Analytical performance evaluation of different test systems on serum creatinine assay.

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.

Performances Evaluation of Four Systems for Homocysteine Determination by LC-MS/MS Reference Method.

BACKGROUND: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method. METHODS: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detection systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and accuracy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS reference method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared. RESULTS: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of variation of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibration, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R² ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum measured by the four detection systems after calibration decreased from 3.76 µmol/L, 0.96 µmol/L, 1.30 µmol/L, -1.56 µmol/L to 0.31 µmol/L, 0.28 µmol/L, 0.4 µmol/L, 0.40 µmol/L, respectively. The relative average bias decreased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration. CONCLUSIONS: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems.

A Pre-Analytical Performance Evaluation for Measurement of Serum Creatinine in a Multicenter Clinical Trial Study.

BACKGROUND: Our multicenter clinical trial study for stage 4 chronic kidney disease (CKD) populations was conducted at 21 centers in China during the period 2011 to 2016. The CKD definition is based on glomerular filtration rate (GFR) values which can be estimated by creatinine-based predictive formulas. The validity and reliability of GFR estimation is thus largely dependent on the accurate and precise serum creatinine (SCr) measurements. As an integral part of this multicenter study, it is important to ensure the precision, accuracy, and center-to-center comparability of the SCr results. METHODS: Prior to initiating the study, we unified the measurement method of SCr determination as an enzymatic method and standardized the procedure in all of the laboratories. Then, the analytical performance of each analyzer at each laboratory was evaluated, including precision, accuracy, and comparability. RESULTS: All within-run and total CVs of the low and high level internal quality control (IQC) were comprised between 0.2% and 4.1% (< 1/3 CLIA'88). Total error of the IQC fall within the maximum 12% at all centers. The analytical bias against the Standard Reference material 967a target was less than ± 0.5% at Central Laboratory, indicating good accuracy. Correlation between the analyzers and the reference method were very high (r > 0.99). Passing-Bablok regression showed no significant deviation from linearity (p > 0.05). Bland-Altman analysis also showed good agreement (≥ 95% of results fell within the 95% limits of agreement). CONCLUSIONS: Performance evaluation helped in addressing preanalytical variations in measurement and gave op-timal quality assurance of laboratory measurement in the context of a multicenter clinical trial study.

Interference of Thalassemias on Hemoglobin A1c Measurement by Ion-Exchange High-Performance Liquid Chromatography Method Tosoh HLC-723 G8.

BACKGROUND: The presence of hemoglobinopathies could interfere with some assays for Hemoglobin A1c (HbA1c) 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 HbA1c 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 HbA1c 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 HbA1c 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 HbA1c using the IE-HPLC method Tosoh G8 and the clinical laboratorial staff should pay close attention.

Clinical Correlates and Reference Intervals for Cystatin C in a Han Population from Southeast China.

BACKGROUND: Cystatin C (CysC) is an endogenous filtration marker for estimation of kidney function. This study aimed to define the reference interval (RI) for serum CysC in a southeast Chinese adult population and to explore the variables that affect serum CysC levels. METHODS: 532 reference individuals (259 male, 273 female, aged 18 - 79 years) were recruited from Guangzhou, China. Multiple regression analysis (MRA) was used to investigate the association between serum CysC levels and clinical factors including age, gender, body mass index, lifestyle, and biochemistry parameters. Reference values were defined using a parametric method according to Clinical and Laboratory Standards Institute guideline (C28A3). RESULTS: The mean serum CysC levels were significantly lower in females than in males (p < 0.001). Serum CysC levels increased with age (~0.047 mg/L increase per decade). MRA demonstrated that serum CysC levels correlated significantly with serum creatinine (Cr), high density lipoprotein (HDL-C), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) concentrations, although their relationships were less prominent than those of gender or age. The RIs for serum CysC levels were calculated at 0.73 - 1.17 mg/L for subjects aged 18 - 49 years and at 0.73 - 1.49 mg/L for those aged 50 - 79 years. CONCLUSIONS: The RIs for serum CysC were established in a southeast Chinese population. In addition to gender and age, serum Cr, HDL-C, ALP, ALB, and UA also influenced serum CysC levels.

Development of reference intervals for serum alkaline phosphatase among adults in Southern China traced to the new IFCC reference measurement procedure.

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.

[Higher procalcitonin level in diabetic nephropathy patients compared with healthy volunteers].

OBJECTIVE: To investigate the difference of procalcitonin (PCT) level between uninfected diabetic nephropathy patients and healthy volunteers. METHODS: This study enrolled 76 patients with diabetes only [DM group, 24 h urinary micro albumin (mALB) < 30 mg/24 h], 81 patients with early DN (EDN group, mALB 30-300 mg/24 h), 87 DN patients (DN group, mALB > or = 300 mg/24 h), and 82 age- and sex-matched healthy volunteers. All the patients were free of systemic infection. PCT levels and various laboratory parameters including metabolic and kidney functions as well as inflammatory element profiles were assessed. RESULTS: The PCT level of DN group was significantly higher than that of healthy control group, DM group and EDN group (P < 0.001 or P < 0.05). Spearman's test showed a significant positive correlation between PCT and serum lactate dehydrogenase (LDH, r = 0.541, P < 0.01), Urine acid (UA) (r = 0. 320, P < 0.01), Urea (r = 0.324, P < 0.01), creatinine (Cr) (r= 0.403, P < 0.01), alpha-hydroxybutyrate dehydrogenase (alpha-HBD) (r = 0.791, P < 0.01) and C-reactive protein (CRP) (r = 0.694, P < 0.001) in diabetic nephropathy patients respectively. CONCLUSION: Serum PCT level of patients with diabetic nephropathy is higher than that of healthy volunteers, which may be associated with minimal inflammation and kidney function damage.

Immunoglobulin A and complement C4 are involved in the progression of liver fibrosis in patients with chronic hepatitis B.

OBJECTIVE: To explore the relationship between immunoglobulin A (IgA), complement C4, and liver fibrosis (L.F.) progression (LFP) in patients with chronic hepatitis B (CHB). METHODS: This is a retrospective cohort study of CHB patients who received liver biopsies. Relevant data, including demographics, clinical serum markers, and immunological results obtained during liver biopsies, were collected and analyzed to assess and verify the relationship between IgA, C4, and LFP. RESULTS: This study included 1,938 CHB patients, of whom 132 received two liver biopsies (group 1). Thirty (22.7%) of these patients were diagnosed with LFP (increase in L.F. stage (Scheuer score F ≥ 1)). IgA (C-IgA) and C4 (C-C4) change values between the first and second biopsies were independent risk factors for LFP. IgA levels increased, and C4 levels decreased during the second liver puncture. The remaining 1,806 patients received one liver puncture (group 2). They were divided into the following subgroups: A (F ≤ 1), B (1 < F ≤ 3), and C (F > 3) to verify whether the same trend was observed by cross-sectional study. IgA levels were highest, and C4 levels were lowest in group C (IgA: C > B > A, p < 0.05; C4: C < B < A, p < 0.05). CONCLUSIONS: The findings of this study suggest that serum IgA and C4 levels are independent risk factors for LFP that could serve as future targets for L.F. management and treatment.

Development of a glucose reference material in human serum for clinical assay standardization.

BACKGROUND: Blood glucose is an important monosaccharide functioning as the main source of energy for the human body. The accurate measurement of blood glucose is crucial for the screening, diagnosis, and monitoring of diabetes and diabetes-associated diseases. To assure the reliability and traceability of blood glucose measurements, we developed a reference material (RM) for use in human serum at two different concentrations, which were certified by the National Institute of Metrology (NIM) as GBW(E)091040 and GBW(E)091043. METHODS: Raw serum samples were collected from residual samples after clinical testing, filtered, and repackaged under mild stirring. The homogeneity and stability of the samples were examined according to ISO Guide 35: 2017. Commutability was evaluated in compliance with CLSI EP30-A. Value assignment was carried out in six certified reference laboratories using the JCTLM-listed reference method for serum glucose. Moreover, the RMs was further applied in a trueness verification program. RESULTS: The developed RMs was homogeneous and commutable enough for clinical use. They were also stable for 24 h at 2-8 â„ƒ or 20-25 â„ƒ and for at least 4 years at - 70 â„ƒ. The certified values were 5.20 ± 0.18 mmol/L and 8.18 ± 0.19 mmol/L (k = 2) for GBW(E)091040 and GBW(E)091043, respectively. The pass rates were evaluated by bias, coefficient of variation (CV), and total error (TE) for 66 clinical laboratories in the trueness verification program were 57.6%, 98.5%, and 89.4% of GBW(E)091040, and 51.5%, 98.5%, and 90.9% of GBW(E)091043, respectively. CONCLUSION: The developed RM could be used for the standardization of reference and clinical systems with satisfactory performance and traceable values, providing strong support for the accurate measurement of blood glucose.

Identification of Copper Metabolism Related Biomarkers, Polygenic Prediction Model, and Potential Therapeutic Agents in Alzheimer's Disease.

BACKGROUND: The underlying pathogenic genes and effective therapeutic agents of Alzheimer's disease (AD) are still elusive. Meanwhile, abnormal copper metabolism is observed in AD brains of both human and mouse models. OBJECTIVE: To investigate copper metabolism-related gene biomarkers for AD diagnosis and therapy. METHODS: The AD datasets and copper metabolism-related genes (CMGs) were downloaded from GEO and GeneCards database, respectively. Differentially expressed CMGs (DE-CMGs) performed through Limma, functional enrichment analysis and the protein-protein interaction were used to identify candidate key genes by using CytoHubba. And these candidate key genes were utilized to construct a prediction model by logistic regression analysis for AD early diagnosis. Furthermore, ROC analysis was conducted to identify a single gene with AUC values greater than 0.7 by GSE5281. Finally, the single gene biomarker was validated by quantitative real-time polymerase chain reaction (qRT-PCR) in AD clinical samples. Additionally, immune cell infiltration in AD samples and potential therapeutic drugs targeting the identified biomarkers were further explored. RESULTS: A polygenic prediction model for AD based on copper metabolism was established by the top 10 genes, which demonstrated good diagnostic performance (AUC values). COX11, LDHA, ATOX1, SCO1, and SOD1 were identified as blood biomarkers for AD early diagnosis. 20 agents targeting biomarkers were retrieved from DrugBank database, some of which have been proven effective for the treatment of AD. CONCLUSIONS: The five blood biomarkers and copper metabolism-associated model can differentiate AD patients from non-demented individuals and aid in the development of new therapeutic strategies.

A population-based characterization study of anti-mitochondrial M2 antibodies and its consistency with anti-mitochondrial antibodies.

OBJECTIVE: This study aims to estimate the prevalence of anti-mitochondrial antibody subtype M2 (AMA-M2) and assess its consistency with AMA in a general population. METHODS: A total of 8954 volunteers were included to screen AMA-M2 using enzyme-linked immunosorbent assay. Sera with AMA-M2 >50 RU/mL were further tested for AMA using an indirect immunofluorescence assay. RESULTS: The population frequency of AMA-M2 positivity was 9.67%, of which 48.04% were males and 51.96% were females. The AMA-M2 positivity in males had a peak and valley value of 7.81% and 16.88% in those aged 40 to 49 and ≥70 years, respectively, whereas it showed a balanced age distribution in females. Transferrin and immunoglobulin M were the risk factors for AMA-M2 positivity and exercise was the only protective factor. Of 155 cases with AMA-M2 >50 RU/mL, 25 cases were AMA-positive, with a female-to-male ratio of 5.25:1. Only 2 people, with very high AMA-M2 of 760 and >800 RU/mL, met the diagnostic criteria of primary biliary cholangitis (PBC), making the prevalence of PBC 223.36 per million in southern China. CONCLUSION: We found that AMA-M2 has a low coincidence rate with AMA in the general population. A new decision-making point for AMA-M2 is needed to improve consistency with AMA and diagnostic accuracy.

Microarray Expression Profile of Exosomal circRNAs from High Glucose Stimulated Human Renal Tubular Epithelial Cells.

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.

A Simplified Herbal Formula Improves Cardiac Function and Reduces Inflammation in Mice Through the TLR-Mediated NF-κB Signaling Pathway.

Nuanxinkang tablet (NXK), a Chinese herbal formula, can improve heart function and quality of life in patients with chronic heart failure (CHF). However, the mechanisms of action of NXK are not fully understood. In this study, we investigated the effects of NXK on inflammation in the CHF mouse model. This model was established by transverse aortic constriction (TAC) and treated with NXK for 8 weeks. Then, the cardiac function and myocardial fibrosis were evaluated. The monocytes/macrophages were evaluated by immunofluorescence. The mRNA levels of IL-1ß, IL-6, TNF-α, ICAM-1, and VCAM-1 were measured by quantitative real-time polymerase chain reaction (qRT-PCR), while TLR4, MyD88, NF-κB p65, P-IκBα, TLR2, TLR7 and TLR9 protein levels were evaluated by Western blot. The results showed that NXK improved the left ventricular ejection fraction (LVEF) and left ventricular end-systolic dimension, reversed myocardial fibrosis, and inhibited pro-inflammatory (CD11b + Ly6C+) monocytes/macrophages in the TAC mouse model. NXK also reduced the mRNA and protein levels of the above markers. Taken together, NXK improved heart function and reduced inflammation through the TLR-mediated NF-κB signaling pathway, suggesting that it might be used as an innovative treatment strategy for CHF.

Simultaneous quantitation of 17 endogenous adrenal corticosteroid hormones in human plasma by UHPLC-MS/MS and their application in congenital adrenal hyperplasia screening.

Accurate investigation of adrenal hormone levels plays a vital role in pediatric endocrinology for the detection of steroid-related disorders. This study aims to develop a straightforward, sensitive UHPLC-MS/MS method to quantify 17 endogenous adrenal corticosteroid hormones in human plasma. These hormones are the main ingredients in the synthetic and metabolic pathways of adrenal corticosteroid hormones. Chromatographic separation was achieved on a C18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode with a run time of 7 min. The samples were extracted by liquid-liquid extraction and required no derivatization. Analytical performance was evaluated, including linearity, analytical sensitivity, accuracy, precision, and specificity. Plasma specimens from 32 congenital adrenal hyperplasia (CAH) patients and 30 healthy volunteers were analyzed to further reveal the diagnostic value of multiple steroid hormones in the synthetic and metabolic pathways of adrenal corticosteroid in CAH diagnosis. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference of isomers or structural analogues. The imprecisions were <10%. The lower limits of quantification varied from 0.05 to 15.0 ng/ml. Good linearity coefficients (r 2 > 0.998) were also obtained for most hormones in the required concentration range, except for 21-deoxycortisol (r 2 = 0.9967) and androstenediol (r 2 = 0.9952). The recoveries for the steroid hormones ranged from 91.7 to 109.8%. We developed the UHPLC-MS/MS method for the simultaneous measurement of steroid hormones. The results showed that measurement of steroid hormones simultaneously could improve the diagnostic efficiency of CAH.

SMG9 Serves as an Oncogene to Promote the Tumor Progression via EMT and Wnt/ß-Catenin Signaling Pathway in Hepatocellular Carcinoma.

Background/Aims: SMG9 participates in the nonsense-mediated mRNA decay process that degrades mRNA harboring nonsense mutations introduced either at the level of transcription or RNA processing. However, little is known about the role of SMG9 in hepatocellular carcinoma (HCC). The objective of this research was to clarify the effects of SMG9 expression on HCC progression. Methods: Microarray data were acquired from NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database to bioinformatically analyze the differential expression of SMG9 between HCC patients and normal controls. SMG9 mRNA level was measured in sixteen sets of fresh tumor tissues and adjacent non-cancerous liver tissues (ANLTs) via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SMG9 protein expression was analyzed in ninety-five sets of paired formalin-fixed and paraffin-embedded tissue specimens by immunohistochemistry (IHC). In addition, clinicopathological features of SMG9 in HCC were checked. For in vitro studies, small interfering RNA (siRNA) was used to silence SMG9 expression for exploring biological functions and underlying mechanisms of SMG9 in SMMC-7721 and HepG2. Results: We found that SMG9 was upregulated in HCC tissues and SMG9 levels were closely related to TNM stage, tumor number and tumor size. Cox regression and Kaplan-Meier proportional hazards analyses showed that high expression of SMG9 was associated with poor patient survival. Furthermore, proliferation, apoptosis resistance, migration and invasion of both SMMC-7721 and HepG2 cells were suppressed by SMG9 inhibition. In addition, EMT and the Wnt/ß-catenin signaling pathway were involved in SMG9-mediated HCC progression. Conclusions: SMG9 may serve as a potential novel prognostic biomarker and therapeutic target in HCC patients.

Inflammation Pharmacological Reaction and YMDD Mutational Patterns in Lamivudine Therapeutics Hepatitis B Virus.

Background/Aims: Emergence of tyrosine-methionine-aspartate-aspartate (YMDD) motif in reverse transcriptase is a serious problem in chronic hepatitis B(CHB) patients after Lamivudine (LAM) therapy. However, the relationship between inflammation pharmacological reaction and YMDD mutational patterns of CHB has not been well-characterized. The aim of this study was to investigate the inflammation pharmacological reaction and different YMDD mutants patterns of CHB patients. Methods: We investigated the inflammation pharmacological reaction and YMDD mutational patterns through biochemical, serological and virological detection among 83 CHB patients, including 25 YMDD mutants, 25 under detection, and 33 control patients without YMDD mutants. Results: Prevalence of YMDD mutation patterns is different. Among 25 YMDD mutants patients, YIDD was the dominant mutation (72%), followed YVDD (16%) and the hybrid YIDD + YVDD (12%). The time course during the YMDD mutations was also different. 52.4% patients developed the mutation less than 12 months after the LAM therapy. Serum hepatitis B virus (HBV) DNA level in patients with YMDD mutants were significantly higher than that in control and negative groups. Serum HbsAg and HbeAg in patients with YMDD mutants were also higher than those in control and negative groups, despite no significant difference was found forserum HbeAb. ALT and AST levels were also significantly higher in mutants group. Conclusions: Illuminating inflammation pharmacological reaction and YMDD mutational patterns of CHB during pathological process may have implications for future therapy in YMDD mutation patients. This may have impact on the choice of treatment strategies for lamivudine-resistant HBV.

Prognostic Value of Inflammatory Indicators in Chronic Hepatitis B Patients With Significant Liver Fibrosis: A Multicenter Study in China.

Diagnosis of significant liver fibrosis is essential to facilitate the optimal treatment decisions and improve prognosis in patients with chronic hepatitis B (CHB). We aimed to evaluate the value of inflammatory indicators and construct a nomogram that effectively predicts significant liver fibrosis among CHB patients. 563 CHB patients from two centers in China from 2014 to 2019 were divided into three cohorts (development, internal validation, and independent validation cohorts), assigned into cases with significant fibrosis (liver fibrosis stages ≥2) and those without. Multiple biochemical and serological inflammatory indicators were investigated. Inflammatory indicators, Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were significantly associated with significant liver fibrosis in CHB patients but limited predictive performance, and then we combined them with prothrombin time activity percentage (PTA) and liver stiffness measurement (LSM) were identified by multivariate logistic regression analysis. Based on these factors, we constructed the nomogram with excellent performance. The area under the receiver operating characteristic curve (AUROC) for the nomogram in the development, internal validation, and independent validation cohorts were 0.860, 0.877, and 0.811, respectively. Our nomogram based on ALT and AST that had excellent performance in predicting significant fibrosis of CHB patients were constructed.

Measurement of HbA1c and HbA2 by Capillarys 2 Flex Piercing HbA1c programme for simultaneous management of diabetes and screening for thalassemia.

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 (ß-) 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 ß-thalassemia trait) and abnormal bands (HbH and/or HbBart's for HbH disease, HbF for ß-thalassemia) may provide valuable information for screening.

Identification of haemoglobin New York by haemoglobin A1c measurement using the Sebia Capillarys 2 Flex Piercing system.

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: ß0-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.

TLR4-HMGB1 signaling pathway affects the inflammatory reaction of autoimmune myositis by regulating MHC-I.

OBJECTIVES: To analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-α, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis. METHODS: We built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-γ on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-γ, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-α and IL-6. RESULTS: The expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all P<0.05). After IFN-γ treatment, the expressions of TLR4, HMGB1, MHC-I, TNF-α and IL-6 in PBMCs significantly increased (all P<0.05). The treatment of anti-TLR4, anti-HMGB1 and anti-MHC-I could significantly downregulate the expression of MHC-I (all P<0.05). In addition, anti-TLR4 and anti-HMGB1 significantly reduced the expression of TNF-α and IL-6 (all P<0.05). CONCLUSIONS: The TLR4-HMGB1 signaling pathway affects the process of autoimmune myositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines.