Despite great improvements in diagnostic imaging procedures and immunologic testing, liver biopsy remains invaluable in establishing diagnoses, in staging, in monitoring the progression of various liver diseases (including chronic hepatitis and primary biliary diseases), and in monitoring the effects of therapy. In liver transplantation patients, day-to-day assessment may be necessary to evaluate immunosuppressive therapy, to detect surgical complications or to identify recurrent disease (3). Similarly, patients with chronic hepatitis due to hepatitis B or hepatitis C virus infection treated with antiviral agents should have pretreatment and posttreatment biopsies to document the initial degree of inflammatory activity and the stage of the disease, as well as to assess the changes that may come with treatment.
TECHNICAL CONSIDERATIONS
Histochemical Stains
Hematoxylin-eosin is the standard stain for the initial study of the liver biopsy. In our practice we routinely prepare two widely separated sections, approximately 100 µm apart, to be stained with hematoxylin-eosin. In addition, a panel of special stains are applied to the intervening sections, including Masson trichrome, reticulin silver stain, periodic acid-Schiff (PAS) after digestion with diastase (dPAS), and Perls Prussian blue stain for iron. Reticulin can be useful in separating capsule tissue from cirrhotic septa, with septa rich in reticulin fibers and capsule lacking them, and Victoria blue for screening for hepatitis B surface antigen (HBsAg) and copper-associated protein. Certain modifications and differences in various departments do exist, however. For example, the orcein stain (Shikata) or aldehyde fuchsin (Gomori) may be used instead of Victoria blue. These stains also demonstrate elastic fibers and phagocytosed material; for specific indications, various immunohistochemical and molecular techniques may have to be applied.
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FIGURE 5.1 Extensive bridging fibrosis and developing cirrhosis is highlighted with the reticulin silver stain (original magnification ×100). |
Trichrome Stains
For the evaluation of type I collagen, Masson trichrome stain or Mallory chromotrope aniline blue are commonly used. Normal liver shows only a small amount of collagen in portal tracts and a narrow rim of collagen around larger terminal hepatic venules. Trichrome stain is useful for determining the presence of increased amount of collagen, as well as for the determination of the extent of fibrosis. Pericellular fibrosis is also easily seen with trichrome stain. Mallory material and giant mitochondria are also highlighted with this stain (18,19).
Reticulin Silver Impregnation
Type III collagen is highlighted by reticulin silver preparation. Reticulin stain is particularly useful in delineating the liver cell plate structure and in studying the overall architectural pattern of the lobule (acinus). Regeneration with increased thickness of liver cell plates, as well as collapse of the underlying reticulin network in acute hepatic necrosis, is also highlighted with reticulin silver preparation (2,6,15,18) (Figs. 5.1, 5.2).
Periodic Acid-Schiff Reaction
The PAS reaction demonstrates mucopolysaccharides of various kinds, including glycogen. Diastase (PAS/D) removes glycogen from the tissue, allowing for the easier recognition of various mucopolysaccharide compounds. For example, digested material in Kupffer cells is highlighted by PAS; the finding of many PAS-positive Kupffer cells can be the only evidence of a recent, but resolved, hepatitis in which the Kupffer cells contain the partially digested hepatocyte cell membrane remnants (Fig. 5.3). Similarly, portal macrophages will react with this method. In general, PAS without diastase is not useful in studying adult liver diseases.
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FIGURE 5.2 Thickening of the liver cell plates in liver regeneration (reticulin silver stain, original magnification × 200). |
PAS/D highlights the characteristic globules of α1-antitrypsin deficiency, characteristically found in zone 1 (periportal/periseptal) hepatocytes.
They have a striking fuchsia color (Fig. 5.3), and their presence can be confirmed with monoclonal antibody.
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FIGURE 5.3 Intracellular globules in α1-antitrypsin deficiency. A. Periodic acid-Schiff (PAS) after digestion with diastase (dPAS, original magnification ×400). B. Immunohistochemical reaction for α1-antitrypsin (avidin-biotin-peroxidase immunoperoxidase, original magnification ×200). |
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FIGURE 5.4 Hepatocytes containing intracytoplasmic hepatitis B surface antigen are stained blue with Victoria blue (original magnification ×200). |
In pediatric pathology, PAS and PASD are helpful in excluding various metabolic diseases, such as glycogenosis IV. Abnormal amylopectinlike glycogen in glycogenosis IV is only partially digested with diastase, and the finding of intense reactivity with PAS and partial and irregular reactivity after diastase digestion is virtually diagnostic. PAS is also helpful in evaluating bile duct basement membrane, especially in destructive biliary diseases, such as primary biliary cirrhosis. In contrast, there is generally no destruction of the basement membrane in primary sclerosing cholangitis.
Victoria Blue
Victoria blue stain is used to screen for HBsAg (Fig. 5.4) and also demonstrates copper-associated protein and elastic fibers. Elastic fibers are present in a cirrhotic septa but not in scars. As another example, collapsed liver parenchyma does not have elastic fibers. The rare developmental disorder termed congenital hepatic fibrosis looks like cirrhosis in biopsy but also lacks elastic fibers. Victoria blue, orcein (Shikata), and Gomori aldehyde fuchsin are equivalent. With orcein, reaction products are brownblack rather than blue, and with aldehyde fuchsin they are fuchsia-purple.
Hemosiderin
Perls stain is used to detect stainable tissue hemosiderin. Semiquantitative analysis can be virtually diagnostic. When the amount of hemosiderin suggests genetic hemochromatosis, quantitative iron analysis and histologic iron index may be needed (see Chapter 15).
Copper
Rhodanine and rubeanic acid are special stains commonly applied in those cases where the accumulation of copper and copper-associated protein is suspected, including Wilson disease and chronic cholestasis. A modification of the rubeanic acid method allows for same-day staining (4). However, these stains are not especially sensitive, and copper assay of hepatic tissue is the standard for establishing the diagnosis of Wilson disease (see Chapter 16).
IMMUNOHISTOCHEMICAL STAINS AND MOLECULAR BIOLOGY
Identification of Viral Material in Tissue
HEPATITIS B. Immunoperoxidase methods using antibodies to both HBsAg and hepatitis B core antigen (HBcAg) are readily available. HBsAg may be present diffusely or focally in the cytoplasm and sometimes may also have membranous expression. HBcAg is expressed in the liver cell nuclei in most cases, but both intranuclear and granular intracytoplasmic expression may be simultaneously present, especially with high levels of viral replication (Fig. 5.5).
The most reliable and highly specific method to demonstrate hepatitis B virus DNA is the polymerase chain reaction (PCR). Patients with hepatitis B virus infection may also be infected with D or delta virus, in the form of superinfection or coinfection. The viral antigen can also be immunohistochemically detected in the tissue and has intranuclear expression very similar to that of HBcAg (18).
HEPATITIS C. There is no reliable immunohistochemical method to demonstrate hepatitis C virus in formalin-fixed paraffin-embedded tissue. A commercially available antibody that gives consistent results in paraffin-embedded tissue, rather than frozen section material, is not available (Fig. 5.6). Hepatitis C virus antigen can be demonstrated in cryostat-prepared sections, but the antibody for this is not widely available. In situ hybridization is useful for detection of replicating and nonreplicating viral proteins. PCR in tissue samples, reverse transcriptase PCR (RT-PCR), and RT-PCR in situ can all be used for the detection of hepatitis C virus RNA, but these techniques are used selectively (12,17).
CYTOKERATINS. Immunohistochemical reactions for low molecular weight (CAM 5.2) and high molecular weight (AE 1/3) keratins are useful in the evaluation of bile ducts and ductules in various conditions, including cholangiopathies and congenital or acquired bile duct paucity, as well as in demonstrating chronic (ductopenic) rejection (Fig. 5.7). The expression of some cytokeratins, particularly CAM 5.2, may be helpful in evaluating liver tumors (8,9,20).
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FIGURE 5.5 Immunohistochemical reactions for A hepatitis B surface antigen and B hepatitis B core antigen; respective intracytoplasmic and intranuclear expression is present (avidin-biotin- peroxidase immunoperoxidase, original magnification ×200). |
One of the newer markers that is helpful in differentiating hepatocellular carcinoma and cholangiocarcinoma or metastatic adenocarcinoma is a hepatocyte monoclonal antibody (Hep, Hepar 1) (15).
Immunofluorescent Studies
Immunofluorescent studies requiring frozen material are only rarely needed for diagnostic purposes. When humoral allograft rejection is suspected, immunofluorescent analysis is needed to demonstrate tissue deposition of immunoglobulin G (IgG), IgA, IgM, C3, and C1q components of complement, as well as fibrinogen. Fibrinogen deposition is also present in cases of eclampsia (Fig. 5.8), preeclampsia, and HELLP (hemolysis, elevated liver function, and low platelets) syndrome.
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FIGURE 5.6 Immunohistochemical reaction for hepatitis C viral (envelope NS4) antigen. Intracytoplasmic expression is seen (avidin-biotin-peroxidase immunoperoxidase, original |
Molecular Pathology
Molecular techniques can demonstrate the causative agent in many viral infections. In situ hybridization is particularly sensitive for the demonstration of cytomegalovirus. The Epstein-Barr virus-associated EBER-1 gene can also be demonstrated in patients with posttransplantation lymphoproliferative disorder (16).
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FIGURE 5.7 Almost complete absence of interlobular bile ducts in this example of ductopenic rejection. Immunoperoxidase method for mixed keratins (AE 1/3 original magnification ×200). |
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FIGURE 5.8 Perisinusoidal fibrinogen deposition in a case of eclampsia (immunofluores- |
Electron Microscopy
Ultrastructural examination is rarely needed in the study of adult liver biopsies. Ultrastructural studies are particularly helpful for metabolic disorders (e.g., in glycogenosis IV (Fig. 5.9)).
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FIGURE 5.9 Non-membrane-bound abnormal glycogen (amylopectin) in a case of glycogenosis IV (electron microscopy, original magnification ×10,000). |
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TABLE 5.1 Histopathologic Considerations in Evaluation and Interpretation of the Liver Biopsy |
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New techniques, including computer-assisted morphometry, flow cytometry, static cytometry image analysis, and neural network analysis can be performed on either fixed or unfixed liver tissue. However, these methods are not widely available and are usually applied for research purposes (1,5,7,13).
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TABLE 5.2 Key Clinical Information for Liver Biopsy Evaluation |
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GENERAL APPROACH TO THE LIVER BIOPSY
A systematic approach to the evaluation of the liver biopsy is vital to ensure that important diagnostic findings are not overlooked. The topographic and biologic relationships of morphologic changes generally provide correct and clinically meaningful diagnoses.
We initially evaluate the liver biopsy without clinical information and subsequently correlate morphologic findings with clinical data. Once the general diagnosis or at least the differential diagnosis has been reached, clinical information is vital. For example, diagnosis may require knowledge of viral serologies, autoantibodies, medications, and imaging studies. Features to be assessed on the liver biopsy are summarized in Table 5.1, and the clinical information that may be useful in liver biopsy interpretation is summarized in Table 5.2. We use a worksheet (Fig. 5.10 for nontumor cases.
Our approach is to, in effect, follow the blood flow. We begin with the portal tract and its component structures, then study the lobular including the limiting plate of hepatocytes, and complete our examination at the terminal hepatic venule (central vein).
Subcapsular liver biopsies, both needle and wedge, may be misleading and exhibit changes not present in the remainder of the parenchyma (9,13). This is particularly important in cases of chronic hepatitis, in which a subcapsular biopsy can be misinterpreted as showing cirrhosis (Fig. 5.11 The subcapsular region is also a common site for bile duct hamartomas (von Meyenburg complex), bile duct adenomas, and peribiliary gland hamartomas (Fig. 5.12). These benign lesions may be misinterpreted as metastatic adenocarcinoma, especially with frozen section examination (Fig. 5.13).
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FIGURE 5.10 Worksheet used to record clinically relevant data and other information in the review of liver biopsies with inflammatory changes. |
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FIGURE 5.11 Subcapsular liver parenchyma showing fibrosis and mild chronic inflammation, imparting a pseudonodule appearance. The findings were present only in this region. The remainder of the liver parenchyma is unremarkable (trichrome, original magnification ×100). |
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FIGURE 5.12 Subcapsular von Meyenburg complex. Irregular bile duct-like structures are in a fibrous background (hematoxylin-eosin, original magnification ×200). |
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FIGURE 5.13 Intraoperative consultation (frozen section) histology section showing subcapsular peribiliary gland hamartoma (hematoxylin-eosin, original magnification ×200). |
REFERENCES
1. An C, Petrovic LM, Reyter I, et al. The application of image analysis and neural network technology to the study of premalignant and malignant liver lesions. Hepatology 1997;26: 1224-1231.
2. Bianchi L. Liver biopsy interpretation in hepatitis. I. Presentation of critical morphological features used in diagnosis (glossary). Pathol Res Pract 1983;178:2-19.
3. Demetris AJ, Belle SH, Hart J, et al. Intraobserver and interobserver variation in the histopathological assessment of liver allograft rejection. Hepatology 1991;14:751-755.
4. Emanuele P, Goodman FD. A simple and rapid stain for copper in liver tissue. Ann Diagn Pathol 1998;2:125-126.
5. Erler BS, Hsu L, Truong HM, et al. Image analysis and diagnostic classification of hepatocellular carcinoma using neural networks and multivariate discriminant functions. Lab Invest 1994;71:446-451.
6. Gerber MA, Thung SN. Histology of the liver. Am J Surg Pathol 1987;11:709-722.
7. Hata K, Van Thiel DH, Herberman RB, et al. Phenotypic and functional characteristics of lymphocytes isolated from liver biopsy specimens from patients with active liver disease. Hepatology 1992;15:816-823.
8. Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma and metastatic adenocarcinoma. Hum Pathol 2002;33:1175-1181.
9. Maharaj B, Maharaj RJ, Leary WP, et al. Sampling variability and its influence on the diagnostic yield of percutaneous needle biopsy of the liver. Lancet 1986;1:523-525.
10. McAffee JH, Keeffe EB, Lee RG, et al. Transjugular liver biopsy. Hepatology 1992;15: 726-732.
11. Nuovo GJ, Lidonnici K, McConnell P, et al. Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol 1993;17:683-690.
12. Orsatti G, Thiese ND, Thung SN, et al. DNA image cytometric analysis of macroregenerative nodules (adenomatous hyperplasia) of the liver: evidence in support of their preneoplastic nature. Hepatology 1993;17:621-627.
13. Petrelli M, Scheuer PJ. Variation in subcapsular liver structure and its significance in the interpretation of wedge biopsies. J Clin Pathol 1967;20:743-748.
14. Petrovic LM. Benign hepatocellular tumors and tumor-like lesions. In: Ferrell LD, ed. Diagnostic Problems in Liver Pathology. London: Hanley and Belfus, 1994.
15. Randhawa PS, Jaffe R, Demetris AJ, et al. Expression of Epstein-Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease. N Engl J Med 1992;327:1710-1714.
16. Savage K, Dhillon AP, Brown D, et al. HCV by PCR of liver in autoimmune hepatitis. Hepatology 1992;16:590.
17. Scheuer PJ. General considerations. In: Scheuer PJ, Lefkowitch JH, eds. Liver Biopsy Interpretation. 5th Ed. London: WB Saunders, 1994:1-10.
18. Snover DC. Technical aspects of the evaluation of liver biopsies. In: Snover, ed. Biopsy Diagnosis of Liver Disease. Baltimore: Williams & Wilkins, 1992:2-23.
19. Van Eyken P, Sciot R, Paterson A, et al. Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study. Hum Pathol 1988;19:562-568.
20. Yamada G, Nishimoto H, Endou H, et al. Localization of hepatitis C viral RNA and capsid protein in human liver. Dig Dis Sci 1993;38:882-887.
