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  SOLP - Guidelines for Diagnosis and Monitoring of Hepatitis
 

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[ Guidelines for Diagnosis and Monitoring of Hepatitis ]
[ References ]

 

Guidelines for Diagnosis and Monitoring of Hepatitis

GUIDELINES - DRAFT 2

Click here for the references

I. Performance Characteristics for Laboratory Tests

A. Aminotransferases

Aspartate aminotransferase (AST, SGOT) and alanine aminotransferase (ALT, SGPT) are widely distributed throughout the body. AST is found primarily in heart, liver, skeletal muscle, and kidney, while ALT is found primarily in liver and kidney, with lesser amounts in heart and skeletal muscle. (1, 2, 3). AST and ALT activity in liver are about 7,000 and 3,000 times serum activities, respectively. (4) ALT is exclusively cytoplasmic; both mitochondrial and cytoplasmic forms of AST are found in all cells. (5) Both AST and ALT require pyridoxal-5í-phosphate (P-5í-P) as a cofactor. The half-life of total AST is 17 ± 5 hours, while that of ALT is 47 ± 10 hours. (6). The half-life of mitochondrial AST averages 87 hours. (7) In adults, AST and ALT are significantly higher in males than in females; for males, peak AST and ALT values are seen in young adults (AST highest from 20-30 years of age, ALT highest from 40-50 years), while in women both AST and ALT increase until about age 50. In both genders, there is a gradual decline in AST and ALT with increasing age after the peak. (8) AST tends to be lower than ALT in both men and women until approximately age 60, when they become roughly equal. Until about age 15, AST tends to be slightly higher than ALT, with the pattern reversing by age 15 in males but persisting till about age 20 in females. (8) Values for AST and ALT are higher in African-American men than for men of European ancestry; values are comparable in women of both ancestries. (9) AST and ALT activities are strongly correlated to weight and body mass index; this is thought to be the major cause of the increase in enzyme activities seen with increasing age. (8, 10) Exercise increases AST more than ALT, especially strenuous or strength exercise. (11, 12) Specimen hemolysis increases aminotransferase activities due to release of enzyme from red cells; RBC AST is about 15x and ALT about 7x plasma activity. Liver disease is the most important cause of increased ALT and a common cause of increased AST. In most types of liver disease, ALT is higher than AST. AST is also commonly increased with muscle injury, both skeletal and cardiac muscle; AST is typically higher than ALT with muscle damage. Hemolytic anemia may cause slight increases in AST, but typically with markedly elevated LDH. In alcohol abuse, AST of mitochondrial origin may be increased due to alcohol-induced release of m-AST in the absence of tissue damage. (13) Rare forms of immunoglobulin-bound AST (macro-AST) and, less frequently, AST (macro-ALT) have been reported to cause persistently elevated enzyme activities. (13a, 13b)

AST and ALT are typically measured by catalytic activity (14); both require pyridoxal-5í-phosphate (P-5í-P) for maximum activity, although the effect of deficient P-5í-P on ALT is greater than that on AST. (15) In renal failure, AST and ALT are significantly lower than in healthy individuals, perhaps due to serum binders of P-5í-P, as total P-5í-P is elevated. (16) In a recent College of American Pathologists (CAP) proficiency survey, the average variation in values between laboratories using the same methods was from 4-9% at aminotransferase values in the normal range. When comparing average results between laboratories using different assays, the range was between 39 and 85 U/L. (17)

Recommendations

Assays for aminotransferases, particularly ALT, should aim for total analytical error of 10% at the upper reference limit to meet clinical usefulness criteria. Standardization of ALT values should aim for bias of 5% between methods and across laboratories; this may require improved analytical techniques for ALT measurement. Once standardization is accomplished, health-related reference limits should be established, defined for age and gender. In individuals engaging in regular exercise, elevated ALT and/or AST should be evaluated by repeat testing after a period of abstinence from exercise; research is needed to determine the appropriate time interval required.

 

B. Alkaline Phosphatase

Alkaline phosphatase (ALP), involved in metabolite transport across cell membranes, is found, in decreasing order of abundance, in placenta, ileal mucosa, kidney, bone, and liver. (18, 19, 20, 21, 22, 23) Bone, liver, and kidney alkaline phosphatase share a common protein structure, coded for by the same gene (24, 25); they differ in carbohydrate content. The half-life of intestinal ALP is minutes, that of bone ALP is one to two days, and placental ALP has a half-life of 7 days. As released, liver isoenzyme has a half-life of three days, but removal of sialic acid shortens this to only a few minutes. (26, 27, 28) Neonates and children, especially adolescents, have significantly higher values than adults due to osteoblastic activity in bone growth. The values in children between 12 and 16 years may be two to three times the upper reference limit for those over age 25 years. Lowest values for ALP occur between the ages of 21 and 60 years; after age 60, there is a gradual increase. Reference limits are higher in men than in women. (29) Fat-rich meals increase ALP in 20% of persons (30); ideally, ALP should be measured fasting. ALP increases in pregnancy due to placental isoenzyme, up to twice baseline by term. Oral contraceptives significantly decrease ALP values. (31) Cholestasis stimulates synthesis of ALP by hepatocytes; bile salts, detergents or other surface-active agents facilitate release of ALP from cell membranes. (32, 33, 34) As a general rule, extrahepatic biliary obstruction leads to higher ALP values than intrahepatic occlusion. (35) Bone diseases with increased osteoblastic activity (most commonly Paget's disease, hyperparathyroidism, and bone malignancies) increase ALP. Unusual isoforms have been described in various malignancies. (36).

The method for total ALP in widest use is the p-nitrophenylphosphate method of Bowers, McComb and Kelly. (37, 38) CAP surveys typically show variation of 5-10% between laboratories using the same manufacturerís assay; agreement of results between laboratories using different manufacturersí assays is not nearly as good. In a recent survey using a sample with ALP approximately twice the upper reference limit, laboratories reported values between 190 U/L and 520 U/L. (39) Complexing agents such as citrate, oxalate, or EDTA give falsely decreased values, as low as zero. Blood transfusion (containing citrate) causes transient decrease in ALP through a similar mechanism.

Separation of tissue nonspecific ALP forms (bone, liver, and kidney) is difficult owing to structural similarity; electrophoresis and isoelectic focusing are the most useful techniques. Bone-specific ALP can be measured by heat inactivation (a poor method), immunologically and by electrophoretic methods. Immunoassays of bone ALP are now available from several sources (40, 41, 42), and can be used to monitor patients with bone disease.

Recommendations

Assays for alkaline phosphatase should aim for total analytical error of 10% at the upper reference limit to meet clinical usefulness criteria. Standardization is not a high priority. Health-related reference limits should be established, defined for age and gender. Specimens for alkaline phosphatase should ideally be obtained in the fasting state; if not, mildly elevated values should be confirmed elevated in the fasting state before further evaluation. Assays for alkaline phosphatase isoenzymes are needed only in patients in whom the source is not obvious from clinical and laboratory features; more commonly, measurement of other associated enzymes (most commonly GGT) is used to confirm hepatic origin of alkaline phosphatase.

C. Bilirubin Measurements

Daily production of unconjugated bilirubin is 250 to 300 mg, mainly from senescent erythrocytes. (43) Clearance at normal values is 5 mg/kg/day, or about 400 mg/day in adults. (44) The half-life of unconjugated bilirubin is < 5 minutes. (45) UDP-glucuronyltransferase rapidly conjugates bilirubin in the liver; conjugated bilirubin is excreted into bile and is essentially absent from blood in normal individuals. Delta bilirubin (d -bilirubin) is produced by reaction of conjugated bilirubin with albumin (46); it has a half-life of about 17-20 days (the same as albumin), accounting for prolonged jaundice in patients recovering from hepatitis or obstruction. (47) Conjugated bilirubin is water soluble; at increased plasma concentrations, it can be detected in urine. Conjugated bilirubin is converted by bacteria in the intestine to colorless, water soluble urobilinogen; some undergoes enterohepatic circulation, where excess can be excreted into urine. The upper reference limits for bilirubin may be as much as 0.7 mg/dL (12.0 m mol/L) higher in young adult men than in women. (29, 48, 49) African-Americans (especially women) have lower bilirubin than those of European, Latin American, or Asian ancestry. (49) Highest values occur at around age 14 for both males and females, and decrease and are stable between the ages of 25 and 65 years; there is a slight increase after age 70. (48) Strenuous exercise and fasting increase unconjugated bilirubin slightly. (50) Hemolysis nearly always produces an increase of unconjugated bilirubin. Increases in conjugated bilirubin usually indicate disease of the liver or bile ducts, and conjugated bilirubin is the most sensitive test for liver dysfunction. With recovery from hepatitis or obstruction, conjugated bilirubin falls quickly, while d -bilirubin declines more slowly. (51) Gilbert's syndrome, found in about 5% of the population causes mild unconjugated hyperbilirubinemia due to impaired UDP-glucuronyltransferase activity. (52) Total bilirubin rarely exceeds 4-5 mg/dL (68-85 m mol/L), even during prolonged fasting, unless other factors increasing bilirubin are also present. (53)

Bilirubin is typically measured using two assays for total and "direct reacting" or direct bilirubin; subtracting direct from total gives "indirect bilirubin". Direct bilirubin measures the majority of d -bilirubin and conjugated bilirubin, and a variable but small percentage of unconjugated bilirubin. (54, 55, 56) High pH or the presence of a wetting agent promotes reaction of unconjugated bilirubin in the "direct" assay. (57) Light can convert unconjugated bilirubin to a photoisomer that reacts directly (58); it also causes total bilirubin to decrease 0.02 mg/dL/hour (0.34 m mol/L/hour). Direct spectrophotometry (dry film methods) measures conjugated and unconjugated bilirubin separately, and calculates d -bilirubin as the difference between these and total bilirubin. Some have suggested conjugated bilirubin is better than "direct" bilirubin to measure recovery from liver disease. (59) In a recent CAP survey, at a total bilirubin concentration of 2.5 mg/dL (38 m mol/L), the average variation in laboratories using the same method was 5%; however, the mean with different methods ranged from 2.0 to 2.7 mg/dL (34-46 m mol/L). At a concentration of 1.5 mg/dL (27 m mol/L), the average variation using the same method was 8%, and the mean with different methods ranged from 0.06 to 1.65 mg/dL (1-28 m mol/L). (60) Urine measurements for bilirubin and urobilinogen are included in routine urinalysis. Limited data from a single study of emergency room patients (61, 62) found positive predictive value of urine urobilinogen and bilirubin was 88% for any liver function test abnormality, but with a sensitivity of only 47%; the authors concluded that urine screening was not justified.

Recommendations

Assays for bilirubin should aim for total analytical error of 10% at the upper reference limit to meet clinical usefulness criteria. Standardization of total and direct bilirubin assays is not as important as for aminotransferase methods, since values are more commonly compared to the patientís previous results. Although total bilirubin is slightly higher in fasting specimens, there is no need to standardize time of collection of samples for bilirubin testing. Health related reference limits should be established, defined for age and gender. Urine bilirubin and urobilinogen assays are not recommended for screening or monitoring purposes.

 

D. Gamma-Glutamyl Transferase (GGT)

Gamma-glutamyltransferase (GGT), a membrane bound enzyme, is present in decreasing order of abundance in proximal renal tubule, liver, pancreas (ductules and acinar cells), and intestine (63, 64, 65) GGT in serum comes primarily from liver. The half-life of GGT in humans is about seven to 10 days; in alcohol-associated liver injury, the half-life increases to as much as 28 days, suggesting impaired clearance. GGT in newborns is about five times adult values, decreasing to adult activities by 6-8 months. Values are approximately 50% higher in males than in females. (66, 67, 68) In African-Americans, GGT is approximately twice that in those of European ancestry. (68) GGT is positively correlated with body mass index, alcohol consumption, (67) cigarette smoking (67a), and physical activity level. (68) GGT tends to increase with time since last food ingestion. (68) In pregnancy, GGT tends to be lower, particularly in the third trimester (69); the sensitivity of GGT for hepatitis also decreases after the first trimester of pregnancy. (69a) GGT is increased by drugs inducing microsomal enzymes, notably anticonvulsants, estrogens, and oral contraceptives. (68, 70) GGT is slightly more sensitive than ALP in obstructive liver disease; 93-100% have increased GGT, while 91% have elevated alkaline phosphatase (71, 72, 72a); GGT, however, is elevated an average of 12 times the upper reference limit, while alkaline phosphatase is increased an average of 3 times the upper reference limit. (72a) GGT appears to increase due to bile acid-induced release of canalicular membrane fragments (with attached GGT) and from increased synthesis, analogous to ALP. (72, 72b) GGT is increased in 80-95% of patients with any form of hepatitis. (73, 73a, 73b) GGT is elevated in the majority of patients with liver tumors, with an average elevation of 14 times normal. (74) GGT is a marker of alcohol intake in many patients with alcohol abuse. (75, 76) After cessation of chronic alcohol intake, GGT may remain elevated for weeks. (76, 77) Patients with diabetes, hyperthyroidism, rheumatoid arthritis and obstructive pulmonary disease often have an increased GGT; the reasons for these findings are largely obscure. After acute myocardial infarction, GGT may remain abnormal for weeks. (78, 79) These other factors cause a low predictive value of GGT for liver disease; in a large study, only 32% of elevated GGT values were due to hepatobiliary disease. (79a)

The IFCC method described by Shaw (80) is used by most laboratories. Precision with activities less than one-half the upper reference limit is about 10%; at about twice the upper reference limit, it is closer to 5%. Average values obtained by different assays show dramatic differences, ranging from 37 to 90 U/L. (81)

Recommendations

Assays for gamma-glutamyl transferase should aim for total analytical error of 15% at the upper reference limit to meet clinical usefulness criteria. Standardization of GGT methods should aim to reduce between-method variation to 10%. Because of intraindividual and diurnal variation, use of fasting morning specimens is recommended. Health related reference limits should be established, defined for race and gender. Because of lack of specificity, routine use of GGT is not recommended.

 

E. Prothrombin Time

Prothrombin time (PT) measures time for clotting of plasma after addition of tissue factor and phospholipid; it is influenced by the activity of factors X, VII, V, II (prothrombin) and I (fibrinogen). All are made by the liver, and three (II, VII, and X) are activated by vitamin K. PT is relatively insensitive to deficiency of any single clotting factor; there is no significant increase until levels fall below 10% of normal. (82) PT is commonly reported in seconds and compared to patient normal values. (83) To minimize variation between reagents, each is assigned an International Sensitivity Index (ISI), compared to a reference method. This allows calculation of an international normalized ratio (INR): . (84) Use of reagents with low ISI improves the reproducibility of INR measurement, and has been advocated for monitoring anticoagulant therapy. (85) INR may not accurately reflect inhibition of coagulation in liver disease. (82, 86, 87, 88, 88a) The effect of ISI is much greater on PT in warfarin use than in liver disease (82); although the reasons for this difference are not known, it may be due to the marked difference in the relative amounts of native prothrombin versus des-g -carboxy prothrombin. Patients on warfarin or with vitamin K deficiency have marked elevation des-g -carboxy prothrombin and decrease in native prothrombin, while patients with acute hepatitis or cirrhosis have decreased native prothrombin but only slight elevation of des-g -carboxy prothrombin. (88b) Specimen storage at room temperature up to three days does not affect PT. (89, 90, 91) Clotting factors are activated at 4oC; prolonged refrigeration falsely lowers PT. (92) Citrate concentration affects PT; when hematocrit is high or tubes are inadequately filled, excess citrate increases PT. (93, 94) A recent consensus conference recommended use of 3.2% (109 mmol/L) sodium citrate for anticoagulation. (92) PT is reproducibly increased, usually at least 3 sec beyond the population mean, in acute ischemic (95, 96) and toxic (97) hepatitis, but is rarely elevated more than three seconds in viral (98) or alcoholic (99, 100) hepatitis. PT is often elevated in obstructive jaundice, and may respond to vitamin K administration. In chronic hepatitis, PT is typically normal, but increases as progression to cirrhosis occurs, and is elevated in cirrhotic patients. (101) Consumptive coagulopathy (as in disseminated intravascular coagulation) and vitamin K deficiency can also prolong PT.

Abnormal PT values are highly dependent on ISI of the reagents used, although normal values are similar. (82) The difference between normal and abnormal is greater using reagents with low ISI, but the effect is much greater for warfarin than for liver disease. (99) Reagents with the same ISI typically give different results on different instruments, even of the same model. (102, 103, 104) In addition, when using reagents with same ISI, the same specimen can give different INRís. (92, 105, 106, 107) The reproducibility of PT results in laboratories using the same instrument and reagents is from 3-8% when prothrombin times are prolonged; variation is greater for INR than it is for the prothrombin time itself. Within a single laboratory, average variation in INR is estimated to be ± 10%. (108) The difference in PT between laboratories using different reagents may be marked; in one study, the average difference was 20%. (105) Recently, use of calibrant plasmas to determine ISI in each laboratory for its own reagents and instrument has been shown to significantly improve reproducibility of INR. (105, 109, 110)

Recommendations

Prothrombin time prolongation has commonly been used as a marker of liver dysfunction; the use of different reagents with different International Sensitivity Index (ISI) makes comparison of results between laboratories impractical. Use of derived numbers, such as percent activity and International Normalized Ratio (INR) appear superior, but little data exists for use of these in liver disease. Additional research on usefulness of derived indices and appropriate ISIís for use in liver disease is needed.

F. Hepatitis Serologic Markers, Immunodiagnostics, and Nucleic Acid Testing

1. Hepatitis A virus (HAV) - IgM antibodies to HAV (anti-HAV IgM) are typically present at onset of symptoms, and remain detectable for 4 to 6 months after infection (110a); IgM anti-HAV may occasionally persist for up to a year after acute infection. (111) Prevalence of HAV RNA is highest when ALT is highest, and mean duration of viremia is 18 ± 14 days. (115) Total anti-HAV persists for long periods after infection, perhaps for life; seroprevalence increases with increasing age, ranging from 11% in children < 5 years to 74% in those > 50 years. (112) HAV vaccine induces detectable IgM and IgG anti-HAV within 2 to 4 weeks of the initial dose of vaccine (112a, 112b) and antibody remains detectable at 5 years in 99% of individuals completing vaccination, with predicted duration of protective antibodies of at least 20 years (112c). There are no commercially available antigen detection tests for HAV but immune electron microscopy (IEM), RIA, and EIA methods have been used to detect HAV antigen in stool filtrates and other specimens (113, 114).

2. Hepatitis B virus - A typical serological and clinical course of acute HBV infection is shown in Figure 1. (116) IgM anti-HBc is usually considered the gold standard for diagnosis of acute hepatitis B (117, 118), but may also be present at fluctuating, low titers in patients with chronic hepatitis B, particularly when patients also have HBeAg, HBV DNA, or episodes of rising ALT indicating reactivation of disease. (119,120) Total anti-HBc typically persists for life. (121) HBsAg is characteristically present and anti-HBs absent at presentation in patients with acute HBV infection, but both are occasionally absent (117), leaving IgM anti-HBc the only marker of infection ("core window"). Isolated anti-HBc may also be a false-positive result. Of those with isolated anti-HBc in low titers, virtually none showed an anamnestic response to vaccination, whereas 50-80% of those with high titers did. (121a, 121b) Convalescence from infection is indicated by loss of HBsAg and development of anti-HBs. Circulating HBV DNA can be found by sensitive PCR amplification methods in a high percentage of patients with negative HBsAg and positive anti-HBs, anti-HBe, and anti-HBc months or years after clinical recovery from acute hepatitis (122, 123) or chronic hepatitis. (124) The significance is not clear, as most viral DNA is found in immune complexes. (122) In the HBeAg positive patient, loss of HBeAg and seroconversion to anti-HBe positivity is typically associated with loss of detectable HBV DNA by non-PCR based methods, normalization of aminotransferases and histologic improvement, implying a low replication state and significant clinical improvement. (118) HBV DNA levels may be useful in following chronic hepatitis B patients receiving antiviral therapy. Loss of detectable HBV DNA by a solution phase hybridization assay is an earlier indicator of response to antiviral therapy than loss of HBeAg (125). Three hybridization assays for detection of serum HBV DNA are commercially available: liquid phase molecular hybridization assay (125), hybrid capture (126), and branched DNA. The manufacturerís stated detection limits for HBV DNA are 3.0 x 106 copies/mL for hybrid capture, 0.7 x106 copies/mL for branched DNA, and 40,000 copies/mL for liquid hybridization. (128) Results are often expressed as pg/mL, which can be determined by dividing the value for copies/mL by 2.85 x 105. PCR is now widely used for detection of HBV DNA in serum (129). A commercial PCR method, with a detection limit of 103 copies/mL is available in Europe. (130) PCR methods detect as few 100 to 1000 copies/ml. Patterns of serological markers in various forms and phases of HBV infection are shown in Table 1. (131)

Table 1. Serological diagnosis of hepatitis B virus infections (modified from Chernesky 1998)

Marker

Incubation

Acute Infection

Past Infection

Chronic Infection

Vaccination

HBsAg

+a

+

-

+

-

HBeAg

+

+

-

+/-

-

HBV DNA

+

+

-

+

-

Anti-HBc

         

IgM

-

+

 

-

-

Total

-

+

+

+

-

Anti-HBe

-

-

+/-

+/-b

-

Anti-HBs

-

-

+

-

+

a+, detectable; -, not detectable; +/- may be detectable.

bPatients with chronic HBV infection usually have detectable HBeAg or anti-HBe.

 

3. Hepatitis C virus - Screening tests for HCV infection detect antibodies to HCV proteins, usually apparent by an average 10 weeks after infection using "second generation" anti-HCV assays (EIA2) with a combination of recombinant HCV core antigens and two nonstructural epitopes from the NS3 and NS4 regions of the genome. (132) Approximately 5 to 8% of infected individuals remain negative for anti-HCV by EIA2 (134). Immunocompromised patients may lack detectable antibodies by EIA2 despite other evidence of active viral infection. A third generation EIA (EIA3) for anti-HCV has been approved by FDA for screening blood products; it contains reconfigured core and NS3 antigens and an additional antigen (NS5) not found in EIA2. EIA3 provides a slight increase in sensitivity and slightly better specificity than EIA2, and shortens time to detection of antibody to an average of 7-8 weeks after infection. (135, 136) Recently, FDA has approved a home use kit for obtaining samples for anti-HCV testing.

Supplemental tests for anti-HCV help resolve false-positive EIA test results. A second-generation recombinant immunoblot assay (RIBA II) contains the same recombinant HCV antigens as EIA2 and recombinant superoxide dismutase (SOD); a positive RIBA II is defined as reactivity against two or more HCV antigens from different regions of the genome without reactivity to SOD. Reactivity to a single HCV antigen and multiband reactivity with reactivity to SOD are considered indeterminate. In populations at high risk for HCV infection less than 1% of EIA2-positive specimens will be false-positives. Additionally, in recently infected individuals, RIBA is positive in only 85% of cases. (139a) Therefore, RIBA II testing in high-risk populations is not necessary for the diagnosis of hepatitis C. (140) A RIBA III (136) has recently been approved by FDA.

Approximately 75-85% of anti-HCV positive persons and >95% of persons with acute or chronic hepatitis C have detectable serum HCV RNA; in those recently infected with HCV, HCV RNA was intermittently present in about 15% of cases. (139a, 140a) HCV RNA can be detected in serum within 1-2 weeks after infection and weeks before ALT elevations or appearance of anti-HCV. (142) Although not FDA-approved, reverse transcription-polymerase chain reaction (RT-PCR) assays for HCV RNA are used commonly in clinical practice; the most sensitive can detect < 100 HCV RNA copies/mL. HCV RNA assays have poor standardization, quantitative assay results may vary up to 1000 fold between different laboratories and using different assays. (143, 143a, 143b) Even in the 1998 surveys, only 70-80% of laboratories reported positive HCV RNA when viral load was 10,000 copies/mL, and only 50% when viral load was 1,000 copies/mL. Using the commercially available RT-PCR method, 24 laboratories reported HCV RNA between 150,000 and 640,000 copies/mL on a specimen with viral load of 500,000 copies/mL, with an average coefficient of variation of 42%. (data from College of American Pathologistís ID surveys, 1998) HCV RNA is not stable unless frozen; serum or plasma should be frozen at Ė70oC within 30 minutes of collection. Quantitative HCV RNA assays, including RT-PCR and branched DNA (bDNA), and non-amplified signal binding amplification (143a, 143b, 146), are less sensitive than qualitative RNA assays with lower limits of detection of 500 copies/ml (RT-PCR) and 200,000 copies/ml (bDNA). Results cannot be directly compared because different standards are used, and both lack sufficient sensitivity to diagnose HCV infection or determine treatment endpoints. There are several distinct genotpyes of HCV, with 1a and 1b the most common in North America. Genotype 1 is associated with poorer response to treatment overall (254), and with need for longer treatment with combined interferon and ribavirin (258, 259) to obtain maximum frequency of response.

4. Hepatitis D virus - HDV is a defective virus that occurs only in the presence of acute or chronic HBV infection; it requires HBV for maturation. Testing for evidence of HDV infection should be considered in HBsAg-positive patients with symptoms of acute or chronic viral hepatitis, particularly with fulminant hepatitis or high risk for HDV infection. The only HDV serological tests commercially available in the US detect total anti-HDV. In patients in whom virus is cleared, antibody typically disappears between 1 and 5 years. (148) In most clinical situations, HBsAg, IgM anti-HBc, and total anti-HDV are adequate to diagnose HDV infection. Patients with acute HDV co-infection are usually positive for IgM anti-HBc, while patients with HDV superinfection are usually negative for IgM anti-HBc.

5. Hepatitis E virus - HEV is an enterically transmitted virus that causes sporadic and epidemic acute hepatitis in the developing countries of the world; it does not cause chronic hepatitis. ELISAís have been developed for diagnostic use (152). An evaluation of multiple methods for detecting anti-HEV antibodies showed significant variation in titers reported, and discordance between methods. (153) While HEV is felt to be rare in the United States, antibody reactive with HEV antigens was found in 15-25% of homosexual men, intravenous drug users, and blood donors in Baltimore, suggesting lack of specificity of assays. (154)

  1. Acute Hepatic Injury

Acute hepatic injury is usually recognized clinically by the presence of jaundice, leading to documentation of significant elevations of AST and ALT. Less commonly, patients with acute liver injury are detected when evaluated for non-specific symptoms and found to have elevated aminotransferases. Rarely, acute liver injury is detected because elevated aminotransferases are noted in an asymptomatic person. Most individuals with acute viral hepatitis are never diagnosed clinically. AST and ALT values are almost never more than 10x the reference limits in any liver diseases except acute and chronic hepatic injury, and alkaline phosphatase is rarely over 3x normal in acute hepatic injury. (160) Rarely (about 1-2% of cases), patients with bile duct obstruction may have AST and/or ALT up to 2000 U/L (72a, 160a); passage of a stone is associated with rapid decline in aminotransferase and bilirubin levels, with aminotransferases returning to reference range by 10 days in the majority of cases even with persistent obstruction (160b). In a patient with jaundice, direct bilirubin < 50% of total suggests a cause other than acute hepatic injury, as only 16% of patients with acute hepatic injury have direct bilirubin < 50% of total bilirubin. (161) The best discriminant values for recognizing acute liver injury appear to be 200 U/L for AST (sensitivity 91%, specificity 95%) and 300 U/L for ALT (sensitivity 96%, specificity 94%). (161) At time of presentation with acute viral hepatitis, maximum AST is > 200 U/L in 88% of cases and over 10x the reference limits in slightly over half of patients. (162) In uncomplicated alcoholic hepatitis, AST and ALT values are almost never over 10x normal, AST/ALT ratio is over 2 in 80%, and a cholestatic picture with elevated alkaline phosphatase is present in 22%. (162a, 162b, 162c, 162d, 163) The frequency of jaundice in patients with acute viral hepatitis differs both by age and etiologic agent. In children with viral hepatitis, jaundice is rare, and when present less severe than in adults; in one study, only 1% of children with acute hepatitis had peak bilirubin > 10 mg/dL (171 m mol/L), while 27% of adults did. (163a) In adults with hepatitis A, about 70% develop jaundice (164), while the frequency is 33-50% in hepatitis B (165, 165a) and 20-33% in hepatitis C (166, 167). There is a direct correlation between age and peak bilirubin in adults. (162) In alcoholic hepatitis, jaundice occurs in 60-70% of cases. (162b, 162c)

Recommendations

Acute hepatitic injury can be reasonably diagnosed by ALT values more than 10x an appropriate upper reference limit and an alkaline phosphatase usually no more than 3x the appropriate upper reference limit in a patient with clinical signs r6tv and symptoms suggesting acute liver disease. Alternatively, acute hepatitic injury can be diagnosed by AST or ALT values more than 2.5 times an appropriate upper reference limit in the presence of jaundice and definable acute symptoms.

 

A. Markers of Severity

Acute viral hepatitis A or B is usually self-limited, with patients recovering completely; in hepatitis C, approximately 85% develop chronic hepatitis. Rarely, acute hepatic injury causes severe liver damage and acute liver failure. Testing should identify patients with the most serious liver injury, at highest risk for liver failure. Aminotransferase activities are more related to the cause of hepatic injury, rather than to severity (Table 2). There is only weak correlation between aminotransferase activities and bilirubin in viral hepatitis (162) and none in ischemic hepatic injury (168). Peak aminotransferase activities bear no relationship to prognosis, and may fall with worsening of the patientís condition. (169) Prothrombin time has been the most important predictor of prognosis, with cutoff times > 4 seconds beyond control, > 20 seconds, or INR > 6.5 identifying patients at high risk of death. (169, 172, 173, 173a, 173b) In alcoholic hepatitis, prothrombin time > 5 seconds beyond control and bilirubin > 25 mg/dL (428 m mol/L ) in a patient > 55 years old or albumin < 2.5 g/dL predict 90% likelihood of death. (87, 162c) In ischemic or toxic hepatic injury, prolongation of prothrombin time is common early after injury, with peak occurring by 24-36 hours and then rapidly returning to normal; marked prolongation by itself does not indicate likelihood of liver failure with these causes of hepatic injury (95, 97), but persistent elevation or rising prothrombin time 4 days after acetaminophen ingestion does. (167a) In most forms of hepatic injury, peak bilirubin is not a good indicator of prognosis. (169) In the absence of other factors affecting results, a total bilirubin > 15 mg/dL (257 m mol/L) or a prothrombin time > 3 seconds above the upper reference limit (118) in patients with viral hepatitis indicates severe liver injury and mandates close monitoring for encephalopathy.

Table 2. Patterns of Laboratory Tests in Types of Acute Hepatic Injury

Disease

Peak ALT (x URL)

AST/ALT Ratio

Peak Bilirubin (mg/dL)

Prothrombin Time Prolongation (s)

Viral Hepatitis

10-40

< 1

< 15

< 3

Alcoholic Hepatitis

2-8

> 2

< 15

Often 3-5

Toxic injury

> 40

> 1 early

< 5

> 5 (transient)

Ischemic injury

> 40

> 1 early

< 5

> 5 (transient)

x- times; URL- upper reference limit

 

Recommendations

The most reliable markers of severity of acute hepatic injury are prothrombin time and total bilirubin. In the absence of other factors affecting results, a total bilirubin > 15 mg/dL or a prothrombin time > 3 seconds above the upper reference limit (in cases of viral hepatitis) indicates severe liver injury. Direct bilirubin is needed to rule out other causes of increased total bilirubin; in hepatitis, the majority of bilirubin should be direct reacting. Transaminases (AST and ALT) correlate weakly with severity of hepatitic injury and falling values may prove misleading.

 

    1. Differential Diagnosis

Initial laboratory evaluation of patients with acute hepatic injury should include testing for antibodies to hepatitis A, B, and C viruses (HAV, HBV, and HCV). IgM anti-HAV is present transiently after acute infection, usually disappearing by 4-6 months (111); total HAV antibodies probably persist for life, and are found in a high percentage of the population (112). IgM anti-HBc and HBsAg are the most reliable tests for acute HBV infection (117, 118); total anti-HBc persists for many years. (121) Other HBV viral markers and antibodies are not of use in the diagnosis of acute HBV infection. There is currently no test to definitively diagnose acute hepatitis C. Anti-HCV is detectable with EIA-2 in only 57% of acute HCV cases at the time of initial enzyme elevation, while HCV RNA is positive in essentially all cases (142), although it is variably present in 15% of cases. (139a, 140a) By the time of clinical presentation, 80-90% will develop detectable anti-HCV. (140a) None of the tests, however, distinguish acute infection from chronic infection. Testing for delta hepatitis virus (HDV) should be limited to patients with positive HbsAg, particularly if accompanied by severe acute hepatitis, high risk factors (IV drug abuse, hemophilia) or a biphasic pattern of illness. (174) Acute exacerbation of chronic hepatitis B can occur when patients become superinfected with HDV. In such a setting, patients may present with a clinical picture resembling severe acute hepatic injury, and fulminant hepatic injury may evolve. (174)

Recommendations

The initial evaluation of patients meeting the case definition of acute hepatitic injury should include a detailed drug history (including over the counter, prescription, and non-traditional drugs) and a panel of viral markers, to include evaluation of IgM anti-HAV, IgM anti-HBc, and HBsAg. Because of the need to give post-exposure prophylaxis to close contacts of hepatitis A patients, turnaround time of IgM anti-HAV should ideally be less than 48 hours. If cost-effective, laboratories may screen with total antibody to HAV and anti-HBc, performing IgM antibodies only if the screen is positive; this will depend on prevalence of IgG antibodies in the population tested. At present, there is no satisfactory test to recognize acute hepatitis C infection; anti-HCV is commonly used for this purpose, but may be negative in 10-20% of patients at time of presentation, and anti-HCV persists in chronic HCV infection.

2. Workup of Patients without Obvious Cause for Acute Hepatic Injury

    1. Ischemic and Toxic Hepatic injury - Values of AST or ALT over 100x normal are rare in viral hepatitis (160, 162), but common in both toxin ingestion, especially acetaminophen (97, 175, 176), and ischemic hepatic injury. (95, 96, 168) In acetaminophen-induced hepatic injury, peak AST is over 3,000 U/L in 90% of cases. (176) In 349 patients hospitalized for acute viral hepatitis, peak AST exceeded 3,000 U/L in only one case. (162) In 56 patients seen in one year with AST > 3,000 U/L, 47 had liver disease; toxic or ischemic hepatic injury was the cause of over 90% of liver cases. (177) In both ischemic and acetaminophen hepatic injury, AST and ALT typically peak early (often in the first 24 hours after admission) with AST initially higher than ALT. After peaking, activities of both fall rapidly; AST may fall by 50% or more in the first 24 hours, (175, 176) and declines more rapidly than ALT due to its shorter half-life. (6) In viral hepatitis, AST and ALT fall more slowly, AST reaching normal values in 22.5 ± 15.7 days and ALT in 27.3 ± 15.5 days. (162) Bilirubin peaks later than enzymes in hepatitis. Once peak bilirubin occurs, bilirubin falls with a biphasic pattern, with a rapid early phase and a later slow clearance due to d -bilirubin. LDH is often higher than AST at presentation in toxic or ischemic hepatic injury (95, 175, 176), while it is increased on initial determination in only 55% of cases of viral hepatitis, with average values being only slightly above the upper reference limit. (162) Rarely, cocaine may cause hepatic injury, usually in patients with coexisting hypotension. (176a.)
    2. Acute Wilsonís Disease Ė Although chronic Wilsonís disease is characterized by reduced ceruloplasmin, reduced serum copper, and increased urine copper excretion (as discussed in more detail later), during acute hepatic injury due to Wilsonís disease, serum and urine copper are often markedly increased (178). Moreover, ceruloplasmin is normal in 60% of patients with fulminant hepatic injury due to Wilsonís disease. (179) Because tests for Wilsonís disease are affected by acute inflammation, an abnormal ceruloplasmin result is helpful while a normal result is not of value. Acute Wilsonís disease is often accompanied by hemolytic anemia and renal insufficiency. (178) In addition, in acute hepatic injury due to Wilsonís disease, there is often a disproportionate increase in bilirubin and a decrease in alkaline phosphatase, which may suggest the diagnosis (180). While the gene for Wilsonís disease has been identified on chromosome 13 (181), at least 30 different mutations of varying types have been linked to the disease, making molecular diagnosis more difficult. (182)
    3. Acute Autoimmune Hepatitis - Although typically considered a chronic disorder, some patients with autoimmune hepatitis may present acutely, in a fashion mimicking acute viral hepatitis. (183, 184) Suggestive features include markers for chronic liver disease (low albumin, ascites), and hypergammaglobulinemia. (185) Specific autoimmune markers such as antinuclear antibodies (ANA) and/or anti-smooth muscle antibodies (ASMA) in titers > 1:80 and absence of evidence for drug or viral hepatitis suggest the diagnosis. (186) ANA is the most sensitive test, positive in about 80% of cases. (187)
    4. Other Causes - Several viruses other than the classical agents (HAV, HBV, HCV, HEV) have been associated with hepatitis, including herpesvirus, cytomegalovirus, enterovirus, coronavirus, reovirus (in neonates), adenovirus, parvovirus B6 (in pediatric populations), varicella-zoster virus, and Epstein-Barr virus. Syphilis, leptospirosis, and toxoplasmosis may also cause hepatic injury, as may other less common infectious agents. Rarely, other disorders including lymphoma, Budd-Chiari syndrome, and venoocclusive disease may present with a picture of acute hepatic injury. In general, hepatic injury associated with these etiologies is either unusual, or is associated with a specific syndrome (chicken pox in varicella-zoster virus, mononucleosis in Epstein-Barr virus). Most patients with other infectious causes of hepatic injury have signs and symptoms that suggest a particular agent as the cause. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are often associated with elevated alkaline phosphatase (92% with EBV 64% with CMV) and minimally elevated aminotransferases. (187aa) Specific diagnosis of infection by other agents should be pursued when the etiology remains unknown after more common causes are excluded, and when establishment of a specific diagnosis appears clinically indicated. Superinfection with other hepatitis viruses may occur in a patient with other forms of hepatic injury; for example, patients with chronic HCV or alcoholic hepatitis may become infected with either HAV or HBV and develop an acute hepatitis due to the new virus. In chronic hepatitis, an acute rise in aminotransferases mimicking acute hepatic injury can occur with clearance of HBeAg (187a) or with emergence of quasispecies of HCV. (187b)

Recommendations

In patients with negative viral markers, if initial AST is > 100x an appropriate upper reference limit, the patient should be evaluated for drug or toxin exposure or ischemia. In patients with lower enzyme levels, testing must exclude the possibility of Wilsonís disease and autoimmune hepatitis, since these require specific treatment. Tests for other infectious agents (Epstein-Barr and Cytomegalovirus, syphilis, toxoplasmosis) may be used if no other causes are evident. Testing for antibody to Hepatitis E is not recommended in the United States unless patients have negative serologies for other viruses and the patient has a history of recent travel to an endemic area.

3. Monitoring

a. Aminotransferases - Aminotransferase activities tend to rise before and peak near onset of jaundice in viral hepatitis, falling gradually from that point on. (188). Decreasing activities indicate lessening of necrosis; values decreasing with clearance rates similar to half-life suggest ischemic or toxic hepatic injury, (95, 96, 176) where damage occurs in a short period of time (189). Activities tend to fall more slowly in viral hepatitis and alcoholic hepatitis, AST decreasing by an average of 11.7% per day and ALT 10.5% per day. (162). In hepatitis A, a secondary rise in enzymes occurs in 5-10% of cases before activities return to baseline, associated with circulating HAV RNA and viral particles in stool, indicating potential for transmission of infection. (190, 190a, 190b). Decreasing activities occur both with recovery and with massive necrosis, making falling activities a poor indicator for monitoring disease recovery. (169) Rapidly falling aminotransferase activities accompanied by rising bilirubin and prothrombin time suggest massive necrosis and impending hepatic failure. Once aminotransferases have shown a consistent pattern of decrease, they need not be checked again until the patient has clinically recovered. Return of aminotransferases to normal is not a reliable sign of recovery in hepatitis C; 49% of those with normal ALT on initial visit after seroconversion developed elevated ALT on subsequent follow-up. (190c)

b. Serum and Urine Bilirubin - Bilirubin peaks later than aminotransferases, often by a week or so, and then gradually decreases. Peak bilirubin over 15-20 mg/dL (257-342 m mol/L) is unusual in viral hepatitis, with only 10-12% of patients with viral hepatitis having peak values over 15 mg/dL (257 m mol/L) and only 4% with peak values over 20 mg/dL (342 m mol/L); higher bilirubin is more common in HBV infection, however, as 29% of HBV cases had peak bilirubin over 15 mg/dL (257 m mol/L). (162, 163a) As total bilirubin declines, the proportion of d -bilirubin increases, often reaching 70-80% of total bilirubin (191, 192, 193). In adults with viral hepatitis, bilirubin remains elevated 30.3 ± 19.7 days after peak levels are reached (162), but clears more quickly in children (162a); jaundice remains more than 6 weeks in 34% of adult HBV cases but in only 15% of other forms of viral hepatitis. (162a) Prolonged elevation of conjugated bilirubin occasionally occurs with viral hepatitis, particularly with HAV, but does not signify a poor prognosis if synthetic function remains intact. (193a) Significant elevation of bilirubin is uncommon in acetaminophen (97) and ischemic hepatic injury; in most cases bilirubin is normal or < 2 mg/dL (34 m mol/L). (95, 96) Once serum bilirubin has begun to decline, there is no reason to measure it again until jaundice has cleared clinically.

c. Coagulation Tests - Elevated prothrombin time is a common finding in ischemic (95, 96) and acetaminophen (97) hepatic injury, often with results > 15 seconds or 4 seconds above the reference limit; results rapidly return to normal. There are no data on the degree of elevation affecting prognosis in ischemic hepatic injury. Elevation of prothrombin time > 15 seconds or more than 4 seconds above reference limits in viral or alcoholic hepatitis is a marker of more severe disease. (99, 100, 162c) In a recent epidemic of hepatitis A, approximately 15% of patients required hospitalization, and 26% of these (or about 4% of all cases) had prothrombin time > 3 seconds above normal. (98) In alcoholic hepatitis, elevated prothrombin time is common, even in mild forms without jaundice where 61% have prothrombin time prolonged an average of 1-3 seconds. (162b, 162c)

Recommendations

Prothrombin time > 15 seconds or > 4 seconds above reference limits, bilirubin > 15 mg/dL (257 m mol/L), or development of encephalopathy identify high risk patients that require close monitoring and consideration of referral to a specialist. In patients with acute hepatitis B, repeat HBsAg should be performed within 6-12 months; if negative and anti-HBs is positive, no further follow-up is needed. If ALT is normal at three months in hepatitis C patients, ALT should be repeated at 6 months and periodically over the next 1-2 years to assure continued normal results.

  1. Chronic Hepatic injury

Chronic hepatic injury is a relatively common disorder with minimal symptoms, yet with long term risk of significant morbidity and mortality. It is defined pathologically by ongoing hepatic necrosis and inflammation in the liver, often accompanied by fibrosis. It may progress to cirrhosis and predisposes to hepatocellular carcinoma. Up to one-third of patients with chronic hepatitis C (the most common form) progress to cirrhosis, a median of 30 years after infection (194), although most studies suggest the figure is closer to 15-20%. (195, 196) Most commonly, it is due to chronic viral infection. In the United States alone, there are an estimated 4 million people positive for anti-HCV; at least 75% have circulating HCV RNA. There are also approximately 1-1.25 million chronic carriers of hepatitis B in the U.S. Clinical findings and laboratory investigation are often adequate to establish the most likely diagnosis; in a study of 90 patients with asymptomatic elevation of aminotransferases, carefully evaluated before undergoing liver biopsy, the clinical diagnosis had a predictive value of 88% for alcoholic hepatitis and 81% for chronic viral hepatitis (before availability of HCV tests); it was less useful for other diseases. (197)

 

Recommendation In the absence of liver biopsy showing chronic hepatitis, one of the following clinical definitions should be used to diagnose chronic hepatitis:

Persistence of increased ALT for more than 6 months after an episode of acute hepatitis. OR

Elevation of ALT (without another explanation) on more than one occasion over a period of 6 months; a shorter time may be appropriate in patients with risk factors for chronic viral hepatitis or clinical signs or symptoms.

Although the definition of chronic hepatic injury by elevated ALT is widely accepted, 15-50% of patients with chronic hepatitis C have persistently normal ALT. (190c, 198, 199. 199a) The majority of these have histologic evidence of chronic hepatitis on biopsy, but, in general, milder inflammation, lower evidence of fibrosis, and lower rates of progression to cirrhosis than do HCV patients with elevated ALT. (166, 199) Recent Centers for Disease Control guidelines do not recommend treatment of patients with HCV and persistently normal ALT. (202) While longer term studies are needed, it appears that the clinical definition proposed will not miss a significant group of patients who require and benefit from treatment.

A. Screening

In reviewing the costs and benefits of detecting chronic hepatic injury, Quinn and Johnston (203) concluded that general screening is not cost effective, and advocate screening only high risk individuals. Of routine laboratory tests, ALT has the highest sensitivity for chronic hepatic injury; ALT is consistently higher than AST with all causes of chronic hepatic injury except alcohol; AST is normal in a significant number of patients. As discussed below, however, ALT may be normal in patients with cirrhosis, while AST remains elevated. Total and direct bilirubin and alkaline phosphatase are normal in essentially all patients, and not useful in screening. (197, 204, 205, 206, 207) If an elevated ALT is found on routine testing, some suggest that ALT should be elevated on repeat testing before further evaluation, since patients with single elevated values are less likely to have liver disease; however, a significant minority of individuals with only one elevated ALT are found to have liver disease. (205, 208). Since ALT is also found in skeletal muscle, it is advisable to consider history of exercise and, if positive, to consider measurement of CK to rule out skeletal muscle origin for ALT. (12, 208)

In patients with risk factors for chronic HBV or HCV infection, however, ALT will not identify all infected individuals. Chronic carriers of HBV have normal ALT, and 15-30% of patients with chronic HCV infection have persistently or intermittently normal ALT. In patients with risk factors for chronic viral infection, HBsAg and anti-HCV should be measured to detect chronic infection. CDC recommends routine testing for HCV in persons with a history of injection drug use, chronic hemodialysis, blood transfusion or organ transplantation prior to 1992, receipt of blood (including health worker percutaneous exposure to blood) from a donor subsequently testing positive for HCV, receipt of clotting factor concentrates produced before 1987, and birth to HCV-positive women as risk factors for chronic hepatitis C. (202) Similar risk factors exist for hepatitis B, along with Asian ancestry. CDC places some categories of individuals in an uncertain category with regard to screening; these include recipients of transplanted tissue, users of illegal drugs that are not injected, history of body piercing or tattooing, multiple sexual partners or sexually transmitted diseases, and long term sex partners of HCV positive individuals. Screening is not recommended for the general population, health care workers, pregnant women, and household contacts of HCV positive individuals.

Recommendations

Screening asymptomatic individuals for chronic hepatitis is only recommended in patients at high risk of having the disease. ALT is the most cost-effective test for screening for chronic hepatitis in patients with a prior history or family history of liver disease, and in patients receiving drugs that may cause liver injury; AST should also be measured with history of alcohol abuse. ALT is inappropriate as a screen for chronic viral infection; specific viral serologies (HBsAg, anti-HCV) should be performed in high risk individuals. There is inadequate information on screening for most congenital diseases causing chronic hepatitis.

 

B. Differential Diagnosis

If history suggests alcohol abuse and/or AST is greater than ALT (especially if > 2x ALT), the most likely diagnosis is alcoholic hepatitis. Virtually no other form of chronic hepatic injury causes AST to be higher than ALT unless cirrhosis develops. (204, 205, 206, 207) While the majority of cases of chronic hepatic injury are caused by viruses, drugs, or ethanol, a number of other disorders may produce chronic hepatic injury. There is little reason to perform additional tests if initial evaluation is consistent with hepatitis B or C or alcoholic hepatitis; in fact, positive test results are most likely to be erroneous. (206, 210) Several studies have found that patients with only slightly elevated (1-2x normal) ALT are more likely to have transient elevation and be found to have no obvious cause for elevation. (197, 206, 208)

Recommendations

Initial evaluation of patients with chronic hepatitic injury should include a detailed drug history (including over the counter, prescription, and non-traditional medications) along with measurement of HBsAg and anti-HCV. Confirmation of exposure to HCV can be made by recombinant immunoblot testing (RIBA) or qualitative PCR for HCV RNA. In patients with persistently elevated ALT, workup should include measurement of antinuclear antibodies (especially if globulins are increased) and iron and iron binding capacity. In patients under age 40, ceruloplasmin should also be measured. In patients negative for these markers, a 1-antitrypsin phenotype may be of use. If these tests are negative or inconclusive, diagnostic liver biopsy should be performed.

 

C. Workup of Patients Without Obvious Cause for Chronic Hepatic injury

 

    1. Nonalcoholic Steatohepatitis (NASH) - Occurrence of chronic liver disease with fatty change, inflammation, and Mallory bodies in patients without alcohol abuse was first recognized in the late 1970ís (211); the term NASH was coined in 1980. (212) NASH is the most common cause of chronic hepatic injury other than viruses and alcohol and the most common cause of cryptogenic cirrhosis (197, 204, 205, 213); although it occurs most commonly in middle aged women with obesity and/or diabetes, it also occurs in men and in patients without these risk factors. (211, 212, 213) Patients with NASH commonly have abnormal lipid profiles, although normal results do not rule out this disease. Although histologically resembling alcoholic hepatitis, it differs in laboratory presentation primarily by having ALT higher than AST (except in patients with cirrhosis) (214, 214a). Weight loss may cause significant improvement in enzyme results; in one study, a 1% reduction in weight caused an average fall of 8.1% in ALT. (215a)
    2. Recommendations

      Although a common cause of chronic hepatitis, nonalcoholic steatohepatitis cannot be diagnosed from laboratory tests. Biopsy is necessary to establish the diagnosis and helps to evaluate the severity of fatty liver. A single study has suggested a trial of weight loss as a diagnostic test, but this has not been confirmed.

    3. Hemochromatosis - An autosomal recessive trait, hemochromatosis is the most common inherited genetic defect in persons of northern European ancestry (approximately 1:200-1:300 in the United States). (216) The vast majority of cases are due to one of two point mutations of the HFE gene on chromosome 6. (217, 218) The majority (60-90%) are homozygous for the C282Y (845A) mutation, with a minority compound heterozygotes for this mutation and the H63D (187G) mutation. Screening involves detection of increased transferrin saturation (saturation = serum iron (Fe) * 100/total iron binding capacity (TIBC)) (219) or low unsaturated iron binding capacity (220). Using transferrin saturation of 62% and elevated ferritin to recognize iron overload, only 23 of 36 patients diagnosed as having iron overload were homozygous for C282Y or compound heterozygous for C282Y and H63D. (221) A recent consensus conference recommends that definitive diagnosis be made by genetic analysis. (222) Several recent publications have shown the feasibility of performing screening for hemochromatosis using transferrin saturation; at this time, most do not recommend screening because of unresolved issues regarding ability to convince young adults to be tested, specificity and reproducibility of screening tests, and questions about natural history of untreated disease. (221, 222, 224, 225) Routine population screening has been advocated by the College of American Pathologists (226), and has been estimated to save $3.19 per blood donor screened. (227)

Recommendations

Testing for hemochromatosis is recommended in patients with unexplained elevation of ALT after evaluation for viral or drug induced chronic hepatitis. Screening of the population may be beneficial. Initial evaluation should consist of fasting serum iron and calculation of transferrin saturation; values over 45% should be followed by analysis for HFE gene mutations.

 

3. Wilsonís Disease - An autosomal recessive disorder, Wilsonís disease is found in about 1 in 30,000 individuals. Due to a chromosome 13 defect in an ATPase needed for copper transport (181), Wilsonís disease may present as liver disease, neurologic problems, or with psychiatric symptoms, almost always before age 40. (178) The most commonly used screening test is low plasma ceruloplasmin; low levels also occur with malnutrition, protein loss, and advanced liver disease, and falsely normal values can occur with pregnancy, estrogen administration, and acute inflammation. (228) Most references report low ceruloplasmin in 95% of homozygotes and 20% of heterozygotes (228, 229, 230); however, one recent study found normal ceruloplasmin in 35% of patients with chronic liver disease due to Wilsonís disease (confirmed by genetic studies in 80%), but in only 15% of patients with Wilsonís without overt liver involvement. (178) Other tests, including free copper in serum, total serum copper, urine copper excretion, and liver copper content may also provide misleading results (178, 231) in Wilsonís disease patients. Multiple tests are frequently needed to establish the diagnosis.

Recommendations

Testing for Wilsonís disease is indicated in patients under age 40 with chronic hepatitic injury or fatty liver, and negative workup for viral hepatitis, drug-induced liver injury, and hemochromatosis. Screening for Wilsonís disease in all patients with chronic hepatitic injury is not indicated. Testing of ceruloplasmin should be the first test performed; there is insufficient evidence as to which type of ceruloplasmin assay should be used. In patients with family history or neurologic or psychiatric symptoms, normal ceruloplasmin should be confirmed with measurement of serum and urine copper. Genetic marker testing may be useful in equivocal cases, but testing must be able to detect multiple mutations in the Wilsonís disease gene.

4. Autoimmune hepatitis - Autoimmune hepatitis is responsible for up to 18% of chronic hepatitis not due to viruses or alcohol. (232) Although several variants have been described (233), only Type 1, found primarily in young women, is common in the United States. Standardized criteria for diagnosis and a scoring system were defined by an international panel. (186) The classic features include chronic hepatic injury (elevated aminotransferases, minimal or no elevation of alkaline phosphatase); hypergammaglobulinemia (at least 1.5 times normal); absence of evidence of viral infection, risk factors for viral infection, or exposure to drugs or alcohol; and positive ANA or ASMA (at least 1:80). (186) The importance of lack of evidence of viral hepatitis is the overlap in serologic test results in chronic HCV and autoimmune hepatitis. Approximately 40% of patients with chronic HCV infection have a positive ANA or ASMA, usually in low titers. (233) In patients with autoimmune hepatitis, false positive anti-HCV has been reported in 60% of patients using second generation tests and 20% in third generation assays (234); anti-HCV typically disappears with successful treatment. (235) In equivocal cases, HCV RNA (or RIBA) can be used to establish the diagnosis. (234)

Recommendations

Autoimmune hepatitis should be suspected in patients with chronic hepatitic injury and increased immunoglobulins and absence of viral markers or risk factors for viral hepatitis. It can be clinically supported by positivity for either anti-nuclear antibodies (ANA) or anti-smooth muscle antibodies (ASMA) in high titers. Testing for anti-liver/kidney microsome (anti-LKM1) may be of use in children, although the variant with this antibody is rare in the United States.

    1. Alpha-1-antitrypsin (A1AT) Deficiency Ė Alpha-1-antitrypsin is the most important protease inhibitor; congenital deficiency occurs in approximately 1 in 1000 to 1 in 2000 persons of European ancestry. The gene for A1AT is located on chromosome 14 (236); deficiency is usually due to a single amino acid substitution that alters carbohydrate binding and impairs release from hepatocytes. (237) The most important deficiency involves homozygosity for the Z variant, termed Pi (for protease inhibitor) ZZ. Although, classically, deficiency is associated with emphysema, neonatal hepatitis (238) and chronic hepatic injury with cirrhosis and hepatocellular carcinoma have also been reported. (239) In neonates with Pi ZZ, almost all have evidence of liver injury at birth; this usually resolves by age 12 year. (238) In adults, 50% of Pi Z positive individuals (either homozygotes or heterozygotes) developed cirrhosis and 31% developed hepatoma. (237) There is also an excess of Pi Z heterozygotes among patients referred for liver transplant, particularly among patients with cryptogenic cirrhosis where approximately 25% of patients are Pi Z positive. (240) There is evidence, however, that AIAT deficiency or heterozygosity for PiZ phenotype may not directly cause liver disease, but increase susceptibility to liver damage by other agents, especially viruses. Two controlled studies have found the same frequency of Pi Z (either homozygous or heterozygous) in patients with liver disease and controls. (241, 242) In a study of 164 patients with Pi Z, 40% had chronic liver disease; 87% were also positive for HCV antibodies or HBV markers. (243) Only 11% of patients with Pi Z and chronic liver disease had no other risk factors. (243) Because A1AT is an acute phase reactant, quantitative levels may be falsely normal with infection or inflammation, and falsely low levels may occur with malnutrition, protein losing states, or end stage liver disease. In one study, quantitative levels were normal in 42% of heterozygous Pi Z patients with liver disease. (244) Testing for A1AT deficiency should be done by phenotype rather than measurement of plasma levels. (237)

Recommendations

The role of alpha-1-antitrypsin deficiency in pathogenesis of liver disease in adults is not clearly defined. Testing for alpha-1-antitrypsin deficiency may be of benefit in patients with chronic hepatic injury and no other apparent cause. Testing is especially important in neonates with evidence of hepatitic injury. Testing should be by determination of phenotype, since evidence suggests that patients homozygous or heterozygous for the PiZ phenotype have an increased risk of liver disease. Screening patients with chronic hepatic injury for alpha-1-antitrypsin deficiency is not recommended.

D. Monitoring

While ALT is the most clinically used laboratory test for monitoring liver injury, there is often considerable fluctuation in enzyme activities over time (particularly in chronic HCV infection, where ALT may be normal despite presence of chronic hepatitis on biopsy) (198, 199). It is important measure ALT repeatedly in chronic HCV before concluding that ALT is normal; 43% of chronically infected individuals have ALT values fluctuating between normal and abnormal, and 16% of those with normal ALT on their first two visits and 11% of those with normal ALT on their first three visits subsequently developed increased ALT. (190c) Enzyme activities do not reflect severity of fibrosis, a major factor in prognosis, and there is at best a weak correlation between ALT activities (245, 246, 247) or, in the case of HCV, HCV RNA levels (247, 248) and histological activity of chronic hepatitis. At best, ALT explains only 30-50% of variation in histologic activity, and there is considerable overlap in values in patients with mild, moderate, or severe activity (245, 246, 247), which relates to rate of progression of fibrosis. (194) With both chronic HBV and HCV, clearance of viral markers is the most reliable method for assessing progression of disease. HBeAg should be rechecked periodically if initially positive; if HBeAg is negative and anti-HBe is positive, HBsAg and anti-HBs should be measured periodically to look for viral clearance. In patients without elevated ALT (chronic carriers), ALT, HBsAg and anti-HBs should be measured periodically to look for development of chronic hepatitis or viral clearance. For HCV, the best indicator of viral clearance is persistent disappearance of HCV RNA by qualitative testing. Decrease in viral load in the absence of clearance is not relaiable evidence of treatment success; even without treatment, HCV RNA loads fluctuate over time, with up to a 3 log difference seen in patients with elevated ALT when HCV RNA was measured monthly. (248a) In contrast, absent HCV RNA 6 months after completion of treatment is associated with only 10% likelihood of recurrent HCV viremia. (248b)

Recommendations

In viral hepatitis, viral markers (HBeAg clearance and anti-HBe appearance indicating loss of HBV replication, HBsAg disappearance and anti-HBs appearance indicating clearance of HBV infection; HCV RNA disappearance for at least 6 months indicating HCV clearance) are the most reliable markers of resolution of hepatitis. In other forms of liver injury, ALT is the best marker of disease activity available, but is of limited utility in predicting severity of inflammation or fibrosis.

IV. Cirrhosis

 

Chronic hepatitis is classified by histology based on activity of inflammation and degree of fibrosis; extent of fibrosis relates to likelihood of developing cirrhosis. Liver fibrosis is associated with deposition of a number of proteins in the liver. Numerous studies of plasma levels of these proteins and their precursors (262, 263, 264, 265, 266, 267, 268, 269, 270, 271) have shown at best a weak correlation between marker levels and extent of fibrosis. Levels reflect activity of fibrosis at the time of sampling, and there is considerable overlap in values with varying degrees of fibrosis. A simple marker, able to be calculated from routine monitoring tests, is the AST/ALT (SGOT/SGPT) ratio. Several studies have shown that, in patients with non-alcoholic hepatitis, the ratio is virtually always < 1, but with progression to cirrhosis the ratio often increases to > 1. (101, 272, 273). In these studies, the specificity of a ratio > 1 was 75-100%, with sensitivity 32-83%. Moreover, in one of the studies (273), the ratio increased with increasing fibrosis score. This appears to be due to a reduction of ALT production in damaged liver. (73) Other routine tests that predict likelihood of cirrhosis are thrombocytopenia and prolonged prothrombin time; using an index of these two variables with AST/ALT ratio gave a sensitivity of 46% and specificity of 98% for cirrhosis, similar to diagnostic performance for AST/ALT ratio (101). Albumin is commonly measured in patients suspected of progrssing to cirrhosis; while it is not as sensitive as the other markers, it is widely used as a marker of severity of synthetic impairment and as part of the Child-Pugh classification of cirrhosis. AFP is more likely to be elevated as degree of hepatic fibrosis increases (273a), especially in cirrhosis; AFP >17.8 ng/mL had a sensitivity of 35%, specificity of 98.6%, and positive predictive value of 97.7% for cirrhosis. (273b)

Recommendations

At present, there is no definitive marker of progression from chronic hepatitis to cirrhosis, other than biopsy. Laboratory markers of fibrosis do not have substantially greater accuracy than either clinical criteria or more simple laboratory tests. Until further data are available, non-specific markers of hepatic function which may indicate progression to cirrhosis (AST/ALT ratio, albumin, prothrombin time, platelet count) should be measured every 3-6 months in patients with chronic hepatitis.

V. Hepatocellular Carcinoma

Primary liver cancer (hepatocellular carcinoma, HCC) is a serious late complication of chronic hepatic injury, particularly in cirrhosis due to HBV, HCV, and hemochromatosis. Infrequently, HCC is seen in patients with chronic HCV and in asymptomatic HBV carriers without cirrhosis. The incidence of HCC has increased by 70% in the United States over the past 20 years, particularly among younger patients. (274) The risk of developing HCC in cirrhosis due to HBV is 15% at 10 years (275), and the projected incidence in HCV is estimated to be similar at 14% at 10 years. (276) In a study of 448 cases of HCC, 75% occurred in patients with cirrhosis; in only 30% was cirrhosis recognized clinically before hepatocellular carcinoma was diagnosed. (277). These data suggest that screening programs, if instituted, must include patients with chronic hepatic injury as well as patients with diagnosed cirrhosis. Other risk factors include male gender and age > 55 years.

The prognosis of patients with HCC detected by development of symptoms is grim, with few patients surviving over 6 months. Detection of small tumors offers the potential for curative resection, and forms the rationale for considering screening. Current practice suggests measurement of a -fetoprotein (AFP) and ultrasound (US) of the liver every 6 months. Unfortunately, AFP interpretation is complicated by intermittent elevations of AFP in patients with chronic hepatic injury, often (but not always) associated with transient increases in ALT. This has caused different groups to adopt differing cutoff values for AFP, making it difficult to compare sensitivity and specificity of AFP between published studies. A Consensus Development workshop recommended screening chronic HBsAg carriers at least once, and preferably twice, yearly with AFP only, while patients with other risk factors (known cirrhosis, family history) should have both AFP and US. (278) The participants felt there was inadequate data to recommend screening in other chronic liver diseases. In chronic liver disease, high risk of HCC is present in patients with hemochromatosis or with cirrhosis due to HBV, HCV, and alcohol abuse. Other causes of liver disease have lower risk of HCC. (279)

In Western countries, the predictive value of AFP is low, often in the range of 10-30%, with sensitivity of AFP between 40-80%. (280, 281, 282, 283, 284, 285) In 147 patients with cirrhosis, none of the 30 patients with HCC had AFP > 105 ng/mL at the time of diagnosis and 60% had AFP < 20 ng/mL; however the frequency of HCC in patients with AFP < 50 ng/mL was 17%, compared with 42% in those with higher AFP. (285) In another study of 260 patients with cirrhosis, HCC developed in 26% of patients with initial AFP < 20 ng/mL, but 46% in those with higher levels. Moreover, those with even transient increases above 100 ng/mL had a significantly higher risk of HCC than those whose AFP was consistently < 20 ng/mL. (286) Sarasin et al. performed decision analysis on published papers of screening for HCC in Western patients with compensated cirrhosis. They concluded that, for patients with a likelihood of survival of 85% at 5 years, screening would likely add 3-9 months to average life expectancy at a cost of $26,000 - $55,000 per year of life gained, figures that compare favorably to those of colon cancer and breast cancer screening. (287) In patients with lower likelihood of survival, screening provided minimal or no gain in life expectancy and does not appear indicated. Collier and Sherman analyzed all published studies and concluded that there is inadequate data to determine the benefit of screening for HCC among patients with chronic liver disease. (288) If screening is used, frequency of testing of every 6 months appears to be optimal based on observed doubling times of HCC, reported to average around 3-5 months (289, 290, 291).

 

Des-g -carboxy prothrombin has also been suggested as a screening test. Although levels are elevated occasionally in chronic liver disease, there is less overlap with values seen in HCC than for AFP. (292, 293) Occasional high levels are encountered in metastatic carcinoma to the liver, but they are usually minimally increased. While des-g -carboxy prothrombin appears less sensitive for HCC than AFP, with sensitivities typically around 50-70%, it is more specific. There is poor correlation between AFP and des-g -carboxy prothrombin, and some tumors are only detected by des-g -carboxy prothrombin. (292, 293, 294, 295, 296) Vitamin K deficiency can also cause significant elevation; repeating testing after administration of vitamin K improves specificity. (292, 293, 297, 298) Recently, a more sensitive immunoassay has shown promise in detection of small HCC, with positivity in 27% of cases compared to 3% with older assays. (298a) Assays for des-g -carboxy prothrombin are not widely available, in contrast to AFP assays. Other laboratory tests, including AFP variants (299) and lectin affinity chromatography of alkaline phosphatase (300) have been evaluated in too few patients to make definitive recommendations. A recent study identified high levels of abnormal forms of GGT in 78 of 91 patients with HCC, but in only 2.5% of 116 patients with other liver diseases. (301)

Recommendations

Screening for hepatocellular carcinoma is of questionable benefit in Western populations. There is evidence that screening detects tumors at an earlier stage than does development of clinical symptoms, but inadequate evidence exists that earlier detection prevents death from hepatocellular carcinoma. Screening should be confined to high risk patients who are candidates for treatment of hepatocellular carcinoma, if detected. If screening is used, measurement of a -fetoprotein and ultrasound at intervals no more frequent than every 6 months is the most commonly accepted strategy. There is little data to support the use of other tests.

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