Immunoassay System Research Articles

Development and production of antibodies against gamma inactivated pathogenic bacterial spores

Abstract Objectives Gram-positive sporulated bacilli can cause many different diseases and isolation from environmental samples is difficult. Therefore, the quick detection and diagnosis of these microorganisms have critical importance because of their potentially harmful situation. However, many accepted diagnostic methods exist, and future technology points to immunoassay systems. Immunological methods to detect biological microorganisms require antigen-specific high-affinity antibodies as key materials. Methods In this study, Bacillus anthracis (34F2 sterne) bacterium, which causes anthrax disease, was chosen as a model organism to develop antibodies against bacterial spores. The produced spores were inactivated with gamma irradiation, and the development of monoclonal antibodies against inactivated spores was performed using hybridoma technology. Also, the polyclonal antibody was successfully obtained by immunizing the rabbit. Indirect and sandwich ELISA tests were performed to determine the antigenic properties of inactivated spores and the specific affinity of the developed antibodies. Results The spores, inactivated with 15 kGy, have the best-preserved surface epitopic regions and were selected as immunogen. Developed monoclonal and polyclonal antibodies were shown that there was no cross-reaction with other Bacillus species. Also, it was demonstrated that these antibodies could detect inactivated spores at a concentration of 105 spores/mL in a sandwich ELISA assay. Conclusions These qualified antibodies obtained will be essential in developing antibody-based diagnostic systems for spore detection from various environmental samples. This study suggests that the inactivated spores are a decent immunogen for generation antibodies and may be a candidate component for live vaccine formulation.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 μL and 100 μL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

Gold Nanoparticle-Decorated Polymer Particles for High-Optical-Density Immunoassay Probes.

To realize a highly sensitive immunoassay, high-optical-density probes conjugated with antibodies for target antigens are needed to increase the detectability of antigen-antibody complex formation. In this work, gold nanoparticle (NP)-decorated polymer (GNDP) particles were successfully prepared by mixing positively charged polymer particles and negatively charged Au NPs. GNDP particles decorated with NPs of 20 nm in size had higher optical density than the original Au NPs and GNDPs decorated with smaller Au NPs. Using GNDP particles as a probe, a highly sensitive immunoassay for influenza H1N1 hemagglutinin was realized with a minimum detectable concentration of 32.5 pg/mL. These results indicate that GNDP particles have high potential as an immunoassay probe that can be used in practical immunoassay systems for detecting a wide variety of antigens.

Evaluation and experience from routine use of chemiluminescence assays for serological screening of blood and plasma donations on the Alinity s system and the Alinity i system, two new fully-automated immunoassay systems in Poland

In Poland, independent evaluations under the auspices of the Institute of Hematology and Transfusion Medicine (IHTM) are mandated for any new device, assay, systems for screening samples from whole blood and plasma donors prior to implementation by Blood Transfusion Center (BTC). In last 5 years, two new systems were introduced to the market by Abbott GmbH, namely the Alinity s and the Alinity i. The evaluations performed for these two systems included the assessment of sensitivity, specificity and precision for each of the four mandatory serological screening markers in Poland: Hepatitis B Surface Antigen (HBsAg), Hepatitis C virus antibodies (Anti-HCV), HIV antibodies (anti-HIV) and Syphilis antibodies (anti-Treponema pallidum, anti-TP). Sensitivity was assessed by testing seroconversion panels, HBsAg international reference standard, well characterized local samples, and dilution panels. Specificity was assessed by testing routine donor samples. The results from Alinity i assays were compared to the results from Abbott ARCHITECT i2000SR and Ortho VITROS 3600 assays, while the results from Alinity s assays were compared to the results of ARCHITECT i2000SR assays. The evaluation of the Alinity s and Alinity i assays for sensitivity (100 %), specificity (99,92–100 %) and precision generated results that were as good as or better than generated by routinely used systems, were within acceptance criteria, and met all requirements for screening blood donor samples in accordance with Polish regulations. The specificity of the assays in routine use by BTCs, analyzed after approximately 150,000 donations on both systems, was comparable to the specificity observed during the evaluations at IHTM.

A microfluidic cover converts a standard 96-well plate into a mass-transport-controlled immunoassay system.

96-well microtiter plates, widely used in immunoassays, face challenges such as prolonged assay time and limited sensitivity due to the lack of analyte transport control. Orbital shakers, commonly employed to facilitate mass transport, offer limited improvements and can introduce assay inconsistencies. While microfluidic devices offer performance enhancements, their complexity and incompatibility with existing platforms limit their wide adoption. This study introduces a novel microfluidic 96-well cover designed to convert a standard 96-well plate to a mass-transport-controlled surface bioreactor. The cover employs microfluidic methods to enhance the diffusion flux of analytes toward the receptors immobilized on the well bottom. Both simulation and experimental results demonstrated that the cover significantly enhances the capture rate of analyte molecules, resulting in increased signal strength for various detection methods and a lower detection limit. The cover serves as an effective add-on to standard 96-well plates, offering enhanced assay performance without requiring modifications to existing infrastructure or reagents. This innovation holds promise for improving the efficiency and reliability of microtiter plate based immunoassays.

Efficient Optimal Cyclic Scheduling of High Throughput Screening System for Enzyme-Linked Immunoassay

Efficient Optimal Cyclic Scheduling of High Throughput Screening System for Enzyme-Linked Immunoassay

Screening and Characterization of Immunobiotic Lactic Acid Bacteria with Porcine Immunoassay Systems.

Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research has allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models, utilizing transfectant cells expressing PRRs along with an established porcine intestinal epitheliocyte (PIE) cell line, have proven to be valuable tools for immunobiotic selection and for gaining insights into the molecular mechanisms responsible for their beneficial effects.

Evaluation of a new Electrochemiluminescence Immunoassay System for Measuring High-Sensitivity Cardiac Troponin T.

The aim of this study was to verify the analytical performance of the UD90DT electrochemiluminescence immunoassay system (the UD90DT system) for measuring high-sensitivity cardiac troponin T (hs-cTnT). According to the Clinical and Laboratory Standards Institute guidelines, the imprecision, linearity, reference interval, limit of blank (LoB), limit of detection (LoD), and functional sensitivity (FS) of hs-cTnT using the UD90DT system were verified. The trueness was validated using the Proficiency Testing (PT) materials. The within-run and between-run coefficients of variations (CVs) of two hs-cTnT levels were 7.2% and 1.5%, and 7.1% and 2.6%, respectively. The biases of the PT samples (n = 6) all fell within the allowable total error. The linearity satisfied the requirements, with a slope of 0.9963 and an R12 value of 0.9998. The hs-cTnT levels of the healthy volunteers (n = 20) ranged from 3.0 ng/L to 7.7 ng/L. All blank calibrator measurements (n = 20) fell within the LoD claim, and none of the samples (n = 25) had a LoB value ≤ 3.0 ng/L. The FS was 5.3 ng/L. Furthermore, a good correlation between the UD90DT system and the Cobas e 601 module was observed for hs-cTnT. The analytical performance of hs-cTnT using the UD90DT system is acceptable and satisfies clinical needs.

Coronavirus Disease 2019 Vaccination Coverage and Seropositivity amongst Nigerians 18 Years Old and Above.

This was a cross-sectional community-based survey to study the prevalence of serum antibodies against the severe acute respiratory syndrome coronavirus 1 (SARS-COV-1) and determine possible source of antibodies as to whether from vaccination or from natural infection as well as attempt to compare antibody levels in response to the different four types of vaccines administered in Nigeria. A cross-sectional community-based study of the prevalence of serum antibodies against all four vaccine types used in Nigeria amongst a representative sample of people aged 18 years and above in the six geopolitical zones of the country using a multistage sampling technique covering 12 states of the country with two states being randomly selected from each geopolitical zone. High-throughput Roche electrochemiluminescence immunoassay system (Elecsys Anti-SARS-COV-1 Cobas) was used for qualitative and quantitative detection of antibodies to SARS-COV-1 in human plasma. There was no statistically significant difference between the proportions with seropositivity for both the vaccinated and the unvaccinated (P = 0.95). The nucleocapsid antibody (anti-Nc) titres were similar in both the vaccinated and the unvaccinated, whereas the Spike protein antibody (anti-S) titres were significantly higher amongst the vaccinated than amongst the unvaccinated. Antibody levels in subjects who received different vaccines were compared to provide information for policy. While only 45.9% of the subjects were reported to have been vaccinated, 98.7% of the subjects had had contact with the SARS-COV-1 as evidenced by the presence of nucleocapsid (NC) antibodies in their plasma. The 1.3% who had not been exposed to the virus, had spike protein antibodies which most likely resulted from vaccination in the absence of NC antibodies. Successive vaccination and booster doses either through heterogeneous or homologous vaccines increased antibody titres, and this stimulation of immune memory may offer greater protection against coronavirus disease 2019.

Effects of cortisol levels on reproductive success in cattle of different temperaments during fixed-time artificial insemination (FTAI)

The present study was designed to assess the effect of stress on the reproductive performance of cattle. For this purpose, a total of 137 cows were divided into two groups: 65 belonging to the Azores Lydia breed and 72 crossing Aberdeen-Angus with Limousine, free of IBR, Chlamydia and BVD diseases and with a body condition score ranging from 2.75 to 4.0. Eight days before starting the experimental procedures, animals passed through the containment sleeve every other day for routine and human presence. For the synchronization protocol, a progesterone-impregnated controlled internal drug release (CIDR®) insert was placed intravaginally, and an injection of GnRH was given. After 7 days, PGF2α was administered, and the CIDR® was removed. Sixty hours after removal, cows were inseminated once after another injection of GnRH to induce ovulation, and pregnancy diagnosis was performed by ultrasonography 30 days after artificial insemination. Peripheral blood was collected in 5 ml tubes with a Z Serum Sep Clot Activator, and cortisol was measured using the IMMULITE 2000 Immunoassay System ®. Our results indicated significant differences (p≤0.05) in the cortisol levels among both groups (beef crossbred cows vs Azores Lydia) of 4.3±0.3 ng/dl and 5.8±0.4 ng/dl, respectively. The pregnancy results were also significantly different: 63.7% vs 45.6%, respectively, for beef crossbred and "Azores Lydia" cows. The present study clearly demonstrated a negative correlation between cortisol levels and pregnancy rates after fixed-time artificial insemination. The low levels of cortisol observed in the animals and particularly in the Azores Lydia breed, when compared to other studies carried out on these animals, must be due to the passage of the animals in the sleeve, allowing them to habituate to a routine, as well as to human presence.

Evaluation of estradiol and progesterone contents of Bach Thao and Boer goats during the oestrous cycle

This paper presents the results of the analysis of estradiol and progesterone contents during the oestrous cycle of Bach Thao and Boer goats at the Center for Research and Development of Large Cattle in Binh Duong province. Blood samples were taken every day between 6 and 8 a.m. for 24 consecutive days. Hormone contents were determined by electroluminescence assay with the Cobas E601 immunoassay system. The results showed that the highest increase in estradiol was observed on day 2 of the cycle with a value of 8.73±0.60 pg/ml in Bach Thao goats and a value of 13.45±1.26 pg/ml in Boer goats. Progesterone concentration reached a peak value of 7.93±0.49 ng/ml on day 14 in Bach Thao goats and reached a peak value of 8.46±0.34 ng/ml on day 13 in Boer goats. The results of this study help determine the time of estrus, choose a timely pairing for effective reproductive management, and serve as a reliable reference when using assisted reproductive tools and strategies to improve reproductive performance in goats.

One-Component Dual-Readout Aggregation-Induced Emission Nanobeads for Qualitative and Quantitative Detection of C-Reactive Protein at the Point of Care.

Fluorescent lateral flow immunoassay (LFA) systems are versatile tools for sensitive and quantitative detection of disease markers at the point of care. However, traditional fluorescent nanoparticle-based lateral flow immunoassays are not visible under room light, necessitate an additional fluorescent reader, and lack flexibility for different application scenarios. Herein, we report a dual-readout LFA system for the rapid and sensitive detection of C-reactive protein (CRP) in clinical samples. The system relied on the aggregation-induced emission nanobeads (AIENBs) encapsulated with red AIE luminogen, which possesses both highly fluorescent and colorimetric properties. The AIENB-based LFA in the naked-eye mode was able to qualitatively detect CRP levels as low as 8.0 mg/L, while in the fluorescent mode, it was able to quantitatively measure high-sensitivity CRP (hs-CRP) with a limit of detection of 0.16 mg/L. The AIENB-based LFA system also showed a good correlation with the clinically used immunoturbidimetric method for CRP and hs-CRP detection in human plasma. This dual-modal AIENB-based LFA system offers the convenience of colorimetric testing and highly sensitive and quantitative detection of disease biomarkers and medical diagnostics in various scenarios.

Biosensors based on potent miniprotein binder for sensitive testing of SARS-CoV‑2 variants of concern.

Theminiprotein binder TRI2-2 was employedas an antibody alternative to build a single antibody-coupled TRI2-2 based gold nanoparticle-based lateral flow immunoassay (AT-GLFIA) biosensor. The biosensor provideshigh specificity and affinity binding between TRI2-2 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) spike antigen receptor binding domain (S-RBD). It also enablesrapid testing of wild-type (WT), B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron) SARS-CoV-2 S-RBD and is at least ~ 16-fold more sensitive than conventional antibody pair-based GLFIA (AP-GLFIA). Besides, we developed a wireless micro-electrochemical assay (WMECA) biosensor based on the TRI2-2, which demonstrates an excellent VOCs testing capability at the pg mL-1 level. Overall, our results demonstrate that integrating miniprotein binders into conventional immunoassay systems is a promising design for improving the testing capabilities of such systems without hard-to-obtain antibody pair, complex reporter design, laborious signal amplification strategies, or specific instrumentation.

Glibenclamide-Loaded Engineered Nanovectors (GNVs) Modulate Autophagy and NLRP3-Inflammasome Activation.

Activation of the NLRP3 inflammasome in response to either exogenous (PAMPs) or endogenous (DAMPs) stimuli results in the production of IL-18, caspase-1 and IL-1β. These cytokines have a beneficial role in promoting inflammation, but an excessive activation of the inflammasome and the consequent constitutive inflammatory status plays a role in human pathologies, including Alzheimer's disease (AD). Autophagic removal of NLRP3 inflammasome activators can reduce inflammasome activation and inflammation. Likewise, inflammasome signaling pathways regulate autophagy, allowing the development of inflammatory responses but preventing excessive and detrimental inflammation. Nanotechnology led to the development of liposome engineered nanovectors (NVs) that can load and carry drugs. We verified in an in vitro model of AD-associated inflammation the ability of Glibenclamide-loaded NVs (GNVs) to modulate the balance between inflammasome activation and autophagy. Human THP1dM cells were LPS-primed and oligomeric Aß-stimulated in the presence/absence of GNVs. IL-1β, IL-18 and activated caspase-1 production was evaluated by the Automated Immunoassay System (ELLA); ASC speck formation (a marker of NLRP3 activation) was analyzed by FlowSight Imaging flow-cytometer (AMNIS); the expression of autophagy targets was investigated by RT-PCR and Western blot (WB); and the modulation of autophagy-related up-stream signaling pathways and Tau phosphorylation were WB-quantified. Results showed that GNVs reduce activation of the NLRP3 inflammasome and prevent the Aß-induced phosphorylation of ERK, AKT, and p70S6 kinases, potentiating autophagic flux and counteracting Tau phosphorylation. These preliminary results support the investigation of GNVs as a possible novel strategy in disease and rehabilitation to reduce inflammasome-associated inflammation.

Analysis on epidemiological characteristics of allergens with allergic skin diseases in a hospital in Beijing City from 2017 to 2021

Objectives: In order to provide valuable information for the diagnosis and treatment of dermatitis, the prevalence rate and trend changes of common allergens in patients with allergic skin diseases in Beijing City were investigated and analyzed. Methods: This study was a retrospective data collection study. A total of 2 822 patients diagnosed with moderate to severe atopic dermatitis with allergen examinations were collected from Peking University First Hospital from 2017 to 2021, most of them were adults, and 83%(2 340/2 822) were over 18 years old. The ImmunoCAP fluorescent enzyme-linked immunoassay system was used to quantify specific IgE. The positive rate of each allergen was counted according to age, gender and year. The epidemiological characteristics and trends were analyzed. Results: In the past five years, 22 503 allergen-specific IgE test data were collected, and the positive rate of allergens in 40%(1 122/2 822) of the 2 822 patients was mainly multiple sensitization, the positive rate of allergens and multiple sensitization rates were the highest in people aged 13-18 years old. The 2 822 patients were dominated by inhaled allergens, and the highest positive rate was inhaled mixture (29.3%). The top five positive rates of inhaled allergens were Dermatophagoides farina (26.7%), Dermatophagoides pteronyssinus (25.5%), Mugwort (23.9%), Birch pollen (19.2%) and Siberian cocklebur (18.2%). The top five positive rates of food allergens were shrimp (15.2%), peanut (14.8%), sesame (14.7%), wheat (13.2%) and milk (13.1%). Analysis of the positive rate of allergens in different age groups showed except for the positive rate of birch pollen peaked at 3-12 years old, other inhaled allergens were the highest food allergens in 13-18 years old. Among food allergens, the positive rates of fx5, milk, egg and wheat were the highest in <3 years old, the positive rate decreased gradually with age, and the positive rate of other food allergens peaked at 13-18 years old, and the shrimp and crab was the highest in adult. The positive rate of various allergens in the 2 822 patients showed little difference between male and female and the male was slightly higher than female. In the past five years, the positive rate of allergens has been on the rise. The changes of inhaled allergens in cat/dog hair dander, Dermatophagoides farina and cicadas were large, while the positive rate of food allergens was stable. Conclusions: The etiology of the allergic skin diseases is complex, which may be related to allergen exposure, heredity, immunity, environmental factors, abnormal skin barrier function, etc. This study showed the distribution of allergens in patients with allergic skin diseases in Beijing City to a certain extent. It provided epidemiological data and clinical evidence for the prevention and treatment of allergic diseases.

Alfaxalone increases measured progesterone concentration in neutered male cats when determined by immunoreactivity.

Alfaxalone is a commonly used anesthetic agent in small animals. In cats, alfaxalone can be administered as an IM agent to achieve clinically useful sedation or anesthesia, negating the need for IV injection in difficult patients. The molecular structure of alfaxalone is similar to the hormone progesterone (P4). It is hypothesized that alfaxalone would cross-react with the assay measuring progesterone causing a false elevation. 8 healthy neutered male, domestic shorthair cats that were privately owned were enrolled in the study. Male neutered cats were administered 3 mg/kg of alfaxalone IM. Blood samples were collected at set time points (baseline, 30 minutes, 60 minutes, 3 hours, 6 hours, and 10 hours after administration), and serum concentrations of progesterone immunoreactivity (IR) were determined using the Siemens Immulite 1000 automated immunoassay system. Statistical analysis was performed with repeated measures ANOVA and a Tukey-Cramer multiple comparisons test. A P value of < .05 was used for significance. Serum progesterone IR was significantly elevated at 30 minutes, 1 hour, and 3 hours (P < .05) when compared to baseline progesterone immunoreactivity. Progesterone immunoreactivity had returned to baseline by 6 hours. This study suggests that alfaxalone administered IM in cats may interfere with immunoassay measurement of serum progesterone for up to 6 hours. Caution should be used when interpreting serum progesterone immunoreactivity results in cats within 4 hours of alfaxalone.

Pro-inflammatory cytokine IL-6 regulates LMO4 expression in psoriatic keratinocytes via AKT/STAT3 pathway.

The transcription factor LIM-only protein 4 (LMO4) is overexpressed in the psoriatic epidermis and regulates keratinocyte proliferation and differentiation. High LMO4 expression levels are induced by interleukin-23 (IL-23) to activate the AKT/STAT3 signaling pathway. Interleukin-6 (IL-6) is mainly involved in regulating T cell functions and development in patients with psoriasis. However, whether LMO4 expression is regulated by IL-6 remains unclear. Therefore, the purpose of this study is to explore the role and molecular mechanisms of IL-6 in regulating LMO4 expression. The interleukin-6 (IL-6) levels in human plasma were determined using a chemiluminescence immunoassay system. A psoriasis-like mouse model was established using imiquimod induction. Epidermal keratinocytes (HaCaT) were cultured in defined keratinocyte-serum-free medium and stimulated by IL-6 alone or with inhibitors. The proteins of interest were detected using western blot analysis, immunofluorescence, and immunohistochemistry. The 5-ethynyl-2'-deoxyuridine assay was used to detect cell proliferation. The results revealed that IL-6 levels were markedly increased in the plasma of patients with psoriasis, compared to healthy control. The high expression of LMO4 was consistent with high levels of IL-6, p-AKT, and p-STAT3 in the lesions of both psoriasis patients and imiquimod-induced psoriasis-like mice. IL-6 activates the AKT/STAT3 signaling pathway, followed by LMO4 high-expression in HaCaT cells. IL-6 induces HaCaT proliferation and differentiation via AKT/STAT3 signaling pathway activation. We think that the high expression of LMO4 in psoriatic keratinocytes requires IL-6 to activate the AKT/STAT3 signaling pathway and leads to epidermal keratinocytes abnormal proliferation and differentiation.

CytogeneticsComparability of Beckman Coulter Access Cardiac Troponin I Assays

Abstract Cardiac troponins (cTn) are the most sensitive and specific biomarkers of myocardial injury. The rise and/or fall of cardiac troponins above the 99th percentile of the upper reference limit (URL) is required in the assessment of acute myocardial infarction. In our institution, high sensitivity troponin I (hsTnI) is performed on four Access 2 Immunoassay Systems and three DXI 800 Access Immunoassay Systems. As a regional medical center, we accept patients from outside hospitals where contemporary cardiac troponin I (cTnI) were performed. The results from different assays and platforms may disagree and lead to incorrect explanation of the results as false rise and/or fall of cTn. To harmonize results among these assays, we analyzed the variation between the two assays of cTnI and hsTnI, and the concordance of same assay hsTnI on different instruments using patient specimens. To investigate the between-method variance, 424 patient specimens with troponin levels ranging from 0-23370 ng/L were used to measure both cTnI and hsTnI on DXI 800 instruments. To investigate the within-method agreement, 43 patient specimens with troponin levels ranging from 2-114394 ng/L were used to measure hsTnI on Access 2 and DXI instruments. The between-method comparison of cTnI and hsTnI on DXI showed a slope of 1.003 with intercept of -43.31 and correlation coefficient (CC) of 0.9590. The mean bias was -5.1%, which was within the analytical imprecision range determined in our laboratory (2.2%-10.7%). However, comparison of cTnI and hsTnI in patients with troponin &amp;lt;20ng/L (n=103), the 99th percentile of the URL for males, showed the slope was 1.325 with intercept of -0.97 and CC of 0.7462. The mean bias was 1.91 (21.53%), which was much larger than the analytical imprecision. This is consistent with the fact that cTnI results are not reliable at the lower level due to the imprecision problem. Comparison of cTnI and hsTnI in patients with troponin &amp;gt;20ng/L (n=321) showed the slope was 1.006 with intercept of –60.09 and CC of 0.9576. The mean bias was -54.30 (-5.23%), which was within the analytical imprecision range. The within-method comparison of hsTnI on Access 2 and DXI 800 showed the slope was 1.138 with intercept of -633.0 and CC of 0.9888. The mean bias was 2126.4 (10.6%), within the analytical imprecision range. These data indicate that cTnI results above the 99th percentile URL are comparable with hsTnI results if both are tested on DXI 800, and hsTnI results tested on Access 2 and DXI 800 are comparable. Thus, to use the cardiac troponins assay properly, we suggest the laboratory report cardiac troponins with the assay and instrument names to reduce confusion during results comparison.

High IL1-RA Plasma Levels at Baseline Allow to Identify Hodgkin Lymphoma Patients with High Risk of Treatment Failure

Introduction: About 10% of localized stages and 20-30% of advanced stages patients (pts) with classical Hodgkin lymphoma (cHL) progress or relapse after the first line of treatment. Therapeutic failures prediction at diagnosis is a major challenge to modulate treatments to minimize acute and long-term toxicities, while having optimal efficacy. The development of prognostic tools is crucial to help therapeutic decision. In 2007, Casasnovas et al. JCO 2007, demonstrated that a plasma cytokine signature composed of IL1-RA, IL-6 and sCD30 could predict pts outcomes and identify pts with high risk of treatment failure. However, it was unusable in routine practice as the ELISA technique used had extensive delay. Since interim PET during induction chemotherapy became the standard approach to guide the therapeutic strategy, and therefore the use of new pre-therapeutic biomarkers deserves to be investigated in the context of PET-guided treatment. As PD-L1, and thymus and activation-related chemokine (TARC) have an important role in cHL physiopathology we analyzed the prognostic impact on progression-free, and overall survival (PFS and OS) of pretherapeutic plasma levels of PD-L1 and TARC in addition to IL1-RA, IL-6, sCD30, in pts treated with a PET-guided strategy. Method: The dosage of IL1-RA, IL-6, PD-L1 and TARC was performed using the ELLA automated immunoassay system (Biotechne) while sCD30 was performed in triplicate using ELISA assay. Samples were prospectively collected (period 2016-2020) within the biocollection BIO-LYMPH (NCT04417803) in the pts treated in the department of Hematology at Dijon Hospital. X tile analysis was used to define the optimal cutoff for survival prediction for each cytokine. Survival functions of pts' subgroups defined by the cytokine circulating levels were estimated using the Kaplan-Meier product limit method and compared using the log rank test. The Cox proportional hazard regression model was used to estimate HRs and associated 95% CI in univariate and multivariate analyses. Results: Among the 105 pts enrolled in this study (Median age: 36 years, range: 17 - 85), 60% were under 45 years, 61% had advanced stage disease, 58% B symptoms, and 39% an IPS &amp;gt;2. 63% of pts had nodular sclerosis HL subtype and 22% had EBV associated HL. Response was assessed at the end of treatment: 89 pts (84 %) achieved a complete response, 4 pts (4%) a partial response, and 9 pts (9%) had progressive disease. With a median follow-up time of 40 months (1-78 months), 13 pts (12 %) relapsed with a median time to relapse of 16 months (range, 4 to 65 months), 14 pts (13 %) died (11 due to cHL progression; 1 due to septic shock; 1 from SARS covid infection; and 1 after a traumatism). While pretherapeutic plasmatic levels of IL-6, sCD30, TARC and PD-L1 did not impact pts survival, IL1-RA was an independent predictor of both PFS and OS. Indeed, 31% of pts harbored high IL1-RA plasma level (≥ 740 pg/ml) and had an unfavorable PFS (5y-PFS: 50.2% vs 86.7%; HR=4.2, CI=2.1-10.3, p=0.0001, figure 1A) and OS (5y-OS 71.4% vs 95%, HR=4.49, CI=1.3-14.2, p=0.006). PET2 positivity (17% of pts) had an unfavorable prognosis value on PFS (5y-PFS 58% vs 86.6%, HR 4.3, CI= 1.4-12.8, p=0.014) but not on OS (5y-OS 91.8% vs 74.1%; HR=3.2 CI=0.7-14.4, p=0.15) independent from pretherapeutic IL1-RA plasma level (PFS multivariate analysis: PET2 positivity HR=2.43, CI=1-6, p= 0.05; High level IL1-RA HR= 3.4, CI=1.5-8.3, p= 0.005). The combination of both PET2 results and pretherapeutic IL1-RA plasma levels allow to stratify pts' outcome into 3 groups (figure 1B): - Pts (57%) with low levels of IL1-RA and negative PET2 had a 5y-PFS of 89.3% and 5y-OS of 95.8%, - Pts with high levels of IL1-RA or positive PET2 (35%) had a 5y-PFS of 57% and 5y-OS of 74.8% whereas - Pts with both (8%) had a 5y-PFS of 42.9% and 5y-OS of 71.4%. The groups with one or 2 prognostic factors (PET2+ or high IL1RA and PET2+ &amp; high IL1RA) had a significantly different PFS compared to the double negative group (1 factor: HR=4.7, CI=1.8-12.2, p=0.0011 and 2 factors: HR= 8.57, CI=2.5-28.3, p=0.0004 respectively). The group with 1 prognosis factor had a significantly different OS (HR=4.1, CI=1.1-15.3, p=0.03) compared to double negative group but not the 2 factors group (HR= 4.9, CI=0.8-30, p=0.08). Conclusion: Pretherapeutic IL1-RA plasma levels allow to stratify the prognosis of pts with cHL treated with first line PET-guided strategies and add prognosis information to PET2 response.

Detection of Listeria Species by Conventional Culture-Dependent and Alternative Rapid Detection Methods in Retail Ready-to-Eat Foods in Turkey.

Foodborne pathogens, like Listeria monocytogenes, continue to inflict substantial financial losses on the food industry. Various methods for detecting Listeria in food have been developed and numerous studies have been conducted to compare the different methods. But, in recent years, new Listeria species have been identified, and currently the genus comprises 26 species. Therefore, it would be a more accurate approach to re-evaluate existing detection methods by considering new species. The present investigation involved the analysis of 42 ready-to-eat (RTE) foods, encompassing a variety of food categories, such as mezes, salads, dairy products, and meat products, with the aim of ascertaining the presence of Listeria. Among the traditional culture-dependent reference methods, the ISO 11290 method was preferred. The process of strain identification was conducted with the API Identification System. Furthermore, to ascertain the existence of L. monocytogenes and Listeria spp., the samples underwent additional analysis employing the VIDAS Immunoassay System, ELISA, and RT-PCR methodologies. Thus, four alternative approaches were employed in this study to compare not only the different methods used to determine Listeria while taking into account the newly identified Listeria species, but also to assess the compliance of retail RTE food items with microbiological criteria pertaining to the genus Listeria. Based on the conducted analyses, L. monocytogenes was conclusively determined to be present in one sample. The presence of Listeria spp. was detected in 30.9% of the samples, specifically in Turkish cig kofte, sliced salami, and salads.