Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

Maintenance of cathepsin D-dependent autophagy-lysosomal function protects against cardiac ischemia/reperfusion injury.

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury.

Molecular epidemiology and characteristics of respiratory syncytial virus among hospitalized children in Guangzhou, China.

BACKGROUND: Human respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection and hospitalization, especially in children. Highly mutagenic nature and antigenic diversity enable the RSV to successfully survive in human population. We conducted a molecular epidemiological study during 2017-2021 to investigate the prevalence and genetic characteristics of RSV. METHODS: A total of 6499 nasopharyngeal (NP) swabs were collected from hospitalized children at Department of Pediatrics, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, China. All NP swab specimens were preliminary screened for common respiratory viruses and then tested for RSV using specific PCR assays. Partial G genes of RSV were amplified for phylogenetic analysis and genetic characterization. RESULTS: The overall detection rate for common respiratory viruses was 16.12% (1048/6499). Among those, 405 specimens (6.20%, 405/6499) were found positive for RSV. The monthly distribution of RSV and other respiratory viruses was variable, and the highest incidence was recorded in Autumn and Winter. Based on the sequencing of hypervariable region of G gene, 93 RSV sequences were sub-grouped into RSV-A (56, 60.2%) and RSV-B (37, 39.8%). There was no coinfection of RSV-A and RSV-B in the tested samples. Phylogenetic analysis revealed that RSV-A and RSV-B strains belonged to ON1 and BA9 genotypes respectively, indicating predominance of these genotypes in Guangzhou. Several substitutions were observed which may likely change the antigenicity and pathogenicity of RSV. Multiple glycosylation sites were noticed, demonstrating high selection pressure on these genotypes. CONCLUSION: This study illustrated useful information about epidemiology, genetic characteristics, and circulating genotypes of RSV in Guangzhou China. Regular monitoring of the circulating strains of RSV in different parts of China could assist in the development of more effective vaccines and preventive measures.

Serum 25-hydroxyvitamin D status of a large Chinese population from 30 provinces by LC-MS/MS measurement for consecutive 3 years: differences by age, sex, season and province.

PURPOSE: We aimed to describe the vitamin D status and its distribution in different age groups, sexes, seasons, and provinces of a large Chinese population. METHODS: This study retrospectively analyzed 1,528,685 results of serum 25-hydroxyvitamin D (25(OH)D) in the central laboratory of KingMed Diagnostics. The samples were from the individuals aged 0-119 years old in 30 provinces of China. Serum 25(OH)D was measured by an accurate commercial liquid chromatography-tandem mass spectrometry (LC-MS/MS) method from January 2017 to December 2019. The subjects were stratified by age, sex, the season of blood collection, and the province of residence. RESULTS: The median 25(OH)D concentration was 25.5 ng/mL (interquartile range (IQR) 18.7-32.7 ng/mL) in males and 20.8 ng/mL (IQR 14.4-28.2 ng/mL) in females. Overall, the median 25(OH)D concentration decreased with age in both males and females. Males had a 0.2-2.4 ng/mL higher median 25(OH)D concentration than females in different age groups. Vitamin D deficiency (25(OH)D < 15 ng/mL for the individuals under 14 years old; < 20 ng/mL for the individuals over 14 years old) was found in 21.3% of males and 43.6% of females. Significant seasonal variation of serum 25(OH)D concentrations was repeatedly observed in 3 years, with median concentration higher in summer (25.3 ng/mL (IQR 19.3-31.9 ng/mL)) and lower in winter (18.5 ng/mL (IQR 12.3-26.6 ng/mL)). Vitamin D status varied by province. The median 25(OH)D concentration was the highest in Hainan (31.0 ng/mL (IQR 24.9-39.2 ng/mL)) and the lowest in Qinghai (14.4 ng/mL (IQR 9.6-20.0 ng/mL)). 25(OH)D2 was detected in 12.2% of the results, and no significant seasonal variation was observed. CONCLUSION: In China, vitamin D deficiency is prevalent in the population participating in clinical vitamin D measurement. Age and sex differences in vitamin D levels were observed in our study. Seasonal variation and provincial differences are important aspects of serum vitamin D status. 25(OH)D2 cannot be ignored entirely in clinical measurement practice in China.

Patient-based real-time quality control for quantitative hepatitis B virus DNA test using moving rate of positive and negative patient results.

OBJECTIVES: Patient-based real-time quality control (PBRTQC) has gained increasing attention in the field of laboratory quality management in recent years. However, PBRTQC has not been reported for use in molecular diagnostics. This study introduces PBRTQC to quantitative hepatitis B virus (HBV) DNA test using moving rate (MR) of positive and negative patient results. METHODS: In contrast to the MR protocols described in other literature, MR protocol for HBV-DNA test has an additional logarithmic transformation and binary conversion steps before using a common statistical process control algorithm, such as the MR. We used all patient test results of HBV-DNA assay from August 2018 to August 2021 at the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, for parameters setting, optimization, and performance validation. The false rejection rate, error detection curves and validation charts were used to assess the MR protocols. RESULTS: The false rejection rates of two MR protocols were both <0.7%. The optimal block sizes for positive and negative errors in each cut-off value were not the same, so we first proposed a combined protocol that used different block size to detect negative and positive errors. It turned out that the combined protocols outperformed the simple protocols for each cut-off value, especially detecting positive errors. CONCLUSIONS: The performances of MR protocols using positive or negative patient results to detect constant errors of HBV-DNA test could meet laboratory requirements. Therefore, we have provided an effective alternative tool for internal quality control in the field of molecular diagnostics.

Development of human serum matrix-based unconjugated estriol-certified reference material by recommended ID-LC-MS/MS reference method.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020.

Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry.

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain 15N isotope-labeled recombinant Cys C (15N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and 15N-Cys C was expressed in M9 medium using 15N as the only nitrogen source. 15N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from 15N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of 15N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of 15N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)2+ was the parent ion of 15N-Cys C and that the secondary ions of 15N-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of 15N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

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. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

Evaluation of AutoLumo A2000 Chemiluminescent Immunoassay for Detection of Respiratory Syncytial Virus and Adenovirus IgM Antibodies.

BACKGROUND: Respiratory syncytial virus and adenovirus are seasonal diseases that cause an enormous burden on health systems. Previously, our lab uses DHI D3-Ultra DFA for detecting antigen of respiratory syncytial virus (RSV) and adenovirus (ADV). This article will evaluate the performance of AutoLumo RSV-IgM and ADV-IgM assays compared to D3-Ultra DFA method. METHODS: We used quality control specimens to evaluate precision, cross-reactivity specimen to evaluate the specificity, exogenous interferent: Hb (1,000 mg/dL); total bilirubin (50 mg/dL), ANA titer (1:10,000), RF (500 IU/mL) to evaluate interference, and paired, nasopharyngeal swab and sera specimens to evaluate clinical sensitivity and specificity. RESULTS: AutoLumo RSV-IgM and ADV-IgM assay show good precision and no cross-reactivity with other pathogen-specific IgM antibodies; no hook effect; exogenous interferent substance: Hb < 1,000 mg/dL; total bilirubin < 50 mg/dL, ANA titer < 1:10,000, and RF < 500 IU/mL showed no interference to RSV-IgM and ADV-IgM antibodies. The paired comparison test showed that RSV-IgM and ADV-IgM appear partly on the fifth day of the disease and peaked on days six to fourteen. CONCLUSIONS: AutoLumo RSV-IgM and ADV-IgM have good performance, but their sensitivities await further improvements.

Performance Evaluation of Antiphospholipid Antibodies on INOVA Quanta Flash System.

BACKGROUND: The diagnosis of antiphospholipid syndrome (APS) relies predominantly on the laboratory measurement of antiphospholipid antibodies (aPLs). We attempt to verify the analytical performance of anticardiolipin antibodies (aCL) IgA/IgG/IgM and anti-ß2-glycoprotein I antibodies (aß2GPI) IgA/IgG/IgM on a high-throughput automated immunoassay platform. METHODS: Limit of blank (LOB), limit of detection (LOD), imprecision, and linearity were calculated according to the corresponding Clinical and Laboratory Standards Institute (CLSI) guidelines protocols. The biological reference intervals (RIs) were verified in healthy individuals. RESULTS: The LoB of aCL IgA/IgG/IgM and aß2GPI IgA/IgG/IgM were 0.000, 1.200, 0.200, and 0.400, 1.250, 0.100, respectively. The LoD were 0.093, 1.715, 0.337 and 0.547, 2.174, 0.185 CU, respectively. All the within-run CVs and total CVs were less than the criterion at 10%. The linear analysis showed a good correlation between the predictive values and observed values with correlation coefficients greater than 0.99. CONCLUSIONS: The BIO-FLASH automated chemiluminescent analyzer performed well in measuring aPLs.

Comparative Performance of Four Nucleic Acid Amplification Tests for SARS-CoV-2 Virus.

BACKGROUND: COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was discovered in 2019 and spread around the world in a short time. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing of COVID-19. Aim of this study was to evaluate the performance of four NAAT assays. METHODS: Limit of detection (LOD), precision, accuracy, analytical specificity and analytical interference studies on four NAATs (Daan, Sansure, Hybribio, and Bioperfectus) were performed according to Clinical Laboratory Standards Institute protocols and guidelines. The four NAATs were compared using 46 clinical samples. RESULTS: The LOD of the N gene for Daan, Sansure, and Hybribio was 500 copies/mL, and that for Bioperfectus was 1,000 copies/mL. The LOD of the ORF1ab gene for Daan, Bioperfectus, and Hybribio was 3,000 copies/mL, and that for Sansure was 2,000 copies/mL. A good precision was shown at the concentration above 20% of the LOD for all four NAATs, with all individual coefficients of variation below 3.6%. Satisfactory results were also observed in the accuracy, analytical specificity, and analytical interference tests. The results of the comparison test showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16/16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity of 81.25% (13/16). However, no significant difference in sensitivity was found between Bioperfectus NAAT and the three other NAATs (p > 0.05). CONCLUSIONS: The four SARS-CoV-2 NAATs showed comparable performance, with the LOD of the N gene lower than the LOD of the ORF1ab gene.

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.

Design and Evaluation of Multiplex One-Step Reverse Transcription PCR-Dipstick Chromatography Method for the Analysis of Seven Respiratory Pathogens.

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)-dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA-DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin-streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR-dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR-dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.

Establishing Classifiers With Clinical Laboratory Indicators to Distinguish COVID-19 From Community-Acquired Pneumonia: Retrospective Cohort Study.

BACKGROUND: The initial symptoms of patients with COVID-19 are very much like those of patients with community-acquired pneumonia (CAP); it is difficult to distinguish COVID-19 from CAP with clinical symptoms and imaging examination. OBJECTIVE: The objective of our study was to construct an effective model for the early identification of COVID-19 that would also distinguish it from CAP. METHODS: The clinical laboratory indicators (CLIs) of 61 COVID-19 patients and 60 CAP patients were analyzed retrospectively. Random combinations of various CLIs (ie, CLI combinations) were utilized to establish COVID-19 versus CAP classifiers with machine learning algorithms, including random forest classifier (RFC), logistic regression classifier, and gradient boosting classifier (GBC). The performance of the classifiers was assessed by calculating the area under the receiver operating characteristic curve (AUROC) and recall rate in COVID-19 prediction using the test data set. RESULTS: The classifiers that were constructed with three algorithms from 43 CLI combinations showed high performance (recall rate >0.9 and AUROC >0.85) in COVID-19 prediction for the test data set. Among the high-performance classifiers, several CLIs showed a high usage rate; these included procalcitonin (PCT), mean corpuscular hemoglobin concentration (MCHC), uric acid, albumin, albumin to globulin ratio (AGR), neutrophil count, red blood cell (RBC) count, monocyte count, basophil count, and white blood cell (WBC) count. They also had high feature importance except for basophil count. The feature combination (FC) of PCT, AGR, uric acid, WBC count, neutrophil count, basophil count, RBC count, and MCHC was the representative one among the nine FCs used to construct the classifiers with an AUROC equal to 1.0 when using the RFC or GBC algorithms. Replacing any CLI in these FCs would lead to a significant reduction in the performance of the classifiers that were built with them. CONCLUSIONS: The classifiers constructed with only a few specific CLIs could efficiently distinguish COVID-19 from CAP, which could help clinicians perform early isolation and centralized management of COVID-19 patients.

Biotin Interference in Susceptible CanAg Ca242 Immunoassays and Elimination Method.

BACKGROUND: High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS). Biotin, as dietary supplement or therapy, may lead to analytical interference in biotin-streptavidin immunoassay. METHODS: Seven concentration gradients of biotin solutions were spiked to three different levels of Ca242 serum samples. All samples were tested by CanAg Ca242 ELISA kit to evaluate the interference from biotin. Serum samples with biotin concentration at 1,000 ng/mL were retested after absorption by streptavidin microparticles or direct analysis on the Mindray CL2000i platform. RESULTS: Our study found that CanAg Ca242 is vulnerable to interference when a sample that contains biotin exceeds 15.63 ng/mL. Biotin interference can result in falsely low results in CanAg Ca242. The effect and extent of biotin interference are, to some extent, dependent on the concentration of serum Ca242 and the concentration of biotin. CONCLUSIONS: CanAg Ca242 is vulnerable to biotin interference. The laboratory can overcome biotin interference on CanAg Ca242 by using a non-biotin streptavidin method or by absorbing biotin with streptavidin-coated microparticles before testing. Clinicians should use caution in interpreting abnormal results in patients who ingest biotin.

Antibodies against atypical pathogens and respiratory viruses detected by Pneumoslide IgM test in adults with community-acquired pneumonia in Guangzhou City.

BACKGROUND: To detect the serum antibodies against respiratory viruses and atypical pathogens in adults with community-acquired pneumonia (CAP) in Guangzhou City (Guangdong province, China). METHODS: A retrospective study was carried out with samples from 685 adults who were admitted with CAP and 108 non-CAP control patients. Atypical pathogens and respiratory viruses in serum were detected using the Pneumoslide IgM test from Vircell, Spain. All patients were divided into 6 groups according to age: 18-24, 25-44, 45-59, 60-74, 75-89, and >90. RESULTS: The total positive rate of CAP was 35.4%, which was highest in the 18-24 age group (P < .05). The highest positive rate, 17.11%, was observed for Mycoplasma pneumoniae (MP). The mean age of MP-infected patients was higher than that of the controls (P < .05). The positive rates for influenza B (INFB), Legionella pneumophila (LP1), Coxiella burnetii (COX), influenza A (INFA), parainfluenza virus (PIV), respiratory syncytial virus (RSV), Chlamydophila pneumoniae (CP), and adenovirus (ADV) were 5.56%, 3.07%, 2.63%, 2.34%, 1.90%, 1.61, 0.88%, and 0.29%, respectively. There were 4.37% of patients with CAP having multiple infections. The main symptoms observed in the 685 CAP patients were cough and sputum production, in 78.4% and 67.4%. Fever was followed by 54% of CAP patients. Dyspnea (39.1%), anorexia (36.8%), increased thirst (26.7%), chills (18.7), headache (14.6%), and nausea (13.1%) were also frequently observed in the CAP patients. CONCLUSIONS: MP infection was the most common in adult CAP patients in Guangzhou City with the highest positive rate in the 18-24 age groups.

Antibiotic Resistance and Molecular Epidemiological Characteristics of Streptococcus agalactiae Isolated from Pregnant Women in Guangzhou, South China.

Streptococcus agalactiae colonization in pregnant women can cause postpartum intrauterine infections and life-threatening neonatal infections. To formulate strategies for the prevention and treatment of S. agalactiae infections, we performed a comprehensive analysis of antibiotic resistance and a molecular-based epidemiological investigation of S. agalactiae in this study. Seventy-two S. agalactiae strains, collected from pregnant women, were subjected to antibiotic susceptibility tests; then, the screened erythromycin and clindamycin nonsusceptible isolates were used for macrolides and clindamycin resistance genes detection, respectively. Detection of resistance genes, serotyping, and determination of virulence genes were performed by polymerase chain reaction. The clonal relationships among the colonized strains were evaluated by multilocus sequence typing. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) mass peak analysis was performed to discriminate the specific sequence types (STs). In our study, 69.4% and 47.2% of the strains were nonsusceptible to erythromycin and clindamycin, respectively; the multidrug resistance rate was 66.7%. All erythromycin nonsusceptible strains harbored resistance genes, whereas only 52.9% of the clindamycin nonsusceptible strains possessed the linB gene. Erythromycin resistance was mainly mediated by the ermB or mefA/E genes. Four serotypes were identified, and the most common serotype was serotype III (52.8%), followed by Ib (22.2%), Ia (18.0%), and II (4.2%). All the strains were divided into 18 STs that were assigned to nine clonal complexes. Most of the major STs were distributed into specific serotypes, including ST19/serotype III, ST17/serotype III, ST485/serotype Ia, ST862/serotype III, and ST651/serotype III. Analysis of virulence genes yielded seven clusters, of which bca-cfb-scpB-lmb (61.6%) was the predominant virulence gene cluster. Among all ST strains distributed in this region, only the ST17 strains had a mass peak at 7620 Da. The outcomes of this study are beneficial for the epidemiological comparison of colonized S. agalactiae in different regions and may be helpful for developing the strategies for the prevention of S. agalactiae infection in Guangzhou. Furthermore, our results show that MALDI-TOF MS can be used for the rapid identification of the ST17 strains.

Optimization of an enzyme-coupling method by spectrophotometer for serum adenosine deaminase: As a candidate reference method.

Adenosine deaminase (ADA) is a key enzyme of adenosine metabolism. There are currently various kits and systems available for ADA measurement, and all yield variable results. This study optimized a reference measurement procedure (RMP) for serum ADA for the standardization of routine methods. ADA coupled with purine-nucleoside-phosphorylase, xanthine-oxidase and peroxidase was selected as the basic method and was optimized using Response Surface Methodology. Then the performance was validated and the results were compared after replication by 3 other reference laboratories. A reference interval was also developed. In addition, this optimized method was applied to calibrate a routine system. The intra-assay precision was 0.44% at both concentrations of 29.8 and 100.4 U/L, and inter-assay precision was 1.01% and 0.95% at 30.1 and 100.3 U/L, respectively. The linearity was up to 351.9 U/L (R2 = 0.9998), with no significant interference or carryover (<5%). A Comparison among 4 reference laboratories showed good reproducibility (R2 ≥ 0.9975). The procedure proved valid for a reference interval of 11.7-38.5 U/L. The mean relative deviation for a routine system was -55.9% and -3.7% before and after calibration. This candidate RMP for serum ADA can potentially be used for standardization of clinical systems.

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

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.