Nongynecologic Fluid and Brushing Cytology

Nongynecologic Fluid and Brushing Cytology A Goyal and TM Elsheikh, Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland, OH, USA ã 2014 Elsevier Inc. All rights reserved.

Glossary Aromatic amines Organic compounds that contain one or more amino groups attached to an aromatic hydrocarbon with one or more benzene rings. Aniline dyes are one example. Barrett’s esophagus A condition wherein the squamous epithelium lining the esophagus is replaced by intestinaltype columnar epithelium. Patients with Barrett’s esophagus have a 30–100-fold increased risk of esophageal adenocarcinoma. Capillary hydrostatic pressure The force exerted by a fluid against the capillary wall; it tends to force fluid out of capillaries. Cytopathology, cytology Synonymous terms for the branch of pathology devoted to the study of cells and/or small tissue fragments obtained using minimally invasive methods for the purpose of clinical diagnosis and patient management. Diff-Quik stain A commonly used proprietary Romanowsky stain performed on air-dried cytological material. It gives good cytoplasmic detail and highlights extracellular substances. Endoscopic retrograde cholangiopancreatography (ERCP) A technique that combines endoscopy and radiography to visualize the biliary and pancreatic ductal systems. Once the major duodenal papilla is visualized endoscopically, a catheter is inserted through it. A dye is injected, and the pancreatic and biliary ductal systems are seen radiographically. Endoscopic ultrasound (EUS) A procedure that combines endoscopy and ultrasound in order to obtain images from the digestive tract and the surrounding tissue and organs. A small ultrasound transducer is installed on the tip of the endoscope, and detailed images are obtained due to close proximity of the transducer to the organs of interest. Flexible bronchoscopy Technique for seeing inside the airways and lungs. Usually, a flexible bronchoscope is used. It contains a fiberoptic system that transmits an image from the tip of the instrument to an eyepiece or video camera at the opposite end. Fluorescence in situ hybridization (FISH) A cytogenetic technique that employs fluorescently labeled probes that are complementary to specific sites in the genome; useful in detecting chromosomal translocations and gene amplifications and deletions. Gout A disease characterized by recurrent bouts of joint inflammation and caused by elevated levels of uric acid in the blood. Uric acid crystals are deposited in the joints, tendons, and surrounding tissues.

Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms

Image analysis A system that extracts quantitative data from objects (usually cells) on a glass slide. Used for DNA content (ploidy) analysis. Cancer cells typically have abnormal DNA content that can be detected using this analysis. In one example, the measurements of optical density are obtained from Feulgen-stained cytological material and analyzed using a histogram. Low-dose spiral computed tomography (CT) scan A low radiation dose CT scan of the lungs that generates a series of detailed cross-sectional images that are used to create a three-dimensional image. Microsatellite analysis Microsatellites are short, repetitive sequences of DNA. Mutations in microsatellites are commonly identified in cancers. Microsatellite alterations can be detected in bladder cancers using the polymerase chain reaction on urine specimens. Oncotic pressure The osmotic pressure of proteins in plasma that pulls water back into the circulatory system. Papanicolaou stain Developed by Dr George Papanicolaou, it consists of a single nuclear stain (hematoxylin) and multiple cytoplasmic stains (orange G and eosin alcohol). Its advantages include enhanced chromatin detail, differential cytoplasmic staining, and cytoplasmic transparency. Pseudogout A form of arthritis caused by the deposition of calcium pyrophosphate crystals in the joints. Radical cystectomy Usually performed for muscle-invasive bladder cancer, this surgical procedure includes the removal of the entire bladder, part of the urethra with adjoining organs, and regional lymph nodes. Septic arthritis Inflammation of a joint usually caused by bacteria, less commonly by a fungus. Telomerase An enzyme that adds DNA sequence repeats to the 30 end of DNA strands in the telomere regions (at the ends of chromosomes). Normally, with each cell division, there is some telomere shortening, hastening cells toward senescence. Cancer cells activate telomerase, allowing them to divide exponentially and become immortal. Telomerase activity assays are used to detect bladder cancer cells in urine samples. ThinPrep™ A common proprietary cytological preparation method (Hologic Inc., Marlborough, MA, United States) that transfers cells in suspension onto a glass slide in a controlled fashion. Upper gastrointestinal tract endoscopy A procedure that uses a lighted, flexible endoscope to see inside the upper gastrointestinal tract (esophagus, stomach, and duodenum).

http://dx.doi.org/10.1016/B978-0-12-386456-7.06503-5

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Nongynecologic fluid and brushing cytology (exfoliative cytology) is the study of cells that are either shed spontaneously or obtained by scraping or brushing an epithelium-lined surface. The Papanicolaou smear is a classic example of exfoliative cytology, wherein cells from the cervix or vagina are sampled using a spatula, endocervical brush, or broom-type device and then transferred onto a slide. Cells from the pancreaticobiliary tract, tracheobronchial tree, and urinary tract can be similarly sampled. The primary purpose of nongynecologic cytology is to detect malignancy, but it can also provide valuable information regarding infectious and inflammatory disorders. The collection of samples for exfoliative cytology is either entirely noninvasive or only minimally invasive. Collecting a sample of voided urine or sputum, for example, is noninvasive. Collecting pleural, pericardial, peritoneal, or cerebrospinal fluid (CSF) is minimally invasive as it requires the introduction of an aspiration needle. Another method for obtaining exfoliated cells is irrigation of the target organ. The latter method applies in the commonly performed bladder washing, bronchial washing, bronchioloalveolar lavage (BAL), and peritoneal washing (Table 1).

Respiratory Specimens Lung cancer accounts for the highest number of cancer deaths and ranks third in the frequency following prostate and breast cancers in the United States. Diagnosis is usually established in symptomatic patients or when an imaging abnormality is detected incidentally on a radiological exam performed for other purposes. The diagnostic modality utilized depends on the size and location of the primary tumor, presence of metastasis, and overall clinical status of the patient; it includes tissue core biopsy and/or fine-needle aspiration (transthoracic or transbronchial), excisional biopsy, and exfoliative cytology. Exfoliative respiratory cytology specimens are considered the least invasive of these techniques and include sputum, bronchial brushings/washings, and BAL. Sputum cytology is the least invasive method for obtaining a diagnosis of lung cancer, but it suffers from low sensitivity. It is particularly useful in patients with central, large tumors (>2.4 cm) in whom semi-invasive procedures such as flexible Table 1

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bronchoscopy and transthoracic needle aspiration may pose a higher risk. The diagnostic accuracy of sputum cytology increases with the number of adequate samples; a minimum of three samples is usually recommended. A sample is considered adequate if it contains numerous alveolar macrophages; otherwise, it is nondiagnostic or unsatisfactory. (These terms are essentially synonymous.) If sufficient spontaneously produced sputum cannot be obtained, sputum production can be induced. Newer imaging modalities such as low-dose spiral computed tomography scan and the detection of molecular abnormalities in sputum have shown promise in the early diagnosis of lung cancer. Flexible bronchoscopy with accompanying sampling procedures (e.g., brushings, washings, and BAL) is most useful in the diagnosis of central tumors; its sensitivity is lower for peripheral lesions, especially those < 2 cm in diameter. Bronchial washings and brushings are usually procured from clinically suspicious areas. Washings consist of repetitive instillation of 3–5 ml of a sterile balanced salt solution through the bronchoscope, and fluid is then reaspirated. For brushings, the surface of the abnormal lesion is scraped with a brush passed through the bronchoscope (Figure 1). Bronchial brushings and washings have a higher sensitivity than sputum cytology and are often combined with forceps biopsies to increase the overall diagnostic yield even more. BAL samples the terminal alveolar spaces (Figure 2). It is most helpful in the diagnosis of opportunistic pulmonary infections, especially in immunosuppressed patients, and it can aid in the diagnosis of diffuse pulmonary conditions such as alveolar proteinosis. To obtain the specimen, the bronchoscope is wedged into a subsegmental bronchus, sequential 20–100 ml of fluid is instilled into the distal airspaces, and the lavage fluid is reaspirated and submitted to a cytology laboratory for examination.

Gastrointestinal Specimens Gastrointestinal tract brushing cytology, especially from the esophagus and bile ducts, is a common specimen. The advantages of brushing cytology over tissue biopsy include sampling of a much larger mucosal surface area, fewer complications,

Types of exfoliative cytology specimens

Respiratory ○ Sputum ○ Bronchial bushings and washings ○ Bronchioloalveolar lavage Gastrointestinal ○ Esophageal and gastric brushings ○ Bile duct and pancreatic duct brushings Urological ○ Voided urine ○ Catheterized urine ○ Bladder washings and brushings ○ Ureter and renal pelvis washings and brushings ○ Ileal conduit urine Effusions ○ Pleural, peritoneal, and pericardial fluids ○ Synovial fluid Peritoneal washings Cerebrospinal fluid

Figure 1 Normal bronchial brushing. Normal bronchial epithelial cells are characterized by a columnar shape, delicate cytoplasm, a conspicuous terminal bar, and prominent cilia (arrowhead). Nuclei are round and smooth in contour, and chromatin is finely granular (Papanicolaou stain, ThinPrep™, 400 ).

Cytopathology | Nongynecologic Fluid and Brushing Cytology

Figure 2 Normal bronchioloalveolar lavage. Numerous round-to-oval, noncohesive alveolar macrophages are characteristic (Papanicolaou stain, ThinPrep™, 400 ).

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Figure 3 Benign bile duct epithelium. The cells are columnar and arranged in tightly cohesive flat sheets, with uniform smoothly contoured nuclei (Papanicolaou stain, ThinPrep™, 400 ).

Urine and Cystoscopic Specimens lower cost, and faster turnaround time to reporting of results. Esophageal brushing is performed during upper gastrointestinal tract endoscopy and is collected from the lesional area. The brush, contained within a sheath, is passed through the endoscopic channel. Esophageal brushing plays an important role in the diagnosis of infections such as candidiasis in both immunocompromised and immunocompetent patients. The value of routine brush cytology in the surveillance of patients with Barrett’s esophagus is debatable. Although cytology has excellent sensitivity in detecting high-grade dysplasia and adenocarcinoma (82%), its sensitivity for detecting low-grade dysplasia is as low as 31%. Histology has a higher accuracy in the diagnosis of low-grade and high-grade dysplasia. The application of molecular diagnostics to cytological material may play an important part in identifying those Barrett’s esophagus patients at risk of progression to cancer and in determining their response to treatment. Malignances of the pancreaticobiliary tract can present as distinct masses or duct strictures and are often associated with a poor prognosis. Radiologically evident pancreatic masses are usually sampled by fine-needle aspiration under endoscopic ultrasound guidance. Pancreatic and bile duct brush cytology plays an important role in the evaluation of pancreatic and bile duct strictures, as histological sampling in this area is difficult due to a high rate of complications and artifacts from tissue crushing and distortion. Establishing a definitive diagnosis of malignancy can aid in the early treatment of a resectable tumor and in the planning of palliative care for patients with unresectable tumors. For pancreatic and bile duct strictures, a brushing obtained by endoscopic retrograde cholangiopancreatography is the preferred sampling method (Figure 3). Bile duct brushing cytology has special value in the surveillance of patients with primary sclerosing cholangitis because of their high risk of cholangiocarcinoma. Early detection of malignancy in these patients may improve survival by enabling liver transplantation. The specificity of brush cytology for the diagnosis of pancreaticobiliary tract malignancy is > 95%, but sensitivity is considerably lower, ranging from 30% to 85%. Fluorescence in situ hybridization (FISH) shows promise as an adjunct test in improving the sensitivity of brush cytology in patients with malignant biliary strictures.

Bladder cancer is the eighth most common cancer in the United States, and 75% of bladder cancers are superficial (noninvasive) urothelial carcinomas. The majority of superficial urothelial carcinomas recur after resection, and approximately 15% progress to invasive disease. Because recurrence is difficult to predict, frequent surveillance is necessary. Urine, a liquid medium that traverses the entire urinary tract from the renal pelvis to the urethra, contains a broad sample of exfoliated urinary epithelial cells and lends itself easily for cytological examination. The majority of urinary tract malignancies are urothelial carcinomas, so the cytological diagnosis of urothelial carcinoma has become the main function of urine cytology. Urine cytology is used to evaluate patients with urinary symptoms (e.g., dysuria), asymptomatic patients with hematuria, and those at high risk for urothelial carcinoma, for example, with exposure to industrial chemicals such as aromatic amines. Currently, it is not cost-effective to screen the general population. Another major indication for urine cytology is the surveillance of patients with a known prior urothelial malignancy, as a complimentary test to cystoscopy and biopsy in detecting small and hidden lesions, for example, in a diverticulum, the ureter, or the renal pelvis. Five types of urine specimens exist: voided urine, catheterized urine, bladder washings and brushings, upper urinary tract brushings and washings, and ileal loop urine. The most inexpensive and widely used specimen is the voided urine. While of acceptable sensitivity for urothelial lesions of the bladder, it is less sensitive for neoplasms of the ureter and urethra, where directed washing and brushing techniques may be required. When the integrity of the lower urinary tract has been surgically disrupted by cystectomy, ileal loop urine specimens are obtained for surveillance.

Voided urine Voided urine is a common initial tool in the assessment of the entire urinary tract (Figure 4). The specimen should be obtained after hydration, 3–4 h after the patient last voided. A ‘first morning’ voided specimen should be avoided, as the integrity of its cellular component is compromised by

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Figure 4 Normal voided urine. There is predominance of intermediate urothelial cells (arrow) and the large ‘umbrella’ cells that line the surface of the urothelium (arrowhead). Urothelial cell cytoplasm has a finely granular and vacuolated appearance. The intermediate cells are polyhedral, with moderate amounts of cytoplasm, round to ovoid nuclei, occasional grooves, fine nuclear chromatin, and small nucleoli. The umbrella/superficial cells possess concave and convex edges corresponding to the inner and luminal edges, respectively. They are large, with abundant cytoplasm, one or multiple round nuclei, evenly distributed chromatin, and occasional prominent nucleoli (Papanicolaou stain, ThinPrep™, 400 ).

Figure 5 Instrumented urine (bladder washing). Urothelial cells are abraded by the instrument (catheter or cystoscope) and exfoliate in cohesive groups. Note the orderly arrangement of the group, capped by umbrella cells (Papanicolaou stain, ThinPrep™, 400 ).

prolonged contact with the toxic milieu of the urine. In women, voided urine can be contaminated by vaginal cells; therefore, to ensure the adequacy of the sample, a midstream (‘clean catch’) specimen is recommended. Three ‘clean catch’ voided urine samples collected over three consecutive days optimize the detection of urothelial malignancies.

Bladder washings and brushings Bladder washings and brushings are performed in patients at high risk for a new or recurrent urothelial cancer or when cystoscopy is performed for other reasons. Washings can be performed through a catheter: multiple pulses of sterile normal saline solution are instilled into the bladder and then recovered. Because these specimens are of much higher cellularity and better cellular preservation than voided urine, they are the preferred sample types for ancillary tests, including FISH and flow cytometry (FCM) (Figure 5). Disadvantages include a risk of infection and instrumentation artifact that may compromise the cytological interpretation.

Ureter and renal pelvis washings and brushings Upper urinary tract (ureter and renal pelvis) washings/brushings are usually employed to determine the nature of a spaceoccupying lesion identified in the upper urinary tract by radiological examination. Retrograde catheterization is performed to obtain washings; brushings can be obtained by direct visualization using a cystoscope. The pitfalls of ureteric/renal washing are similar to those of bladder washing. Brushing cytology is more accurate for the diagnosis of upper urinary tract neoplasms than other cytological techniques.

Figure 6 Ileal conduit urine. The intestinal glandular cells exhibit marked degeneration. They appear round (rather than columnar, their in situ appearance), with vacuolated cytoplasm, nuclear pyknosis, and karyorrhexis, and resemble macrophages (Papanicolaou stain, ThinPrep™, 400 ).

Catheterized urine Catheterized urine is a collection of a urine sample from an indwelling catheter. Although the specimen tends to be highly cellular, the cells are subject to marked degeneration and slough off in clusters, which can pose problems in the cytological diagnosis.

Ileal conduit urine Radical cystectomy is the standard treatment for muscleinvasive bladder cancer. Following cystectomy, an ileal conduit (‘neobladder’) is created whereby a segment of ileum is reanastomosed to the ureters and urethra, respectively (Figure 6). It is important to screen these patients because of their increased risk for developing cancers of the urinary tract.

Cytopathology | Nongynecologic Fluid and Brushing Cytology

Ancillary procedures The invasive nature of cystoscopy and the limited sensitivity of cytology in detecting low-grade urothelial carcinoma have led to a search for more sensitive, noninvasive testing methods. A variety of nonmorphological techniques have been developed to enhance the early recognition of recurrent urothelial malignancy. These techniques include assays of nuclear matrix protein 22 (NMP22), fibrin/fibrinogen degradation products, telomerase, FCM and image analysis for ploidy, cell cycle regulators, bladder tumor antigen (BTA) tests, FISH testing for a variety of chromosomal abnormalities, and microsatellite analysis. Currently, of these techniques, BTA and FISH represent the most utilized techniques, while microsatellite analysis is the most promising.

Effusions The pleural, pericardial, and peritoneal spaces are virtual cavities lined by a serous membrane that has a visceral and parietal aspect. These spaces normally enclose a small amount of fluid, just enough for the lubrication of the apposing surfaces. The cells lining these membranes are derived from the embryonic mesoderm and are known as mesothelial cells. Fluid in body cavities collects from the filtration of the plasma through the capillaries of the serous membranes and is dependent on the balance between the hydrostatic pressure, oncotic pressure, capillary permeability, and resorption of fluid through the veins and lymphatics (Figure 7). Any alteration in this balance results in an increased amount of fluid, termed an effusion. Effusions are broadly classified as transudates (arising due to imbalance between hydrostatic pressure and oncotic pressure) and exudates (usually due to vessel damage resulting in increased capillary permeability). As a general rule, malignancies result in the development of exudates (Table 2). Fluid is collected by inserting a needle into the respective cavity and can be sent for cytological, biochemical, and/or microbiological analysis. The peritoneal cavity can be accessed in women from the cul-de-sac (rectouterine pouch) through the vagina. It is recommended that at least 30–50 ml of fluid be sent

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for cytological analysis. The examination of multiple specimens increases the detection rate of malignancy, but approximately 90% of malignant effusions are diagnosed on the first specimen. Cytology is superior to pleural biopsy in diagnosing malignancy (sensitivity ¼ 71% vs. 35%) and is more cost-effective. False-positive diagnoses occur in less than 1% of cases and are mostly due to mesothelial atypia in the setting of pulmonary infarction, chemotherapy, acute pancreatitis, ovarian fibroma, cirrhosis, renal failure, and AIDS. Most effusions are due to benign etiologies such as congestive heart failure, nephrotic syndrome, cirrhosis, pancreatitis, and trauma. The predominant cellular components of benign effusions are mesothelial cells and histiocytes. Variable numbers of inflammatory cells, including lymphocytes, neutrophils, and eosinophils, may be also seen (Figure 8). Most patients with a malignant effusion have an established cancer diagnosis. A malignant effusion, however, is the initial manifestation of disease in 7–14% of cases, especially in Table 2

Transudate versus exudate

Characteristic Protein content Specific gravity Lactate dehydrogenase Pathogenesis

Important causes

Transudate 1

Exudate

< 3 g dl <1.015 <200 U l1

> 3 g dl1 >1.015 >200 U l1

Increased hydrostatic pressure Or Decreased oncotic pressure Or Both Congestive heart failure Nephrotic syndrome Cirrhosis Hypoproteinemia

Increased capillary permeability Or Decreased lymphatic resorption Or Both Malignant neoplasms Infections Autoimmune disorders Uremia Trauma

Lymphatic obstruction

Tumor cells Capillary Increase in hydrostatic pressure

Decrease in oncotic pressure

Increase in capillary permeability

Pathogenesis of Effusions

Figure 7 Schematic representation of the pathogenesis of effusions. The development of an effusion is governed by alterations in capillary hydrostatic pressure and plasma oncotic pressure. In the case of a malignant effusion, tumor cells cause lymphatic obstruction and damage capillary walls, leading to increased capillary permeability. Adapted from Kumar, V., Abbas, K.A., Fausto, N., et al. (Eds.), 2010. Robbins and Cotran Pathologic Basis of Disease, eighth ed. Philadelphia, Elsevier, with permission.

Figure 8 Benign pleural effusion. Mesothelial cells (in the center of the field) are often separated by a slit-like space or ‘window’ (arrowhead). They exhibit an outer lacy cytoplasm (arrow) and an inner dense cytoplasm. Nuclear chromatin is finely distributed, and nucleoli are not prominent. The background shows lymphocytes and histiocytes (Papanicolaou stain, ThinPrep™, 400 ).

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patients with mesothelioma and cancers of the lung and ovary. The most common malignancies causing pleural effusions are breast, lung, and gastrointestinal tract cancers and lymphomas/ leukemias. The most common malignancies associated with peritoneal effusions are ovarian, gastrointestinal tract, pancreatic, and breast cancers and lymphomas/leukemias.

Peritoneal Washings Peritoneal washings are not effusions but they are considered here because their cytological constituents are similar to those of pleural, pericardial, and peritoneal effusions. Peritoneal washings are obtained during surgery, most often for gynecologic and primary peritoneal cancers. Washings are particularly important in women with cancers of the ovary and fallopian tube because a positive result upstages the disease. Staging of endometrial cancers is not affected by the results of washing cytology, although some gynecologic oncologists believe that the presence of malignant cells adversely impacts prognosis. The peritoneal washing is performed by instilling 50– 200 ml of sterile physiological solution into different areas, including the pelvis, the right and left paracolic gutters, and the undersurface of the diaphragm, and then recollecting the fluid. A normal washing shows the sheets of mesothelial cells admixed with histiocytes (Figures 9 and 10). Diagnostic challenges include distinguishing low-grade malignancies from reactive mesothelial proliferations, endometriosis, and endosalpingiosis. High-grade malignancies are easier to recognize due to their greater degree of atypia.

Synovial Fluid Arthrocentesis (aspiration of fluid from a joint), along with synovial fluid analysis, is used in evaluating joint effusions or inflamed joints. It is especially useful in diagnosing septic and gouty arthritis. Normal synovial fluid is almost acellular, whereas septic arthritis is associated with numerous acute inflammatory cells. A Gram stain and culture studies help identify the causative organisms. Polarized light microscopy can be performed to look

Figure 9 Pelvic washing. Sheets of benign mesothelial cells (Papanicolaou stain, ThinPrep™, 200 ).

for crystals such as monosodium urate in gout and calcium pyrophosphate dihydrate in pseudogout.

Cerebrospinal Fluid CSF is formed by the choroid plexuses of the lateral and fourth ventricles through filtration of plasma across the capillaries and by active secretion. It circulates in the subarachnoid space between the pia mater and the arachnoid mater (the leptomeninges) and is reabsorbed into the venous circulation through the arachnoid granulations. CSF is most often sampled by lumbar puncture, through intervertebral space L3/L4 or L4/ L5, and it can be submitted for biochemical, microbiological, and cytological examination. Normal cellular components of CSF include lymphocytes and monocytes, ranging from 0 to 5 cells ml1. Red blood cells may be seen, but this is usually secondary to peripheral blood contamination. The malignant neoplasms with the greatest predilection for leptomeningeal metastases include lung cancer (especially small cell carcinoma and adenocarcinoma), breast cancer, melanoma, lymphoma, and leukemia. CSF cytology is instrumental in the diagnosis of such metastases. Primary brain tumors such as medulloblastoma may also involve the leptomeninges and shed cells into the CSF. The sensitivity of CSF cytology in detecting malignancy is approximately 60% and increases with the number of samples, the amount of specimen (> 10 ml is ideal), and the extent of leptomeningeal involvement. CSF cytology is also very useful in the diagnosis of nonneoplastic conditions such as meningitis. Acute bacterial meningitis shows numerous neutrophils, and offending organisms can be identified by microbiological cultures. Cryptococcus neoformans, an important cause of fungal meningitis, shows characteristic budding yeast forms. Aseptic or viral meningitis has nonspecific findings, including a predominance of lymphocytes and/or neutrophils.

Specimen Processing and Preparation Good cytopreparatory technique is essential for accurate cytological diagnosis. The most commonly employed specimen processing methods are direct smears, cytocentrifuge

Figure 10 Pelvic washing. Collagen balls (center of the field) are commonly encountered in pelvic washings. They are composed of a core of collagen and surrounded by mesothelial cells. They have no known pathological significance (Papanicolaou stain, ThinPrep™, 200 ).

Cytopathology | Nongynecologic Fluid and Brushing Cytology

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Specimen Processing and Preparation

95% Alcohol

(a) Direct smears

Air dry

Brushing

(b)

Cytospin preparation

Diff-quik stain

Decant supernatant

Centrifuge Cytolyt

Cytocentrifuge

Cytofunnel

(c)

Papanicolaou stain

Cytospin

Decant supernatant

Liquid-based preparation

Centrifuge

Cytolyt

Sure path Preservcyt

(d)

Cell block

Thin prep

Centrifuge

Cytolyt

Add plasma

Filtration

Clot Add thrombin forms

Add formalin

Cyto Sedimentation rich

Decant supernatant

Cell Load Cassette block in processor

Figure 11 Specimen processing and preparation. Nongynecologic, exfoliative cytology specimens can be prepared by different methods. (a) Direct smears are prepared from brushings by rolling the end of the brush on a glass slide and fixing it in 95% alcohol for Papanicolaou staining or air-drying it for a Romanowsky-type stain. For processing the samples, different techniques can be employed. Usually, the sample is received fresh or in some type of transport medium. After initial centrifugation, the sediment is processed using a cytocentrifuge (yielding cytospins); or (b) the ThinPrep™ processor, which uses vacuum to trap cells on a polycarbonate filter that is pressed against a slide; or (c) the cell block method (e.g., the thrombin-clot method, whereby thromboplastin and plasma are added to the specimen to form a clot that is formalin-fixed and processed for histological examination); or (d) the SurePath™ method. For the latter, the specimen is received in CytoRich fixative (ethanol-based), and slides prepared by centrifugation with a density gradient reagent.

preparations, liquid-based preparations, and cell block sections (Figure 11). Brushings can be collected directly into fixative solutions such as CytoLyt solution or 50% alcohol. Body fluids may be sent fresh to the laboratory and can be refrigerated at 4  C until fixation. With brushings, the brush is cut off from its shaft, placed in fixative fluid, and transported to the laboratory. Concentration techniques are especially useful with specimens that are naturally of low cellularity, such as urine and CSF. One or more of the following methods may be utilized for cytopreparation.

Direct Smears Direct smears can be prepared from brushings by rolling the end of the brush on a glass slide and immediately fixing the slide(s) in 95% alcohol (or by spray fixing). The slides are

subsequently stained with a Papanicolaou stain and submitted for cytological examination.

Cytocentrifuge Preparation (Cytospin™) A cytocentrifuge takes cells in fluid suspension and transfers them onto a slide. The cytocentrifuge equipment, in addition to the centrifuge itself, consists of a plastic funnel, a filter card (which adsorbs excess fluid), and a glass slide (with a ‘well’ etched on it). A few drops of a precentrifuged specimen are placed in the funnel, and the centrifugal force of the centrifuge transfers the cells, which are denser than the fluid medium, onto the slide. The slide is either immediately fixed in 95% alcohol for Papanicolaou staining or air-dried for the DiffQuik (or other Romanowsky-type) stain.

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Liquid-Based Preparations With the successful application of liquid-based preparation (LBP) technology to the gynecologic Pap test, it has been increasingly employed for nongynecologic specimens as well. Compared to cytospin preparations, LBP has several advantages, including improved cellular distribution with minimal overlap, optimized cellular preservation and nuclear chromatin detail, and a cleaner background. ThinPrep™ and SurePath™ are the most common LBP methodologies. The ThinPrep™ and SurePath™ methods are based on filtration and density gradient sedimentation, respectively.

Cell Block Sections Cell blocks are useful adjuncts to a cytospin and LBP; they are not usually prepared as the sole cytological preparation because they require more time. Cell blocks are ideal for demonstrating architectural detail and for ancillary testing such as immunohistochemistry (IHC) and molecular studies. They are routinely employed in the evaluation of body fluids and occasionally for other exfoliative cytology specimens. The fluid sample is centrifuged to a cell pellet, after which several methods of cell block preparation can be employed. One of the more common is the thrombin-clot method: it involves congealing the cell sediment into a clot by adding a drop or two each of thromboplastin and discarded human plasma.

The clot is then formalin-fixed and paraffin-embedded for histological examination.

Applications to Diagnosis Reactive Changes Urothelial cells, mesothelial cells, bronchial cells, and pancreatic/bile duct epithelial cells all show reactive changes in response to noxious stimuli such as infection, inflammation, radiation, and chemotherapy (Figure 12(a)–12(d)). Cytologically, reactive changes are characterized by a low nuclear-tocytoplasmic ratio, evenly distributed chromatin, smooth nuclear membranes, and prominent nucleoli. Radiation and chemotherapeutic agents cause marked cytomegaly, multinucleation, polychromasia of the cytoplasm, cytoplasmic and nuclear vacuolation, and a smudgy nuclear hyperchromasia. Reactive changes can be so pronounced that it may be difficult to rule out a malignancy.

Infections Viral infections Several viral infections are associated with characteristic cytopathic effects, on the basis of which they can be readily diagnosed on exfoliative cytology specimens. Herpes simplex and

Figure 12 Reactive changes. (a) Urothelial cells. (b) Mesothelial cells. (c) Bile duct epithelium. (d) Bronchial cells. Reactive change is characterized by the maintenance of a low nuclear-to-cytoplasmic ratio. In addition, the chromatin is evenly distributed, the nucleoli become prominent, and the nuclear membranes remain smooth (Papanicolaou stain, ThinPrep™, 400 ).

Cytopathology | Nongynecologic Fluid and Brushing Cytology

cytomegalovirus cytopathic changes can be seen in BALs, esophageal brushings, and urine specimens, especially in immunosuppressed patients (Figures 13 and 14). Adenovirus infection manifests as intranuclear inclusions in bronchial epithelial cells and a detachment of their cilia (‘ciliocytophthoria’). Cellular changes associated with human papillomavirus infection are occasionally seen in urine specimens, but they are most often attributable to contamination from a genital tract lesion (Figure 15). Polyomaviruses are an important cause of nephropathy in renal allograft recipients. The majority of cases are due to the BK polyomavirus, although JC polyomavirus is implicated in some cases. They cause a primary infection during childhood, enter a latency phase, and reactivate with changes in immune status. Infected renal tubular and urothelial cells have a characteristic cytomorphology in urine and are known colloquially as ‘decoy cells.’ Decoy cells were first identified in the 1950s

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by Mr. Andrew Ricci, a senior cytotechnologist at the Memorial Hospital for Cancer in New York. He termed them after the decoy ducks used in hunting because these infected cells can be easily mistaken for cancer cells (Figure 16). Urine cytology is an easy method for diagnosing polyoma viral infection and has a very high negative predictive value in excluding BK virusassociated nephropathy. Cytological recognition of BK viral cytopathic changes may not be specific for nephropathy, however: it may simply represent viral reactivation. Other screening methods for BK virus include polymerase chain reaction (PCR) serum assays, but the gold standard for a definitive diagnosis of BK virus-associated nephropathy is renal biopsy.

Figure 15 Human papillomavirus cytopathic effect in urine. The cells exhibit enlarged, hyperchromatic nuclei surrounded by a sharply punched-out cytoplasmic halo with thick cytoplasmic edges. Binucleation is observed in some cells (Papanicolaou stain, ThinPrep™, 400 ).

Figure 13 Herpes simplex (esophageal brushing). Herpetic cytopathic effect is evidenced by nuclear enlargement, ground-glass chromatin, margination of chromatin, nuclear molding, and multinucleation (upper right). This is in contrast to the normal squamous cells in the lower left side of the field (Papanicolaou stain, ThinPrep™, 400 ).

Figure 14 Cytomegalovirus (bronchioloalveolar lavage). The infected cell is enlarged and shows a basophilic intranuclear inclusion (arrow). Small cytoplasmic inclusions are also identifiable (Papanicolaou stain, ThinPrep™, 400 ).

Figure 16 Polyomavirus (voided urine). Viral effect is characterized by an enlarged nucleus with a smooth nuclear membrane and a groundglass, homogeneous inclusion. Due to their high nuclear-to-cytoplasmic ratio, these cells were once mistaken for carcinoma cells, hence the term ‘decoy cells’ (Papanicolaou stain, ThinPrep™, 400 ).

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Bacterial infections In patients with tuberculosis, mycobacteria can sometimes be recovered from sputum, bronchial washings, BALs, urine, CSF, and synovial fluid. For sputum, a minimum of three early morning specimens should be collected on three successive days to enhance the recovery of mycobacteria. Acid-fast stains can highlight mycobacteria, but the sensitivity of cytology is limited compared to microbiological studies. Current microbiological methods involve a combination of phenotypic and molecular assays for the rapid identification of mycobacteria. Nocardia spp. are opportunistic pathogens encountered in immunocompromised patients, especially those on long-term corticosteroid therapy or with chronic lung disease. They are thin, beaded, branched, filamentous rods with the modified Kinyoun acid-fast stain. Actinomyces israelii is a normal inhabitant of the tonsillar region and commonly seen as a harmless contaminant in respiratory specimens (Figure 17). The demonstration of tissue invasion is required for the diagnosis of a pathogenic actinomycosis infection.

Figure 18 Candida (esophageal brushing). These fungi show a mixture of yeast forms (round) and pseudohyphae (elongated). This pattern is often referred to as ‘spaghetti and meat balls’ (Papanicolaou stain, ThinPrep™, 400 ).

Fungal infections Respiratory exfoliative cytology specimens, especially BAL, play an important role in detecting fungal infections in both immunocompetent and immunosuppressed individuals. Esophageal brushings aid in the diagnosis of esophageal candidiasis (Figure 18). Candida spp. seen in respiratory specimens, on the other hand, are frequently a result of contamination from oral flora. Systemic candidiasis is observed in patients with debilitating conditions such as the blast crises of leukemias and following organ transplantation. Pneumocystis jiroveci (formerly P. carinii) is a yeast-like fungus that causes pneumonia with widespread pulmonary infiltrates in immunocompromised patients, especially those with AIDS. It is best seen in BAL specimens, which reveal foamy alveolar casts containing the Pneumocystis cysts. These can be visualized with the Grocott methenamine silver (GMS) stain (Figure 19(a) and 19(b)).

Figure 17 Actinomyces (esophageal brushing). These long filamentous bacteria appear in clumps resembling ‘cotton balls.’ They are normal inhabitants of the oral cavity and are encountered in respiratory and gastrointestinal specimens (Papanicolaou stain, ThinPrep™, 400 ).

Figure 19 (a) Pneumocystis jiroveci. This infection appears as foamy alveolar casts in bronchoalveolar lavage specimens (Papanicolaou stain, ThinPrep™, 400 ). (b) The organisms are highlighted with a Grocott methenamine silver stain, appearing as black, cup- or boat-shaped structures (GMS stain, ThinPrep™, 400 ).

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Figure 20 Aspergillus spp. (bronchial washing). Aspergillus spp. appear as septate hyphae with dichotomous branching, often at a 45 angle, highlighted here by a Grocott methenamine silver stain (ThinPrep™, 400 ).

Aspergillus spp. can cause bronchopulmonary and disseminated disease in immunosuppressed hosts. They are characterized by septate, regular hyphae with dichotomous branching at a 45 angle (Figure 20). Fruiting bodies are seen in lung cavities connected to open bronchi and exposed to air. Certain species produce oxalic acid and birefringent calcium oxalate crystals. Zygomycetes have broad, aseptate, ribbonlike hyphae with 90 branching. They can cause pulmonary disease and are seen in exfoliative cytology specimens. Histoplasma and Blastomyces mainly cause pulmonary disease and are seen most often on BAL specimens. Histoplasma capsulatum infection is endemic in the Ohio, Mississippi, and Missouri river valleys in the United States. Intracellular and extracellular yeast forms are best highlighted by the GMS stain (Figure 21(a) and 21(b)). Blastomyces dermatitidis shows broad-based budding yeast forms. Coccidioides spp. are endemic in the southwestern United States, Mexico, and Central and South America. Two different species have been identified: Coccidioides immitis (California species) and Coccidioides posadasii (non-California species). Most patients are asymptomatic; disseminated disease develops in < 5% of patients. The lungs are the most affected, but infection can involve other organs including the lymph nodes, skin, spleen, liver, kidneys, bones, joints, and meninges and the central nervous system. Diagnostic specimens include sputum, BAL, transtracheal aspirates, pleural fluid, and lung tissue, and, in extrapulmonary sites, skin and bone biopsies, pus from abscesses, joint fluid, and CSF. Fungal identification is usually performed by biopsy, culture, and DNA probes. The diagnostic fungal forms are in the form of spherules filled with endospores, best seen with the GMS stain. C. neoformans causes meningitis, pneumonia, and disseminated disease and causes disease in both immunocompetent and immunosuppressed individuals. It is considered one of the AIDS-defining infections. Cryptococcus consists of irregularly sized, encapsulated yeast forms with teardrop budding (Figure 22(a)–22(c)).

Figure 21 Histoplasma capsulatum (BAL). (a) Tiny yeast forms accumulate in the cytoplasm of alveolar macrophages (Papanicolaou stain, ThinPrep™, 400 ). The organisms are highlighted with a Gomori methenamine silver stain (b) (ThinPrep™, 400 ).

Parasites and protozoa Trichomonas vaginalis is a flagellated protozoan and the causative agent of trichomoniasis, the most common sexually transmitted disease. Most patients are asymptomatic, but mild to severe inflammation, with pruritus, dysuria, and dyspareunia, and a foul-smelling discharge can occur. It is most often diagnosed via cervical (‘Pap’) smear or wet mount of a genital discharge. The organism is rarely encountered in urine specimens as a contaminant from the genital tract, but primary bladder infections do occur. Newer methods of testing include rapid antigen testing and the PCR.

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Schistosoma haematobium is a trematode infestation commonly found in Egypt, India, Portugal, and Africa. The eggs are laid in the pelvic and bladder veins, and the larvae infect the bladder. Due to chronic irritation, these patients are prone to the development of squamous cell carcinoma of the bladder. The eggs are occasionally seen in urine samples and have a characteristic terminal spine. Strongyloides stercoralis is a nematode that causes disseminated infection in immunocompromised individuals. Filariform larvae are found in sputum and bronchial washing/BAL specimens.

Tumors Squamous cell carcinoma In the respiratory tract, the majority of squamous cell carcinomas are central in location, thus enabling their diagnosis in many instances by sputum and bronchial brushing cytology (Figure 23). The diagnosis of a moderately or poorly differentiated squamous carcinoma is usually easily made in cytological specimens. Atypical squamous metaplasia, however, a benign condition in the setting of lung injury due to pulmonary infarction, fungal infection, radiation injury, or diffuse alveolar damage, is a cytological mimic of well-differentiated squamous carcinoma and thus a potential pitfall. Similarly, esophageal squamous carcinomas are readily diagnosed by esophageal brushing cytology. Squamous differentiation is associated with some urothelial carcinomas and can be detected in voided and instrumented urine specimens. Pure squamous carcinomas of the urinary tract, however, are extremely rare except where S. haematobium is endemic. Squamous cell carcinomas of pulmonary, esophageal, or gynecologic origin are occasionally detected in pleural and ascites fluids (Figure 24).

Urothelial carcinoma According to the 2004 World Health Organization (WHO) classification, urothelial neoplasms are subclassified into invasive and noninvasive urothelial neoplasms (Table 3). These are further divided into flat and papillary lesions. Papillary urothelial lesions include papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade urothelial carcinoma, and high-grade urothelial carcinoma. Papillomas are biologically benign. PUNLMP lacks the capacity for invasion or metastasis but is associated with a 25–45% risk for recurrence. The risk of progression is less than 8% and usually secondary to the development of high-grade carcinoma. PUNLMP and papilloma are virtually impossible to recognize by cytology. The majority of low-grade urothelial neoplasms do not demonstrate invasion of the bladder muscular wall or metastases. Moreover, they appear to remain stable over the course of the

Figure 22 Cryptococcus neoformans (cerebrospinal fluid). (a) The organism manifests as yeast forms of different sizes. On Pap stain, they are visualized as negative images (arrowhead) (Papanicolaou stain, ThinPrep™, 400 ). (b) The organisms are best highlighted with a Grocott methenamine silver stain. Narrow-based budding is characteristic of this organism, resulting in a ‘teardrop’ appearance. (c) Mucicarmine stains the capsule red, further emphasizing its budding nature (c) (ThinPrep™, 400 ).

Figure 23 Squamous cell carcinoma (bronchial brushing). Malignant keratinized squamous cells derived from a squamous cell carcinoma show abnormal shapes and nuclei (Papanicolaou stain, direct smear, 400 ).

Cytopathology | Nongynecologic Fluid and Brushing Cytology

Figure 24 Squamous cell carcinoma (pleural fluid). In comparison with the mesothelial cells, these cells are much larger; they have abnormal chromatin distribution and macronucleoli. Squamous cell carcinomas are uncommon in effusion specimens (Papanicolaou stain, ThinPrep™, 400 ).

Table 3 2004 WHO classification of papillary urothelial neoplasms (modified)

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Figure 25 High-grade urothelial carcinoma (bladder washing). The tumor cells are described as ‘cercariform,’ having a cytoplasmic tail (bearing resemblance to cercariae larval forms). They show a high nuclear-to-cytoplasmic ratio, nuclear hyperchromasia and pleomorphism, and abnormal chromatin distribution (Papanicolaou stain, ThinPrep™, 400 ).

Noninvasive urothelial neoplasms Papilloma Papillary urothelial neoplasm of low malignant potential Low-grade urothelial carcinoma High-grade urothelial carcinoma Urothelial carcinoma in situ Invasive urothelial carcinoma Low-grade urothelial carcinoma High-grade urothelial carcinoma Source: Adapted from Eble, J.N., Sauter, G., Epstein, J.I., Sesterhenn, I.A. (Eds.), 2004. World Health Organization Classification of Tumors. Pathology and Genetics of Tumors of the Urinary System and Male Genital Organs. IARC Press, Lyon, with permission.

patient’s lifetime, and additional lesions that may develop most likely represent new primaries rather than a recurrent low-grade papillary neoplasm. High-grade urothelial cancers show marked cytological atypia and include carcinoma in situ (CIS) and high urothelial carcinoma (Figures 25 and 26). Lowgrade urothelial carcinomas demonstrate only mild-tomoderate atypia (Figure 27(a) and 27(b)). The cytological diagnosis of high-grade urothelial carcinoma and CIS is highly accurate, with a sensitivity of at least 79% and a negative predictive value of over 90%. Urothelial CIS is difficult to detect by cystoscopy because it is virtually indistinguishable from cystitis. Urine cytology is therefore the best method to diagnose CIS (Figure 28(a) and 28(b)). Lowgrade urothelial carcinomas have relatively low diagnostic sensitivity (20–50%) because they are often difficult to distinguish cytologically from instrumentation artifact, stone atypia, and other benign mimics.

Adenocarcinoma Adenocarcinoma is the most common malignancy encountered in cytological specimens. Because a BAL samples the alveolar spaces, it has a particularly high sensitivity for detecting tumors with a lepidic pattern (growth along the alveolar

Figure 26 High-grade urothelial carcinoma (renal pelvic brushing). The tumor cells in this example form three-dimensional groups. The cells have a high nuclear-to-cytoplasmic ratio, irregular nuclear membranes, and abnormal chromatin distribution (Papanicolaou stain, ThinPrep™, 400 ).

walls), including adenocarcinoma in situ (Figure 29). Highgrade invasive adenocarcinoma of the lung is relatively easy to diagnose, but low-grade adenocarcinomas can be hard to distinguish from reactive type II pneumocytes, which are associated with diffuse alveolar damage, pulmonary infarction, and pneumonia. Adenocarcinoma in an effusion may be the initial presentation of a cancer of unknown primary origin (Figure 30). Adenocarcinomas and high-grade dysplasias of the esophagus, usually in the setting of Barrett’s esophagus, have similar features on brushing cytology. Pancreatic and bile duct brushings can sample ductal adenocarcinomas of the pancreas and cholangiocarcinomas (Figure 31). Pure adenocarcinoma of the urinary bladder is rare, but urothelial carcinomas can exhibit focal glandular differentiation.

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Figure 27 Low-grade urothelial carcinoma. (a) Bladder washing specimen with a monomorphic population of neoplastic cells with eccentrically placed nuclei. (b) Catheterized urine specimen with clusters and single atypical urothelial cells with eccentric nuclei and mild atypia (Papanicolaou stain, ThinPrep™, 400 ). (c) Histological section of renal pelvis tumor showing papillary architecture, increased epithelial thickness, and loss of polarity. The neoplastic cells are similar in appearance to the cytological specimens (H&E, 400 ).

Small cell carcinoma Small cell carcinoma is a high-grade neuroendocrine carcinoma, most commonly seen in respiratory specimens but occasionally in effusions and urine specimens (Figure 32). Sputum and bronchial brushing and washing cytology are useful tools in establishing the diagnosis, because small cell carcinoma tends to present as a central mass in or adjacent to the hilum. Cytology shows small, highly atypical cells with prominent nuclear molding and streaking (Figure 33). Small cell carcinomas in fluid specimens most commonly arise from the lung but may rarely originate from the cervix, pancreas, colon, or head and neck. Rarely, small cell carcinoma arises as a primary malignancy of the urinary tract. The major entities to be considered in the differential diagnosis are lymphoma and poorly differentiated non-small cell carcinoma, in which case IHC is required for a definitive diagnosis.

Mesothelioma Pleural and peritoneal fluid cytology plays a major role in the diagnosis of malignant mesothelioma (Figure 34). An effusion that is suspicious for malignant mesothelioma usually prompts a pleural (or peritoneal, as the case may be) biopsy for a

definitive diagnosis. Some of the challenges encountered in effusion cytology include the distinction of metastatic adenocarcinoma from reactive mesothelial cells and mesothelioma. The latter differential diagnosis is aided by IHC in conjunction with clinical and radiological correlation.

Lymphoma/leukemia Lymphomas cause up to 15% of malignant effusions. Of all lymphomas, T-cell lymphoblastic lymphomas have a special propensity for serosal involvement. High-grade lymphomas such a diffuse large B-cell lymphoma and Burkitt lymphoma are relatively easy to recognize by cytomorphology (Figure 35). Low-grade lymphomas like follicular lymphoma and small lymphocytic lymphoma are hard to distinguish from reactive lymphoid proliferations by cytomorphology alone; in such cases, immunophenotyping by FCM or immunocytochemistry can be essential to demonstrate B-cell clonality. The cytological differential diagnosis includes small round blue cell tumors (in children), small cell carcinoma, and melanoma. CSF may also be involved by lymphoma. Lymphomas with a predilection for leptomeningeal metastases include diffuse large B-cell lymphoma, lymphoblastic lymphoma, and Burkitt lymphoma.

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Figure 30 Adenocarcinoma (pleural fluid). In contrast to the benign mesothelial cell (top left corner), the tumor cells are in large, tightly cohesive clusters. They exhibit high nuclear-to-cytoplasmic ratios, irregular chromatin distribution, and prominent nucleoli (Papanicolaou stain, ThinPrep™, 400 ).

Figure 28 Urothelial carcinoma in situ. (a) This voided urine specimen contains malignant urothelial cells, identified as such because of their abnormally shaped, hyperchromatic nuclei. Note the irregular nuclear membranes (arrowhead) (Papanicolaou stain, ThinPrep™, 400 ). (b) The corresponding histological section shows a noninvasive flat carcinoma in situ. Note the high nuclear-to-cytoplasmic ratios and hyperchromatic nuclei (H&E, 400 ).

Figure 29 Adenocarcinoma (BAL). Cohesive clusters of this welldifferentiated adenocarcinoma demonstrate cellular monomorphism, pale chromatin, and nuclear grooves. The smaller cells in the background are alveolar macrophages (Papanicolaou stain, ThinPrep™, 400 ).

Figure 31 Adenocarcinoma (bile duct brushing). In contrast to the benign ductal cells (bottom half), the tumor cells (top half) show architectural disorganization (as manifested by nuclear overlapping and crowding), nuclear pleomorphism, and abnormal chromatin distribution (Papanicolaou stain, ThinPrep™, 400 ).

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Figure 32 Small cell carcinoma (pleural fluid). The malignant cells have a minimal amount of cytoplasm and hyperchromatic nuclei with high nuclear-to-cytoplasmic ratios, in contrast to the mesothelial cells and histiocytes in the background (left lower corner) (Papanicolaou stain, ThinPrep™, 400 ).

Figure 33 Small cell carcinoma (bronchial brushing). The malignant cells show prominent nuclear streaking and molding. The nuclei have a mixture of fine and coarse nuclear chromatin (often referred to as ‘salt and pepper’) (Papanicolaou stain, direct smear, 400 ).

Acute lymphoblastic and myeloid leukemias can also involve the CSF (Figure 36). Adjunctive studies including FCM, IHC, and molecular studies such as FISH and the PCR are often essential complements to cytology for the definitive diagnosis and classification of lymphoma/leukemia.

Sarcoma Sarcomas are rarely encountered in exfoliative cytology. They may involve the serosal surfaces in patients with a known primary tumor and widespread disease. Comparison of the cytology to the original tumor and the use of ancillary studies such as IHC aid in establishing the diagnosis. Leiomyosarcomas, rhabdomyosarcomas, liposarcomas, synovial sarcomas, epithelioid hemangioendotheliomas, osteosarcomas, and chondrosarcomas have been reported in effusions.

Figure 34 Mesothelioma (pleural fluid). The specimen can be highly cellular, with large clusters containing more than 50–100 tumor cells. The clusters have scalloped borders, and the neoplastic cells retain their mesothelial appearance. The degree of cytological atypia is variable but usually mild (as in this case) (Papanicolaou stain, ThinPrep™, 400 ).

Figure 35 Large cell lymphoma (pericardial fluid). These large atypical lymphoid cells proved to be monoclonal B cells by flow cytometry (Papanicolaou stain, ThinPrep™, 400 ).

Metastatic tumors The majority of patients with a malignant effusion have a wellestablished history of a previous malignancy, the most common being carcinomas of the lung, breast, ovary, pancreas, gastrointestinal tract, kidney, prostate, and endometrium and melanoma (Figure 37(a) and 37(b)). In the event of an unknown primary, a panel of immunohistochemical stains, in conjunction with the clinical and radiological information, helps in determining the primary site. Prostate and colon cancer can directly invade the bladder wall and exfoliate cells in urine. CSF cytology aids in diagnosis of metastatic tumors that have predilection for leptomeningeal metastases, such as the lung, breast, and melanoma.

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Figure 36 Acute lymphoblastic leukemia (cerebrospinal fluid). The sample consists of lymphoid blasts (Papanicolaou stain, ThinPrep™, 400 ).

Ancillary Studies Special Stains and Microbiology Cultures Special stains in exfoliative cytology are used predominantly for the identification of microorganisms. These include stains for acid-fast bacilli (Ziehl–Neelsen stain) and fungal organisms (GMS stain), which can be performed on cell block sections, cytospins, or LBP. Fresh, unfixed material can be sent in sterile containers for microbiological culture studies. These are especially useful in the analysis of BAL and CSF specimens.

Immunohistochemistry Immunohistochemical stains detect specific antigens on the surface of cells and thus help determine their lineage. The antigen binds to an antibody or a series of antibodies, which are detected using an enzymatic reaction that forms a colored product. The streptavidin–biotin–peroxidase technique is a commonly employed method. IHC is ideally performed on formalin-fixed, paraffin-embedded cell block material, but they can also be performed on cytospins and LBP. Some of the important applications of these stains include distinguishing reactive mesothelial cells and mesothelioma from adenocarcinoma and determining the primary site of malignancy in body fluid specimens (Figure 38(a)–38(c)).

Flow Cytometry Flow cytometric analysis for the diagnosis of lymphoma and leukemia consists of immunophenotyping cells utilizing a series of cell surface markers tagged with different fluorescent dyes. FCM uses a panel of appropriately selected monoclonal antibodies and measures multiple fluorescence and light scatter-associated parameters from individual cells as they flow in single file through a light excitation source. It can confirm the morphological impression of a reactive or neoplastic lymphoproliferative process and aid in the interpretation of cytologically equivocal cases. The determination of clonality essentially rests on the identification of light chain restriction in B-cell lymphomas and aberrant surface markers in T-cell lymphomas. The analysis of fresh

Figure 37 Melanoma (pleural fluid). (a) Melanoma cells often have markedly prominent nucleoli (arrowhead) (Papanicolaou stain, ThinPrep™, 400 ). (b) The same tumor cells are readily seen on this cell block section (arrowheads) (H&E, 400 ). The diagnosis is confirmed by positive immunostaining with S-100 (c) and HMB-45 (d) (immunostain, cell block, 400 ).

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Figure 38 Adenocarcinoma (pleural fluid). (a) These tumor cells have high nuclear-to-cytoplasmic ratios, abnormal chromatin distribution, and prominent nucleoli (Papanicolaou stain, ThinPrep™, 400 ). As shown by immunohistochemistry on cell block sections, the tumor cells are positive for MOC-31 (membranous staining (b)) and CEA (cytoplasmic staining (c) (immunostain, cell block, 400 ).

fluids by FCM is best performed within 48 h of specimen procurement.

Molecular Studies A growing armamentarium of molecular studies can be applied to exfoliative cytology specimens, including respiratory, pleural and peritoneal fluids, CSF, and urine. These include FISH, mutational analysis, and PCR. Due to advances in the understanding of the molecular biology of lung cancers – and the downstream treatment implications – non-small cell carcinomas on cytological material need to be further classified as adenocarcinomas and squamous cell carcinomas. Some lung adenocarcinomas harbor mutations in the epidermal growth factor receptor (EGFR), rendering them susceptible to tyrosine

kinase inhibitors; others exhibit anaplastic lymphoma kinase (ALK) gene rearrangements and respond to ALK inhibitors like crizotinib. A cell block or smear from a fluid specimen with sufficient quantity and density of tumor cells can be employed for EGFR mutational analysis and FISH for the ALK gene rearrangement. FISH for cytogenetic abnormalities and PCR for the demonstration of clonal gene rearrangements can also be performed on cytological material for the diagnosis of lymphoproliferative disorders. UroVysion™ (Abbott Molecular, Inc.) is a multiprobe FISH assay and the most widely employed molecular test in exfoliative nongynecologic cytology. It is approved by the Food and Drug Administration for the surveillance of patients with a previously diagnosed urothelial cancer and for patients with

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Figure 39 UroVysion™ findings in bladder cancer. (a) Note the abnormal cell (arrowhead) with aneusomy of chromosomes 3 (red), 7 (green), and 17 (aqua). (b) In contrast to the cell at the bottom, the cell at the top exhibits homozygous loss of 9p21 (gold).

hematuria. It identifies aneuploidy of chromosomes 3, 7, and 17 and loss of the 9p21 locus (deletion of the p16 gene), aberrations frequently seen in urothelial cancer (Figure 39(a) and 39(b)). UroVysion™ has higher sensitivity than cytology for all grades and stages of urothelial cancer. In patients under surveillance, FISH may be positive when the cytology is negative and no visible lesions are detected. These cases are termed ‘anticipatory positives,’ and up to 65% of such patients develop tumors on follow-up.

Further Reading Respiratory Specimens Rivera, M.P., Mehta, A.C., American College of Chest Physicians, 2007. Initial diagnosis of lung cancer: ACCP evidence-based clinical practice guidelines (second ed.). Chest 132 (Suppl. 3), 131S–148S. Suen, K.C., Abdul-Karim, F.W., Kaminsky, D.B., Layfield, L.J., Miller, T.R., Spires, S.E., et al., 1999. Guidelines of the Papanicolaou Society of Cytopathology for the examination of cytologic specimens obtained from the respiratory tract. Papanicolaou Society of Cytopathology Task Force on Standards of Practice. Diagn. Cytopathol. 21 (1), 61–69. Idowu, M.O., Powers, C.N., 2010. Lung cancer cytology: potential pitfalls and mimics— a review. Int. J. Clin. Exp. Pathol. 3 (4), 367–385. Witt, B.L., Wallander, M.L., Layfield, L.J., Hirschowitz, S., 2012. Respiratory cytology in the era of molecular diagnostics: a review. Diagn. Cytopathol. 40 (6), 556–563. Saad, R.S., Silverman, J.F., 2010. Respiratory cytology: differential diagnosis and pitfalls. Diagn. Cytopathol. 38 (4), 297–307. National Lung Screening Trial Research Team, Aberle, D.R., Adams, A.M., Berg, C.D., Black, W.C., Clapp, J.D., et al., 2011. Reduced lung-cancer mortality with low-dose computed tomographic screening. N. Engl. J. Med. 365 (5), 395–409.

Katz, R.L., Zaidi, T.M., Fernandez, R.L., Zhang, J., He, W., Acosta, C., et al., 2008. Automated detection of genetic abnormalities combined with cytology in sputum is a sensitive predictor of lung cancer. Mod. Pathol. 21 (8), 950–960. Travis, W.D., Brambilla, E., Noguchi, M., Nicholson, A.G., Geisinger, K.R., Yatabe, Y., et al., 2011. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma. J. Thorac. Oncol. 6 (2), 244–285. Gastrointestinal Specimens Geisinger, K.R., Teot, L.A., Richter, J.E., 1992. A comparative cytopathologic and histologic study of atypia, dysplasia, and adenocarcinoma in barrett’s esophagus. Cancer 69 (1), 8–16. Kumaravel, A., Lopez, R., Brainard, J., Falk, G.W., 2010. Brush cytology vs. endoscopic biopsy for the surveillance of Barrett’s esophagus. Endoscopy 42 (10), 800–805. Saad, R.S., Mahood, L.K., Clary, K.M., Liu, Y., Silverman, J.F., Raab, S.S., 2003. Role of cytology in the diagnosis of Barrett’s esophagus and associated neoplasia. Diagn. Cytopathol. 29 (3), 130–135. Selvaggi, S.M., 2004. Biliary brushing cytology. Cytopathology 15 (2), 74–79. Athanassiadou, P., Grapsa, D., 2008. Value of endoscopic retrograde cholangiopancreatography-guided brushings in preoperative assessment of pancreaticobiliary strictures: what’s new? Acta Cytol. 52 (1), 24–34. Barr Fritcher, E.G., Kipp, B.R., Voss, J.S., Clayton, A.C., Lindor, K.D., Halling, K.C., et al., 2011. Primary sclerosing cholangitis patients with serial polysomy fluorescence in situ hybridization results are at increased risk of cholangiocarcinoma. Am. J. Gastroenterol. 106 (11), 2023–2028. Boberg, K.M., Jebsen, P., Clausen, O.P., Foss, A., Aabakken, L., Schrumpf, E., 2006. Diagnostic benefit of biliary brush cytology in cholangiocarcinoma in primary sclerosing cholangitis. J. Hepatol. 45 (4), 568–574. Lewis, J.T., Talwalkar, J.A., Rosen, C.B., Smyrk, T.C., Abraham, S.C., 2010. Precancerous bile duct pathology in end-stage primary sclerosing cholangitis, with and without cholangiocarcinoma. Am. J. Surg. Pathol. 34 (1), 27–34. Kipp, B.R., Fritcher, E.G., Clayton, A.C., Gores, G.J., Roberts, L.R., Zhang, J., et al., 2010. Comparison of KRAS mutation analysis and FISH for detecting pancreatobiliary tract cancer in cytology specimens collected during endoscopic retrograde cholangiopancreatography. J. Mol. Diagn. 12 (6), 780–786.

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Cytopathology | Nongynecologic Fluid and Brushing Cytology

Kipp, B.R., Stadheim, L.M., Halling, S.A., Pochron, N.L., Harmsen, S., Nagorney, D.M., et al., 2004. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am. J. Gastroenterol. 99 (9), 1675–1681. Urine and Cystoscopic Specimens Chou, R., Dana, T., 2010. Screening adults for bladder cancer: a review of the evidence for the U.S. preventive services task force. Ann. Intern. Med. 153 (7), 461–468. Felknor, S.A., Delclos, G.L., Lerner, S.P., Burau, K.D., Wood, S.M., Lusk, C.M., et al., 2003. Bladder cancer screening program for a petrochemical cohort with potential exposure to beta-naphthylamine. J. Occup. Environ. Med. 45 (3), 289–294. Olfert, S.M., Felknor, S.A., Delclos, G.L., 2006. An updated review of the literature: risk factors for bladder cancer with focus on occupational exposures. South. Med. J. 99 (11), 1256–1263. Kaufman, D.S., Shipley, W.U., Feldman, A.S., 2009. Bladder cancer. Lancet 374 (9685), 239–249. Layfield, L.J., Elsheikh, T.M., Fili, A., Nayar, R., Shidham, V., Papanicolaou Society of Cytopathology, 2004. Review of the state of the art and recommendations of the Papanicolaou Society of Cytopathology for urinary cytology procedures and reporting: the Papanicolaou Society of Cytopathology practice guidelines task force. Diagn. Cytopathol. 30 (1), 24–30. Renshaw, A.A., 2000. Subclassifying atypical urinary cytology specimens. Cancer 90 (4), 222–229. Brimo, F., Vollmer, R.T., Case, B., Aprikian, A., Kassouf, W., Auger, M., 2009. Accuracy of urine cytology and the significance of an atypical category. Am. J. Clin. Pathol. 132 (5), 785–793. Dodd, L.G., Johnston, W.W., Robertson, C.N., Layfield, L.J., 1997. Endoscopic brush cytology of the upper urinary tract. Evaluation of its efficacy and potential limitations in diagnosis. Acta Cytol. 41 (2), 377–384. Thiryayi, S.A., Rana, D.N., 2012. Urine cytopathology: challenges, pitfalls, and mimics. Diagn. Cytopathol. 40 (11), 1019–1034. Koss, L.G., 2005. On decoy cells. Acta Cytol. 49 (3), 233–234. Maia, T.M., Silva, S.F., Silva, S.L., Holanda, M.C., Nascimento, J.M., Ferreira, M.V., 2011. Polyomavirus-infected decoy cells in cytocentrifuged urine cytology specimens from renal transplant recipients. Acta Cytol. 55 (5), 445–448. Cimbaluk, D., Pitelka, L., Kluskens, L., Gattuso, P., 2009. Update on human polyomavirus BK nephropathy. Diagn. Cytopathol. 37 (10), 773–779. Boldorini, R., Brustia, M., Veggiani, C., Barco, D., Andorno, S., Monga, G., 2005. Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients. Acta Cytol. 49 (3), 235–243. Effusions Ehya, H., 1991. Effusion cytology. Clin. Lab. Med. 11 (2), 443–467. Husain, A.N., Colby, T.V., Ordo´n˜ez, N.G., Krausz, T., Borczuk, A., Cagle, P.T., et al., 2009. Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the international mesothelioma interest group. Arch. Pathol. Lab. Med. 133 (8), 1317–1331.

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Relevant Websites http://www.cytologystuff.com/study/nongyn_atlas.htm – HOLOGIC. http://www.thinprep.com/hcp/lab_professionals/thinprep_non_gyn.html – ThinPrep Pap Test: Patient Homepage.