COVID-19 promotes endothelial dysfunction and thrombogenicity: role of proinflammatory cytokines/SGLT2 prooxidant pathway.

BACKGROUND: COVID-19 is associated with an increased risk of cardiovascular complications. Although cytokines have a predominant role in endothelium damage, the precise molecular mechanisms are far from being elucidated. OBJECTIVES: The present study hypothesized that inflammation in patients with COVID-19 contributes to endothelial dysfunction through redox-sensitive SGLT2 overexpression and investigated the protective effect of SGLT2 inhibition by empagliflozin. METHODS: Human plasma samples were collected from patients with acute, subacute, and long COVID-19 (n = 100), patients with non-COVID-19 and cardiovascular risk factors (n = 50), and healthy volunteers (n = 25). Porcine coronary artery endothelial cells (ECs) were incubated with plasma (10%). Protein expression levels were determined using Western blot analyses and immunofluorescence staining, mRNA expression by quantitative reverse transcription-polymerase chain reaction, and the level of oxidative stress by dihydroethidium staining. Platelet adhesion, aggregation, and thrombin generation were determined. RESULTS: Increased plasma levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1 were observed in patients with COVID-19. Exposure of ECs to COVID-19 plasma with high cytokines levels induced redox-sensitive upregulation of SGLT2 expression via proinflammatory cytokines IL-1ß, IL-6, and tumor necrosis factor-α which, in turn, fueled endothelial dysfunction, senescence, NF-κB activation, inflammation, platelet adhesion and aggregation, von Willebrand factor secretion, and thrombin generation. The stimulatory effect of COVID-19 plasma was blunted by neutralizing antibodies against proinflammatory cytokines and empagliflozin. CONCLUSION: In patients with COVID-19, proinflammatory cytokines induced a redox-sensitive upregulation of SGLT2 expression in ECs, which in turn promoted endothelial injury, senescence, platelet adhesion, aggregation, and thrombin generation. SGLT2 inhibition with empagliflozin appeared as an attractive strategy to restore vascular homeostasis in COVID-19.

COVID-19 exposure in SARS-CoV-2-seropositive hospital staff members during the first pandemic wave at Strasbourg University Hospital, France.

OBJECTIVES: Strasbourg University Hospital faced an important COVID-19 first wave from early March 2020. We performed a longitudinal prospective cohort study to describe clinical and virological data, exposure history to COVID-19, and adherence to strict hygiene standards during the first pandemic wave in 1497 workers undergoing a SARS-CoV-2 serological test at our hospital, with a follow up of serology result three months later. PATIENTS AND METHODS: A total of 1497 patients were enrolled from April 6 to May 7, 2020. Antibody response to SARS-CoV-2 was measured, and COVID-19 exposure routes were analyzed according to SARS-CoV-2 serological status. RESULTS: A total of 515 patients (34.4%) were seropositive, mainly medical students (13.2%) and assistant nurses (12.0%). A history of COVID-19 exposure in a professional and/or private setting was mentioned by 83.1% of seropositive subjects (P<0.05; odds ratio [OR]: 2.5; 95% confidence interval [CI]: 1.8-3.4). COVID-19 exposure factors associated with seropositive status were non-professional exposure (OR: 1.9, 95% CI: 1.3-2.7), especially outside the immediate family circle (OR: 2.2, 95% CI: 1.2-3.9) and contact with a COVID-19 patient (OR: 1.6; 95% CI: 1.1-2.2). Among professionally exposed workers, systematic adherence to strict hygiene standards was well observed, except for the use of a surgical mask (P<0.05, OR: 1.9, 95% CI: 1.3-2.8). Of those who reported occasionally or never wearing a surgical mask, nurses (25.7%), assistant nurses (16.2%), and medical students (11.7%) were predominant. CONCLUSION: Infection of staff members during the first pandemic wave in our hospital occurred after both professional and private COVID-19 exposure, underlining the importance of continuous training in strict hygiene standards.

Evolution of antibody responses up to 13 months after SARS-CoV-2 infection and risk of reinfection.

BACKGROUND: Assessment of the kinetics of SARS-CoV-2 antibodies is essential in predicting risk of reinfection and durability of vaccine protection. METHODS: This is a prospective, monocentric, longitudinal, cohort clinical study. Healthcare workers (HCW) from Strasbourg University Hospital were enrolled between April 6th and May 7th, 2020 and followed up to 422 days. Serial serum samples were tested for antibodies against the Receptor Binding Domain (RBD) of the spike protein and nucleocapsid protein (N) to characterize the kinetics of SARS-CoV-2 antibodies and the incidence of reinfection. Live-neutralization assays were performed for a subset of samples before and after vaccination to analyze sensitivity to SARS-CoV-2 variants. FINDINGS: A total of 4290 samples from 393 convalescent COVID-19 and 916 COVID-19 negative individuals were analyzed. In convalescent individuals, SARS-CoV-2 antibodies followed a triphasic kinetic model with half-lives at month (M) 11-13 of 283 days (95% CI 231-349) for anti-N and 725 days (95% CI 623-921) for anti-RBD IgG, which stabilized at a median of 1.54 log BAU/mL (95% CI 1.42-1.67). The incidence of SARS-CoV-2 infections was 12.22 and 0.40 per 100 person-years in COVID-19-negative and COVID-19-positive HCW, respectively, indicating a relative reduction in the incidence of SARS-CoV-2 reinfection of 96.7%. Live-virus neutralization assay revealed that after one year, variants D614G and B.1.1.7, but less so B.1.351, were sensitive to anti-RBD antibodies at 1.4 log BAU/mL, while IgG ≥ 2.0 log BAU/mL strongly neutralized all three variants. These latter anti-RBD IgG titers were reached by all vaccinated HCW regardless of pre-vaccination IgG levels and type of vaccine. INTERPRETATION: Our study demonstrates a long-term persistence of anti-RBD antibodies that may reduce risk of reinfection. By significantly increasing cross-neutralizing antibody titers, a single-dose vaccination strengthens protection against variants. FUN1DING: None.

[Myocardial injury in coronavirus disease 19 (Covid-19): main pathophysiological mechanisms and clinical utility of cardiac biomarkers]. / Atteintes myocardiques au cours de la maladie à coronavirus 19 (Covid-19) : principaux mécanismes physiopathologiques et utilité clinique des biomarqueurs cardiaques.

Covid-19 is responsible for myocardial injury in many infected patients, which is associated with severe disease and critical illness. The mechanisms by which SARS-CoV-2 may cause myocardial damage involve direct effect of the virus in cardiac cells and indirect effect due to the clinical consequences of Covid-19. Cardiomyocytes are well known to express Angiotensin-Converting Enzyme-2 receptors (ACE-2) to facilitate the virus cell entry, which could explain the occurrence of myocarditis, functional alterations in the myocardium, and more rarely, myocardial infarction. Myocardial injury may also be secondary to systemic inflammation or coagulopathy due to complicated Covid-19. The existence of a cardio-intestinal axis with alteration of tryptophan metabolism in the small bowel leading first to colitis and then to systemic inflammation has also been evoked to explain the myocardial injury. Morphological and metabolic disturbances of the heart during the Covid-19 are associated with elevated concentrations of cardiac blood biomarkers, mainly troponins and natriuretic peptides. The determination of these biomarkers has proven to be very useful for diagnosis, prognosis, and risk stratification. Indeed, recent data demonstrated that about 20% of infected patients admitted to the hospital have elevated troponin or BNP levels, and Covid-19 patients with elevated troponin concentrations beyond the diagnostic threshold (99th percentile) were associated with a higher risk of in-hospital mortality. In conclusion, after more than a year of a unique global pandemic, it is now clearly established that myocardial injury during Covid-19 is frequent and strongly contributes to the severity of the disease. Cardiac alterations secondary to direct infection of cardiac cells by SARS-CoV-2 or to the clinical consequences of Covid-19 are associated with elevated levels of cardiac biomarkers in blood, whose measurement is crucial in clinical decision making.

Sirolimus Pharmacokinetics Variability Points to the Relevance of Therapeutic Drug Monitoring in Pediatric Oncology.

Sirolimus is widely used in transplantation, where its therapeutic drug monitoring (TDM) is well established. Evidence of a crucial role for sirolimus in the PI3K/AkT/mTor pathway has stimulated interest in its involvement in neoplasia, either as monotherapy or in combination with other antineoplastic agents. However, in cancer, there is no consensus on sirolimus TDM. In the RAPIRI phase I trial, the combination sirolimus + irinotecan was evaluated as a new treatment for refractory pediatric cancers. Blood sampling at first sirolimus intake (D1) and at steady state (D8), followed by LC/MS2 analysis, was used to develop a population pharmacokinetic model (Monolix® software). A mono-compartmental model with first-order absorption and elimination best fit the data. The only covariate retained for the final model was "body surface area" (D1 and D8). The model also demonstrated that 1.5 mg/m2 would be the recommended sirolimus dose for further studies and that steady-state TDM is necessary to adjust the dosing regimen in atypical profiles (36.4% of the population). No correlation was found between sirolimus trough concentrations and efficacy and/or observed toxicities. The study reveals the relevance of sirolimus TDM in pediatric oncology as it is needed in organ transplantation.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat.

BACKGROUND: Empagliflozin (empa), a selective sodium-glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its' lean control. METHODS: Lean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis. RESULTS: Empa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats. CONCLUSION: Empa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

Optimizing Hydroxyurea Treatment for Sickle Cell Disease Patients: The Pharmacokinetic Approach.

BACKGROUND: Hydroxyurea (HU) is a FDA- and EMA-approved drug that earned an important place in the treatment of patients with severe sickle cell anemia (SCA) by showing its efficacy in many studies. This medication is still underused due to fears of physicians and families and must be optimized. METHODS: We analyzed our population and identified HU pharmacokinetic (PK) parameters in order to adapt treatment in the future. Working with a pediatric population, we searched for the most indicative sampling time to reduce the number of samples needed. RESULTS: Nine children treated by HU for severe SCA were included for this PK study. HU quantification was made using a validated gas chromatography/mass spectrometry (GC/MS) method. Biological parameters (of effectiveness and compliance) and clinical data were collected. None of the nine children reached the therapeutic target defined by Dong et al. as an area under the curve (AUC) = 115 h.mg/L; four patients were suspected to be non-compliant. Only two patients had an HbF over 20%. The 2 h sample was predictive of the medication exposure (r2 = 0.887). CONCLUSIONS: It is urgent to be more efficient in the treatment of SCA, and pharmacokinetics can be an important asset in SCA patients.

A novel machine learning-derived decision tree including uPA/PAI-1 for breast cancer care.

Background uPA and PAI-1 are breast cancer biomarkers that evaluate the benefit of chemotherapy (CT) for HER2-negative, estrogen receptor-positive, low or intermediate grade patients. Our objectives were to observe clinical routine use of uPA/PAI-1 and to build a new therapeutic decision tree integrating uPA/PAI-1. Methods We observed the concordance between CT indications proposed by a canonical decision tree representative of French practices (not including uPA/PAI-1) and actual CT prescriptions decided by a medical board which included uPA/PAI-1. We used a method of machine learning for the analysis of concordant and non-concordant CT prescriptions to generate a novel scheme for CT indications. Results We observed a concordance rate of 71% between indications proposed by the canonical decision tree and actual prescriptions. Discrepancies were due to CT contraindications, high tumor grade and uPA/PAI-1 level. Altogether, uPA/PAI-1 were a decisive factor for the final decision in 17% of cases by avoiding CT prescription in two-thirds of cases and inducing CT in other cases. Remarkably, we noted that in routine practice, elevated uPA/PAI-1 levels seem not to be considered as a sufficient indication for CT for N≤3, Ki 67≤30% tumors, but are considered in association with at least one additional marker such as Ki 67>14%, vascular invasion and ER-H score <150. Conclusions This study highlights that in the routine clinical practice uPA/PAI-1 are never used as the sole indication for CT. Combined with other routinely used biomarkers, uPA/PAI-1 present an added value to orientate the therapeutic choice.

Commutability Assessment of Candidate Reference Materials for Pancreatic α-Amylase.

BACKGROUND: Measurement standardization of the catalytic concentration of α-amylase in serum is based on 3 pillars: the primary reference measurement procedure (PRMP), reference laboratories, and suitable certified reference materials (CRMs). Commutability is a prerequisite when using a CRM for calibration and trueness control of routine methods or for value transfer from the PRMP to end-user calibrators of routine methods through a calibration hierarchy. METHODS: We performed a commutability study with 30 serum pools and 5 candidate reference materials (RMs) for pancreatic α-amylase using an automated version of the PRMP and 5 different routine methods. Four candidate RMs had an artificial matrix, each with a different composition, and 1 candidate RM was based on human serum. Data were analyzed according to a linear regression analysis with prediction interval as described in the Clinical and Laboratory Standards Institute guideline EP30-A and a difference in bias analysis as described in the recommendations of the IFCC Working Group on Commutability. RESULTS: The commutability profile of the 4 candidate RMs with an artificial matrix was variable. Only 1 candidate RM, with human serum albumin in the matrix, showed a good profile like that of the candidate RM based on serum. The comparison of both commutability assessment approaches indicated some differences because of inconclusive results for the difference in bias approach, suggesting a large uncertainty on the commutability assessment. CONCLUSIONS: A CRM for pancreatic amylase in an artificial matrix can be commutable for routine methods using the same substrate as the PRMP, but the matrix composition is crucial.

Rifampicin reverses nicardipine effect inducing uncontrolled essential hypertension.

Dihydropyridine calcium-channel blockers are a known substrate for the cytochrome P450 isoform 3A4. Rifampicin, an antitubercular agent, is one of the most potent inducers of hepatic and intestinal CYP3A4 thus increasing dihydropyridine metabolism. We report a case of a 67-year-old hypertensive female treated with a four-drug antihypertensive regimen including a dihydropyridine (nicardipine 50 mg bid), who was admitted for septic arthritis of the knee requiring antibiotic treatment with teicoplanin 400 mg od and rifampicin 600 mg bid. Six days after rifampicin initiation, she presented with Posterior Reversible Encephalopathy Syndrome due to uncontrolled hypertension. We hypothesized that disequilibrium of previously controlled hypertension was partially due to nicardipine ineffectiveness. Plasma nicardipine concentration was assessed through high-performance liquid chromatography 5 hours after coadministration of the two drugs and proved undetectable.

Prognosis of patients with severe hyponatraemia is related not only to hyponatraemia but also to comorbidities and to medical management: results of an observational retrospective study.

BACKGROUND: The true cause of death in severe hyponatraemic patients remains controversial. The present study aimed to analyse the relationship between comorbidity, medical management and prognosis in severe hyponatraemic patients. METHODS: Medical records of all patients hospitalised in our institution in 2012 with a plasma sodium ≤120 mmol/l were retrospectively analysed. RESULTS: One hundred forty-seven of 64 723 adult patients (0.2 %) were identified with severe hyponatraemia. In-hospital mortality rate was 24.5 and 50.3 % after a median follow-up of 431 days. Patients with plasma sodium <110 mmol/l had less comorbidity (Charlson Comorbidity Index 2.2 ± 1.9 vs. 4.0 ± 3.1 (plasma sodium 110-115 mmol/l) and 4.2 ± 3.1 (plasma sodium 116-120 mmol/l); P = .02)) and a small trend for less mortality, respectively 40.0, 51.2 and 52.3 % (P = .64). At discharge, nonsurvivors and survivors had similar plasma sodium with 58.3 % of nonsurvivors being normonatraemic. Urine analysis was performed in 74.2 % of cases and associated with lower in-hospital mortality (20.2 % vs. 36.8 %, P = .05). In multivariate Cox analysis, mortality was significantly associated with plasma sodium normalisation (HR 0.35, P < 0.001), urine analysis (HR 0.48, P = .01), Charlson Comorbidity Index (HR 1.23, P < .001) and serum albumin (HR 0.88, P < .001). CONCLUSION: Mortality in severe hyponatraemia appears mainly due to comorbidities although the latter are potentiated by hyponatraemia itself and its management thereby exacerbating the risk of death.

An Innovative Fluorescent Semi-quantitative Methylation-specific PCR Method for the Determination of MGMT Promoter Methylation is Reflecting Intra-tumor Heterogeneity.

High grade gliomas (HGG) are usually associated with a very dismal prognosis, which was moderately improving in the last decade with the introduction of the alkylating agent temozolomide in their treatment. The methylation status of MGMT (O6 methylguanine DNA-methyltransferase) promoter is one of the strongest predictive and prognostic factors for the patient chemoresponse. For instance, the molecular method of assessment for MGMT promoter status is not standardized. In this background, we developed a fluorescent capillary gel electrophoresis-based methylation specific-PCR. This technique allowed a semi-quantitative estimate of the relative ratio between methylated and unmethylated alleles. The efficacy and accuracy of the technique was assessed in a retrospective cohort of 178 newly diagnosed adult HGGs, who were homogeneously treated. First, we analyzed the impact on survival of different cut-off points in the MGMT promoter methylation and, to go further, we correlated these different rates to other well-known prognostic molecular factors involved in adult HGGs. This strategy allowed to validate our technique as a very sensitive technique (detection of a low methylation percentage, < 5%), which was feasible in fresh-frozen as well as in FFPE samples and had the propensity to detect intra-tumor heterogeneity. This technique identified a new sub-group of anaplastic oligodendrogliomas or oligoastrocytomas defined by a minor methylation and a worse outcome and, therefore, will help to substratify accurately into more homogeneous subgroups of methylated tumors.

Mitomycin C Pharmacokinetics as Predictor of Severe Neutropenia in Hyperthermic Intraperitoneal Therapy.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is an approach to overcome peritoneal carcinomatosis from colorectal adenocarcinoma. Mitomycin C (MMC) is frequently used but not devoid of toxicity, of which the most common and feared is neutropenia. Our study explores the clinical and surgical risk factors of neutropenia and a possible link between MMC pharmacokinetics and neutropenia as HIPEC's supervention. METHODS: A total of 45 patients undergoing CRS-HIPEC for peritoneal carcinomatosis of colorectal origin between 2004 and 2010 were followed. For each patient, MMC was measured in plasma at different times during HIPEC and the area under the MMC concentration-time curve (MMC-AUC) was calculated. RESULTS: The incidence of neutropenia was 40 %. No demographic, clinical, or surgical factors increased the risk of neutropenia. However, we found that the occurrence of neutropenia and its gravity increased in direct correlation with an increase in MMC plasma concentration 30 min (T30) and 45 min (T45) after the start of HIPEC. The same correlation was observed between the MMC-AUC and the risk of neutropenia. CONCLUSIONS: Neutropenia is a frequent complication associated with MMC-HIPEC. The results of our study indicate the feasibility and the potential benefit of a protocol including the MMC dosage at T30 after the start of HIPEC. A threshold of 572 µg/L gives a predictive sensitivity of 86 % and a specificity of 80 %. These results must be considered in the management of patients undergoing MMC-HIPEC in order to place high-risk patients under neutropenic monitoring while the other patients can undergo simple hematological monitoring.

[Clinical laboratory medicine: continuous amelioration with a book of objectives and satisfaction survey]. / Amélioration continue du DES de biologie médicale à l'aide d'un carnet de stage et d'un questionnaire d'évaluation de la satisfaction.

We report in this publication the use of two educational tools, a questionnaire of satisfaction and a training book, to improve the training of students during their internship in clinical laboratory at the "Pôle de biologie des Hôpitaux universitaires de Strasbourg" in France. First, the ongoing training was assessed by the interns with a questionnaire measuring satisfaction. The analysis of this questionnaire identified four key points to improve: 1) define the teaching objectives, 2) organize the training with a schedule, 3) revise certain teaching methods and 4) ensure better integration of the students in the team of medical biologists. After this assessment, we implemented a training book to answer these four points. Indeed, the training book presents the objectives, the schedule of training, and how to validate the educational objectives. A new assessment was performed again using the same methodology. Results showed an improvement in student satisfaction from 74 to 88 %. The questionnaire of satisfaction and the training book are presented in this article. The aim of the assessment of training combined with the training book is to incite the actors of the training (students and teachers) to continually improve the training. The objectives of the Pôle de Biologie are to obtain an 80 % satisfaction rate during the 6 months trainings and to reduce or eliminate dissatisfaction, and finally to ensure the validation by students of 80 to 100 % of their predetermined objectives.

Analysis of the RNA chaperoning activity of the hepatitis C virus core protein on the conserved 3'X region of the viral genome.

The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3'X genomic region required for genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled 16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1 and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop-loop interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during HCV replication.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)).

Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °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 γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

Traceability of values for catalytic activity concentration of enzymes: a Certified Reference Material for aspartate transaminase.

BACKGROUND: A new reference material for the liver enzyme aspartate transaminase (AST) (L-aspartate: 2-oxoglutarate-aminotransferase, EC 2.6.1.1), also called aspartate aminotransferase (ASAT), has been developed under the code ERM-AD457/IFCC. This certified reference material (CRM) for AST has been produced from a human type recombinant AST expressed in Escherichia coli and a buffer containing bovine serum albumin, and has been lyophilised. METHODS: The homogeneity and the stability of the material have been tested and the catalytic activity concentration has been characterised by 12 laboratories using the reference procedure for AST at 37 degrees C from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). RESULTS: The certified catalytic activity concentration and certified uncertainty of AST in the reconstituted material are (1.74+/-0.05) microkat/L or (104.6+/-2.7) U/L (with a coverage factor k=2; 95% confidence interval). CONCLUSIONS: Both the certified value and uncertainty are traceable to the International System of Units (SI). The material is aiming to control the IFCC reference procedure for AST at 37 degrees C, which will then be used to assign values to calibrants and control materials. The present paper highlights the scientific challenges and innovations which were encountered during the development of this new CRM.

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.