Commercial Immunoassay Research Articles

Comprehensive assessment of cardiovascular-kidney-metabolic (CKM) syndrome: Novel tools for assessment of cardiovascular risk and kidney outcomes in long-term kidney transplant patients.

Cardiovascular (CV)-kidney-metabolic (CKM) syndrome, a newly defined entity, offers a framework for assessing CV risk. Emerging evidence suggests that histone deacetylase sirtuin-1 (SIRT1) and adipokine chemerin hold promise as CKM markers. This study aimed to explore the relationship between CKM stage, clinical parameters, and both novel and established markers of CV and renal risk. A cohort of 102 patients with long-term, stable kidney transplant (KTx) (>24 months) and median (interquartile range) follow-up of 83 (42-85) months was recruited alongside 32 healthy controls. Patients were classified into modified CKM stages following the American Heart Association guidance. Serum high-sensitivity interleukin-6 (hsIL-6), chemerin, and SIRT1 were measured using commercial immunoassay kits. The incidence of CV events (CVE), CV mortality, and permanent transfer to dialysis therapy were primary endpoints. CKM stage was associated with higher risk of CVE/CV death (HR 95% CI, 3.79 [1.16-12.42]; P = 0.03) and allograft loss (HR 95% CI, 2.05 [1.17-3.57]; P = 0.01). Elevated sirtuin-1 was associated with significantly lower risk of CV event/death (HR 95% CI, 0.11 [0.11-0.89]; P = 0.04), whereas high chemerin status was tied to dialysis risk (HR 95% CI, 5.77 [1.96-17.00]; P = 0.001) alone. In contrast to sirtuin-1, hsIL-6 and chemerin showed incremental changes across more advanced CKM stages, though statistically significant for hsIL-6 alone. In age-adjusted models for CV disease, independent associations with diabetes and total cholesterol were noted. Classifying patients based on modified CKM stages offers valuable prognostic insights for stable KTx recipients, particularly when assessing reno-cardiovascular risk. The investigated serum markers may serve as supplemental tools for refining risk stratification.

Monoclonal-Antibody-Based Immunoassays for the Mycotoxins NX-2 and NX-3 in Wheat.

The fungal infestation of crops can cause major economic losses. Toxins produced by the causative fungi (mycotoxins) represent a potential safety hazard to people and livestock consuming them. One such mycotoxin is deoxynivalenol (DON, also known as vomitoxin), a trichothecene associated with Fusarium Head Blight of wheat. DON is commonly found in cereal crops worldwide. A group of trichothecene mycotoxins closely related to DON, the NX toxins, have been reported to occur in the northeastern United States and southern Canada. While many commercial immunoassays are available to detect DON, there are no rapid screening assays for the NX toxins. We describe the development and isolation of three monoclonal antibodies (mAbs) specific towards two NX toxins: NX-2 and NX-3. The mAbs did not recognize DON or several other closely related trichothecenes. One of the mAbs was selected for development of an enzyme-linked immunosorbent assay (ELISA) for NX-2 and NX-3 in wheat. The dynamic ranges for the assay were 7.7 to 127 μg/kg for NX-2 and 59 μg/kg to 1540 μg/kg for NX-3 in wheat. Recoveries from spiked wheat averaged 84.4% for NX-2 and 99.3% for NX-3, with RSDs of 10.4% and 11.3%, respectively (n = 24). The results suggest that this assay can be used to screen for NX toxins in wheat at levels relevant to human food and animal feed safety.

Serum Levels of Zinc, Albumin, Interleukin-6 and CRP in Patients with Unipolar and Bipolar Depression: Cross Sectional Study.

Unipolar (UD) and bipolar depression (BDD) show a high degree of similarity in clinical presentations, which complicates the differential diagnosis of these disorders. The aim of this study was to investigate the serum levels of interleukin 6 (IL-6), C-reactive protein (CRP), albumin (Alb), and zinc (Zn) in patients with UD, BDD, and healthy controls (HC). A total of 211 samples were collected: 131 patient samples (65 UD and 68 BDD) and 80 HC. The Montgomery-Asberg Depression Rating Scale (MADRS), along with the Hamilton Depression Rating Scale (HAMD-17), were administered to patient groups to evaluate symptoms. A cross-sectional study was performed to analyse the serum levels of IL-6, CRP, albumin, and zinc. The concentration of CRP was determined using the immunoturbidimetry method, zinc using the colorimetric method, and albumin using the colorimetric method with bromocresol green on the Alinity c device. IL-6 cytokine concentration in serum samples was ascertained using a commercial enzyme immunoassay, ELISA. We found no significant differences in serum concentrations of zinc, albumin, CRP, and IL-6 between the groups of patients with unipolar and bipolar depression. There was a significant statistical difference (p < 0.001) between serum levels of all investigated parameters in both groups of depressed patients in comparison with HC. Furthermore, correlations with specific items on HAMD-17; (namely, hypochondrias, work and activities, somatic symptoms-general, and weight loss) and on MADRS (concentration difficulties, lassitude) were observed in both patient groups. These findings confirm the presence of low-grade inflammation in depression, thus adding better insight into the inflammation hypothesis directed to explain the aetiology of depressive disorders. Our results do not indicate potential biomarkers for distinguishing between unipolar and bipolar depression.

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Validating a Non-Invasive Method for Assessing Cortisol Concentrations in Scraped Epidermal Skin from Common Bottlenose Dolphins and Belugas.

Society is showing a growing concern about the welfare of cetaceans in captivity as well as cetaceans in the wild threatened by anthropogenic disturbances. The study of the physiological stress response is increasingly being used to address cetacean conservation and welfare issues. Within it, a newly described technique of extracting cortisol from epidermal desquamation may serve as a non-invasive, more integrated measure of a cetacean's stress response and welfare. However, confounding factors are common when measuring glucocorticoid hormones. In this study, we validated a steroid hormone extraction protocol and the use of a commercial enzyme immunoassay (EIA) test to measure cortisol concentrations in common bottlenose dolphin (Tursiops truncatus) and beluga (Delphinapterus leucas) epidermal samples. Moreover, we examined the effect of sample mass and body location on cortisol concentrations. Validation tests (i.e., assay specificity, accuracy, precision, and sensitivity) suggested that the method was suitable for the quantification of cortisol concentrations. Cortisol was extracted from small samples (0.01 g), but the amount of cortisol detected and the variability between duplicate extractions increased as the sample mass decreased. In common bottlenose dolphins, epidermal skin cortisol concentrations did not vary significantly across body locations while there was a significant effect of the individual. Overall, we present a contribution towards advancing and standardizing epidermis hormone assessments in cetaceans.

Insight into the status of plasma renin and aldosterone measurement: findings from 526 clinical laboratories in China.

Accurate measurements of renin and aldosterone levels play an important role in primary aldosteronism screening, which is of great importance in the management and categorization of hypertension. The objective of this studyis to investigate the current status of plasma renin and aldosterone measurements in China, which is achieved by analyzing the results of 526 clinical laboratories nationwide for three pooled fresh plasma samples derived from more than 2,000 patients. Renin and aldosterone in three pooled plasma samples were measured four times in 526 laboratories employing various measurement systems. The inter- and intra-laboratory %CV were calculated and compared. To determine the source of the substantial inter-laboratory %CV,laboratories were categorized according to the measurement systems they are using, and both the inter- and intra-measurement-system %CV were calculated and compared. Regarding renin, the majority of laboratories use four primary commercial immunoassays. However, for aldosterone, in addition to commercial immunoassays, laboratory-developed liquid chromatography-tandem mass spectrometry (LC-MS) methods are also used by laboratories. The median values of intra-laboratory %CVs, intra-measurement-system %CVs, inter-laboratory %CVs, and inter-measurement systems %CVs varied between 1.6 and 2.6 %, 4.6 and 14.9 %, 8.3 and 25.7 %, and 10.0 and 34.4 % for renin, respectively. For aldosterone, these values ranged from 1.4 to 2.2 %, 2.5-14.7 %, 9.9-31.0 %, and 10.0-35.5 %, respectively. The precision within laboratories and measurement systems for plasma renin and aldosterone measurements is satisfactory. However, the comparability between laboratories using different measurement systems remains lacking, indicating the long way to achieve standardization and harmonization for these two analytes.

Point-of-Care Detection of HER2 and CA 15-3 in Breast Cancer Patients: Dual-Channel Biosensor Implementation

Breast cancer remains a considerable health challenge, affecting numerous individuals annually. This research introduces an innovative method for detecting breast cancer utilizing dual-channel test strips capable of simultaneously assessing two key biomarkers—HER2 and CA 15-3. The test strip utilized in this study is not only cost-effective but also entirely non-invasive. The reusable device employs a printed circuit board with metal-oxide-semiconductor field-effect transistor amplification and Arduino-based control to convert voltage signals from test strips into digital readings efficiently. The device utilizes double-pulse measurement instead of direct current, effectively mitigating the screening effect. The detection limit for both biomarkers is exceptionally low at 10−15 g ml−1, surpassing commercial enzyme-linked immunoassay kits by four orders of magnitude. The sensor demonstrates remarkable sensitivity, with 78/dec for HER2 and 56/dec for CA 15-3. Human sample tests were conducted to validate the efficacy of the dual-channel strip, successfully distinguishing between healthy and cancerous groups. The results reveal significant p-values for both HER2 and CA 15-3 tests, underscoring the significance of this research. Note that this is a rapid testing process, completed in less than 2 s. These findings offer a promising avenue for swift and accurate breast cancer detection, furnishing crucial insights for early diagnosis and subsequent treatment.

Detection of intrathecal IgG antibody for varicella and measles diagnosis by evaluation and comparison of a commercial IgG chemiluminescent immunoassay with two ELISAs.

Analyse alternative methods of intrathecal antibody detection by comparing chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) techniques to determine if CLIA can replace ELISA in the diagnosis of CNS infections. A panel of 280 paired samples-cerebrospinal fluid (CSF) and serum-with known antibody reactivities (Varicella, n = 60; Measles, n = 120) and negative samples (n = 100) were used to evaluate the performance of six serological test kits (Enzygnost, VirClia®, and Serion ELISA (Measles and Variella). For Measles virus IgG, the VirClia® IgG monotest revealed 97% and 94% positive and negative agreement to the Enzygnost as reference test,respectively. In contrast, Serion ELISA kits yielded values of 18% and 90%. For theVaricella Zoster virus (VZV) IgG, the VirClia® IgG monotest showed 97% and 90% positive and negative agreement compared to Enzygnost. The Serion ELISA kits showed values of 55% and 86%, respectively. ROC analysis revealed that the areas under the curve for Measles and VZV IgGs were 0.7 and 0.852, respectively, using the Serion kit, and 0.963 and 0.955, for Vircell S.L CLIA technique. VirClia® monotest values were calculated using an antibody index cut-off of 1.3. The findings indicate that CLIA testing can improve antibody detection in CSF samples, aiding the diagnosis of infectious neurological impairments.

Orthostatic hypotension is associated with higher levels of circulating endostatin.

The pathophysiology of orthostatic hypotension (OH), a common clinical condition, associated with adverse outcomes, is incompletely understood. We examined the relationship between OH and circulating endostatin, an endogenous angiogenesis inhibitor with antitumour effects proposed to be involved in blood pressure (BP) regulation. We compared endostatin levels in 146 patients with OH and 150 controls. A commercial chemiluminescence sandwich immunoassay was used to measure circulating levels of endostatin. Linear and multivariate logistic regressions were conducted to test the association between endostatin and OH. Endostatin levels were significantly higher in OH patients (59 024 ± 2513 pg/mL) vs. controls (44 090 ± 1978pg/mL, P < 0.001). A positive linear correlation existed between endostatin and the magnitude of systolic BP decline upon standing (P < 0.001). Using multivariate analysis, endostatin was associated with OH (adjusted odds ratio per 10% increase of endostatin in the whole study population = 1.264, 95% confidence interval 1.141-1.402), regardless of age, sex, prevalent cancer, and cardiovascular disease, as well as traditional cardiovascular risk factors. Circulating endostatin is elevated in patients with OH and may serve as a potential clinical marker of increased cardiovascular risk in patients with OH. Our findings call for external validation. Further research is warranted to clarify the underlying pathophysiological mechanisms.

Exploring cell-free assays for COVID-19 serosurvey

Serosurveys to monitor immunity toward COVID-19 in the population are primarily performed using an ELISA to screen samples for SARS-CoV-2 antibodies, followed by confirmation by a virus neutralization test, which is considered the Gold Standard. However, virus neutralization test may not be feasible for some laboratories because of the requirement for specific facilities and trained personnel. In an attempt to address this limitation, we evaluated three cell-free methods as potential alternatives for assessing SARS-CoV-2 seroprevalence in human population from plasma. We report the establishment of two inhibition ELISAs designed to detect anti-Spike RBD IgG antibodies and a microsphere quantitative suspension array technology assay, based on the Luminex xMAP platform, to measure the presence of antibodies against various SARS-CoV-2 antigens, including anti-RBD. These methods were also compared to a commercial chemiluminescent immunoassay designed for anti-RBD antibodies detection and to the combined ELISA + virus neutralization test strategy. These cell-free assays performed equally to estimate the percentage of positive and negative samples and could be used to determine the prevalence of SARS-CoV-2 antibodies in human population, at least in cohort with high-expected prevalence, without the use of seroneutralization assay.

One Health approach for Brucella canis: Serological and molecular detection in animal-hoarding individuals and their dogs.

Animal hoarding disorder (AHD) is classified as a psychiatric obsessive-compulsive condition characterized by animal accumulation and often accompanied by unsanitary conditions and animal cruelty. Although AHD may increase pathogen transmission and spread, particularly for zoonotic diseases, human and dog exposure in such cases has yet to be fully established. Accordingly, this study aimed to assess Brucella canis in 19 individuals with AHD (11 households) and their 264 dogs (21 households) in Curitiba, the eighth largest city in Brazil, with approximately 1.8 million habitants. Anti-B. canis antibodies were detected by the 2-mercaptoethanol microplate agglutination test (2ME-MAT) and by a commercial lateral flow immunoassay (LFIA), while molecular detection of previously positive seropositive samples was performed by conventional PCR. Although all the human samples were 2ME-MAT negative, 12/264 (4.5%, 95% Confidence Interval: 2.0-7.0%) dog samples were 2ME-MAT and LFIA positive, with 2ME-MAT titers ranging from 20 to 640. At least one dog in 4/21 (19.0%, 95% CI: 2.0-46.0%) households was seropositive. Despite the absence of seropositivity in individuals with AHD and the comparatively low seroprevalence in dogs, B. canis circulation and outbreaks should be considered in such human populations due to the high burden and recurrent character of B. canis exposure in high-density dog populations and the constant introduction of susceptible animals.

Combining Bioorthogonal Chemistry with Fluorescent Silica Nanoparticles for the Ultrasensitive Detection of the HIV-1 p24 Antigen.

Early detection and viral concentration monitoring of human immunodeficiency virus in resource-poor settings are important to control disease spread and reduce mortality. Nucleic acid amplification tests are expensive for low-resource settings. Lateral flow antibody tests are not sensitive if testing is performed within 7-10 days, and these tests are not quantitative. We describe a signal enhancement technique based on fluorescent silica nanoparticles and bioorthogonal chemistries for the femtomolar detection of the HIV-1 p24 antigen. We developed a magnetic bead-based assay, wherein we used fluorescent-dye-encapsulated silica nanoparticles as reporters. The number of reporters was increased by using bioorthogonal chemistry to provide signal enhancement. The limit and range of detection of the sandwich immunoassay using alternating multiple layers for p24 in human serum were found to be 46 fg/mL (1.84 fM) and 46 fg/mL to 10 ng/mL, respectively. This simple assay was 217-fold higher in sensitivity compared to that of commercial enzyme-linked immunoassays (limit of detection of 10 pg/mL).

Performance of eight commercial immunoassays for the detection of cytomegalovirus-specific IgM antibodies in pregnancy - no test fits all needs.

Detection of cytomegalovirus (CMV)-specific immunoglobulin M (IgM) antibodies as first-line serologic diagnosis plays an important role in identifying CMV primary infection during pregnancy. The performance characteristics of eight commercially available CMV IgM assays were compared. Sensitivity and IgM antibody kinetics were assessed using 100 acute phase and follow-up sera from 39 pregnant women with a well-defined onset of CMV primary infection. Specificity was analyzed using 50 well-characterized serum samples from pregnant women not infected or latently infected with CMV and from patients with other acute infections. Until 12 weeks after the onset of primary infection, four assays showed sensitivities of 100%, whereas the others had individual gaps to detect all primary infections in this time period. All assays showed a time-dependent decrease of IgM levels. More than 12 weeks after the onset of infection, the IgM-positive rates varied considerably between tests. The specificity was between 92% and 98% in all but one assay. The observed differences in the performance characteristics must be taken into account in CMV screening and diagnosis of primary infection during pregnancy.

SARS-CoV-2 infections among pregnant women, 2020, Finland-Cross-testing of neutralization assays.

We studied the development of the severe acute respiratory syndrome-related coronavirus(SARS-CoV-2) pandemic in southern Finland in 2020 and evaluated the performance of two surrogate immunoassays for the detection of neutralizing antibodies (NAbs). The data set consisted of 12 000 retrospectively collected samples from pregnant women in their first trimester throughout 2020. All the samples were initially screened for immunoglobulin G (IgG) with SARS-CoV-2 spike antibody assay (EIM-S1, Euroimmun) followed by confirmation with nucleocapsid antibody assay (Architect SARS-CoV-2, Abbott). Samples that were reactive (positive or borderline) with both assays were subjected to testing with commercial surrogate immunoassays of NeutraLISA (EIM) and cPassTM (GenScript Biotech Corporation) by using pseudoneutralization assay (PNAbA) as a golden standard. No seropositive cases were detected between January and March. Between April and December, IgG (EIM-S1 and Abbott positive) and NAb (PNAbA positive) seroprevalences were between 0.4% and 1.4%. NeutraLISAshowed 90% and cPass 55% concordant results with PNAbA among PNAbA negative samples and 49% and 92% among PNAbA positive samples giving NeutraLISA better specificity but lower sensitivity than cPass. To conclude, seroprevalence in pregnant women reflected that of the general population but the variability of the performance of serological protocols needs to be taken into account in inter-study comparison.

Standardisation and harmonisation of thyroid-stimulating hormone measurements: historical, current, and future perspectives.

Thyroid-stimulating hormone (TSH) is an important clinical marker in the diagnosis and management of thyroid disease. TSH measurements are reported in milli-International Units per Litre (mIU/L), traceable to a World Health Organisation (WHO) reference material. There is a wide variety of commercial immunoassays for TSH measurements available, which have historically been poorly harmonised due to a lack of commutability of the WHO reference materials with patient samples. This led to the recent development of a serum-based reference panel for TSH, traceable to the WHO reference material, available via the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC), aimed at harmonisation of TSH immunoassays. This report describes recent developments in the TSH reference system, including establishment of the4th WHO International Standard for TSH, and aims to clarify the relationship between the available reference materials and their intended uses. This 4th WHO IS is widely available and defines the unit of TSH activity, therefore its continued existence is of paramount importance, however it continues to show a lack of commutability with patient in many TSH immunoassays. This makes the C-STFT TSH panel, albeit available in restricted numbers, a critical resource to ensure better TSH assay harmonisation.

Facile and Ultrasensitive Food Allergen Quantification Using Microzone Paper-Based Mass Spectrometric Immunoassay.

Highly sensitive and rapid measurement of food allergens is essential to avoid unanticipated food allergies and to determine whether cross-contamination occurs in the food industry. Commercial immunoassay kits offer high specificity and convenience for allergen detection but still suffer limited quantitative sensitivity, accuracy, and stability based on the optical readout. In this work, a paper-based mass spectrometric immunoassay platform was constructed to achieve facile and highly sensitive quantification of peanut allergen, which combined the advantages of good specificity and accurate quantification from mass spectrometry and simplicity from a paper-based immunoassay. In this platform, a novel quaternary ammonium-based mass tag and a paper chip with a microzone were designed and developed, contributing to a large signal enhancement. This method was able to detect Ara h1 with a linear range of 0.1-100 ng mL-1 and a detection limit of 0.08 ng mL-1 in milk matrices. It has also been successfully applied to the accurate quantification of Ara h1 in six milk-related beverages, two biscuits, and two candy bars with complicated matrices and presented a low-concentration quantitation capability. This method gives a new type of mass spectrometric immunoassay for rapid and ultrasensitive allergen regulation in the food industry and for individual allergen differentiation research.

An LC-MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays.

The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking. The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated. No significant matrix effect or interference wasobserved with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method. The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Analytical and biological validation of a noninvasive measurement of glucocorticoid metabolites in feces of Geoffroy's spider monkeys (Ateles geoffroyi).

We report on an analytical and biological validation of a commercial cortisol enzyme immunoassay (EIA)to measure glucocorticoids (GC)in feces of Geoffroy's spider monkeys (Ateles geoffroyi). Validation of endocrinological methods for each sample matrix and study species is crucial to establish that the methods produce reliable results. For the analytical validation of the EIA, we assessed parallelism, accuracy, and precision. We carried out a biological validation based on three well-studied GC patterns with the following predictions: (1) increased fecal GC metabolite (fGCM)concentrations after veterinary intervention; (2) increased fGCM concentrations during early morning hours; and (3) higher fGCM concentrations during gestation than in other female reproductive states. For the first prediction, we sampled feces of two zoo-housed females 2 days before, the day of, and 2 days after a veterinary intervention. For the second prediction, we analyzed 284 fecal samples collected from 12 wild males using a linear mixed model (LMM).For the third prediction, we analyzed 269 fecal samples of eight wild females using an LMM. Analytical validation revealed that the EIA showed parallelism, was accurate, and precise within each assay. However, there was elevated variation in between-assay precision. The biological validation supported all predictions: (1) the two zoo-housed females showed a substantial increase in fGCM concentrations 2.5and 11 h after veterinary intervention; (2) there was a negative effect of sample collection time on fGCM concentrations (i.e., higher concentrations during early morning); (3) gestating females had significantly higher fGCM concentrations than lactating females. Thus, we analytically validated the commercial EIA and, despite between-assay variation, we were able to find three biologically relevant GC signals in captive and wild settings, and in males and females. We are therefore confident that the method can be used to noninvasively address behavioral endocrinology questions in Geoffroy's spider monkeys.

A comparative analysis of blood-borne infections among sickle cell anemia patients and first-time donors in Gabon

Managing sickle cell disease often requires transfusions, exposing multi-transfused sickle cell patients to a heightened risk of transfusion-transmitted infections. This study aimed to assess the seroprevalence of the human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV) in multi-transfused sickle cell patients and first-time donors in Libreville. The serological testing for HBsAg, anti-HIV, and anti-HCV antibodies was conducted using commercial enzyme immunoassays and confirmed by the COBAS modular analyzer (Roche Diagnostics). The seroprevalence of HIV, HCV antibodies, and HBsAg was 4%, 10%, and 10%, respectively, in multi-transfused sickle cell patients and 5%, 0%, and 8% in first-time donors. Interestingly, HIV and HBsAg seroprevalence were similar in both groups, indicating that transfusion was not associated with these infections. However, HCV antibody seroprevalence was significantly higher in multi-transfused sickle cell patients than in first-time donors (10% vs. 0%, p&lt;0.001). Furthermore, the presence of anti-HCV antibodies in multi-transfused sickle cell patients was significantly associated with the number of donations received (7.20±2.37 vs. 3.96±2.06, p=0.042). These findings suggest that while blood transfusion is not a significant risk factor for HIV and HBsAg transmission, it may increase the risk of HCV transmission, particularly in multi-transfused sickle cell patients.

Validation of an automated sample preparation module directly connected to LC-MS/MS (CLAM-LC-MS/MS system) and comparison with conventional immunoassays for quantitation of tacrolimus and cyclosporin A in a clinical setting

BackgroundTherapeutic drug monitoring (TDM) systems generally use either liquid chromatography/tandem mass spectrometry (LC-MS/MS) or immunoassay, though both methodologies have disadvantages. In this study, we aimed to evaluate whether a CLAM-LC-MS/MS system, which consists of a sample preparation module directly connected to LC-MS/MS, could be used for clinical TDM work for immunosuppressive drugs in whole blood, which requires a hemolytic process. For this purpose, we prospectively validated this system for clinical measurement of tacrolimus and cyclosporin A in patients’ whole blood. The results were also compared with those of commercial immunoassays.MethodsWhole blood from patients treated with tacrolimus or cyclosporin A at the Department of Nephrology and Departments of Rheumatology, Kanazawa University Hospital, from May 2018 to July 2019 was collected with informed consent, and drug concentrations were measured by CLAM-LC-MS/MS and by chemiluminescence immunoassay (CLIA) for tacrolimus and affinity column-mediated immunoassay (ACMIA) for cyclosporin A. Correlations between the CLAM-LC-MS/MS and immunoassay results were analyzed.ResultsTwo hundred and twenty-four blood samples from 80 patients were used for tacrolimus measurement, and 76 samples from 21 patients were used for cyclosporin A. Intra- and inter-assay precision values of quality controls were less than 7%. There were significant correlations between CLAM-LC-MS/MS and the immunoassays for tacrolimus and cyclosporin A (Spearman rank correlation coefficients: 0.861, 0.941, P < 0.00001 in each case). The drug concentrations measured by CLAM-LC-MS/MS were about 20% lower than those obtained using the immunoassays. CLAM-LC-MS/MS maintenance requirements did not interfere with clinical operations. Compared to manual pretreatment, automated pretreatment by CLAM showed lower inter-assay precision values and greatly reduced the pretreatment time.ConclusionsThe results obtained by CLAM-LC-MS/MS were highly correlated with those of commercial immunoassay methods. CLAM-LC-MS/MS offers advantages in clinical TDM practice, including simple, automatic pretreatment, low maintenance requirement, and avoidance of interference.