Pooled Serum Research Articles

Hemolysis, icterus and lipemia interfere with the determination of two oxidative stress biomarkers in canine serum

BackgroundOxidative stress has been proven to play a role in numerous human and canine diseases. Among the biomarkers of oxidative stress, Thiobarbituric Acid Reactive Substances (TBARS) and Total Antioxidant Status (TAS) are two of the most widely used. Preanalytical factors are crucial for obtaining accurate results in these assays. Hemolysis, icterus and lipemia (HIL) are common sources of preanalytical errors in the laboratory; however, limited information is available regarding the considerations for canine specimens. Therefore, the objective of this study was to evaluate the potential interferences of HIL in the determination of TBARS and TAS in canine serum.MethodsSolutions of pooled canine serum samples were prepared by adding increasing concentrations of hemolysate, bilirubin and a synthetic lipid emulsion. TBARS and TAS were determined, and biases from the control value caused by the interfering substances were calculated.ResultsHemolysis, icterus and lipemia induced significant interferences on TBARS and TAS, albeit to varying degrees depending on the specific biomarker and interfering substance. TBARS appeared to be more susceptible to interferences in this study. Slight hemolysis, moderate icterus and slight lipemia caused notable deviations in TBARS values, surpassing the acceptable threshold for interference. TAS assay was also affected by HIL, although to a lesser extent compared to TBARS. Significant biases from TAS control value were observed when icterus was moderate, and when hemolysis and lipemia were more pronounced.ConclusionsIn light of our results, we conclude that hemolyzed, icteric and lipemic specimens are not suitable for TBARS and TAS determination in canine serum. Our findings hold considerable practical utility, as a simple visual inspection would be sufficient for identifying and excluding such specimens.

Allergy patient-specific IgE antibody shows significantly stability during 3 months of storage at multiple temperatures from -80 to 25°C.

The detection of allergen-specific IgE antibodies is an important biomarker for the diagnosis and treatment monitoring of allergic diseases. And the pre-analytical phase is an important part of the overall quality of the laboratory. In this study, 44 patients with allergic diseases (including 23 patients with allergic rhinitis, 12 patients with allergic rhinitis and asthma, and 9 patients with allergic dermatitis) were included in the outpatient center of the Department of Allergy, the First Affiliated Hospital of Guangzhou Medical University. We mixed the serums of the above 44 patients (approximately 0.8 ml of serum volume per patient) into a large volume of serum pool (about 35 ml in total) and divided into 26 parts. And 26 serum samples were stored at 4 different temperatures for 90 days to observe the stability of sIgE antibodies to 16 allergens in serum. The results show that serum sIgE antibody titers in patients with allergic diseases show significant stability during 90 days of storage, even at room temperature. Good stability even after up to 10 freeze-thaw cycles under low temperature storage conditions.

Comparison between Pooled Sera and Coomercial Serum on the Accuracy of Triglyceride Assessment

The increase in dyslipidemia cases every year requires the Clinical Chemistry laboratory to carry out quality control which is influenced by an assessment of accuracy and accuracy. These measurements were used with control materials. So far, in laboratories, there are still many commercial serum control materials used as controls that are used daily in triglyceride tests, and rarely use pooled sera. In fact, there are several advantages in using pooled sera as a control material. This type of analytic survey research with a comparative study method. The sampling technique used is purposive sampling. Data collection techniques were carried out using observation sheets. Data were processed computerized using univariate analysis and bivariate analysis using independent t-tests. The results showed that the average triglyceride examination value with Pooled Sera control material was 94.67 mg/dL with a Standard Deviation of 2.52, a coefficient value variance of 2.66%. While the results of triglyceride examination with Commercial Serum control material were 79.13 mg/dL with a Standard Deviation of 3.57, the coefficient of variance was 4.51%. It was concluded that there is a significant difference between triglyceride examination using Pooled Sera and Commercial Serum. It is recommended that there be developments in medical laboratory technology in hospitals, making this research a reference in educational institutions, and further research on other parameters between Pooled Sera ingredients and other Serum.

Anorexia nervosa and adrenal hormones: a systematic review and meta-analysis.

Anorexia nervosa is a primary psychiatric disorder characterized by self-induced negative energy balance. A number of hormonal responses and adaptations occur in response to starvation and low body weight including changes in adrenocortical hormones. Our objective was to systematically review adrenocortical hormone levels in anorexia nervosa. We searched MEDLINE and EMBASE for studies that reported at least one adrenocortical hormone, including dehydroepiandrosterone (DHEA), DHEA-sulphate (DHEA-S), progesterone, 17-hydroxyprogesterone, pregnenolone, cortisol (serum, urine, cerebrospinal fluid, and hair sample), aldosterone, androstenedione, and testosterone in patients with anorexia nervosa and normal-weight healthy controls from inception until October 2021. Means and standard deviations for each hormone were extracted from the studies to calculate a mean difference (MD). A pooled MD was then calculated by combining MDs of each study using the random-effects model. We included a total of 101 studies with over 2500 females with anorexia nervosa. Mean cortisol levels were significantly higher in anorexia nervosa as compared to normal-weight controls for multiple forms of measurement, including morning cortisol, 12-hour and 24-hour pooled serum cortisol, 24-hour urine cortisol, and after an overnight dexamethasone suppression test. In contrast, mean serum total testosterone and DHEA-S levels were significantly lower among patients with anorexia nervosa. Women with anorexia nervosa have higher cortisol levels and lower DHEA-S and testosterone levels compared to women without anorexia nervosa. This finding is important to consider when evaluating low-weight women for disorders involving the adrenal axis, especially Cushing's syndrome.

Novel Per- and Polyfluoroalkyl Substances Discovered in Cattle Exposed to AFFF-Impacted Groundwater.

The leaching of per- and polyfluoroalkyl substances (PFASs) from Australian firefighting training grounds has resulted in extensive contamination of groundwater and nearby farmlands. Humans, farm animals, and wildlife in these areas may have been exposed to complex mixtures of PFASs from aqueous film-forming foams (AFFFs). This study aimed to identify PFAS classes in pooled whole blood (n = 4) and serum (n = 4) from cattle exposed to AFFF-impacted groundwater and potentially discover new PFASs in blood. Thirty PFASs were identified at various levels of confidence (levels 1a-5a), including three novel compounds: (i) perfluorohexanesulfonamido 2-hydroxypropanoic acid (FHxSA-HOPrA), (ii) methyl((perfluorohexyl)sulfonyl)sulfuramidous acid, and (iii) methyl((perfluorooctyl)sulfonyl)sulfuramidous acid, belonging to two different classes. Biotransformation intermediate, perfluorohexanesulfonamido propanoic acid (FHxSA-PrA), hitherto unreported in biological samples, was detected in both whole blood and serum. Furthermore, perfluoroalkyl sulfonamides, including perfluoropropane sulfonamide (FPrSA), perfluorobutane sulfonamide (FBSA), and perfluorohexane sulfonamide (FHxSA) were predominantly detected in whole blood, suggesting that these accumulate in the cell fraction of blood. The suspect screening revealed several fluoroalkyl chain-substituted PFAS. The results suggest that targeting only the major PFASs in the plasma or serum of AFFF-exposed mammals likely underestimates the toxicological risks associated with exposure. Future studies of AFFF-exposed populations should include whole-blood analysis with high-resolution mass spectrometry to understand the true extent of PFAS exposure.

Validation of metrological traceability of the new generation of Abbott Alinity alkaline phosphatase assay.

Recently, Abbott Diagnostics marketed a new generation of Alinity enzyme assays, introducing a multiparametric calibrator [Consolidated Chemistry Calibrator (ConCC)] in place of or in addition to factor-based calibrations. For alkaline phosphatase (ALP), both calibration options are offered, i.e.,with ConCC (ALP2) and with an experimental calibration factor (ALP2F). Both options are declared traceable to the 2011 IFCC reference measurement procedure (RMP). Before to replace the old generation (ALP1) with the new one, we decided to validate the trueness of ALP2/ALP2F. Three approaches were employed: (a)preliminary comparison on 48 native frozen serum samples with ALP1, of which traceability to RMP was previously successfully verified; (b)examination of three banked serum pools (BSP) with values assigned by RMP; (c)direct comparison with RMP on a set of 24 fresh serum samples. Bias estimation and regression studies were performed, and the standard measurement uncertainty associated with ALP measurements on clinical samples (uresult) was estimated and compared with established analytical performance specifications (APS). ConCC commutability was also assessed. A positive proportional bias was found with both ALP2 and ALP2F when compared to ALP1 and RMP. This positive bias was confirmed on BSP: in average, +13.1 % for ALP2 and +10.0 % for ALP2F, respectively. uresult were 13.28 % for ALP2 and 10.04 % for ALP2F, both not fulfilling the minimum APS of 4.0 %. Furthermore, ConCC was not commutable with clinical samples. Our results unearth problems in the correct implementation of traceability of Alinity ALP2/ALP2F, with the risk for the new assay to be unfit for clinical purposes.

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers.

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.

Effects of N-acetyl-L-tryptophan on desorption of the protein-bound uremic toxin indoxyl sulfate and effects on uremic sarcopenia.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that causes uremic sarcopenia. IS has poor dialysis clearance; however, the addition of a binding competitor improves its removal efficiency. Dialysis experiments were performed using N-acetyl-l-tryptophan (L-NAT) instead of l-tryptophan (Trp) using pooled sera obtained from dialysis patients. The molecular structures of L-NAT and Trp were similar to that of IS. Therefore, we examined whether Trp and L-NAT were involved in muscle atrophy in the same manner as IS by performing culture experiments using a human myotube cell line. The removal efficiency of L-NAT was the same as that of Trp. However, L-NAT concentrations in the pooled sera increased at the end of the experiment. Trp (1 mM) decreased the area of human myocytes, similar to IS, whereas L-NAT did not. L-NAT is a binding competitor with the ability to remove protein-bound IS while preventing sarcopenia.

CD4 T lymphocyte subsets display heterogeneous susceptibility to apoptosis induced by serum from patients with systemic lupus erythematosus

BackgroundSerum from systemic lupus erythematosus (SLE) patients has been shown to induce T-lymphocyte (TL) apoptosis. Given that different cells of the immune system display different sensitivity to apoptosis, we set to evaluate the in vitro effect of SLE serum on regulatory T-cells (Treg), Th17, Th1 and Th2 from SLE patients and healthy controls.MethodsPeripheral blood mononuclear cells from SLE patients or normal controls were exposed to a pool of sera from SLE patients or normal controls. Annexin V was used to label cells in apoptosis or necrosis. Annexin V-labeled Treg, Th17, Th1 and Th2 cells were determined using flow cytometry.ResultsTotal CD3 + and CD4 + cells from SLE patients showed higher frequency of spontaneous apoptosis/necrosis, whereas Th1 cells from SLE patients presented reduced spontaneous apoptosis/necrosis rate as compared with cells from controls. Incubation with SLE serum induced increased frequency of apoptotic/necrotic CD3 + , CD4 + and Th2 cells from normal controls or from SLE patients as compared with cultures incubated with normal human serum (NHS) or without human serum at all. Incubation with SLE serum did not increase the apoptosis/necrosis rate in Th1 or Th17 cells. Treg cells from SLE patients were more prone to apoptosis/necrosis induced by SLE serum than Treg cells from normal individuals. Th1, Th2, and Th17 cells presented increased apoptosis rates in cultures without human serum.ConclusionOur findings indicate that the serum of patients with active SLE stimulates apoptosis of CD4 + T cells in general and exhibit differentiated effects on CD4 + T-cell subsets.

Association of Serum Very-Long-Chain Saturated Fatty Acids With Changes in Insulin Sensitivity and β-Cell Function: The Prospective Metabolism and Islet Cell Evaluation (PROMISE) Cohort.

A unique group of circulating very long-chain saturated fatty acids (VLCSFA), including arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0), have been associated with lower risk of type 2 diabetes, although associations with early metabolic risk phenotypes preceding type 2 diabetes have received limited study. We aimed to examine the associations of VLCSFA with longitudinal changes in insulin sensitivity and beta-cell function in a cohort at risk of type 2 diabetes. VLCSFA in the four main serum pools (phospholipid, triacylglycerol, cholesteryl ester, and non-esterified fatty acid) were extracted from fasting baseline samples (n=467). Generalized Estimating Equations were used to determine the associations between VLCSFA and changes over 9 years in validated indices of insulin sensitivity (HOMA2-%S and ISI) and beta-cell function (IGI, IGI/IR and ISSI-2). Associations of VLCSFA with outcomes were strongest in the triacylglycerol lipid pool: 20:0 was positively associated with both insulin sensitivity and beta-cell function (5.01% increase in HOMA2-%S and 4.01-6.28% increase in IGI/IR and ISSI-2 per SD increase in 20:0); 22:0 was positively associated with insulin sensitivity with 6.55% increase in HOMA2-%S and a 5.80% increase in ISI per SD increase in 22:0. Lastly, 24:0 was positively associated with insulin sensitivity and beta-cell function (7.94-8.45% increase in HOMA2-%S and ISI, and 4.61-6.93% increase in IGI/IR and ISSI-2 per SD increase in 24:0). Fewer significant associations were observed in the cholesteryl ester and non-esterified pools. Overall, our results indicate positive longitudinal associations of VLCSFA with insulin sensitivity and beta-cell function, especially within the triacylglycerol pool.

Development of a model immunofiltration assay using a conjugate based on horseradish peroxidase

The aim of the study was to optimize the conditions for a model immunoassay in the immunofiltration format using diagnostic reagents based on horseradish peroxidase. Residual blood serum samples from patients in the red zone with a verified diagnosis of a new coronavirus infection were used as positive sera, and blood sera obtained before 2019 were used as negative samples. The procedure of immunofiltration analysis was carried out using a pool of negative and positive blood sera. Studies were carried out to optimize the analysis procedure and increase the significant characteristics of the test. Results. It has been shown that the addition of sodium dodecyl sulfate to a final concentration of 50 M in the substrate buffer makes it possible to achieve a higher analytical signal and a stable result 10 minutes after the end of the analysis procedure. Such conditions of immunofiltration analysis as dilutions of the diagnostic reagent, the volume of the introduced sample and the amount of the S-protein of the coronavirus applied to the nitrocellulose membrane were optimized. It has been determined that using immunofiltration analysis it is possible to detect antibodies against the coronavirus S-protein in a dilution of a serum sample of more than 1/1000. The results of immunofiltration analysis reproduce the results of ELISA.

Methodological considerations in determining sex steroids in children: comparison of conventional immunoassays with liquid chromatography-tandem mass spectrometry.

In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays. The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3). For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9±1.2 and 33.9±1.6 pmol/L (group mean±standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3±18.1 and 45.7±19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06±0.00 to 1.00±0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12±0.11 and 0.85±0.23 nmol/L. For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended.

Novel recombinant proteins and peptides from Clonorchis sinensis and Opisthorchis viverrini for liver fluke exposure ELISA

Human serum samples from individuals living in Vietnam and Taiwan suspected of past Clonorchis sinensis or Opisthorchis viverrini infection were screened using several novel peptides and recombinant liver fluke proteins to determine if any consistent patterns could be discerned and used as the basis for future liver fluke ELISA development. Absorbance values at 405 nm were compared to those of pooled unexposed normal human serum and analyzed for statistical significance. The data exhibited some interesting patterns consistent with egg antigen sequestration in the gut possibly leading to lower serum antibody levels and potential regional exposure differences between Vietnamese and Taiwanese subjects. In particular, antibodies against Cathepsin B and B2 peptides, as well as a partial Cahedrin Domain peptide may be elevated in some Taiwanese serum samples while antibodies against recombinant Clonorchis egg protein and Hepatocellular Carcinoma Peptide Antigen 59 may be elevated in some samples from both Taiwan and Vietnam. The data appear to suggest that some of the novel recombinant protein and peptide antigens selected and tested herein warrant further study with larger sample sizes as possible targets for detecting anti-liver fluke antibodies by ELISA from humans suspected of liver fluke infections.

Artificially raised creatinine concentrations due to analytical interference for samples contaminated with total parenteral nutrition fluid.

A sample received in the laboratory from a patient receiving total parenteral nutrition (TPN) indicated that the patient may have renal dysfunction, but the results were not considered to be reliable enough to report. Investigations using a reference method for measurement of creatinine confirmed positive interference in the creatinine assay and distribution of samples via an External Quality Assessment (EQA) Scheme showed that this positive interference was method dependent. Residual TPN fluid (Nutriflex Lipid Special) left in the bag after the patient had completed the infusion was collected and added to a patient serum pool in increasing amounts and distributed to different laboratories for analysis of creatinine and glucose through an EQA Scheme. Positive interference in a number of different creatinine assays was identified as a result of a component in the TPN fluid. Positive interference from high concentrations of glucose has been demonstrated to be a cause for falsely high results in Jaffe creatinine assays. The concern would be that a sample contaminated with TPN fluid would have both abnormal electrolytes and creatinine concentrations and give the impression that the patient was in renal failure due to analytical interference in the creatinine assay and laboratory staff need to be aware of this problem.

Deep Characterization of Serum Metabolome Based on the Segment-Optimized Spectral-Stitching Direct-Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Approach.

Direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FTICR MS) shows great promise for metabolomic analysis due to ultrahigh mass accuracy and resolution. However, most of the DI-FTICR MS approaches focused on high-throughput metabolomics analysis at the expense of sensitivity and resolution and the potential for metabolome characterization has not been fully explored. Here, we proposed a novel deep characterization approach of serum metabolome using a segment-optimized spectral-stitching DI-FTICR MS method integrated with high-confidence and database-independent formula assignments. With varied acquisition parameters for each segment, a highly efficient acquisition was achieved for the whole mass range with sub-ppm mass accuracy. In a pooled human serum sample, thousands of features were assigned with unambiguous formulas and possible candidates based on highly accurate mass measurements. Furthermore, a reaction network was used to select confidently unique formulas from possible candidates, which was constructed by unambiguous formulas and possible candidates connected by the formula differences resulting from biochemical and MS transformation. Compared with full-range and conventional segment acquisition, 8- and 1.2-fold increases in observed features were achieved, respectively. Assignment accuracy was 93-94% for both a standard mixture containing 190 metabolites and a spiked serum sample with the root mean square mass error of 0.15-0.16 ppm. In total, 3534 unequivocal neutral molecular formulas were assigned in the pooled serum sample, 35% of which are contained in the HMDB. This method offers great enhancement in the deep characterization of serum metabolome by DI-FTICR MS.

Commutability evaluation of candidate reference materials and ERM-DA470k/IFCC for immunoglobulin M using two international approaches.

This study aimed to assess the commutability of frozen pooled human serum (PHS), high concentration of Immunoglobulin M (IgM) pure diluted materials (HPDM), commercialized pure materials (CPM), and dilutions of ERM-DA470k/IFCC in IgM detection using the CLSI and IFCC approaches, to support standardization or harmonization of IgM measurement. Twenty-four serum samples, relevant reference materials (PHS, HPDM, CPM), and different ERM-DA470k/IFCC dilutions were analyzed in triplicate using six routine methods. The commutability of the relevant reference materials was carried out following CLSI EP30-A and IFCC bias analysis. According to the CLSI approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 13, 15, 13, and 8 of 15 assay combinations, respectively. Using the IFCC approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 11, 9, 15, and 10 of 15 assay combinations, respectively. The ERM-DA470k/IFCC dilutions with D-PBS and RPMI-1640 Medium were commutable on 13 of 15 assay combinations according to CLSI and were commutable on all 15 assay combinations using IFCC approach. High concentration of PHS were commutable on all six detection systems using the CLSI approach. Low and medium concentration of PHS showed unsatisfied commutability. HPDM, not CPM have good commutability, has the potential to become reference materials. ERM-DA470k/IFCC diluted with different medium showed different commutability.

Growth, allergen profile and microbiome studies in Dermatophagoides pteronyssinus cultures

Mites are mass-cultured to manufacture allergen extracts for allergy diagnostics and therapeutic treatment. This study focused on characterizing the growth, the allergen profile, and the microbiome of Dermatophagoides pteronyssinus cultures. Mite population, protein profile, total protein content and major allergen levels (Der p 1, Der p 2, Der p 23) were monitored at different times of three independent cultures. The allergenicity was studied by immunoblot using a pool of sera from allergic patients. Mite microbiome was characterized by sequencing the 16S rRNA gene from 600 adult mites from the last day of the culture. Endotoxin content was also analyzed. The cultures had a fast and unrelenting evolution. Mite density, total protein content, major allergen levels and the allergenicity were increased progressively during the cultures. Regarding the microbiome studies, the results confirm the presence of non-pathogenic bacteria, being firmicutes and actinobacteria the most common bacterial taxa, with a very low content of Gram-negative bacteria and endotoxin content. The allergenicity and levels of the main allergens in the mite cultures are objective methods useful to monitor the mite culture that help to produce standardized allergen extracts. The high presence of Gram-positive bacteria found limits the possibility for vaccine contamination by bacterial endotoxins.

Development of a metabolite calculator for diagnosis of pancreatic cancer.

Carbohydrate antigen (CA) 19-9 is a known pancreatic cancer (PC) biomarker, but is not commonly used for general screening due to its low sensitivity and specificity. This study aimed to develop a serum metabolites-based diagnostic calculator for detecting PC with high accuracy. A targeted quantitative approach of direct flow injection-tandem mass spectrometry combined with liquid chromatography-tandem mass spectrometry was employed for metabolomic analysis of serum samples using an Absolute IDQ™ p180 kit. Integrated metabolomic analysis was performed on 241 pooled or individual serum samples collected from healthy donors and patients from nine disease groups, including chronic pancreatitis, PC, other cancers, and benign diseases. Orthogonal partial least squares discriminant analysis (OPLS-DA) based on characteristics of 116 serum metabolites distinguished patients with PC from those with other diseases. Sparse partial least squares discriminant analysis (SPLS-DA) was also performed, incorporating simultaneous dimension reduction and variable selection. Predictive performance between discrimination models was compared using a 2-by-2 contingency table of predicted probabilities obtained from the models and actual diagnoses. Predictive values obtained through OPLS-DA for accuracy, sensitivity, specificity, balanced accuracy, and area under the receiver operating characteristic curve (AUC) were 0.9825, 0.9916, 0.9870, 0.9866, and 0.9870, respectively. The number of metabolite candidates was narrowed to 76 for SPLS-DA. The SPLS-DA-obtained predictive values for accuracy, sensitivity, specificity, balanced accuracy, and AUC were 0.9773, 0.9649, 0.9832, 0.9741, and 0.9741, respectively. We successfully developed a 76 metabolome-based diagnostic panel for detecting PC that demonstrated high diagnostic performance in differentiating PC from other diseases.

Solid-phase immunoextraction followed by liquid chromatography-tandem mass spectrometry for the selective determination of thyroxine in human serum

In this work, an analytical method based on solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) has been developed for the selective determination of thyroxine (T4) in human serum. For this purpose, two immunosorbents (ISs) specific to T4 were synthesized by grafting two different T4-specific monoclonal antibodies on a cyanogen bromide (CNBr)-activated-Sepharose® 4B solid support. The grafting yields obtained from the immobilization of each antibody on the CNBr-activated-Sepharose® 4B were over 90%, demonstrating that most of the antibodies were covalently bound to the solid support. The SPE procedure was optimized by studying the retention capability and selectivity of the two ISs in pure media fortified with T4. Under the optimized conditions, high elution efficiencies were achieved in the elution fraction for both specific ISs (i.e., 85%), whereas low ones were obtained in the control ISs (ca. 2%), showing the selectivity of the specific ISs. The ISs were also characterized by studying extraction and synthesis repeatability (RSD <8%), and capacity (104 ng of T4 per 35 mg of ISs, i.e., 3 μg g−1). Finally, the methodology was applied to a pooled human serum sample in order to study its analytical utility and accuracy. Relative recovery (RR) values between 81 and 107% were obtained, showing no matrix effects during the global methodology. Furthermore, the need to perform the immunoextraction was evidenced by comparing the LC-MS scan chromatograms and RR values with and without applying the immunoextraction procedure on a serum sample submitted to protein precipitation. This works exploits, for the first time, the use of an IS on the selective determination of T4 in human serum samples.

Proteomic-based identification of APCS as candidate protein for diagnosis of patients exhibiting anti-tubercular drug induced liver injury

Traditional markers evaluate anti-tubercular drug-induced liver injury (AT-DILI). However, these markers have certain limitations and studies are in progress to characterize AT-DILI at an early stage. In the present study, 40 patients were categorized and equally distributed into healthy controls, newly diagnosed tuberculosis (TB), TB without hepatotoxicity and TB with hepatotoxicity groups based on their conventional liver function tests. Relative protein quantification was performed on depleted pooled serum samples of each representative group by LC–MS/MS, and validation of shortlisted protein was done by ELISA. Levels of all analysed biochemical parameters showed a statistical increment in the hepatotoxicity group compared to the other three groups, representing AT-DILI. Comparative proteomic analysis between TB with hepatotoxicity versus TB without hepatotoxicity groups highlighted 24 significant differentially expressed proteins, including PROS1, KNG1, CFH, LCAT, APCS and ADIPOQ. Identified proteins were involved in complement activation, triglyceride-rich lipoprotein particle remodelling and pathways comprising complement, coagulation cascades and cholesterol metabolism. Based on functional relevance, the serum amyloid P component (APCS) was shortlisted for validation, and it showed a similar trend as observed in the discovery phase with 100% sensitivity and 87% specificity; however, findings need exploration in larger cohorts.