can be observed in the urinary sediment.[3]

Cytology has few opportunities for detection of cells from the renal epithelium (glomerular or tubular cells) in the urine, except when they are forming a part of the epithelial cylinders or in pathologies such as nephrotic syndrome, tubular acute necrosis, tubular-interstitial diseases[4] or renal transplant rejection.[5]

The cellular alterations produced in renal lithiasis or in n class="Disease">bladder diverticula, as well as the changes caused by certain viral processes or by the effects of drugs or radiotherapy can be erroneously diagnosed as a case of malignancy.

The carcinogenesis of urothelial tumors, mainly of the urinary bladder, is associated with the exposure of industry workers to known carcinogens (cadmium and aromatic amines in coloring industries).[6]

About 95% of the carcinomas of the lower urinary tract are of the urothelial type. The squamous carcinomas and the adenocarcinomas have a low frequency. In addition, a majority of the tumors develop in the bladder. Although most invasive urothelial carcinomas are accompanied or preceded by papillary tumors, the invasion takes place from the flat variant of the tumors. The grading of carcinomas (I, II, III) reflects the degree of cytological abnormality. The morphology of the cells of grade I is similar to that of normal cells. When tumors reach grade III, they are invasive and can prove to be fatal to the patient within a few years. The poor prognosis of papillary carcinomas grade III is explained because these tumors are often accompanied by a flat form of carcinoma in situ[7] [Table 1].

Table 1

Non-tumoral and tumoral pathologies of the urinary tract

Non-tumoral and n class="Disease">tumoral pathologies of the urinary tract

The cytological diagnosis of these neoplastic cells is very difficult in low-grade tumors. The sensitivity of cytology for the diagnosis of bladder cancer is not very high; this is mainly due to the minimal morphological differences between the normal cells and the cellular groups exfoliated from low-degree tumors. With the purpose of increasing the sensitivity of cytology, different methods have been designed, for example the assay for tumor-associated bladder antigen, the assay for protein from the nuclear matrix, and the Fluorescent in situ hybridization (FISH) technique.[89]

The aim of this study was to show the high correlation that exists between cytological examination of urinary samples observed in fresh urine sediments and urinary deposits stained with Papanicolaou's method. The observation of fresh urine sediment can identify different pathologies that will subsequently be corroborated by Pap staining. In this article we describe the criteria that can guide the correct diagnosis, increasing the sensitivity of urinary cytological examination.

Materials and Methods

The wet mount sediments of freshly voided urine samples were evaluated along with smears of sediment stained with Papanicolaou method. Some of these samples were also stained using the Giemsa technique. The study was conducted over three years. Of 5,200 patients without any tumor or urinary pathologies, 45 were selected, and of 350 patients with lower urinary tract tumors we chose 36 (20: Low-grade urothelial tumors; 16: high-grade urothelial tumors, squamous carcinomas and adenocarcinomas); (confidence interval 15%, confidence level 95%).

In this retrospective analysis, only patients with a clinical history suggestive of an urinary pathology were included, while cases with inconclusive or mixed pathologies, were excluded from the study. Controls were selected from among the population without pathologies of the urinary tract, as evidenced by urine analysis and clinical examination.

The final diagnosis was established through an evaluation of the clinical history, follow-up of the patients, cystoscopy, and bladder biopsies.

The urine samples, gathered after three or four hours after the last micturition, were centrifugated at 250 g, accumulating three sediments in the same tube. The accumulated sediments were re-suspended by soft blows to the centrifugal tube and spread on a microscope slide, where they were left to concentrate, but they were not allowed to reach total dryness. Then the slides were carefully submerged in 96˚ ethanol to fix the sediments for a minimum of 30 minutes. Finally, they were stained using the Papanicolaou method.[10]

The fresh urine sediments smears were examined through an optical microscope or a phase contrast microscope. The Giemsa stain was carried out on urinary sediments completely dried and fixed with methanol for two minutes.

Statistical analyses were used to calculate the size of the populations and the margin of error.[11] The margin of error in extrapolating the results to the population was 12% for tumoral or no n class="Disease">tumoral pathologies.

Results

Non-tumoral pathologies

Kidney lithiasis

Fresh smears: In the microscopic examination of the urinary sediment we observed flat groups of small urothelial cells, umbrella cells, and an abundant quantity of leukocytes and red cells.

Giemsa stain: We saw a predominance of small cells and some umbrella cells (of the ureters, bladder or urethra), some of which presented reactive changes: hyperchromatic nuclei with homogeneous chromatin, which may present with regular chromocenters [Figure 2].

Figure 2

Image of a non-tumoral pathology in a case of renal lithiasis. (a) Fresh smear. Reactive urothelial cells (arrow), (Optical microscopy, ×400). (b) Umbrella cell with big nuclei, (Giemsa stain, ×400). (c) Group of umbrella cells showing anisokaryosis. (d) Group of small cells from the deep layers of urothelium (thick arrow) and umbrella cells, which present reactive changes (thin arrow), (Giemsa stain, ×100)

Image of a non-tumoral pathology in a case of n class="Disease">renal lithiasis. (a) Fresh smear. Reactive urothelial cells (arrow), (Optical microscopy, ×400). (b) Umbrella cell with big nuclei, (Giemsa stain, ×400). (c) Group of umbrella cells showing anisokaryosis. (d) Group of small cells from the deep layers of urothelium (thick arrow) and umbrella cells, which present reactive changes (thin arrow), (Giemsa stain, ×100)

Viral pathologies

Fresh smears: The cells had a high N/C ratio. We observed some ‘empty’ nuclei, with clumps of chromatin on their membranes.

Giemsa stained smears: We observed nuclei with high N/C ratio and basophilic amorphous inclusions that occupied the entire nucleus or were located on the nuclear membranes, obscuring the chromatic structure. These morphological characteristics were due to infection by the Polyomaviruses [Figure 3].

Figure 3

Non-tumoral pathology corresponding to Polyomavirus. (a and b) Cells with high N/C ratio. Nuclei of ‘empty’ aspect (thin arrow), with clumps of chromatin on their membranes (thick arrow) Fresh smear (Optical microscopy, ×400). (c and d) Cells with high N/C ratio and blurred chromatin (white arrow), sometimes presenting clumps on the membranes, characteristic of Polyomavirus infection. Notice that some cells have a cytoplasmic tail like a ‘comet’ (black arrow) (Giemsa stain, ×500)

Non-tumoral pathology corresponding to n class="Species">Polyomavirus. (a and b) Cells with high N/C ratio. Nuclei of ‘empty’ aspect (thin arrow), with clumps of chromatin on their membranes (thick arrow) Fresh smear (Optical microscopy, ×400). (c and d) Cells with high N/C ratio and blurred chromatin (white arrow), sometimes presenting clumps on the membranes, characteristic of Polyomavirus infection. Notice that some cells have a cytoplasmic tail like a ‘comet’ (black arrow) (Giemsa stain, ×500)

Replacement of bladder by ileocolonic tissue

Fresh smear: Showed abundant cells, with similar size, were predominantly clustered to the leukocytes, with necrotic changes. The leukocytes were very few.

Papanicolaou stained smear: Showed small necrotic cells, isolated and in compact groups, predominantly flat; these cells did not come from the urothelium. There were few leukocytes and an absence of neoplastic cells. The necrotic cells were interpreted as derived from the ileocolonic epithelium, in agreement with the information of the clinical history [Figure 4].

Figure 4

Images of cystectomy and bladder replacement. (a and b) Tissue fragments of difficult interpretation: Cells of similar size exfoliate in compact and two-dimensional groups. (Fresh smear. Optical microscopy, ×400); (c and d) Cells from the replacement of bladder by ileocolonic tissue, with necrotic changes. In C, the necrotic isolated cell (thick arrow) confirms the cylindrical origin of the group. Observe the cilia in the apical edge of the cell (thin arrow). (Pap, ×400)

Tumoral pathologies

Low-grade urothelial tumors

Fresh smear: Showed groups of small cells, some of them with a three-dimensional aspect.

Papanicolaou stained smear: Showed abundant cells, exfoliated, in compact groups, with uniform nuclei, showing bi- and three-dimensional aspects (papillary arrangement), and slight hematuria. Smears were compatible with a low-grade bladder tumor [Figure 5].

Figure 5

Vesical tumor. Image of cells in a papillary arrangement of a benign aspect. (a) Bi-dimensional image of a compact cellular group with high N/C ratio, powdery chromatin, and visible nucleoli. (b) Papillary cellular group of cells with light anisokaryosis. (Fresh smear. Optical microscopy, ×400). (c) Bi-dimensional cellular group of cells with similar size, powdery chromatin, and small nucleoli. Observe the similarity with the image in (a). (d) Papillary cellular group of a benign aspect. Images compatible with low-grade vesical tumor (Pap, ×400)

High-grade urothelial tumors

Fresh smears: Showed big isolated cells, with a high N/C ratio, multinucleation, signs of cannibalism, and macronucleoli. Microhematuria was observed.

Papanicolaou-stained smears: Showed isolated cells of a neoplastic aspect, presenting hyperchromatism, multinucleation, macronucleoli, and signs of cannibalism. Some cases showed bizarre cellular shapes. The smears were compatible with high-grade n class="Disease">bladder tumor [Figure 6].

Figure 6

Vesical cancer. high-grade vesical tumor. (a) Big multinucleated cell. (b) Big multinucleated cell, with macronucleoli (thick arrrows) and cannibalism (thin arrow) (Fresh smear. Optical microscopy, ×400). (c) Multinucleated neoplastic cell. (d) Multinucleated cell, with hyperchromatism and signs of cannibalism (arrow) (Pap, ×400)

Vesical cancer. high-grade n class="Disease">vesical tumor. (a) Big multinucleated cell. (b) Big multinucleated cell, with macronucleoli (thick arrrows) and cannibalism (thin arrow) (Fresh smear. Optical microscopy, ×400). (c) Multinucleated neoplastic cell. (d) Multinucleated cell, with hyperchromatism and signs of cannibalism (arrow) (Pap, ×400)

Non-urothelial tumors

Squamous carcinomas

Fresh smears: A high refringence of cells was observed, with predominance of fibrillar shapes, sometimes grouped in a squamous pearl [Figure 7].

Figure 7

Keratinizing Squamous Carcinoma. (a) Tapered cells coiled as an onion, forming a squamous pearl (arrow). (b-d) Spindle-like cells. Observe the refringence due to the keratin present in the cells. (Fresh smear. Optical microscopy, ×400)

Keratinizing Squamous Carcinoma. (a) Tapered cells coiled as an n class="Species">onion, forming a squamous pearl (arrow). (b-d) Spindle-like cells. Observe the refringence due to the keratin present in the cells. (Fresh smear. Optical microscopy, ×400)

Papanicolaou stained smear: Showed keratinizing neoplastic cells, with bizarre shapes (spindle-like cells, tadpole cells, squamous pearls), compatible with keratinizing n class="Disease">squamous carcinoma [Figure 8].

Figure 8

Keratinizing Squamous Carcinoma. (a) Tadpole cell intensely keratinized (arrow). (b) Big fibrillar and multinucleated cell (arrow) and a tadpole cell (thick arrow). (c) Keratinized and multinucleated cells. (d) Group of keratinized cells, some of them with fibrillar shape (Pap, ×300)

Adenocarcinomas

Fresh smears: Showed three-dimensional compact groups, presenting with anisokaryosis and marked vacuolization [Figure 9].

Figure 9

Adenocarcinoma. Compact and three-dimensional cellular group presenting megakaryosis, high N/C ratio, and marked vacuolization (Fresh smear. Optical microscopy, ×400)

Papanicolaou-stained smears: Showed groups of neoplastic cells showing vacuolization, three-dimensional shapes, and ‘signet ring’ cells, compatible with n class="Disease">adenocarcinoma [Figure 10].

Figure 10

Adenocarcinoma. (a) Neoplastic cell with macronucleoli (thin arrow) and gross nuclear membrane (thick arrow). (b) ‘Signet ring’ cell (arrow) including other neoplastic cells of the same origin, (c and d) Three-dimensional cellular groups with irregular chromatin and macronucleoli. In d a big ‘signet ring’ cell is visible (arrow) (Pap, a ×400, b;c;d: ×500)

Discussion

The meticulous observation of fresh urinary sediments allowed the identification of diverse cellular types associated with varied pathologies. The inflammatory changes were frequently related to infections of the urinary tract and to nephrolithiasis. The inflammatory cells presented nuclei with irregular chromocenters, thick nuclear membrane, areas of clear chromatin, and clumps of heterochromatin. The limits between euchromatin and heterochromatin became blurred because of denaturation of the histones associated with DNA. All these changes, described in stained smears, could be detected in fresh smears with high sensitivity.

The typical image of the normal nucleus of a urothelial cell (small nucleoli, fine chromatin, and thin and homogeneous nuclear membrane), was replaced in the inflammatory cell by irregular chromatin, chromocenters of various sizes, and thicker nuclear membranes. Another characteristic of the inflammatory reactions of the lower urinary tract was the great cellularity observed, especially in cases of renal lithiasis, where one could observe urothelial cells from all the layers of transitional epithelium. In this pathology, the umbrella cells were frequently multinucleated, with big nuclei and inflammatory signs, as shown in Figure 2.

The polyomaviruses can be activated, either without apparent cause or following immunodepression due to chemotherapy, transplants, or acquired immunodeficiency syndrome (AIDS).[12] This virus produces nuclear lysis, transforming the cellular nuclei into amorphous hyperchromatic masses that can occupy the totality of the nuclei or be arranged as peripheral or central clumps (decoy cells).[1314]

Figure 3 corresponds to adult immunosuppressed patients infected by polyomavirus, where there was a high correlation between the images of the fresh and Papanicolaou-stained smears. To date, only one report has been published about immunocompetent children with polyomavirus infection.[15]

A cystectomy and bladder replacement was carried out using intestinal segments (ileocolonic neobladder)[16] in a patient with chronic bladder inflammation, hemorrhagic cystitis, and fibrosis. The patient arrived at our hospital and was treated with antibiotics, but the urine sediments did not show any inflammatory reaction. It was very important to make a correct differentiation between exfoliated cells of the neobladder and the grouping of neutrophils. Cells from the neobladder exfoliated in compact and two-dimensional groups, while groups of neutrophils were three-dimensional. The stained samples presented the typical aspect of the cylindrical ileocolonic epithelium, with some cilia in the isolated cells. These cells presented necrotic changes due to their contact with an adverse environment [Figure 4].

Approximately 95% of the urinary bladder tumors are of the urothelial type.[17] Although numerous criteria exist to classify these tumors, we use a simplified criterion, which allows a good correlation between cytology and histology — the division in low and high degree tumors. Low-grade tumors correspond to grade I and II of the most used histological classifications. As shown in Figure 5, the fresh urinary smears present compact groups of cells, with similar shape and size, homogeneous and powdery chromatin, and small nucleoli. The Papanicolaou stain confirms these findings.

The sensitivity in the detection of low-grade tumors is low, due to the similarity of the cellular groups to others from benign pathologies.[1819]

High-grade urothelial tumors are aneuploid and are detected with a high sensitivity, as compared to those of a low grade. The cells are predominantly isolated, present with anisokaryosis, high N/C ratio, bizarre shapes, and multinucleation in the fresh smears. The cells stained by the Papanicolaou method show nuclear hyperchromatism, irregular chromatin, with big irregular chromocenters, clear areas of different size, and big nucleoli in some of them[2021] [Figure 6].

The urothelial tumors of the n class="Gene">papillary type (low-grade) are accompanied with a certain frequency of carcinoma in situ, a carcinoma that has a great probability of transforming into an invading cancer. The carcinoma in situ is a non papillary, non-exophytic tumor, generally not detectable by cystoscopy. In these cases the examination of the urine stained by the Papanicolaou method is very important, because it can detect cells exfoliated from carcinoma in situ, which are characterized by the high N/C ratio, megakaryosis, irregular and hyperchromatic chromatin or with so high a content of chromatin that the nuclei appear as ‘drops of Indian ink’. The finding of these malignant cells in the urinary sediment, associated with negative cystoscopy, is an indication to take multiple vesical biopsies, to search for the intraepithelial lesion.[22]

Squamous carcinomas of the bladder are not frequent, although there are endemic zones in the world for this disease, related to the presence of the parasite Schistosoma haematobium in the contaminated waters (for example, in the proximities of the river Nile).[23] Such keratinized tumors show refringent neoplastic cells in the fresh smears [Figure 7]. This carcinoma develops the well-known bizarre forms: Spindle-like cells, tadpole cells, and squamous pearls. The images of the fresh smears show a strong correlation with the stained samples [Figure 8].

The least frequent among urinary bladder cancers was adenocarcinoma (2% of the malignant tumors). The fresh smears in Figures 9 show cellular groups that suggest the presence of this pathology owing to the presence of very vacuolated cells, arranged in three-dimensional form, in a compact group. The Papanicolaou stain confirmed the diagnosis of adenocarcinoma, showing the typical three-dimensional vacuolated cellular groups, with irregular chromatin and macronucleoli [Figure 10].

The main findings in the fresh urinary sediments of this study can be summarized as follows:

Cells from benign pathologies show a conserved N/C ratio according to the layer of the urothelium in which they originate, with an irregular chromocenters, and thickened nuclear membrane, except in the Polyomavirus infection, where the cells have a high N/C ratio and the nuclei present an ‘empty’ aspect, with clumps of chromatin on their membranes.

The low-grade urothelial tumors show compact cellular groups in bi-dimensional or n class="Gene">papillary arrangements, with a high N/C ratio, powdery chromatin, and visible nucleoli.

The high-grade urothelial tumors show predominantly isolated cells, with anisokaryosis, high N/C ratio, bizarre shapes, and multinucleation.

The keratinizing squamous carcinoma presents with squamous pearls, spindle-like cells, and tadpole cells with a large refringence.

Vesical adenocarcinoma is characterized by compact and three-dimensional cellular groups presenting with megakaryosis, high N/C ratio, and marked vacuolization.

We can conclude that this study demonstrates the high correlation between the cytological examination of urine specimen in fresh smears and in the Papanicolaou-stained smears. For this reason, the identification of cells of the urinary tract in samples observed in fresh sediments is important as a primary diagnostic procedure between non-tumoral and tumoral pathologies, with a later diagnostic confirmation with the Papanicolaou method.

The study was made possible due to the interaction of nephrologists, urologists, and cytologists. The limitations were: A lack of data on patients who did not continue their treatment and the incorrect manner in which the urine samples were collected; in both cases these patients were excluded from the study. Another limitation was the margin of error of 12% for the extrapolation of results. To diminish this value in half, it should triple the cases analyzed.[11] Studies should continue in order to reduce the margin of error.

A possible limitation of this study is related to the low sensitivity of the cytology regarding low-grade bladder tumors. However, it is possible to increase the sensitivity of the cytodiagnosis if, instead of single cells, one looks for groups of three-dimensional urothelial cells, with powdery chromatin. These originate in low-grade tumors.

We hope this new vision of the urinary sediment will allow an improvement in patient care and a change in health policy, because it would avoid expensive studies and delay of treatment. For example, the diagnosis of Polyomavirus in renal transplant patients, which is usually done by polymerase chain reaction (PCR), an expensive methodology, could be replaced in many cases with the observation of fresh material and confirmation by the Papanicolaou method.

The next step will be the training of professionals and technicians in the recognition of these diseases, and making the urologists and nephrologists in our community hospitals aware of the conclusions of this study.

The methodological proposal presented in this article aims to establish an early cytodiagnosis, which may help diminish the morbidity and mortality associated with this type of pathology of the urinary tract.