Pool Of Serum Samples Research Articles

B-034 Performance Evaluation of the Total Bile Acids (TBA) Assay on Mindray BS-2800M Analyzer

Abstract Background Bile acids (BA) are synthesized in the liver and secreted into bile. The function of BA is to promote absorption of lipids including fat-soluble vitamins. During liver diseases such as viral hepatitis and liver injury by hepatotoxic drugs, and cholestasis condition, BA secretion to bile is impaired, causing serum BA level to rise. Therefore, the determination of serum BA can be used as a sensitive indicator of liver function. TBA reagent contains NADH that requires pH ≥ 9 to be stable that is usually difficult to be maintained during reagent opened and stored on the analyzer. Meanwhile, 3α-Hydroxysteroid Dehydrogenase (3α-HSD) is required for the reaction. 3α-HSD is sensitive to pH condition. Minor changes in pH results in significant changes of enzyme activity. We formulated our TBA reagent with prolonged stability when reagent is onboard on the analyzer. This study evaluated the performance of the new Mindray TBA assay on Mindray BS-2800M. Methods TBA assay was based on the method with coupled Thio-NAD and NADH reaction for BA. The R1 contains Thio-NAD. The R2 contains NADH and 3α-HSD. R1 is incubated with serum sample containing BA for 5 min at 37°C and R2 is then added. During further incubation in the presence of Thio-NAD, 3α-HSD converts BA to 3-ketosteroids and Thio-NADH. The reaction is reversible, and 3α-HSD can convert 3-keto steroids and Thio-NADH to BA and Thio-NAD. In the presence of excess NADH, enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 412 nm. Two-point-line calibration is used by this assay. The TBA concentrations for the test samples are obtained by the absorbances of test samples from the calibration line. The performances of precision, linearity, onboard stability, interference (hemolysis, lipid and bilirubin), correlation are evaluated following CLSI protocols. Results The precision study was run according to EP05A3 with two commercial controls (about 21 and 36μmol/L) and two serum sample pools(about 9.9 and 24.2 μmol/L)on the Mindray BS-2800M system. The repeatability and within-lab CVs ranged from 0.74% to 1.97%, respectively. The linearity of the assay was determined as from 2.0 μmol/L to 180 μmol/L. The reagent onboard stability is for 28 days without recalibration that is superior to TBA reagents from other manufacturers. The interference of hemolysis, lipid and bilirubin are with less than ±10% impact with hemolysate Hemoglobin up to 500 mg/dL, Intralipids up to 500 mg/dL, and Conjugated bilirubin up to 30 mg/dL, Unconjugated bilirubin up to 30mg/dL. This assay (Y) also correlated well with SEKISUI TBA assay (x), (Y = 0.9965X + 0.2694, r= 0.9993). Conclusions From this evaluation, we concluded that Mindray TBA assay can measure serum TBA precisely and accurately with good performances, especially for onboard stability. It is suitable for use in routine clinical laboratories.

A-096 A New Direct Bilirubin (DBIL) Assay with Enhanced Performance in Anti-hemolysis Interference on Mindray BS-2800M Analyzer

Abstract Background Serum bilirubin could be used to assess liver functions. Due to clinical importance, it is highly desired to have an accurate bilirubin test. However, there are circumstances where error could occur in bilirubin test results. In particular, the hemolysis interference is one of the major problems that could mislead the direct bilirubin results because of their relatively high occurring frequencies in the clinical laboratories. To resolve this issue and improve the accuracy for the direct bilirubin, we developed a new reagent formulation for DBIL assay that minimizes hemolysis interference. In addition, this reagent also shown good performance characteristics on Mindray BS-2800M Clinical Chemistry analyzer. Methods The new DBIL assay is a vanadate-oxidation method that bilirubin is gradually transformed to biliverdin because of oxidizing agent vanadate, resulting in decreasing of absorbance at 450 nm. Two-level calibration is used for this assay. DBIL concentrations are obtained from the calibration line with signals from the patient samples in the assay. Following CLSI protocols, basic assay performances were evaluated: interference, precision, linearity, method comparison and low end detection (LOB, LOD, LOQ). Results Hemolysis interference was tested in parallel with DBIL reagents from different IVD companies, Roche, Wako and Chinese domestic main-player companies, A, B, and C. The results showed that the reagents of A and B companies were significantly suffered from hemolysis interference with maximum bias exceeded -30%. Although there are some effects on the anti-hemolysis with DBIL reagents of Wako and C company, the DBIL reagent of Mindray was the best with less than -8% of impact when hemolysate hemoglobin was up to 1000 mg/dL (H index). The Diazo DBIL assay from Roche shown issue with hemolysis interference. The interference to our new DBIL assay from lipid (up to 1000 mg/dL) and ascorbic acid (up to 30 mg/dL) were also with less than ±10% impact. The precision study was run according to EP05A3 with two commercial controls (∼ 10 and 30 µmol/L) and three serum sample pools (∼ 6.8,17.1 and 342 µmol/L). The CVs of repeatability were ranged from 0.54% to 0.58% for controls and 0.24% to 0.88% for serum pools, respectively. The within-lab precision was below 2.5%. The analytical linearity was determined as from 0.66 µmol/L to 439.66 µmol/L. For low-end detection, the LOB, LOD and LOQ were 0.5,1.0 and 1.7 µmol/L, respectively. In addition, the Mindray (y) correlated well with the DBIL assay of Wako (x) reagent on Mindray BS-2800M system (y): y =0.9847 x+0.1301 (r =0.9997, n =400, sample range: 1.08 - 430.00 μmol/L). Conclusions The new Mindray direct bilirubin assay shown a good anti-hemolysis interference performance on BS-2800M system and demonstrated good performances in other assay characteristics. It is therefore suitable for use in clinical laboratories.

B-048 Performance Evaluation of the New Anti-Streptolysin-O Assay on Mindray BS-2800M System

Abstract Background Elevated serum concentrations of Anti- Streptolysin-O(ASO) can be used to provide serologic evidence of past or present infection by β-Hemolytic Streptococci. Increasing serum concentrations of ASO antibodies are produced in response to ASO exotoxins secreted by the bacteria. Measurement of ASO antibody levels in serum can be used as an aid in the diagnosis of diseases such as glomerulonephritis, rheumatic fever, bacterial endocarditis, tonsillitis, and scarlet fever, etc. The New ASO assay has been evaluated on Mindray BS-2800M that is an automated, high throughput, and low cross-contamination system. The evaluation of this assay included precision, linearity, dilution accuracy, interference, high dose hook, and method comparison studies. Methods The new ASO reagent contains R1 buffer and R2 with latex particles conjugated with in-house made ASO antigen. The R1 is incubated with human serum sample containing ASO antibodies for 5 min at 37°C and then R2 is added. After another 5 min of incubation, immune-aggregation complex is formed. The increased turbidity is measured at 570 nm. By constructing a six-level calibration curve (water is used as reagent blank) from the absorbances of calibrators, the ASO antibody concentration of the sample is determined. Results The precision study was run according to EP05A3 with two commercial controls (approximately 80 and 300 IU/mL) and two serum sample pools (approximately 150 and 400 IU/mL) on the Mindray BS-2800M system. The repeatability and within-lab CVs ranged from 0.8% to 2.0% and 2.0% to 6.0%, respectively. The analytical linearity of the assay was determined as from 20 IU/mL to 1000 IU/mL with the limit of blank of 2 IU/mL and limit of detection of 10 IU/mL. The dilution accuracy was evaluated by diluting high ASO level samples at upper region of assay range using saline with 5-fold and run neat and diluted samples. The deviation in recoveries between diluted sample after calculation and the neat sample was found to be less than ±10%. The new ASO assay showed <±10% interference with bilirubin (conjugated or free) up to 40 mg/dL,hemolysate hemoglobin up to 1000 mg/dL,Intralipid up to 1000 mg/dL,and Triglycerides up to1000 mg/dL. The assay on the Mindray BS-2800M system(y) correlated well with the ASO assay(x) on the Beckman AU5800 system:y = 1.01 x + 1.55(r = 0.9514,n = 95,range:50–971 IU/mL). No prozone was observed with the assay on the Mindray BS-2800M system up to the highest ASO antibody concentration of 5000 IU/mL. Conclusion We conclude that the New ASO assay with in-house developed ASO antigen raw material, when used on the Mindray BS-2800M system, can measure serum ASO antibody concentrations precisely and accurately over a broad range. It is suitable for use in routine clinical laboratories.

Analytical evaluation and Sigma metrics of 6 next generation chemistry assays on the Abbott Architect system.

We evaluated analytical and Sigma performance for 6 next generation chemistry assays on the Abbott Architect c8000 system. Albumin with bromocresol purple or green, amylase, cholesterol, total protein, and urea nitrogen were analyzed using photometric technology. Analytical performance goals were defined based on Accreditation Canada Diagnostics (ACD) and Clinical Laboratory Improvement Amendments (CLIA). Precision study consisted of testing 2 quality control concentrations and 3 patient serum sample pools, twice a day in quintuplicate over 5days. Linearity testing consisted of 5-6 concentrations of commercial linearity materials. We tested a minimum of 120 serum/plasma specimens on the new and current Architect methods for comparison. We assessed accuracy with reference materials for 5 assays, and a calibration standard for cholesterol. Bias from the reference standard target value was used for Sigma metric analysis. Observed total imprecision of the assays ranged from 0.5 to 4%, meeting pre-defined goals. Linearity was acceptable over the tested range. Measurements on the new and current Architect methods were comparable. Accuracy ranged from 0 to 2.0% absolute mean difference from target value. All 6 next generation clinical chemistry assays demonstrated Six Sigma quality, using CLIA standards. Applying ACD recommendations, 5 assays showed Six Sigma, while cholesterol showed Five Sigma performance.

Simultaneous determination of 35 organochlorine pesticides and polychlorinated biphenyls in the serum of the general population in Wuhan by solid phase extraction-gas chromatography-tandem mass spectrometry

有机氯农药(OCPs)和多氯联苯(PCBs)是两类重要的持久性有机污染物,可在环境介质中长期存在,并通过多种途径进入人体,导致人体的高暴露风险。OCPs和PCBs对人体存在诸多健康危害,精准定量人体内OCPs和PCBs的暴露水平是健康效应评价的关键。该研究基于固相萃取-气相色谱-串联质谱联用技术(SPE-GC-MS/MS)建立了同时检测100 μL血清中35种OCPs和PCBs的分析方法。血清样品经尿素沉淀蛋白后,采用Oasis® HLB小柱净化,正己烷-二氯甲烷混合溶液(1∶1, v/v)洗脱,氮吹近干,正己烷定容,多反应监测(MRM)模式检测,内标法定量分析。结果表明,OCPs和PCBs在0.05~50.0 ng/mL范围内线性关系良好,检出限在1.2~71.4 ng/L之间。35种目标分析物的加标回收率在72.6%~142%之间,相对标准偏差小于25%。利用所建立的方法检测了武汉市普通人群血清样本中OCPs和PCBs的浓度水平,结果表明武汉市普通人群广泛暴露于OCPs和PCBs,且以OCPs为主。有8种OCPs和7种PCBs检出率高于50%,其中p,p'-滴滴伊、p,p'-滴滴滴和甲氧滴滴涕检出率达100%,非类二噁英PCBs是PCBs的主要成分。血清中OCPs浓度随年龄增长呈升高趋势,在60岁以上存在性别差异;不同性别、年龄人群血清中PCBs浓度无统计学差异。该方法样本用量少,操作简便,具有较高的准确度和精密度,适用于环境健康研究中大量人群血清样本中痕量OCPs和PCBs的生物监测。

Seroprevalence of pestivirus infections is low in Belgian small ruminant flocks and is significantly associated with the presence of cattle

A study was implemented to estimate the pestivirus seroprevalence in sheep and goats in Belgium, to identify circulating species and to check for a potential association between seropositivity of small ruminants and presence of cattle in the same farm. It was based on the testing of serum samples and bulk tank milk samples (BTM) collected in sheep and goat flocks in 2018–2019 all over the country. 7460 serum samples collected from 410 flocks were tested by a commercial ELISA able to detect antibodies (Ab) against Border Disease Virus (BDV), and Bovine Viral Diarrhea Virus (BVDV). BTM samples (n = 144) were collected from dairy flocks in November 2019 and tested with the same Ab ELISA. ELISA positive serum samples were also tested by virus neutralization test (VNT) for neutralizing antibodies against BDV, BVDV-type1 and BVDV-type2. Virological tests (RT-PCR) were performed on pools of serum samples from pestivirus-exposed flocks with at least two seropositive animals and on all Antibody-positive BTM samples. Information about serum and milk samples (identification, test results, farm of origin and location, presence of cattle) were gathered in animal-level and farm-level databases.Based on this study, the apparent animal seroprevalence for pestiviruses in small ruminant flocks in Belgium in 2018 was estimated to be 0.87 % (95 % C.I. [0.68 %–1.11 %]). The prevalence of flocks exposed to pestivirus (i.e. with at least one seropositive animal) was estimated to be 8.5 % (95 % C.I. [6.4 % - 11.6 %]). In exposed flocks, the average within-flock seroprevalence was 9.9 %. In dairy sheep and goats, the estimated proportion of exposed flocks in 2019, based on the detection of pestivirus antibodies in the bulk tank milk, was 9.7 % [5.9 %–15.7 %]. All PCR tests were negative, indicating the likely absence of active pestivirus circulation in these flocks. Although the observed pestivirus seroprevalence was found to be low in Belgian small ruminants, this study also showed, based on VNT results, that they are exposed to several pestivirus species: BDV, BVDV-1 and BVDV-2. 22.4 % of the farms included in the serological survey were holding both a small ruminant flock and a cattle herd, hence with a potential risk of contact between the two species. There was a significant positive association between pestivirus seropositivity in the sheep/goat flocks and the presence of a cattle herd in the same farm (OR = 2.42 (95 %C.I. [1.18–4.94]) but this association was not found for Ab-positive BTM in dairy flocks.

Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin.

Biotin >20ng/mL may interfere with the Elecsys® Troponin T-high sensitive assay (cTnT-hs; Roche Diagnostics International Ltd). We evaluated the performance of an updated assay, cTnT-hs*, which was designed to reduce biotin interference. cTnT-hs* assay performance was assessed using up to two applications (18min/9min) on three analyzers (cobas e 411/cobas e 601/cobas e 801). Biotin interference was determined by measuring recovery in an 11-sample series dilution with biotin ranging from 0-3600ng/mL. Repeatability/reproducibility were evaluated in five serum sample pools (n=75 each). Method comparisons tested: cTnT-hs* vs. cTnT-hs (18min/cobas e 601); cTnT-hs* assay 18 vs. 9min (cobas e 601); cTnT-hs* (18min) on cobas e 601 vs. cobas e 411 and cobas e 601 vs. cobas e 801. Concordance at the 99th percentile decision limit between cTnT-hs* and cTnT-hs (9min/cobas e 601) was calculated using 300 lithium-heparin plasma samples and a 14ng/L assay cutoff. cTnT-hs* assay (18min/cobas e 601) recovery was≥96% for biotin≤1250ng/mL. Across all applications/analyzers, coefficients of variation for repeatability/reproducibility with the cTnT-hs* assay were <5% in most serum sample pools (mean cardiac troponin T: 8.528-9484ng/L). High correlation (Pearson's r=1.000) was demonstrated for all method comparisons. Concordance at the 99th percentile decision limit was high between the cTnT-hs* and cTnT-hs assays. The updated cTnT-hs* assay may provide greater tolerance to biotin interference, and shows good analytical and clinical agreement/concordance with the previous cTnT-hs assay.

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics.

Background Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat worldwide as, after initial reports in China, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre-analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management. Methods The imprecision of the MAGLUMI™ 2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from <5 days up to 26-30 days. Results Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was <4% and <6% for IgM and IgG, respectively, whilst intermediate imprecision was <6%). In addition, results of dilution and recovery studies were satisfactory. The kinetics of COVID-19 antibodies confirmed previously reported findings, showing a rapid increase of both IgM and IgG after 6-7 days from the symptom onset. IgG had 100% sensitivity on day 12, whilst 88% was the higher positive rate achieved for IgM after the same time interval. Conclusions The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.

Performance evaluation of Siemens Atellica enhanced estradiol assay.

Serum estradiol (E2) levels may be used in the diagnostic and/or monitoring of a broad variety of clinical conditions. The aims of this study were to evaluate the Siemens enhanced estradiol assay (eE2) on Atellica IM 1600 (Siemens Healthineers) and to perform a sample comparison with the Siemens ADVIA Centaur XP (Siemens Healthineers). Within-day and between-day coefficient of variation (CV) were determined using serum sample pools and quality control materials. Six serum samples with decreasing concentrations of E2 were used to assess the limit of quantification. Linearity was evaluated using two different serum samples. Accuracy was evaluated by measuring three certified reference materials. Passing-Bablok regression and Bland-Altman plot were used for comparing Atellica and Centaur XP in 119 serum samples ranging from 45 to 10,059pmol/L. Within-day and between-day imprecision was <6.6%. Accuracy was <6.0% for all values except for 114pmol/L (22%). The study of limit of quantification resulted in an interday imprecision <20%. High correlation between measured and expected estradiol dilution results was observed (R=0.99), with recoveries ranging from 77 to 93%. Comparison study showed good agreement and no significant bias. The study shows that Atellica eE2 assay presents acceptable imprecision and accuracy and correlates well with Centaur XP.

Distribution of Psychotropic Drugs Into Lipoproteins.

The aim of this pilot study was to investigate whether psychotropic drugs frequently analyzed in a routine therapeutic drug monitoring laboratory bind to low-density lipoproteins/very-low-density lipoproteins (LDL/VLDL) in human serum. Drug concentrations in 20 serum sample pools containing one psychotropic drug each, and in the LDL/VLDL fractions extracted from the same samples, were measured by triple quadrupole liquid chromatography tandem mass spectrometry. The membrane permeability of the drugs was measured using a Parallel Artificial Membrane Permeability Assay. Of the 20 antidepressants, antipsychotics, and antiepileptics examined, 7 drugs were detected in both the pooled serum samples and in the LDL/VLDL fraction. Binding of drugs to LDL/VLDL significantly correlated with high octanol: water partition coefficient (logP), high degree of protein binding, and a low polar surface area. The drugs found in LDL/VLDL, with the exception of aripiprazole, were also characterized by high or intermediate membrane permeability. The present results indicate that psychotropic drugs with certain characteristics bind to LDL/VLDL in blood. This further implies that lipoproteins could play an important role in drug transport.

Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera.

Background International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies. Methods Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization. Results A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 μL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation. Conclusions This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

Serum microRNA-193b as a promising biomarker for prediction of chemoradiation sensitivity in esophageal squamous cell carcinoma patients.

Esophageal squamous cell carcinoma (ESCC) is the most predominantly occurring type of esophageal cancer worldwide. Locally advanced ESCC patients are treated by neoadjuvant chemoradiation for tumor downstaging prior to tumor resection. Patients receiving this treatment have an increased expectation of cure via the following tumor resection and have better survival outcomes. However, not all patients respond well to chemoradiation and poor responders suffer from treatment-associated toxicity and complications without benefits. No method is currently available to predict patient chemoradiation response and to exclude poor responders from ineffective treatment. To address this clinical limitation, the present study aimed to identify non-invasive biomarkers for predicting patient chemoradiation response. Due to the features of microRNA (miRNA) in cancer diagnosis, prognosis and treatment response prediction, serum miRNA arrays were performed to identify potential miRNA(s) that may be used for chemoradiation response prediction in ESCC. Using an miRNA array to compare pre-treatment serum sample pools from 10 good responders and 10 poor responders, the present study identified miR-193b, miR-942 and miR-629* as candidate miRNAs for predicting chemoradiation response. Subsequent validation using reverse transcription-quantitative polymerase chain reaction confirmed that miR-193b, however not miR-942 and miR-629*, were significantly increased in sera from 24 good responders, compared with 23 poor responders. Further analyses using the receiver operating characteristic curve revealed a strong predictive power of serum miR-193b on discriminating good responders from poor responders to chemoradiation. In addition, a high serum level of miR-193b was significantly associated with better survival outcomes. Therefore, serum miR-193b may be considered a promising biomarker for predicting chemoradiation response and post-therapy survival of ESCC patients.

Identification of reference genes for circulating microRNA analysis in colorectal cancer.

Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control. Seven candidate miRNAs chosen from profiling results as well as three previous reported reference miRNAs were validated using qPCR in 30 colorectal cancer patients and 30 healthy individuals, and thereafter analyzed by statistical algorithms BestKeeper, geNorm and NormFinder. Taken together, hsa-miR-93-5p, hsa-miR-25-3p and hsa-miR-106b-5p were recommended as a set of suitable reference genes. More interestingly, the three miRNAs validated from 485 miRNAs are derived from a single primary transcript, indicting the cluster may be highly conserved in colorectal cancer. However, all three miRNAs differed significantly between healthy individuals and non-small cell lung cancer or breast cancer patients and could not be used as reference genes in the two types of cancer.

Cratylia mollis lectin nanoelectrode for differential diagnostic of prostate cancer and benign prostatic hyperplasia based on label-free detection

The research for new biomarkers of cancer has studied the role of fetuin glycoprotein on the metastatic disease diagnosis. Cratylia mollis is a lectin with high finity to fetuin, and used here to differentiate prostate cancer and benign prostatic hyperplasia. A label-free electrochemical nanosensor based on assembled carboxylated carbon nanotubes (COOH-CNTs) and poly-L-lysine (PLL) film was developed and applied to serum samples of prostate cancer positive for Gleason score. The electrode analytical response to fetuin in PBS samples, obtained by square wave voltammetry, exhibited a linear range from 0.5 to 25µgmL−1, with a high correlation coefficient (r=0.994, p<0.001) and low limit of detection (0.017µgmL−1). The lectin nanoelectrode showed a good repeatability (1.24% RSD) and reproducibility (4.24% RSD). A pool of serum samples from prostate cancer patients with known the Gleason score were tested showing a significant statistically correlation. Thus, the lectin nanoelectrode was able to distinguish the degree of staging prostate cancer, providing the diagnostic differentiation of benign and malign hyperplasia. To the best of our knowledge, it is the first biosensor for this application using a lectin.

Alternative Processing of the U2 Small Nuclear RNA Produces a 19–22nt Fragment with Relevance for the Detection of Non-Small Cell Lung Cancer in Human Serum

RNU2 exists in two functional forms (RNU2-1 and RNU2-2) distinguishable by the presence of a unique 4-bases motif. Detailed investigation of datasets obtained from deep sequencing of five human lung primary tumors revealed that both forms express at a high rate a 19–22nt fragment (miR-U2-1 and -2) from its 3′ region and contains the 4-bases motif. Deep sequencing of independent pools of serum samples from healthy donors and lung cancer patients revealed that miR-U2-1 and -2 are pervasively processed in lung tissue by means of endonucleolytic cleavages and stably exported to the blood. Then, microarrays hybridization experiments of matched normal/tumor samples revealed a significant over-expression of miR-U2-1 in 14 of 18 lung primary tumors. Subsequently, qRT-PCR of miR-U2-1 using serum from 62 lung cancer patients and 96 various controls demonstrated that its expression levels identify lung cancer patients with 79% sensitivity and 80% specificity. miR-U2-1 expression correlated with the presence or absence of lung cancer in patients with chronic obstructive pulmonary disease (COPD), other diseases of the lung – not cancer, and in healthy controls. These data suggest that RNU2-1 is a new bi-functional ncRNA that produces a 19–22nt fragment which may be useful in detecting lung cancer non-invasively in high risk patients.

Simultaneous quantification of serum phytosterols and cholesterol precursors using a simple gas chromatographic method

AbstractDetermination of the main phytosterols (Ps, β‐sitosterol and campesterol) and cholesterol precursors (desmosterol and lathosterol) in human serum using a simple GC‐FID method has been validated. Direct saponification, without lipid extraction, sterols extraction, and further derivatization was applied to samples prior to GC analysis. To evaluate the method, a pool of serum samples from eight healthy women was used. Good linearity (r&gt;0.99) was found in the assay range: β‐sitosterol (0.99–17.82 µg/mL), campesterol (0.14–10.8 µg/mL), desmosterol (0.17–2.6 µg/mL), and lathosterol (0.6–5.97 µg/mL). Limits of detection (ng/mL) were: 86 (β‐sitosterol), 42 (campesterol), 4 (desmosterol), and 44 (lathosterol). Accuracy, estimated by recovery assays (%), were: 113 (β‐sitosterol), 114 (campesterol), 111 (desmosterol), and 102 (lathosterol). Within and between precision values (%), expressed as the relative SD (RSD), were: 2.6 and 8.1 (β‐sitosterol), 1.6 and 7.2 (campesterol), 2.1 and 7.9 (desmosterol), and 4.1 and 5.8 (lathosterol), respectively. The developed methodology allowed fast (1‐day analysis) and reliable quantification of sterols in serum, required a small volume of sample and reduced use of solvents. It therefore could be used in clinical assays for the determination of serum sterols, as in evaluating the pharmacological response to lipid‐lowering agents, and in assessing biological responses to Ps‐enriched diets.Practical applications: This methodology allows fast and reliable quantification of sterols in serum, requiring a small volume of sample and reduced use of solvents. It can be used as a routine method for the quantification of phytosterols and cholesterol precursors in clinical assays, and it is also suitable for monitoring biological responses to health‐promoting phytosterol‐enriched diets.

Characterization of Novel Leishmania infantum Recombinant Proteins Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human Visceral Leishmaniasis

To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4-93.0% and 36.4-97.2%) and specificities (76.1-100% and 90.4-97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.

Discovery and Validation of Serum Protein Changes in Type 1 Diabetes Patients Using High Throughput Two Dimensional Liquid Chromatography-Mass Spectrometry and Immunoassays

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

The Usefulness of the Combination of Free CA 15.3 and CA 15.3-IGM Complexes for Breast Cancer Diagnosis

Background Several studies have shown that the main circulating biomarkers of liver and colorectal cancer can be detected in the bloodstream and are also associated with immunoglobulin M to form stable complexes. These immune complexes show increased capacity of discrimination between cancer patients and healthy controls if combined with the free biomarker form. Within the context of the Project FIRB 2003 - Nanosized Cancer Polymarker Biochip - we wanted to investigate if IgM complexes have importance also in breast cancer. We focused our study on the immune complexes between IgM and CA 15.3 because free CA 15.3 is the most commonly used breast cancer biomarker in clinical practice. However, this biomarker alone lacks satisfactory sensitivity especially in early cancer detection. Aim The aim of our study was to assess the occurrence of immune complexes between CA 15.3 and IgM in sera from patients with primary breast cancer and in sera from healthy controls to evaluate its putative diagnostic value compared with the diagnostic value of free CA 15.3. Methods A total of 130 serum samples were obtained from 56 healthy women (mean age±SD, 45±8.23 years) and 74 women with stage l and II breast cancer (mean age±SD, 59±13.6 years) before any treatment, either surgical or chemo-therapeutic. Serum samples were collected, aliquoted and stored at-80°C in the centralized biobank of the project according to very stringent standard operating procedures (SOPs) distributed by the coordinating unit. To evaluate the presence of CA 15.3-lgM immune complexes, we developed and validated a novel enzyme-linked immunosorbent assay (ELISA) with a polyclonal rabbit anti-human CA 15.3 antibody (Abcam) as the catcher antibody. CA 15.3-lgM was detected with peroxidase-conjugated anti-human IgM and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide as substrate (Sigma Aldrich, Italy). The levels of CA 15.3-lgM were expressed in arbitrary units per mL (AU/mL) by interpolation on a calibration curve obtained by serial dilution of a reference calibrator purified by gel filtration chromatography from a pool of serum samples with high levels of CA 15.3-lgM. Serum levels of free CA 15.3 were assessed in parallel on each sample using an automated immunoassay system (ADVIA Centaur-Siemens Diagnostics) and expressed in U/mL. Results To discriminate between cancer patients and healthy controls, we used as cutoff values 31.5 U/mL for free CA 15.3 (corresponding to the cutoff used in the clinical routine) and 794 AU/mL for CA 15.3-lgM (representing the 95th percentile of the distribution of serum levels of CA 15.3-lgM in healthy controls). By using these cutoff values, we obtained a sensitivity of 1 0% (7/74 cases) and a specificity of 95% (53/56 controls) for CA 15.3-lgM. The sensitivity and specificity of free CA15.3 were 7% (5/74 cases) and 1 00% (56/56 controls), respectively. Interestingly, the serum levels of the two biomarkers did not overlap, so their combination at 95% specificity identified 12/74 cases (16.2%). When we took a cutoff of 22 U/ mL for free CA15.3 (the 95th percentile of its distribution in healthy controls), we calculated a sensitivity of 26% (1 9/74 cases) and a specificity of 95% (53/56 controls); its combination with CA 15.3-lgM had a sensitivity of 34% (25/74 cases) and a specificity of 90% (50/56 controls). Conclusions These results demonstrate for the first time the presence of CA 15.3-lgM in the bloodstream of patients with breast cancer. In addition, our data suggest that CA 15.3-lgM is a complementary serological marker to free CA 15.3 and the combination of these biomarkers could improve the diagnosis of breast cancer.

Feasibility of Ribavirin Therapeutic Drug Monitoring in Hepatitis C

Ribavirin, a nucleoside analog, is administered in combination with interferon to patients with chronic hepatitis C. To evaluate the feasibility of ribavirin therapeutic drug monitoring, we investigated the influence of blood collection and preanalytical conditions on ribavirin concentrations and compared the results obtained from interlaboratory blind tests by 3 laboratories using different analytical techniques. On 3 occasions, blank serum samples spiked with ribavirin and pooled serum samples from patients on ribavirin were sent to the 3 laboratories. Two analytical techniques were based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and 1 on high-performance liquid chromatography with UV detection (HPLC-UV), with protein precipitation or solid-phase extraction, all validated according to international guidelines. Inter- and intra-batch mean relative errors ranged from -7.4% to +10.3% and from -10.3% to +7.4%, respectively. Relative standard deviations were <13.5% and <10.6%, respectively. Linearity, assessed blindly, between 125 and 4550 ng/mL was excellent (r > 0.991) for all 3 methods. The 2 LC-MS/MS techniques were slightly less precise and accurate than HPLC-UV, perhaps because the internal standard used was not a ribavirin isotope. Accurate and precise LC-MS/MS and HPLC-UV methods developed in 3 different laboratories provided excellent and consistent results to blind tests for ribavirin determination in spiked serum samples and pools of serum samples from patients with chronic hepatitis C virus infection.