Validation of metrological traceability of the new generation of Abbott Alinity alkaline phosphatase assay.

OBJECTIVES: Recently, Abbott Diagnostics marketed a new generation of Alinity enzyme assays, introducing a multiparametric calibrator [Consolidated Chemistry Calibrator (ConCC)] in place of or in addition to factor-based calibrations. For alkaline phosphatase (ALP), both calibration options are offered, i.e., with ConCC (ALP2) and with an experimental calibration factor (ALP2F). Both options are declared traceable to the 2011 IFCC reference measurement procedure (RMP). Before to replace the old generation (ALP1) with the new one, we decided to validate the trueness of ALP2/ALP2F. METHODS: Three approaches were employed: (a) preliminary comparison on 48 native frozen serum samples with ALP1, of which traceability to RMP was previously successfully verified; (b) examination of three banked serum pools (BSP) with values assigned by RMP; (c) direct comparison with RMP on a set of 24 fresh serum samples. Bias estimation and regression studies were performed, and the standard measurement uncertainty associated with ALP measurements on clinical samples (uresult) was estimated and compared with established analytical performance specifications (APS). ConCC commutability was also assessed. RESULTS: A positive proportional bias was found with both ALP2 and ALP2F when compared to ALP1 and RMP. This positive bias was confirmed on BSP: in average, +13.1 % for ALP2 and +10.0 % for ALP2F, respectively. uresult were 13.28 % for ALP2 and 10.04 % for ALP2F, both not fulfilling the minimum APS of 4.0 %. Furthermore, ConCC was not commutable with clinical samples. CONCLUSIONS: Our results unearth problems in the correct implementation of traceability of Alinity ALP2/ALP2F, with the risk for the new assay to be unfit for clinical purposes.

A Hidden Gem: Highlighting the Indispensable Capabilities and History of the DoD Cholinesterase Monitoring Program and DoD Cholinesterase Reference Laboratory.

The DoD Cholinesterase Monitoring Program and Cholinesterase Reference Laboratory have safeguarded U.S. government employees in chemical defense for over five decades. Considering Russia's potential deployment of chemical warfare nerve agents in Ukraine, it is critical to maintain a robust cholinesterase testing program and its efficiency presently and in future.

The effect of the immunoassay curve fitting routine on bias in troponin.

OBJECTIVES: Unlike many dose-response curves used in clinical chemistry, the immunoassay curve used to quantitate measurands is often sigmoidal rather than linear. Consequently, a more complex curve fitting model is required. Various models are available, but they can introduce bias, and there can be little awareness of why this error can be introduced. CONTENT: These curve-fitting models include those based on the law of mass-action, empirical models such as splines or linearization models such as the log/logit function. All these models involve assumptions, which can introduce bias as the dose-response curve is 'forced' to fit or minimize the distance between the standard concentration points to the theoretical curve. The most common curve fitting model is the four or five parameter model, which uses four or five parameters to fit a sigmoidal curve to a set of standard points. SUMMARY AND OUTLOOK: Measurement of cardiac troponin is an important element in establishing a diagnosis of acute myocardial infarction. We use troponin, a cardiac biomarker, to demonstrate the potential effect of the bias that the curve fit could introduce. Troponin is used for both rule-in and rule-out decisions at different concentrations and at either end of the dose-response curve. The curve fitting process can cause lot-to-lot reagent (and calibrator) variation in immunoassay. However, laboratory staff need to be aware of this potential source of error and why it occurs. Understanding how the error occurs leads to a greater awareness of the importance of validating new reagent/calibrator assessment using patient samples with concentrations at crucial decision points.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

A Novel Fecal Elastase Assay for the Detection of Pancreatic Exocrine Insufficiency.

BACKGROUND: Fecal pancreatic elastase 1 (FPE1) is an established screening test for pancreatic exocrine insufficiency (PEI), a condition that is underdiagnosed and if not treated may cause significant morbidity. The aim of this study was to compare a new FPE1 machine based CLIA kit to an ELISA assay which is considered the de facto gold standard in our laboratory for FPE1 measurement. METHODS: Levels of FPE1 from the 227 stool samples were analyzed by the ScheBo ELISA kit and the CLIA Liaison XL system simultaneously with the same cutoff values for both assays. Performance of the Liaison XL system was assessed by calculating sensitivity, specificity, and accuracy. RESULTS: The comparison between the Liaison XL system performance and the ScheBo ELISA kit as reference revealed a sensitivity, specificity, and accuracy of 86.8%, 94.3%, and 92.1%, respectively, using a cutoff of 100 µg FPE1/g stool. When the cutoff is 200 µg FPE1/g stool the sensitivity, specificity, and accuracy were 86.6%, 97.1%, and 90.7%, respectively. Furthermore, linear correlation of FPE1 levels between the two assays were found to be significant by Pearson's correlation coefficient test (R = 0.85, p-values < 0.0001). CONCLUSIONS: The Liaison XL system showed good laboratory performance with our pre-determined cutoff values when compared to our previous assay. An important advantage of this system is its semi-automated mechanism that enables large scale analysis of FPE1. In addition to that, the Liaison XL system is ideal for both qualitative and quantitative analysis of FPE1 allowing for its application to the clinical setting.

Assessment of urine sample quality by the simultaneous measurement of urinary γ-glutamyltransferase and lactate dehydrogenase enzyme activities: possible application to unravel cheating in drugs of abuse testing.

OBJECTIVES: Evaluation of the simultaneous measurement of urinary γ-glutamyltransferase (γGT) and lactate dehydrogenase (LDH) to discriminate fresh from previously frozen specimens in urine drug monitoring. METHODS: Two widely available photometric tests (Siemens Healthineers Atellica) were used to determine the range of urinary γGT and LDH excretion and to study the decay in urinary enzyme activity under various storage conditions (room temperature, 4-8 °C, -18 °C, -80 °C). From these data, cut-off values were established and evaluated in split (fresh/frozen) specimens. RESULTS: Both assays allow robust, reliable, and simultaneous determination of urinary γGT and LDH. In healthy subjects, the 95% reference intervals for enzyme activity in native urine were γGT: 24.4-100.4 U/g Crea (creatinine) and LDH: 2.5-45.8 U/g Crea. Frozen storage for at least 7 days at -18 °C resulted in a loss of activity to less than 50% in both enzymes. Cut-offs for frozen samples were γGT≤33.2 U/g Crea and LDH≤ 8.4 U/g Crea. When applied to 100 sample pairs (fresh/frozen), 86.5% (173/200) of the measurements were conclusive and the combination of concordant enzyme measurements (low γGT/low LDH or high γGT/high LDH) was able to predict the mode of storage with a sensitivity of 96.3% and a specificity of 96.7%. CONCLUSIONS: The additional measurements of urinary γGT and LDH can be used to detect previously frozen urine specimens. A simple protocol is proposed to provide additional information on sample quality when deceit is suspected. The procedure can be easily integrated into the standard workflow of urinary drug monitoring.

Comparison of fecal calprotectin and pancreatic elastase assays based on proficiency testing results.

BACKGROUND: Fecal calprotectin and fecal pancreatic elastase assays are not standardized because of a lack of suitable reference material. Laboratories may have difficulty in switching assays because different manufacturers do not compare well with each other despite having similar reference intervals. Data from proficiency testing performed in Germany (Fecal Diagnostics 01 Survey, INSTAND eV) were investigated to understand how results differed across eight calprotectin and five pancreatic elastase manufacturers. METHODS: Data were collected from participating laboratories in external quality assessment schemes from 2015 to 2020 for calprotectin and 2017 to 2020 for pancreatic elastase. The manufacturer group mean values and standard deviations were calculated. Reference points were created for each external quality assessment scheme by calculating the average of all manufacturer group means. Deming regression analyses were used to observe the differences across manufacturers. RESULTS: The slopes of the Deming regression spanned 0.37-1.91 for calprotectin and 0.84-1.33 for pancreatic elastase. The calprotectin assays had a high degree of variability in quantitative results by manufacturer. However, pancreatic elastase assays appear to be harmonized across the different manufacturer when considering the qualitative interpretation. CONCLUSIONS: Both calprotectin and pancreatic elastase assays could be improved by standardization efforts. Given the clinical utility and our data demonstrating high inter-manufacturer variability, calprotectin should be prioritized over pancreatic elastase in standardization efforts.

A problem of proportions: estimates of metabolic associated fatty liver disease and liver fibrosis in Australian adults in the nationwide 2012 AusDiab Study.

Metabolic Associated Fatty Liver Disease (MAFLD) is the most common cause of liver disease in Australia, but prevalence data are limited. We aimed to describe the frequency of alanine aminotransferase (ALT) elevation, and MAFLD within a large prospective Australian cohort. Cross-sectional analysis of the 2012 survey of the Australian Diabetes, Obesity and Lifestyle (AusDiab) study which included 4747 Australian adults (aged 34-97 yrs) was performed. Frequency of ALT elevation (men ≥ 40 IU/L, women ≥ 30 IU/L) and MAFLD (Fatty Liver Index (FLI) > 60 alongside metabolic risk factors) was determined and risk of advanced fibrosis stratified using the BARD score. Elevated ALT was found in 13% of the cohort, including 22% of people with diabetes, 18% with obesity, and 17% with the metabolic syndrome. 37% of the cohort had MAFLD, and those with MAFLD were more likely to be older (OR 1.01 per 1 year (95% CI 1.00-1.02)), male (OR 1.37 (95% CI 1.17-1.59)), have ALT elevation (OR 3.21 (95% CI 2.59-3.99)), diabetes (OR 3.39 (95% CI 2.61-4.39)), lower HDL-C (OR 0.15 per 1 mmol/L (95% CI 0.12-0.19)), higher diastolic blood pressure (OR 1.05 per 10 mmHg (95% CI 1.05-1.06)), a sedentary lifestyle (OR 1.99 (95% CI 1.59-2.50)) and less likely to have tertiary education (OR 0.81 (95% CI 0.7-0.94) compared to those without MAFLD. Of those with MAFLD, 61% had a BARD score suggesting risk of advanced fibrosis and 22% had an elevated ALT. Over 10% of this Australian cohort had elevated ALT, and 37% had MAFLD, with many at risk for advanced fibrosis.

The association between elevated lipid profile and liver enzymes: a study on Bangladeshi adults.

Dyslipidemia, a major contributor to cardiovascular diseases, is rapidly increasing in Asian countries including Bangladesh. In addition to the cardiovascular system, abnormal lipid levels are also known to cause complications in renal and hepatic systems. The data regarding dyslipidemia and its relationship with liver enzymes are scarce for the Bangladeshi population. Therefore, this study was conducted to estimate the prevalence of dyslipidemia and determine the relationship between lipid profile and liver enzymes in Bangladeshi adults. A total of 405 participants (318 males and 87 females) were enrolled in the study. Serum levels of TG, TC, LDL, HDL and liver enzymes including ALT, AST, GGT and ALP were analyzed using standard methods. Dyslipidemia and liver function tests abnormalities were defined according to the international standard guidelines. The association between elevated lipid profile markers and liver enzyme abnormalities was assessed by logistic regression analysis. Overall, the prevalence of elevated TG, TC, LDL and low HDL were 30.9%, 23.7%, 26.2% and 78.8%, respectively. On the other hand, the prevalence of elevated liver enzymes ALT, AST, GGT and ALP were 18.8%, 21.6%, 12.9% and 21.9%, respectively. Dyslipidemia and liver enzyme abnormalities were higher in diabetic and hypertensive participants than in the healthy participants. About 61% of participants with dyslipidemia had at least one or more elevated liver enzymes. In regression analysis, an independent association was observed between serum GGT and all lipid components. In conclusion, a high prevalence of dyslipidemia and liver enzyme abnormalities were observed among the study participants. Of the four liver enzymes, the serum levels of GGT showed an independent association with all lipid components. Moreover, this study indicates that subjects with dyslipidemia often have a higher chance of having liver diseases than subjects with no dyslipidemia. However, large-scale prospective studies are needed to understand the underlying mechanisms of lipid-induced hepatic dysfunction in the Bangladeshi population.

Abnormal transaminase and lipid profiles in coexisting diseases in patients with fatty liver: a population study in Sichuan.

Among chronic liver diseases, fatty liver has the highest incidence worldwide. Coexistence of fatty liver and other chronic diseases, such as diabetes, hepatitis B virus (HBV) and Helicobacter pylori (Hp) infection, is common in clinical practice. The present study was conducted to analyze the prevalence and association of coexisting diseases in patients with fatty liver and to investigate how coexisting diseases contribute to abnormal transaminase and lipid profiles. We enrolled participants who were diagnosed with fatty liver via ultrasound in the physical examination center of West China Hospital. Multivariable logistic regression was used to determine the adjusted odds ratios (ORs). We found that 23.6% of patients who underwent physical examinations were diagnosed with fatty liver. These patients had higher risks of metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and hypertension and a lower risk of HBV infection. The risks of Hp infection and hyperthyroidism did not statistically differ. When fatty liver coexisted with T2DM, MetS and thyroid dysfunction, it conferred a higher risk of elevated transaminase. Fatty liver was positively correlated with triglycerides, cholesterol and low-density lipoprotein cholesterol (LDL-C) and negatively correlated with HBV; thus, HBV had a neutralizing effect on lipid metabolism when coexisting with fatty liver. In conclusion, patients with fatty liver that coexists with T2DM, MetS and thyroid dysfunction are more prone to elevated transaminase levels. Patients with both fatty liver and HBV may experience a neutralizing effect on their lipid metabolism. Thus, lipid alterations should be monitored in these patients during antiviral treatment for HBV.

Results of Early Transplantation for Alcohol-Related Cirrhosis: Integrated Addiction Treatment With Low Rate of Relapse.

BACKGROUND & AIMS: In 2018, our team initiated a prospective pilot program to challenge the paradigm of the "6-month rule" of abstinence for patients with alcohol-related liver disease (ALD) requiring transplant. Our pilot involved an in-depth examination of patients' alcohol use, social support, and psychiatric comorbidity, as well as the provision of pre- and post-transplantation addiction treatment. METHODS: Patients with ALD were assessed for inclusion in the pilot by a multidisciplinary team. Relapse prevention therapy was provided directly to all patients deemed to meet the program's inclusion criteria. Random biomarker testing for alcohol was used pre and post transplantation. RESULTS: We received 703 referrals from May 1, 2018 to October 31, 2020. After fulfilling the program's criteria, 101 patients (14%) were listed for transplantation and 44 (6.2%) received transplants. There were no significant differences in survival rates between those receiving transplants through the pilot program compared with a control group with more than 6 months of abstinence (P = .07). Three patients returned to alcohol use during an average post-transplantation follow-up period of 339 days. In a multivariate analysis, younger age and lower Model for End-Stage Liver Disease scores at listing were associated with an increased likelihood of a return to alcohol use (P < .05); length of abstinence was not a predictor. CONCLUSIONS: Our prospective program provided direct monitoring and relapse prevention treatment for patients with ALD and with less than 6 months of abstinence and resulted in a reduction of post-transplantation return to drinking. This pilot study provides a framework for the future of more equitable transplant care.

Dealing with redundant gamma glutamyl transpeptidase in primary care, when requested along with alkaline phosphatase.

The use of gamma glutamyl transpeptidase (GGT) levels as screening test for liver function is controversial. The GGT main utility is in cases in which alkaline phosphatase (ALP) is elevated. We aimed to investigate the request over time for alanine amino transferase (ALT), ALP and GGT, study the effect of a new demand management (DM) intervention for optimal GGT measurement in primary care. Our descriptive study was conducted from January 2010 to December 2020. The intervention was established in November 2019 and consisted of the laboratory information system would automatically remove GGT, if the test had been ordered simultaneously with ALP and there was no prior pathological result on record. We counted the absolute number of measured ALT, ALP and GGT, and calculated the ratios for each of the three markers related to creatinine, and GGT related to ALT in a monthly basis. The number of measured GGT increased slightly and progressively along the study until October 2019, when a decrease was observed. The ALT and ALP request from primary care also increased slightly along years. However, the GGT/ALT ratio never reached the 0.2 goal. Out of the 57,614 GGT requested in primary care patients, 38,167 (66.2%) were not measured. 7633.4€ were saved in reagent. The DM intervention to reduce the measurement of GGT when requested redundantly with ALP in primary care was successful, and the results have been maintained over time as observed by monitoring the GGT/CREA and GGT/ALT indicator results.

Indoleamine 2,3 dioxygenase (IDO) level as a marker for significant coronary artery disease.

BACKGROUND: Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) degradation, is modulated by inflammation, and is regarded as a key molecule driving immunotolerance and immunosuppressive mechanisms. Little is known about IDO activity in patients with active coronary artery disease (CAD). METHODS: We prospectively enrolled patients who were scheduled to undergo coronary angiography. Measurement of IDO, high-sensitivity troponin T (hs-TnT), and high-sensitivity C-reactive protein (hs-CRP) levels was performed at baseline, and IDO activity was monitored at the 6-month follow-up. RESULTS: Three hundred and five patients were enrolled. Ninety-eight patients (32.1%) presented with recent acute coronary syndrome (ACS). Significant difference in IDO, kynurenine, and hs-TnT between patients with and without significant CAD was observed. Baseline IDO activity, kynurenine level, and hs-TnT level were all significantly higher in significant CAD patients with 3-vessel, 2-vessel, and 1-vessel involvement than in those with insignificant CAD [(0.17, 0.13, and 0.16 vs. 0.03, respectively; p = 0.003), (5.89, 4.58, and 5.24 vs. 2.74 µM/g, respectively; p = 0.011), and (18.27, 12.22, and 12.86 vs. 10.89 mg/dL, respectively; p < 0.001)]. One-year mortality was 3.9%. When we compared between patients who survived and patients who died, we found a significantly lower prevalence of left main (LM) disease by coronary angiogram (6.1% vs. 33.3%, p = 0.007), and also a trend toward higher baseline kynurenine (5.07 vs. 0.79 µM/g, p = 0.082) and higher IDO (0.15 vs. 0.02, p = 0.081) in patients who survived. CONCLUSION: Immunometabolic response mediated via IDO function was enhanced in patients with CAD, and correlated with the extent and severity of disease. Patients with LM disease had higher 1-year mortality. Lower level of IDO, as suggested by inadequate IDO response, demonstrated a trend toward predicting 1-year mortality. Trial registration TCTR Trial registration number TCTR20200626001. Date of registration 26 June 2020. "Retrospectively registered".

A new method for anti-negative interference of calcium dobesilate in serum creatinine enzymatic analysis.

BACKGROUND: Serum creatinine is a widely used biomarker for evaluating renal function. Sarcosine oxidase enzymatic (SOE) analysis is currently the most widely used method for the detection of creatinine. This method was negatively interfered with by calcium dobesilate, causing pseudo-reduced results. The aim of this study was to explore a new method to alleviate the negative interference of this drug on creatinine detection. METHOD: We formulated eight drug concentrations and 12 creatinine concentrations from serum. The SOE method, the new method, and the Jaffe method were used for detection in five systems. Creatinine biases were analyzed under the conditions with or without the interference of calcium dobesilate, at consistent or inconsistent creatinine concentrations. Creatinine concentrations were also analyzed at three medical decision levels (MDLs). RESULTS: Calcium dobesilate had negative interference in creatinine SOE analysis. With the increase in calcium dobesilate concentrations, the negative bias increases. The new BG method showed an anti-negative interference effect. In the Roche system, the BG method reduced the negative bias from -71.11% to -16.7%. In the Abbott system, bias was reduced from -45.15% to -2.74%. In the Beckman system, the bias was reduced from -65.36% to -7.58%. In the Siemens system, the bias was reduced from -58.62% to -7.58%. In the Mindray system, the bias was reduced from -36.29% to -6.84%. CONCLUSION: The new method alleviated the negative interference of calcium dobesilate in creatinine SOE detection. The negative bias could be reduced from -60% or -70% to less than -20%.

Diagnostic value of gamma-glutamyl transpeptidase to alkaline phosphatase ratio combined with gamma-glutamyl transpeptidase to aspartate aminotransferase ratio and alanine aminotransferase to aspartate aminotransferase ratio in alpha-fetoprotein-negative hepatocellular carcinoma.

BACKGROUND: The purpose of the study was to evaluate the diagnostic value of gamma-glutamyl transpeptidase to alkaline phosphatase ratio (GAPR) combined with gamma-glutamyl transpeptidase to aspartate aminotransferase ratio (GAR) and alanine aminotransferase to aspartate aminotransferase ratio (AAR) in alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). METHODS: A total of 925 AFP-negative patients, including 235 HCC patients, 213 chronic hepatitis (CH) patients, and 218 liver cirrhosis (LC) patients, as well as 259 healthy controls were enrolled in this study. The differences of laboratory parameters and clinical characteristics were analyzed by Mann-Whitney U or Kruskal-Wallis H-test. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of GAPR, GAR, and AAR in AFP-negative HCC (AFP-NHCC) patients. RESULTS: GAPR, GAR, and AAR were important parameters closely related to AFP-NHCC. The combination of GAPR, GAR, and AAR was most effective in differentiating AFP-NHCC group from control group (AUC = 0.875), AFP-negative CH group (AUC = 0.733), and AFP-negative LC group (AUC = 0.713). GAPR combined with GAR and AAR exhibited a larger AUC than single ratio or pairwise combination for distinguishing AFP-NHCC group with TNMⅠstage, BCLC stage A, and tumor size less than 3 cm. The diagnostic value of GAPR combined with GAR and AAR was higher in AFP-NHCC and was also reflected in the TNM stage, Barcelona Clinic Liver Cancer (BCLC) stage and tumor size. CONCLUSIONS: GAPR combined with GAR and AAR were effective diagnostic markers of AFP-NHCC, especially in patients with good liver function, early stage or small size.

Pyruvate dehydrogenase deficiency disease detected by the enzyme activity of peripheral leukocytes.

BACKGROUND: Pyruvate dehydrogenase complex (PDHC) deficiency is a common neurodegenerative disease associated with abnormal mitochondrial energy metabolism. The diagnosis of PDHC is difficult because of the lack of a rapid, accurate, and cost-effective clinical diagnostic method. METHODS: A 4-year-old boy was preliminarily diagnosed with putative Leigh syndrome based on the clinical presentation. PDHC activity in peripheral blood leukocytes and a corresponding gene analysis were subsequently undertaken. Sodium pyruvate 1-13 C was used for the analysis of PDHC activity in peripheral leukocytes. The genes encoding PDHC were then scanned for mutations. RESULTS: The results showed that the corresponding PDHC activity was dramatically decreased to 10.5 nmol/h/mg protein as compared with that of healthy controls (124.6 ± 7.1 nmol/h/mg). The ratio of PDHC to citrate synthase was 2.1% (control: 425.3 ± 27.1). The mutation analysis led to the identification of a missense mutation, NM_000284.4:g214C>T, in exon 3 of PDHC. CONCLUSION: The peripheral blood leukocyte PDHC activity assay may provide a practical enzymatic diagnostic method for PDHC-related mitochondrial diseases.