Commutability Assessment of Candidate External Quality Assessment Materials for Aminotransferase Activity Measurements Based on Different Approaches in China.

BACKGROUND: Using commutable external quality assessment (EQA) materials is important for monitoring successful harmonization efforts. We assessed the commutability of four human serum pool (HSP) preparations to identify candidate EQA materials for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity measurement. METHODS: One set each of 85 clinical samples (CSs) was collected for ALT and AST activity measurement. The 15 candidate EQA materials included four types of HSP preparations (A to D): materials A, C, and D contained human original recombinant (HOR) aminotransferases; materials B was mixed leftover samples. The CSs and 15 candidate EQA materials were analyzed using seven routine assays, and the ln-transformed results were analyzed in 21 assay pairs. Commutability was assessed using Deming regression, with a 95% prediction interval (CLSI approach) and the difference in bias with an error component model (International Federation of Clinical Chemistry and Laboratory Medicine [IFCC] approach). RESULTS: For ALT, all materials were commutable for 14-21 assay pairs according to the CLSI and IFCC approaches. For AST, B01-03 showed commutability for 14-21 assay pairs, and C01-03 and D01-03 showed commutability for no less than 10 assay pairs according to the two approaches. A01-06 were commutable for 9-16 assay pairs according to the CLSI approach, but for 6-9 assay pairs according to the IFCC approach. CONCLUSIONS: Mixed leftover samples showed desirable commutability characteristics as candidate EQA materials for routine aminotransferase activity measurements. Human serum bases supplemented with HOR were commutable for most routine ALT activity measurements.

New trimester-specific reference intervals for clinical biochemical tests in Taiwanese pregnant women-cohort of TMICS.

BACKGROUND: Because there are no published biochemical reference intervals (RI) for pregnant Taiwanese women, we used an established islandwide birth cohort, the Taiwan Maternal and Infant Cohort Study, to establish RIs for important biochemical parameters in women during their 3rd trimester in Taiwan. Additionally, we compared the differences in these biochemical parameters between early third trimester (weeks 28 to 31) and late third trimester (weeks 37 to 40) of pregnant women as well as the differences in them between the third trimester and after delivery. METHODS: Between 2012 and 2015, we recruited a total of 2,136 pregnant women from nine hospitals located in northern (n = 3), central (n = 3), southern (n = 2), and eastern Taiwan (n = 1) to receive regular prenatal health examinations during their third trimester (weeks 28 to 40). After exclusion, samples obtained from 993 eligible pregnant women were analyzed. RESULTS: There were increases in both lower and upper normal limits for blood neutrophil, thyroid profile (triiodothyronine (T3) and thyroxine (T4)), testosterone, estradiol, and progesterone and decreases for RBC, hemoglobin (Hb), alanine aminotransferase (ALT) and creatinine (Cr) during their third trimesters. Women in their late third trimester (n = 378) had higher median RBC, Hb, aspartate aminotransferase (AST), Cr, thyroid-stimulating hormone (TSH), testosterone, estradiol, and progesterone and lower median platelet and insulin, compared with those in their early third trimester (n = 490). Twenty-three of the women had both third trimester and post-pregnancy data. After delivery, the women had lower median AST, ALT, insulin, T3, T4, testosterone, estradiol, and progesterone and higher median Cr, free T4, FSH, and luteinizing hormone (LH), compared to their third trimesters. CONCLUSIONS: Gestation-related changes in important biochemical parameters should be considered when evaluating clinical laboratory values in pregnant women.

The potential of reducing AST testing in hospital settings.

BACKGROUND: Aspartate aminotransferase (AST) is regularly ordered with alanine aminotransferase (ALT) to assess liver integrity. In many situations, AST testing provides little or no added clinical value, since ALT is more specific and the both enzyme activities highly correlate. The objective of this study is to determine the potential reduction in AST testing, if not performed when ALT results are within reference intervals (RI). METHODS: Results for patients >18 years of age for both AST and ALT from the same specimen were obtained for the period January 1, 2017 - December 31, 2017. We calculated frequency of AST and ALT results that had various combinations of results within and above the RI. We also investigated the clinical locations of origin for the samples. RESULTS: In total 87,704 paired samples with both AST and ALT test results were recovered. The total of 73.2% of AST tests for males and 66.9% for females would be eliminated if we performed AST testing only when ALT was increased. However, 7.4% of elevated AST tests would be missed for males and 3.8% for females due to ALT being within limits. Specifically in the outpatient clinics, 79% male and 73% females paired enzyme results were within RI. Only 4% of males and 3% of females had paired results where ALT was within RI while AST > RI. CONCLUSIONS: The rate of test results with increased AST while ALT is within the RI is low enough to recommend limiting AST testing only to cases where ALT is above the RI. Our recommendation for AST restriction is to begin with the hospitals outpatient clinics.

An Assessment of Upper Limits of Normal for ALT and the Impact on Evaluating Natural Course of Chronic Hepatitis B Virus Infection in Chinese Children.

BACKGROUND: The current upper limits of normal (ULN) for serum alanine aminotransferase (ALT) are increasingly challenged. We aimed to re-evaluate the ULN for ALT and assess the potential impact on the classification of natural course of chronic hepatitis B virus (HBV) infection in children. METHODS: Laboratory data obtained from three hospitals in China were retrospectively analysed. In total, 2054 children with chronic HBV infection and 8149 healthy children at age ≤18 years were included in the study. RESULTS: Age-specific and gender-specific ULNs for ALT, at averages of 30 U/L for boys and 24 U/L for girls, were calculated from the data of healthy children. Using the revised ULNs vs. the current ULNs (40-50 U/L), 31-60% vs. 9-17% of the 2054 HBV-infected children had an abnormal result as seen in their ALT baseline analysis, and the highest abnormality rate was seen in the infants. Data of 516 HBV-infected children were applied for the classification of clinical phase, 28.8% vs. 19.8% of the children were classified into the phases of hepatitis B e antigen (HBeAg-)positive/negative hepatitis. During a median follow-up of 62 months, 39 of 153 children underwent HBeAg seroconversion, whereas 3 of them had persistently "normal" ALT, according to the current ULN. CONCLUSIONS: The revision of ULN for ALT in children substantially impacts the classification of the natural course of chronic HBV infection. Mild ALT fluctuation is common during the stage childhood, suggesting a need to rethink the current conceptions of immune tolerance and natural course of chronic HBV infection in the children.

Updated thresholds for alanine aminotransferase do not exclude significant histological disease in chronic hepatitis C.

BACKGROUND AND AIM: Histological changes in hepatitis C virus (HCV)-infected patients with persistently normal alanine aminotransferase (PNALT) have not been evaluated for updated upper limits of normal (ULN; ≤ 19/30 U/L for females/males). We assessed significant fibrosis (≥ F2, METAVIR) in patients with PNALT and persistently elevated alanine aminotransferase (PEALT). PATIENTS AND METHODS: Nine hundred and twenty consecutive, unselected HCV patients were stratified into four groups: Group I: (n = 124) PNALT within the updated ULN [0.5 × ULN (corresponding to ≤ 19 U/L) for females; 0.75 × ULN (corresponding to ≤ 30 U/L) for males]; Group II (n = 173): PNALT ≤ 1 × ULN but greater than Group I; Group III (n = 313): PEALT 1-2 × ULN; and Group IV (n = 310): PEALT > 2 × ULN. PNALT was defined as ≥ 3 determinations within the normal range over ≥ 6 months. RESULTS: Advanced ≥ F3 and ≥ F2 fibrosis increased incrementally across Groups I; II; III; and IV: 24.2 and 45.2%; 25.4 and 56.1%; 36.1 and 64.2%; and 50 and 77.1% respectively (P<0.0001 for both). Multivariable logistic regression analysis identified age [odds ratio (OR), 1.05; 95% confidence intervals (CI): 1.02-1.08; P<0.0001], alanine aminotransferase (ALT) groups (OR 1.38; 95% CI: 1.03-1.83; P = 0.030), presence of moderate-severe steatosis (OR 2.70; 95% CI: 1.19-6.15; P = 0.018) and ≥ A2 necroinflammation (OR 17.9; 95% CI: 8.88-36.20; P < 0.0001) as independent predictors of ≥ F2 fibrosis. Updated ULN for ALT were better at excluding ≥ F2 fibrosis compared with traditional ULN (90.6 vs. 74.2%, P = 0.0041) but less specific (20.8 vs. 44%, P = 0.0007) with similar positive/negative predictive values. CONCLUSIONS: HCV patients with 'updated' normal ALT have the lowest prevalence of significant fibrosis, although utilizing these levels without resorting to biopsy would miss significant fibrosis in almost one-half of such patients.

Case definition and phenotype standardization in drug-induced liver injury.

Drug-induced liver injury (DILI) is the most frequent reason cited for the withdrawal of approved drugs from the market and accounts for up to 15% of the cases of acute liver failure. Investigators around the globe have begun to identify and study patients with DILI; several large registries and tissue banks are being established. In order to gain the maximum scientific benefit from these efforts, the definitions and terminology related to the clinical phenotypes of DILI must be harmonized. For this purpose, an international DILI Expert Working Group of clinicians and scientists reviewed current DILI terminology and diagnostic criteria so as to develop more uniform criteria that would define and characterize the spectrum of clinical syndromes that constitute DILI. Consensus was established with respect to the threshold criteria for definition of a case as being DILI, the pattern of liver injury, causality assessment, severity, and chronicity. Consensus was also reached on approaches to characterizing DILI in the setting of chronic liver diseases, including autoimmune hepatitis (AIH).

Enhancing the utility of alanine aminotransferase as a reference standard biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specifically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualification model.

Intermethod calibration of alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) results: application to Fibrotest and Actitest scores.

Two multi-component scores (Fibrotest and Actitest) have been proposed to evaluate liver fibrosis or necro-inflammatory lesions as an alternative to liver biopsy. This approach requires standardization of alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) determinations. For this purpose, ALT and GGT values were assigned to a multi-enzyme material using the appropriate primary reference procedure. This material was used as a common calibrator for sera from 20 patients with viral hepatitis. Measurements were carried out in 11 laboratories, using their own automated routine methods and compared to results obtained using the primary reference procedure. The expression of results in multiples of the upper reference limit worsened the inter-laboratory variation for both enzymes. The multi-enzyme material was commutable for ALT and GGT determination carried out with six analytical systems. Common calibration significantly improved inter-laboratory consistency, which finally reached 1.8% and 3.3% for ALT and GGT, respectively. For each enzyme, it also permitted the retention of a common reference interval for a set of calibrated methods and the improvement of inter-laboratory coherency of Fibrotest and Actitest scores.

Metrological traceability of values for catalytic concentration of enzymes assigned to a calibration material.

BACKGROUND: The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. METHODS: A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. RESULTS: Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. CONCLUSION: The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.

New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects.

Consensus among clinical chemists has dictated a change in reference temperature for enzyme catalytic concentrations from 30 to 37 degrees C. Consequently, International Federation of Clinical Chemistry (IFCC) reference procedures have been redefined at the latter temperature. Acceptance in practice of these new procedures requires well-established reference values and clinical decision limits, but the establishment of reference values is complex. Therefore, as a provisional approach and to facilitate early application of the new IFCC procedures, we report our experience gained with them in the transfer of values from the consensus methods used hitherto in Germany to the new procedures. The preliminary upper reference limits were determined for catalytic activity concentrations of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) in human sera. Since enzyme measurements are almost always made on sera from non-ambulant subjects, we have used hospital patients aged 17 years and older as the subjects of our study. The catalytic activity concentrations obtained by measurements with the German consensus methods for the respective enzyme were chosen in combination with additional enzymes of similar diagnostic relevance to classify patients' samples as part of the respective reference collective. Measurements for the determination of the upper reference limits were performed manually by use of the primary reference procedures at the measurement temperature 37 degrees C according to IFCC, and also by employing mechanized measurements adapted to the reference procedures. The upper reference limits were calculated as the 97.5th percentile of the reference collectives and determined separately for women and men: ALT: 34 U/l (female) and 45 U/l (male); AST: 31 U/l (female) and 35 U/l (male); CK: 145 U/l (female) and 171 U/l (male); gamma-GT: 38 U/l (female) and 55 U/l (male); LDH: 247 U/l (female) and 248 U/l (male).

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.

This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase accord.

This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat/l (186 U/l +/- 4 U/l), for alanine aminotransferase and 1.68 microkat/l +/- 0.07 microkat/l (101 U/l +/- 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.

Factors associated with serum alanine transaminase 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.

In clinical research, the definition of the upper limit of normal (ULN) alanine transaminase (ALT) is never detailed. However, such a definition can vary and may have practical consequences. Our aim was to assess factors associated with serum ALT activity in apparently healthy subjects and then to apply seven different definitions of ULN in three different populations so as to assess the prevalence of subjects with normal ALT among blood donors and among hepatitis C patients before (normal ALT hepatitis C patients) and after treatment (interferon [IFN] responders). ALT measurements were performed in the same laboratory using the same technique; 1,033 donors were prospectively investigated, 186 patients with hepatitis C never treated and 40 patients treated with 3 MU three times per week of IFN-alpha for at least 6 months. The seven definitions (D) of ULN were: D1: 95th percentile of ALT; D2: 95th percentile after separating males and females; D3: males and females separately, ULN=10 (mean of log10 ALT + 1.96 SD); D4: ULN=45 IU/L given by the manufacturer; D5: mean + 1 SD after exclusion of the 5% extreme values; D6: 95th percentile after separating subjects with body mass index (BMI) under or equal to the median (23); and D7: 95th percentile after separating subjects according to BMI and sex. BMI and male sex were independently associated (P < .0001; logistic regression) with ALT, without an association with alcohol. The range of ULN varied from 26 IU/L in females (D5) to 66 IU/L in males with BMI >23 (D7). Depending on the definition, the prevalence of blood donors with normal ALT varied from 82% to 96%, i.e., a range of 14%; that of hepatitis C patients with normal ALT varied from 16% to 27%, i.e., a range of 11%; the prevalence of IFN responders varied from 25% to 42%, i.e., a range of 17%. Definitions of normal ALT values should be adjusted for sex and BMI to reduce artificial heterogeneity in blood donor selection and in hepatitis C clinical studies.

Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material.

We present the results of a study on performance of a reference material for alanine aminotransferase (ALT, EC 2.6.1.2) and the corresponding IFCC-approved method in an interlaboratory trial involving 13 laboratories. The ALT material was partly purified from pig heart (specific activity, 150 kU/g) and was essentially free of six potentially contaminating enzyme activities, including aspartate aminotransferase (EC 2.6.1.1). The partly purified ALT was lyophilized in a triethanolamine-buffered matrix, pH 6.4, containing bovine serum albumin and saccharose. Under these conditions, the predicted yearly loss of activity was 0.02% at 4 degrees C and < 0.01% at -20 degrees C. The final blank-corrected results of the accepted set of data gave a mean (SD) of 128.5 (5.1) U/L. The among-laboratory SD was 4.6 U/L and the within-laboratory SD was 2.0 U/L. The certified ALT catalytic concentration in the reconstituted material was 129 U/L with a 0.95 confidence interval of +/- 4 U/L.

Mathematical model for comparing proficiency testing results across analytical methods.

Proficiency testing (PT) programs may fail to establish a range of acceptable performance in a challenge when an insufficient number of participants use a particular method. In this study I analyzed a mathematical approach to establish acceptable performance in alanine aminotransferase (ALT; EC 2.6.1.2) PT challenges. This approach was derived from the mathematical model used in establishing a nationwide ALT standardization system for blood-collection facilities in 1988. A ratio of results was derived between each method in the PT program and an arbitrarily chosen "standard" method. The intent of this approach was to transform a target value for a challenge as determined in the "standard" method to units applicable to methods utilized less often. However, the high degree of variability over time in the ratios for some methods precluded general application of this approach. Therefore, although this simple mathematical method was successful in implementing an ALT standardization system across different methods, a derivation of this approach did not afford comparison of results in PT challenges.