Atherosclerosis (AS) is characterized by chronic inflammation, which is a significant pathogenic factor of cardiovascular diseases (CVDs). Alleviating endothelial dysfunction and monocyte adhesion are effective ways to halt the development of AS. Membrane Spanning 4-Domains A6A (MS4A6A) is associated with inflammation and primarily regulates immunity and cell signaling. These processes are closely related to the occurrence of AS. However, the specific mechanism remains unclear. In this study, bioinformatics analysis revealed that MS4A6A expression was elevated in human atherosclerotic plaques. Western blot, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence analyses confirmed that MS4A6A expression correlated with the severity of AS and was significantly expressed in endothelial cells. We determined that MS4A6A was upregulated in atherosclerotic lesions of high-cholesterol diet (HFD) ApoE-/- mice. In a cellular model using human umbilical vein endothelial cells (HUVECs) stimulated with oxidized low-density lipoprotein (ox-LDL), MS4A6A expression exhibited a temporal and concentration-dependent upregulation. Silencing MS4A6A reduced endothelial dysfunction and monocyte adhesion, decreasing the expression of inflammatory factors, adhesion molecules, and reactive oxygen species (ROS). The pathway inhibitor Bay 11-7085 (irreversible inhibitor of IxBalpha phosphorylation) using IκB kinase (IKK) silencing showed that MS4A6A promotes endothelial dysfunction and monocyte adhesion by regulating the IKK/NF-kappaB pathway. This study demonstrated for the first time that MS4A6A facilitates endothelial dysfunction and monocyte adhesion by modulating the IKK/NF-κB signaling pathway, thereby promoting the progression of AS. This study provides a theoretical foundation for utilizing MS4A6A as a biomarker and potential therapeutic target for the prevention and treatment of AS.

Background Diabetic nephropathy (DN) is the most intractable complication of diabetes. Despite decades of research, accurate diagnostic markers and effective therapeutic drugs are still elusive. Abnormal copper metabolism is also implicated in diabetes and its complications. This study aims to identify copper metabolism-related biomarkers and potential drugs for DN. Methods DN datasets and copper metabolism-related genes (CMGs) were obtained from Gene Expression Omnibus (GEO) and GeneCards. Differentially expressed CMGs (DE-CMGs) were identified using the limma package and the Venn algorithm. Functional enrichment analysis and protein-protein interaction (PPI) network were performed to identify candidate hub genes. The single gene with an area under the receiver operating characteristic (ROC) curve > 0.7 was identified as a potential diagnostic biomarker of DN. Finally, these biomarkers were validated by quantitative real-time polymerase chain reaction (qRT-PCR) in high-glucose-treated human proximal tubular (HK-2) cells. These validated hub genes were used to construct a combined prediction model, confirmed by additional GSE30528 and GSE30529 datasets. The correlation analysis between the expression level of the hub genes and the estimated glomerular filtration rate (eGFR) was carried out. Additionally, immune cell infiltration and potential target drugs were investigated for these biomarkers. Results Five hub genes associated with copper metabolism, namely CD36, CCL2, CASP3, LPL, and APOC3, were identified as biomarkers for the early diagnosis of DN. Utilizing multiple biomarkers enhanced diagnostic accuracy and specificity. CD36, CCL2, and CASP3 correlated negatively with eGFR levels, while LPL and APOC3 correlated positively. Additionally, these hub genes were significantly linked to various immune cell types, including macrophages M1 and M2, T cells, gamma delta resting dendritic cells, neutrophils, and NK cells. Furthermore, 15 agents targeting these biomarkers were retrieved from the DrugBank database. Conclusion Our study identified key genes possibly related to copper metabolism in the pathological mechanism of DN that could serve as novel targets for the diagnosis and therapy of DN.

Introduction: Circular RNA (circRNAs) are a type of non-coding RNA (ncRNAs) with a wealth of functions. Recently, circRNAs have been identified as important regulators of diabetic kidney disease (DKD), owing to their stability and enrichment in exosomes. However, the role of circRNAs in exosomes of tubular epithelial cells in DKD development has not been fully elucidated. Methods: In our study, microarray technology was used to analyze circRNA expression in cell supernatant exosomes isolated from HK-2 cells with or without high glucose (HG) treatment. The small interfering RNAs (siRNA) and plasmid overexpression were used to validate functions of differentially expressed circRNAs. Results: We found that exosome concentration was higher in HG-stimulated HK-2 cells than in controls. A total of 235 circRNAs were significantly increased and 458 circRNAs were significantly decreased in the exosomes of the HG group. In parallel with the microarray data, the qPCR results showed that the expression of circ_0009885, circ_0043753, and circ_0011760 increased, and the expression of circ_0032872, circ_0004716, and circ_0009445 decreased in the HG group. Rescue experiments showed that the effects of high glucose on regulation of CCL2, IL6, fibronetin, n cadherin, e cadherin and epcam expression can be reversed by inhibiting or overexpressing these circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses indicated that circRNA parental genes are associated with glucose metabolism, lipid metabolism, and inflammatory processes, which are important in DKD development. Further analysis of circRNA/miRNA interactions indicated that 152 differentially expressed circRNAs with fold change (FC) >= 1.5 could be paired with 43 differentially expressed miRNAs, which are associated with diabetes or DKD. Discussion: Our results indicate that exosomal circRNAs may be promising diagnostic and therapeutic biomarkers, and may play a critical role in the progression of DKD.

Objectives: To investigate the influence of preservation methods and processes on the plasma interleukin-6 (IL-6) stability. Methods: Lithium-heparin plasma was collected from female patients: 32 female patients with invasive breast neoplasms and 20 healthy females. Each sample was divided into three tubes. Samples were stored at different temperatures or at different times. The concentration of IL-6 was detected. Results: IL-6 levels in patients were not altered significantly compared to the control group after storage at 4 degrees C or 25 degrees C for 12 h. However, IL-6 levels were significantly higher compared to controls (p<0.05) after storage at 25 degrees C for 48 h. IL-6 levels in patients with breast neoplasms were significantly higher compared to the control group (p<0.05) when stored at 4 degrees C after 12 h. IL-6 levels in patients with breast neoplasm increased more than 10-folds compared to the control group after only 2 h storage at 25 degrees C. Conclusions: Concentrations of IL-6 in breast neoplasms samples significantly change under different storage conditions. Pretreatment needs to be standardized for blood sample handling procedure. Comparison of different storage conditions of IL-6 levels may not be reliable.

Background Serum creatinine (SCr) is a useful diagnostic marker for the assessment of renal function. Accurate quantitation of SCr is clinically important in calculation of glomerular filtration rate (GFR). Method To confirm whether there are differences in SCr between enzymatic kits of different manufacturers, the analytical performance of the matched and open test system in the measurement of SCr was evaluated. The analytical performance evaluation was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Precision, accuracy, linearity, dilution, lower limit of measurement and analytical interference were studied between the two test systems. Results The performance of SCr from the open test system was in compliance with the matched test system with good precision, accuracy, and linearity. In presence of most common interferents, both test systems could lead to accurate creatinine results except for the existence of specified drugs. For dobutamine, the open test system showed better anti-interference performance than the matched system. Conclusion This study provides referable opinions for clinical laboratory selection on the test system and a framework for future analogous studies based on different test systems.

目的 探讨样本不同保存时间和温度对电化学发光法(ECLIA)检测白细胞介素-6(IL-6)结果的影响.方法 收集广州中医药大学第二附属医院2019年9-12月健康人群20名(健康对照组)、乳腺癌术后化疗患者30例和10例脓毒症患者肝素锂抗凝全血标本,采用ECLIA检测IL-6浓度.样本采集后立即离心检测,结果记录为初始值(0h),样本离心后分离血浆,分别在室温25℃和4℃保存1、2、4、8、12、24、48、72 h后上机检测,记录结果并进行统计分析.结果 健康对照组,在4℃环境下保存72h内IL-6的浓度未出现明显变化;在室温25℃条件下保存48 h后,IL-6浓度显著升高,差异有统计学意义(P＜0.05).乳腺癌术后化疗后患者IL-6的浓度在4℃条件下保存12h与0h差异无统计学意义(P＞0.05),而保存24h后IL-6的浓度显著升高,差异有统计学意义(P＜0.05);在25℃条件下保存2 h后IL-6的浓度显著升高,差异有统计学意义(P＜0.05).脓毒症患者的低值IL-6浓度组在25℃环境下保存12 h后与4℃比较差异有统计学意义(P＜0.05);而高值IL-6浓度组在4℃与25℃的条件下保存比较差异无统计学意义(P＞0.05).结论 不同的保存条件和时间对IL-6的浓度会产生一定的影响,不能及时检测的标本建议保存在更低的温度,同时尽快检测,以保证结果的可靠性和准确性.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11–101.61%. The relative bias between this candidate RMP and the established RMP was 2.76–1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials.

Background The detection of the Epstein–Barr capsid antigen (VCA) immunoglobulin A (IgA) is widely used in the diagnosis of nasopharyngeal carcinoma (NPC), but a reference standard for evaluating the presence of VCA-IgA is not yet available. Therefore, a reference standard is urgently needed for a uniform and quantitative detection of VCA-IgA. Methods A mixed reference serum from three NPC patients diluted with healthy subject serum was made as a potential first international standard for VCA-IgA. VCA-IgA was detected in twenty NPC patients by four ELISA kits and two chemiluminescent immunoassays kits using the reference as a calibration curve. The performance of these six kits was evaluated, and the quantitative results were compared. Results Our results showed a good linearity of the reference in different kits. Without reference, the difference of the total coefficient of variation (from 3.98 to 43.11%) and Within-run coefficient of variation (from 2.47 to 19.66%) was large in the 6 kits. The positive and negative coincidence rate between the 6 kits and indirect immunofluorescence for NPC diagnosis was 75% overall agreement, but a difference among the six kits was found, ranging from 55 to 90%. The concentration of VCA-IgA in the 20 NPC samples led in the division into three categories such as negative, low, or medium/high positive, but these concentrations were significantly different within these three categories depending on the kit used of the 6 considered. However,a good correlation (R 2 = 0.986) was observed between Antu and Beier ELISA kits. Conclusions The reference serum mightbe used as a reference standard for a better comparison of the results from different kits/laboratories. However, the quantitative results of some kits are still inconsistent due to the diversity of VCA antigens.

Background As Epstein–Barr capsid antigen (VCA) immunoglobulin A (IgA) detection is widely used for nasopharyngeal carcinoma (NPC),many immunoassays are available for VCA- IgA detection. Methods We evaluated the performance of different immunoassays for the quantitative detection of VCA-IgA. Twenty NPC patients were detected VCA-IgA by four ELISA kits and twochemiluminescent immunoassays(CLIA) kits using the reference as a calibration curve. The performance of six kits were evaluated, and the quantitative results were compared. Results Our results shown the reference has good linearity in different kits. Without reference, the difference of total CV (from 3.98% to 43.11%) and Within-run CV(from 2.47% to 19.66%) was large in 6 kits. The positive and negative coincidence rate between 6 kits and IFA for NPC diagnosis was 75% overall agreement, but there was a difference among the six kits ranged from 55%-90%. Concentration of samplesdivided into three categories was shown significant difference in certain subgroup of 6 kits. Almost all assays gave a correlation coefficient lower than 0.80, except for Antu and Beier kit and Tarcine kit and New Industries kit. Conclusions The reference serum may be used as a reference standard and better compared results from different methods/laboratories.Despite the use of our reference, the quantitative results of each kit are still quite different.We should try to unify the diversity between different antigens to promote the development of VCA-related research in the further.

Background: The presence of hemoglobinopathies could interfere with some assays for Hemoglobin A(1c) (HbA(1c)) measurement; therefore, the effect of thalassemia on ion-exchange high-performance liquid chromatography (IEHPLC) method Tosoh HLC-723 G8 (Tosoh G8) was evaluated. Methods: A total of 43 normal controls and 101 thalassemia patients were quantified by Premier Hb9210 and Tosoh G8 (variant-mode) systems. At the same time, 7 normal controls and 8 thalassemia patients were confirmed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method for verification. Results: For normal controls, the HbA(1c) values of Tosoh G8 system (y) showed great correlation and agreement with those from Premier Hb9210 (x) (y = 0.9688x + 0.2151, r = 0.9951; mean difference 0.02 +/- 0.30%), and no significant relative bias above 7% was observed; the HbA(1c) values obtained by Tosoh G8 were consistent with the IFCC targets (relative bias < +/- 6%) in all of the samples. However, for thalassemia, the correlation between Tosoh G8 (y) and Premier Hb9210 (x) became relatively low (y = 0.8079x + 1.2897, r = 0.7780); the HbA ie values of 91.1% of the samples (92/101) obtained by Tosoh G8 were higher than those by Premier Hb9210 (mean difference 0.33 +/- 0.48%) and a significant positive bias above 7% was noticed in 43.3% (45/101) thalassemia patients; when compared with the IFCC targets, the 87.5% (7/8) relative bias was > +/- 6%. Conclusions: Thalassemia could directly affect the measurement of HbA(1c) using the IE-HPLC method Tosoh G8 and the clinical laboratorial staff should pay close attention.

Introduction Thalassemia could interfere with some assays for haemoglobin A1c (HbA1c) measurement, therefore, it is useful to be able to screen for thalassemia while measuring HbA1c. We used Capillarys 2 Flex Piercing (Capillarys 2FP) HbA1c programme to simultaneously measure HbA1c and screen for thalassemia. Materials and methods Samples from 498 normal controls and 175 thalassemia patients were analysed by Capillarys 2FP HbA1c programme (Sebia, France). For method comparison, HbA1c was quantified by Premier Hb9210 (Trinity Biotech, Ireland) in 98 thalassaemia patients samples. For verification, HbA1c from eight thalassaemia patients was confirmed by IFCC reference method. Results Among 98 thalassaemia samples, Capillarys 2FP did not provide an HbA1c result in three samples with HbH due to the overlapping of HbBart’s with HbA1c fraction; for the remaining 95 thalassaemia samples, Bland-Altman plot showed 0.00 ± 0.35% absolute bias between two systems, and a significant positive bias above 7% was observed only in two HbH samples. The HbA1c values obtained by Capillarys 2FP were consistent with the IFCC targets (relative bias below ± 6%) in all of the eight samples tested by both methods. For screening samples with alpha (α-) thalassaemia silent/trait or beta (β-) thalassemia trait, the optimal HbA2 cut-off values were ≤ 2.2% and > 2.8%, respectively. Conclusions Our results demonstrated the Capillarys 2FP HbA1c system could report an accurate HbA1c value in thalassemia silent/trait, and HbA2 value (≤ 2.2% for α-thalassaemia silent/trait and > 2.8% for β-thalassemia trait) and abnormal bands (HbH and/or HbBart’s for HbH disease, HbF for β-thalassemia) may provide valuable information for screening.

Haemoglobinopathies may interfere with the haemoglobin A1c (HbA1c) measurement, leading to incorrect diagnosis and inappropriate treatment. It is essential that HbA1c assays are capable of identifying haemoglobinopathies. We report two cases of haemoglobin New York (HbNY) discovered through HbA1c analysis using capillary electrophoresis (Capillarys 2 Flex Piercing [C2FP], Sebia). We used these samples to evaluate the ability of three other HbA1c assays to identify this variant: ion-exchange high-performance liquid chromatography (Variant II Turbo [VII-T], Bio-Rad); boronate affinity high-performance liquid chromatography (Ultra2, Trinity Biotech) and immunoassay (Cobas c501 Tina-quant Generation 3, Roche Diagnostics). Each method was used for HbA1c assay of in samples from two cases of heterozygous haemoglobinopathy: β0-thalassemia/HbNY (Case 1) and HbA/NY (Case 2). Only the C2FP system detected HbNY (an additional peak appeared between HbA1c and HbA0). Clinical laboratories should be aware of the limitations of their HbA1c assay methods especially in geographic areas, where haemoglobinopathy prevalence is high.

BACKGROUND:Serum alkaline phosphatase (ALP) plays a critical role in the diagnosis of various diseases, and the establishment of relevant, reliable reference intervals (RI) is key to avoiding misdiagnoses. In 2011, IFCC published the new reference measurement procedure (RMP) for the determination of serum ALP in which one of the main modifications was the measuring temperature of the assay. Here, the new RMP was used to help establish RIs for serum ALP concentrations in healthy Chinese Han.METHODS:Volunteer individuals in Guangdong province, China (n=1622) were screened by questionnaire and laboratory testing for eligibility as a reference. Blood (20 mL) was collected and samples were measured by the Roche Modular system using the new RMP for the serum ALP compatible method. Partitioning of values by gender and/or age was evaluated with a standard normal deviate test after removing outliers. A simple non-parametric method for a two-sided 95% distribution of reference values was calculated.RESULTS:Serum ALP concentrations were obtained from the cohort of eligible reference individuals (n=658). The RI for serum ALP in males age 18-79 years was 48-131 U/L. Females were partitioned into two age groups based on statistical analysis, 18-49 years and 50-79 years, and the RIs derived were 40-106 U/L and 57-159 U/L, respectively.CONCLUSIONS:RIs for serum ALP for Chinese Han individuals in between the ages of 18 and 79 years were determined and required partitioning due to the higher ALP values of females age 50-79 years.

Introduction Haemoglobin A1c (HbA1c) is widely used in the management of diabetes. Therefore, the reliability and comparability among different analytical methods for its detection have become very important. Materials and methods A comparative evaluation of the analytical performances (precision, linearity, accuracy, method comparison, and interferences including bilirubin, triglyceride, cholesterol, labile HbA1c (LA1c), vitamin C, aspirin, fetal haemoglobin (HbF), and haemoglobin E (Hb E)) were performed on Capillarys 2 Flex Piercing (Capillarys 2FP) (Sebia, France), Tosoh HLC-723 G8 (Tosoh G8) (Tosoh, Japan), Premier Hb9210 (Trinity Biotech, Ireland) and Roche Cobas c501 (Roche c501) (Roche Diagnostics, Germany). Results A good precision was shown at both low and high HbA1c levels on all four systems, with all individual CVs below 2% (IFCC units) or 1.5% (NGSP units). Linearity analysis for each analyzer had achieved a good correlation coefficient (R2 > 0.99) over the entire range tested. The analytical bias of the four systems against the IFCC targets was less than ± 6% (NGSP units), indicating a good accuracy. Method comparison showed a great correlation and agreement between methods. Very high levels of triglycerides and cholesterol (≥ 15.28 and ≥ 8.72 mmol/L, respectively) led to falsely low HbA1c concentrations on Roche c501. Elevated HbF induced false HbA1c detection on Capillarys 2FP (> 10%), Tosoh G8 (> 30%), Premier Hb9210 (> 15%), and Roche c501 (> 5%). On Tosoh G8, HbE induced an extra peak on chromatogram, and significantly lower results were reported. Conclusions The four HbA1c methods commonly used with commercial analyzers showed a good reliability and comparability, although some interference may falsely alter the result.

OBJECTIVE:To establish of blood beta hydroxybutyrate (βOHB) threshold for diagnosing type 2 diabetes ketoacidosis (DKA) and explore the relationship between βOHB levels and the severity of DKA.METHODS:Correlation analysis was performed between serum βOHB and [HCO(3)] in type 2 diabetic patients admitted in the emergency department in the past year. Regression equation was used to calculate the concentration of βOHB corresponding to a [HCO(3)] level of 18.0, 15, and 10.0 mmol/L, and βOHB concentration corresponding to a [HCO(3)] level of 18.0 mmol/l was used as the DKA diagnostic threshold.RESULTS:The serum βOHB level and [HCO3] concentration showed a good correlation (R²=0.7023, P<0.001). βOHB concentrations that corresponded to a [HCO(3)] level of 18.0, 15, and 10.0 mmol/L were 3.0, 4.70, and 7.5 mmol/L, respectively, in accordance with the severity of DKA. Combined with the blood glucose concentration ≥ 13.9 mmol/L, a blood βOHB≥3.0 mmol/L showed a sensitivity of 99%, specificity of 86%, and total effectiveness of 92.81% for diagnosing DKA.CONCLUSION:A serum βOHB level above 3.0 mmol/L can be used as the diagnostic threshold of DKA. βOHB can serve as an index for assessing the severity of DKA.