Consistency and diagnostic accuracy of 4 assays in the detection of the total and free prostate-specific antigen.

Background: The lack of interchangeability among prostate-specific antigen (PSA) assays causes difficulties in clinical interpretation. The currently available mainstream assays for PSA were based on Western populations, but it is not clear whether these assays yield different results in prostate cancer (PCa) screening in Chinese populations. Methods: A total of 163 men with a total PSA (tPSA) level of 2-10 µg/L scheduled for prostate biopsy were enrolled in this study. The levels of the tPSA and free PSA (fPSA) were detected by the Beckman (using the Hybritech calibration), Roche, Abbott, and Mindray (using the World Health Organization's calibration). Methodological comparison were performed according to EP9-A3 of the Clinical and Laboratory Standards Institute, USA. With pathological diagnosis as the gold standard, the predictive accuracy of the biomarkers was quantified as the area under the receiver operating characteristic curve (AUC) for each of the four methods. Results: A total of 32 PCa patients and 131 patients with benign prostate disease were included in this study. The tPSA levels detected by the Roche, Abbott, and Mindray showed good consistency with Beckman but not of the fPSA levels. Compared to the Beckman tPSA values, the measured values were 1.1% higher for Roche, 2.1% higher for Abbott, and 6.7% higher for Mindray. The fPSA levels measured by Roche, Abbott, and Mindray were 5.4% lower, 22.1% higher, and 4.6% higher than Beckman, respectively. When the tPSA was 4 ng/mL, the diagnostic performance (sensitivity, specificity, positive predictive value, negative predictive value, and missed diagnosis rate) was similar among these 4 assays. When the %fPSA was 16%, Abbott had the highest missed diagnosis rate (37.50%), while Mindray had a sensitivity of 81.25%, the highest negative predictive value (93.88%), and the lowest missed diagnosis rate (18.75%). Conclusions: When the tPSA level is 2-10 ng/mL, the Mindray, like the Roche and Abbott, has good consistency with the Beckman in detecting tPSA, which makes it possible to relieve the pressure of clinical interpretation. However, 4 assays using the same %fPSA cut-off may lead to diverse missed diagnosis rates for PCa screening in China.

Analytical Evaluation and Reference Interval Investigation for Chinese Han Population in Wuhan of CA72-4 Performed on an Architect i2000sr System.

BACKGROUND: Carbohydrate antigen 72-4 (CA72-4) is a mucin-like, tumor-associated glycoprotein. Its elevation in serum has been found to be associated with various carcinomas. The purpose of this study was to evaluate the analytical performance of the Architect chemiluminescent immunoassay CA72-4 on the Architect i2000sr platform and to determine the reference interval (RI) of CA72-4 for the Chinese Han population in the city of Wuhan. METHODS: We evaluated the precision, analytical sensitivity, linearity and interference according to the guidelines of the Clinical Laboratory Standardization Institute. Additionally, the Abbott Architect i2000sr CA72-4 assay was compared to the Roche Cobas E602 CA72-4 assay. A total of 448 healthy individuals were selected to determine the RIs of CA72-4. RESULTS: The assay had an imprecision of 1.38% - 2.63% within runs, 1.41% - 2.73% between runs and a total imprecision of 2.54% - 4.10%. The limit of blank, limit of detection and limit of quantitation concentrations were 0.19 U/mL, 0.21 U/mL, and 0.21 U/mL. Linearity was confirmed across the entire measurable range. Additionally, the carryover was less than 0.065%. Interferences with hemoglobin, conjugated bilirubin and lipemia were acceptable with changes of less than 10%. A correlation coefficient of 0.9450 was determined through the method comparison experiment (95% CI 0.9318 - 0.9557). The RI for serum CA72-4 in the Han population was established as an upper limit, 11.13 U/mL (90% CI 9.39 - 12.99) by using the Abbott Architect i2000sr system. CONCLUSIONS: This study establishes a RI for the Chinese Han population in Wuhan using the Abbott Architect i2000sr CA72-4 assay and demonstrates an analytical performance for clinical use.

Clinical Performance of SARS-CoV-2 IgG and IgM Tests Using an Automated Chemiluminescent Assay.

Serology tests for viral antibodies provide an important tool to support nucleic acid testing for diagnosis of the novel coronavirus disease 2019 (COVID-19) and is useful for documenting previous exposures to SARS-CoV-2, the etiological agent of COVID-19. The sensitivities of the chemiluminescent SARS-CoV-2 IgG/IgM immunoassay were assessed by using serum samples collected from 728 patients testing positive for SARS-CoV-2 RNA. The specificity was evaluated on a panel of 60 serum samples from non-COVID-19 patients with high levels of rheumatoid factor, antinuclear antibody, or antibodies against Epstein-Barr virus (EBV), cytomegalovirus (CMV), mycoplasma pneumonia, human respiratory syncytial virus (RSV), adenovirus, influenza A or influenza B. The imprecision and interference were assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A2 and EP7-A2, respectively. Sensitivities between 1 and 65 days after onset of symptoms were 94.4% and 78.7%, for IgG and IgM test, respectively. The sensitivity increased with the time after symptom onset, and rose to the top on the 22nd to 28th days. The total imprecision (CVs) was less than 6.0% for IgG and less than 6.5% for IgM. Limited cross-reactions with antibodies against EBV, CMV, mycoplasma pneumonia, human RSV, adenovirus, influenza A or influenza B were found. These data suggested the chemiluminescent SARS-CoV-2 IgG and IgM, assay with reliable utility and sensitivity, could be used for rapid screening and retrospective surveillance of COVID-19.

TERT-rs33963617 and CLPTM1L-rs77518573 reduce the risk of non-small cell lung cancer in Chinese population.

BACKGROUND: Genome-wide association studies (GWAS) have identified 5p15.33 as a susceptible locus for lung cancer. However, for non-small cell lung cancer (NSCLC), low-frequency risk variants in this region have not been systematically studied. We intended to explore the associations between low-frequency variants on 5p15.33 and NSCLC using a next-generation sequencing based approach in this study. METHODS: We have acquisited the variation spectrum of 400 NSCLC patients on 5p15.33 by sequencing the targeted region before. Candidate variants were primarily selected by restricting the minor allele frequency (MAF 1-5%) and then by comparing their frequency in 400 NSCLC patients with 1008 East Asians from The genome Aggregation Database (gnomAD). The associations between candidate variants and NSCLC were discovered and replicated in two case-control sets: discovery stage with 960 cases and 916 controls, and replication stage with 1596 cases and 1614 controls in total. RESULTS: Five low-frequency variants were selected as our candidates and subsequent association analyses showed that 2 polymorphisms were significantly associated with risk of NSCLC, including rs33963617 (OR = 0.63, 95% CI: 0.53-0.76, P = 3.80 × 10-7) in TERT and rs77518573 (OR = 0.73, 95% CI: 0.63-0.84, P = 2.00 × 10-5) in upstream of CLPTM1L. When stratified by histologic subtype, a significant association was only investigated in adenocarcinoma for rs77518573. We also observed an obvious cumulative effect of the two significant variants. CONCLUSIONS: We newly identified two NSCLC related variants on chromosome 5p15.33. Both TERT-rs33963617 and CLPTM1L-rs77518573 conferred reduced risk for NSCLC in Chinese Han population.

Functional polymorphisms on chromosome 5p15.33 disturb telomere biology and confer the risk of non-small cell lung cancer in Chinese population.

The chromosome 5p15.33 has been reported as a susceptibility locus for lung cancer. However, causal variants in this region have not been fully uncovered. In this study, we intended to identify functional polymorphisms associated with non-small cell lung cancer (NSCLC) susceptibility in Chinese population. A targeted sequencing on 5p15.33 region was conducted in 400 NSCLC cases. We selected candidate variants by comparing genotypic frequency with data from 1000 Genomes Project, and their associations with NSCLC were validated in 985 cases and 970 controls. The relationships between risk variants and telomere length were evaluated in 774 healthy subjects. Luciferase assays and electrophoretic mobility shift assays (EMSA) were performed to explore potential functions and reveal carcinogenic mechanisms. As a result, we identified 1478 variants through targeted sequencing and selected 17 candidates. Four polymorphisms exhibited prominent associations with lung cancer risk, including rs7726159 (OR = 1.34, 95%CI: 1.18-1.52, P = 7.78 × 10-6 ), rs10054203 (OR = 1.29, 95%CI: 1.13-1.46, P = 1.37 × 10-4 ), rs2736107 (OR = 1.28, 95%CI: 1.11-1.47, P = 5.14 × 10-4 ), and rs2853677 (OR = 1.23, 95%CI: 1.08-1.39, P = 0.002). The minor allele of rs7726159 and rs10053203 were associated with long telomeres (P = 0.008 and 0.036, respectively). Mechanistically, the rs7726159-A increased TERT transcription through mediating allele-specific MYC binding. In conclusion, the functional variant rs7726159 confers lung cancer susceptibility might by affecting MYC binding and inducing telomere lengthening, which provides a new insight into the crucial role of telomere biology in tumorigenesis.

Analysis of 2 Reverse Syphilis Testing Algorithms in Diagnosis of Syphilis: A Large-Cohort Prospective Study.

Background: Two serologic syphilis screening algorithms recommended by the US Centers for Disease Control and Prevention (US CDC) and the European Centre for Disease Prevention and Control (ECDC), respectively, are commonly used for syphilis screening; however, which one is optimal remains to be determined. Methods: We conducted a prospective study of 119891 subjects to analyze the consistency of the US CDC- and ECDC-recommended algorithms. The US CDC-recommended algorithm begins with a treponemal immunoassay, followed by a rapid plasma reagin (RPR) test. RPR-nonreactive samples are confirmed by the Treponema pallidum particle agglutination assay (TPPA). The ECDC-recommended algorithm begins with a treponemal immunoassay, followed by a confirmatory treponemal test. If the confirmatory test is reactive, a quantitative nontreponemal assay is used to assess the disease activity and treatment response. In the present study, a total of 119891 serum samples from a large hospital (sixth largest in China) were included, and each sample was screened with a chemiluminescent immunoassay (CIA). CIA-reactive samples were then simultaneously tested with RPR and TPPA. The consistency of these 2 algorithms was determined by calculating the percentage of agreement and κ coefficient. Results: The overall percentage of agreement and κ value between these 2 algorithms were 99.996% and 0.999, respectively. The positivity rate for syphilis as determined by the US CDC- and ECDC-recommended algorithms was 1.43% and 1.42%, respectively. Conclusions: Our results suggest that the US CDC-recommended algorithm and the ECDC-recommended algorithm have comparable performances for syphilis screening in low-prevalence populations.

Analytical validation and investigation on reference intervals of aldosterone and renin in Chinese Han population by using fully automated chemiluminescence immunoassays.

BACKGROUND: The aldosterone/renin ratio (ARR) is recommended to screen for primary aldosteronism (PA) in hypertension. We estimated fully automated chemiluminescence immunoassays (CLIA) for plasma aldosterone concentrations (PAC) and plasma direct renin concentrations (PRC) and investigated their reference intervals in Chinese Han population. METHODS: PAC and PRC were measured on a fully automated analyzer (LIAISON XL, DiaSorin, Italy). Performance characteristics were estimated according to CLSI approved guidelines. 328 healthy individuals were selected for reference intervals investigation. Results simultaneously tested by CLIA and radioimmunoassays were reviewed from 123 patients with hypertension and/or adrenal space-occupying lesion. PAC/PRC ratio (ARRprc) was compared to PAC/plasma renin activity (PRA) ratio (ARRpra). RESULTS: Within-laboratory imprecision was 5.6%-6.7% for PAC and 3.0%-3.3% for PRC. The LoQ was 72.2 pmol/L for PAC and 1.27 mIU/L for PRC. Linearity was excellent in the range of concentrations between 94 and 2708 pmol/L for PAC and 1.3-461.8 mIU/L for PRC. Interferences of hemoglobin, unconjugated bilirubin and lipaemia could be acceptable, but not of conjugated-bilirubin when renin and aldosterone at low concentrations. The central 95% reference intervals for males: PAC: 76-722 pmol/L, PRC: 3.3-92.7 mIU/L, ARR: 2.2-46.0 pmol/mIU; for females: PAC: 85-1010 pmol/L, PRC: 3.7-99.8 mIU/L, ARR: 3.6-68.4 pmol/mIU. Upper reference limits for ARR of younger and older men were lower than women. ARRprc and ARRpra showed almost perfect agreement (kappa = 0.815) for screening PA. CONCLUSION: The DiaSorin tests are valuable analytical options for PAC and PRC measurements. We recommend sex-specific and age-specific reference intervals of these items should be estimated.

AMP-activated protein kinase alleviates extracellular matrix accumulation in high glucose-induced renal fibroblasts through mTOR signaling pathway.

BACKGROUND/AIMS: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK) has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. METHODS: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f). RESULTS: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1(312) (encoding constitutively active AMPKα1) whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1). In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. CONCLUSION: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

The SNP rs402710 in 5p15.33 is associated with lung cancer risk: a replication study in Chinese population and a meta-analysis.

BACKGROUND: Lung cancer is the most commonly diagnosed cancer and leading cause of cancer mortality in the world. A single nucleotide polymorphism (SNP), rs402710, located in 5p15.33, was firstly identified to be associated with the lung cancer risk in a genome-wide association study. However, some following replication studies yielded inconsistent results. METHODOLOGY AND FINDINGS: A case-control study of 611 cases and 1062 controls in a Chinese population was conducted, and then a meta-analysis integrating the current and previously published studies with a total 31811 cases and 36333 controls was performed to explore the real effect of rs402710 on lung cancer susceptibility. Significant associations between the SNP rs402710 and lung cancer risk were observed in both case-control study and meta-analysis, with ORs equal to 0.77 (95%CI = 0.63-0.95) and 0.83 (95%CI = 0.81-0.86) in dominant model, respectively. By stratified analysis of our case-control study, the associations were also observed in never smoker group and non-small cell lung cancer(NSCLC) group with ORs equal to 0.71 (95%CI = 0.53-0.95) and 0.69 (95%CI = 0.55-0.87), which was remarkable that larger effect of the minor allele T was seen in the two groups than that in overall lung cancer. Besides, the sensitive and cumulative analysis indicated the robust stability of the current results of meta-analysis. CONCLUSION: The results from our replication study and the meta-analysis provided firm evidence that rs402710 T allele significantly contributed to decreased lung cancer risk, and the case-control study implied that the variant may yield stronger effect on NSCLC and never smokers. However, the mechanism underlying the polymorphism conferring susceptibility to lung cancer is warranted to clarify in the follow-up studies.

Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: initiated by hypoxia inducible factor 1α, presented by exosomes.

Excessive tumor necrosis factor-α (TNF-α) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-α is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-α release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-α expression of cardiomyocytes, the role of hypoxia inducible factor-1α (HIF-1α) in TNF-α regulation and potential secretory pathway of TNF-α. Elevated TNF-α expression and HIF-1α activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-α mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1α small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1α protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-α gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-α promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-α within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1α initiates expression of TNF-α, mediated by exosomes in cardiomyocytes.

The SNP rs961253 in 20p12.3 is associated with colorectal cancer risk: a case-control study and a meta-analysis of the published literature.

BACKGROUND: Colorectal cancer (CRC) is the third common cancer and the fourth leading cause of cancer death worldwide. A single nucleotide polymorphism (SNP), rs961253 located in 20p12, was firstly described to be associated with the increased risk of CRC in a genome-wide association study; however, more recent replication studies yielded controversial results. METHODOLOGY/PRINCIPAL FINDINGS: A hospital-based case-control study in a Chinese population was firstly performed, and then a meta-analysis combining the current and previously published studies were conducted to explore the real effect of rs961253 in CRC susceptibility. In the Chinese population including 641 cases and 1037 controls, per-A-allele conferred an OR of 1.60 (95% CI = 1.26-2.02) under additive model. In the meta-analysis including 29859 cases and 29696 controls, per-A-allele have an OR of 1.13 (95% CI = 1.09-1.18) under a random-effects model due to heterogeneity (P = 0.019). Nevertheless, the heterogeneity can be totally explained by ethnicity, with the tau(2) reduced to 0 after including ethnicity in meta-regression model. In stratified analysis by ethnicity, per-A-allele had ORs of 1.34 (95% CI = 1.20-1.50) and 1.11 (95% CI = 1.08-1.14) for Asian and European, respectively, without heterogeneity. Modest influence of each study was observed on overall estimate in sensitive analysis, and evident tendency to significant association was seen in cumulative analysis over time, together indicating the robust stability of the current results. CONCLUSIONS/SIGNIFICANCE: The results from our study and the meta-analysis provided firm evidence that rs961253 significantly contributed to CRC risk in both Asian and European population.

[Experimental study of targeting MMP-9 deoxyribozyme role of adhesion and migration in human lung adenocarcinoma cancer cell.].

BACKGROUND: Deoxyribozyme has high biocatalytic activity and sequence specificity against the target mRNA and inhibits gene expression at mRNA level. The aim of this study is to investigate the impact of targeting MMP-9 deoxyribozyme to cell adhesion and migration in human lung adenocarcinoma cell line A549. METHODS: The targeting MMP-9 deoxyribozyme was designed by oligofectamine into human lung adenocarcinoma cell line A549. The expression of MMP-9 in cell was detected by Western blot. The cell adhesion and migration after the intervention of deoxyribozyme was observed. RESULTS: After targeting MMP-9 deoxyribozyme intervention, the expression of MMP-9 in the cells compared with the control group was significantly lower (P <0.01), at the same time, the rate of cell adhesion and migration were significantly decreased. CONCLUSIONS: Targeting MMP-9 deoxyribozyme inhibited the expression of MMP-9 in human lung adenocarcinoma cell line A549 and effectively prevent cell adhesion and migration.