European Biological Variation Study (EuBIVAS): within- and between-subject biological variation estimates of ß-isomerized C-terminal telopeptide of type I collagen (ß-CTX), N-terminal propeptide of type I collagen (PINP), osteocalcin, intact fibroblast growth factor 23 and uncarboxylated-unphosphorylated matrix-Gla protein-a cooperation between the EFLM Working Group on Biological Variation and the International Osteoporosis Foundation-International Federation of Clinical Chemistry Committee on Bone Metabolism.

We have calculated the biological variation (BV) of different bone metabolism biomarkers on a large, well-described cohort of subjects. BV is important to calculate reference change value (or least significant change) which allows evaluating if the difference observed between two consecutive measurements in a patient is biologically significant or not. INTRODUCTION: Within-subject (CVI) and between-subject (CVG) biological variation (BV) estimates are essential in determining both analytical performance specifications (APS) and reference change values (RCV). Previously published estimates of BV for bone metabolism biomarkers are generally not compliant with the most up-to-date quality criteria for BV studies. We calculated the BV and RCV for different bone metabolism markers, namely ß-isomerized C-terminal telopeptide of type I collagen (ß-CTX), N-terminal propeptide of type I collagen (PINP), osteocalcin (OC), intact fibroblast growth factor 23 (iFGF-23), and uncarboxylated-unphosphorylated Matrix-Gla Protein (uCuP-MGP) using samples from the European Biological Variation Study (EuBIVAS). METHODS: In the EuBIVAS, 91 subjects were recruited from six European laboratories. Fasting blood samples were obtained weekly for ten consecutive weeks. The samples were run in duplicate on IDS iSYS or DiaSorin Liaison instruments. The results were subjected to outlier and variance homogeneity analysis before CV-ANOVA was used to obtain the BV estimates. RESULTS: We found no effect of gender upon the CVI estimates. The following CVI estimates with 95% confidence intervals (95% CI) were obtained: ß-CTX 15.1% (14.4-16.0%), PINP 8.8% (8.4-9.3%), OC 8.9% (8.5-9.4%), iFGF23 13.9% (13.2-14.7%), and uCuP-MGP 6.9% (6.1-7.3%). CONCLUSIONS: The EuBIVAS has provided updated BV estimates for bone markers, including iFGF23, which have not been previously published, facilitating the improved follow-up of patients being treated for metabolic bone disease.

Minding the gap between cortisol levels measured with second-generation assays and current diagnostic thresholds for the diagnosis of adrenal insufficiency: a single-center experience.

PURPOSE: The current cut-offs for the diagnosis of adrenal insufficiency (AI) have been established using outdated immunoassays. We compared the cortisol concentrations measured with Roche Cortisol I (R1), the newly available Roche Cortisol II (R2), and liquid chromatography tandem mass spectrometry (LC-MS/MS), the gold standard procedure to measure steroids in patients undergoing the corticotropin (ACTH) test. METHODS: We enrolled 30 patients (age 47 ± 21 years) referred to undergo the ACTH test (1 or 250 µg). Cortisol was measured at 0, 30, and 60 min after stimulation with R1, R2, and LC-MS/MS. AI was diagnosed for R1-stimulated peak cortisol concentrations < 500 nmol/L. RESULTS: Mean cortisol concentrations measured with R2 and LC-MS/MS were comparable, while mean cortisol concentrations measured by R1 were higher than those of both R2 and LC-MS/MS (respectively, basal 411 ± 177, 287 ± 119, and 295 ± 119 nmol/L; at 30 min, 704 ± 204, 480 ± 132, and 500 ± 132 nmol/L; at 60 min, 737 ± 301, 502 ± 196, and 519 ± 201 nmol/L, p ≤ 0.01 for R1 vs. both R2 and LC-MS/MS at each point). Considering the 500 nmol/L cortisol peak cut-off, AI was diagnosed in 5/30 patients using R1 and in 12/30 using R2 (+ 140%). Based on the correlation between R1 and R2, the threshold of 500 nmol/L became 351 nmol/L (12.7 µg/dL) when cortisol was measured with R2, and 368 nmol/L (13.3 µg/dL) with LC-MS/MS. CONCLUSIONS: The use of more specific cortisol assays results in lower cortisol concentrations. This could lead to misdiagnosis and overtreatment when assessing AI with the ACTH test if a different cut-off for cortisol peak is not adopted.

Characteristics of 1573 healthcare workers who underwent nasopharyngeal swab testing for SARS-CoV-2 in Milan, Lombardy, Italy.

OBJECTIVES: The management of healthcare workers (HCWs) exposed to confirmed cases of coronavirus disease 2019 (COVID-19) is still a matter of debate. We aimed to assess in this group the attack rate of asymptomatic carriers and the symptoms most frequently associated with infection. METHODS: Occupational and clinical characteristics of HCWs who underwent nasopharyngeal swab testing for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a university hospital from 24 February 2020 to 31 March 2020 were collected. For those who tested positive and for those who tested positive but who were asymptomatic, we checked the laboratory and clinical data as of 22 May to calculate the time necessary for HCWs to then test negative and to verify whether symptoms developed thereafter. Frequencies of positive tests were compared according to selected variables using multivariable logistic regression models. RESULTS: There were 139 positive tests (8.8%) among 1573 HCWs (95% confidence interval, 7.5-10.3), with a marked difference between symptomatic (122/503, 24.2%) and asymptomatic (17/1070, 1.6%) workers (p < 0.001). Physicians were the group with the highest frequency of positive tests (61/582, 10.5%), whereas clerical workers and technicians had the lowest frequency (5/137, 3.6%). The likelihood of testing positive for COVID-19 increased with the number of reported symptoms; the strongest predictors of test positivity were taste and smell alterations (odds ratio = 76.9) and fever (odds ratio = 9.12). The median time from first positive test to a negative test was 27 days (95% confidence interval, 24-30). CONCLUSIONS: HCWs can be infected with SARS-CoV-2 without displaying any symptoms. Among symptomatic HCWs, the key symptoms to guide diagnosis are taste and smell alterations and fever. A median of almost 4 weeks is necessary before nasopharyngeal swab test results are negative.

Impact of reference materials on accuracy in clinical chemistry.

The analytical accuracy of the results of routine clinical chemistry measurements is contributed by a two-steps mechanism, involving transferring trueness from a higher metrological and monitoring the time-stability of trueness itself. In both operations, different materials are used: however, accuracy in the routine assay of genuine patient samples has to be the end product of this overall process. To such an aim, the materials must show an intermethod behavior similar to that of patient sera, i.e., they have to show commutability. Definitions of commutability and methods for assessing such a property are mentioned. The following aspects of lack of commutability of materials are then discussed: frequency; effects on the measured interlaboratory variability; and effects on the recalibration of analytical systems. The causes giving rise to lack of commutability are neither clear or easy to be shown. Matrix effect is one of the main causes; also, differences in the characteristics of the component being measured are often responsible for noncommutability of materials for enzyme activity measurements. Examples of these two different situations are given. It is concluded that, for an efficacious overall quality assurance process, either a set of minimally processed patient sera or commutable reference materials are to be used in the operations concerned with the control of trueness. An additional alternative approach is based on the use of materials with system-specific assigned values.

Diagnostic value of four assays for lipase determination in serum: a comparative reevaluation.

We assessed the diagnostic value of four commercially available methods for determining pancreatic lipase (LPS) in serum (the turbidimetric procedure from Boehringer, two enzymatic approaches from Kodak and Poli, and an immunochemical assay) in a population of 46 hospitalized patients with acute abdominal pain. In 31 cases (67.4%), the final diagnosis was acute pancreatitis. When evaluated by means of receiver-operating characteristic (ROC) curves, no significant differences were found among the procedures. Concerning clinical efficiency, all the assays had values equal to or greater than 90%. Using the calculation of the overlap index (OI) as a statistical approach to quantify the clinical utility of various LPS assays, the test having the greatest potential for differentiating between patients with and without acute pancreatitis was the turbidimetric assay (OI = 0.14).

Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608).

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was > 99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43,650 and 41,700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at -20 degrees C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2+/-1.8 U/L (1.12+/-0.03 microkat/L) when measured, at 30 degrees C, by the Recommended Method of the International Federation of Clinical Chemistry.

Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Effects of suprofen on renal function in patients with rheumatoid arthritis.

Suprofen is a new potent analgesic with antiinflammatory properties that appears to inhibit prostaglandin synthetase in a tissue-selective manner, having relatively little effect on the kidneys of experimental animals. The effects were studied of one week of treatment of rheumatoid arthritis patients with suprofen or ibuprofen on Na+ and K+ excretion, creatinine clearance, urinary enzymes that are markers for tubular damage, and urinary prostaglandins such as PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin). Neither compound caused changes in renal function related to the week of treatment, but significant decreases in prostaglandins were observed: this change was fully reversible after discontinuation of the drug.

Determination of serum cholesterol on the Reflotron (BBR) dry chemistry analyser. Assessment of accuracy.

This investigation is aimed at assessing the accuracy of S-cholesterol determination by means of the Reflotron, an analytical system based upon the dry chemistry approach. Two sets of sera, one of them including specimens with elevated concentrations of urea, creatinine, glucose, urate or bilirubin, were assayed in parallel with the Refloton system, with a class-A "reference" method (including alkaline hydrolysis and extraction) and with a routine liquid chemistry enzymatic method. Results for both sets of sera indicated good agreement of the tested system with the routine method, whilst a small proportional negative bias was observed in comparison with the "reference" method. The use of the reference method as a basis for comparison allows more conclusive statements about accuracy to be made.

Improved direct specific determination of serum iron and total iron-binding capacity.

Serum iron is released from transferrin and reduced at pH 1.7 by treating serum with a 10 g/L ascorbic acid solution in 0.1 mol/L HCl. When ferrozine is added to this reagent, it forms a complex with iron that is as intensely colored as at higher pH values, and under these conditions no turbidity is produced. The second major interference, that from copper, is eliminated by adding 1 g of thiosemicarbazide per liter, which at a low pH forms a stable, uncolored complex with copper without affecting the reaction of ferrozine with iron.

Pediatric emergency laboratory.

The laboratory contribution in the care of a seriously ill child is essential to plan and organize the therapy after the first-step emergency care and to know the aethiology of the illness. The most acute syndromes in pediatric emergency care are: coma, convulsions, dehydration, metabolic disequilibrium, hypovolemic or anaphylactic shock, serious infectious diseases and chemical or drug poisoning. The laboratory tests which have to be available within few minutes are blood cell count, hemogasanalysis, sodium, potassium and calcium, glucose. Total proteins, serum creatinine and urea, bleeding tests, blood smear, sedimentation rate, ALT, AST, osmolality, urinary electrolytes and creatinine and cerebrospinal fluid examination should be available within sixty minutes. New accurate and rapid techniques and instrumentations make easier the diagnostic and therapeutical approach to pediatric emergency.

Establishing a reference system in clinical enzymology.

The goal of standardization for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the procedure is carried out. To pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has launched a project to establish a reference system in clinical enzymology. This system is based on three hinges: a) extensively evaluated and carefully described reference procedures, b) certified reference materials and c) a network of reference laboratories operating in a highly controlled manner. The original IFCC-recommended procedures for alanine aminotransferase, aspartate aminotransferase, creatine kinase, gamma-glutamyltransferase, lactate dehydrogenase and alpha-amylase have been slightly modified to optimize them at 37 degrees C, with the definition of detailed operating procedures. A group of laboratories perform these procedures manually, with self-made reagents on carefully calibrated instruments. Partially purified and stabilized materials, prepared in the past by the Community Bureau of Reference, have been re-certified by these laboratories for alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase activities. Using these materials and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Thus, clinical laboratories, which will use routine procedures with these validated calibrators to measure human specimens, can finally obtain values which are traceable to reference procedures.

Automation in urinalysis: evaluation of three urine test strip analysers.

A clinical laboratory evaluation was conducted on the Clinitek Auto 2000, the Super Aution Analyzer and the Urotron RL9 for the determination of glucose, protein, pH, blood, ketone-bodies and bilirubin.Precision of the systems was tested using three commercial control urine materials, and reported as the percentage of times the instrument repeats a certain value. Good repeatability was obtained with all the instruments.Accuracy of the systems was evaluated by comparison with quantitative procedures, and to check agreement between methods yielding semi-quantitative and quantitative results, ranges of acceptability were defined, based on the criteria reported in a previous paper [2]. It was then found that 87.5 to 98.9% of results from the Urotron RL9 and the Clinitek Auto 2000 were acceptable. With the Super Aution Analyzer the level of agreement was apparently lower because of the higher number of concentration steps used by this instrument.

Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.

Optimization of the urinary acid alpha-glucosidase determination.

The optimization of the method for acid alpha-glucosidase determination (EC 3.2.1.3) in human urines, employing the synthetic substrate 4-nitrophenyl-alpha-D-glucopyranoside, is reported. Storage conditions of the specimens and their pretreatment were particularly investigated. The precision of the whole analytical procedure (including gel filtration) is good (within-run CV = 7.4% for normal samples and 3.7% for elevated ones). The correlation with the method using maltose as substrate is excellent (y = -0.01 + 0.13 x; r = 0.9893).

Multicentre evaluation of the Monarch (IL) clinical chemistry analyser.

A multicentre evaluation of the Monarch centrifugal analyser is reported. Precision, linearity and accuracy were assessed by comparison with routine methods. Calibration stability, photometric and dispensing accuracy, and carry-over related to samples and reagents were also evaluated. The overall performance of the instrument was good, showing an excellent photometric and dispensing accuracy, absence of sample-dependent carry-over, and almost negligible reagent carry-over. Good precision, linearity and correlation with routine methods were found for the parameters tested. The instrument is reliable and is now used as the routine clinical chemistry analyser in two of the three laboratories taking part in the evaluation.

Commutability of control materials in glycohemoglobin determinations.

The intermethod variabilities of control materials and patient blood samples for the measurement of glycohemoglobin were compared. Sets of 50 blood samples and 15 control materials were analyzed by HPLC and affinity and immunochemical methods. For each pair of methods, the distances of the materials from the regression line of patient blood results (expressed as normalized residuals) were calculated. Only two of 15 controls had normalized residuals exceeding 3 standard deviations from the regression line. Total hemoglobin (Hb) content, Hb derivatives, and cellulose acetate electrophoresis demonstrated that only a minority of controls could be considered similar to patients' blood samples. We selected Menarini's and our home-prepared controls to simulate calibration of the different techniques by these materials. Intermethod calibration succeeded mostly in harmonizing results obtained by HPLC methods. On the contrary, calibration of the immunochemical techniques (Boehringer and Roche) did not improve intermethod agreement to a clinically useful level.

Behavior of frozen serum pools and lyophilized sera in an external quality-assessment scheme.

To further improve analytical accuracy in clinical chemistry, proficiency testing needs amelioration in the quality of materials tested and in target value assignment. To obtain information on the actual state-of-the-art in the Lombardy region of Italy, and to examine the behavior of different types of control materials (fresh-frozen human sera and lyophilized materials), we developed the following experimental design. Two human serum pools and two lyophilized sera were distributed to 32 laboratories for determination of glucose, creatinine, cholesterol, sodium, potassium, and gamma-glutamyltransferase (gamma-GT). Each analyte was measured in triplicate on each of 3 days. Target values for the controls were obtained with Reference Methods. The results show a good intralaboratory precision for every component but some accuracy problems for glucose, creatinine, cholesterol, and gamma-GT. Lyophilized materials showed some commutability problems for glucose, electrolytes, and gamma-GT, mainly with dry chemistry technology.