Symposium on Diseases of the Liver

Diagnostic Methods in Liver Disease Martin Black, M.D.*

Investigation of patients with liver disease has undergone substantial change in little over a decade, owing to massive advances in technology and instrumentation. Improved understanding of the pathogenesis and pathophysiology of liver diseases has resulted from, and contributed to, a more sophisticated and scientific approach. This improved diagnostic capability should now insure that virtually no patient need undergo an unnecessary exploratory laparotomy in order to diagnose a nonsurgical hepatic disorder, and conversely that the patient with surgically remediable disease should be readily recognized and the appropriate therapeutic procedure demonstrated. Progress in immunoserologic techniques, which has followed Blumberg's first description of a plasma antigen associated with viral hepatitis,9 has permitted a better understanding of the epidemiology, biology, and biochemistry of at least one form of viral hepatitis. Simultaneously, the increased availability and expert examination of tissue obtained by percutaneous needle biopsy of the liver has demonstrated the many faces of hepatitis and its sequelae.26 The responsibility of the attending physician has thus extended well beyond primary diagnosis, to embrace the long-term supervision of continuing or progressive disease.

GENERAL CONSIDERATIONS Although the resources currently available for diagnosis of liver disease include some very sophisticated equipment, many patients do not require comprehensive work-up. Investigation must be individualized, with the physician relying on his experience to choose the direction and the scope of the studies carried out. Several levels of inquiry can be recognized, ranging from the differential diagnosis of jaundice, which might require all modalities of investigation to be employed, to screening for evidence of hepatic metastases, which could be satisfied by some specified laboratory tests and the use of radionuclide imaging. Familiarity with the various techniques allows one to judge the relative value of any specific procedure in the clinical problem being faced. "'Associate Professor of Medicine and Pharmacology, and Head, Liver Unit, Temple University Health Sciences Center, Philadelphia, Pennsylvania

Medical Clinics of North America- Vol. 59, No. 4, July 1975

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LABORATORY INVESTIGATIONS The laboratory has long held center-stage in the investigation of suspected liver disease, and though its limitations in permitting accurate diagnosis are increasingly recognized, it still offers the physician the most convenient and least invasive method of determining the general category of liver disease present. A great number of laboratory tests have been developed over the years, from the nonspecific serum turbidity/fiocculation tests to the relatively sophisticated serum enzyme determinations, but in the author's opinion maximum information is obtained from just a few such studies. These include serum bilirubin (with fractionation into direct-reacting and indirect-reacting moieties), alkaline phosphatase, 5 '-nucleotidase, glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), and prothrombin time. Determination of serum albumin and serum protein electrophoresis provide important additional information and are discussed in the section on Immunoserology. Bilirubin, the end-product of heme catabolism in the body, is the pigment whose accumulation in the plasma in the face of impaired hepatic metabolism or excretion produces the easily recognizable jaundice-frequently the first manifestation of liver disease. It may be present in the plasma as free (unconjugated) bilirubin or bilirubin glucuronide, and is measured by coupling with a diazo reagent such as the diazonium salts of sulfanilic acid or ethylanthranilate and quantitating the azopigment(s) spectrophotometrically. Rapidity of diazo-coupling in serum specimens is related to the water-solubility of the pigments, being fairly rapid with the glucuronide but very slow with unconjugated bilirubin. This difference forms the basis for fractionation of the serum total bilirubin into the direct-reacting moiety (bilirubin glucuronide, to a greater or lesser extent) and the indirect-reacting moiety. The latter (theoretically, unconjugated bilirubin) is calculated by subtracting the figure for the direct-reacting fraction from that of the total bilirubin, which itself is determined by carrying out the diazo-coupling after the addition to the serum of an "accelerator" such as alcohol or diphyllin. Several methods are available for these measurements, the most commonly used one in the United States probably being the MalloyEvelyn method,41 but none is totally satisfactory. In general, these methods tend to overestimate the amount of unconjugated bilirubin present when the specimen contains substantial amounts of bilirubin glucuronide, and also to overestimate the amount of conjugated bilirubin present in specimens which contain predominantly unconjugated bilirubin. In the majority of the normal population the serum total bilirubin does not exceed 1 mg per dl, and the direct-reacting fraction is less than 0.3 mg per dUB A small number of otherwise normal subjects (possibly as high as 8 to 10 per cent of the population31 ) have mildly elevated values for the serum total bilirubin, the increase being entirely accounted for by unconjugated bilirubin in the specimen. These subjects may be classified as cases of Gilbert's syndrome (benign unconjugated hyperbilirubinemia) and probably represent the lowermost pole of the normal distribution of hepatic bilirubin UDP-glucuronyl transferase (UDPGT)

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1017 in the Their importance lies in the totally benign nature of the hyperbilirubinemia present; correct diagnosis (which requires fractionation of the serum total bilirubin) ensures that the patient does not suffer the emotional trauma of believing he has a serious disease. Jaundice resulting from increased amounts of direct-reacting bilirubin in the blood is a much more common clinical problem. The increase may be related to diffuse parenchymal injury (such as in viral hepatitis) or to cholestatic syndromes including diseases of the extrahepatic biliary tree. In either case, there will be accompanying abnormalities of some or all of the laboratory investigations discussed below. The conjugated hyperbilirubinemia of the Dubin-Johnson syndrome, however, is not accompanied by other abnormal studies. The ·alkaline phosphatases are a group of enzymes distributed widely in the body and generally involved in membrane transport processes. In the plasma, alkaline phosphatase activity derives predominantly from the contributions of isoenzymes from liver, intestinal tract, and bone,47 but during pregnancy, placental alkaline phosphatase boosts plasma levels as much as two-fold.45 In the nonpregnant subject, disease of either liver or bone can result in an increase in plasma alkaline phosphatase activity, and a number of procedures have been developed in order to differentiate the sources of the increased enzyme. 46 . 47 However, simultaneous determination of 5 ' -nucleotidase (or leucine aminopeptidase or gamma glutamyl transpeptidase) will usually establish whether an elevated serum alkaline phosphatase indicates hepatobiliary disease, and the need for fractionation into isoenzymes is rarely required. The enzyme is localized in the hepatocyte in the plasma membranes, particularly in the area surrounding the bile canaliculus.67 It is most likely to leak into the circulation and produce high plasma levels when there is disease affecting the biliary passages, be they intrahepatic or extrahepatic. Glutamic-oxalocetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) are two enzymes that transfer an amino group from glutamic acid to oxaloacetic acid and pyruvic acid respectively to yield aspartic acid and alanine. They are present in the mitochondria of many tissues including the liver and are released into the blood fbllowing damage to liver cells. n . 72 A rise in the serum concentration of GOT may also occur in myocardial infarction, skeletal muscle disease, hemopoietic disturbances, etc., whereas increases of serum GPT are virtually diagnostic of liver disease. Tandem determination of the serum transaminases ensures that the advantage of the increased sensitivity of the SGOT is balanced by the greater specificity of the SGPT. Several other enzymes are released into the blood following livercell injury, and their determination provides information similar to that obtained from the transaminases. However, they do not appear to offer any significant advantage over the SGOT and SGPT, and none has achieved any widespread popularity. Measurement of the prothrombin time provides useful information on the liver's ability for synthesis (to synthesize clotting factors), and is widely used as a critical hepatic function for assessing prognosis in acute hepatocellular disease (height of the plasma bilirubin is another). Abnormalities of the prothrombin time indicate impaired hepatic synthesis of some or all of the clotting factors 11, V, VII, IX, and X.51 Failure population.s

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to readily correct this abnormality by administration of vitamin K indicates that severe hepatocellular disease is present, and that the impaired absorption of the fat-soluble vitamin from the gastrointestinal tract (which usually occurs in the presence of jaundice) is only partially responsible for the clotting abnormality. The information provided by these investigations will generally allow the physician to make a broad classification of the disease being investigated. Jaundice in the presence of markedly increased values for the SGOT and SGPT (e.g., more than 1000 LU. per liter) but with relatively minor rises in the alkaline phosphatase and 5' -nucleotidase suggests that there has been necrosis of liver-cells, and this pattern of enzyme abnormality is referred to as "hepatocellular." On the other hand, jaundice which is accompanied by lesser elevations of the SGOT and SGPT (e.g., 300 LU. per liter or less) but with more striking rises in the alkaline phosphatase and 5'-nucleotidase is more likely to be due to disturbances of bile secretion or flow. This pattern of enzyme abnormality is referred to as cholestatic, and may be due to intrahepatic disease ("intrahepatic cholestasis") or to diseases of the extrahepatic biliary passages ("extrahepatic cholestasis"). In either type of liver injury the height of the serum bilirubin level gives a rough indication of the severity of the disease. The ability to clear the dye bromsulphthalein (BSP) or indocyanine green (leG) from the plasma after intravenous injection is one of the most sensitive measures of hepatic function. It is often utilized to render a judgment on the presence of liver disease when other biochemical indices are normal, particularly in systemic diseases which are known to affect the liver. Dye clearance is usually expressed as the per cent retained in the plasma at 30 to 45 minutes following the injection of a bolus dose of 2 or 5 mg per kg of body weight. Normally, less than 5 per cent remains at 45 minutes using the 5 mg per kg dose, although Zieve and Hill73 showed that many normal subjects gave results modestly in excess of that figure. Refinement of the technique, as proposed by Wheeler and co-workers,7° involves infusion of BSP over a prolonged period of study and provides additional information on the basis for abnormal plasma retention. Thus, impairment of hepatic uptake and metabolism of the dye (e.g., because of parenchymal disease) results in reduction of BSP storage (BSP s), while abnormalities of bile secretion (e.g., cholestatic disorders, or Dubin-Johnson syndrome) are reflected in a decrease of the BSPTm , the threshold maximum for BSP secretion. Biochemical investigation of the urine for the presence of bilirubin or urobilinogen are useful office procedures but do not play a significant role in the subsequent evaluation of the patient. There is some theoretical basis for urine urobilinogen determination being of value in the differential diagnosis of cholestatic jaundice, but experience suggests that results are too unreliable for routine use.

IMMUNOSEROLOGY Serum protein electrophoresis with accurate calculation of albumin and globulin fractions has replaced the older flocculation and turbidity tests, which largely reflect changes in these serum constituents. Quanti-

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tation of serum albumin provides information on the presence of subacute or chronic hepatic disease in terms of the synthesizing ability of the liver, but may be normal in a "well-compensated" cirrhosis. Decreased values in the ascites-forming patient may be more a reflection of increased "albumin space" than impaired production.37 Recent observations have also indicated that serum albumin levels may parallel drug hydroxylating capability.43 Progressive increases in the serum beta and gamma globulins occur as a result of protracted hepatic inflammation. 23 Typically, some patients with chronic active hepatitis have very marked increases in the gamma globulin fraction,49 raising the possibility that the hepatic inflammation results from an autoimmune process. 57 Determination of the immunoglobulins shows that it is predominately IgG that is increased in such patients, though lesser elevations of IgA and IgM are often present. 18 Similar increases in serum concentrations of the immunoglobulins occur in some patients with alcohol-related liver disease,18 and thus appear as nonspecific indicators of hepatic inflammation of diverse etiology. A notable exception to this pattern occurs in patients with primary biliary cirrhosis, which is characterized by early and marked increases in IgM levels in the presence of only moderately elevated values for IgG and IgA.18 A further piece of information to be gained from serum protein electrophoresis, namely the amount of alpha-l globulin present, has recently been shown to have important implications as far as the liver is concerned. This globulin fraction comprises predominantly alpha-l antitrypsin,62 the serum protein with protease inhibitor activity, which can be more accurately measured by immunodiffusion techniques. Alpha-l antitrypsin content is an inherited charactelistic, and in some patients (homozygous Pi ZZ ) is virtually absent from the serumP Such patients have an increased susceptibility to infection and a high incidence of cirrhosis and hepatocellular carcinoma.5 Examination of serum for antibodies against a variety of normal body tissues affords additional opportunities to diagnose or to categorize liver disease. Antimitochondrial antibodies are non-organ specific antibodies, which recent studies suggest react with antigens localized to the inner mitochondrial membrane. 4 ,6 These antibodies occur in 90 to 95 per cent of patients with primary biliary cirrhosis,58 and in a compatible clinical setting may be diagnostic. Anti-smooth muscle and antinuclear antibodies are less specific markers of liver disease, but have been suggested as criteria for the diagnosis of one type of chronic active hepatitis,u Positive L.E. cells may also be associated with this type of disease.4o A recent addition to this area of study has been the antimicrosomal antibody which shows a generally similar patient distribution as that seen with anti-smooth muscle and anti-nuclear antibodies. 52 Examination of serum for the presence of HBsAg, the marker for infection with hepatitis B virus (HBV) is an important component in the investigation of any patient with suspected liver disease. A variety of methods have been described for its detection, but the most commonly used ones are radioimmunoassay (RIA)38 and counter-immunoelectrophoresis (CIEP).31 The antigen can be detected by those means for 1 to 2 weeks before, and after, the onset of clinical disease due to hepatitis B

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virus infection, and in about 90 per cent of cases becomes undetectable thereafter.50 In these patients anti-HBsAg becomes detectable, using a passive hemagglutination technique,66 some 6 weeks after the onset of clinical disease, and remains detectable for 2 years or longer. 32 Persistence of HBsAg in the remaining 10 per cent of patients is associated with transition of the acute hepatic disease into chronic persistent or chronic active hepatitis. 50 Demonstration of HBsAg in the sera of some patients with hepatocellular carcinoma, particularly in Africa65 or the Far East,64 has also implicated the virus in an oncogenic role. The antigen may also be found in asymptomatic blood donors, where it may acompany mild chronic persistent hepatitis, inactive cirrhosis, or, at times, no liver disease at all.S 9 In addition to the presence of HBsAg in the blood, patients who have been acutely infected with the hepatitis B virus develop an antibody to the core portion of the virus particle, this being referred to as anti-HBcAg.24 This antibody becomes detectable during the acute phase of the clinical disease, at a time when there is active virus replication, and remains detectable long after HBsAg has disappeared from the serum. It can also be detected in chronic carriers of HBsAg, and in somehemophilic patients who have anti-Hb,Ag in the serum.24 Active virus replication during acute hepatitis B virus infection also releases DNA polymerase into the serum, and this too can be detected by appropriate methods.29 Alpha-fetoprotein is a fetal globulin which normally disappears from the circulation shortly after birth. Early reports indicated that the protein could be detected in the sera of some patients with hepatocellular carcinoma.!' 2 The value of this potential diagnostic aid was clouded, however, by other reports which cast doubt on its specificity by demonstrating the presence of alpha-fetoprotein in other hepatic disorders and in some non-hepatic tumors.42 More sensitive assays for alpha-fetoprotein now indicate that it is certainly detectable in many hepatic and nonhepatic disorders, but is present in much greater quantity in some patients with hepatocellular carcinoma, where it may offer some diagnostic value. 55 Additional areas of use which are presently being studied include monitoring the treatment of hepatocellular carcinoma30 and assessing prognosis of fulminant hepatic necrosis. Lipoprotein X is an atypical lipoprotein which appears in the blood of patients with cholestatic liver disease and can be detected by agar gel immunoelectrophoresis.56 Initial hopes that the presence or absence of lipoprotein X could help differentiate between extrahepatic and intrahepatic cholestasis (respectively) have not, however, been confirmed by subsequent study~4

RADIOLOGY Cholangiography Visualization of the extrahepatic bile ducts by some form of cholangiography is an essential part of the work-up of a patient with cholestatic liver disease. The intravenous route of dye administration is the simplest method of obtaining a cholangiogram, but will fail to visualize in patients with serum bilirubin levels much in excess of 2 mg per dl. In such patients the clinical course will dictate whether an alterna-

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1021 tive form of cholangiography is attempted or whether the intravenous study is postponed until the biochemical evidences of cholestasis have remitted. Two general approaches to visualization of the extrahepatic bile ducts in the jaundiced patient have been developed in the last few years. The first type of procedure involves transhepatic cannulation of the ducts and direct injection of dye into the ducts. This can be achieved in a number of ways, including the percutaneous route,20.22 via minilaparotomy61 or laparoscopy,33 and via the transjugular approach.53 For all these procedures it is necessary that results of tests of coagulation be within normal limits. All are more likely to succeed in the patient with dilated ducts, and because of the potential hazard of bile peritonitis (which may be less than is generally feared22 . 34 ), access to immediate surgery ought to be assured. The other type of approach utilizes transduodenal cannulation of the common duct via a duodenoscope, and retrograde injection of dye. 14 This procedure has only recently been introduced, but promises to replace transhepatic cannulation in a considerable number of patients. The greatest advantage may lie in the ability to demonstrate disease of the extrahepatic ducts in patients with coexisting disease of the intrahepatic ducts, preventing them from becoming dilated and able to be cannulated by the transhepatic approach.16

Angiography Catheterization of the hepatic artery, the portal vein (via the umbilical vein, or by trans-splenic injection) and the hepatic veins facilitates angiographic investigation of the hepatic arterial, portal venous, and hepatic venous circulations. Hepatic arteriography is an important part of the delineation of a space-occupying mass in the liver. Hepatocellular carcinomas are usually demonstrated during the arterial phase of the study,69 while metastatic tumors may be more easily detected during the capillary and venous phases.3 Large cysts or abscesses remain avascular throughout the study, and their size is reflected by the extent of stretching of intrahepatic vessels. 3 Portal venography is most commonly employed in the investigation of patients with portal hypertension, and is combined with manometric measurement of portal pressure. The size and location of varices and collateral vessels can be demonstrated, and patency of the portal vein confirmed for possible decompressive surgery.19 Examination of the intrahepatic portal venous system distinguishes between the cirrhotic pattern and that which is seen in noncirrhotic portal hypertension. lo Hepatic venography combined with inferior venacavography is also commonly accompanied by manometric investigation. Its major role lies in the investigation of the cause of hepatic venous outflow block presenting with the Budd-Chiari syndrome.t2

Gastrointestinal Radiology Radiologic investigation of the esophagus is the traditional method for demonstrating esophageal varices, and remains a useful adjunct to the diagnosis of these structures. However, the advent of safe endoscopy has led to greater faith being placed on the endoscopic diagnosis and has shown that the radiologic diagnosis is unreliable. 15

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Examination of the duodenal loop by a conventional radiologic study of the upper gastrointestinal tract or by hypotonic duodenography35 offers a valuable approach to the diagnosis of lesions in the head of the pancreas which present with cholestasis.

Isotope Scanning Following the intravenous administration of one of a number of isotopically labelled materials (radionuclides), an image of the liver can be obtained using a scanning collimator-head or a scinti-camera.68 The radionuclides used are taken up either by the Kupffer cells (e.g., 9!Yfcsulphur colloid, 198 Au-colloidal gold, 67 Ga-citrate, etc.) or by the hepatocytes (131 I-rosebengal); the former are used when a mass lesion in the liver is suspected, while the latter is usually employed in the differential diagnosis of cholestasis, particularly in the neonate. Typically, an intrahepatic mass appears as an area of decreased activity in the liver scan.36 However, 67Ga-citrate is often preferentially taken up by hepatocellular carcinomas and abscesses, and these may appear as "hot-spots" on the gallium scan.39 In the presence of diffuse parenchymal disease, hepatic uptake of all radionuclides is generally poor, much of the radioactivity being taken up by the spleen and bone marrow. Accordingly, the isotope scan has some value in the documentation of chronic liver disease, but is unreliable in disclosing mass lesions in that situationP Ultrasound Examination This form of investigation has recently become available and offers diagnostic capabilities in the same general area as isotope scanning.44 Mass lesions within the liver can be identified, and frequently it is possible to determine whether the mass is cystic or solid. Refinement of ultrasound examination with "gray-scale" display has enhanced the diagnostic potential of the technique. 63

BIOPSY AND LAPAROSCOPY Percutaneous biopsy of the liver and light microscopic examination of the tissue has been singularly responsible for much that is now known of the natural history and outcome of hepatic inflammatory diseases. It represents the most accurate diagnostic tool that the hepatologist possesses, but needs to be interpreted in light of the available clinical, biochemical, and other informationP There are virtually no clinical situations in which liver biopsy examination is not an important component of the investigation of a patient with suspected liver disease. The only contraindication to its being carried out are abnormalities of coagulation (viz., prothrombin content less than 26 per cent, severe thrombocytopenia), and the suspected presence in the liver of a highly vascular tumor or a hydatid cyst. Maximum information to be gained from light microscopic examination of a liver biopsy specimen requires utilization of several special stains including hematoxylin and eosin, trichrome (or Masson), reticulin, and iron. It also requires great familiarity with the appearances of hepatic diseases;7 and expertise in the area is not lightly acquired.

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1023 Combination of liver biopsy with laparoscopic view of the liver is widely used in European countries for the diagnosis of diffuse hepatic parenchymal diseases. Here in the United States laparoscopy has not proved popular for that purpose, but it has become a valuable aid in the diagnosis of focal diseases of the liver. The laparoscopist is able to view the lesion and can direct the biopsying needle to the portion of the liver where the lesion is placed. 28 Augmentation of light microscopic examination of liver biopsies by electron-microscopic examination has not proved as helpful diagnostically as similar investigation of the kidney. Initial claims that abnormalities of the bile canaliculi seen in cholestasis caused by testosterone and related compounds were specific for that condition have since been disproved, and presently there are few pathologic conditions of the liver for which electron-miscrosopic examination is of more than research interest.60 An additional role for liver biopsy has appeared in recent times. Biochemical analyses of hepatic tissue have become technically feasible and assays for specific enzymes offer diagnostic opportunities.7 • 25

SUMMARY At the present time the biochemical, serologic, and technological resources available in academic centers for the diagnosis of hepatic diseases are impressive. In that type of setting, and using the appropriate combination of diagnostic procedures discussed here, it is possible to determine with a considerable degree of accuracy the nature of the hepatic disease present, its extent, and, to a greater or lesser degree, its prognosis. Investigation in less favored centers must settle for diagnostic short-cuts and inevitably a lesser degree of accuracy. In either case, the state of the art is weighted heavily in favor of diagnosis and against successful therapy of most of the disease disclosed to be present. Temple University Health Sciences Center Philadelphia, Pennsylvania 19140