Martha Bishop Pitman


Cytological analysis of the liver involves the aspiration of cells from focal mass lesions or from brushing the lining of strictured intrahepatic ducts primarily for the purpose of determining the presence or absence of malignancy. Fine needle aspiration (FNA) of focal mass lesions can be performed percutaneously or with endoscopic ultrasound guidance.1,2 Intrahepatic ducts are brushed with the aid of endoscopic retrograde cholangiography.3,4

Needle aspiration of the liver was performed as long ago as 1833 when Roberts and Biett reported its use in the treatment of hepatic abscesses and echinococcal cysts.5,6 Although the use of fine needles to obtain tissue for cytological diagnosis was first used by Lucatello in 1895 (cited in Lundquist 1971),7 it was a technique refined in the 1920s by Martin and Ellis at the Memorial Hospital in New York.8 Its utility in the liver was highlighted by Lundquist who published his experience using aspiration cytology in the evaluation of neoplastic and non-neoplastic disorders of the liver.7,9,10 FNA biopsy is now the diagnostic procedure of choice for the diagnosis of focal mass lesions and is considered accurate and safe when performed and interpreted by experienced radiologists and pathologists.1,1113

The diagnostic accuracy rate of FNA is reported at greater than 85% in most series.14 False negative FNAs are most often due to sampling error. Proper specimen processing and staining optimises the preservation and presentation of the cells and is an important factor in reducing interpretation error. Routine supplementation of FNA with a cell block of tissue fragments or core biopsy is better than either method alone, especially for benign neoplasms and poorly differentiated neoplasms that require ancillary studies.1517


Contraindications for FNA of the liver are few but include a non-correctable bleeding diathesis, the lack of a safe access route, and an uncooperative patient1,11 For EUS guided FNA, gastrointestinal obstruction is an absolute contraindication because of the risk of intestinal perforation.18


With modern-day techniques, complications of FNA are uncommon. The most common complications include pain and haemorrhage.14 The haemorrhage can be intraperitoneal, subcapsular, or intrahepatic and, if intrahepatic, can lead to haemobilia. There is a low risk of haematogenous dissemination of malignant cells after liver biopsy19,20 as well as ‘seeding’ of the biopsy tract, complications that tend to be related to larger needle size.14,21

Pre-biopsy assessment

Pre-biopsy assessment of patients undergoing FNA of the liver includes evaluation of the coagulation system to prevent excessive bleeding, generally complete blood count, prothrombin time and partial thromboblastin time.1 Patients who are receiving intravenous sedation should not eat at least 6 hours before the biopsy.1 Local anaesthetic is generally given down to the liver capsule. Discussing the procedure fully with the patient reduces patient anxiety, improves patient cooperation and increases the likelihood of a successful FNA procedure.

Guidance systems

Percutaneous computed tomographic (CT) or ultrasound (US) guidance are the guidance systems used for most liver FNAs.1,2224 Endoscopic ultrasound guidance (EUS) is increasingly used for those lesions accessible through a transgastric approach, mostly in the left lobe.2,2528 Factors influencing the choice of the guidance system include the size, location and visibility of the mass, in addition to the experience and preference of the operator.1,11 Ultrasound provides real-time needle visualisation, flexible patient positioning, variable imaging of the lesion, and is performed without ionising radiation.1 CT facilitates biopsy of small, deep seated lesions not well demonstrated on ultrasound. CT more precisely demonstrates the anatomic relationships of a given lesion compared to US, improves the definition of tissue components and vascularity, and provides for accurate localisation of the needle tip immediately prior to sampling without the transmission of potential impediments such as drains, bone and gas.

Bile duct brushing

Biliary brushing cytology of suspicious biliary strictures is key to making an early diagnosis. Unfortunately, despite specificity of >95%, the technique has a low sensitivity ranging from 17–83%.3 Low sensitivity can be attributed to difficult access, desmoplasia, associated inflammation (stents, primary sclerosing cholangitis ), scant specimen cellularity and poor cellular preservation and preparation.4,35,36 If sufficient cells are present and the cells are properly preserved and prepared for cytological evaluation (direct smears, cytospins or liquid-based cytology (LBC)), the criteria for malignancy are universal: high N:C ratio, prominent nucleoli, nuclear membrane abnormalities and hyperchromasia yielding 100% specificity for malignancy.37 LBC processing alone or in addition to direct smears has improved sensitivity and accuracy in some studies, but cytologists must be familiar with alterations to morphology and background elements that may make malignancy appear more subtle in some cases.3840

Elevation in serum tumour markers CEA and CA19-9 have higher sensitivity for detecting carcinoma, but lower specificity (i.e. false positive tests).37 Adding digital image analysis35 and fluorescence in situ hybridisation (FISH)41 show promise in improving the diagnostic value of biliary brush cytology.

Specimen processing

As mentioned above, it is desirable to obtain both smears and cell block preparations in all FNAs of the liver. Smears are made from the aspiration part of the procedure using a small needle (<22 gauge) that provides a rapid means of evaluating the specimen, not only for cellular adequacy but frequently for diagnosis. Multiple FNAs can be performed with minimal morbidity. If well-fixed, adequately smeared slides are difficult to obtain, the aspirate can be expressed into a preservative and submitted to the laboratory as a liquid-based specimen for processing by either the ThinPrep® (Cytyc Corporation, Marlborough, MA) or Sure Path™ (TriPath Imaging, Burlington, NC) methods. Cell blocks are made from FNA rinsings, any tissue fragments that are obtained, and dedicated CNBs using a spring loaded 18–20 gauge CNB gun such as the ASAP Biopsy System (Meditech/Boston Scientific Corp, Watertown, MA) or the Coaxial Temno Biopsy System (Allegiance Healthcare Corp, McGaw Park, IL). This material provides a formalin fixed, paraffin embedded tissue sample from which special stains and immunohistochemical studies can be readily obtained. It also provides, in many cases, the architecture necessary for a specific diagnosis, particularly in benign liver lesions.

CNB specimens can also be used for rapid interpretation by touching the core to a glass slide in a touch prep fashion.42 Despite the presence of thick, three-dimensional tissue fragments and the probability of some air-drying artifact due to the inherent time delay in preparing the slide, architectural clues may still be readily apparent for rapid diagnosis.

The cytopathologist is an important part of the overall team approach to FNA of the liver. The presence of a cytopathologist at the time of the FNA increases the overall accuracy of the procedure.13,17,43 The time of the actual biopsy, when additional tissue is still readily available, is the time to evaluate the specimen for adequacy and to triage the tissue for special studies such as flow cytometry or electron microscopy studies. If a cytopathologist or cytotechnologist is not available to assist in the preparation of the specimen, it is imperative that the radiologist learn how to make proper smears. The most cellular specimen is useless if inadequately prepared for optimal interpretation.

Tumour classification

The most recent WHO classification of tumours of the liver is presented in Table 8.1.44 Correct diagnosis is imperative for proper patient management. Pyogenic abscesses are typically drained,45,46 and smaller tumours are being treated with ablation techniques such as alcohol and thermal ablation.47 Chemotherapy and radiation protocols require a tissue diagnosis and targeted gene therapy is under investigation.48,49

Table 8.1 WHO histological classification of tumours of the liver and intrahepatic bile ducts

Epithelial tumours
Hepatocellular adenoma (liver cell adenoma)
Focal nodular hyperplasia
Intrahepatic bile duct adenoma
Intrahepatic bile duct cystadenoma
Biliary papillomatosis
Hepatocellular carcinoma (liver cell carcinoma)
Intrahepatic cholangiocarcinoma (peripheral bile duct carcinoma)
Bile duct cystadenocarcinoma
Combined hepatocellular and cholangiocarcinoma
Undifferentiated carcinoma
Non-epithelial tumours
Lymphangioma and lymphangiomatosis
Infantile haemangioendothelioma
Epithelioid haemangioendothelioma
Embryonal sarcoma (undifferentiated sarcoma)
Miscellaneous tumours
Solitary fibrous tumour
Yolk sac tumour (endodermal sinus tumour)
Kaposi sarcoma
Rhabdoid tumour
Haemopoietic and lymphoid tumours
Secondary tumours
Epithelial abnormalities
Liver cell dysplasia (liver cell change)
Large cell type (large cell change)
Small cell type (small cell change)
Dysplastic nodules (adenomatous hyperplasia)
Low grade
High grade (atypical adenomatous hyperplasia)
Bile duct abnormalities
Hyperplasia (bile duct epithelium and peribiliary glands)
Dysplasia (bile duct epithelium and peribiliary glands)
Intraepithelial carcinoma (carcinoma in situ)
Miscellaneous lesions
Mesenchymal hamartoma
Nodular transformation (nodular regenerative hyperplasia)
Inflammatory pseudotumour

(From Hamilton SR, Aaltonen LA. Tumours of the liver and intrahepatic bile ducts. In: Hamilton SR, Aaltonen LA (eds) Pathology & Genetics Tumours of the Digestive System. Albany: WHO Publications Center; 2001: 158.)

Normal morphology

The liver is a complex organ with functional lobular units of hepatic parenchyma anchored by portal tracts containing branches of the hepatic artery, hepatic portal vein and bile duct. Sinusoids are lined by a discontinuous layer of endothelial cells that separate hepatic plates of 1–2 cells thick and that terminate in the central vein. Sinusoidal endothelial cells differ from vascular endothelial cells. Unlike the endothelial cells of true vessels, sinusoidal endothelial cells are not supported by a basement membrane and do not express factor VIII, Ulex europaeus or CD 34.50 Transformation of sinusoids with the acquisition of these properties leads to ‘capillarisation’ of the sinusoids, changes that are exploited in both histological and cytological evaluation for diagnosis (see below).

In order to appreciate an abnormality in liver FNA, it is imperative that the cytopathologist understands the components of the ‘normal’ liver.

Cytological findings of ‘normal liver’

The normal hepatocyte is a large polygonal cell with:

Benign bile duct epithelial cells present as:

Endothelial cells of sinusoidal spaces and Kupffer cells are rarely appreciated in benign lesions.


Intracytoplasmic pigments in cytology preparations differ in appearance depending on the stain. Lipofuscin, which constitutes the debris of intracellular lysosome breakdown, appears as a fine golden, granular, relatively non-refractile pigment in alcohol fixed, Papanicolaou (PAP) stained smears and is typically concentrated around the nucleus. This pigment is generally very common in the FNA of older adults and its absence should increase suspicion of a neoplasm on FNA of a mass lesion. Lipofuscin will stain darkly with a Fontana Mason stain creating a potential diagnostic pitfall with a metastatic melanoma.51

Bile pigment is produced by hepatocytes and is virtually pathognomonic of hepatocellular carcinoma when recognised within malignant cells. Bile appears as coarse, irregular, rather amorphous, non-refractile green to golden brown globular intracytoplasmic and extra-cytoplasmic deposits on Papanicolaou stain (Fig. 8.5). With Giemsa–Romanowsky stain, bile has a dark green to black hue. The distribution of the pigment is dependent on the degree of cholestasis, with pools of bile within canalicular spaces apparent in cases of extrahepatic obstruction (Fig. 8.6).

Iron or haemosiderin is a coarse, brown-black refractile pigment with Papanicolaou stain (Fig. 8.7A). While FNA can confirm the presence of heavy iron overload, it cannot replace CNB for histology and biochemical analysis to answer the clinical question of haemochromatosis. Malignant hepatocytes loose their ability to retain iron, and in the setting of haemachromatosis induced cirrhosis, where reactive hepatocyte atypia may be quite marked, it is helpful to use a special stain for iron like Prussian blue to highlight cells or clusters of cells without staining (Fig. 8.7B).52

Non-neoplastic conditions


Ruptured splenic tissue following trauma or splenectomy can result in implantation or auto-transplantation of splenic tissue in the peritoneal cavity and even within parenchymal organs like the liver mimicking a neoplastic process, including hepatocellular carcinoma and especially metastatic malignancy given the typical multiple implants.55,56 Although most patients are asymptomatic, these mass forming lesions are picked up incidentally and may be investigated by FNA. Smears that are cellular and well-preserved recapitulate the normal spleen demonstrating a mixture of normal haemopoietìc cells in a bloody background supported by a reticular network57 (Fig. 8.11). Lymphoid dominant FNAs, however, introduce small blue cell tumours into the differential diagnosis including small cell lymphoma and small cell carcinoma. An important clue to the diagnosis is a history of splenectomy and/or trauma.


Abscess formation in the liver is often suspected radiologically from the characteristic double target sign on computed tomography; however, organised abscesses can mimic hepatic tumours leading to FNA.53 Pyogenic abscess is the most common and percutaneous biopsy is performed for tissue confirmation, culture and drainage.45,59 FNA is an acceptable means of diagnosis for all abscess sizes, but for therapeutic drainage, FNA is recommended only for small abscesses <50 mm; larger abscesses require percutaneous catheter drainage for complete management.45 Smears are dominated by nonspecific acute inflammatory cells and cellular debris. Cultures are helpful for identification of bacterial organisms.

Amoebic abscess is 10 times more common in men than in women and is rare in children.60 The distinction between pyogenic and amoebic abscess is important for both therapy and prognosis. Pyogenic abscesses have a 20–60% morbidity due to an association with systemic infection, the tendency to produce multiple liver abscesses and an association with an older patient age.61 Amoebic abscesses tend to occur as solitary liver masses in young patients, rarely require drainage and respond to metronidazole therapy.60,61 Most patients with hepatic amoebiasis do not demonstrate intestinal symptoms, but hepatomegaly with point tenderness over the liver, below the ribs or in the intercostal spaces are typical presenting symptoms.60 Identification of organisms in aspiration fluid of hepatic abscesses is uncommon and diagnosis rests on the constellation of presenting symptoms, high risk factors, typical radiological findings of a solitary right lobe mass and positive serum antibodies which are present in 70–80% of patients.60 The single cell trophozoites of E. histolytica with round nucleus, condensed peripheral chromatin and central small nucleolus with foamy cytoplasm resemble histiocytes and can be easily overlooked.

Other infectious organisms may be recognised by unique characteristics such as the ‘sulphur granules’ of actinomyces and ‘anchovy paste’ smears (Fig. 8.13), echinococcal hooklets or scolices (Fig. 8.14) or laminated cyst wall (Fig. 8.15) in hydatid disease.46

Granulomatous inflammation may be related to infection such as with fungal or acid fast organisms, but the presence of granulomata in a liver FNA is in no way diagnostic of an infectious aetiology. The presence of granulomata may be related to many conditions including primary hepatobiliary disorders, sarcoidosis, and tumours such as lymphoma and metastatic carcinoma. Hepatic sarcoidosis is one of the most common causes of non-caseating granulomata of the liver.62 Cytologically granulomata are composed of clusters of epithelioid histiocytes with oval to elongated, sometimes twisted nuclei and visible but indistinct, non-phagocytic, cytoplasm (Fig. 8.16). Special stains such as Grocott methenamine silver (GMS) and Ziehl Neelsen (ZN) for acid fast bacilli (AFB) can be performed on smears but are easier to perform on cell block preparations.

Bile duct hamartoma

Bile duct hamartoma may present as a mass lesion mimicking a neoplasm, and as a result, may be encountered on FNA.63 This lesion is often aspirated at the time of diagnostic EUS-FNA of pancreatic masses, so care must be taken to distinguish gastrointestinal contamination from bile duct epithelium as a means of ensuring the aspirate is representative of the lesion. Smears contain a predominant population of benign appearing bile duct epithelial cells often in the form of flat monolayered sheets of glandular cells with small uniformly spaced round nuclei (Fig. 8.17). Luminal edges with scant but visible non-mucinous cytoplasm are present. Benign hepatocytes are uncharacteristically few or absent as would be expected for a benign hepatic lesion such as focal nodular hyperplasia. Aspirates of bile duct adenoma have a similar appearance. Cell block preparations are helpful in rendering a diagnosis (Fig. 8.18).

Mesenchymal hamartoma

Hepatic mesenchymal hamartoma is a benign mass forming lesion of malformed bile ducts and myxoid mesenchyme diagnosed in mostly male (70%) infants and children less than 5 years of age with rare cases reported in adults.64,65 The aetiology is unclear with theories ranging from congenital plate abnormality to true neoplasm. These typically predominantly cystic masses are usually resected in childhood and complete resection is curative. Although serological tumour markers are usually normal, elevated AFP and atypical FNA cytology have been reported to lead to a preoperative diagnosis of hepatoblastoma, the primary tumour in the differential diagnosis.12,66

Reports of the cytological features are few12,67,68 FNA produces scant smears composed of a loose myxoid mesenchymal stroma with benign-appearing spindle cells and sheets of benign glandular epithelium that reflects the histology of disorganised loose myxoid mesenchymal tissue surrounding variably sized benign bile ducts and occasionally hepatocytes (Fig. 8.19). Pseudocysts occur in the mesenchyme and are not lined by epithelial cells. Extramedullary haematopoesis may be noted. The patient’s young age, male gender, cystic radiological appearance and benign-appearing myxoid spindled and glandular cell proliferation should lead to the correct diagnosis.

Inflammatory pseudotumour

Inflammatory pseudotumour (IPT) of the liver is an uncommon, benign, mass-forming proliferation of mixed inflammatory cells and histiocytes dominated by polyclonal plasma cells infiltrating a stroma of fibroblasts, myofibroblasts and collagen. These lesions are reported in patients of both genders, but mostly males (70%), and all ages from infancy to the eighth decade.69 The heterogeneity of these proliferations has led to many synonymous names, including plasma cell granuloma, inflammatory myofibrohistiocytic proliferation, fibroxanthoma and inflammatory myofibroblastic tumour. Although some reports of clonality and association with the Epstein–Barr virus have supported a neoplastic process in some cases,70 the aetiology in most hepatic IPT is inflammatory or infectious.71 More recently, pseudotumour is considered to be part of the IgG4 related sclerosing disease, often associated with autoimmune pancreatitis.7276

These lesions are benign, but rare cases of biological aggressiveness and malignant transformation have been reported.77

Diagnosis by FNA is challenging due to the non-specific and variable nature of the mixed inflammatory sample.78,79 Smears are often cellular composed of cohesive networks of spindle mesenchymal cells enmeshed with mixed inflammatory cells and foamy (xanthomatous) histiocytes that also populate the background. Plasma cells are usually prominent and neutrophils and eosinophils are minor components (Fig. 8.20). The spindle cell component may also be dominant and single spindle cells are noted. Atypia, especially in the mesenchymal cells and histiocytes, can be a pitfall.78 The differential diagnosis includes spindle cell lesions of the liver including gastrointestinal stromal tumours and sarcomas. The typical high cellularity of the inflammatory component should preclude a false positive interpretation.

Benign neoplasms


This benign mesenchymal neoplasm is purported to arise from the perivascular epithelioid cell (PEC) and has been classified as a PEComa, one of several benign neoplasms of the tuberous sclerosis complex.82 It is composed of varying combinations of fat, smooth muscle and vessels. When the fatty component is readily recognised radiologically, histologically and cytologically, the diagnosis is relatively straightforward (Fig. 8.22). In fact, it is usually the paucity of fat in the neoplasm that leads to a diagnostic dilemma and subsequent FNA.83,84 The histological and cytological features of this tumour are similar in the liver and the kidney (see Ch. 11). Diagnostic difficulty on cytology arises when the fatty component is scant or focal and not sampled, and when solid epithelioid areas predominate (Fig. 8.23)85,86 Cell block preparations provide not only architectural clues, but tissue for ancillary testing. Positive staining with HMB-45 confirms the diagnosis.87 Other immunohistochemical markers that label this neoplasm include vimentin, desmin, actin and endothelial markers such as Factor VIII.88


Haemangiomas constitute the most common benign neoplasm of the liver. They occur in all ages and both genders. These mass lesions are generally small and asymptomatic, but can occasionally be large (>5 cm) and cause symptoms. Although radiological diagnosis has improved with enhanced imaging techniques, most are found incidentally during work-up for other conditions, including staging of malignancy, and can be difficult to distinguish from a metastasis radiologically.89,90 Cytologically, FNA smears are frequently considered unsatisfactory or non-diagnostic due to either the aspiration of blood only, or the presence of nonspecific appearing connective tissue. It is this loose, rather than dense fibrous type connective tissue and smooth muscle fragments associated with blood and few to no background hepatocytes that should alert the pathologist to the diagnosis of haemangioma in the proper clinical setting. Cell block preparation of a core needle biopsy is crucial in making a specific diagnosis (Fig. 8.24).

Hepatobiliary cystadenoma

This benign cystic tumour is a solitary, multiloculated cystic neoplasm of the liver that is histologically similar to cysts found in the pancreas and ovary. Women are almost exclusively affected. These neoplasms can become very large and patients usually present with abdominal pain. The neoplasm does not communicate with the biliary system. The locules are filled with fluid that varies from thin and clear to bloody and turbid. The cyst lining is mucinous but the cells can become attenuated or denuded from fluid pressure. The characteristic subepithelial ovarian type stroma is not typically sampled on FNA which has been rarely reported.91,92 The cytology is similar to mucinous cystic neoplasm of the pancreas, often scantily cellular and nondiagnostic on its own. Cyst fluid with foamy histiocytes and mucinous epithelium, even if very scant, are sufficient for diagnosis in the appropriate clinical setting (Fig. 8.25) Recognition of the malignant counterpart, hepatobiliary cystadenocarcinoma, is possible if the cytology is overtly malignant, but less than malignant cytology does not exclude malignancy and, as such, the treatment of choice is total resection.92

Jun 8, 2017 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Liver
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