Non-Kolbe electrolysis of N-protected-α-amino acids: a standardized method for the synthesis of N-protected (1-methoxyalkyl)amines

A high-yielding, standardized, electrochemical method for the synthesis of N-protected (1-methoxyalkyl)amines using commercially available, user-friendly kit – not only for “electro-curious” chemists.

Harmonization of PD-L1 Immunohistochemistry and mRNA Expression Scoring in Metastatic Melanoma: A Multicenter Analysis

Background: Melanoma is a type of cancer with robust response to immunotherapy. Programmed death ligand 1 (PD-L1) testing is not required to treat patients, but most studies have demonstrated correlations between PD-L1 expression and treatment response using various assays and scoring methods. This multicenter study aimed to harmonize PD-L1 immunohistochemistry (IHC) and scoring in melanoma. To provide a reference for PD-L1 expression independent of the IHC protocol, PD-L1 mRNA expression was determined via RNAscope, then compared to IHC.Methods: Standardized PD-L1 assays (22C3, 28–8, SP142, and SP263) and laboratory-developed tests (clone QR1 and 22C3) were evaluated on three IHC platforms using a training set of 7 cases. mRNA expression was determined via RNAscope (CD274/PD-L1 probe) and analyzed by image analysis. PD-L1 IHC findings were scored by seven blinded pathologists using the tumor proportion score (TPS), combined positive score (CPS), and MELscore. After the study, a standardized method was proposed; this was validated by three blinded pathologists on a set of 40 metastatic melanomas that were stained using three protocols.Results: Concordances among various antibody/platforms were high across antibodies (e.g., intraclass correlation coefficient [ICC] > 0.80 for CPS), except for SP142. Two levels of immunostaining intensities were observed: high (QR1 and SP263) and low (28–8, 22C3, and SP142). Reproducibilities across pathologists were higher for QR1 and SP263 (ICC ≥ 0.87 and ≥ 0.85 for TPS and CPS, respectively). QR1, SP263, and 28-8 showed the highest concordance with mRNA expression (ICC ≥ 0.81 for CPS). Accordingly, we proposed a standardized method for PD-L1 immunodetection and scoring, then tested it on 40 metastatic melanomas. This method included analysis of specimen quality (e.g., host-tumor interface and pigmentation). Concordances among antibodies were excellent for all criteria, and concordances among pathologists were better for the MELscore than for other scores.Conclusion: Harmonization of PD-L1 staining and scoring in melanomas with good concordance is achievable using the PD-L1 IHC protocols applied to other cancers; this reproducible approach can simplify daily practice. Our proposal for a PD-L1 standardized methodology has higher reproducibility across pathologists for clinical and research use.

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Rationale: Primary cardiomyocytes are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified cardiomyocytes at any postnatal age, without using different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of cardiomyocytes in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Hearts of C57BL/6J mice were cannulated using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates (postnatal day 0-2, P0-2). Hearts were then subjected to Langendorff retrograde perfusion and enzymatic digestion. Cardiomyocytes were isolated after subsequent tissue disaggregation and filtration, in high yield (1.56-2.2x106 cardiomyocytes/heart) and viability (~70-100%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal, cardiomyocytes relative to non-myocytes, allowed enrichment by differential centrifugation. Cardiomyocytes from all ages were further purified by immunomagnetic bead-based depletion of non-myocytes. Together, these procedures resulted in the isolation of highly purified cardiomyocytes (~94%) within 1 hour, enabling experiments using individual replicates. For example, RNA-sequencing of cardiomyocytes purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters and sex differences for nine differentially expressed genes (FDR<0.005). In situ fixation via coronary perfusion, performed immediately after tissue digestion, preserved the cytoarchitecture of isolated cardiomyocytes (yielding ~94% rod-shaped cardiomyocytes at all ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of cardiomyocyte area and nucleation state. Conclusion: The procedures developed here provide a universal protocol for the rapid isolation and purification of high-quality cardiomyocytes from hearts of any postnatal age, even those of neonates, thereby enabling direct comparisons between individual hearts.

Standardization of the method of contrast-enhanced ultrasound of kidneys in patients with type 2 diabetes

Aim of the research. Standardization of the quantitative assessment of the contrast-enhanced ultrasound examination of the kidneys in patients with type 2 diabetes.Materials and methods. On the basis of the Fundamental research laboratory “Diagnostic researches and minimally invasive technologies”, of the Federal State Budgetary Educational Institution of Higher Education “Smolensk State Medical University” of Smolensk State Medical University, the Ministry of Health of the Russian Federation, 12 patients with type 2 diabetes were examined in 2020. The age of the examined patients was 36–64 years old. Average age – 44 ± 1.8 years, of which 7 women (58.33%) and 5 men (41.67%). All patients were examined using a single diagnostic algorithm, which included 2 stages: 1 stage. Ultrasound examination of the kidneys (Aloka Hitachi Arietta 850 (Hitachi Medical Corporation, Japan) in B-mode; stage 2 – contrast-enhanced ultrasound examination (CEUS) of the kidneys. All patients were divided into 2 groups: the 1st group consisted of patients with type 2 diabetes type (n = 6), who underwent CEUS with subsequent assessment of the renal parenchyma in different areas of the cortex and medulla; group 2 consisted of patients with type 2 diabetes (n = 6) who underwent CEUS with subsequent standardized assessment in the indicated 5-th points of the arterial bed and 3 points of the venous bed.Results and discussions. Taking into account the current epidemiological situation in 2020, the number of performed radiation diagnostic methods, such as X-ray and computer examinations of the chest organs, significantly increased the overall background of radiation exposure on the bulk of patients. Taking into account the anxiety index of patients, ionizing research methods for the diagnosis of angionephrosclerosis are undesirable. The standardized method for quantitative parameters of CEUS showed good agreement (0.61–0.76). In turn, the assessment of the quantitative parameters of renal CEUS when using the routine technique showed low consistency (0.21–0.3). Cohen's kappa (CK) scores of 0 to 0.2 indicate a lack of agreement among clinicians; 0.21–0.4 – weak consistency; 0.41–0.6 – moderate agreement between doctors; 0.61–0.8 – good consistency; 0.81–1.0 – complete consistency.The circulatory system of the kidneys is characterized by the presence of a developed capillary network and, accordingly, low peripheral resistance, which confirms the importance of a standardized approach in conducting this study. In the context of the prevailing epidemiological circumstances, we understand that it is necessary to choose a reference research method that would allow us to accurately assess the characteristics of the experimental evaluation of this method. As a result of renal C EUS using a routine technique, hypoperfusion of the right kidney was revealed in 50% (n = 3) cases and the data were interpreted as normal in 50% (n = 3) cases. As a result of the renal CEUS using the proposed method, pronounced hypoperfusion of the right kidney was revealed in 100% (n = 6) cases. Dynamic nephroscintigraphy was used as a reference method to determine the functional work of the kidneys. According to the results of dynamic nephroscintigraphy of the kidneys, hypoperfusion of the right kidney was revealed in all cases in both groups of patients.Conclusion. 1. The standardized method for assessing the quantitative parameters of the kidney CEUS is more reproducible by the ultrasound doctors than the routine one. 2. Enhanced ultrasound can be used to assess the efficacy of hypoperfusion with the maximum efficacy of an equivalent dose per patient.

Comparison of Serum, Plasma, and Liver Zinc Measurements by AAS, ICP-OES, and ICP-MS in Diverse Laboratory Settings

Progress improving zinc nutrition globally is slowed by limited understanding of population zinc status. This challenge is compounded when small differences in measurement can bias the determination of zinc deficiency rates. Our objective was to evaluate zinc analytical accuracy and precision among different instrument types and sample matrices using a standardized method. Participating laboratories analyzed zinc content of plasma, serum, liver samples, and controls, using a standardized method based on current practice. Instrument calibration and drift were evaluated using a zinc standard. Accuracy was evaluated by percent error vs. reference, and precision by coefficient of variation (CV). Seven laboratories in 4 countries running 9 instruments completed the exercise: 4 atomic absorbance spectrometers (AAS), 1 inductively coupled plasma optical emission spectrometer (ICP-OES), and 4 ICP mass spectrometers (ICP-MS). Calibration differed between individual instruments up to 18.9% (p < 0.001). Geometric mean (95% CI) percent error was 3.5% (2.3%, 5.2%) and CV was 2.1% (1.7%, 2.5%) overall. There were no significant differences in percent error or CV among instrument types (p = 0.91, p = 0.15, respectively). Among sample matrices, serum and plasma zinc measures had the highest CV: 4.8% (3.0%, 7.7%) and 3.9% (2.9%, 5.4%), respectively (p < 0.05). When using standardized materials and methods, similar zinc concentration values, accuracy, and precision were achieved using AAS, ICP-OES, or ICP-MS. However, method development is needed for improvement in serum and plasma zinc measurement precision. Differences in calibration among instruments demonstrate a need for harmonization among laboratories.

Uncertainty in the standardized method "Characterization of the acoustic properties of road surfaces" by CEN TC227 WG5

TC227 of CEN has developed a method to determine the effect of the road pavement on the sound emission of road vehicles. The proposed methods can be applied to define the acoustic label value of a generic or proprietary pavement type, to check compliance of a pavement with the specifications for that pavement type and to monitor the development of the acoustic properties over the lifetime of the product. With the procedure one can additionally derive the coefficients for the pavement correc tion in the noise emission formulae for road vehicles in the CNOSSOS-EU calculation model. The application of the method exhibits a limited accuracy. The paper investigates the sources of uncertainty of the standardized method and combine the contributions into a single overall uncertainty according to the procedures laid down in Guide 98-3 of ISO. The uncertainty is determined for each of the listed application areas. From the uncertainty analysis the major contributions are identified. Improvement of the method shall focus on only these contributions.