Fresh Serum Research Articles

Impact of heat-inactivation on the detection of SARS-CoV-2 IgM and IgG antibody by ELISA

BackgroundTo establish a safe and accurate method for detecting SARS-CoV-2 IgM and IgG, we assessed the impact of sera after heat-inactivation on the SARS-CoV-2 IgM and IgG levels measured by ELISA-immunoassay. MethodsThe serum samples of 62 patients with COVID-19 and 18 healthy controls were collected in Hankou’s Hospital of Wuhan from February 27 to March 6, 2020. Before and after the samples were inactivated, the levels of IgM and IgG antibodies were measured. ResultsThe indexes of antibodies after inactivated were significantly higher than those in fresh sera, while the positive rates in all participants or in patients with COVID-19 did not change. The positive coincidence rate, negative coincidence rate and total coincidence rate of IgM antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively (κ = 0.971, P < 0.001), while those for IgG antibodies were 98.21% (55/56), 91.67% (22/24) and 98.75% (79/80) respectively (κ = 0.910, P < 0.001). These results showed a good consistency. ConclusionsHeating-activation does not decrease the diagnostic efficacy of SARS-CoV-2 IgM or IgG antibodies. Sera inactivated by heating at 56 °C for 30 min should be recommended to minimize the risk of virus contamination of laboratory staff.

Investigation of promoter methylation of MCPH1 gene in circulating cell-free DNA of brain tumor patients.

Despite advanced diagnostic and therapeutic techniques, many brain tumors are still diagnosed at high grades and, therefore finding novel molecular markers may assist in early detection and reducing brain tumors-related mortality rate. Owing to the previous reports on the importance of MCPH1 gene in tumorigenesis, the present study was aimed to study the promoter methylation of MCPH1 gene in paired circulating cell-free DNA (cfDNA) and tumor tissues of brain tumor patients. Fourteen fresh paired serum and tumor tissue samples in addition to 18 isolated serum samples were collected from patients affected by different grades of brain tumor. Genomic DNA and cfDNA was isolated from tissue and serum samples using QIAamp DNA Mini Kit Norgen Bioteck Kit, respectively. Methylation DNA immunoprecipitation Real-time polymerization chain reaction (MeDIP-Real-time PCR) was performed on isolated DNA samples using EpiQuik MeDIP Ultra Kit and specific primer pairs. cfDNA quantity was determined through Real-time PCR analysis using specific primer pairs designed for GAPDH gene. MCPH1 was methylated in 54% of cfDNA samples which was significantly associated with tumor grade, as well (P-value = 0.02). The methylation rate of MCPH1 was found as 78% in the tissue samples which was meaningfully associated with tumor grade (P-value = 0.03). Moreover, methylation of the MCPH1 gene was consistent in 57% of the same cfDNA and tissue samples. Methylation of MCPH1 gene in neither tumor tissues nor cfDNA was not correlated with age and sex of the patients. Due to the conformity of methylation of MCPH1 gene in cfDNA and tissue samples in more than half of the enrolled patients, especially in higher grades of tumors, it seems that MCPH1 promoter methylation could be a potential epimarker in not only detection of brain tumors but also in response to chemo- and radiotherapy which warranted further assessment.

Tomato juice as a potential replacement for human serum in germ tube test assay

Candida albicans (C. albicans) is the most common opportunistic fungal pathogen often isolated from clinical samples. Traditionally, the differentiation of C. albicans from other Candida species is performed through the germ tube test using human serum. The aim of this study was to assess the use of tomato juice of “Thilina” variety for the differentiation of C. albicans from other non albicans species. This study was carried out using 66 Candida isolates including five Candida standards. Candidal suspension (100 μl) equals to 0.5 McFarland was mixed with 1 ml of fresh human serum and 1 ml of tomato juice, separately. Test tubes were incubated at 37 oC and the observations were made at 15 minute intervals for two hours. The tomato juice exhibited 97% sensitivity, 100% specificity, 97% positive predictive value, 100% negative predictive value and higher positive correlation (R2 > 0.6) in producing results. There was no statistically significant difference (P = 0.05) in the meantime of initial germ tube production when tomato juice was compared with the routine serum medium. For the first time the study strongly suggests that human serum can be replaced by juice of “Thilina” variety of tomato for the differentiation of C. albicans from non albicans species.

Utility of non-serum liquid media against conventional human serum in germ tube production test

: , a predominant yeast pathogen can be easily distinguished from other non-albicans species in a diagnostic laboratory based on their property to produce germ tubes using human serum as a routine substrate. As usage of human serum is accompanied with risk of transmission of blood-borne viral infections, several other media were employed by many clinical laboratories. The aim of this study was to evaluate the efficacy of four commonly used non-serum liquid media relative to fresh human serum in germ tube (GT) productivity among various Candida isolates. A total of 146 Candida isolates (96 ; 47 ; 3 ) identified by conventional methods were assessed for germ tube production in five types of media namely peptone water, Mueller Hinton broth, Trypticase soy broth, Brain Heart infusion broth and fresh human serum . Results obtained in different media for germ tube production in all Candida isolates were analysed for sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.All the Candida isolates showed 100% specificity and PPV in all the five-liquid media. Highest performance in germ tube production (96%) in isolates was found in Trypticase soy broth followed by fresh human serum (95%). Trypticase soy broth was shown to have 96% sensitivity and 97% accuracy for germ tube formation comparable to human serum with 95% sensitivity and 96.5% accuracy. Trypticase soy broth can be suggested as a choice medium for GT production in diagnostic mycology.

Detection of immunoregulatory rheumatoid factor

Regulatory rheumatoid factor (regRF) represents a separate rheumatoid factor population that might be potentially used for clinical diagnostics, whereas regRF-producing lymphocytes serve as a promising therapeutic target for autoimmune diseases. Hence, it is worth outlining conditions allowing to reliably detect regRF in humans and laboratory animals. The method of agglutination with tanned IgG-loaded red blood cells was used to measure level of regulatory rheumatoid factor, because it allows to identify solely regRF as compared to the latex fixation method detecting both the regulatory rheumatoid factor as well as the rheumatoid factor associated with rheumatic diseases. It was found that fresh or frozen serum should be used to detect regRF in rats. Freshly purified rat blood plasma stabilized with heparin, sodium citrate or EDTA did not allow to detect regRF, potentially being linked to the presence of fibrinogen known to demonstrate IgG binding activity. In addition, heparin itself also complicates regRF detection in rats. In humans, regRF was reliably detected in pre-frozen plasma stabilized with sodium citrate. Finally, false-negative results were obtained in a small number of cases while using blood serum for detecting regRF in humans.

Lyophilized Autologous Serum Eyedrops: Experimental and Comparative Study

To analyze the biological stability of autologous serum eyedrops after lyophilization. Prospective, comparative experimental study. This was a comparative study with serum obtained from 12 healthy volunteers. The concentrations of different epitheliotropic factors (eg, transforming growth factor-β [TGF-β1], epidermal growth factor [EGF], platelet-derived growth factor AB [PDGF-AB], and albumin) were measured in fresh and lyophilized serum. The samples were studied after serum preparation (fresh serum) and immediately after saline solution reconstitution of lyophilized serum (0), 15, and 30days later. The biological effects of both serum samples were also compared on conjunctival and corneal cell cultures. The pH, osmolarity, and serum density were also determined. No significant differences were found in the concentration of growth factors between fresh serum and re-dissolved serum samples after lyophilization. The concentration of growth factors remained stable during 1month at 4°C in re-dissolved lyophilized form with saline solution. No differences were found related to osmolarity, pH, and density between fresh and lyophilized serum. In addition, no differences were found on the conjunctival and corneal cells proliferation and differentiation in cells cultures between either serum preparation. The properties of autologous serum remain after lyophilization. The lyophilized serum can be easily stored without temperature restrictions and easily reconstituted for preparation of eyedrops for standard clinical use.

Determination of lithium in dried blood spots and dried plasma spots by graphite furnace atomic absorption spectrometry: Method development, validation and clinical application

Therapeutic drug monitoring (TDM) has become a standard of care for the mood stabilizer lithium (Li+). Dried Blood Spots (DBS) and Dried Plasma Spots (DPS) are promising alternative sampling strategies for TDM, which allows simple and cost-effective logistics in many settings, particularly in Developing Countries. DBS and DPS are of particular interest to Li + TDM for allowing the estimation of Li + erythrocyte levels. Thus, the aim of this study was to develop and validate an assay for the determination of Li+ in DBS and DPS by Graphite Furnace Atomic Absorption Spectrometry (GFAAS), and to evaluate its application in a clinical setting. Li+ was extracted from one 8 mm DBS disc punch with nitric acid 4.5% and from one 6 mm DPS disc punch with diluent solution (HNO3 1% + Triton 0.1%) and injected into GFAAS. The method was applied to Li + TDM in 43 patients with mood disorder. The assays were linear from 0.10 to 3.0 mEq L−1 (r > 0.99), precise, with CV 3.6–7.2% for DBS and 4.6–9.3% for DPS samples, and accurate, with accuracy values of 97–109% and 98–106% for DBS and DPS samples, respectively. Li+ was stable in dried samples during twenty days at up to 42 °C. The DBS assay accuracy and recovery were not influenced by blood hematocrit. The patients presented Li + serum concentrations of 0.18–1.1 mEq L−1 and 0.17 to 0.92 mEq L−1 in DBS and 0.15 to 0.99 mEq L−1 in DPS samples. DPS had comparable Li + concentrations to the ones found in fresh serum samples. With DBS samples it was possible to estimate the Li + erythrocyte to plasma concentration ratio (LiR). The findings of this study support the clinical application of DBS and DPS samples for the TDM of Li+.

Evaluation of interference factors in creatinine measurement by using new guideline CLSI EP7-A3

Objective To investigate the interfering factors in the determination of creatinine(Cr) using the American Clinical Laboratory Standards Association (CLSI) EP7-A3 document. Methods According to the CLSI EP7-A3 document, fresh serum (no hemolysis, lipemia, and jaundice) was used on the day of the experiment and confirmed the interfering substances through the pairing difference experiment and the point-to-point analysis method was used in the dose effect experiment to clarify the difference of interfering substances. Results Triglyceride (16.94 mmol/L), dobutamine hydrochloride (4.01 μmol/L), ascorbic acid (298 μmol/L) did not interfere with the determination of Cr. Free bilirubin (684 μmol/L), conjugated bilirubin (684 μmol/L), calcium hydroxybenzene sulfonate (144 μmol/L) and hemoglobin (10 g/L) were used as the maximum concentrations of interferences for the dose effect test, the results showed that the above interferences had negative interference on the determination of Cr. Conclusion According to EP7-A3, it is valuable to evaluate the interference factors of creatinine determination. Key words: Interference experiment; Creatinine

Sampling and processing blood samples within the South American Youth/Child cARdiovascular and Environmental (SAYCARE) Study

Obesity and overweight in children and adolescents is increasing rapidly worldwide; however, scarce data have been reported from South America countries. With the purpose of assessing hyperlipidemia, insulin resistance and chronic inflammation, the evaluation of blood biomarkers such as glucose, lipoproteins and chronic inflammation proteins is required. In the context of the SAYCARE study, in children and adolescents (3 to 18 years) from seven South American cities, our aim was to assess the impact of pre analytical conditions on different biomarkers evaluated in 474 fresh serum samples, in different country centers. We also evaluated the stability according to time and frozen storage within this study across the concordance of the results obtained from the 49 blood samples measured in three different centers. Significant correlations as well as concordance were observed in TG, Total-C, HDL-C and glucose between Buenos Aires and São Paulo. The samples evaluated in Teresina and São Paulo presented similar results, with exception of total cholesterol. We observed acceptable concordance between Buenos Aires vs São Paulo and Teresina vs São Paulo, suggesting that samples could be processed in each of these centers. This concordance is a consequence of the strict pre analytical conditions previously established in the SAYCARE study.

Distribution of Red Blood Cell Alloantibodies Among Transfusion-Dependent β-Thalassemia Patients in Different Population of Iran: Effect of Ethnicity

The best approach for prevention of alloimmunization in β-thalassemia (β-thal) patients is perfect matching of all red blood cell (RBC) antigens associated with clinically significant antibodies, but this is expensive and may limit the blood supply. Knowing the most common alloantibodies in transfusion-dependent β-thal patients make it possible to establish more cost-effective matching strategies for high-risk antigens. With this in mind, we intended to determine the most common alloantibodies in different parts of Iran. A total of 480 alloimmunized β-thal major (β-TM) patients who were referred to the Tehran Adult Thalassemia Clinic in Tehran, Iran from all provinces between 2015 and 2017, were included in this study. Antibody screening was performed on the fresh serum of all patients. Subsequently, the specification of antibodies was identified using a panel of recognized blood group antigens. Anti-K was the most common alloantibody detected in β-TM patients in all regions of Iran. The prevalence of this antibody reached to 37.7% in the western area, but in southeastern region, anti-E was predominant. Interestingly, the rare alloantibody anti-Kpa was detected with a high prevalence in the western region. The antibodies against E and D antigens were also encountered with high prevalence in most regions of the country. The present study demonstrated the distribution of alloantibodies in alloimmunized transfusion-dependent β-thal patients from diverse ethnic and racial backgrounds of the Iranian population. The results of this study can be used as a basis to establish cost-effective RBC phenotyping and matching strategies for high-risk antigens in donors and chronic transfusion recipients in different regions of Iran.

Cholesterol grafted cationic lipopolymers: Potential siRNA carriers for selective chronic myeloid leukemia therapy.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

Effects of acetyl-L-carnitine on the recovery of hindlimb movement afterspinal cord injury in rats and its mechamism

To observe the effects of different doses of acetyl-L-carnitine (ALC) on hindlimb motor function and spinal cord tissue structure in rats with spinal cord injury. The study will provide theoretical and experimental evidences for acetyl-L-carnitine's clinical treatment. Fifty-five SD rats aged 8-10 weeks were randomly divided into high, medium and low-dose drug intervention (SCI + ALC) group, injury group (SCI) and sham group for behavioral evaluation, MAD and SOD detection, as well as HPLC detection and HE staining. BBB scores and Rivlin experiments were performed to evaluate hindlimb motor function in each group. The morphology and structure of spinal cord tissue was detected by HE staining. Another 9 rats were randomly divided into Sham group, SCI group and ALC group for TUMEL detection of apoptosis. The BBB scores of the high, medium, and low dose SCI+ALC groups were significantly higher than those in the SCI group. The medium and high-dose ALC groups had significant differences (P＜0.01), and the hindlimb motor function was significantly improved in rats. The maximum tilt angle of the Rivlin experiment was observed. The SCI+ALC group had a significantly increased angle compared with the SCI group (P＜0.05), the medium and high-dose ALC group had a significant difference (P＜0.01). Compared with the SCI group, the tissue structure of ALC high-dose group was improved significantly, the number of inflammatory cells and red blood cells was decreased, and the nucleolus of the nerve cells was unclear. The SOD activity of the SCI+ALC group was significantly higher than that of the SCI group, while the MDA content was significantly decreased(P＜0.05), the middle and high dose ALC groups were significantly different (P＜0.01). HPLC chromatogram showed that the SCI+ALC fresh serum sample and the ALC standard solution had the same absorption spectrum at 260 nm, while the Sham group and SCI group serum samples did not show spectral values there, which indicated that the same substance as the standard existed in the sample of SCI+ALC group. TUNEL staining showed that the apoptosis signal was occasionally seen in the sham group, and the apoptosis signal was significantly decreased in the ALC high-dose group compared with the SCI group(P＜0.05). ALC can promote the recovery of hindlimb motor function in rats with spinal cord injury, inhibit oxidative stress and apoptosis, and repair the damaged spinal cord tissue.

Prevalence and Associated Risk Factors of Strongyloides sp. Infection in Diabetic Patients in the Central Part of Mazandaran, Northern Iran

Background: Strongyloides stercoralis, a soil transmitted helminth, is well known as a potentially fatal parasite in immunosuppressed patients. Objectives: The current study aimed to evaluate the prevalence of strongyloidiasis in diabetic patients, in the central parts of Mazandaran province, Iran, using coprological examination and enzyme-linked immunosorbent assay (ELISA). Methods: Fresh stool and serum samples were obtained from diabetic patients. The stool samples were examined using direct smear and formalin-ether concentration methods. The serum samples were tested for the existence of Strongyloides sp. antibodies using a commercial diagnostic kit (Strongyloides - ELISA). Results: The overall prevalence rates were 13.3% (4/30) and 25.6% (46/180) using parasitological and ELISA methods, respectively. The sensitivity and specificity of ELISA compared with stool examination were 100% and 84.6%, respectively. The seroprevalence rate of this infection was higher in females (27.3%) than males (19.5%), and among participants living in rural regions (31%) compared with urban areas (20%). The prevalence rate of Strongyloides sp. infection was higher in patients with diabetic foot (39.1%) compared with cases with non-diabetic foot (23.6%). It also was higher in insulin dependent patients (29.9%) compared with non-insulin dependent subjects (23%). However, these differences were not statistically significant which may have resulted from the small sample size. Conclusions: Our findings demonstrated a high seroprevalence of Strongyloides sp. infection in diabetic patients. Furthermore, this is the first seroprevalence study of strongyloidiasis in diabetic patients from Iran. It seems that the ELISA technique can be used for the diagnosis of individual cases and is a good screening assay to rule out strongyloidiasis in diabetic patients.

Current status of the reference system for measurement of serum high- and low-density lipoprotein cholesterol

Decreased serum high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein cholesterol (HDL-C) concentrations are important risk factors for cardiovascular diseases. Achieving accurate and comparable HDL-C and LDL-C results is the primary requirement for the prevention and treatment of cardiovascular diseases, in which reference system including reference method, reference materials and standardization programs are needed. The current reference method for HDL-C and LDL-C measurement is the β-quantification method developed by the United States Center for Disease Control and Prevention (CDC), a multi-step procedure involving ultracentrifugation, chemical precipitation, and cholesterol analysis by CDC reference method. This method uses a large amount of serum and is technically demanding which limited its application in standardization. The reduced volume β-quantification method uses a small volume of serum sample, is simple and high through put and is more applicable. However, both methods are sensitive to serum matrix. Commutability of reference materials should be evaluated when they are used to calibrate routine methods. Fresh (non-frozen) serum samples should be used for a split sample comparison between reference and direct methods. In addition, development of reference measurement procedure based on ultracentrifugation and preparation of commutable reference materials are the important work in lipoprotein standardization. Key words: Cholesterol,HDL; Cholesterol,LDL; Ultracentrifugation; Reference standards; Reproducibility of results

Normalization of the immune system of large cattle with the help of “Immunopheron” and “Ribotan” medicines

This article presents the results of studying the effect of immunostimulants “Immunoferon” and “Ribotan” on the indices of specific and non-specific resistance of calves. To this end, according to the principle of analogues, 3 groups of calves were formed, with 5 animals each. Calves of the third group served as a control. In the blood of calves, the content of immunoglobulins A, M, G was determined on the analyzer “Uniplan”. The number of T-lymphocytes in the blood was determined by the method of spontaneous rosette formation with erythrocytes of a ram, B-lymphocytes - by the method of complementary rosette formation using standard rabbit hemolysin and as a complement - fresh serum of cows. The bactericidal activity of the blood serum (BASK) was determined by O.V. Smirnova and TA. Kuzminoy, lysozyme activity of blood serum (LASK) - according to KA. Kagramanova and Z.V. Ermolieva, phagocytic index and number (FH and FI) - according to V.S. Gostevu. Experiments showed that the calves of the experimental groups showed an increase in serum levels of immunoglobulin A, M and G, as well as bactericidal and lysozyme activity of blood serum, a phagocytic number and an index. The relative and absolute levels of B-lymphocytes and T-lymphocytes were also significantly higher in the calves of the test groups than in the control group. And the most significant changes were noted with the use of the drug “Immunoferon”.

Inhibition of Human Serum Paraoxonase-I with Antimycotic Drugs: In Vitro and In Silico Studies.

Drugs show their pharmacological effects by inhibiting or activating enzymes. Therefore, enzyme inhibitors have an essential place in the drug design for numerous different sorts of diseases, i.e., cardiovascular, cancer, metabolic, and neurological. The purpose of the current study was to contribute to the growing drug discovery and development area by analyzing functions and drug interactions of paraoxonase-I. For this purpose, the PON1 enzyme was purified from fresh human serum using simple, rapid, and different chromatographic techniques. Then, it was tested for the inhibitory effects of some antimycotic drugs on the PON1. Also, molecular docking analyses of each drug were carried out to determine the binding interactions on the active site of the PON1 enzyme. It was identified that the purified enzyme had the specific activity of 3880.83 EU/mg proteins and the molecular weight of 43 kDa by SDS-PAGE. IC50 values for PON1 were in the range of 0.037 ± 0.001-5.728 ± 0.043 mM. Ki constants for caspofungin acetate, amphotericin B, anidulafungin, and fluconazole were determined to be 0.0105 ± 0.0015 mM, 0.3206 ± 0.0196 mM, 0.1674 ± 0.0233 mM, and 2.5464 ± 0.1655 mM, respectively. The inhibition mechanism of amphotericin B was non-competitive, whereas anidulafungin was mixed type, and the others were competitive.

Murine T Cell Maturation Entails Protection from MBL2, but Complement Proteins Do Not Drive Clearance of Cells That Fail Maturation in the Absence of NKAP.

Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.

A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78–100% and 79.06–100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

Comparison between the triglycerides determination by HPLC in China and reference procedure used in the isotope dilution mass spectrometry CDC CRMLN

Objective To evaluate the comparability and consistency of two kinds of triglycerides reference methods, one of which is the isotope dilution liquid chromatography-mass spectrometry (LC/MS) in the Cholesterol Reference Method Laboratory Network (CRMLN), the other isthehigh-performance liquid chromatography (HPLC) method for triglyceride detection in China. Methods 52 fresh frozen sera with triglycerides levels among 0.45-4.52 mmol/L were determined by LC/MS and HPLC. After evaluation the precision and accuracy of the two methods, a series of analyses were conducted including plotting to scatter plots and deviation graphs, testing outliers, selecting the best fitting regression models and calculating the regression equations and parameters, and so on. The expected deviation at the level of medical decision of triglycerides and the 95% confidence range were statistically analyzed. Results For HPLC method, the CV of instrument measurement was 0.29%(0%-1.16%), the total CV of samples measurement was 0.54%(0.04%-1.28%), and the average bias of the reference materials was 0.22% (-0.43%-0.68%). ForLC/MSmethod,the CV of instrument measurement was 0.55%(0%-1.68%), the total CV of samples measurement was 0.79%(0%-1.93%), and the average bias of the NIST reference materials was 0.09%(-0.73%-1.29%). No outlier was found from the scatter plots and the statistical analysis and the linear regression was fitted to analyze the results of the two methods. The linear regression parameters of two methods for 52 fresh frozen human sera were as follows: the slope was 0.998 8, the standard error of slope was 0.003 5, the intercept was 0.003 7mmol/L, the standard error of intercept was 0.003 0 mmol/L, the standard error of Y-estimate was 0.023 6 mmol/L, and the correlation coefficient was 0.999 7. Compared with the LC/MS method, the absolute deviation of fresh sera by HPLC method was -0.001 mmol/L (-0.070-0.056 mmol/L), with a relative deviation of 0.13% (-2.01-2.83%). T-test showed no statistically significant difference between the two methods. The expected deviations at the triglycerides medicine decision level were within the 95% confidence range, and the expected deviations were far less than the allowable error. Conclusions The HPLC method of triglyceridesdetetion has good consistency and comparability with LC/MS method as one of the reference methods of CRMLN. Because of the advantages of HPLC method such as low cost, simplicity, less technical need, and better precision, HPLC method is expected to play an important role in the process of standardization and traceability of serum triglycerides. Key words: Triglycerides; Chromatography, high pressure liquid; Chromatography, liquid; Tandem mass spectrometry; Isotopes

Inhibition Effects of Phenolic Compounds on Human Serum Paraoxonase-1 Enzyme

Metabolic processes in living organisms are closely related to the catalytic activity of enzymes. Inhibition or induction of enzymes leads to toxicities and metabolic interactions. This studyaims to contribute to the growing drug design field by studying PON1-phenolic compound interactions. For this purpose, the paraoxonase-1 enzyme was purified from fresh human serum byusing rapid and different chromatographic techniques. Additionally, it was investigated the inhibitory effects of some phenolic substances on the PON1 and was found that the purified enzyme had themolecular weight of 43 kDa and the specific activity of 3945.15 EU mg-1. These compounds showed potent inhibition against PON1, especially homovanillic acid exhibited a significant inhibition profileagainst PON1 with an IC50 value of 13.84±0.08 mM. Ki constants were 6.10±0.26 mM for homovanillic acid and 16.96±0.76 mM for phloridzin dihydrate. Homovanillic acid had competitiveinhibition while the phloridzin dihydrate inhibited the PON1 as non-competitive. Also, molecular docking computations were performed by using the Glide XP mode. Glide energy of the homovanillicacid determined to be -23.95 kcal mol-1.