Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests.
Knowledge in the field of hepatocyte injury has increased rapidly, expanding the number of tests available for diagnosing and monitoring viral, metabolic, and immunologic etiologies of hepatocyte injury. At the same time, changes in the healthcare environment and Medicare reimbursement policies have made it important to have useful guidelines to follow in diagnosis and monitoring of patients with liver disease. The National Academy of Clinical Biochemistry (NACB) has, for several years, developed evidence-based laboratory medicine practice guidelines for the diagnosis and monitoring of various disorders. The American Association for the Study of Liver Diseases (AASLD) also publishes clinical practice guidelines for treatment of patients with liver disease. The present guidelines represent a consensus of both guideline committees. They have been reviewed and approved by the AASLD Council.
These guidelines, intended for use by physicians and laboratories, suggest preferable approaches to the diagnostic aspects of care. These guidelines are intended to be flexible, in contrast with "standards of care", which are inflexible policies to be followed in almost every case. The guidelines presented have been developed in a manner consistent with the AASLD Policy Statement on Development and Use of Practice Guidelines. Specific recommendations are based on relevant published information. In an attempt to standardize recommendations, the Practice Guidelines Committee of AASLD has adopted modified categories of the Quality Standards of the Infectious Diseases Society of America. These categories (Table 1) are reported with each recommendation, using the Roman numerals I-IV to determine quality of evidence on which recommendations are based and the letters A-E to determine the strength of recommendation. Because of the nature of these guidelines, only categories B and E are used in the recommendations.
The NACB developed a committee in March 1998, composed of two biochemists (D.D., J.L.), two virologists (D.G., F.N.), and two hepatologists (R.K., L.S.), to develop guidelines for diagnosis and monitoring of hepatic injury. After an initial meeting to determine areas that should be addressed by the guidelines, a comprehensive literature search was conducted of English-language articles in Index Medicus from 1966 to 1998, with "Knowledge Finder" as a search engine. Key search words included hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), hepatitis G, TT virus, alcoholic hepatitis, [[alpha].sub.1]-antitrypsin, Wilson's disease, hemochromatosis, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, cirrhosis, hepatocellular carcinoma, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), [gamma]-glutamyltransferase (GGT), bilirubin, ammonia, and laboratory analytical performance goals. Compound searches were performed using the terms "fibrosis markers and chronic hepatitis", "HCV and genotype", and "prothrombin time (PT) and liver disease". For several of the key words, the search was repeated in August 1999 for articles from 1998 and 1999. The filter was set for fuzzy logic for word matching and to select the top 1000 matches in order of relevance. All titles with high or moderate relevance values were reviewed, and if they appeared to address the topics selected, the abstracts were reviewed to select articles for further study. A total of >750 articles were selected for review; additional references were selected from the bibliographies of the articles selected. Assessment of strength of the evidence for each recommendation was based on the criteria of AASLD (Table 1).
The guidelines were reviewed in a multistep process. An initial draft was prepared by the committee and reviewed by 11 experts: 8 in the field of hepatology, and 3 in laboratory medicine. On the basis of the comments received, a second draft of the guidelines was prepared and presented to the AASLD Practice Guidelines Committee and NACB board of directors. A third draft was then prepared and posted on the NACB website for open comments, and presented in a 2-day, NACB-sponsored symposium at the AACC Annual Meeting in July 1999. A transcript of the comments made at the symposium was reviewed by the committee, and modifications to the guidelines were again presented to the AASLD Practice Guidelines Committee and the NACB board of directors for final comments. The guidelines were then reviewed and approved by the AASLD Council. These guidelines represent the product of the final modifications based on those comments.
The following serum tests should be used to evaluate patients with known or suspected liver disease: AST, ALT, ALP, total bilirubin, direct bilirubin, total protein, and albumin (IIIB, E). A panel with all of these tests is currently approved by the Health Care Financing Administration for Medicare reimbursement. Total protein is not discussed extensively in this guideline because of its limited utility in evaluation of liver status; its primary utility as a liver-related test is in allowing recognition of increased y-globulins, to aid in recognition of patients at increased likelihood of having autoilnmune chronic hepatitis.
PERFORMANCE SPECIFICATIONS FOR LIVER TESTS
A recent conference, sponsored by the IFCC, WHO, and the International Union for Pure and Applied Chemistry, addressed strategies for establishing performance specifications for laboratory tests (5, 6). Performance specifications for laboratory tests can be established by different methods, including (in decreasing order of importance) medical outcome studies, data on biological variation, opinions of clinicians or professional societies, or data from proficiency testing or government directives (7). Goals should specify acceptable imprecision (degree of reproducibility of measurement), bias (difference between measured results and the true value), and total error (defined as bias + 1.65 x imprecision). When goals are derived from biological data, the target for imprecision is less than one-half of the intraindividual variation (difference between measurements in a single person; [CV.sub.i]) for the test, whereas the target for bias is less than one-fourth of the average intraindividual and interindividual (difference in values within a population of individuals; [CV.sub.g]) variation, calculated as 1/4 [([CV.sub.i.sup.2] + [CV.sub.i.sup.2]]).sup.1/2] (8). Table 2 summarizes published data on performance specifications and within-laboratory precision for liver-related tests. Throughout this report, goals are defined based on the upper reference limit for all tests except albumin; there is no clinical significance to most occurrences of low concentrations of enzymes, bilirubin, or ammonia.
Reference intervals refer to the range of values for a laboratory test seen in a specific population, typically described by upper and lower reference limits. Reference intervals serve as a means for physicians to compare results in their patients to expected values in particular settings. Most commonly, reference intervals are derived from samples of presumably healthy individuals and defined as the central 95% of results from healthy volunteers, tested under defined conditions (for example, fasting, drawn in the morning, with the patient seated for 10-15 min). Reference intervals may be established for different groups by partitioning the values to account for differences between groups of individuals, such as between men and women or between children and adults.
Partitioning is necessary when data show that results are significantly different between particular subgroups. Although individual laboratories seldom perform such extensive studies to establish reference limits, the validity of the limits used must be verified by testing a small number of healthy individuals to assure that the reference limits suggested in studies performed by the manufacturers of methods or reagents or published in the literature are acceptable for the population tested by the laboratory.
For a few tests, reference limits are based on data associated with risk of developing particular disease manifestations or complications. For example, upper reference limits for cholesterol have been established based on risk of development of atherosclerosis in prospectively followed individuals. Similarly, upper reference limits for fasting glucose have been defined by risk of developing diabetic complications in several cross-sectional surveys of apparently healthy individuals. For such health-derived reference limits to be used, there must be a high degree of comparability of results between laboratories, a process termed harmonization. In the case of cholesterol, this was accomplished by use of fresh serum samples, with results determined by a reference method, to prepare calibration curves for methods used in individual laboratories.
For most of the liver-associated tests discussed here, there are few data to suggest a risk-based reference limit. In the case of ALT, however, there are data to suggest that a value of 45 U/L in men is a clinically useful upper reference limit. Several studies, discussed in Part 11 of the Guidelines (9), have shown that treatment of chronic HCV infection is not indicated if ALT is within the reference interval, although this is controversial. The outcomes for treated patients with only slight increases in ALT (1-1.3 times the upper reference limit of 45 U/L) are similar to those for patients with larger increases in ALT activity (10). Furthermore, studies of blood donors (before availability of HCV tests) found that risk of transmission of hepatitis increased markedly in donors with ALT even slightly above the reference limit of 45 U/L, whereas there was no difference in likelihood in those with activities in the top one-third of the reference interval compared with those with lower ALT activities (11,12). To use a single reference limit, harmonization of ALT methods among laboratories would be required. A pilot project using ALT reference material RM8430 showed an ability to reduce the range of ALT activities obtained by laboratories using a single analytical method (13).
AST (EC 184.108.40.206) and ALT (EC 220.127.116.11) are widely distributed throughout the body. AST is found primarily in heart, liver, skeletal muscle, and kidney, whereas ALT is found primarily in liver and kidney, with lesser amounts in heart and skeletal muscle (14-16). The AST and ALT activities in liver are ~7000- and 3000-fold higher than serum activities, respectively (17). ALT is exclusively cytoplasmic; both mitochondrial and cytoplasmic forms of AST are found in all cells (18). The half-life of total AST in the circulation is 17 [+ or -] 5 h, whereas that of ALT is 47 [+ or -] 10 h (19). The half-life of mitochondrial AST averages 87 h (20). In adults, AST and ALT activities are substantially higher in males than in females and vary with age (Figs. 1 and 2) (21,22). Until approximately age 15, AST activity is slightly higher than that of ALT, with the pattern reversing by age 15 in males but persisting until age 20 in females (21). In adults, AST activity tends to be lower than that of ALT until approximately age 60, when the activities become roughly equal. An informal survey of attendees at a recent national meeting indicated that one-half of laboratories have gender-based reference intervals for AST and ALT, and fewer have age-adjusted reference intervals. Because upper reference limits vary little between the ages of 25 and 60, age-adjusted reference limits need not be used for this population, which comprises most persons with chronic liver injury. Separate reference limits are needed for children and older adults; these may require national efforts to obtain enough samples from healthy individuals to accurately determine reference limits.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Liver disease is the most important cause of increased ALT activity and a common cause of increased AST activity. Several factors other than liver disease must be considered in interpreting AST and ALT activities; these are summarized in Table 3. In most types of liver disease, ALT activity is higher than that of AST; exceptions include alcoholic hepatitis and Reye syndrome. The reasons for the higher AST activity in alcoholic hepatitis appear to be multiple. Alcohol increases mitochondrial AST activity in plasma, whereas other causes of hepatitis typically do not (23). Most forms of liver injury decrease hepatocyte activity of both cytosolic and mitochondrial AST, but alcohol produces a decrease only in cytosolic AST activity (24). Pyridoxine deficiency, common in alcoholics, decreases hepatic ALT activity (25), and alcohol induces release of mitochondrial AST from cells without visible cell damage (26).
AST and ALT typically are measured by catalytic activity (27); both require pyridoxal-5'-phosphate (P-5'-P) for maximum activity, although the effect of deficient P-5'-P on ALT is greater than the effect on AST (28). In renal failure, AST and ALT are significantly lower than in healthy individuals, perhaps because of serum binders of P-5'-P, as total P-5'-P is increased; decreased free P-5'-P reduces enzymatic activity (29). In a recent College of American Pathologists (CAP) proficiency survey, the average variation in values between laboratories using the same methods was 4-9% at aminotransferase values in the reference interval. When average results between laboratories using different assays were compared, the range was 39-85 U/L for the same specimen (30). Unexpectedly abnormal results are often normal on repeat testing (31,32). Because of marked differences between laboratories, harmonization of methods is a priority. Alternative methods to minimize differences between laboratories, such as expressing results as multiples of the reference limit (33), have been shown to minimize between-laboratory variation (34).
Current target values for performance goals for total error in ALT activity measurements are 20% [Clinical Laboratory Improvement Act of 1988 amendments (CLIA)] and 32% (based on biological variation in healthy individuals). Data from outcome studies are not available for most laboratory tests for liver evaluation, with the exception of ALT. Few data exist on the biological variation of ALT in chronic hepatitis, particularly hepatitis C, although it is commonly stated that ALT results are highly variable. In a study of 186 patients with confirmed chronic HCV infection, the average intraindividual CV was 39%. Although the majority of patients had fluctuations in enzyme activities over time, approximately onethird had ALT that varied little over time, with an average CV of 23% (D. Dufour, unpublished observations). As discussed earlier, there is evidence that distinguishing mildly increased ALT activity from normal ALT activity has clinical relevance (10-12). Thus, accurate determination of ALT at the reference limit is critical for correct treatment of patients with HCV infection.
The consensus of the authors and the AASLD Practice Guidelines committee is that performance criteria for ALT should be defined at the upper reference limits and that current performance goals are inadequate for clinical use. The data in patients with stable ALT suggest that a total error of <10% is required at the upper reference limits for accurate detection of patients who may benefit from treatment for HCV. Current data on within-laboratory precision (Table 2) suggest that this target cannot be met by current methods. It will likely be necessary to develop a standardization program for ALT measurements, similar to that used for creatine kinase MB. This may require use of other methods, such as immunoassay, to achieve the necessary total error target for management of patients with chronic hepatitis.
Performance goals for total error in AST activity measurement are 15-20%, by both CLIA requirements and based on biological variation. These meet the perceived needs of clinicians for diagnosis and management of liver disease (35). Performance goals are not as critical for AST as for ALT; a lower percentage of AST results are abnormal in chronic HCV compared with ALT (33% vs 71%). AST seldom is abnormal (6%) when ALT is normal, except in cirrhosis (D. Dufour, unpublished observations). Performance goals based on biological variation (Table 2) are adequate for clinical use, and precision goals for AST seem capable of meeting clinically relevant performance needs (35).
* Assays for ALT activity should have total analytical error of [less than or equal to]10% at the upper reference limit (IIIB). Current published performance goals for AST, with total error of 15-20%, are adequate for clinical use (IIIB).
* Standardization of ALT values between methods and across laboratories is a priority need for patient care. Until standardization is accomplished, use of normalized results should be considered (IIIB).
* At a minimum, laboratories should have separate upper reference limits for adult males and females; reference limits should also be established for children and adults over age 60 by cooperative efforts (IIB).
* Unexpectedly increased ALT and/or AST should be evaluated by repeat testing; in individuals engaging in strenuous exercise, it should be repeated after a period of abstinence from exercise. Research is needed to determine the appropriate time interval required (IIIB and IIIE).
ALP (EC 18.104.22.168), which is involved in metabolite transport across cell membranes, is found (in decreasing order of abundance) in placenta, ileal mucosa, kidney, bone, and liver (36-41). The bone, liver, and kidney ALP isoenzymes share a common protein structure coded for by the same gene (42,43), but they differ in carbohydrate content. The half-life of the liver isoenzyme is 3 days (44-46). Age- and gender-related changes in ALP upper reference limits are illustrated in Fig. 3. Interpretation of ALP results using appropriate reference populations is particularly important in children; reference limits differ little in adult males and females between the ages of 25 and 60. After age 60, reference limits increase in women, although studies have not consistently evaluated subjects for the presence of osteoporosis, which can increase ALP activity in serum (47). Separate reference intervals are required for children and for pregnant women.
Cholestasis stimulates synthesis of ALP by hepatocytes, and bile salts facilitate release of ALP from cell membranes (48-50). Other factors affecting ALP are summarized in Table 4.
The method for total ALP in widest use is the p-nitrophenylphosphate method of Bowers and co-workers (51,52). CAP surveys typically show variations of 5-10% between laboratories using the same manufacturer's assay; assays using different manufacturers' reagents vary widely (53). Complexing agents such as citrate, oxalate, or EDTA bind cations such as zinc and magnesium, necessary cofactors for ALP activity measurement, causing falsely decreased values as low as zero in plasma samples collected with the agents. Blood transfusion (containing citrate) causes transient decrease in serum ALP through a similar mechanism.
[FIGURE 3 OMITTED]
Separation of tissue-nonspecific ALP forms (bone, liver, and kidney) is difficult because of structural similarity; high-resolution electrophoresis and isoelectric 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 (54-56) and can be used to monitor patients with bone disease. Because there is good agreement between increases in ALP of liver origin and increases in the activity of other canalicular enzymes such as GGT, measurement of GGT activity is a good indication of a liver source, but does not rule out coexisting bone disease (57).
In contrast to most enzymes, intraindividual variation in ALP is low, averaging slightly more than 3% (Table 2). The current average within-laboratory imprecision of 5% is close to recommended performance specifications; a total error of 10-15% would meet health-based target values of 12%. The CLIA-specified total error range of 30% appears too wide for clinical use and should be narrowed.
* Assays for ALP activity should have total analytical error of [less than or equal to]10-15% at the upper reference limit (IIIB).
* Separate reference limits should be provided for children, based on age and gender, and for pregnant women. A single reference interval is adequate for adults over age 25 (IIB).
* Specimens for ALP activity should be obtained in the fasting state; if not, mildly increased patient values should be reevaluated in the fasting state before further evaluation (11B and IIE).
* Assays for ALP isoenzymes or measurement of other associated enzymes (such as GGT) are needed only when the source is not obvious from clinical and laboratory features (111B and IIIE)
GGT (EC 22.214.171.124), a membrane-bound enzyme, is present (in decreasing order of abundance) in proximal renal tubule, liver, pancreas (ductules and acinar cells), and intestine (58-60). GGT activity in serum comes primarily from liver. The half-life of GGT in humans is ~7 to 10 days; in alcohol-associated liver injury, the half-life increases to as much as 28 days, suggesting impaired clearance (61). Age- and gender-related differences in GGT are summarized in Fig. 4. In adult men, a single reference interval is adequate between the ages of 25 and 80. Although upper reference limits are approximately twofold higher in those of African ancestry, information on racial characteristics is not commonly provided to laboratories; it would thus be difficult for laboratories to report values with the appropriate race-based reference range. In women and children, GGT upper reference limits increase gradually with age and are considerably lower than those in adult men. Separate reference limits should be established for men and women and for different age ranges in women and children. In children, this will probably require a cooperative effort of laboratories to obtain adequate numbers of specimens from healthy children.
[FIGURE 4 OMITTED]
GGT is slightly more sensitive than ALP in obstructive liver disease. GGT is increased an average of 12-fold above the upper reference limit in 93-100% of those with cholestasis, whereas ALP is increased an average of 3-fold above the upper reference limit in 91% of the same group (57, 62, 63). GGT appears to increase in cholestasis by the same mechanisms as does ALP (63, 64). GGT is increased in 80-95% of patients with any form of acute hepatitis (65-67). Other factors that affect GGT activity are summarized in Table 5. 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 (68, 69). These other factors cause a low predictive value of GGT (32%) for liver disease (70).
The IFCC method described by Shaw et al. (71) is used by most laboratories. Precision with activities less than one-half the upper reference limit is ~10%; at approximately twice the upper reference limit, it is closer to 5%. Mean values obtained in adults by different assays show dramatic differences, ranging from 37 to 90 U/L (72). Performance goals for GGT are based primarily on biological variation, with total error tolerance limits of ~20%. These are adequate for clinical purposes, given the limited clinical utility of GGT measurements.
* Assays for glutamyltransferase activity should have total analytical error of [less than or equal to]20% at the upper reference limit (IIIB).
* Use of fasting morning specimens is recommended (IIB).
* Although a single upper reference limit is appropriate for adult men, separate reference limits (based on age) are needed for children and adult women (IIB).
* Because of lack of specificity, GGT should be reserved for specific indications such as determining the source of an increased alkaline phosphatase (IIIB and IIIE).
[FIGURE 5 OMITTED]
Daily production of unconjugated bilirubin is 250-350 mg, mainly from senescent erythrocytes (73). Clearance at normal values is 5 mg * [kg.sup.-1] * [day.sup.-1], or ~400 mg/day in adults; the rate does not increase significantly with hemolysis (74). The half-life of unconjugated bilirubin is <5 min (75). UDP-glucuronyltransferase catalyzes rapid conjugation of bilirubin in the liver; conjugated bilirubin is excreted into bile and is essentially absent from blood in healthy individuals. [delta]-Bilirubin, sometimes termed biliprotein, occurs when conjugated bilirubin covalently binds to albumin (76); it has a half-life of ~17-20 days (the same as albumin), which accounts for the prolonged jaundice in patients recovering from hepatitis or obstruction (77). Age- and gender-related changes in bilirubin reference limits are illustrated in Fig. 5. Increases in conjugated bilirubin are highly specific for disease of the liver or bile ducts (78). Increased conjugated bilirubin may also occur with impaired energy-dependent bilirubin excretion in sepsis and total parenteral nutrition and after surgery (79). With recovery from hepatitis or obstruction, conjugated bilirubin falls quickly, whereas [delta]-bilirubin declines more slowly (80). Gilbert syndrome, found in ~5% of the population, causes mild unconjugated hyperbilirubinemia because of impaired UDP-glucuronyltransferase (EC 126.96.36.199) activity (81). Total bilirubin rarely exceeds 68-85 [micro]mol/L (4-5 mg/dL), even during prolonged fasting, unless other factors that increase bilirubin are also present (82). Other factors affecting bilirubin are summarized in Table 6.
Bilirubin typically is measured using two assays for total and "direct reacting" or direct bilirubin; subtracting direct from total gives "indirect bilirubin". The direct bilirubin assay measures the majority of [delta]-bilirubin and conjugated bilirubin and a variable but small percentage of unconjugated bilirubin (83-85). High pH or the presence of a wetting agent promotes reaction of unconjugated bilirubin in the direct assay; the reagent for direct bilirubin should have at least 50 mmol/L HCl to prevent measurement of unconjugated bilirubin (86). Light can convert unconjugated bilirubin to a photoisomer that reacts directly (87); it also causes total bilirubin to decrease by 0.34 [micro]mol * [L.sup.-1] * [h.sup.-1] (0.02 mg * [dL.sup.-1] * [h.sup.-1]). Direct spectrophotometry (dry film methods) measures conjugated and unconjugated bilirubin and calculates [delta]-bilirubin as the difference between the sum of these and total bilirubin. Some authors have suggested that conjugated bilirubin is better than direct bilirubin to measure recovery from liver disease (88).
In a recent CAP survey, at a total bilirubin concentration of 38 [micro]mol/L (2.5 mg/dL), the average variation in laboratories using the same method was 5%; however, the mean with different methods ranged from 34 to 46 [micro]mol/L (2.0-2.7 mg/dL). At a concentration of 27 [micro]mol/L (1.5 mg/dL), the average variation using the same method was 8% (89).
Performance goals for total bilirubin measurement allow 20% (CLIA) to 30% (biological variation) total error (Table 2). Clinicians felt that a 23% change in bilirubin at the upper reference limits indicated a significant change in condition (Table 2) (35). Thus, CLIA performance goals appear to meet clinical performance needs. Few data exist on performance goals for direct bilirubin. There is poor reproducibility of direct bilirubin between laboratories as a result of a variety of factors (90, 91). Because conjugated bilirubin is essentially absent from normal serum (80), it is reasonable to expect that laboratories should report direct bilirubin of [less than or equal to]1.7 [micro]mol/L (0.1 mg/dL) in virtually all healthy individuals.
* Assays for total bilirubin should have a total analytical error of [less than or equal to]20% [or 6.8 [micro]mol/L (0.4 mg/dL)] at the upper reference limit (IIIB).
* Separate upper reference limits should be used for total bilirubin in men and women. Although total bilirubin upper reference limits decline with age in adults, there is little significance to slight increases in bilirubin, and separate adult age-adjusted upper reference limits are not needed. In children, separate reference intervals should be used (IIIB).
* There are no data on analytical performance goals for direct or conjugated bilirubin. Laboratories should assure that direct bilirubin measurements are <1.7 [micro]mol/L (0.1 mg/dL) in most healthy individuals. Additional data are needed on performance goals in patients with increased conjugated bilirubin (IIIB).
Albumin is the most abundant plasma protein and is produced by hepatocytes. The rate of production is dependent on several factors, including the supply of amino acids, plasma oncotic pressure, concentrations of inhibitory cytokines (particularly interleukin-6), and the number of functioning hepatocytes (92). Albumin functions as the major determinant of plasma oncotic pressure and serves as a transport protein for drugs, hormones, and waste products such as bilirubin; it also serves as a source of amino acids for the synthesis of other proteins. The half-life of plasma albumin typically is ~19-21 days. Plasma albumin concentrations are low in neonates, typically 28-44 g/L (2.8-4.4 g/dL). By the first week of life, adult values of 37-50 g/L (3.7-5.0 g/dL) are reached, increasing to 45-54 g/L (4.5-5.4 g/dL) by age 6 and remaining at these concentrations through young adulthood before declining to typical adult values. There is no significant difference in reference limits between males and females (93). Increases of serum albumin typically are secondary to hemoconcentration caused by dehydration, prolonged tourniquet use during collection, or specimen evaporation (92). The main causes for decreased albumin include protein loss (nephrotic syndrome, burns, protein losing enteropathy), increased albumin turnover (catabolic states, glucocorticoids), decreased protein intake (malnutrition, very low protein diets), and liver disease (92).
Plasma albumin seldom is decreased in acute hepatitis because of its long half-life, but in chronic hepatitis albumin gradually falls with progression to cirrhosis (92). Albumin concentrations are a marker of decompensation and prognosis in cirrhosis (94).
Albumin is most commonly measured by dye-binding methods, particularly bromcresol green and bromcresol purple; currently, ~50% of laboratories use each method. Bromcresol green methods may overestimate albumin (95), although differences between the two methods are small (92). Bromcresol purple underestimates albumin in renal failure (96,97) and in patients with increased [delta]-bilirubin (98, 99), making this method unsuitable for patients with jaundice. Protein electrophoresis and staining may overestimate albumin because of higher binding of the dye to albumin than to other proteins (92). Immunoassays for albumin are available but not widely used in plasma (100). Results from recent CAP surveys indicate that average variation in albumin among laboratories using the same method is low (2-3%), although there are significant differences between laboratories using different methods (101). Such differences appear less dramatic when fresh specimens rather than CAP survey materials are used.
Performance goals for albumin measurement based on biological variation are typically ~4%, whereas CLIA allows an error of 10%. The clinical uses of albumin measurements for liver disease are primarily in recognition of cirrhosis and in determining its severity; these require significant changes from reference limits. Data from CAP surveys indicate that only 2% of laboratories can meet the error limits based on biological variation. The opinion of the committee is that the CLIA performance goals are adequate for clinical purposes.
* Total error of <10% at the lower reference limit is adequate for clinical purposes; performance goals based on biological variation, although an ideal goal for measurement, cannot be met by most laboratories (IIIB).
* Assays for albumin in patients with liver disease should use bromcresol green. Bromcresol purple and electrophoresis determinations of albumin may be inaccurate in patients with liver disease (IIB).
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 of these factors are made by the liver, and three (II, VII, and X) are activated by vitamin K by the addition of a second, [gamma]-carboxyl group onto glutamic acid residues. Immunoassays are available to measure "proteins induced by vitamin K antagonists (PIVKA)", the most common measuring des-[gamma]-carboxy prothrombin (102). PT is relatively insensitive to deficiency of any single clotting factor; there is no significant increase until the concentration of any one factor falls below 10% of normal (103).
PT is commonly reported in seconds and compared to patient reference values (104). To minimize variation between reagents, each is assigned an International Sensitivity Index (ISI), compared to a reference method. The lower the amount of tissue factor (which initiates clotting in the assay), the lower the ISI value and the longer the PT. To adjust for differences in the ISI of reagents, a derived term, the international normalized ratio (INR), is used; the value is calculated as:
INR = [(P[T.sub.patient]/P[T.sub.control mean]).sup.ISI]
(105). For example, a sample with PT of 20 s with high ISI reagents had a PT of 40 s when tested with low ISI reagents, but the INR was essentially identical with both reagents (103). INR thus normalizes results in a patient on warfarin, despite differences in the ISI of reagents used. The use of reagents with low ISI improves the reproducibility of INR measurements, making the use of low ISI reagents ideal for monitoring anticoagulant therapy (106).
The effect of ISI is much greater on PT in warfarin use than in liver disease, so that the INR does not accurately reflect inhibition of coagulation in liver disease (103,107-109). In liver disease, a decrease in the ISI of the reagents used produces only a slight increase in PT. For example, a sample from a patient with liver disease, tested with three differing ISI reagents, had INR values that varied between 1.86 and 2.90 although the difference in PT was only 3.6 s (103). If reagents with a low ISI are used, the INR markedly overestimates the degree of coagulation impairment in liver disease. A possible cause for the discrepancy in INR utility between warfarin use and liver disease is the marked difference in the relative amounts of native prothrombin vs des-[gamma]-carboxy prothrombin present in the two conditions. Patients on warfarin or with vitamin K deficiency have markedly increased des-[gamma]-carboxy prothrombin and decreased native prothrombin, whereas patients with acute hepatitis or cirrhosis have decreased native prothrombin but only slightly increased des-[gamma]-carboxy prothrombin (110). Some preparations of tissue factor are inhibited by des-[gamma]-carboxy prothrombin (110).
PT is reproducibly increased, usually at least 3 s beyond the population mean, in acute ischemic (111,112) and toxic (113) hepatitis, but it rarely is increased >3 s in acute viral (114) or alcoholic (115, 116) hepatitis. PT often is increased in obstructive jaundice and may respond to parenteral vitamin K administration. In chronic hepatitis, PT typically is within reference limits, but it increases as progression to cirrhosis occurs and is increased in cirrhotic patients (117). Other factors affecting PT are summarized in Table 7.
Abnormal PT values are highly dependent on the ISI of the reagents used, although reference values are similar (103). Reagents with the same ISI typically give different results on different instruments, even of the same model (118-120). In addition, when reagents with the same ISI are used, a specimen can yield different INRs (106,121-123). The variability (CV) of PT results among laboratories using the same instrument and reagents is 3-8% when PTs are prolonged, and variation is greater for the INR than it is for the PT itself. Within a single laboratory, the average variation (CV) in INR is estimated to be 10% (124). The difference in PT between laboratories using different reagents may be marked; in one study, the average difference was 20% (122). Recently, use of calibrant plasmas to determine the ISI in each laboratory for its own reagents and instrument has been shown to significantly improve agreement of INR values between laboratories (122,125,126).
* PT in seconds rather than the INR should be used to express results of PT in patients with liver disease (IIIB); however, this does not standardize results between laboratories (IIB).
* Additional research into standardization of reagents and use of derived indices (percentage of activity, INR) in liver disease is needed (IVB).
N[H.sub.3] is a product of amino acid metabolism and is cleared primarily by urea synthesis in the liver. Helicobacter pylori in the stomach appears to be an important source of N[H.sub.3] in patients with cirrhosis (127,128). In liver disease, increased N[H.sub.3] typically is a sign of hepatic failure. High concentrations are seen with deficiency of urea cycle enzymes (129), in Reye syndrome (130), and with acute or chronic hepatic encephalopathy (131,132). Mild increases in plasma N[H.sub.3] are seen in patients with chronic hepatitis, in proportion to the extent of disease (133). The use of N[H.sub.3] for monitoring of patients with encephalopathy is controversial; some studies have shown good correlation of N[H.sub.3] concentrations with degree of encephalopathy (130, 132), whereas others have not (134). N[H.sub.3] appears to enhance the effects of [gamma]-aminobutyric acid (135) and to increase benzodiazepine receptors (136); both [gamma]-aminobutyric acid and benzodiazepines have been implicated in the pathogenesis of hepatic encephalopathy. On the other hand, clinical features seen in persons with isolated hyperammonemia are not identical to those of hepatic encephalopathy (137). Factors affecting N[H.sub.3] are summarized in Table 8. Specimens should have plasma separated from cells within 1 h of collection to avoid artifactual increases in N[H.sub.3]; in patients with liver disease, separation within 15 min is ideal (138,139).
Several methods have been used to measure N[H.sub.3] (138), with enzymatic assays currently the most widely used. One manufacturer uses slide technology with alkaline pH to convert N[H.sub.4.sup.+] + to N[H.sub.3] and then measures N[H.sub.3] with bromphenol blue. Reproducibility within laboratories using the same method averages 10-20%, with mean values using different methods differing by <10% on average (140).
* Measurement of plasma N[H.sub.3] for diagnosis or monitoring of hepatic encephalopathy is not routinely recommended in patients with acute or chronic liver disease; it may be useful in patients with encephalopathy of uncertain etiology (IIIB).
* Ideally, arterial, rather than venous, specimens should be used (IIB).
* Plasma should be separated from cells within 15 min of collection to prevent artifactual increases in N[H.sub.3] (IIB).
HEPATITIS SEROLOGIC MARKERS AND NUCLEIC ACID TESTING
HAV. IgM antibodies to HAV (anti-HAV IgM) typically are present at onset of symptoms and remain detectable for 3-6 months after infection in most patients, but they persist for >200 days in 13.5% of cases and may remain up to 420 days (141,142). Prevalence of HAV RNA in blood is highest when ALT is highest, and the mean duration of viremia is 18 days (143). Total anti-HAV antibodies persist for long periods after infection, perhaps for life (144); seroprevalence increases with increasing age, ranging from 11% in children <5 years to 74% in adults >50 years (145). HAV vaccine induces detectable anti-HAV antibodies within 2-4 weeks of the initial dose of vaccine (146,147), and the antibodies remain detectable at 5 years in 99% of individuals completing vaccination (148). There are no commercially available antigen detection tests for HAV, but immune electron microscopy and immunoassay methods have been used to detect HAV antigen in stool filtrates and other specimens (149,150).
* Anti-HAV IgM should be used to diagnose acute HAV infection (IB); HAV RNA tests are needed only for research purposes (IIIB).
* Total antibody should be used for determining immune status for HAV (IB).
HBV. Hepatitis B is a DNA virus with several protein antigens that can induce antibody responses. The most abundant, HBV surface antigen (HBsAg) is produced in excess along with viral particles and during nonreplicative phases of infection. HBV core antigen and e antigen (HBcAg and HBeAg) are produced by the same region of the viral genome and are found in infectious particles. A typical serological and clinical course of acute HBV infection is shown in Fig. 6 (151). IgM antibody to HBcAg (anti-HBc) usually is considered the gold standard for diagnosis of acute hepatitis B (152,153), but it may also be present at fluctuating, low titers in patients with chronic hepatitis B, particularly when patients also have positive plasma HBeAg and episodes of rising ALT, indicating reactivation of disease (153-155). Total anti-HBc typically persists for life (156). HBsAg is characteristically present and anti-HBs absent at presentation in patients with acute HBV infection, but both are occasionally absent (152,153), leaving IgM anti-HBc the only marker of infection ("core window"). Isolated positive anti-HBc also may represent low-level viremia, loss of anti-HBs many years after recovery, or a false-positive result (153, 156-159). Two factors are associated with likelihood of false-positive results: low anti-HBc reactivity and absence of anti-HBs in sensitive radioimmunoassays. In several studies, virtually none of those with low anti-HBc activity and negative anti-HBs showed an anamnestic response to a single injection of HBsAg vaccine, whereas 35-40% of those with weakly positive anti-HBs and 50-80% of those with high anti-HBc activity responded (157,159-161). Convalescence from infection is indicated by loss of HBsAg and development of anti-HBs. Concomitant HBsAg and anti-HBs may be seen in a small number of patients with chronic HBV infection. This phenomenon appears to be particularly common in patients on maintenance hemodialysis (7%) compared with other HBsAg-positive patients (2%) (162). The presence of anti-HBs in these settings does not appear to have clinical importance. Patterns of serological markers in various forms and phases of HBV infection are shown in Table 9 (163). Examples of discordant or unusual hepatitis profiles are given in Table 10. Tests with discordant results should be repeated, and testing for additional serological markers may be indicated to establish the correct diagnosis (164).
[FIGURE 6 OMITTED]
* Tests for HBsAg, anti-HBs, and anti-HBc should be performed for diagnosis of current or past HBV infection. In suspected acute HBV infection, tests for IgM anti-HBc should be utilized (IB).
* HBeAg and anti-HBe should be measured only when indicated based on results of the initial tests (IIIB and IIIE).
* In patients with discordant results, tests should be repeated; persistently discordant results should be evaluated by a hepatologist or gastroenterologist (IIIB).
In the HBeAg-positive patient, loss of HBeAg and seroconversion to anti-HBe positivity typically is associated with loss of detectable HBV DNA by methods other than PCR, normalization of aminotransferases, and histologic improvement, implying a low replication state and significant clinical improvement (153). HBV DNA concentrations are useful in monitoring 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 (164). Several assays for detection of serum HBV DNA are available commercially; detection limits are given in Table 11. There currently is no standardization of HBV DNA assays between laboratories. Circulating HBV DNA can be found by sensitive PCR amplification methods in some patients with negative HBeAg and positive anti-HBs, anti-HBe, and anti-HBc months or years after clinical recovery from acute (165,166) or chronic (167) hepatitis. The significance is not clear because most viral DNA is found in immune complexes (165) and may not represent the entire genome. Similarly, in patients with chronic HCV, HBV viral DNA can be found (using sensitive PCR methods) in both liver and serum, particularly in patients with anti-HBc as an isolated HBV marker (157 158). Thus, there are few data on which to determine desirable lower limits of detection for HBV DNA measurements.
* Quantitative HBV DNA, HBeAg, and anti-HBe measurements should be used for monitoring response to antiviral therapy (IB).
* An international standard for HBV DNA tests should be established and manufacturers should calibrate methods against it (IIIB).
* Tests for HBV DNA should be quantitative, and the clinically useful dynamic range for HBV DNA tests should be defined (IIIB).
HCV. Screening tests for HCV infection detect antibodies to HCV proteins, usually apparent by an average of 80 days (range, 33-129 days) after infection, measured by second-generation anti-HCV enzyme immunoassays (EIA-2) (168). Immunocompromised patients and those on dialysis may rarely lack detectable antibodies by EIA-2 despite other evidence of active viral infection (169). A third-generation EIA (EIA-3) for anti-HCV has been approved by the Food and Drug Administration (FDA) for screening of blood products; it contains reconfigured core and NS3 antigens and an additional antigen (NS5) not found in EIA-2. EIA-3 provides a slight increase in sensitivity but lower specificity than EIA-2, and shortens the time to detection of antibody to an average of 7-8 weeks after infection (170-172). The FDA has approved a method for home use for obtaining samples for anti-HCV testing. In patients who have cleared HCV from the circulation, titers of anti-HCV antibodies gradually fall (173,174). In one study of persons with posttransfusion HCV infection, 6% were negative for all HCV markers, including anti-HCV antibodies, 17 years after infection (175). In evaluating possible perinatal transmission of HCV, maternal antibody clears by 12 months in 90% of uninfected infants and by 18 months in 100% (176). Approximately 90% of infected infants have detectable plasma HCV RNA by 3 months of age (177).
Supplemental tests for anti-HCV help resolve suspected false-positive EIA test results. Recombinant immunoblot assays (RIBAs) contain the same HCV antigens as do the EIA tests, along with superoxide dismutase (SOD; EC 188.8.131.52). A positive RIBA 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 or multiband reactivity with reactivity to SOD is considered indeterminate. Individuals with isolated positivity for either C100 or 5-1-1 antigen rarely have evidence of active HCV infection, whereas either C22- or C33-indeterminate patterns predict active HCV infection in 25-50% of individuals (178-180). In populations at high risk for HCV infection, <1% of EIA-2-positive specimens will be false positives. Additionally, in recently infected individuals, RIBA results are positive in only 85% of cases (181). Therefore, RIBA testing in high-risk populations is not necessary for the diagnosis of hepatitis C (182). Although the overall pattern does not differ among the common genotypes (183), antibody responses to two of the four antigens included (core and NS4) are significantly lower in patients infected with genotypes other than 1 (179). Isolated positivity for NS5 in the RIBA-3 is virtually never associated with presence of HCV RNA, suggesting that it is the cause of the reduced specificity with EIA-3 (184,185). Indeterminate RIBA-3 results (attributable to antigens other than NS5) are associated with HCV viremia in ~50% of cases; HCV RNA-positive patients with indeterminate RIBA results are more likely to be immunosuppressed than those with positive RIBA results (180). At present, there are no commercially available antigen tests for HCV, although a highly sensitive assay for HCV core protein has been developed (186).
Detection of active HCV infection depends on detection of HCV RNA. HCV RNA can be detected in serum within 1-2 weeks after acute infection, weeks before ALT becomes abnormal and before the appearance of anti-HCV antibodies (173). The time course of typical HCV infection is illustrated in Fig. 7. Although not FDAapproved, reverse transcription-PCR assays for HCV RNA are used frequently in clinical practice; the most sensitive can detect <100 HCV RNA copies/mL. HCV RNA assays are not standardized, and quantitative assay results may vary significantly between different laboratories using different assays (187,188). EDTA and sodium citrate plasma are preferred specimens for HCV RNA tests. Heparinized plasma is inhibitory for many nucleic acid amplification assays, and serum specimens provide suboptimal stability unless serum is frozen soon after specimen collection. HCV RNA is very susceptible to degradation by the high activities of RNase present in blood; therefore, serum specimens for HCV RNA should be centrifuged as soon as possible after clot formation. If centrifugation is performed immediately, <10% of HCV RNA is lost even if the plasma or serum is not separated from the formed elements for up to 6 h (189). If a serum separator tube is used, specimens are stable after centrifugation for up to 24 h (189). Short-term (<7 days) storage of serum or plasma at 4 [degrees]C is acceptable. Once frozen, samples are stable through at least three freeze-thaw cycles (189-191).
Quantitative HCV RNA assays often are less sensitive than qualitative RNA assays using the same technology, but this is not universal; in the case of PCR, this may be related to the larger sample size used for amplification. The current version of the branched DNA assay is the least sensitive, with a lower limit of detection of 200 000 copies/mL, although a third-generation assay with significantly lower cutoff values will soon be available. Results cannot be directly compared because different standards are used. A WHO HCV RNA international standard (genotype 1) for HCV RNA for nucleic acid amplification assays is now available (192). There are few data on clinically desirable lower detection limits for HCV RNA in untreated patients. In patients receiving treatment, lack of detectable HCV RNA in assays with lower detection limits <1000 copies/mL 6 months after treatment is associated with 90-95% likelihood of permanent clearance of the virus (193). In terms of upper detection limit, data from treatment studies show that risk of treatment failure is associated with HCV RNA >3.2 x [10.sup.6] copies/ mL (194).
[FIGURE 7 OMITTED]
* EIA screening tests for HCV antibody are adequate for diagnosis of past or current HCV infection in a patient population with a high prevalence of disease; supplemental testing is not needed in such patients. If confirmation of active infection is required, HCV RNA should be used (IIB and IIE).
* Supplemental anti-HCV tests (RIBAs) should be used in populations with low prevalence of disease or to confirm prior infection by HCV in a patient who is HCV RNA negative (IIIB and IIIE).
* Improved intermethod agreement and precision are needed for HCV RNA tests; methods should use a standard such as that developed by WHO (IIB)
* Specimens for HCV RNA should be collected either as EDTA or citrated plasma or be centrifuged promptly to prevent falsely low results (IIB).
* Assays for HCV RNA should ideally have a dynamic range from <1000 copies/mL to >3.2 x [10.sup.6] copies/mL (IIB).
There are six major genotypes and >90 subtypes of HCV, which vary in their world-wide distribution. In addition, HCV has a high rate of spontaneous mutation, producing discrete "quasispecies" that vary from one individual to the next (195). Genotypes 1a and 1b account for approximately two-thirds of HCV infections in the United States; genotype 1 represents 90-95% of infections in African Americans compared with ~60% in white patients (196,197). Genomic amplification and sequencing followed by sequence comparison and phylogenetic tree construction is the reference method for genotype determination (198). A variety of genotype screening assays have been described, including PCR using genotype-specific primers (199201), restriction fragment length polymorphism of amplified sequences (199, 202, 203), and a commercially available line probe assay (204-206). These methods compare favorably with the commonly used reference methods for determining HCV genotypes (207).
* Genotype assays should reliably differentiate all six major genotypes and distinguish genotype 1a from 1b (IIIB and IIIE).
HDV. HDV is a defective virus that replicates 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 hepatitis, particularly in those with fulminant hepatitis or where there is a high risk for HDV infection (such as in injection drug abusers). The only HDV serologic tests widely available commercially detect total anti-HDV. In patients in whom virus is cleared, antibody typically disappears between 1 and 5 years (208). In most clinical situations, HBsAg, IgM anti-HBc, and total anti-HDV are adequate to diagnose HDV infection. Patients with acute HDV co-infection usually are positive for IgM anti-HBc, whereas patients with HDV superinfection usually are negative for IgM anti-HBc. There is inadequate information on performance of HDV tests to make performance recommendations.
HEV. 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. In the United States, HEV infections have been seen rarely as a cause of hepatitis, predominantly among those who have traveled to endemic areas, although at least one case has occurred without a history of travel (209). Immunoassays have been developed for diagnostic use (210). An evaluation of multiple methods for detecting anti-HEV antibodies showed significant variation in titers reported and discordance between methods, although tests detecting antibodies to ORF2 were most accurate (211). Antibodies reactive with HEV antigens were found in 15-25% of homosexual men, intravenous drug users, and blood donors in Baltimore, MD, suggesting lack of specificity of assays (212); antibody to HEV antigens is found commonly in rats and pigs in the United States (213, 214).
* Assays for HEV antibodies should detect antibodies to the ORF2 antigen to assure adequate clinical specificity (IIIB and IIIE).
Development and publication of these guidelines was supported by grants from Abbot Diagnostics; Diasorin, Inc.; Bayer Diagnostics (formerly Chiron Diagnostics); Irmogenetics, Inc.; and Ortho Clinical Diagnostics. The following individuals reviewed the guidelines at various stages of their development and offered helpful comments and modifications: Miriam Alter, Henry C. Bodenheimer, Thomas D. Boyer, Max A. Chernesky, Gary L. Davis, Jean C. Edmond, Stuart C. Gordon, Norman D. Grace, F. Blaine Hollinger, Donald M. Jensen, Lawrence A. Kaplan, Jacob Korula, Karen Lindsay, Brian J. McMahon, Jan M. Novak, Melissa Palmer, Eve A. Roberts, James R. Spivey, Thomas A. Shaw-Stiffel, and Myron Warshaw. Specific comments were provided by the following individuals during open discussion at the AACC Annual Meeting: Ed Ashwood, Bill Brock, Thomas Burgess, Jack Goldberg, Ajit Golwikar, Neal Greenberg, Michael Heinz, Richard Horowitz, Graham Johns, Ronald Lee, Steve Lobell, Greg Post, Phil Rosenthal, Norbert Tietz, Mark Walter, Earl Weissman, William Winter, and Jeffery Young.
Received March 24, 2000; accepted October 6, 2000.
(1.) Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999;341: 556-62.
(2.) Staadatmand F, Stinson FS, Grant BF, Dufour MC. Liver cirrhosis mortality in the United States, 1970-1995. Surveillance Report No. 49. US Washington, DC: Department of Health and Human Services, Public Health Service, National Institutes of Health, 1999:32pp.
(3.) Davis GL, Albright JH, Cook SF, Rosenberg D. Projecting the future healthcare burden from hepatitis C in the United States. Hepatology 1998;28:390A.
(4.) El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999;340:745-50.
(5.) Westgard JO, Seehafer JJ, Barry PL. European specifications for imprecision and inaccuracy compared with operating specifications that assure the quality required by US CLIA proficiency-testing criteria. Clin Chem 1994;40:1228-32.
(6.) Fraser CG, Kallner A, Kenny D, Petersen PH. Introduction: strategies to set global quality specifications in laboratory medicine. Scand J Clin Lab Invest 1999;59:477-8.
(7.) Kaplan LA. Determination and application of desirable analytical performance goals: the ISO/TC 212 approach. Scand J Clin Lab Invest 1999;59:479-82.
(8.) Fraser CG. The application of theoretical goals based on biological variation data in proficiency testing. Arch Pathol Lab Med 1988;112:404-5.
(9.) Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. II. Recommendations for use of laboratory tests in screening, diagnosis, and monitoring. Clin Chem 2000;46:2050-2068.
(10.) Gordon SC, Fang JWS, Silverman WL, McHutchison JG, Albrecht JK. The significance of baseline serum alanine aminotransferase on pretreatment disease characteristics and response to antiviral therapy in chronic hepatitis C. Hepatology 2000;32:400-4.
(11.) Alter HJ, Purcell RH, Holland PV, Ailing DW, Koziol DE. Donor transaminase and recipient hepatitis-impact on blood transfusion services. JAMA 1981;246:630-4.
(12.) Aach RD, Szmuness W, Mosley JW, Hollinger FB, Kahn RA, Stevens CE, et al. Serum alanine aminotransferase of donors in relation to the risk of non-A, non-B hepatitis in recipients: the Transfusion-Transmitted Viruses Study. N Engl J Med 1981;304: 989-94.
(13.) Lott JA, Tholen DW, Massion CG. Proficiency testing of enzymes. Charting the way toward standardization. Arch Pathol Lab Med 1988;112:392-8.
(14.) Adolph L, Lorenz R. Enzyme diagnosis in diseases of the heart, liver, and pancreas. New York: S Karger, 1982:9-27.
(15.) Jung K, Mildner D, Jacob B, Scholz D, Precht K. On the pyridoxal-5'-phosphate stimulation of AST and ALT in serum and erythrocytes of patients undergoing chronic hemodialysis and with kidney transplants. Clin Chim Acta 1981;115:105-10.
(16.) Wroblewski F. The clinical significance of alterations in transaminase activities of serum and other body fluids. Adv Clin Chem 1958;1:313-51.
(17.) Lott JA, Wolf PL. Alanine and aspartate aminotransferase (ALT and AST). Clinical enzymology: a case-oriented approach. Chicago: Year Book Medical Publishers, 1986:111-38.
(18.) Rej R. Measurement of aminotransferases. Part 1. Aspartate aminotransferase. CRC Crit Rev Clin Lab Sci 1984;21:99-106.
(19.) Price CP, Alberti KGMM. Biochemical assessment of liver function. In: Wright R, Alberti KGMM, Karran S, Millward-Sadler GH, eds. Liver and biliary disease--pathophysiology, diagnosis, management. London: WB Saunders, 1979:381-416.
(20.) Panteghini M. Aspartate aminotransferase isoenzymes. Clin Biochem 1990;23:311-9.
(21.) Siest G, Schiele F, Galteau M-M, Panek E, Steinmetz J, Fagnani F, Gueguen R. Aspartate aminotransferase and alanine aminotransferase activities in plasma: statistical distributions, individual variations, and reference values. Clin Chem 1975;21:1077-87.
(22.) Siest G, Henry J, Schiele F, Young DS. Interpretation of clinical laboratory tests: reference values and their biological variation. Foster City, CA: Biomedical Publications, 1985:459pp.
(23.) Nalpas B, Vassault A, Le Guillou A, Lesgourgues B, Ferry N, Lacour B, Berthelot P. Serum activity of mitochondrial aspartate aminotransferase: a sensitive marker of alcoholism with or without alcoholic hepatitis. Hepatology 1984;4:893-6.
(24.) Pol S, Nalpas B, Vassault A, Bousquet-Lemercier B, Franco D, Lacour B, et al. Hepatic activity and mRNA expression of aspartate aminotransferase isoenzymes in alcoholic and nonalcoholic liver disease. Hepatology 1991;14:620-5.
(25.) Ludwig S, Kaplowitz N. Effect of serum pyridoxine deficiency on serum and liver transaminases in experimental liver injury in the rat. Gastroenterology 1980;79:545-9.
(26.) Zhou S-L, Gordon RE, Bradbury M, Stump D, Kiang C-L, Berk PD. Ethanol up-regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase in Hep G-2 cells. Hepatology 1998;27:1064-74.
(27.) Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem 1978;24:58-73.
(28.) Vanderlinde RE. Review of pyridoxal phosphate and the transaminases in liver disease. Ann Clin Lab Sci 1986;16:79-93.
(29.) Allman MA, Pang E, Yau DF, Stewart PM, Tiller DJ, Truswell AS. Elevated plasma vitamers of vitamin B6 in patients with chronic renal failure on regular hemodialysis. Eur J Clin Nutr 1992;46: 679-83.
(30.) College of American Pathologists. AST and ALT. Chemistry survey 1997. Northfield, IL: College of American Pathologists, 1997:710;15-8.
(31.) Hultcrantz R, Glaumann H, Lindberg G, Nilsson LH. Liver investigation in 149 asymptomatic patients with moderately elevated activities of serum aminotransferases. Scand J Gastroenterol 1986;21:109-13.
(32.) Kundrotas LW, Clement DJ. Serum alanine aminotransferase (ALT) elevation in asymptomatic US Air Force basic trainee blood donors. Dig Dis Sci 1993;38:2145-50.
(33.) Dybkaer R, for the International Federation of Clinical Expert Panel on Theory of Reference Values and International Committee for Standardization in Haematology Standing Committee on Reference Values. Approved recommendation (1987) on the theory of reference values, part 6. Presentation of observed values related to reference values. Labmedica 1988;(Apr/May): 27-30.
(34.) Lott JA, Tholen DW, Massion CG. Determination of reference ranges for serum enzymes via a large interlaboratory survey. Arch Pathol Lab Med 1987;111:9-15.
(35.) Skendzel LP, Barnett RN, Platt R. Medically useful criteria for analytic performance of laboratory tests. Am J Clin Pathol 1985;83:200-5.
(36.) Crofton PM. Biochemistry of alkaline phosphatase isoenzymes. CRC Crit Rev Clin Lab Sci 1982;16:161-94.
(37.) Posen S. Alkaline phosphatase. Ann Intern Med 1967;67:183-94.
(38.) Fishman WH. Alkaline phosphatase isoenzymes: recent progress. Clin Biochem 1990;23:99-104.
(39.) Kaplan MM. Alkaline phosphatase. N Engl J Med 1972;62:45268.
(40.) Crofton PM. Biochemistry of alkaline phosphatase isoenzymes. Crit Rev Clin Lab Sci 1982;16:161-94.
(41.) McComb RB, Bowers GN Jr, Posen S. Alkaline phosphatase. New York: Plenum Press, 1979:525-786.
(42.) Moss DW. Multiple forms of acid and alkaline phosphatases: genetics, expression and tissue modification. Clin Chim Acta 1986;161:123-35.
(43.) Weiss MJ, Ray K, Henthorn PS, Lamb B, Kadesch T, Harris H. Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 1988;263:12002-10.
(44.) Clubb JS, Neale FC, Posen S. The behavior of infused placental alkaline phosphatase in human subjects. J Lab Clin Med 1965; 66:493-507.
(45.) Kleerekoper M, Horne M, Cornish CJ, Posen S. Serum alkaline phosphatase after fat ingestion: an immunological study. Clin Sci 1970;38:339-45.
(46.) Posen S, Grundstein HS. Turnover of skeletal alkaline phosphatase in humans. Clin Chem 1982;28:153-4.
(47.) Kress BC, Mizrahi IA, Armour KW, Marcus R, Emkey RD, Santora AC. Use of bone alkaline phosphatase to monitor alendronate therapy in individual postmenopausal osteoporotic women. Clin Chem 1999;45:1009-17.
(48.) Moss DW. Physicochemical and pathophysiological factors in the release of membrane-bound alkaline phosphatase from cells. Clin Chim Acta 1997;257:133-40.
(49.) Schaeler R. The mechanism of the increase in the activity of liver alkaline phosphatase in experimental cholestasis: measurement of an increased enzyme concentration by immunochemical titration. Z Klin Chem Klin Biochem 1975;13:277-81.
(50.) Schlaeger R, Haux D, Kattermann R. Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis: significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver. Enzyme 1982;28:3-13.
(51.) Bowers GN Jr, McComb RB, Kelley ML. Measurement of total alkaline phosphatase activity in human serum. Sel Methods Clin Chem 1977;8:31-9.
(52.) Bowers GN Jr, McComb RB. Measurement of total alkaline phosphatase activity in human serum. Clin Chem 1975;21: 1988-95.
(53.) College of American Pathologists. Alkaline phosphatase. Chemistry survey 1997. Northfield, IL: College of American Pathologists, 1997:11-4.
(54.) Gomez B Jr, Ardakani S, Ju J, Jenkins D, Cerelli MJ, Daniloff GY, Kung VT. Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity in serum. Clin Chem 1995;41: 1560-6.
(55.) Martin M, Van Hoof V, Couttenye M, Prove A, Blockx P. Analytical and clinical evaluation of a method to quantify bone alkaline phosphatase, a marker of osteoblastic activity. Anticancer Res 1997;17:3167-70.
(56.) Woitge HW, Seibel MJ, Ziegler R. Comparison of total and bone-specific alkaline phosphatase in patients with nonskeletal disorders or metabolic bone diseases. Clin Chem 1996;42: 1796-804.
(57.) Anciaux ML, Pelletier AG, Attali P, Meduri B, Liguory C, Etienne JP. Prospective study of clinical and biochemical features of symptomatic choledocholithiasis. Dig Dis Sci 1986;31:449-53.
(58.) Miura T, Matsuda Y, Tsuji A, Katunuma N. Immunological cross-reactivity of [gamma]-glutamyl transpeptidase from human and rat kidney. J Biochem (Tokyo) 1981;89:217-22.
(59.) Tate SS, Meister A. [gamma]-glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem 1981;39:35768.
(60.) Jung K, Wischke UW. Electrophoretic variants of alanine aminopeptidase, alkaline phosphatase, and [gamma]-glutamyl transferase in urine. Clin Chem 1984;30:856-9.
(61.) Orrego H, Blake JE, Israel Y. Relationship between [gamma]-glutamyl transpeptidase and mean urinary alcohol levels in alcoholics while drinking and after alcohol withdrawal. Alcohol Clin Exp Res 1985;9:10-3.
(62.) Sheeman M, Maythorn P. Predictive value of [gamma]-glutamyl transpeptidase in various liver diseases. In: Goldberg DM, Werner M, eds. Progress in clinical enzymology. New York: Masson, 1979: 184-7.
(63.) Ratanasavanh D, Tazi A, Gaspart E. Hepatic [gamma]-glutamyl transferase release: effect of bile salt and membrane structure modification. Adv Biochem Pharamacol 1982;3:93-103.
(64.) Itoh S, Nakajima M. Liver [gamma]-glutamyl transferase activity in viral liver disease. Digestion 1986;33:121-5.
(65.) Schmidt E, Schmidt FW. Progress in the enzyme diagnosis of liver disease: reality or illusion. Clin Biochem 1990;23:375-82.
(66.) Nemesanszky E, Lott JA. [gamma]-Glutamyltransferase and its isoenzymes: progress and problems. Clin Chem 1985;31:797-803.
(67.) Betro MG, Oon RC, Edwards JB. [gamma]-Glutamyl transpeptidase in diseases of the liver and bone. Am J Clin Pathol 1973;60: 672-8.
(68.) Hedworth-Whitty RB, Whitfield JB, Richardson RW. Serum [gamma]-glutamyltranspeptidase activity in myocardial ischaemia. Br Heart J 1967;29:432-8.
(69.) Goldberg DM. Functional and clinical aspects of [gamma]-glutamyl-transpeptidase and its isoenzymes: a critical review of recent progress. In: Griffiths JC, ed. Clinical enzymology. New York: Masson Publishing USA, 1979:123-54.
(70.) Burrows S, Feldman W, McBride F. Serum [gamma]-glutamyl transpeptidase. Evaluation in screening of hospitalized patients. Am J Clin Pathol 1975;64:311-4.
(71.) Shaw LM, Stromme JH, London JL, Theodorsen L. International Federation of Clinical Chemistry. Scientific Committee, Analytical Section. Expert Panel on Enzymes. IFCC methods for measure ment of enzymes. Part 4. IFCC methods for [gamma]-glutamyltransferase [([gamma]-glutamyl)-peptide: amino acid [gamma]-glutamyltransferase, EC 184.108.40.206]. Clin Chim Acta 1983;135:315f-38f.
(72.) College of American Pathologists. Gamma glutamyl transferase. Chemistry survey 1997. Northfield, IL: College of American Pathologists, 1997:33-4.
(73.) Chowdhury JR, Wolkoff AW, Chowdhury NR, Arias IM. Hereditary jaundice and disorders of bilirubin metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Stanbury JB, Wyngaarden JB, Fredrickson DS, eds. The metabolic and molecular bases of inherited disease, Vol. 2. New York: McGraw-Hill, 1995:2161-208.
(74.) Berk PD, Martin JF, Blaschke TF, Scharchmidt BF, Plotz PH. Unconjugated hyperbilirubinemia: physiologic evaluation and experimental approaches to therapy. Ann Intern Med 1975;82: 552-70.
(75.) Bloomer JR, Berk PD, Vergalla J, Berlin NI. Influence of albumin on the extravascular distribution of unconjugated bilirubin. Clin Sci Mol Med 1973;45:517-21.
(76.) McDonagh AF, Palma AA, Lauff JJ, Wu TW. Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat. J Clin Invest 1984;74:763-70.
(77.) Fevery J, Blanckaert N. What can we learn from analysis of serum bilirubin. J Hepatol 1986;2:113-21.
(78.) Berk PD, Noyer C. Clinical chemistry and physiology of bilirubin. Semin Liver Dis 1994;14:346-55.
(79.) Zimmerman HJ. Intrahepatic cholestasis. Arch Intern Med 1979; 139:1038-45.
(80.) Van Hootegem P, Fevery J, Blanckaert N. Serum bilirubins in hepatobiliary disease: comparison with other liver function tests and changes in the portobstructive period. Hepatology 1985;5: 112-7.
(81.) Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N Engl J Med 1995;333:1171-5.
(82.) Thomsen HF, Hardt F, Juhl E. Diagnosis of Gilbert's syndrome. Scand J Gastroenterol 1981;16:699-703.
(83.) Lo DH, Wu TW. Assessment of the fundamental accuracy of the Jendrassik-Grof total and direct bilirubin assays. Clin Chem 1983;29:31-6.
(84.) ArvanD, Shirey TL. Conjugated bilirubin: a better indicator of impaired hepatobiliary excretion than direct bilirubin. Ann Clin Lab Sci 1984;15:252-9.
(85.) Doumas BT, Wu T-W, Jendrzejcaz B. Delta bilirubin: absorption spectra, molar absorptivity, and reactivity in the diazo reaction. Clin Chem 1987;33:769-74.
(86.) Doumas BT, Wu T-W. The measurement of bilirubin fractions in serum. Crit Rev Clin Lab Sci 1991;28:415-46.
(87.) Ihara H, Aoki Y, Aoki T, Yoshida M. Light has a greater effect on direct bilirubin measured by the bilirubin oxidase method than by the diazo method. Clin Chem 1990;36:895-7.
(88.) Kubasik NP, Mayer TK, Bhaskar AG, Sine HE, D'Souza JP. The measurement of fractionated bilirubin by Ektachem film slides-method validation and comparison of conjugated bilirubin mea surements with direct bilirubin in obstructive and hepatocellular jaundice. Am J Clin Pathol 1985;84:518-23.
(89.) College of American Pathologists. Total bilirubin. Chemistry survey 1997. Northfield, IL: College of American Pathologists, 1997:18-20.
(90.) Chan K-M, Scott MG, Wu T-W, Clouse RE, Calvin DR, Koenig J, et al. Inaccurate values for direct bilirubin with some commonly used direct bilirubin procedures. Clin Chem 1985;31:1560-3.
(91.) Lott JA, Doumas BT. "Direct", total bilirubin tests: contemporary problems. Clin Chem 1993;39:641-7.
(92.) Doumas BT, Peters T. Serum and urine albumin: a progress report on their measurement and clinical significance. Clin Chim Acta 1997;258:3-20.
(93.) Dufour DR. Gender related differences in liver function and integrity tests [Abstract]. Clin Chem 1998;44(Suppl 6):A137.
(94.) Bonis PA, Tong PA, Tong MJ, Blatt LM, Conrad A, Griffith JL. A predictive model for the development of hepatocellular carcinoma, liver failure, or liver transplantation for patients presenting to clinic with chronic hepatitis C. Am J Gastroenterol 1999;94: 1605-12.
(95.) McGinlay JM, Payne RB. Serum albumin by dye-binding: bromcresol green or bromcresol purple? Ann Clin Biochem 1988;25: 417-21.
(96.) Maguire GA, Price CP. Bromcresol purple method for serum albumin gives falsely low values in patients with renal insufficiency. Clin Chim Acta 1986;155:83-8.
(97.) Beyer C, Boekhout M, van Iperen H. Bromcresol purple dyebinding and immunoturbidimetry for albumin measurement in plasma or serum of patients with renal failure. Clin Chem 1994;40:844-5.
(98.) Bush V, Reed RG. Bromcresol purple dye-binding methods underestimate albumin that is carrying covalently bound bilirubin. Clin Chem 1987;33:821-3.
(99.) Ihara H, Nakamura H, Aoki Y, Aoki T, Yoshida M. Effects of serum-isolated vs. synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin Clin Chem 1991; 37:1269-72.
(100.) Pascucci MW, Grisley DW, Rand RN. Electroimmunoassay of albumin in human serum: accuracy and long-term precision. Clin Chem 1983;29:1787-90.
(101.) College of American Pathologists. Albumin. Chemistry survey set C-B. Northfield, IL: College of American Pathologists, 1999:6-8.
(102.) Liebman HA, Furie BC, Tong MJ, Blanchard RA, Lo KJ, Lee SD, et al. Des-[gamma]-carboxy (abnormal) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med 1984;310: 1427-31.
(103.) Ts'ao C, Swedlund J, Neofotistos D. Implications of use of low international sensitivity index thromboplastins in prothrombin time testing. Arch Pathol Lab Med 1994;118:1183-7.
(104.) Peters RH, van den BesselaarAMHP, Olthuis FM. Determination of the mean normal prothrombin time for assessment of international normalized ratios. Usefulness of lyophilized plasma. Thromb Haemost 1991;66:442-5.
(105.) WHO Expert Committee on Biological Standardization. 33rd report. WHO Tech Rep Ser 1983;687:81-105.
(106.) Taberner DA, Poller L, Thomson JM, Darby KV. Effect of international sensitivity index (ISI) of thromboplastins on precision of international normalised ratios (INR). J Clin Pathol 1989;42: 92-6.
(107.) Johnson M, Harrison L, Moffatt K, Wilian A, Hirsh J. Reliability of the international normalized ratio for monitoring the induction phase of warfarin: comparison with the prothrombin time ratio. J Lab Clin Med 1996;128:214-7.
(108.) Kovacs MJ, Wong A, MacKinnon K, Weir K, Keeney M, Boyle E, Cruickshank M. Assessment of the validity of the INR system for patients with liver impairment. Thromb Haemost 1994;71:727-30.
(109.) Robert A, Chazouilleres O. Prothrombin time in liver failure: time, ratio, activity percentage, or International Normalized Ratio? Hepatology 1996;24:1392-4.
(110.) Blanchard RA, Furie BC, Jorgensen M, Kruger SF, Furie B. Acquired vitamin K-dependent carboxylation deficiency in liver disease. N Engl J Med 1981;305:242-8.
(111.) Dufour DR, Teot L. Laboratory identification of ischemic hepatitis (shock liver) [Abstract]. Clin Chem 1988;34:1287.
(112.) Fuchs S, Bogomolski-Yahalom V, Paltiel O, Ackerman Z. Ischemic hepatitis: clinical and laboratory observations of 34 patients. J Clin Gastroenterol 1998;26:183-6.
(113.) Singer AJ, Carracio TR, Mofenson HC. The temporal profile of increased transaminase levels in patients with acetaminophen-induced liver dysfunction. Ann Emerg Med 1995;26:49-53.
(114.) Willner IR, Uhl MD, Howard SC, Williams EQ, Riely CA, Waters B. Serious hepatitis A: an analysis of patients hospitalized during an epidemic in the United States. Ann Intern Med 1998;128: 111-4.
(115.) Mendenhall CL. Alcoholic hepatitis. The VA Cooperative Study Group on Alcoholic Hepatitis. Clin Gastroenterol 1981;10:417-41.
(116.) O'Grady JG, Alexander GJM, Hayllar KM, Williams R. Early indicators of prognosis in fulminant hepatic failure. Gastroenterology 1989;97:4439-45.
(117.) Bonacini M, Hadi G, Govindarajan S, Lindsay KL. Utility of a discriminant score for diagnosing advanced fibrosis or cirrhosis in patients with chronic hepatitis C infection. Am J Gastroenterol 1997;92:1302-4.
(118.) Poller L, Triplett DA, Hirsh J, Carroll J, Clarke K. The value of plasma calibrants in correcting coagulometer effects on international normalized ratios. An international multicenter study. Am J Clin Pathol 1995;103:358-65.
(119.) Davis KID, Danielson CFM, May LS, Han Z-Q. Use of different thromboplastin reagents causes greater variability in international normalized ratio results than prolonged room temperature storage of specimens. Arch Pathol Lab Med 1998;122:972-7.
(120.) Cunningham MTA, Johnson GF, Pennell BJ, Olson JD. The reliability of manufacturer-determined, instrument specific international sensitivity index values for calculating the international normalized ratio. Am J Clin Pathol 1994;102:128-33.
(121.) Fairweather RB, Ansell J, van den Besselaar AM, Brandt JT, Bussey HI, Poller L, et al. College of American Pathologists Conference XXXI on laboratory monitoring of anticoagulant therapy: laboratory monitoring of oral anticoagulant therapy. Arch Pathol Lab Med 1998;122:768-81.
(122.) Stevenson KJ, Craig S, Dufty JMK, Taberner DA. System ISI calibration: a universally applicable scheme is possible only when coumarin plasma calibrants are used. Br J Haematol 1997;96:435-41.
(123.) Kitchen S, Walker ID, Woods TAL, Preston FE. Thromboplastin related differences in the determination of international normalized ratio: a cause for concern? Thromb Haemost 1994;72: 426-9.
(124.) Lassen JF, Kjeldsen J, Antonsen S, Petersen PH, Brandslund I. Interpretation of serial measurements of international normalized ratio for prothrombin times in monitoring oral anticoagulant therapy. Clin Chem 1995;41:1171-6.
(125.) Poller L. Screening INR deviation of local prothrombin time systems. J Clin Pathol 1998;51:356-9.
(126.) Johnson M, Brigder M. A cross-Canada survey of prothrombin time testing. Does the establishment of local ISI values improve the accuracy of International Normalized Ratio reporting? Am J Clin Pathol 1998;110:683-90.
(127.) Miyaji H, Ito S, Azuma T, Ito Y, Yamazaki Y, Ohtaki Y, et al. Effects of Helicobacter pylori eradication therapy on hyperammonemia in patients with liver cirrhosis. Gut 1997;40:726-30.
(128.) Zullo A, Rinaldi V, Hassan C, Folino S, Winn S, Pinto G, et al. Helicobacterpylori and plasma ammonia levels in cirrhotics: role of urease inhibition by acetohydroxamic acid. Ital J Gastroenterol Hepatol 1998;30:405-9.
(129.) Batsshaw ML. Inborn errors of urea synthesis. Ann Neurol 1994;35:133-41.
(130.) Heubi JE, Daugherty CC, Partin JS, Partin JC, Schubert WK. Grade 1 Reye's syndrome-outcome and predictors of progression to deeper coma grades. N Engl J Med 1984;311:1539-42.
(131.) Stahl J. Studies of the blood ammonia in liver disease-its diagnostic, prognostic, and therapeutic significance. Ann Intern Med 1963;58:1-23.
(132.) Butterworth RF, Giguere JF, Michaud J, Lavoie J, Layrargues GP. Ammonia: key factor in the pathogenesis of hepatic encephalopathy. Neurochem Pathol 1987;6:1-12.
(133.) Muting D, Kalk JF, Fischer R, Wuzel H, Reikowski J. Hepatic detoxification and hepatic function in chronic active hepatitis with and without cirrhosis. Dig Dis Sci 1988;33:41-6.
(134.) McClain CJ, Zieve L, Doizaki WM, Gilberstadt S, Onstad GR. Blood methanetriol in alcoholic liver disease with and without hepatic encephalopathy. Gut 1980;21:318-23.
(135.) Jones EA, Basile AS. The involvement of ammonia with the mechanisms that enhance GABA-ergic neurotransmission in hepatic failure. Adv Exp Med Biol 1997;420:75-83.
(136.) Norenberg MD, Itzhak Y, Bender AS. The peripheral benzodiazepine receptor and neurosteroids in hepatic encephalopathy. Adv Exp Med Biol 1997;420:95-111.
(137.) Chamuleau RAFM, Vogels BAPM. Hyperammonemia without portal systemic shunting does not resemble hepatic encephalopathy. Adv Exp Med Biol 1997;420:173-83.
(138.) Huizenga JR, Tangerman A, Gips CH. Determination of blood ammonia in biological fluids. Ann Clin Biochem 1994;31:529-43.
(139.) da Fonseca-Wollheim F. Preanalytical increase of ammonia in blood specimens from healthy subjects. Clin Chem 1990;36: 1483-7.
(140.) College of American Pathologists. Ammonia. Chemistry survey set C-B. Northfield, IL College of American Pathologists, 1999: 73.
(141.) Stapleton JT. Host immune response to hepatitis A virus. J Infect Dis 1995;175(Suppl 1):S9-14.
(142.) Kao HW, Ashcaval M, Redeker AG. The persistence of hepatitis A IgM antibody after acute clinical hepatitis A. Hepatology 1984;4:933-6.
(143.) Fujiwara K, Yokosuka O, Ehata T, Imazeki F, Saisho H, Miki M, Omata M. Frequent detection of hepatitis A viral RNA in serum during the early convalescent phase of acute hepatitis A. Hepatology 1997;26:1634-49.
(144.) Skinhoj P, Mikkelsen F, Hollinger FB. Hepatitis A in Greenland: importance of specific antibody testing in epidemiologic surveillance. Am J Epidemiol 1977;105:140-7.
(145.) Koff RS. Seroepidemiology of hepatitis A in the United States. J Infect Dis 1995;171(Suppl 1):S19-23.
(146.) Jilg W, Bittner R, Bock HL, Clemens R, Schatzl H, Schmidt M, et al. Vaccination against hepatitis A: comparison of different short-term immunization schedules. Vaccine 1992;10(Suppl 1): S126-8.
(147.) Goubau P, Gan Gerven V, Safary A. Effect of virus strain and antigen dose on immunogenicity and reactogenicity of an inactivated hepatitis A vaccine. Vaccine 1992;10(Suppl 1):S114-8.
(148.) Totos G, Gizaris V, Papaevangelou G. Hepatitis A vaccine: persistence of antibodies 5 years after the first vaccination. Vaccine 1997;15:1252-3.
(149.) Feinstone SM, Kapikian AZ, Purcell RH. Hepatitis A: detection by immune electron microscopy of virus-like antigen associated with acute illness. Science 1973;182:1026-8.
(150.) Hollinger FB, Bradley DW, Maynard JE, Dreesman GR, Melnick JL. Detection of hepatitis A viral antigen by radioimmunoassay. J Immunol 1975;115:1464-6.
(151.) Hollinger FB, Dreesman GR. Hepatitis viruses. In: Rose NR, de Macario EC, Folds JD, Lane HC, Nakamura RM, eds. Manual of clinical laboratory immunology, 5th ed. Washington: American Society for Microbiology, 1997:702-18.
(152.) Lemon SM, Gates NL, Simms TE, Bancroft WH. IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 1981;143:803-9.
(153.) Hollinger FB. Hepatitis B virus. In: Fields BN, Knipe DM, Hawley PM, eds. Fields virology, 3rd ed. Philadelphia: Lippincott-Raven, 1996:2739-807.
(154.) Czaja AJ, Shiels MT, Taswell HF, Wood JR, Ludwig J, Chase RC. Frequency and significance of immunoglobulin M antibody to hepatitis B core antigen in corticosteroid-treated severe chronic active hepatitis B. Mayo Clin Proc 1988;63:119-25.
(155.) Sjogren M, Hoofnagle JH. Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis. Gastroenterology 1985;89:252-8.
(156.) Seeff LB, Beebe GW, Hoofnagle JH, Norman JE, Buskell-Bales Z, Waggoner JG, et al. A serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the United States Army. N Engl J Med 1987;316:965-70.
(157.) Silva AE, McMahon BJ, Parkinson AJ, Sjogren MH, Hoofnagle JH, DeBisceglie AM. Hepatitis B virus DNA in persons with isolated antibody to hepatitis B core antigen who subsequently received hepatitis B vaccine. Clin Infect Dis 1998;26:895-7.
(158.) Cacciola I, Pollicino T, Squadrito G, Grenzia G, Orlando ME, Raimondo G. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 1999;341:22-6.
(159.) McMahon BJ, Parkinson AJ, Helminiak C, Wainwright RB, Bulkow L, Kellerman-Douglas A, et al. Response to hepatitis B vaccine of persons positive for antibody to hepatitis B core antigen. Gastroenterology 1992;103:590-4.
(160.) Aoki SK, Finegold D, Kuramoto IK, Douville C, Richards C, Randell R, et al. Significance of antibody to hepatitis B core antigen in blood donors as determined by their serologic response to hepatitis B vaccine. Transfusion 1993;33:362-7.
(161.) Draelos M, Morgan T, Schifman RB, Sampliner RE. Significance of isolated antibody to hepatitis B core antigen determined by immune response to hepatitis B vaccination. JAMA 1987;258: 1193-5.
(162.) Foutch PG, Carey WD, Tabor E, Cianflocco AJ, Nakamoto S, Smallwood LA, Gerety RJ. Concomitant hepatitis B surface antigen and antibody in thirteen patients. Ann Intern Med 1983;99:460-3.
(163.) Chernesky MA, Gretch D, Mushahwar IK, Swenson PD, Yarbough P0. Cumitech 18A. Laboratory diagnosis of the hepatitis viruses. Young S, coordinating ed. Washington, DC: American Society for Microbiology, 1998.
(164.) Hollinger FB, Dienstag JL. Hepatitis B and D viruses. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of clinical microbiology, 7th ed. Washington: ASM Press, 1999: 1773pp.
(165.) Yotsuyanagi H, Yasuda K, lino S, Moriya K, Shintani Y, Fujie H, et al. Persistent viremia after recovery from self-limited acute hepatitis B. Hepatology 1998;27:1377-82.
(166.) Cabrerizo M, Bartolome J, De Sequera P, Caramelo C, Carreno V. Hepatitis B virus DNA in serum and blood cells of hepatitis B surface antigen-negative hemodialysis patients and staff. J Am Soc Nephrol 1997;8:1443-7.
(167.) Loriot MA, Marcellin P, Walker F, Boyer N, Degott C, Randrianatoavina I, et al. Persistence of hepatitis B virus DNA in serum and liver from patients with chronic hepatitis B after loss of HBsAg. J Hepatol 1997;27:251-8.
(168.) Alter HJ. New kit on the block: evaluation of second-generation assays for detection of antibody to the hepatitis C virus. Hepatology 1992;15:340-53.
(169.) Lu RH, Hwang SJ, Chan CY, Chang FY, Lee SD. Quantitative measurement of serum HCV-RNA in patients with chronic hepatitis C: comparison between Amplicor HCV monitor system and branched DNA signal amplification assay. J Clin Lab Anal 1998; 12:121-5.
(170.) Barrera JM, Francis B, Ercilla G, Nelles M, Achord D, Darner J, Lee SR. Improved detection of anti-HCV in post-transfusion hepatitis by a third-generation ELISA. Vox Sang 1995;68:15-8.
(171.) Uyttendaele S, Claeys H, Mertens W, Verhaert H, Vermylen C. Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sang 1994;66:15-8.
(172.) Gretch D. Diagnostic tests for hepatitis C. Hepatology 1997; 26(Suppl 1):43S-7S.
(173.) Barrera JM, Bruguera M, Ercilla MG, Gil C, Celis R, Gil MP, et al. Persistent hepatitis C viremia after acute self-limited posttransfusion hepatitis. Hepatology 1995;21:639-44.
(174.) Beld M, Penning M, van Putten M, Lukashov V, van den Hoek A, McMorrow M, Goudsmit J. Quantitative antibody responses to structural (core) and nonstructural (NS3, NS4, and NS5) hepatitis C virus proteins among seroconverting injecting drug users: impact of epitope variation and relationship to detection of HCV RNA in blood. Hepatology 1999;29:1288-98.
(175.) Seeff LB. Mortality and morbidity of transfusion-associated non-A, non-B hepatitis and type C hepatitis: and NHLBI multicenter study [Abstract]. The NHLBI Study Group. Hepatology 1994;20:204A.
(176.) Mast AF, Hwang L-Y, Seto D, Nolte FS, Kelly MG, Alter MJ. Perinatal hepatitis C virus transmission: maternal risk factors and optimal timing of diagnosis [Abstract]. Hepatology 1999;30: 499A.
(177.) Thomas SL, Newell ML, Peckham CS, Ades AE, Hall AJ. Use of polymerase chain reaction and antibody tests in the diagnosis of vertically transmitted hepatitis C virus infection. Eur J Clin Microbiol Infect Dis 1997;16:711-9.
(178.) Busch M, Tobler L, Quan S, Wilber JC, Johnson P, Polito A, et al. A pattern of 5-1-1 and c100-3 only on hepatitis C (HCV) recombinant immunoblot assay does not reflect HCV infection in blood donors. Transfusion 1993;33:84-8.
(179.) Rossini A, Gazzola GB, Ravaggi A, Agostinelli E, Biasi L, Albertini A, et al. Long-term follow-up and infectivity in blood donors with hepatitis C antibodies and persistently normal alanine aminotransferase levels. Transfusion 1995;35:108-11.
(180.) Pawlotsky J, Bastie A, Pellet C, Remire J, Darthuy F, Wolfe L, et al. Significance of indeterminate third-generation hepatititis C virus recombinant immunoblot assay. J Clin Microbiol 1996;34: 80-3.
(181.) Villano SA, Vlahov D, Nelson KE, Cohn S, Thomas DL. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. Hepatology 1999;29:908-14.
(182.) Pawlotsky J-M, Lonjon I, Hezode C, Raynard B, Darthuy F, Remire J, et al. What strategy should be used for diagnosis of hepatitis C virus infection in clinical laboratories? Hepatology 1998;27: 1700-2.
(183.) Pawlotsky JM, Roudot-Thoraval F, Pellet C, Aumont P, Darthuy F, Remire J, et al. Influence of hepatitis C virus (HCV) genotypes on HCV recombinant immunoblot assays. J Clin Microbiol 1995;33: 1357-9.
(184.) Vernelen K, Claeys H, Verhaert AH, Volckaerts A, Vermylen C. Significance of NS3 and NS5 antigens in screening for HCV antibody [Letter]. Lancet 1994;343:853.
(185.) LaPerche S, Courouce A-M, Lemaire J-M, Coste J, Defer C, Cantaloube J-F. GB virus type C/hepatitis G virus infection in French blood donors with anti-NS5 isolated reactivities by recombinant immunoblot assay for hepatitis C virus. Transfusion 1999;39:790-1.
(186.) Aoyagi K, Ohue C, lida K, Kimura T, Tanaka E, Kiyosawa K, Yagi S. Development of a simple and highly sensitive enzyme immunoassay for hepatitis C virus core antigen. J Clin Microbiol 1999;37:1802-8.
(187.) Ravaggi A, Biasin MR, Infantolino D, Cariani E. Comparison of competitive and non-competitive reverse transcriptiohn-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA. J Virol Methods 1997;65A:123-9.
(188.) Lunel F, Cresta P, Vitour D, Payan C, Dumont B, Frangeul L, et al. Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and Monitor assays. Hepatology 1999; 29:528-35.
(189.) Davis GL, Lau JY, Urdea MS, Neuwald PD, Wilber JC, Lindsay K, et al. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification assay: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology 1994;19:1337-41.
(190.) Cuypers HTM, Bresters D, Winkel IN, Reesink HW, Weiner AJ, Houghton M, et al. Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus. J Clin Microbiol 1992;30: 3220-4.
(191.) Wang J-T, Wang, T-H, Sheu J-C, Lin SM, Lin JT, Chen DS. Effects of anticoagulants and storage of blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus. J Clin Microbiol 1992;30:750-3.
(192.) Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology assays for HCV RNA. Vox Sang 1999;76:149-58.
(193.) Camma C, Giunta M, Pinzello G, Morabito A, Verderio P, Pagliaro L. Chronic hepatitis C and interferon alpha: conventional and cumulative meta-analyses of randomized clinical trials. Am J Gastroenterol 1999;94:581-95.
(194.) Poynard T, McHutchison J, Goodman Z, Ling M-H, Albrecht J. Is an "a la carte" combination interferon [alpha]-2b plus ribavirin regimen possible for first line treatment in patients with chronic hepatitis C? The ALGOVIRC Project Group. Hepatology 2000;31:211-8.
(195.) Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin Liver Dis 1995;15: 41-63.
(196.) Reddy KR, Hoofnagle JH, Tong MJ, Lee WM, Pockros P, Heathcote EJ, et al. Racial differences in response to therapy with interferon in chronic hepatitis C. Consensus Interfereon Study Group. Hepatology 1999;30:787-93.
(197.) McHutchison JG, Poynard T, Gordon SC, Dienstag J, Morgan T, Yao R, et al. The impact of race on response to anti-viral therapy in patients with chronic hepatitis C [Abstract]. Hepatology 1999; 30:302A.
(198.) Forns X, Bukh J. Methods for determining the hepatitis C virus genotype. J Viral Hepat 1998;4:1-19.
(199.) Germer JJ, Rys PH, Thorvilson JN, Pershing DH. Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test. J Clin Microbiol 1999;37:2625-30.
(200.) Stuyver L, Wyseur A, van Arnhem W, Lunel F, Laurent-Puig P, Pawlotsky JM, et al. Hepatitis C virus genotyping by means of a 5'-UR/core line probe assays and molecular analysis of untypeable specimens. Virus Res 1995;38:137-57.
(201.) Giannini C, Thiers V, Nousbaum JB, Stuuyver L, Maertens G, Brechot C. Comparative analysis of two assays for genotyping hepatitis C virus based on genotype-specific primers or probes. J Hepatol 1995;23:246-53.
(202.) Marshall DJ, Heisler LM, Lyamichev V, Murvine C, Olive DM, Ehrlich GD, et al. Determination of hepatitis C virus genotype in the United States by cleavage fragment length polymorphism analysis. J Clin Microbiol 1997;35:3156-62.
(203.) Davidson F, Simmonds P, Ferguson JC, Jarvis LM, Dow BC, Follett EA, et al. Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region. J Gen Virol 1995;76:1197-204.
(204.) Pawlotsky JM, Prescott L, Simmonds P, Pellet C, Laurent-Puig P, Labonne C, et al. Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay. J Clin Microbiol 1997;35:1734-9.
(205.) Toyoda H, Fukuda Y, Hayakawa T, Kumada T, Nakano S, Takamatsu J, Saito H. Presence of multiple genotype-specific antibodies in patients with persistent infection with hepatitis C virus (HCV) of a single genotype: evidence for transient or occult superinfection with HCV of different genotypes. Am J Gastroenterol 1999;94:2230-6.
(206.) Lau JY, Davis GL, Prescott LE, Maertens G, Lindsay KL, Qian K, et al. Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Hepatitis Interventional Therapy Group. Ann Intern Med 1996;124:868-76.
(207.) Lau JY, Mizokami M, Kolberg JA, Davis GL, Prescott LE, Ohno, et al. Application of six hepatitis C genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis 1995;171:282-9.
(208.) Rizetto M, Ponzetto A, Forzani I. Epidemiology of hepatitis delta virus: overview. Prog Clin Biol Res 1991;364:1-20.
(209.) Erker JC, Dessai SM, Schlauder GG, Dawson GJ, Mushawar IK. A hepatitis E variant from the United States: molecular characterization and transmission in cynomolgus macaques. J Gen Virol 1999;80:681-90.
(210.) Yarbough PO, Tam AW, Gabor K, Garza E, Mockli RA, Palings I, et al. Assay development of diagnostic tests for hepatitis E. In: Nishioka K, Suzuki H, Mishiro S, Oda T, eds. Viral hepatitis and liver disease. Tokyo: Springer-Verlag, 1994:367-70.
(211.) Mast EE, Alter MJ, Holland PV, Purcell RH. Evaluation of assays for antibody to hepatitis E virus by a serum panel. Hepatitis E Virus Antibody Serum Panel Evaluation Group. Hepatology 1998; 27:857-61.
(212.) Thomas DL, Yarbough PO, Vlahov D, Tsarev SA, Nelson KE, Saah AJ, Purcell RH. Seroreactivity to hepatitis E virus in areas where the disease is not endemic. J Clin Microbiol 1997;35:1244-7.
(213.) Kabrane-Lazizi Y, Fine JB, Elm J, Glass GE, Higa H, Diwan A, et al. Evidence for widespread infection of wild rats with hepatitis E virus in the United States. Am J Trop Med Hyg 1999;61:331-5.
(214.) Meng XJ. Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. J Med Virol 1999;59:297-302.
(215.) Ricos C, Alvarez V, Cava F, Garcia-Lario JV, Hernandez A, Jimenez CV, et al. Current databases on biological variation: pros, cons and progress. Scand J Clin Lab Invest 1999;59:491-500.
(216.) Ross JW, Lawson NS. Analytic goals, concentration relationships, and state of the art for clincal laboratory precision. Arch Pathol Lab Med 1995;119:495-513.
(217.) Cordoba J, O'Riordan K, Dupuis J, Borensztajin J, Blei AT. Diurnal variation of serum alanine transaminase activity in chronic liver disease. Hepatology 1999;28:1724-5.
(218.) Rivera-Coll A, Funtes-Arderiu X, Diez-Noguera A. Circadian rhythms of serum concentrations of 12 enzymes of clinical interest. Chronobiol Int 1993;10:190-200.
(219.) Fraser CG. Biological variation in clinical chemistry. An update: collated data, 1988-1991. Arch Pathol Lab Med 1992;116: 916-23.
(220.) Holzel WGE. Intra-individual variation in serum from patients with chronic liver diseases. Clin Chem 1987;33:1133-6.
(221.) Fraser CG, Cummings ST, Wilkinson SP, Neville RG, Knox JD, Ho O, MacWalter RS. Biological variability of 26 clinical chemistry analytes in elderly people. Clin Chem 1989;35:783-6.
(222.) Manolio TA, Burke GL, Savage PJ, Jacobs DR Jr, Sidney S, Wagenknecht LE, et al. Sex- and race-related differences in liver-associated serum chemistry tests in young adults in the CARDIA study. Clin Chem 1992;38:1853-9.
(223.) Salvaggio A, Periti M, Miano L, Tavanelli M, Mazurati D. Body mass index and liver enzyme activity in serum. Clin Chem 1991;37:720-3.
(224.) Piton A, Poynard T, Imbert-Bismut F, Khalil L, Delattre J, Pelissier E, et al. Factors associated with serum alanine aminotransaminase activity in healthy subjects: consequences for the definition of normal values, for selection of blood donors, and for patients with chronic hepatitis C. MULTIVIRC group. Hepatology 1998;27: 1213-9.
(225.) Robinson D, Whitehead TP. Effect of body mass and other factors on serum liver enzyme levels in men attending for well population screening. Ann Clin Biochem 1989;26:393-400.
(226.) Nuttall FQ, Jones B. Creatine kinase and glutamic oxoacetic transaminase activity in serum: kinetics of change with exercise and effect of physical conditioning. J Lab Clin Med 1968;51: 257-61.
(227.) Dufour DR. Effects of habitual exercise on routine laboratory tests [Abstract]. Clin Chem 1998;44(Suppl 6):A136.
(228.) Dickerman RD, Pertusi R, Zachariah NY, Dufour DR, McConathy WJ. Anabolic steroid induced hepatotoxicity: is it overstated? Clin J Sports Med 1999;9:34-9.
(229.) Ono T, Kitaguchi K, Takehara M, Shiiba M, Hayami K. Serumconstituents analyses: effect of duration and temperature of storage of clotted blood. Clin Chem 1981;27:35-8.
(230.) Williams KM, Williams AE, Kline LM, Dodd RY. Stability of serum alanine aminotransferase activity. Transfusion 1987;27:431-3.
(231.) DiMagno EP, Corle D, O'Brien JF, Masnyk IJ, Go VL, Aamodt R. Effect of long-term freezer storage, thawing, and refreezing on selected constituents of serum. Mayo Clin Proc 1989;64:1226-34.
(232.) Prasad R, Firkins K, Fiorello J. Stability of AST and ALT assays in Tris buffers. Clin Chem 1990;36:131-2.
(233.) Prasad R, Welch C. Effect of pyridoxal 5-phosphate on the stability of alanine aminotransferase. Clin Chem 1992;38: 2340-1.
(234.) Litin SC, O'Brien JF, Pruett S, Forsman RW, Burritt MF, Bartholomew LG, Baldus WP. Macroenzyme as a cause of unexplained elevation of aspartate aminotransferase. Mayo Clin Proc 1987;62:681-7.
(235.) Mifflin TE, Bruns DE, Wrotnoski U, MacMillan RH, Stallings RG, Felder RA, Herold DA. University of Virginia case conference. Macroamylase, macro creatine kinase, and other macroenzymes. Clin Chem 1985;31:1743-8.
(236.) Bayer PM, Hotschek H, Knoth E. Intestinal alkaline phosphatase and the ABO blood group system: a new aspect. Clin Chim Acta 1980;108:81-7.
(237.) Reynoso G, Elias EG, Mittelman A. The contribution of the intestinal mucosa to the total serum alkaline phosphatase activity. Am J Clin Pathol 1971;56:707-12.
(238.) Domar U, Karpe F, Hamsten A, Stigbrand T, Olivecrona T. Human intestinal alkaline phosphatase; release to the blood is linked to lipid absorption, but removal from the blood is not linked to lipoprotein clearance. Eur J Clin Invest 1993;23:753-60.
(239.) Gordon T. Factors associated with serum alkaline phosphatase level. Arch Pathol Lab Med 1993;117:187-90.
(240.) Young DS. Effects of preanalytical variables on clinical laboratory tests, 2nd ed. Washington, DC: AACC Press, 1997:1285pp.
(241.) De Flamingh JP, van der Merwe JV. A serum biochemical profile of normal pregnancy. S Afr Med J 1984;65:552-5.
(242.) Dufour DR. Effects of oral contraceptives on routine laboratory tests [Abstract]. Clin Chem 1998;44(Suppl 6):A137.
(243.) Nilssen O, Helge-Forde O, Brenn T. The Tromso Study-distribution and population determinants of [gamma]-glutamyltransferase. Am J Epidemiol 1990;132:318-26.
(244.) Schiele F, Guilmin A-M, Detienne H, Siest G. [gamma]-glutamyltransferase activity in plasma: statistical distributions, individual variations, and reference intervals. Clin Chem 1977;23: 1023-8.
(245.) Combes B, Shore GM, Cunningham FG, Walker FB, Shorey JW, Ware A. Serum [gamma]-glutamyl transpeptidase activity in viral hepatitis: suppression in pregnancy and by birth control pills. Gastroenterology 1977;72:271-4.
(246.) Young DS. Effect of drugs on clinical laboratory tests, 5th ed. Washington, DC: AACC Press, 2000:2200pp.
(247.) Whitehead TP, Clarke CA, Whitfield AGW. Biochemical and haematological markers of alcohol intake. Lancet 1978;1:978-81.
(248.) Belfrage P, Berg B, Hagerstrand I, Nilsson-Ehle P, Tomqvist H, Wiebe T. Alterations of lipid metabolism in healthy volunteers during long-term ethanol intake. Eur J Clin Invest 1977;127: 127-31.
(249.) Moussavian SN, Becker RC, Piepmeyer JL, Mezey E, Bozian RC. Serum [gamma]-glutamyl transpeptidase and chronic alcoholism-influence of alcohol ingestion and liver disease. Dig Dis Sci 1985; 30:211-4.
(250.) Barrett PVD. Bilirubinemia and fasting [Letter]. N Engl J Med 1970;283:823.
(251.) Dufour DR. Effects of food ingestion on routine laboratory tests [Abstract]. Clin Chem 1998;44(Suppl 6):A136.
(252.) Carmel R, Wong ET, Weiner JM, Johson CS. Racial differences in serum total bilirubin levels in health and in disease (pernicious anemia). JAMA 1985;253:3416-8.
(253.) Van den Besselaar AMHP, Halem-Visser LP, Loeliger EA. The use of evacuated tubes for blood collection in oral anticoagulant control. Thromb Haemost 1983;40:676-7.
(254.) Raskob GE, Durica SS. Owen WL, Comp PC. Monitoring low-dose warfarin therapy by a central laboratory and implications for clinical trials and patient care: the Coumadin Aspirin Reinfarction (CARS) Pilot Study Group. Am J Cardiol 1996;78:1074-6.
(255.) Baglin T, Luddington R. Reliability of delayed INR determination: implications for decentralized anticoagulant care with off-site blood sampling. Br J Haematol 1997;96:431-4.
(256.) Dwyre AL, Giles AR, Key LA, Butler JR, Beck LR, Callahan JB. The effects of sodium citrate anticoagulant concentration, storage on or off cellular matrix, and specimen age on prothrombin time INR using a low ISI (1.06) rabbit brain thromboplastin [Letter]. Thromb Haemost 1995;73:1237.
(257.) Adcock DM, Kressen DC, Marlar RA. Effect of 3.2% vs. 3 8% sodium citrate on routine coagulation testing Am J Clin Pathol 1997;107:105-10.
(258.) Diaz J, Tornel PL, Martinez P. Reference intervals for blood ammonia in healthy subjects, determined by microdiffusion [Letter]. Clin Chem 1995;41:1048.
(259.) Agroyannis B, Tzanatos H, Fourtounas C, Kopelias I, Katsoudas S, Chondros K. Arteriovenous difference of blood ammonia in uremic patients under hemodialysis. Artif Organs 1998;22: 703-5.
(260.) Derave W, Bouckaert J, Pannier JL. Gender differences in blood ammonia response during exercicse. Arch Physiol Biochem 1997;105:203-9.
(261.) Howanitz JH, Howanitz PJ, Skrodzki CA, Iwanski JA. Influences of specimen processing and storage conditions on results for plasma ammonia. Clin Chem 1984;30:906-8.
(262.) da Fonseca-Wollheim F. Deamidation of glutamate by increased plasma [gamma]-glutamyltransferase is a source of rapid ammonia formation in blood and plasma specimens. Clin Chem 1990;36: 1479-82.
(263.) Davies SM, Szabo E, Wagner JE, Ramsay NK, Weisdorf DJ. Idiopathic hyperammonemia: a frequently lethal complication of bone marrow transplantation. Bone Marrow Transplant 1996; 17:1119-25.
(264.) Xu SR, Yao EG, Dong ZR, Liu RS, Pan L, Lin FR, et al. Plasma ammonia in patients with acute leukemia. Chin Med J 1992; 105:713-6.
(265.) Altunbasak S, Baytok V, Tasouji M, Herguner O, Burgut R, Kayrine L. Asymptomatic hyperammonemia in children treated with valproic acid. J Child Neurol 1997;12:461-3.
(266.) Shepard RL, Kraus SE, Babayan RK, Sirosky MB. The role of ammonia toxicity in the post transurethral prostatectomy syndrome. Br J Urol 1987;60:349-51.
 Nonstandard abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; NACB, National Academy of Clinical Biochemistry; AASLD, American Association for the Study of Liver Diseases; HAV, hepatitis A virus; HDV, hepatitis D virus; HEV, hepatitis E virus; ALT, alkaline aminotransferase (EC 220.127.116.11); AST, aspartate aminotransferase (EC 18.104.22.168); ALP, alkaline phosphatase (EC 22.214.171.124); GGT, [gamma]-glutamyltransferase (EC 126.96.36.199); PT, prothrombin time; P-5'-P, pyridoxal-5'-phosphate; CAP, College of American Pathologists; CLIA, Clinical Laboratory Improvement Act of 1988 amendments; 181, international sensitivity index; INR, international normalized ratio; HBsAg, hepatitis B virus surface antigen; HBcAg, hepatitis B virus core antigen; HBeAg, hepatitis B virus e antigen; RIBA, recombinant immunoblot assay; SOD, superoxide dismutase (EC 188.8.131.52); EIA, enzyme immunoassay; and FDA, Food and Drug Administration.
D. ROBERT DUFOUR,  * JOHN A. LOTT,  FREDERICK S. NOLTE,  DAVID R. GRETCH,  RAYMOND S. KOFF,  and LEONARD B. SEEFF 
 Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Washington, DC 20422, and Department of Pathology, George Washington University School of Medicine, Washington, DC 20037.
 Department of Pathology, The Ohio State University College of Medicine, Columbus, OH 43210.
 Departments of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322.
 Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA 98104-2499.
 Department of Medicine, University of Massachusetts Medical Center, Worchester, MA 06155.
 Hepatitis C Programs, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, and Georgetown University School of Medicine, Washington, DC 20037.
An Approved Guideline of the National Academy of Clinical Biochemistry "Laboratory Medicine Practice Guidelines" and the American Association for the Study of Liver Diseases.
Presented in part at the American Association for Clinical Chemistry Annual Meeting, July 25-26, 1999, New Orleans, LA.
A complete monograph of these guidelines will be published by the National Academy of Clinical Biochemistry. Reprints are not available from the authors.
* Address correspondence to this author at: Pathology and Laboratory Medicine Service-113, VA Medical Center, 50 Irving Street NW, Washington, DC 20422. Fax 202-745-8284; e-mail firstname.lastname@example.org.
Table 1. AASLD categories reflecting evidence supporting guidelines (A-E) and quality of evidence on which recommendation is based (I-IV). Category Explanation I Evidence from multiple well-designed randomized controlled clinical trials, each involving a number of patients to be of sufficient statistical power II Evidence from at least one large well-designed clinical trial with or without randomization, from cohort or case-controlled analytical studies, or well-designed metaanalysis III Evidence based on clinical experience, descriptive studies, or reports of expert committees IV Not rated A Survival benefit B Improved diagnosis C Improvement in quality of life D Relevant pathophysiologic parameters improved E Impacts cost of healthcare Table 2. Performance specifications and precision for liver tests (%). Source Type ALT AST Performance specifications CLIA Mandate TE (a) = 20 TE = 20 European (5) Biological I = 13.6 I = 7.2 variation B=13.6 B=6.2 TE=36 TE=18 Ricos et Biological I = 12.2 I = 6.0 al. (215) variation B=12.2 B=5.4 TE=32 TE=15 Skendzel et Clinician NS TE = 26 al. (35) opinion Within-laboratory imprecision, % Lott et al. (13) Proficiency tests 8 9 Ross et al. (216) Proficiency tests NS NS Source Type ALP GGT Performance specifications CLIA Mandate TE = 30 European (5) Biological I = 3.4 NS variation B=6.4 TE=12 Ricos et Biological I = 3.2 I = 6.9 al. (215) variation B=6.4 B=10.8 TE=12 TE=22 Skendzel et Clinician NS NS al. (35) opinion Within-laboratory imprecision, % Lott et al. (13) Proficiency tests 5 6 Ross et al. (216) Proficiency tests NS NS Source Type Albumin Bilirubin Performance specifications CLIA Mandate TE = 10 TE = 20 or 0.4 mg/dL European (5) Biological I = 1.4 I = 11.3 variation B=1.1 B=9.8 TE=3.4 TE=28 Ricos et Biological I = 1.6 I = 12.8 al. (215) variation B=1.3 B=10 TE=3.9 TE=31 Skendzel et Clinician NS TE = 23 al. (35) opinion Within-laboratory imprecision, % Lott et al. (13) Proficiency tests NS NS Ross et al. (216) Proficiency tests 4.40 8.9 (a) TE, total error; I, imprecision or degree of reproducibility; B, bias or difference from correct result; NS, not specified. Table 3. Factors affecting AST and ALT activity, other than liver injury. Factor AST ALT Reference(s) Time of day 45% variation 217, 218 during day; highest in afternoon, lowest at night Day-to-day 5-10% variation 10-30% 219-221 from one day variation from to next one day to next Race/gender 15% higher in 222 African-American men BMI (a) 40-50% higher 40-50% higher with 21, 223-225 with high BMI high BMI Meals No effect No effect 21 Exercise Threefold increase 20% lower in those 225-228 with strenuous who exercise at exercise usual levels than in those who do not exercise or exercise more strenuously than usual Specimen Stable at room Stable at room 229-233 storage temp for 3 days, temperature for in refrigerator 3 days, in for 3 weeks (<10% refrigerator decrease); stable for 3 weeks for years frozen (10-15% (10-15% decrease) decrease); marked decrease with freezing/thawing Hemolysis, Significant Moderate increase hemolytic increase attributable to anemia release from red cell Muscle Significant Moderate increase 228 injury increase Other Macroenzymes Macroenzymes 23, 235 Factor AST Comments Time of day No significant difference between 0900 and 2100; similar in liver disease and health Day-to-day 5-10% variation Similar in liver from one day disease and health, to next and in elderly and young Race/gender 15% higher in No significant African-American difference between men African-American, other women BMI (a) 40-50% higher Direct with high BMI relationship between weight and AST, ALT Meals No effect Exercise Threefold increase Effect of exercise seen with strenuous predominantly in men; minimal exercise difference in women (,10%). Enzymes increase more with strength training Specimen Stable at room Stability based on serum storage temp for 3 days, separated from cells; stable in refrigerator for 24 h in whole blood, for 3 weeks (<10% marked increase after 24 h decrease); stable for years frozen (10-15% decrease) Hemolysis, Significant Dependent on degree of hemolytic increase hemolysis; usually severalfold anemia lower than increases in LDH Muscle Significant Related to amount injury increase of increase in CK Other Macroenzymes Typically stable increase, affects only AST or ALT Table 4. Factors affecting ALP activity, other than liver injury. Factor Change Reference(s) Comments Day-to-day 5-10% 219-221 Similar in liver disease and health, and in elderly and young Food ingestion Increases as 236-239 In patients much as 30 U/L of blood groups B and O; remains increased up to 12 h; attributable to intestinal isoenzyme Race/gender 15% higher in 222 African-American men; 10% higher in African-American women BMI (a) 25% higher with 239 increased BMI Exercise No significant 225, 227 effect Specimen Stable for up 231 storage to 7 days in refrigerator, months in freezer Hemolysis Hemoglobin inhibits 240 enzyme activity Pregnancy Increases up to 241 Attributable two-to three-fold to placental in third trimester isoenzyme Smoking 10% higher 222, 239 Oral 20% lower 242 contraceptives Other High in bone disease, 240 Can be tumors producing separated ALP; low after from liver severe enteritis causes by (in children)and ALP in hypophosphatasia isoenzymes and/or normal GGT (a) BMI, body mass index. Table 5. Factors affecting GGT, other than liver injury. Factor Change Reference(s) Comments Day-to-day 10-15% 219-221 Similar in liver disease and health, and in elderly and young Race Approximately double 222 Similar in African Americans differences in adult males, females BMI (a) 25% higher with mild 223 Effect similar increase in BMI; in adult males, 50% higher with females BMI .30 Food Decreases after 243 ingestion meals; increases with increasing time after food ingestion Exercise No significant 243 effect Specimen Stable for up 240 storage to 7 days in refrigerator, for months in freezer Pregnancy 25% lower 244, 245 during early pregnancy Drugs Increased by 246 Values up to carbamazepine, 2 times cimetidine, reference furosemide, limits are heparin, common, may be isotretinoin, up to 5 times methotrexate, reference oral contraceptives, limits, phenobarbital, especially phenytoin, with phenytoin valproic acid Smoking 10% higher with 243 1 pack/day; approximately double with heavier smoking Alcohol Direct relationship 243, 247-249 May remain consumption between alcohol increased for intake and GGT weeks after cessation of chronic and alcohol intake (a) BMI, body mass index. Table 6. Factors affecting bilirubin, other than liver injury. Factor Change Day-to-day 15-30% Food ingestion Bilirubin increases an average of one- to twofold with fasting up to 48 h Race 33% lower in African-American men, 15% lower in African-American women Exercise 30% higher in men Light exposure Up to 50% decrease in 1 h Pregnancy Decreases 33% by second trimester Hemolysis Cross-reacts in some assays Oral contraceptives 15% lower Hemolytic anemia Increases in unconjugated bilirubin Factor Reference(s) Comments Day-to-day 219 Food ingestion 250, 251 Averages 20-25% higher after overnight fast than after meals Race 222, 252 Compared with values in other racial/ethnic groups Exercise 227 No significant effect in women Light exposure 87 Affects unconjugated bilirubin more than direct-reacting bilirubin Pregnancy 241 Similar in second, third trimester Hemolysis 240 Hemoglobin absorbs light at the same wavelength as bilirubin, falsely increasing results Oral contraceptives 242 Hemolytic anemia 240 Table 7. Factors affecting PT. Factor Change Reference(s) Specimen No change at room temperature 121, 253-255 storage up to 3 days; refrigeration falsely shortens PT Citrate 3.2% citrate minimizes 121 concentration problems compared with 3.8% citrate Inadequate tube Falsely increases PT 256, 257 filling High hematocrit Falsely increases PT 256, 257 Other factors Warfarin, malabsorption, vitamin K deficiency, drugs that decrease vitamin K production (especially antibiotics, fibric acid derivatives), and consumptive coagulopathy increase PT Table 8. Factors affecting [NH.sub.3], other than liver injury. Factor Change Age Four- to eightfold higher in neonates; two- to threefold higher in children <3 years; reaches adult concentrations by adolescence Specimen Arterial higher than venous; source differences greater in renal, hepatic disease; capillary blood falsely increased because of [NH.sub.3] in sweat if skin inadequately cleaned Exercise Increases up to threefold after exercise Smoking Increases 10 mmol/L after 1 cigarette Delay in analysis [NH.sub.3] increases because of metabolism by red blood cells: 20% in 1 h and 100% by 2 h Other factors Increased in acute leukemia, blood transfusion, bone marrow transplantation, portal-systemic shunts, gastrointestinal bleeding, or high protein intake Medications Valproic acid and glycine (in irrigation fluids used in prostate, endometrial resection) increase [NH.sub.3] production Factor Reference(s) Age 258 Specimen 131, 138, 259 source Exercise 260 Smoking 138 Delay in analysis 139, 261, 262 Other factors 263, 264 Medications 265, 266 Factor Comments Age Specimen Arterial [NH.sub.3] correlates better with change source in liver function than venous [NH.sub.3]; tourniquet use, clenching fist increase venous [NH.sub.3] Exercise Increase greater in males than in females Smoking Delay in analysis Use of ice water, rapid centrifugation, and separation of plasma minimize increases; rate of increase higher in liver disease because of high GGT activity in specimens, releasing [NH.sub.3] from peptides Other factors Medications Table 9. Serologic diagnosis of HBV infections. (a) Marker Incubation Acute Past infection infection HBsAg + (b) + - HBeAg + + - HBV DNA + + [+ or -] (c) Anti-H Bc IgM - + - Total - + + Anti-HBe - - [+ or -] Anti-HBs - - + Marker Chronic Vaccination infection HBsAg + - HBeAg [+ or -] - HBV DNA + - Anti-H Bc IgM [+ or -] (d) Total + - Anti-HBe [+ or -] (e) - Anti-HBs - + (a) Modified from Chernesky et al. (163). (b) +, detectable; -, not detectable; [+ or -], may be detectable. (c) PCR methods. (d) May be positive in 10-15% patients with reactivation of infection. (e) Patients with chronic HBV infection usually have detectable HBeAg or anti-HBe. Table 10. Discordant or unusual hepatitis B serologic profiles requiring further evaluation. * HBsAg positive/anti-HBc negative * HBsAg, anti-1-113s, and anti-HBc positive * Anti-HBc positive only * Anti-HBs positive only in a nonimmunized patient * HBsAg negative/HBeAg positive * Positive for HBeAg and anti-HBe * Total anti-HBc negative/IgM anti-HBc positive Table 11. Lower detection limits of HBV DNA assays. Method Detection limit, copies/mL (a) Hybrid capture 3.0 x [10.sup.6] Branched DNA 0.7 x [10.sup.6] Liquid hybridization 4.0 x [10.sup.4] PCR [10.sup.2]-[10.sup.3] (a) Results can also be expressed as ng/L of HBV DNA by dividing by 2.85 x [10.sup.5]
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||National Academy of Clinical Biochemistry|
|Author:||Dufour, D. Robert; Lott, John A.; Nolte, Frederick S.; Gretch, David R.; Koff, Raymond S.; Seeff, Le|
|Article Type:||Clinical report|
|Date:||Dec 1, 2000|
|Next Article:||Diagnosis and monitoring of hepatic injury. II. Recommendations for use of laboratory tests in screening, diagnosis, and monitoring.|