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Background: FibroTest, a noninvasive method of measuring biomarkers of liver fibrosis, is an alternative to liver biopsy for determining the severity of chronic hepatitis C virus (HCV) infection. We compared the 5-year prognostic value of the FibroTest with biopsy staging for predicting cirrhosis decompensation and survival in patients with chronic HCV infection., Methods: Fibrosis stage was assessed on the same day by FibroTest and biopsy in a prospective cohort of 537 patients. Disease classification at baseline was 157 patients with severe fibrosis (FibroTest >0.58), 137 with moderate fibrosis (FibroTest 0.32-0.58), and 243 with no or minimal fibrosis (FibroTest <0.32)., Results: In 64 untreated patients with severe fibrosis, survival without HCV complications was 73% [95% confidence interval (CI), 59%-086%; 13 complications], and survival without HCV-related death was 85% (95% CI, 73%-96%; 7 HCV deaths). Survival rates were higher in patients with moderate fibrosis, [99% (95% CI, 97%-100%; 1 complication; P <0.001) and 100% (no HCV death; P <0.001) for patients with and without HCV-related complications, respectively], and in patients with minimal fibrosis [100% (no complication; P <0.001 vs severe) and 100% (no HCV death; P <0.001 vs severe), respectively]. FibroTest was a better predictor than biopsy staging for HCV complications, with area under the ROC curves (AUROC) = 0.96 (95% CI, 0.93%-0.97%) vs 0.91 (95% CI, 0.85%-0.94%; P = 0.01), respectively; it was also a better predictor for HCV deaths: AUROC = 0.96 (95% CI, 0.93%-0.98%) vs 0.87 (95% CI, 0.70%-0.94%; P = 0.046), respectively. The prognostic value of FibroTest was still significant (P <0.001) in multivariate analyses after taking into account histology, treatment, alcohol consumption, and HIV coinfection., Conclusion: The FibroTest measurement of HCV biomarkers has a 5-year prognostic value similar to that of liver biopsy.

Background: Acetaminophen was falsely detected in the plasma of a severely jaundiced patient, and a methodologic interference from bilirubin was suspected., Methods: Acetaminophen was measured by an enzymatic method (GDS Diagnostics). The putative bilirubin interference was investigated in 12 hyperbilirubinemic specimens and in bilirubin linearity calibrators. The analytical method was modified to correct for background absorbance at a second wavelength. Hyperbilirubinemic specimens were fortified with acetaminophen to assess the effect of the interference on acetaminophen measurements., Results: Acetaminophen was detected in 12 specimens from hyperbilirubinemic patients without a history of recent acetaminophen exposure. Dilution of hyperbilirubinemic specimens produced a nonproportional decrease in apparent acetaminophen concentrations, and no acetaminophen was detected when bilirubin was <50 mg/L. Background correction at a second wavelength failed to compensate for the interference. Although erroneous acetaminophen concentrations were detected in all specimens with high bilirubin, acetaminophen measurements in fortified specimens were accurate., Conclusion: The data are consistent with bilirubin interference in the enzymatic and/or chromogenic reactions involved in the acetaminophen method.

Background: Biochemical liver function tests are widely used in the clinic and are some of the most frequently used tests in screening for diseases in older age groups. The aim of the present study was to estimate the relative importance of genetic and environmental factors to variations in these tests among the elderly., Methods: We conducted a survey among Danish twins, 73-102 years of age, identified in the population-based Danish Twin Registry. Among the 2749 individuals in the study population, an interview was conducted with 79%. From these, a blood sample was collected from 290 same-sex twin pairs, total of 580 subjects, within a 6-month period and analyzed for alanine aminotransferase (ALT), lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), bilirubin, and albumin. The interview included questions about alcohol consumption and body mass index (BMI; self-calculated height and weight). Heritability (proportion of the population variance attributable to genetic variation) was estimated using structural-equation analyses before and after correction for alcohol consumption and BMI., Results: Structural-equation analyses revealed a substantial heritability (35-61%) for the four biochemical liver function tests: ALT, GGT, LDH, and bilirubin. The remaining variation could be attributed to individuals' nonfamilial environments. Adjustment for alcohol consumption and BMI had no influence on the heritability for ALT, GGT, LDH, and bilirubin. For albumin, two models fit equally well before adjustment for alcohol and BMI: a model including additive genetic and nonshared environmental factors (AE), and a model including shared and nonshared environmental factors (CE). After adjustment, the model including shared and nonshared environment was clearly the best fitting model., Conclusions: For both males and females, the effect of genetic factors on the biochemical liver function tests ALT, GGT, LDH, and bilirubin is substantial and accounts for one-third to two-thirds of the variation among individuals 73-102 years of age. The heritability is equal for males and females and does not change notably after controlling for alcohol consumption and BMI. For albumin, no major impact of genetic factors was found independent of BMI and alcohol consumption. An understanding of the genetic mechanisms underlying biochemical liver function tests among the very old may be of value in the interpretation of these tests in this age group.

Background: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated., Methods: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation., Results: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination., Conclusions: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.

Background: LDL-cholesterol (LDL-C) concentrations currently are determined in most clinical laboratories using the Friedewald calculation. This approach has several limitations and may not always meet the current total error recommendation in LDL-C measurement of

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

We compared the effect of light on direct-reacting bilirubin (DBIL) measurement by the bilirubin oxidase (EC 1.3.3.5; BOX) method and by the Jendrassik-Gróf diazo method. DBIL concentrations determined by the BOX method in the sera of hyperbilirubinemic infants treated with phototherapy yielded falsely higher values than those by the direct diazo method. A similar tendency was noted when DBIL concentrations in infants' sera irradiated with light in vitro were determined by both methods, although by HPLC none of these sera had detectable DBIL (i.e., conjugated plus delta bilirubin). In general, DBIL concentrations after photoirradiation remained unchanged when measured by the diazo method, but significantly increased when the BOX method was used. Indeed, photoirradiation gave rise to material that acted like a photobilirubin product, which was oxidized at pH 3.7 and therefore was measured as DBIL. Such false increases in DBIL values generated by the BOX method may have clinical diagnostic implications in monitoring jaundiced neonates and in differentiating between physiological jaundice and incipient pathological jaundice.

Delta bilirubin (B delta), isolated from serum, has an absorption maximum near 440 nm and a molar absorptivity of 72,000 L mol-1cm-1 in either Tris HCl (0.1 mol/L, pH 8.5) or phosphate (0.13 mol/L, pH 7.4) buffer. This absorptivity exceeds by approximately 50% and 59%, respectively, that of unconjugated bilirubin in the same buffers. This finding suggests that substantial errors can be incurred in direct spectrophotometry of bilirubins in serum. In the total diazo (TBIL) assay (Clin Chem 1985;31:1779-89), the color yield from B delta increases by 10% as the final diazo concentration is increased from 0.27 to 0.81 mmol/L. In the direct (DBIL) assay, if done in HCl (50 mmol/L), B delta yields approximately 15% more color as the diazo concentration is increased from 0.51 to 1.53 mmol/L, whereas in acetate buffer (0.4 mol/L, pH 4.7) the corresponding color yield is 25% greater. However, the absolute color yield for the reaction in HCl exceeds that in acetate buffer. In both the TBIL and the DBIL assay, B delta reacts slowly, nearly complete reaction requiring 10 min. Thus, B delta may be seriously underestimated in diazo (especially DBIL) methods in which short reaction times (20 s to 1 min) are used.

A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.

I evaluated the diagnostic value of routinely ordered liver-function tests in 175 biopsy-proven cases of hepatic disease by use of stepwise discriminant analysis. The tests studied-total and "direct" bilirubin, alkaline phosphatase, lactate dehydrogenase, and aspartate aminotransferase-correctly classified 45-73% of cases, depending on the homogeneity of the diagnostic groups. Aspartate aminotransferase and alkaline phosphatase were the best discriminators. When all tests were used in the most homogeneous groups (tumors, cirrhosis, and hepatitis), there was a stepwise improvement in diagnostic accuracy from 51 to 73%.

We developed an enzymatic method for measuring direct-reacting bilirubin (DBIL) in serum. At pH 4.5, bilirubin oxidase (BOX) oxidizes mono-conjugated bilirubin, di-conjugated bilirubin, and most of the delta-bilirubin to biliverdin. The resulting decrease in absorbance at 460 nm is linearly related to the concentration of DBIL in serum. Mean DBIL values in the 51 patients' sera examined by the BOX method and a diazo procedure (Clin Chem 1982;28:2305) were 45.4 and 42.8 mg/L, respectively. For the same samples, mean values for DBIL and conjugated bilirubin by the Kodak "Ektachem" methods were 50.2 and 24.8 mg/L, respectively. Hemoglobin, up to 1.5 g/L, does not interfere. Unconjugated bilirubin reacts negligibly. Day-to-day CVs were 2.2% and 2.4% at DBIL concentrations of 37 and 74 mg/L, respectively.

This newly developed and highly specific and sensitive procedure was applied to the determination of unconjugated bilirubin and its ester conjugates in rat serum and human amniotic fluid. Bilirubin conjugates in biological samples are converted to methyl esters by alkaline methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are fractionated by "high-performance" liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2 mumol/L for unconjugated bilirubin, 0.07 to 34.6 mumol/L for the C-8 monoconjugate, 0.06 to 69.3 mumol/L for the C-12 monoconjugate, and 0.17 to 43.7 mumol/L for the di-conjugate fraction. The detection limit was 0.03 mumol/L for unconjugated bilirubin and for each monoconjugate, and 0.1 mumol/L for the di-conjugated pigment.

This paper describes a fast kinetic method for the simultaneous determination of unconjugated and conjugated bilirugin in the same reaction solution. A stopped-flow mixing system with a stabilized photometer and small computer is used to mix sample and reagent rapidly and to record 250 data points during a 700-ms reaction time, and a regression program is used to resolve these kinetic data into the concentrations of unconjugated and conjugated bilirubin. Data are reported for synthetic single and two-component samples and for serum samples. Kinetic results for synthetic mixtures and serum samples are compared with results obtained by a conventional two-step procedure. Regression equations show good linearity between kinetically determined absorbance changes and concentration, good agreement between taken and found values for total, unconjugated, and conjugated bilirubin for synthetic samples in human serum albumin, and a good correlation between kinetic and equilibrium results for these species in sera. Regression slopes for kinetic vs. equilibrium assay results for total, unconjugated, and conjugated bilirubins in sera were 1.01 +/- 0.05, 1.04 +/- 0.03 and 0.91 +/- 0.04, respectively, with intercepts of 6.6,--2.7, and 3.8 micromol/liter, and standard errors of estimate of 28, 14, and 20 micromol/liter. These data reflect uncertainties in both the kinetic and equilibrium methods.

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum. By contrast, the DBIL reaction in acetate buffer (pH 4.75) is quantitative for DTB in human serum (or its albumin), but less so in bovine serum (or its albumin). DTB is water soluble, is relatively stable when lyophilized with human serum, and is determined with such high precision in the above-mentioned endpoint assays that it may be a suitable surrogate calibrator for conjugated bilirubin.

Dexamethasone or prednisolone, added in vitro to bilirubin-containing amniotic fluid, produces a time-dependent decrease in the 450-nm absorbance of the pigment. Neither the chemical determination of bilirubin in amniotic fluid nor the lecithin/sphingomyelin ratio as determined by thin-layer chromatography is affected by these glucocorticoids. The effect probably is not a result of displacement of bilirubin from its binding sites on albumin, because the absorbance of a solution of crystalline bilirubin at 450 nm is unaffected by added bovine serum albumin. Light scattering of amniotic fluid increases slightly when dexamethasone is added, whether or not low concentrations of bilirubin (less than 1.6 mumol/L) are present. Thus the effect on absorbance evidently is not ascribable to supersaturation and formation of a colloidal sol of bilirubin particles. This direct interference of glucocorticoids with the spectrophotometry of bilirubin in amniotic fluid prompts cautious interpretation of such data as an index to the severity of hemolytic disease of the fetus, specifically in cases of Rhesus-isoimmunization that are being treated with glucocorticoids.

The EXPERT consultation system-building tool, a knowledge-based artificial intelligence program developed at Rutgers University, has been applied to the development of a laboratory consultation system facilitating sequential laboratory testing and interpretation. Depending on the results of a basic panel of laboratory tests, the system requests that specific secondary tests be performed. Input of these secondary findings can result in requests for tertiary testing, to complete the database necessary for interpretation. Interpretation of all results is based upon final inferences from the collected findings through a series of rules, a hierarchical network that yields an efficient production system not easily obtained through conventional programming. The rules included in this model are based upon initial results for total protein, calcium, glucose, total bilirubin, alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, thyroxin, hemoglobin, mean corpuscular volume, and the concentrations of four drugs. Pertinent clinical history items included are jaundice, diabetes, thyroid disease, medications, and ethanol. Implementing this system in a laboratory-based accelerated testing program involving outpatients maximized the effective use of laboratory resources, eliminated useless testing, and provided the patient with low-cost laboratory information.

We evaluated an enzymatic procedure for the automated determination of serum and urinary creatinine. The procedure was sensitive and precise for serum creatinine concentrations as low as 3 mg/L, and the standard curve was linear to 200 mg/L. Measurements of creatinine in clinical serum and urine specimens agreed with those obtained by two other enzymatic methods and four picric acid methods. There was no interference from picrate-reactive substances tested. Among other substances, bilirubin (50-100 mg/L) interfered negatively with the assay, decreasing apparent creatinine concentrations by as much as 6 mg/L. The advantages of specificity, speed, and convenience make this enzymatic procedure an attractive primary or secondary method for creatinine determinations in clinical laboratories.

We discuss the relative merits of absorption and first- and second-derivative spectra for the simultaneous quantification of bilirubin and hemoglobin, and evaluate single-, two-, and multiwavelength methods. Although both species can be quantified from single- or two-wavelength absorption data, lipids or other absorbing or light-scattering components introduce systematic errors that can be substantially decreased by using first- or second-derivative spectra. Multi-wavelength data-processing methods with derivative spectra permit quantification of components with overlapping spectra and decrease the random error usually associated with derivative methods. A typical least-squares equation for quantifying bilirubin in the presence of hemoglobin and bovine serum albumin from multi-wavelength second-derivative data is y(computed) = 0.999x(prepared) + 0.00 mg/L.

A colorimetric peroxidase-coupled procedure for determination of creatinine in human serum and urine is described. A 30-s sample pre-treatment with bilirubin oxidase eliminates interference from endogenous bilirubin. The 4-aminoantipyrine-2-hydroxy-3,5-dichlorobenzenesulfonate chromogen system of this method is about fourfold more sensitive than current procedures that involve monitoring NAD(P)H and nearly fivefold more sensitive than the traditional picrate procedures. Incorporation of a sample blank eliminates positive interference from endogenous creatine. Results of the proposed procedure are somewhat lower than those of the common kinetic or equilibrium picrate techniques, as would be expected because of the effects of the well-known interferences in the latter methods.

By means of gel-filtration of bilirubin/albumin mixtures, it is shown that unconjugated bilirubin remains completely linked to albumin (both human and bovine) in tetraborate buffer (pH 9.3), protein-free bilirubin appearing only when the bilirubin/albumin molar ratio exceeds two. On the other hand, bilirubin is completely set free from its protein link in the caffeine reagent. Additional chromatographic and spectrophotometric evidence is reported indicating the formation of a low-affinity complex between bilirubin and caffeine. These data explain why the spectrophotometric properties of bilirubin/albumin mixtures are matrix-dependent if measured in the tetraborate buffer but are no longer so when measured in the caffeine reagent. The relevance of these findings to the spectrophotometric "direct" assay of bilirubin in serum is discussed with reference to the occurrence of "delta-bilirubin" in pathological sera: this tightly protein-bound bilirubin fraction does not split in the presence of caffeine.

The hexokinase method (I) for glucose in biological fluids, proposed by the FDA as a potential national standard method, is compared with the Eyetone/Dextrostix/Control method (II) for measurement of glucose in whole blood. I used a new, two-step calibration procedure based on a 1.30 g/liter standard for the II system. The correlation coefficient for results by the two methods was 0.99 for 142 assays in duplicate. Recovery values were near 100% (I, range 100.0 to 106.2%; II, range 100.8 to 105.1%), and the coefficients of variation were respectively +/- 4.1% and +/- 4.5%. Considering the close correlation between the two methods, together with the simplicity, speed, and convenience of II, I conclude that it has great clinical utility, particularly in emergency situations. The precision and accuracy of II depends greatly on proper calibration and control procedures and on adequate training of the operator.