High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

Tumor Biomarker In-Solution Quantification, Standard Production, and Multiplex Detection.

The diagnosis and monitoring of cancer have been facilitated by discovering tumor "biomarkers" and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and "direct, in-solution" ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker "standards", paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers.

Evaluation of Abbott Architect, Siemens Immulite, bioMerieux Vidas, and Euroimmune assays for determination of Epstein-Barr virus serological diagnosis.

Serological tests detecting antibodies for Epstein-Barr virus (EBV) antigens are frequently used to define infection status. Several new automated assays are available for this purpose. We compared the performance of Architect, Immulite, Vidas, and Euroimmune immunofluorescence assays (IFA)/enzyme-linked immunosorbent assays (ELISA) for the detection of EBV viral capsid antigen (VCA) immunoglobulin M (IgM), VCA IgG, Epstein-Barr nuclear antigen (EBNA)-1 IgG. The routine diagnosis of EBV in our laboratory is done by anti-EBV VCA IgM IFT, anti-EBV VCA IgG IFT, and anti-EBNA-1 IgG ELISA (Euroimmune) Kits. Samples were tested with EBV Kits of Architect, Immulite, and Vidas for anti-VCA IgM, anti-VCA IgG, and anti-EBNA-1 IgG. The agreement between assays was calculated for each marker individually and for the determination of the EBV infection profile, based on the combination of three markers. BIOCHIP Sequence EBV (Avidity test) and/or EUROLINE EBV Profile 2 (IgG/IgM) were used as confirmatory assays to resolve discrepancies. The best concordance for VCA IgM detection was between Immulite and Vidas; for VCA IgG and EBNA-1 IgG were between Architect and Vidas. The sensitivities and specificities for VCA IgM were 97% and 88% for IFA, 100% and 94% for Architect, 100% and 99% for Vidas, and 100% and 100% for Immulite, respectively. The most problematic marker was EBNA-1 IgG with a 68.1% specificity by Immulite. Vidas panel had a perfect performance (100%) for determining all EBV profiles. Overall, evaluated assays had comparable performance. There were more discordant VCA IgG and EBNA-1 IgG results than VCA IgM results. The agreement between Architect and Vidas was better than other assays.

Comparisons of plasma aldosterone and renin data between an automated chemiluminescent immunoanalyzer and conventional radioimmunoassays in the screening and diagnosis of primary aldosteronism.

Determining values of plasma renin activity (PRA) or plasma active renin concentration (ARC), plasma aldosterone concentration (PAC), and aldosterone-to-renin ratio (ARR) is essential to diagnose primary aldosteronism (PA), but it takes several days with conventional radioimmunoassays (RIAs). Chemiluminescent enzyme immunoassays for PAC and ARC using the Accuraseed® immunoanalyzer facilitated the determination, but relations between Accuraseed® immunoanalyzer-based and RIA-based values in samples of PA confirmatory tests and adrenal venous sampling remained to be elucidated. We addressed this issue in the present study. This is a prospective, cross-sectional study. ARC and PAC values were measured by the Accuraseed® immunoanalyzer in samples, in which PRA and PAC values had been measured by the PRA-FR® RIA and SPAC®-S Aldosterone kits, respectively. The relations between Accuraseed® immunoanalyzer-based and RIA-based values were investigated with regression analyses. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was determined by the receiver operating characteristic analysis. After log-log transformations, linear relations with high coefficients of determination were observed between Accuraseed® immunoanalyzer-based and RIA-based data of renin and aldosterone. Following the PA guidelines of Japan Endocrine Society, Accuraseed® immunoanalyzer-based cutoffs were calculated from the regression equations: the basal PAC for PA screening >12 ng/dL, PAC for the saline infusion test >8.2 ng/dL, ARC for the furosemide-upright test <15 pg/mL, and ARR for the captopril challenge test >3.09 ng/dL per pg/mL. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was >2.43 ng/dL over pg/mL not to overlook bilateral PA patients. The present study provided conversion formulas between Accuraseed® immunoanalyzer-based and RIA-based values of renin, aldosterone, and ARR, not only in basal samples but also in samples of PA confirmatory tests and adrenal venous sampling. Although validation studies are awaited, the present study will become priming water of harmonization of renin and aldosterone immunoassays.

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays.

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Evaluation of the diagnostic performances of two commercially available assays for the detection of enteric adenovirus antigens.

The performance of 2 antigenic commercial assays for enteric adenovirus (AdV) infection, bioNexia Rota-Adeno ImmunoChromatographic Tests (ICT) and LIAISON® Adenovirus ChemiLuminescence Immuno Assays (CLIA), was evaluated on 321 stools from children hospitalized for acute gastroenteritis in Palermo, Italy, using a Real time-PCR (Rt-PCR) as reference method. The CLIA showed higher sensitivity (77% vs 60%), accuracy (94.4 vs 90.9) and concordance (k: 0.81 vs 0.67) with respect to ICT, despite equivalent specificity (98.8%). Using the Ct values of the Rt-PCR as a proxy of the fecal viral load, similar Ct values (mean 9.32 vs 9.89) were observed among the true positive samples, whilst a significant difference (P < 0.05) was observed in false negative samples of CLIA (mean Ct 25.68) and ICT (mean Ct 19.87). Cross-reactivity with other enteric viruses was not observed. These results indicate that both the assays tested are suitable for diagnosis of AdV gastroenteritis.

Quantification of bacteria by in vivo bioluminescence imaging in comparison with standard spread plate method and reverse transcription quantitative PCR (RT-qPCR).

In vivo bioluminescence imaging (BLI) offers a unique opportunity to analyze ongoing bacterial infections qualitatively and quantitatively in intact animals over time, leading to a reduction in the number of animals needed for a study. Since accurate determination of the bacterial burden plays an essential role in microbiological research, the present study aimed to evaluate the ability to quantify bacteria by non-invasive BLI technique in comparison to standard spread plate method and reverse transcription quantitative PCR (RT-qPCR). For this purpose, BALB/c mice were intranasally infected with 1 × 105 CFU of bioluminescent Streptococcus pneumoniae A66.1. At day 1 post-infection, the presence of S. pneumoniae in lungs was demonstrated by spread plate method and RT-qPCR, but not by in vivo BLI. However, on the second day p.i., the bioluminescent signal was already detectable, and the photon flux values positively correlated with CFU counts and RT-qPCR data within days 2-6. Though in vivo BLI is valuable research tool allowing the continuous monitoring and quantification of pneumococcal infection in living mice, it should be kept in mind that early in the infection, depending on the infective dose, the bioluminescent signal may be below the detection limit.

Isothermal chemiluminescent assay based on circular stand-displacement polymerization reaction amplification for cel-miRNA-39-3p determination in cell extracts.

A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10-13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR.

Optimization of the detection of inhibitory autoantibodies against the VWF-cleaving protease ADAMTS13 with an automated chemiluminescent ADAMTS13 activity immunoassay.

INTRODUCTION: Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosILR AcuStar ADAMTS13 Activity assay. METHODS: The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours incubation at 25°C, 37°C, or 37°C after addition of Ca2+ to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosILR AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the TechnozymR ADAMTS13 activity ELISA. RESULTS: The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura. CONCLUSION: Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca2+ can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosILR AcuStar ADAMTS13 Activity assay.

Inhibitory anti ADAMTS13 antibodies with a new rapid fully automated CLiA assay.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by severe ADAMTS13 deficiency. The acquired form is associated with autoantibodies directed against ADAMTS13. Both noninhibitory and inhibitory autoantibodies can be detected by ELISA assay, while only inhibitory autoantibodies are detected by Bethesda assay. Due to its short TAT and good performance, chemiluminescence (CliA) ADAMTS13 activity (HemosIL Acustar) has proven to be a good choice in the diagnosis of TTP in emergency settings. Aim of this study was to analyse the performance of the CliA ADAMTS13 activity assay in detecting inhibitory ADAMTS13 antibodies using the Bethesda assay. METHODS: A method comparison study was performed on 69 stored samples: 11 acute TTPs, 38 TTP follow-ups, 5 TTP relapses, 1 congenital TTP, 10 HUS, 4 suspected TTPs. We retrieved the results of tests previously run in ELISA for both activity and autoantibodies. At the same time, we reran new tests including ELISA and CliA activity, ELISA autoantibodies, and ELISA and CliA Bethesda assays on thawed frozen samples. RESULTS: Very good correlation was observed between ELISA and CliA activity assay results (r = 0.96) and between archived ELISA and CliA activity results (r = 0.93). Agreement between the anti-ADAMTS13 assays ranged from good (k = 0.63) to very good (k = 0.92). CONCLUSIONS: CliA and ELISA Bethesda assays showed very good agreement with samples run at the same time using ELISA ADAMTS13-autoantibody assay. Albeit more expensive, the CliA Bethesda assay identified inhibitory anti-ADAMTS13 within almost the same TAT as ELISA, but with better automation and limited operator involvement.

Correlation of SARS-CoV-2 Neutralizing Antibodies to an Automated Chemiluminescent Serological Immunoassay.

INTRODUCTION: Neutralizing antibodies (NAbs) are capable of binding to a virus to render it incapable of infection. The ability of commercially available SARS-CoV-2 serological tests to detect NAbs has not been widely reported. We sought to correlate the antibodies detected by an automated chemiluminescent immunoassay with NAbs. METHODS: Residual serum samples from 35 patients that had a positive antibody test using the LIAISON® SARS-CoV-2 S1/S2 IgG chemiluminescent immunoassay and 2 antibody-negative control sera were tested for NAbs using a plaque reduction neutralization test (PRNT). RESULTS: NAbs were detected in 66% (23/35) of the antibody-positive samples. The immunoassay signal value ranged from 21.7 to 131.3 AU/mL (median, 90.5) with significant correlation between it and the PRNT (r = 0.61, P = 0.002). In the samples without NAbs, the immunoassay signal ranged from 16.3 to 66.2 AU/mL (median, 27.2). An immunoassay signal cutoff of >41 AU/mL was 91% sensitive and 92% specific for the detection of NAbs. DISCUSSION: It is important that correlates of immunity to SARS-CoV-2 be identified and NAbs are considered to be central indicators of such. PRNT is the gold-standard test for identifying NAbs but it cannot be used for large-scale testing of populations. It is necessary to establish relationships between it and widely used commercial serological assays for SARS-CoV-2.

Diagnostic accuracy of serological tests and kinetics of severe acute respiratory syndrome coronavirus 2 antibody: A systematic review and meta-analysis.

This study aimed to assess the diagnostic test accuracy (DTA) of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) serological test methods and the kinetics of antibody positivity. Systematic review and meta-analysis were conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline. We included articles evaluating the diagnostic accuracy of serological tests and the kinetics of antibody positivity. MEDLINE through PubMed, Scopus, medRxiv and bioRxiv were sources of articles. Methodological qualities of included articles were appraised using QUADAS-2 while Metandi performs bivariate meta-analysis of DTA using a generalized linear mixed-model approach. Stata 14 and Review Manager 5.3 were used for data analysis. The summary sensitivity/specificity of chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) were 92% (95% CI: 86%-95%)/99% (CI: 97%-99%), 86% (CI: 82%-89%)/99% (CI: 98%-100%) and 78% (CI: 71%-83%)/98% (95% CI: 96%-99%), respectively. Moreover, CLIA-based assays produced nearly 100% sensitivity within 11-15 days post-symptom onset (DPSO). Based on antibody type, the sensitivity of ELISA-total antibody, CLIA-IgM/G and CLIA-IgG gauged at 94%, 92% and 92%, respectively. The sensitivity of CLIA-RBD assay reached 96%, while LFIA-S demonstrated the lowest sensitivity, 71% (95% CI: 58%-80%). CLIA assays targeting antibodies against RBD considered the best DTA. The antibody positivity rate increased corresponding with DPSO, but there was some decrement when moving from acute phase to convalescent phase of infection. As immunoglobulin isotope-related DTA was heterogeneous, our data have insufficient evidence to recommend CLIA/ELISA for clinical decision-making, but likely to have comparative advantage over RT-qPCR in certain circumstances and geographic regions.

Research and discussion on the evaluation scheme of reagent lot-to-lot differences in 16 chemiluminescence analytes, established by the EP26-A guidelines of the CLSI.

BACKGROUND: Verification of new reagent lots is a part of the crucial tasks in clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) EP26-A guideline provides laboratories with an evaluation method for reagent verification. The purpose of this study was to compare the performance of EP26-A with our laboratory reagent lot verification protocol and get the final scheme. METHOD: 16 chemiluminescence analytes including estradiol (E2), progesterone (P), ferritin (FER), cortisol (COR),carbohydrate antigen 153 (CA153), and free prostate-specific antigen (FPSA). were prospectively evaluated in two reagent lots. The laboratory's lot verification process included evaluating 5 patient samples with the current and new lots and acceptability according to a predefined criteria. For EP26-A, method imprecision data and critical differences at medical decision points were important factors affecting the sample size requirements and rejection limits. RESULT: The number of samples required for EP26-A was 3 to 12, of which P, CA153, and FPSA had increased by more than 5 samples compared with the current protocol. Of the 16 chemiluminescence analytes, 11 had higher rejection limits when using EP26-A than the current laboratory scheme. Our current protocol and EP26-A were in agreement in 32 of the 32 (100%) paired verifications. CONCLUSION: The EP26-A protocol is an important tool to find the differences between reagent lots, and it makes up for the loopholes in the statistical efficiency, sample concentration and quantity, and the selection of rejection limits in the current protocol.

Multicenter evaluation of two chemiluminescence and three lateral flow immunoassays for the diagnosis of COVID-19 and assessment of antibody dynamic responses to SARS-CoV-2 in Taiwan.

This multicenter, retrospective study included 346 serum samples from 74 patients with coronavirus disease 2019 (COVID-19) and 194 serum samples from non-COVID-19 patients to evaluate the performance of five anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests, i.e. two chemiluminescence immunoassays (CLIAs): Roche Elecsys® Anti-SARS-CoV-2 Test (Roche Test) and Abbott SARS-CoV-2 IgG (Abbott Test), and three lateral flow immunoassays (LFIAs): Wondfo SARS-CoV-2 Antibody Test (Wondfo Test), ASK COVID-19 IgG/IgM Rapid Test (ASK Test), and Dynamiker 2019-nCoV IgG/IgM Rapid Test (Dynamiker Test). We found high diagnostic sensitivities (%, 95% confidence interval [CI]) for the Roche Test (97.4%, 93.4-99.0%), Abbott Test (94.0%, 89.1-96.8%), Wondfo Test (91.4%, 85.8-94.9%), ASK Test (97.4%, 93.4-99.0%), and Dynamiker Test (90.1%, 84.3-94.0%) after >21 days of symptom onset. Meanwhile, the diagnostic specificity was 99.0% (95% CI, 96.3-99.7%) for the Roche Test, 97.9% (95% CI, 94.8-99.2%) for the Abbott Test, and 100.0% (95% CI, 98.1-100.0%) for the three LFIAs. Cross-reactivity was observed in sera containing anti-cytomegalovirus (CMV) IgG/IgM antibodies and autoantibodies. No difference was observed in the time to seroconversion detection of the five serological tests. Specimens from patients with COVID-19 pneumonia demonstrated a shorter seroconversion time and higher chemiluminescent signal than those without pneumonia. Our data suggested that understanding the dynamic antibody response after COVID-19 infection and performance characteristics of different serological test are crucial for the appropriate interpretation of serological test result for the diagnosis and risk assessment of patient with COVID-19 infection.

High-Performance Cataluminescence Sensor Based on Nanosized V2O5 for 2-Butanone Detection.

The development of high-performance sensors is of great significance for the control of the volatile organic compounds (VOCs) pollution and their potential hazard. In this paper, high crystalline V2O5 nanoparticles were successfully synthesized by a simple hydrothermal method. The structure and morphology of the prepared nanoparticles were characterized by TEM and XRD, and the cataluminescence (CTL) sensing performance was also investigated. Experiments found that the as-prepared V2O5 not only shows sensitive CTL response and good selectivity to 2-butanone, but also exhibits rapid response and recovery speed. The limit of detection was found to be 0.2 mg/m3 (0.07 ppm) at a signal to noise ratio of 3. In addition, the linear range exceeds two orders of magnitude, which points to the promising application of the sensor in monitoring of 2-butanone over a wide concentration range. The mechanism of the sensor exhibiting selectivity to different gas molecules were probed by quantum chemistry calculation. Results showed that the highest partial charge distribution, lowest HOMO-LUMO energy gap and largest dipole moment of 2-butanone among the tested gases result in it having the most sensitive response amongst other VOCs.

Verification of the Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) reference values in Croatian children and adolescents.

INTRODUCTION: The aim of this study was to examine whether the Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) could be applied to Croatian children and adolescents. MATERIALS AND METHODS: A total of 295 outpatient healthy children and adolescents of age 1 to 18 were selected using the direct a posteriori sampling method. According to current guidelines, 20 samples were tested for each of a total of 51 reference intervals for ferritin, cortisol, dehydroepiandrosterone sulfate, follicle stimulating hormone, lutein stimulating hormone, prolactin, progesterone, sex hormone binding globulin, thyroid stimulating hormone, total testosterone, total thyroxine and total triiodothyronine. Serum samples were analysed on the Beckman Coulter DxI600 immunoassay analyser by chemiluminescence immunoassay method. A reference interval was adopted if < 10% of the results fall outside CALIPER reference interval range. For analytes in which this criterion is not met in the first set of samples, a new set of 20 samples were collected. RESULTS: After the first set of measurements, 96% of all tested reference intervals were adopted for use. The additional sets of 20 reference subjects were tested for only two reference intervals; follicle stimulating hormone for female aged 1 to 9 years, and irrespective of the gender, sex hormone binding globulin for children aged 8 to 11 years. All results of additional samples were within the specified interval limits. CONCLUSIONS: CALIPER reference intervals for ferritin and 11 hormones defined for Beckman Coulter DxI600 immunoassay analyser can be implemented into the Croatian laboratories and clinical practice.

Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence.

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Interleukin 8 Association with respiratory syncytial virus bronchiolitis: a systematic review and meta-analysis.

Infants with the respiratory syncytial virus (RSV) and human rhinovirus respiratory infection (HRV) produce inflammatory interleukins (ILs) in the respiratory epithelium. The aim of this study was to evaluate the levels of interleukin-8 in RSV negative and RSV positive patients. This study search was conducted without a time limit until 2020 through the databases of PubMed, Wiley, Springer, ScienceDirect and Google Scholar search engines, by two researchers independently. The random-effects model was used to compare of interleukin-8 in RSV negative vs. RSV positive patients, using Revman software version 5 meta-analysis software. Totally, 921 patients were evaluated (207 RSV-negative and 714 RSV-positive). The mean concentration of IL8 in RSV positive patients was 15.02 pg/ml (95% CI: 13.68- 16.35%).  According to the meta-analysis results, the standardized mean difference (SMD) of IL8 concentration between RSV-positive and negative patients was 6.31 pg/ml) (95% confidence interval: 2.50- 10.13%). subtotal analysis of the IL8 laboratory assessment method revealed that there was no significant SMD deference in the studies that have used chemiluminescence (P=0.21). while IL8 concentrations were significantly higher in RSV positives in ELISA and Magnetic bead-based assays (P<0.05).  It appears that RSV positive patients may have greater levels of IL8 than RSV negative ones; whereas the synthesis of IL8 tends to be more secreted into the nasopharyngeal space; whereas the evaluation approach can also affect the results.

Calcitonin levels by ECLIA correlate well with RIA values in higher range but are affected by sex, TgAb, and renal function in lower range.

Calcitonin (CT) is a marker for both initial diagnosis and monitoring of patients with residual or recurrent medullary thyroid carcinoma (MTC). In Japan, serum CT had been measured by radioimmunoassay (RIA) until recently. Electrochemiluminescence immunoassay (ECLIA) became commercially available in 2014, and this technique is now the only method used to examine CT concentration. The purposes of this study were to investigate the correlations between the CT concentration measured with ECLIA (ECLIA-CT) and RIA (RIA-CT) and to explore the clinical characteristics of patients with elevated ECLIA-CT. CT concentrations of 348 sera samples from 334 patients with various thyroid disorders including nine MTC were measured using both assays. The correlation analysis revealed an excellent correlation between ECLIA-CT and RIA-CT among the cases with CT level >150 pg/mL by both assays (rs = 0.991, p < 0.001). However, 63% of all samples exhibited undetectable ECLIA-CT, while their RIA-CTs were measured between 15 and 152 pg/mL. The ECLIA-CTs in all patients who underwent total thyroidectomy for non-MTC showed low concentrations. High ECLIA-CT was observed in patients with MTC or pancreas neuroendocrine tumor. ECLIA-CT was also increased in 14 other male patients with non-MTC, including four with renal failure. Multivariate logistic regression analysis showed that male sex, negative TgAb, and lower estimated glomerular filtration rate were independent factors to predict detectable ECLIA-CT (≥0.500 pg/mL). These results indicate that ECLIA-CT correlates well with RIA-CT in higher range and is affected by sex, TgAb, and renal function.