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.

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.

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.

Certification of the mass concentration of creatine kinase isoenzyme 2 (CK-MB) in the reference material BCR 608.

We describe the certification of a mass concentration value in the already prepared creatine kinase-2 reference material (BCR 608). Creatine kinase-2 was purified from human heart. The purified enzyme was diluted in order to measure its protein concentration by the Doetsch method. A protein concentration value of 124.30+/-13.17 mg/l was assigned to the stock solution of purified creatine kinase-2. This stock solution was diluted in 25 mmol/l piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) pH 7.2, containing 2 mmol/l ADP, 5 mmol/l 2-mercaptoethanol, 154 mmol/l sodium chloride and 50 g/l human albumin to obtain a stable liquid standard of known creatine kinase-2 mass concentration (80.36 microg/l). This standard was then used to recalculate the creatine kinase-2 mass concentration measured in the BCR 608 material by immunoassay. The mass concentration of creatine kinase-2 in samples of reconstituted BCR 608 was certified to be 93.30+/-9.65 microg/l.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 917 analysis system.

The new selective access analysis system BM/Hitachi 917 was evaluated in an international multicentre study, mainly according to the ECCLS protocol for the evaluation of analysers in clinical chemistry. Forty-three different analytes, covering 56 different methods--enzymes, substrates, electrolytes, specific proteins, drugs and urine applications--were tested in seven European clinical chemistry laboratories. Additionally, the practicability of the BM/ Hitachi 917 was tested according to a standardized questionnaire. Within-run CVs (median of 3 days) for enzymes, substrates and electrolytes were <2% except for creatine-kinase MB isoform and lipase at low concentration. For proteins, drugs and urine analytes the within-run CVs were < 4% except for digoxin and albumin in urine. Between-day median CVs were generally < 3% for enzymes, substrates and electrolytes, and < 6% for proteins, drugs and urine analytes, except for lipase, creatine kinase and MB isoform, D-dimer, glycosylated haemoglobin, rheumatoid factors, digoxin, digitoxin, theophylline and albumin in urine in some materials. Linearity was found according to the test specifications or better and there were no relevant effects seen in drift and carry-over testing. The interference results clearly show that also for the BM/Hitachi 917 interference exists sometimes, as could be expected because of the chemistries applied. It is a situation that can be found in equivalent analysers as well. The accuracy is acceptable regarding a 95-105% recovery in standard reference material, with the exception of the creatinine Jaffé method. Most of the 160 method comparisons showed acceptable agreement according to our criteria: enzymes, substrates, urine analytes deviation of slope +/- 5%, electrolytes +/- 3%, and proteins and drugs +/- 10%. The assessment of practicability for 14 groups of attributes resulted in a grading of one-three scores better for the BM/Hitachi 917 than the present laboratory situation. In conclusion, the results of the study showed good analytical performance and confirmed the usefulness of the system as a consolidated workstation in medium-sized to large clinical chemistry laboratories.

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.

Multicentre evaluation of KONE Optima analysis system.

Optima from KONE Instruments Corporation is a new selective laboratory analyzer for turbidimetric, colorimetric and ion selective electrode measurements. Overall analytical performance of Optima and reagents provided by KONE was evaluated according to ECCLS guidelines, in a multicentre study involving four different laboratories, including substrates (cholesterol, high-density lipoprotein-cholesterol, creatinine), specific proteins (transferrin, IgG), enzyme activities (gamma-glutamyltransferase, alanine aminotransferase) and electrolytes (sodium, potassium, chloride). The results obtained attest good precision of the system for all the analytes tested: the range of within-run CV is 0.5 %-4.3 %, and range of between-day CV % is 0.8 %-7.9 % (median of four laboratories). Except for total cholesterol (5 % overestimation compared to the reference method) accuracy of measurement is adequate. Creatinine and uric acid assays were subjects of interference (defined as deviation > 10 % from target value) by bilirubin and haemoglobin respectively.

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.

Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay.

A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were < or = 1.87%; total CVs were < or = 3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method - 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below +/-5% as long as triglyceride concentrations are < or = 10 g/L and in case of moderate hypercholesterolemia.

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.

European multicentre evaluation of the Super Aution SA-4220 urinalysis analyser.

A multicentre evaluation of the urine test strip analyser Super Aution-4220 was carried out in six laboratories. The analytical performance of the instrument with regard to imprecision, linearity, detection limit, drift, carry-over and method comparison was studied. Using the Aution stick 8 test strip the pH, glucose, protein, ketones, bilirubin, blood, urobilinogen and leukocyte esterase were analysed. Specific gravity measurements were performed by refractive index method. Within-run and between-run imprecision determined at three levels of analyte were good. No carry-over was observed. Obtained results were linear through all the described analytical range. No significant drift was detected. Method comparison with some quantitative methods was performed and showed a good correlation with most of the analytes. The study of interferences showed minor interferences by common therapeutic drugs with the measurement of some analytes. During the assessment period of about 6 months no breakdown occurred in any laboratory. The Super Aution urine analyser appeared to be a highly automated analyser of urinary test strips. The operation was simple and the maintenance required only a few minutes a day.

Point: status of lipid and lipoprotein standardization.

Cholesterol and triglyceride standardization procedures have been used extensively and continuously since the 1950s. Definitive and Reference Methods, as well as primary and secondary standards, have been developed and maintained as the basis for evaluating the accuracy of results by various methods in many laboratories. But, although standardization efforts for apolipoprotein A-I and B measurements have been reported in detail in the scientific literature, much less has been reported in the area of total and lipoprotein cholesterol and triglyceride standardization efforts. Standardized cholesterol and triglyceride concentrations, determined in multiple large epidemiological and clinical studies, have been instrumental to the National Cholesterol Education Program panels that have assessed the lipoprotein values associated with risk of coronary disease, and have determined the cutpoints that are now used extensively by physicians to guide diagnosis and treatment of individual patients.

Creatinine measurement proficiency testing: assignment of matrix-adjusted ID GC-MS target values.

The results of an external quality-assessment experiment for serum creatinine measurement are described. Fifty-one laboratories performed quintuplicate analyses during three different analytical runs on six lyophilized sera and two frozen human serum pools. Isotope dilution gas chromatography-mass spectrometry (ID GC-MS) target values were assigned to all the materials. Intralaboratory within- and between-run imprecision results were very similar for all the materials tested (CV < or = 2.20% and < or = 4.70%, respectively). The overall imprecision obtained was high (CV 6.5-20.0%) because of increased interlaboratory-intermethod variability. A significant positive bias (+ 9.2-+43.7%) was found for all the materials at lower creatinine concentration. By using two human sera at different concentrations, we could calculate the constant and the proportional calibration bias displayed by each peer group. The majority of the lyophilized materials showed a behavior divergent from the frozen pools, indicating matrix-related problems. We propose a new algorithm for calculating matrix bias correction factor instrument-reagent specific for each material.

Accuracy of cholesterol measurements in Italian clinical laboratories. Joint project GISSI prevention--Italian Society of Clinical Biochemistry. SIBioC GISSI Prevenzione Group.

We report the results of an external quality assessment scheme for serum total cholesterol measurement involving about 100 Italian laboratories participating in an epidemiological study of post myocardial infarction. Two frozen human serum pools with Abell-Kendall assigned values are distributed quarterly at the laboratories (up to now seven events occurred); the obtained results are evaluated and discussed. In one exercise (# 5) duplicated measurements were repeated on three different days. Eighty-five to 98% of the laboratories obtained results within the total error limits (+/- 8.9%). But, while precision (calculated on the six replicates of exercise # 5) is good (90% of the laboratories obtained CV < 3%), inaccuracy problems are evident in every event. Indeed the mean bias from the reference method value ranged from 1.54 and 3.49% in the various events.

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.

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.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 747 analysis system.

Analytical performance and practicability of the new Boehringer Mannheim/Hitachi 747 analysis system were assessed in a multicentre evaluation involving four laboratories. The analytical performance was evaluated according to a protocol similar to the ECCLS guidelines and comprised 13 analytes including enzymes, substrates and electrolytes. About 65,000 results were obtained within three months. The evaluation was planned and supported by a program system called "Computer Aided Evaluation". Acceptance criteria have been established for judging the results. The median of the within-run coefficients of variation (CVs) in control sera of all methods was below 1%, being far below the acceptance limit of 2%. The median of CVs of between-days imprecision was below 2% (acceptance criterion 3%). The high degree of precision prompted us to set up a biometrical model suitable for the differentiation between deviant points, outliers and measurements that can still be explained by the system performance. No relevant drift effects were observed during eight hours. The methods were linear over a wide range, avoiding rerun analysis in most cases. No sample-related carry-over was found. Reagent-dependent carry-over outside the acceptance limits was measured from uric acid to phosphorus to a slight extent, and from triacylglycerols to lipase, as well as from total protein to bilirubin to a perceptible degree. It can be avoided by separating these reagent combinations in the channel arrangement. Taking a systematic deviation of more than 10% as unacceptable, four of the 13 analytes suffered from interference by haemoglobin, one by bilirubin and one by turbidity. The Boehringer Mannheim/Hitachi 747 analysis system is capable of determining serum indices which in combination with the interferogram allow an assessment of the interference. With the exception of chloride the recovery of the assigned values for all control sera showed values between 95 and 105%. Out of 40 method comparison studies for enzymes and substrates, 31 yielded regression equations with less than 5% proportional errors and less than 5% constant errors. Deviations exceeding these acceptance criteria can be explained by differences in the reagent formulation, in the method employed or in calibration. The agreement of the ISE method comparisons was within a +/- 5% deviation over a wide analytical range. Practicability of the Boehringer Mannheim/Hitachi 747 analysis system was assessed with the help of a questionnaire, in which properties of the instrument were quantified, thus permitting a relatively objective rating. The 190 questions were placed in 14 groups, each dealing with an attribute of the instrument.(ABSTRACT TRUNCATED AT 400 WORDS)