Traceability validation of six enzyme measurements on the Abbott Alinity c analytical system.

Background Laboratory professionals should independently verify the correct implementation of metrological traceability of commercial measuring systems and determine if their performance is fit for purpose. We evaluated the trueness, uncertainty of measurements, and transferability of six clinically important enzyme measurements (alanine aminotransferase [ALT], alkaline phosphatase [ALP], aspartate aminotransferase [AST], creatine kinase [CK], γ-glutamyltransferase [γGT], and lactate dehydrogenase [LDH]) performed on the Abbott Alinity c analytical system. Methods Target values and associated uncertainties were assigned to three pools for each enzyme by using the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference measurement procedures (RMPs) and the pools were then measured on the Alinity system. Bias estimation and regression studies were performed, and the uncertainty associated with Alinity measurements was also estimated, using analytical performance specifications (APS) derived from biological variability of measurands as goals. Finally, to validate the transferability of the obtained results, a comparison study between two Alinity systems located in Milan, Italy, and Bydgoszcz, Poland, was carried out. Results Correct implementation of traceability to the IFCC RMPs and acceptable measurement uncertainty fulfilling desirable (ALP, AST, LDH) or optimal APS (ALT, CK, γGT) was verified for all evaluated enzymes. An optimal alignment between the two Alinity systems located in Milan and Bydgoszcz was also found for all enzyme measurements. Conclusions We confirmed that measurements of ALT, ALP, AST, CK, γGT, and LDH performed on the Alinity c analytical system are correctly standardized to the IFCC reference measurement systems and the system alignment is consistent between different platforms.

Letter to the editor regarding "GRAS from the ground up: Review of the Interim Pilot Program for GRAS notification" by.

Present letter is aimed at clarifying some critical points highlighted by Hanlon et al. regarding the common knowledge element of the safety of food enzymes in support of their GRAS designation. Particularly, we outline the development of peer-reviewed, generally recognized safety evaluation methodology for microbial enzymes and its adoption by the enzyme industry, which provides the US FDA with a review framework for enzyme GRAS Notices. This approach may serve as a model to other food ingredient categories for a scientifically sound, rigorous, and transparent application of the GRAS concept.

Hipertransaminasemia secundaria a macroaspartato aminotransferasa / Secondary hypertransaminasemia to macro-aspartate aminotransferase

No disponible

The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes.

BACKGROUND: A multicenter study conducted in Southeast Asia to derive reference intervals (RIs) for 72 commonly measured analytes (general chemistry, inflammatory markers, hormones, etc.) featured centralized measurement to clearly detect regionality in test results. The results of 31 standardized analytes are reported, with the remaining analytes presented in the next report. METHOD: The study included 63 clinical laboratories from South Korea, China, Vietnam, Malaysia, Indonesia, and seven areas in Japan. A total of 3541 healthy individuals aged 20-65 years (Japan 2082, others 1459) were recruited mostly from hospital workers using a well-defined common protocol. All serum specimens were transported to Tokyo at -80°C and collectively measured using reagents from four manufacturers. Three-level nested ANOVA was used to quantitate variation (SD) of test results due to region, sex, and age. A ratio of SD for a given factor over residual SD (representing net between-individual variations) (SDR) exceeding 0.3 was considered significant. Traceability of RIs was ensured by recalibration using value-assigned reference materials. RIs were derived parametrically. RESULTS: SDRs for sex and age were significant for 19 and 16 analytes, respectively. Regional difference was significant for 11 analytes, including high density lipoprotein (HDL)-cholesterol and inflammatory markers. However, when the data were limited to those from Japan, regionality was not observed in any of the analytes. Accordingly, RIs were derived with or without partition by sex and region. CONCLUSIONS: RIs applicable to a wide area in Asia were established for the majority of analytes with traceability to reference measuring systems, whereas regional partitioning was required for RIs of the other analytes.

[The preanalytical stage of under measuring of concentration of catalytic activity of enzymes: the characteristics and tasks of standardization].

The article explains the importance of standardization, the development and maintenance of rules and recommendations regulating the conditions and order of implementation of particular parts of preanalytical stage. The order of these conditions is noted including the rules stated and published in such normative documents as national standards GOST R 15 189-2009, GOST R 53079.4-2008, GOST R ISO 6710-209 and in the recommendations of foreign National societies ofclini-cal chemistry and laboratory medicine. These requirements concern all the analytes, enzymes included. The cited data have a practical significance for acquisition of reliable results in everyday functioning of laboratories. Enough to mention data concerning the anticoagulants influence on catalytic concentration of enzymes, the most often measured concentrations of alpha-amylase, lipase, amynotransferase, alkaline and acid phosphatase, creatine kinase, lactate dehydrogenase, choline esterase, hemolysis impact, the increased concentration of bilirubin and hyperlipemia in samples and significance of measurement of indices of serum and plasma as well. The possible mechanisms of impact of these interferents on the results of measurement of catalytic concentration of enzymes are discussed.

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 µm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 µm or 3 µm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.

Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

IFCC reference procedures for measurement of the catalytic concentrations of enzymes: corrigendum, notes and useful advice. International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)--IFCC Scientific Division.

The primary reference measurement procedures (PRMPs) for the international standardization of catalytic concentration measurements of alpha-amylase, alanine aminotransferase, aspartate aminotransferase (AST), creatine kinase (CK), gamma-glutamyltransferase and lactate dehydrogenase have been performed in reference laboratories for several years. The IFCC Committee on Reference Systems for Enzymes and two reference laboratories, with official accreditation for the PRMPs, have collected useful information on some of the steps of the reference procedures that require special attention. This document comprises errata corrige for minor mistakes in published PRMPs for AST and CK. Several notes on the PRMPs are emphasized. This includes details that are very important for improved standardization, and general suggestions for reducing measurement uncertainty.

Effect of waist circumference on reference intervals of liver-related enzyme tests in apparently healthy adult Mexican Americans, black and white Americans.

BACKGROUND: We used a more comprehensible and more physiologically valid indicator of obesity-related liver disease, waist circumference (WC), rather than BMI to study the effect of obesity on liver enzymes, easily measurable surrogates of liver disease. METHODS: WC, liver enzymes and pertinent demographic data were abstracted from NHANES III. After exclusion criteria were applied, we stratified the populations by sex and ethnicity and graphed the percentage of elevated enzymes per WC interval. RESULTS: There was a significant relationship between enzyme activity and WC (p<0.001 for ALT, p<0.01 for ALP, p<0.01 for AST, p=0.02 for GGT and p<0.05 for LD), and the relationship was stronger in females (p<0.002) and Mexican Americans (p<0.001). The enzyme-WC relationship is much weaker in non-Hispanic black females and males. CONCLUSION: As increases in liver enzyme activity can indicate potentially reversible liver disease, our graphs should be used by physicians to motivate obese, apparently healthy Mexican American and non-Hispanic white patients with elevated liver enzymes to reduce their WC.

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.

External Quality Assessment in The Netherlands: time to introduce commutable survey specimens. Lessons from the Dutch "Calibration 2000" project.

The performance of suitable secondary reference material for the use of trueness control of six routinely measured clinical enzymes in the Dutch External Quality Assessment (EQA) scheme is described. The reference material of choice was selected using the split-patient-sample between-field method (twin study) design as described in an earlier study of the Calibration 2000 project in The Netherlands. This material, which was proven to be commutable for all wet chemistry systems, was implemented as the national enzyme calibrator. It consisted of a cryo-protected lyophilised serum with additions of recombinant human enzymes. Various batches of the frozen version of this material without cryo-protection additive, called native EQA samples, were used in the general EQA scheme for performance evaluation. The results of Calibration 2000 calibrated and non-Calibration 2000 calibrated laboratories were compared for both the regular (spiked with non-human enzymes) and native EQA samples in terms of precision and bias with established reference method values for the native samples. The regular samples showed mean between-laboratory CV ranges for all six enzymes involved (low-high) of 5.5-10.3% for the non-calibrated users vs. 4.6-10.8% for the calibrated users. For the native samples these respective ranges were 5.2-9.9% vs. 2.2-4.9%. Without exception, the group of Calibration 2000 calibrated users showed the lowest bias against the reference method values. Regular EQA samples (spiked with non-human enzymes) showed poorer performance than native samples and are not suitable for accuracy assessment purposes, the main aim of EQA schemes. Native samples that are commutable should be used for trueness control in current EQA schemes.

Study on OELs for enzyme-containing detergent in China.

This study is aimed at setting occupational exposure levels for total detergent dust and enzymes in detergent industries. The study population consisted of 795 workers from four enzyme-containing detergent manufacturing plants (A1, A2, B1 and B2), and 156 control workers from an electronic assembly factory. Work environment monitoring was conducted using high volume of air sampler fro measuring the concentration of total dust (mg/m3), and analyzing the level of enzyme (ng/m3) by ELISA method. A standard questionnaires, pulmonary function test, and skin prick test are used to assess health effects. The levels of detergent total dust varied from 0.2 mg/m3 to 12.54 mg/m3. For enzyme levels, in A1, B1 and B2, the concentration ranged from non-detectable to 9.92 ng/m3 and in A2, the concentration was analyzed by enzyme activity methods and was expressed as Gu/m3 (1 Gu/m3 = 16 ng/m3). The concentration is between 0.16-31.36 ng/m3. Non-specific irritation rates in exposed workers were significantly higher than that in controls. Based on the data collected from A1, B1 and control plants, 95% benchmark dose lower bound were calculated as 1.17 mg/m3. The difference of pulmonary function between exposed workers and controls is not significant. The results of SPT showed that neither Savinase- nor Alcalase-induced sensitization was found in controls. The prevalence rates of sensitization for Savinase and Alcalase were ranged between 3.2% and 31% in all enzyme-containing detergent manufacturers investigated. No case of occupational asthma was observed. For total dust, 1 mg/m3 is suggested as permissible concentration-time weighted average (PC-TWA), and 2 mg/m3 as permissible concentration-short term exposure limit (PC-STEL). For the enzyme Subtilisins, 15 ng/m3 is suggested as PC-TWA, and 30 ng/m3 as PC-STEL.

PDBSprotEC: a Web-accessible database linking PDB chains to EC numbers via SwissProt.

A mapping between chains in the Protein Databank and Enzyme Classification numbers is invaluable for research into structure-function relationships. Mapping at the chain level is a non-trivial problem and we present an automatically updated Web-server, which provides this link in a queryable form and as a downloadable XML or flat file.

Nomenclatura de Enzimas

Contém informação sobre nomenclatura e classificação de enzimas, seguindo as recomendações do Nomenclature Committee da International Union of Biochemistry and Molecular Biology e informações de como sugerir novas enzimas para a lista ou correções nos nomes já existentes.

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 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.

The tortuous road to the adoption of katal for the expression of catalytic activity by the General Conference on Weights and Measures.

BACKGROUND: The "unit" for "enzymic activity" (U = 1 micromol/min) was recommended by the International Union of Biochemistry and Molecular Biology (IUB) in 1961 and is widely used in medical laboratory reports. The general trend in metrology, however, is toward global standardization through defining units coherent with the International System of Units (SI). APPROACH: Several proposals were advanced from the IFCC, International Union of Pure and Applied Chemistry, and IUB regarding the definition for enzymic activity as well as the terms for kind-of-quantity, units, symbol, and dimension. In 1977, international agreement was reached between these bodies and WHO that "catalytic activity" (z), of a catalyst in a given system is defined by the rate of conversion in a measuring system (in mol/s) and expressed in "katal" (symbol, kat; equal to 1 mol/s). The katal is invariant of the measurement procedure, but the numerical quantity value is not. Gaining support for the katal from the final arbiter, the General Conference on Weights and Measures, was slow, but Resolution 12 of 1999 adopted the katal (symbol, kat) as a special name and symbol for the SI-derived unit, mol/s, used in measuring catalytic activity. CONCLUSIONS: Laboratory results for amounts of catalysts, including enzymes, measured by their catalytic activity can now officially be expressed in katals and are traceable to the SI provided that the specified indicator reaction reflects first-order kinetics. The conversion from "unit" is: 1 U = 16.667 x 10(-9) kat. Further derived quantities have coherent units such as kat/L, kat/kg, and kat/kat = 1.

[The existence of contamination in enzymes used for the isolation of Aspergillus and Candida species DNA]. / Aspergillus ve Candida cinsi DNA'sinin izolasyonunda kullanilan enzimlerde kontaminasyon varliginin arastirilmasi.

Molecular biological methods which are widely used in different fields, have been replaced with conventional diagnostic tests for the early diagnosis of invasive fungal infections, recently. Polymerase chain reaction (PCR) is one of these methods, with high specificity and sensitivity, which is accepted throughout the world. However, the enzymes that are used for the isolation of target DNA, may be contaminated with the gene sequences of some other fungal species in the preparation steps and may affect the PCR results. The studies showed that, the contamination of these enzymes with any type of fungi during their production steps, leads false positive results in PCR tests. So, the additional studies are recommended for minimizing the contamination. The aim of this study was to search whether the enzymes necessary for the isolation of fungal target DNA, used in PCR, are contaminated or not. For this purpose, five different enzymes namely, Zymolase 20T (from Arthrobacter luteus, two examples from different companies), lyticase (from Arthrobacter luteus), lysing enzyme (from Trichoderma harzianum) and proteinase K (from Tritrachium album) have been investigated for the presence of contamination. As a result, both of zymolase 20T enzymes were found to be contaminated from some fungal species with the demonstration of 18S rRNA gene sequences, while the other enzymes were found non-contaminated. By the help of this method, the most suitable enzyme for PCR was chosen and fungal contamination was prevented.