CHAPTER 10 Liver and spleen
Clinical aspects
Liver
The place of FNA in the investigative sequence
Malignancy in the liver, primary or metastatic, is usually inoperable at the time of diagnosis and, as such, portends an ominous prognosis. A diagnostic modality such as FNA, which offers accuracy without significant complications and which requires minimal intervention at low cost, warrants consideration early in the investigative sequence. Portal vein FNA sampling is feasible in patients with hepatocellular carcinoma (HCC) and may be considered as a staging option.1–3 FNA may become useful after local ablative therapy of liver tumors to determine residual viable tumor.4,5 In this era of liver transplantation there is some reluctance in performing FNA in patients who are being worked up for a transplant because of the risk of needle track implantation in the setting of immunosuppression, although this is disputed by some.6–10 Single or multiple focal abnormalities demonstrated by palpation, ultrasonography (US), computed tomography (CT) or nuclear scan constitute the main indications for FNA of the liver. In industrialized countries, metastatic tumor deposits are the most common cause of focal abnormalities. In developing countries, however, hepatocellular carcinoma (HCC) is a major health problem, and is more likely to be encountered in FNA. Approximately 80% of the annual global burden of 560 000 new cases of HCC occurs in Africa and Asia. In these areas, etiology is frequently the hepatitis B virus, with superadded aflatoxin ingestion. HCC in developed countries arises in a background of cirrhosis consequent on chronic alcohol abuse or, more recently, hepatitis C virus.11
Some workers have advocated the use of FNA in diffuse parenchymal liver disease.12–14 Most physicians feel that cytological methods lack the precision necessary to be of diagnostic value in processes such as cirrhosis, hepatitis and drug-induced effects.15,16 The pathologist must nevertheless be familiar with the hepatic changes which may be present as a local reaction adjacent to space-occupying lesions, and with the severe parenchymal abnormality which may represent the cirrhotic background of HCC. The cytological features of normal liver, and various changes associated with diffuse chronic liver disease have been described.12–14,16–19
FNA can diagnose certain diffuse processes such as hemosiderosis, amyloidosis and myeloid metaplasia. Metastatic melanoma or carcinoma, particularly of breast and lung, and malignant lymphoma can occasionally cause hepatomegaly by diffuse infiltration, without demonstrable focal abnormalities. These infiltrates may be sampled and can be diagnosed by FNA techniques. FNA has also been used for monitoring liver transplants.20–25
Accuracy of diagnosis
Most studies comparing core needle biopsy and FNA favor the fine needle for focal liver disease,26–28 although not all,29 and generally a combination of the two yields the best results.28,30 Diagnostic sensitivity of FNA in malignancy is usually to around 90% (range 67–100%).26,28,30–36 Cell typing can sometimes be problematic in smears and many centers therefore advocate the additional use of cell blocks for histological assessment and providing multiple sections for immunocytochemistry.32,37 The use of cell blocks improved reported accuracy of hepatocellular carcinoma typing from around 85%38,39 to 95%.40,41 Certain groups report the combined use of aspirates and tissue cores for more definitive diagnosis of focal liver lesions.30 Recent use of the endoscopic ultrasound guided approach to FNA of the liver appears not only to maintain the high sensitivity and specificity of liver FNA, but also to identify and target lesions too small to be detected by conventional transabdominal ultrasound and CT.42–44 Whichever modality is used for sampling, we strongly advise the on-site participation of personnel from cytopathology to ensure optimal handling of the sample, rapid interpretation, and triage of the specimen as required.
Complications
No deaths and only one significant complication, an intrahepatic hematoma, occurred in an early report of 2611 cases.45 A later summary of 7500 FNAs from 11 series recorded no deaths.46 In an extensive literature review and questionnaire study,47 Smith collated fatalities following abdominal percutaneous FNA. Of 21 deaths involving liver FNA, he noted that 17 were due to hemorrhage. A needle larger than 0.8 mm (21 gauge) was used in seven. Three of the other 10 followed FNA of vascular tumors (hemangioma 1, angiosarcoma 2). Fatalities due to other causes were rare (sepsis 2, carcinoid crisis 1, uncertain 1). More recently, two deaths due to hemorrhage from among 1750 US guided FNA has been reported.48
Allegations of needle track spread of tumor have always dogged FNA, and the liver is no exception in this regard. Of recent concern has been a number of literature reports of subcutaneous seeding of needle tracks from otherwise operable HCCs.47,49–51 Whereas in some of these reports the procedures comply in every respect with the usual safety precautions advised (0.8-mm/21-gauge needles or smaller, few needle passes, traversal of normal liver parenchyma between abdominal wall and tumor),52 other reports often included in discussion of this issue have used 18-gauge needles,53 have traversed one lobe to reach a lesion in the other lobe and have not defined needle size,54 or are series in which cutting55 or Trucut56 needles have been used. Cytopathologists and radiologists performing hepatic FNA should discuss and be very clear about the caliber of needles used.
In those cases in which potentially resectable or transplantable HCC is being investigated, local opinion might preclude preoperative sampling.9 This is despite studies that show FNA has no significant adverse effect on operability, extrahepatic metastases or long-term survival.6,8,57 Experience with cases of needle track spread has shown that local treatment by resection has been successful for isolated subcutaneous tumor deposits.47,49,53–56 In the vast majority of hepatic aspiration procedures, physicians and patients can be assured of very low complication rates (hemorrhage 0.006–0.031%, needle track spread 0.007%).47
Contraindications
Hemorrhagic diathesis and anticoagulation are contraindications, and the prothrombin time and the platelet count should be checked before the FNA. The patient should be kept under observation for a few hours after biopsy to monitor pulse, blood pressure and abdominal pain. Hydatid cyst was formerly considered to be a relative contraindication in view of the risk of an anaphylactic reaction.58 However, no allergic reactions have been observed in two series of 11 hydatid cysts aspirated inadvertently.59,60 The single documented case of anaphylactic shock, which was reversed, occurred when an 18-gauge needle was used.58 The risk of hemorrhage by needling of vasoformative tumors exists, but is low if correct techniques are used. This is no longer considered an absolute contraindication.61–64
Technical considerations
Whenever possible, the biopsy should be directed rather than blind. If a mass cannot be clearly felt, the biopsy must be guided by radiological imaging. US is the most commonly used modality.31,32 More recently, clinicians and radiologists have been using endoscopic ultrasound to target some liver lesions, in particular those involving the hilum and left lobe. Great technical expertise is required for good results, and is acquired after significant experience.43,44,65–67
Ideally, a cytopathologist or cytologist attends the FNA procedure for rapid review to confirm diagnostic material and to triage the material.68 Rapid staining (Diff-Quik or H&E) allows immediate screening of samples. The parallel use of MGG and Pap staining is strongly recommended for permanent smears of liver aspirates. Most adjunctive cytochemical or immunocytochemical tests are more easily performed on cell blocks, but some may be useful in smears or in cytocentrifuged and thin-layer preparations. Additionally, demonstration of architectural patterns in sections of tissue fragments increases diagnostic accuracy significantly.37,40,69–72 If immediate assessment suggests an acute, chronic or granulomatous inflammatory process, material for microbiological assessment should be collected in a sterile container. If a lymphoproliferative disorder is suggested, a separate needle pass should be collected into RPMI or normal saline for flow cytometry. Cytogenetic FISH testing, e.g. for specific translocations, can be performed on cytospin preparations and molecular studies, e.g. for lymphoma clonality, can be conducted on material from cell blocks. Electron microscopy may also contribute to type-specific tumor diagnosis.70,73 These adjunctive techniques ensure that liver aspirates are highly suited to sophisticated cytohistological interpretation. A core biopsy should occasionally be requested, particularly if architectural assessment is crucial to the diagnosis e.g. hepatic adenoma versus hepatocellular carcinoma. If radiology staff are to prepare the sample they should be trained, appropriate consumables should be supplied and collection of material into fluid for a cell block or cytospin preparations should be encouraged.
Cytological findings
Normal structures (Fig. 10.1)
In smears, normal hepatocytes form irregular, cohesive sheets and narrow cords 1–2 cells thick (Fig. 10.1A). Single cells are infrequent. The cells are large, round or distinctly polygonal in outline with abundant granular cytoplasm, sometimes vacuolated, staining eosinophilic with H&E, blue with Papanicolaou or gray–blue with MGG (Fig. 10.1B). Nuclei are centrally located, round, with finely granular chromatin which is evenly distributed. Binucleate cells are common. Nucleoli are central and usually small although prominent. The cell membrane is indistinct. Normal hepatocytes may display mild to moderate anisokaryosis due to polyploidy, increasing with the patient’s age. Occasional nuclei may be very large in non-neoplastic liver tissue. Coarse, granular intracytoplasmic pigment is commonly present in hepatocytes. It stains green–black with MGG, brown with H&E and is probably lipofuscin rather than bile. Kupffer cells can usually be found and appear as single, bare, comma-shaped nuclei between the hepatocytes. Bile duct epithelial cells form small, regular monolayered sheets. They are palisaded when seen from the side. The cytoplasm is minimal, nuclear chromatin is granular and nucleoli are inconspicuous. These are few in number in samples from normal liver tissue.
Diffuse parenchymal disease (Figs 10.2–10.9)
Cytologic features
Fig. 10.5 Reticulin stain
Intact although disorganized sinusoidal network identified in cirrhosis (cell block reticulin, HP).
As already stated, FNA is not primarily suited to diagnosis of non-neoplastic, parenchymal liver disease.15 However, it is essential that the cytologist recognizes these changes, as they may occur in the region of space-occupying lesions and may themselves mimic neoplasia.
The presence of parenchymal disease is indicated by decreased cohesion of hepatocytes. Smears may be hypercellular and appear quite pleomorphic due to anisocytosis. The hepatocytes are swollen to a variable degree, and the cytoplasm stains less uniformly than usual, paler at the periphery of the cell. Cell membranes which are indistinct in normal hepatocytes become more clearly visible (Fig. 10.2A). Fatty change is commonly seen as intracytoplasmic vacuoles of variable size displacing the nucleus to the periphery. Despite nuclear pleomorphism and binucleation, the chromatin pattern remains finely granular. Intranuclear cytoplasmic invaginations are seen in some nuclei, large prominent nucleoli in others (Fig. 10.2B). There are no atypical bare hepatocyte nuclei in the background. Liver cell necrosis may be obvious in severe disease. Acidophilic bodies and alcoholic hyaline are difficult to identify in cytological preparations.12,75 The number of lymphocytes and of Kupffer cells is increased. The Kupffer cells often appear swollen and may cluster. The number of sheets of bile duct epithelium is often increased, particularly in cirrhosis (Fig. 10.3).
The degree of parenchymal abnormality varies widely. The reactive change commonly seen adjacent to a neoplasm is an example of mild change, including some dissociation and swelling of hepatocytes and mild to moderate anisokaryosis. In cirrhosis, which may mimic neoplasia radiologically as well as cytologically,76 there is a marked increase in the number of Kupffer cells and of bile duct epithelial cells, fatty change, and dissociation of hepatocytes. Bile stasis is easily recognized as casts of dense material between hepatocytes, staining black–green with MGG (Fig. 10.4), yellow–green with Pap or H&E. Nuclear changes and nucleolar enlargement may be quite prominent. In so-called liver cell dysplasia associated with cirrhosis, there is a dominant background of normal hepatocytes among which are scattered atypical cells with considerably enlarged nuclei. The fibrosis of cirrhosis is not often evident on routinely stained smears, but can be demonstrated on smears or on cell block material using a connective tissue stain. Normal liver will demonstrate a fine regular network of reticulin. Cirrhosis is characterized by a disorganized but still intact network (Fig. 10.5). HCC typically shows fragmentation or a complete absence of reticulin framework.77,78
Hemosiderosis is recognized by the demonstration of coarse, refractile hemosiderin pigment – golden brown in Papanicolaou, black in Romanowsky stains – in the hepatocytes (Fig. 10.6) and in the Kupffer cells. It is not always easy to distinguish hemosiderin from lipofuscin or even from bile in routinely stained smears; a suitable special stain such as Prussian blue should be used if hemosiderosis is suspected.14,79 Further, with malignant transformation, iron pigment is lost. Prussian blue negative clusters can be regarded with suspicion in the setting of a mass arising in a background of siderosis.80 Amyloidosis is not common but may cause massive hepatomegaly and is diagnosable cytologically. Amyloid appears in smears as a dense amorphous, waxy material, which stains pink with H&E (Fig. 10.7), magenta with MGG and orangeophilic with Papanicolaou stain. Its presence should be confirmed with special stains such as Congo red. The hepatocytes often appear atrophic.81–84
The finding of granulomatous inflammation may either be incidental or represent the lesion being aspirated but does not permit a specific diagnosis.85 Cytological features include loose aggregates of epithelioid histiocytes which have large elongated plump nuclei, finely granular chromatin, small distinct nucleoli, abundant, pale-staining cytoplasm, Langhans and/or foreign body type giant cells and non-specific inflammation (Fig. 10.8). Caseous necrosis may be seen. The etiology most likely to present as a mass, particularly in the Third World, is infection such as mycobacteria.86 Material should be collected appropriately for culture and special stains to demonstrate the acid-fast bacilli.12 Failing this, PCR for mycobacteria can be performed on fresh or fixed material. The list of differential diagnoses for granulomata in the liver is long and their demonstration in FNA smears requires clinical correlation and further investigation to determine their exact etiology.
Non-neoplastic focal lesions
Cysts
Congenital (developmental) cysts
An aspirate from a congenital cyst, in reality a dilatation of a biliary duct, consists of thin, clear fluid but may be bloody or bile stained. A few uniform, cuboidal epithelial cells, single or in monolayered sheets, and some macrophages with vacuolated cytoplasm may be found in the smears (Fig. 10.9A).87 The columnar cells may balloon if degenerate. The ciliated hepatic foregut cyst can be diagnosed cytologically by observing clusters of ciliated columnar cells in the cyst milieu (Fig. 10.9B).88,89
Criteria for diagnosis
Hydatid cyst
Routine aspiration of a hydatid cyst is not recommended because of the theoretical risk of anaphylaxis should the cyst contents spill into the abdomen.62,90
Abscess (Fig. 10.11)
Purulent material aspirated from a focal lesion should always be subjected to microbiological investigation. Routine smears should also be prepared and screened for neoplastic cells, as tumor metastases, for example from a primary bowel adenocarcinoma, can undergo central necrosis, simulating an abscess. Pyogenic abscess demonstrates a marked neutrophil infiltrate, with necrotic debris.93 Hepatocytes from the periphery of the abscess may show considerable atypia.76 The predominant organism in pyogenic abscesses is Klebsiella.93 Amebic abscess due to Entamoeba histolytica, with contents likened to anchovy paste (thick reddish-brown semi-fluid material), shows abundant necrosis with fewer inflammatory cells. Trophozoites are not found in the central necrotic area; they should be sought in the viable periphery of the abscess (Fig. 10.11).93,94 They are globular organisms, with an eccentric spherical nucleus showing a central clear zone with a dot-like karyosome and margination of chromatin. The cytoplasm is vacuolated and contains ingested red blood cells. Amebae stain positive with PAS. They resemble foamy macrophages and may be missed if not suspected. The diagnosis of hepatic actinomycosis by FNB has been reported.95,96 It occurs in a background of systemic actinomycosis, secondary to pulmonary or uterine primary sources of infection.
Other non-neoplastic lesions
Focal fatty change in the liver can simulate neoplasia on radiologic imaging, and diagnosis by FNA is of practical importance.97,98 It is less common than the diffuse form and the etiology is uncertain. The hepatocytes have normal nuclei and the majority contain multiple or single clear vacuoles, bile duct epithelium may be present and the reticulin framework may be distorted by the fat droplets.
Inflammatory pseudotumor is a distinct entity increasingly being identified radiologically and thus entering the ambit of cytodiagnosis. The liver is probably the second most common site after lung for its occurrence.99 It is a localized lesion of uncertain etiology, representing an atypical immune response or a hypersensitivity reaction to antigen. Histological variants include inflamed granulation tissue, xanthogranuloma, hyaline sclerosing variant and plasma cell granuloma. FNAC can be expected to be equally variable. Hepatocytes and bile duct cells, which can show atypia and plasma cells, lymphocytes, neutrophils ± eosinophils and/or xanthoma cells in a background of fibroblasts, create a mixed picture which should not be overdiagnosed as malignant. Single large abnormal cells with lobulated vesicular nuclei resembling Reed-Sternberg cells have been reported.100–102
Nodular extramedullary hemopoiesis occurs typically in the setting of myeloproliferative disease and is recognized by the criteria of the triple cell lines – normoblasts, promyelocytes and megakaryocytes.103–105 The last mentioned could be mistaken for bizarre malignant cells.
Primary hepatic tumors of epithelial origin
Hepatobiliary cystadenoma
These solitary multiloculated lesions may reach sizes over 20 cm. They are encountered almost exclusively in females, generally of middle age.107,108 They are composed of a bland, cuboidal to columnar, nonciliated mucin-secreting epithelium, overlying dense ovarian-like stroma. The cyst contents are mucoid and may show raised CEA and CA 19-9 levels on biochemical testing.87,107,109
Tumors have a definite malignant potential and any solid, nodular or polypoid region seen on imaging should be sampled.110–114
Liver cell adenoma (Fig. 10.13)
Cytodiagnosis of this entity can hardly be made with confidence by cytology alone and a core biopsy can provide additional architectural information. The cytological pattern must be correlated with clinical presentation and radiological findings.115 A solitary liver mass found in a young woman with a history of oral contraceptive usage is most likely a hepatocellular adenoma, whereas a mass in an older patient with cirrhosis is more likely either a regenerative nodule or a hepatocellular carcinoma.
The differences in cytological patterns between adenoma and well-differentiated carcinoma can be subtle and will be discussed in the section on hepatocellular carcinoma. The cytoplasm is more abundant, less fragile and better defined with more distinct cell borders in adenoma, the nuclear : cytoplasmic ratio is consistently low, and single, bare neoplastic nuclei are less apparent (Fig. 10.13). Marked anisokaryosis is not a feature. Adenoma and FNH both comprise benign hepatocytes. In the absence of abundant bile duct epithelium, which favors FNH, distinction between them is not possible.41,106
Hepatocellular carcinoma (Figs 10.14–10.27)
Fig. 10.14 Hepatocellular carcinoma
Cellular smears with irregular large fragments, clusters and dispersed cells (H&E, LP).
Fig. 10.18 Hepatocellular carcinoma
Endothelial cell envelopes seen as thin spindle nuclei surrounding tumor fragments (H&E, HP).
Criteria for diagnosis
The characteristic findings relate to (1) cell groupings, (2) relationship to endothelium and (3) cell morphology.39,41,72,79,116–132
The features in any individual FNA sample are extremely variable and largely dictated by tumor differentiation.116,117,127,132 As the degree of differentiation decreases, the cells become more obviously malignant and their resemblance to hepatocytes decreases.
Smears are typically cellular with large fragments, clusters and dispersed cells (Fig. 10.14). Cell groupings are classically trabecular (Fig. 10.15A), particularly in better-differentiated tumors. Acinar arrangements may be seen in up to 40% of HCC (Fig. 10.15B).72 With decreasing differentiation, smaller sheets and single-lying cells become more frequent. A reticulin stain on smears or cell block material may highlight the widened trabeculae and/or acinar structures or the reduced or absent reticulin (Fig. 10.16). Endothelial relationships to HCC cell groups are an integral part of the diagnosis. Endothelial cells of sinusoidal capillaries may traverse (Fig. 10.17) or enclose (Fig. 10.18) trabeculae or separate tumor cell groupings.128 This important diagnostic criterion is diminished and then lost with decreasing differentiation.
The cells resemble hepatocytes, thus demonstrating polygonal outlines and dense, granular cytoplasm, but with increased nucleocytoplasmic ratios over normal counterparts (Fig. 10.19). Centrally placed nuclei, characterized by granular to coarse chromatin, show cytoplasmic invaginations (intranuclear inclusions) in up to 70% of cases and large central nucleoli in up to 60% of cases. One of the most outstanding cytological appearances in HCC is the presence of many stripped malignant nuclei with identical characteristics, lying free in the background between cell groups (Fig. 10.20).126 The presence among malignant cells of occasional tumor giant cells or of bizarre hepatocytes would seem to favor primary liver carcinoma as they are rare in metastatic carcinoma.72,131 Bile pigment within the cytoplasm and between the cells proves the hepatocellular origin of the tumor, but is demonstrable in only 25% of cases (Fig. 10.21). Fouchet’s reagent counterstained with hematoxylin/Sirius red stains bile intensely green (Fig. 2.19). Contaminant benign hepatocytes and bile duct epithelium may be collected from the surrounding non-neoplastic liver, particularly in the setting of cirrhosis. They usually lie apart from the tumor cells.
Cytoplasmic vacuolation is pronounced in clear cell variants of HCC (Fig. 10.22).133 The clear cytoplasm is due to accumulation of glycogen or fat, and mucin stains are usually negative.134,135 This is not a defined subtype but rather a morphological pattern usually associated with a component of ‘conventional’ HCC. Its importance lies in distinguishing it from metastatic clear cell malignancies including carcinomas of renal, adrenal, pancreatic and ovarian origin, and sarcomas.133
The fibrolamellar subtype of HCC occurs as a distinct clinicopathologic variant in the second and third decades of life, without associated parenchymal liver disease and is therefore more often resectable. The neoplastic cells are intermingled with bundles of spindle-shaped fibroblasts and fragments of lamellar collagen (Fig. 10.23A). The tumor cells, readily recognized as of hepatocellular origin, are very large and may be pleomorphic. They have a polygonal shape, abundant, dense, granular cytoplasm, large vesicular nuclei, and large, single, central nucleoli (Fig. 10.23B). Trabeculae and peripheral endothelial wrapping are not observed in this otherwise well-differentiated variant, as the cell population is dispersed or adherent to the fibrous tissue. Intranuclear cytoplasmic inclusions are plentiful and there may be intracytoplasmic hyaline inclusions and pale bodies.136–138
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