OBJECTIVE: The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking.
METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated.
RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method.
CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Street food outlets are characterised by poor microbiological quality of the food and poor hygiene practices that pose a risk to consumer health. The aim of the study was to evaluate the hygiene of surfaces in food trucks (FT) using the reference method together with alternatives such as PetrifilmTM and the bioluminescence method. TVC, S. aureus, Enterobacteriaceae, E. coli, L. monocytogenes, and Salmonella spp. were assessed. The material for the study consisted of swabs and prints taken from five surfaces (refrigeration, knife, cutting board, serving board, and working board) in 20 food trucks in Poland. In 13 food trucks, the visual assessment of hygiene was very good or good, but in 6 FTs, TVC was found to exceed log 3 CFU/100 cm2 on various surfaces. The assessment of surface hygiene using various methods in the food trucks did not demonstrate the substitutability of culture methods. PetrifilmTM tests were shown to be a convenient and reliable tool for the monitoring of mobile catering hygiene. No correlation was found between the subjective visual method and the measurement of adenosine 5-triphosphate. In order to reduce the risk of food infections caused by bacteria in food trucks, it is important to introduce detailed requirements for the hygiene practices used in food trucks, including techniques for monitoring the cleanliness of surfaces coming into contact with food, in particular cutting boards and work surfaces. Efforts should be focused on introducing mandatory, certified training for food truck personnel in the field of microbiological hazards, appropriate methods of hygienisation, and hygiene monitoring.

In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF).
To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode.
A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high).
The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.

OBJECTIVE: The aim of the current study was to establish a reliable candidate reference method for serum 25-hydroxyvitamin D [25(OH)D] measurement and to assess the commutability of multiple control materials among liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.
METHODS: Serum 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] together with spiked internal standards were extracted with a one-step approach and then analyzed by LC-MS/MS. The commutability assessment for 25(OH)D was conducted according to the Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol. 25(OH)D concentrations in 5 levels of unprocessed serum pools, 7 levels of serum pools spiked with 25(OH)D3 or 25(OH)D2, 3 levels of commercial control materials, 2 levels of spiked bovine serum, and 4 levels of external quality assessment (EQA) materials were measured along with 30 single-donor samples using the candidate reference method and two routine LC-MS/MS methods.
RESULTS: The candidate reference method could separate 25(OH)D2 and 25(OH)D3 from 14 potential interfering compounds completely within a 9-min analysis time. Good method precision was obtained, and measurement results on certified reference material NIST SRM 972a were within the uncertainty of the certified values. All candidate materials were assessed commutable for LC-MS/MS methods.
CONCLUSIONS: The candidate reference method for serum 25(OH)D measurement is precise, accurate, and robust against interferences and can provide an accuracy base for routine methods. The multiple alternative control materials with commutability among LC-MS/MS methods will facilitate the further standardization for serum 25(OH)D measurement.

Blood glucose measurement is one of the most commonly performed clinical diagnostic tests used to monitor glycaemia in several animal diseases. Usually, these laboratory analyses are performed on blood venous samples in remote laboratories, and the results are delayed, at best. The use of portable glucometers could evidently solve many constraints but veterinary-use glucometers are not usually available. The present study aimed to compare blood glucose levels obtained by Bionime glucometer to the reference method using glucose oxidase. Venous blood was collected from a total number of 140 healthy dogs (72 males and 68 females), of different breeds (28 German Shepherd, 27 Pitt bull, 21 Boxer, 24 Rottweiler and 40 cross-bred dogs) and different ages (range: 3 months-14 years) for glucose measurement using the reference laboratory method. Capillary blood samples were used to conduct a glucose measurement with a human-use glucometer. Our results revealed that there was no significant difference between the mean capillary blood glucose (CBG) measured with the human-use glucometer (5.06 ± 0.84 mmol/L) and the mean venous blood glucose (VBG) measured with the laboratory reference method (4.90 ± 0.73 mmol/L) (p = 0.42). Similarly, there was no significant difference of the mean CBG and VBG in male dogs (5.11 ± 0.88 and 4.97 ± 0.75 mmol/L, respectively) and female dogs (5.01 ± 0.81 and 5.07 ± 0.72 mmol/L, respectively) (p = 0.73 and 0.21, respectively), and no correlation to neither age (5.43 ± 0.90 and 5.20 ± 0.70 mmol/L in 3 to 6 month-old dogs, 5.03 ± 0.82 and 4.94 ± 0.79 mmol/L in 6 months to 1 year-old, 4.94 ± 0.67 and 5.13 ± 0.66 mmol/L in 1 to 4 year-old dogs; 4.88 ± 0.94 and 4.80 ± 0.75 mmol/L in dogs older than 4 years, respectively, p < 0.05), nor to breed (4.94 ± 1.01 and 4.99 ± 0.79 mmol/L in German Shepherd, 5.13 ± 0.84 and 4.99 ± 0.79 mmol/L in Pitt Bull, 5.07 ± 0.94 and 5.07 ± 0.77 mmol/L in Boxer, 5.40 ± 0.59 and 5.48 ± 0.55 mmol/L in Rottweiler and 4.89 ± 0.75 and 4.77 ± 0.59 mmol/L in cross-bred dogs, respectively, p < 0.05). The present study confirms that human glucometer can be used to measure glucose in dogs with a good accuracy.

UNASSIGNED: In 2009, the Japan Society of Clinical Chemistry (JSCC) recommended a reference method for the measurement of serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels. This automated method uses cholesterol esterase-cholesterol dehydrogenase to measure cholesterol levels in fractions obtained after ultracentrifugation and dextran sulfate/magnesium chloride precipitation. In the present study, using fresh samples, we compared the LDL-C and HDL-C levels measured using this method with those measured using the traditional Centers for Disease Control and Prevention (CDC)-beta-quantification (BQ) method.
UNASSIGNED: and methods: Using both the JSCC and CDC-BQ methods, LDL-C/HDL-C levels were measured in 47 non-diseased and 126 diseased subjects, whose triglyceride levels were lower than 11.29 ​mmol/L (1000 ​mg/dL).
UNASSIGNED: For LDL-C, the equation of the line representing the correlation between the two methods was y ​= ​0.991x + 0.009 ​mmol/L; r ​= ​0.999; and Sy/x ​= ​0.025 ​mmol/L, where x is the mean LDL-C level measured using the CDC-BQ method. Similarly, for HDL-C, the equation of the line representing the correlation between the two methods was y ​= ​0.988x + 0.041 ​mmol/L, r ​= ​0.999, and Sy/x ​= ​0.019 ​mmol/L, where x is the mean HDL-C level measured using the CDC-BQ method.
UNASSIGNED: The JSCC method agreed with the CDC-BQ method in cases of both non-diseased and diseased subjects, including those with dyslipidemia.

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Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization.
A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed.
A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64).
Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.

Nitric oxide (NO) and its by-products are important biological signals in human physiology and pathology particularly in the vascular and immune systems. Thus, in situ determination of the NO-related molecule (NOx) levels using embedded sensors is of high importance particularly in the context of cellular biocompatibility testing. However, NOx analytical reference method dedicated to the evaluation of biomaterial biocompatibility testing is lacking. Herein, we demonstrate a PAPA-NONOate-based reference method for the calibration of NOx sensors. After, the validation of this reference method and its potentialities were demonstrated for the detection of the oxidative stress-related NO secretion of vascular endothelial cells in a 3D tissue issued from 3D printing. Such NOx detection method can be an integral part of cell response to biomaterials. Graphical abstract.

OBJECTIVE: To compare fosfomycin susceptibility testing with the commercial agar dilution (AD) test, AD Fosfomycin (Liofilchem, Roseto degli Abruzzi, Italy) and the reference AD method, using a collection of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa clinical isolates.
METHODS: The collection included 119 carbapenemase-producing Enterobacterales, 53 Enterobacterales producing acquired AmpC-type and/or extended-spectrum β-lactamases and 38 carbapenemase-producing P. aeruginosa, including representatives of different high-risk clones. AD Fosfomycin and AD reference method (ISO 20776-1:2019) were performed starting from the same microbial suspension. Results were interpreted according to EUCAST clinical breakpoints (10.0). Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization.
RESULTS: Of 172 Enterobacterales, 143 (83.1%, including 92.9% (52 of 56) of the NDM-producers and 84.2% (48 of 57) of the KPC-producers) were susceptible to fosfomycin using reference AD. A CA of 91.9% (158 of 172; 95% CI 87.1%-95.3%) and an EA of 92.5% (136 of 147; 95% CI 87.4%-96.0%), respectively, were calculated for the commercial AD Fosfomycin test, with 9.8% (14 of 128) of major errors and no very major errors (0 of 29). Overall, 86.8% (33 of 38) of P. aeruginosa showed a fosfomycin MIC ≤128 mg/L using reference AD. An EA of 84.8% (95% CI 66.3%-92.0%) was calculated for the commercial AD Fosfomycin test, with a CA of 100% (95% CI 93.6%-100%) when considering a tentative breakpoint at 128 mg/L.
CONCLUSIONS: AD Fosfomycin showed an overall good concordance compared with reference AD.