Fresh Serum Research Articles

Complement activation by IgM autoantibodies linked to immune-mediated neuropathies depends on C2.

Complement factor C2 is a potential therapeutic target in immune-mediated neuropathies. However, literature suggests that classical complement pathway activation may proceed to C3 in the absence of C2, a so-called "C2 bypass." Here, we evaluated a C2 bypass mechanism during complement activation by pathogenic human IgM from patients with immune-mediated neuropathies. IgM autoantibodies from 51 patients with multifocal motor neuropathy (MMN) or anti-myelin-associated glycoprotein (MAG) neuropathy (AMN) were used to activate complement in exvivo disease models. C2 bypass was evaluated using C2-depleted (C2D) serum and a therapeutic anti-C2 antibody. In two different disease models of MMN, IgM anti-GM1 and IgM anti-GM2 autoantibodies from MMN patients were bound to induced pluripotent stem cell-derived motor neurons and Schwann cells, respectively, and fixed C3 upon incubation with fresh serum. C3 fixation was inhibited by anti-C2 and did not occur with C2D serum. Similarly, in an AMN model, IgM anti-MAG antibodies were incubated with fresh serum fixed C3, which in all cases was abrogated in the absence of C2 or in the presence of anti-C2. In exvivo disease models of MMN and AMN, complement activation by IgM autoantibodies from 51 patients was in all cases dependent on C2 and was inhibited by an antihuman C2 antibody. No evidence of a C2 bypass mechanism was found.

Chondrocyte Response to Fresh Autologous Conditioned Serum Versus Freeze-Dried Allogenic Conditioned Serum.

To investigate the cytokine release profile and histological response of human cartilage after exposure to autologous conditioned serum (ACS) and freeze-dried allogenic conditioned serum (FD-CS). Cartilage explants were collected from 6 patients undergoing total knee arthroplasty. ACS and FD-CS were created from patient serum samples. Cartilage samples were divided into 6 groups: (1) untreated control, (2) ACS, (3) FD-CS, (4) untreated interleukin (IL)-1β (5 ng/ml), (5) IL-1β + ACS, and (6) IL-1β + FD-CS. After 12 days, cartilage samples were analyzed with glycosaminoglycan (GAG) concentration normalized to wet weight while comparing cytokine concentrations, and histological scoring. There was a significant decrease in pathology scoring for ACS (P = 0.0368) and FD-CS (P = 0.0368) in the IL-1β injury groups compared with the untreated IL-1β insult group. ACS and FD-CS significantly mitigate the IL-1β induced increase in basic fibroblast growth factor (bFGF) (P = 0.0009 and P = 0.0002, respectively). FD-CS showed a significant decrease in IL-1β concentration in the presence of IL-1β insult compared with the untreated IL-1β group (P < 0.0001). ACS-treated samples had significantly higher concentration of tumor necrosis factor (TNF)-α independent of IL-1β when compared with samples not treated with biologics (P = 0.0053). Explanted osteoarthritic cartilage responds favorably and equivalently to treatment with ACS and FD-CS from a histological perspective. Both ACS and FD-CS were able to mitigate the IL-1β-induced increases in bFGF and FD-CS lowered IL-1β concentration while increasing interleukin-1 receptor antagonist (IL-1Ra) concentration. Although the cytokine profile of cartilage tissue explants treated with FD-CS appears to be different than that of ACS, this difference does not seem to affect biologic activity of FD-CS.

Relationship between serum iron, zinc, calcium, and HIF-1a-comparative analysis of 2 regions and 4 ethnic groups in China.

Altitude illness has serious effects on individuals who are not adequately acclimatized to high-altitude areas and may even lead to death. However, the individualized mechanisms of onset and preventive measures are not fully elucidated at present, especially the relationship between altitude illness and elements, which requires further in-depth research. Fresh serum samples were collected from individuals who underwent health examinations at the two hospitals in Xining and Sanya between November 2021 and December 2021. The blood zinc (Zn), iron (Fe), and calcium (Ca) concentrations, as well as hypoxia-inducible factor 1-alpha (HIF-1α) concentrations, were measured. This study conducted effective sample size estimation, repeated experiments, and used GraphPad Prism 9.0 and IBM SPSS version 19.0 software for comparative analysis of differences in the expression of elements and HIF-1α among different ethnic groups, altitudes, and concentration groups. Linear regression and multiple linear regression were employed to explore the relationships among elements and their correlation with HIF-1α. This study included a total of 400 participants. The results from the repeated measurements indicated that the consistency of the laboratory test results was satisfactory. In terms of altitude differences, except for Fe (p = 0.767), which did not show significant variance between low and high altitude regions, Zn, Ca, and HIF-1α elements all exhibited notable differences between these areas (p < 0.0001, p = 0.004, and p < 0.0001). When grouping by the concentrations of elements and HIF-1α, the results revealed significant variations in the distribution of zinc among different levels of iron and HIF-1α (p < 0.05). The outcomes of the linear regression analysis demonstrated that calcium and zinc, iron and HIF-1α, calcium and HIF-1α, and zinc and HIF-1α displayed substantial overall explanatory power across different subgroups (p < 0.05). Finally, the results of the multiple linear regression analysis indicated that within the high-altitude population, the Li ethnic group in Sanya, and the Han ethnic group in Sanya, the multiple linear regression model with HIF-1αas the dependent variable and elements as the independent variables exhibited noteworthy overall explanatory power (p < 0.05). The levels of typical elements and HIF-1α in the blood differ among various altitudes and ethnic groups, and these distinctions may be linked to the occurrence and progression of high-altitude illness.

Relationship between fresh single embryo morphology scores and serum HCG values at 14 days and 14–18-day doubling values

Research questionWhat is the effect of embryo morphology score on 14-day β-HCG levels and 14–18-day β-HCG doubling values, and do they have differences in day-3 embryo or day-5 blastocyst transfers? DesignRetrospective analysis of 4434 fresh cycles of single embryo transfers (SET) with β-HCG ≥15 mIU/ml on day 14 after transfer via IVF and ICSI. The correlation between embryo morphology score and 14-day β-HCG was examined. Doubling of 14–18 day β-HCG was analysed in 2628 cycles to determine correlations with embryo morphology score. ResultsIn day-3 SET, number of embryonic cells was positively correlated with 14-day post-transfer β-HCG values (R = 0.076; P = 0.013). No significant correlation was observed between the grade of the transferred embryos and the 14–18-day serum β-HCG doubling values. In day-5 single blastocyst transfers, the degree of blastocyst expansion, trophoblast cell and inner cell mass (ICM) grades demonstrated a significant positive correlation with 14-day post-transfer β-HCG (P < 0.001, P = 0.014, P = 0.003). Degree of blastocyst expansion was significantly correlated with 14–18-day β-HCG doubling values (R = –0.051, P = 0.027). Grades of the ICM and trophoblast cells showed no significant correlation with 14–18-day β-HCG doubling values. ConclusionIn fresh SET, embryo morphology score influences 14-day β-HCG values in day-3 embryos and day-5 blastocyst transfers. Embryo morphology score in day-3 SET does not affect 14–18-day β-HCG doubling values. Degree of blastocyst expansion significantly affects 14–18-day β-HCG doubling values in day-5 blastocyst transfers.

Integrating Patient-Based Real-Time Quality Control (PBRTQC) in a New Field: Inter-Comparison between Biochemical Instrumentations with LDL-C.

Patient-based real-time quality control (PBRTQC) can be a valuable tool in clinical laboratories due to its cost-effectiveness and constant monitoring. More focus is placed on discovering and improving algorithms that compliment conventional internal control techniques. The practical implementation of PBRTQC with a biochemical instrument comparison is lacking. We aim to evaluate PBRTQC's efficacy and practicality by comparing low-density lipoprotein cholesterol (LDL-C) test results to ensure consistent real-time monitoring across biochemical instrumentations in clinical laboratories. From 1 September 2021 to 30 August 2022, the First Affiliated Hospital of Xi'an Jiaotong University collected data from 158,259 both healthy and diseased patients, including 84,187 male and 74,072 female patients, and examined their LDL-C results. This dataset encompassed a group comprising 50,556 individuals undergoing health examinations, a group comprising 42,472 inpatients (IP), and a group comprising 75,490 outpatients (OP) for the PBRTQC intelligent monitoring platform to conduct daily tests, parameter configuration, program development, real-time execution, and performance validation of the patients' data. Moreover 40 patients' LDL-C levels were assessed using two biochemical analyzers, designated as the reference and comparator instruments. A total of 160 LDL-C results were obtained from 40 both healthy and diseased patients, including 14 OP, 16 IP, and 10 health examination attendees, who were selected to represent LDL-C levels broadly. Two biochemical instruments measured LDL-C measurements from the same individuals to investigate consistency and reproducibility across patient statuses and settings. We employed exponentially weighted moving average (EWMA) and moving median (MM) methods to calculate inter-instrument bias and ensure analytical accuracy. Inter-instrument bias for LDL-C measurements was determined by analyzing fresh serum samples, different concentrations of quality control (QC), and commercialized calibrators, employing both EWMA and MM within two assay systems. The assessment of inter-instrumental bias with five different methods adhered to the external quality assessment standards of the Clinical Laboratory Center of the Health Planning Commission, which mandates a bias within ±15.0%. We calculated inter-instrument comparison bias with each of the five methods based on patient big data. The comparison of fresh serum samples, different concentrations of QC, commercialized calibrators, and EWMA were all in the permissive range, except for MM. MM showed that the bias between two biochemical instruments in the concentration ranges of 1.5 mmoL/L-6.2 mmoL/L exceeded the permissible range. This was mainly due to the small number of specimens, affected by variations among individual patients, leading to increased false alarms and reduced effectiveness in monitoring the consistency of the inter-instrumental results. Moreover, the inter-comparison bias derived from EWMA was less than 3.01%, meeting the 15% range assessment criteria. The bias result for MM was lower than 24.66%, which was much higher than EWMA. Thus, EWMA is better than MM for monitoring inter-instrument comparability. PBRTQC can complement the use of inter-comparison bias between biochemical analyzers. EWMA has comparable inter-instrument comparability monitoring efficacy. The utilization of AI-based PBRTQC enables the automated real-time comparison of test results across different biochemical instruments, leading to a reduction in laboratory operating costs, enhanced work efficiency, and improved QC. This advanced technology facilitates seamless data integration and analysis, ultimately contributing to a more streamlined and efficient laboratory workflow in the biomedical field.

ABO Blood System: Biosynthesis of Agglutinogenic Alkaline and Non-Agglutinogenic Acid Glycotopes of A and B Antigens at Different pHs of the Culture Medium.

The biosynthesis of agglutinogenic and adsorbing groups A and B glycotopes of the erythrocyte's membrane is mediated by the activity of specific glycosyltransferases. This study aimed to assess the nature of the biosynthesis of A and B antigenic glycotopes, depending on the pH of the medium during the cultivation of erythrocytes, and the antigenic (transferase) characteristics of the donor serum of the other group. Monoclonal antibodies (Mabs) were obtained from IGBRL under Program IV of the International Workshop on Monoclonal Antibodies and Red Blood Cell Antigens. Biosynthesis was performed using erythrocytes, fresh serum, medium 199, and an antibiotic solution. The agglutinogenic characteristics of 11 out of 33 samples changed by the end of the cultivation period due to the acquisition of additional agglutinogen corresponding to the donor serum. None of the samples lost their inherent agglutinogen due to its absence in the donor serum. Four of six samples of O(I) erythrocytes acquired the ability to be agglutinated by anti-A reagents, especially by the polyclonal anti-A, and the manifestation of agglutination depended on the reaction time. Two of the three samples with initial A(II) agglutinogenic specificity added to the donor serum with Bc'+ characteristic of the erythrocytes acquired this characteristic. However, none of the five A(II)Ac'+ samples cultured in the serum of Ac'-O(I)Ac'-Bc'+ and O(I)Ac'-Bc'- donors lost their inherent earlier Ac'+ characteristic. The investigation of the inhibitory ability of alkaline and acidic glycoconjugates isolated from membranes revealed that alkaline Alp-00 and Alp-1 glycotopes isolated from glycolipids had the highest inhibitory activity, and the degree of inhibition of polyclonal anti-A antibodies was even higher than that of monovalent BRIC-131. This study showed the possibility of the biosynthesis of specific non-agglutinogenic A and B glycotopes under the influence of a different group's serum as a source of the corresponding transferase.

A rare cause of Acquired Hemolytic Anemia in a child; Donath-Landsteiner antibody

Abstract Introduction/Objective INTRODUCTION: The Donath-Landsteiner (DL) antibody is known to cause paroxysmal cold hemoglobinuria (PCH), a rare form of intravascular hemolytic anemia which is precipitated by an IgG biphasic hemolysin with specificity to the P blood group antigen and most commonly reported in children. The DL test is a serologic test specifically examines, the biphasic nature of the antibody activity. Methods/Case Report CASE PRESENTATION: A 4-year-old previously healthy boy presented at an academic medical center with rhinorrhea and nasal congestion. His family reported that his urine became darker almost coke colored after his symptoms started. His initial vitals; temperature of 37.1 C, heart rate of 140, blood pressure of 122/87 and O2 saturation of 96% on room air. Laboratory investigations showed a hemoglobin level of 4.2 g/dL, hematocrit level of 12.5%, platelets count of 229 K/uL, indirect bilirubin of 6.2mg/dL, haptoglobin &amp;lt;30 mg/dL, LDH &amp;gt;1800 U/L. Direct antiglobulin test was positive with complement only. Thermal Amplitude and indirect Donath-Landsteiner tests were both positive. Respiratory viral panel was positive for Rhinovirus. The antibody screen was negative, and the patient was provided supportive care including as needed RBC transfusion, warming of fluids and body. By day day 9 patient Hgb stabilized, LDH, bilirubin and haptoglobin normalized and his urine turned yellow color and was discharged in stable condition. Results (if a Case Study enter NA) NA Conclusion CONCLUSIONS PCH is classified as a cold autoimmune hemolytic anemia caused by an IgG antibody that sensitizes RBCs at cold temperatures by attaching complement to the RBCs, causing intravascular hemolysis upon rewarming. The DL test for PCH involves in vitro testing for hemolysis with the patient's serum, washed group-O cells expressing the P antigen, and fresh normal serum. To prevent autoadsorption of the antibody and hemolysis, blood should be collected in prewarmed tubes that do not contain anticoagulants and kept at 37°C during the collection procedure. A high index of suspicion especially in pediatric population should be maintained after a viral infection. The treatment is usually supportive.

Integration of gut microbiome and serum metabolome revealed the effect of Qing-Wei-Zhi-Tong Micro-pills on gastric ulcer in rats

Ethnopharmacological relevanceQing-Wei-Zhi-Tong Micro-pills (QWZT) is herbal compound used in the treatment of GU, whose functions include clearing the stomach and fire, softening the liver and relieving pain. However, its mechanistic profile on host intestinal microbiota and metabolism has not been determined. Aim of the studyThe present study aimed to observe the healing effect of QWZT on acetic acid-induced gastric ulcer in a rat model and to preliminarily elucidate its possible therapeutic mechanism from the perspective of host intestinal microbiota and metabolism. Materials and methodsThe Wistar male rats (7 weeks old; weight 180–200 g) were randomly divided into normal control group (NC), acetic acid-induced gastric ulcer group (GU), and QWZT treatment group (High dose: 1250 mg/kg/day, Middle dose: 625 mg/kg/day, Low dose: 312.5 mg/kg/day) of 6 rats each. An acetic acid-induced gastric ulcer rat model was constructed based on anatomical surgery. QWZT (High dose, Middle dose, and Low dose) was used to treat gastric ulcer rats for 7 days by gavage. At the end of treatment, the body weight, macroscopic condition of gastric tissue ulcers, pathological changes (HE staining), inflammatory factors, oxidative stress factors, and endocrine factors were assessed in each group of rats. Fresh feces and serum from each group of rats were collected for microbiome and metabolome analysis on the machine, respectively. Drug-disease common targets and functional pathways were captured based on network pharmacology. The complex network of Herbs-Targets-Pathways-Metabolites-Microbiota interactions was constructed. Ultimately, Fecal Microbiota Transplantation (FMT) evaluated the contribution of gut microbiota in disease. ResultsQWZT increased the abundance of beneficial bacteria (Bacteroides, Alloprevotella, Rikenellaceae_RC9_gut_group, Lactobacillus, Lachnospiraceae_NK4A136_group, Parabacteroides, etc.), reduced the abundance of harmful bacteria (Micromonospora, Geobacter, Nocardioides, and Arenimonas, etc.), reduced the levels of inflammatory mediators (12,13-EpOME, 9,10-Epoxyoctadecenoic acid, SM(d18:1/16:0) and Leukotriene A4, etc.), restored host metabolic disorders (Linoleic acid metabolism, Glycerophospholipid metabolism, and Arachidonic acid metabolism), and regulated the level of cytokines (IL-6, TNF-a, SOD, MDA, PEG-2 and NO), ultimately exerting an anti-ulcer effect. Apart from that, FMT improved acetic acid-induced gastric ulcers in rats. ConclusionQWZT improved acetic acid-induced gastric ulcers in rats by remodeling intestinal microbiota and regulating host metabolism. This work may promote the process of developing and utilizing clinical applications of QWZT.

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.

A pre-oxidized Eu-probe doped into bio-MOF-1 for ascorbic acid emission “off-on” detection in human serum

Ascorbic acid (or widely known as vitamin C, VC) is an essential antioxidant and a free radical scavenger in human body. The appeal for reliable fluorescent nanosensors always promotes the development of VC probe. In this work, a pre-oxidized Eu-probe (denoted as Eu-NO9) was synthesized. Without VC, Eu-NO9 was nearly non-emissive owing to the inefficient ligand energy transfer (ET) to Eu ion (caused by mismatched ligand level and long distance to Eu, as revealed by single crystal analysis and emissive parameters). By adding VC, the pre-oxidized ligand was deoxidized and its ET to Eu ion became efficient (confirmed by electrochemical analysis), with Eu(III) red emission intensity obviously increased. Then Eu-NO9 was doped into a porous host bio-MOF-1 for ascorbic acid detection (denoted as Eu-NO9@MOF). The molecular sieving effect of bio-MOF-1 improved sensing selectivity, and bio-MOF-1 blue emission (421 nm) was applied as a reference for Eu(III) red emission. Linear working curves were obtained within a wide working region of 0–100 μM, with LOD of 1.7 μM. A short response time of 192 s at 25 °C was confirmed. Practical sensing plates were prepared and found applicable for VC detection in fresh human serum. The advantage of this work was the combination of a pre-oxidized probe and a porous host which gave emission “turn on” fluorescence sensing for VC with good selectivity, linear calibration curve and wide working region.

P-604 Inhibin B in Low Ovarian Reserve Patients: Correlation With Ovarian Reserve Markers and Oocyte Yield

Abstract Study question Is there a correlation between Inhibin B (InhB) serum levels and oocyte yield after ovarian stimulation (OS) in women with low ovarian reserve? Summary answer InhB presents a positive relationship with oocyte yield during OS in women with AMH&amp;lt;1.1ng/mL. What is known already InhB is produced by granulosa cells in the ovary with the goal of suppressing FSH production. Assay and intercycle variability have been the main limitations affecting specificity and reproducibility of InhB as a marker of ovarian reserve or oocyte yield after OS. This fact is attributed to low specific antibodies for the molecule in previous assays, exhibiting cross-reactivity with other glycoproteins in the transforming growth factor beta family. High specific ELISA assay,measuring bioavailable InhB (bio-InhB), may provide more accurate results, specially important in poor responders, as results may involve clinical decisions on whether or not to proceed with OS. Study design, size, duration Prospective observational study measuring bio-InhB in 72 women with low ovarian reserve, performing OS for IVF/ICSI, at a tertiary referral fertility center, from February 2019 to December 2021. Low ovarian reserve was defined as AMH serum levels &amp;lt;1.1ng/ml (Elecsys, Roche), following Bologna criteria. All patients performed OS for IVF/ICSI in a GnRH-antagonist protocol. Participants/materials, setting, methods On day 2/3 of cycle, before starting OS, AFC, FSH, LH, estradiol, progesterone and AMH (Elecsys, Roche) were measured. Extra serum samples were frozen at -20C for subsequent bio-InhB analysis (AnshLabs, Texas). Ethical approval was obtained (Research Ethics Committee-REFA033c) and informed consent was signed for all participants. Main results and the role of chance Patient characteristics were [Median(IQR)]: age: 39(36-42)years, BMI: 28.51(26.1-30.1) Kg/m2, AFC: 5.5(4-7), FSH:8.23(6.4-12.5) mIU/mL, LH: 6.54(4.51-8.91) mIU/mL, Estradiol: 41.18(28.47-56.44) pg/mL, AMH: 0.64(0.29-0.81)ng/mL, InhB: 62.69(33.78-97.92) pg/mL. Stimulation outcomes were: follicle number on day of trigger (Fdot): 5(3-7), cumulus-oocyte-complexes (COC’s): 3(2-4.5) andmetaphase II oocytes (MII): 3(1-4). Inh-B showed significant positive correlation (Spearman correlation) with AMH (rs = 0.35, p = 0.003), although was better with AFC (rs = 0.43, p &amp;lt; 0.001). Moreover, Inh-B serum levels presented positive association with oocyte yield: Fdot (rs = 0.41, p &amp;lt; 0.001), COC’s (rs = 0.36, p = 0.002) and MII (rs = 0.37, p = 0.002). AUROC analysis was performed to investigate which model predicted better a bad outcome (defined as &amp;lt; =3COC’s after OS) in low ovarian reserve women. No significant benefit was observed when Inh-B was added to AMH+AFC model (AUROC AMH+AFC: 0.757; AUROC AMH+AFC+Inh-B: 0.759, p = 0.843). Limitations, reasons for caution Although clinical assessment was performed in the same centre following the same methodology, inter-observer variability for AFC is a limitation. Besides, fresh AMH serum samples were analysed using Elecsys assay, yet frozen serum samples were shipped to Ansh Lab (Texas) for batched Inh-B analysis. Wider implications of the findings Bio-InhB is correlated to oocyte yield during OS. Although no additional benefit for prediction in women with AMH&amp;lt;1.1ng/mL was observed when added to other currently used markers, future research should explore the utility of bio-inhB on other outcomes associated to ovarian reserve, as embryo quality and ploidy. Trial registration number Not applicable

O-067 Does the measurement of different AMH isoforms in poor responder patients improve the prediction of ovarian response?

Abstract Study question Does the measurement of AMH isoforms in low ovarian reserve patients improve the prediction of the number of oocytes after ovarian stimulation (OS)? Summary answer AMH assays measuring a broader range of AMH isoforms combined with antral follicle count (AFC) improve the prediction of oocytes collected after OS. What is known already Granulosa cells secrete AMH as a non-active prohormone (proAMH). After cleavage, it forms a complex (AMHN,C) which activates the receptor (AMHR2). Circulating AMH is a mixture of isoforms (proAMH, AMHN,C and other sub-fragments) after proteolysis, targeted by the monoclonal antibodies included in the AMH assays commonly used in clinical practice, potentially affecting the quantification of the hormone. Novel AMH assays include antibodies directed towards a broader range of the molecule. They may provide more accurate and reliable results, especially important in poor responders, as results may involve clinical decisions on whether or not to proceed with OS. Study design, size, duration Prospective observational study measuring AMH isoforms using five different AMH assays, including 72 women with low ovarian reserve performing OS for IVF/ICSI at a tertiary referral fertility center, from February 2019 to December 2021. Low ovarian reserve was defined as AMH serum levels &amp;lt;1.1ng/ml (Elecsys, Roche), following Bologna criteria. All patients performed OS for IVF/ICSI with antagonist protocol. Participants/materials, setting, methods On day 2/3 of cycle, prior to initiating the OS, AFC, serum FSH, LH, estradiol, progesterone and AMH levels were measured using Elecsys assay (Roche). Extra serum samples were frozen at -20C for subsequent analysis, using four different AMH assays (AnshLabs, Texas): AL-196 (PCOCheck ELISA), AL-124 (PicoAMH ELISA), AL-105 (US-AMH ELISA) and AL-133 (Total Mature-AMH ELISA). Ethical approval was obtained (Research Ethics Committee-REFA033c) and informed consent was signed by all participants. Main results and the role of chance Patient characteristics were [Median(IQR)]: age:39(36-42)years, BMI:28.51(26.1-30.1) Kg/m2, AFC:5.5(4-7), FSH:8.23(6.4-12.5) mIU/mL, LH 6.54(4.51-8.91) mIU/mL, Estradiol: 41.18(28.47-56.44)pg/mL, AMH-Elecsys: 0.64(0.29-0.81)ng/mL, AL-196:0.64(0.39-0.99)ng/mL, AL-124:0.79(0.49-1.24ng/mL, AL-105: 0.89(0.57-1.39)ng/mL, AL-133:1.06(0.63-1.59)ng/mL. Stimulation outcomes were: follicle number on the day of trigger (Fdot):5(3-7), cumulus-oocyte-complexes (COC’s):3(2-4.5) and metaphase II oocytes (MII):3(1-4). Spearman correlation was performed between AMH assays, AFC and OS outcomes. Elecsys revealed a good correlation with the other AMH assays (rs = 0.60-0.63,p&amp;lt;0.001), however, it was higher among ELISA assays (rs = 0.954-0.993,p&amp;lt;0.001). All AMH assays showed a significant positive correlation with AFC, Fdot, COC’s and MII. However, AL-196 showed the highest correlation (rs = 0.524, rs = 0.615, rs = 0.594, rs = 0.595, respectively;p&amp;lt;0.001). Different models combining one AMH assay plus AFC were created to predict COC’s and MII: AFC+AL-196 revealed the best prediction (Adjusted R2=0.474[p &amp;lt; 0.001] and 0.485[p &amp;lt; 0.001], respectively) when compared to the same AMH assay alone. AUROC analysis was performed to investigate which model predicted better &amp;lt; =3COC’s after OS. Although all models demonstrated fair values, AUROC was better for AFC+AL-196 model (AUROC:0.796), yet no significant differences were seen when compared to AUROC for AFC+AMH-Elecsys (p = 0.39). AFC and AMH were good predictors for COC’s collected [RR(95%CI):1.49(1.11-2.04),p&amp;lt;0.01; RR(95%CI):1.26(1.13-1.49),p&amp;lt;0.001, respectively). AFC+AMH-Elecsys predicted COC’s better than AFC alone (R2=0.47 vs 0.29, respectively;Performance-Score:42.56% vs 13.21%, respectively). AFC+AL-196 had the highest performance (R2=0.599;Performance-Score:90.12%). Limitations, reasons for caution Although clinical assessment was performed in the same centre following the same methodology, inter-observer variability for AFC is a limitation. Besides, fresh AMH serum samples were analysed using Elecsys assay, whereas frozen serum samples were shipped to Ansh Lab (Texas) for batched analysis. Wider implications of the findings For patients with serum AMH&amp;lt;1.1ng/mL, a wide range of oocytes might be expected after OS. Novel AMH assays, targeting a wider broad of the molecule, together with AFC, can be used for these patients to obtain a better prediction for clinical outcomes and anticipate very poor response. Trial registration number Not applicable

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

Correlation analysis for alterations of intestinal flora in hepatocellular carcinoma patients: combinatorial detection of Coriobacterium, Atopobium, Coprococcus and Veillonella dispar may be a new method for HCC diagnosis.

Introduction. Hepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world. Due to the characteristics of low early diagnosis rate, high malignancy and rapid progression, the majority of diagnosed patients are in the middle or late stage. Accumulating evidence reveals that intestinal flora imbalance will aggravate HCC by disturbing immune regulation, especially interleukin expression. Therefore, intestinal flora-based methods have the potential to be new diagnostic or therapeutic methods for HCC.Hypothesis. Compositions of intestinal florae were different between HCC patients and healthy people. Further, intestinal florae may alleviate or aggravate HCCs.Methods. To determine which intestinal florae and interleukin aggravate HCCs, we studied the differences in intestinal florae composition and interleukin (IL) indices between HCC patients and healthy people. A total of 64 HCC patients and 24 healthy people were recruited, and their fresh stool samples and serum samples were collected for 16S rRNA sequencing and metabolite index measurement.Results. Data showed that 484 operational taxonomic units (OTUs) and 476 OTUs were detected in the HCC and control groups, respectively. From the phylum level to the species level, 5, 6, 10, 15, 23 and 19 colonies showed differential abundance between the HCC group and healthy people. Moreover, interleukin-6 expression and interleukin-10 expression were significantly different between two groups. Of note, differences of Coriobacterium, Atopobium and Coprococcus at genus level and Veillonella dispar at species level in two groups were significantly related to IL-6 and IL-10.Conclusion. The abundance of intestinal florae in the HCC group was different from the control group. Additionally, combinatorial detection of Coriobacterium, Atopobium and Coprococcus at genus level and V. dispar at species level may be a new method for HCC diagnosis.

Method Comparison and Workflow Differences Using the Same Free Light Chain Assay on 2 Analyzer Platforms.

The Freelite assay (The Binding Site) is utilized to quantify serum immunoglobulin free light chains (sFLC), which is crucial for diagnosing and monitoring plasma cell dyscrasias (PCDs). Using the Freelite test, we compared methods and evaluated workflow differences across two analyzer platforms. sFLC concentrations were measured in 306 fresh serum specimens (cohort A) and 48 frozen specimens with documented sFLC >20 mg/dL (cohort B). Specimens were analyzed on the Roche cobas 8000 and Optilite analyzers using the Freelite κ and λ assays. Performance was compared using Deming regression. Workflow was compared by assessing turnaround time (TAT) and reagent usage. For cohort A specimens, Deming regression revealed a slope of 1.04 (95% CI, 0.88-1.02) and an intercept of -0.77 (95% CI, -0.57 to 1.85) for sFLCκ and a slope of 0.90 (95% CI, -0.04 to 1.83) and intercept of 1.59 (95% CI, -3.12 to 6.25) for sFLCλ. Regression of the κ/λ ratio revealed a slope of 2.44 (95% CI, 1.47-3.41) and intercept of -8.13 (95% CI, -16.82 to 0.58) with a concordance kappa of 0.80 (95% CI, 0.69-0.92). The proportion of specimens with TAT >60 min was 0.33% and 8% for the Optilite and cobas, respectively (P < 0.001). The Optilite required 49 (P < 0.001) and 12 (P = 0.016) fewer tests for sFLCκ and sFLCλ relative to the cobas. Cohort B specimens showed similar but more dramatic results. Analytical performance of the Freelite assays was comparable on the Optilite and cobas 8000 analyzers. In our study, the Optilite required less reagent, had a slightly reduced TAT, and eliminated manual dilutions for samples with sFLC concentrations >20 mg/dL.

Sustained release of GLP-1 analog from γ-PGA-PAE copolymers for management of type 2 diabetes.

GLP-1 has been clinically exploited for treating type 2 diabetes, while its short circulation half-life requires multiple daily injections to maintain effective glycemic control, thus limiting its widespread application. Here we developed a drug delivery system based on self-assembling polymer-amino acid conjugates (γ-PGA-PAE) to provide sustained release of GLP-1 analog (DLG3312). The DLG3312 loaded γ-PGA based nanoparticles (DLG3312@NPs) exhibited a spherical shape with a good monodispersity under transmission electron microscope (TEM) observation. The DLG3312 encapsulation was optimized, and the loading efficiency was as high as 78.4±2.2%. The transformation of DLG3312@NPs to network structures was observed upon treatment with the fresh serum, resulting in a sustained drug release. The in vivo long-term hypoglycemic assays indicated that DLG3312@NPs significantly reduced blood glucose and glycosylated hemoglobin level. Furthermore, DLG3312@NPs extended the efficacy of DLG3312, leading to a decrease in the dosing schedule that from once a day to once every other day. This approach combined the molecular and materials engineering strategies that offered a unique solution to maximize the availability of anti-diabetic drug and minimize its burdens to type 2 diabetic patients.

Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma

AbstractSmall extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA‐ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non‐platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo.

Germ Tube Induction Test Comparing Total of Six Liquid and Three Solid Media in Candida albicans

Invasive candidiasis (IC) has a high mortality rate of 70%, thus diagnosis should be established without delay. Given its fast result, serological test such as β-d-glucan (BDG) test is one alternative diagnosis modalities. However, it lacks specificity. Candida albicans germ tube antibody (CAGTA) test is an alternative serological test which has a high sensitivity of 76.2% and specificity of 80.3%. Manufacturing CAGTA serological test requires provision of specific germ tube antigen. In this study, various culture media were tested to find the best media for germ tube induction. This study was an experimental in vitro study. The number and length of the germ tube were recorded in two- and three-hour incubation periods. A total of six samples containing one C. albicans ATCC 90028, four C. albicans wild type strains, and one C. krusei wild type strain were used. Nine media were tested to induce germ tube formation: human and sheep serum, fetal bovine serum, mueller hinton agar and broth, tryptic soy agar and broth, brain heart infusion agar and broth. At both incubation periods, the medium with the highest number of germ tube was human serum (p=0.001 and p=0). The longest germ tube was found in sheep serum at two-hour incubation period (p=0.005). Mueller hinton broth (MHB) showed comparable results with human and sheep serum (p&gt;0.05). Human serum is a superior inducer of morphogenesis. However, the use of MHB is recommended in this study, since provision of fresh human and sheep serum on a regular basis is impractical.

Long-term stability of thyroid peroxidase antibody (anti-TPO) in serum in the Danish General Suburban Population Study.

We evaluated the long-term stability of thyroid peroxidase antibody (anti-TPO). In the Danish General Suburban Population Study (GESUS), serum samples were biobanked at-80°C during 2010-2013. In a paired design with 70 subjects, we compared anti-TPO (30-198U/mL) measured on fresh serum on Kryptor Classic in 2010-2011 (anti-TPOfresh) with anti-TPO remeasured on frozen serum (anti-TPOfrozen) on Kryptor Compact Plus in 2022. Both instruments used the same reagents and the anti-TPOn automated immunofluorescent assay, which was calibrated against the international standard NIBSC 66/387, based on the Time Resolved Amplified Cryptate Emission (TRACE) technology from BRAHMS. Values greater than 60U/mL are regarded as positive in Denmark with this assay. Statistical comparisons included Bland-Altman, Passing-Bablok regression, and Kappa statistic. The mean follow-up time was 11.9 years (SD: 0.43). For anti-TPOfrozen vs. anti-TPOfresh, the line of equality waswithin the confidence interval of the absolute mean difference [5.71 (-0.32; 11.7)U/mL] and the average percentage deviation [+2.22% (-3.89%; +8.34%)]. The average percentage deviation of 2.22% did not exceed analytical variability. Passing-Bablok regression revealed both a statistically significant systematic and proportional difference: Anti-TPOfrozen=-22.6+1.22*(anti-TPOfresh). Frozen samples were correctly classified as positive in 64/70 (91.4%; Kappa=71.8%). Anti-TPO serum samples in the range 30-198U/mL were stable after 12-years of storage at-80°C with an estimated nonsignificant average percentage deviation of +2.22%. This comparison is based on Kryptor Classic and Kryptor Compact Plus, which used identical assays, reagents, and calibrator, but for which the agreement in the range 30-198U/mL is unclarified.

ADAPTATION OF A POINT-OF-CARE CANINE PROGESTERONE TEST FOR USE OF PARTURITION PREDICTION IN CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS): PROOF OF CONCEPT.

In the Asian elephant (Elephas maximus), the levels of progesterone products 5α-pregnane, 3α-hydroxypregnane, and 17α-progesterone are elevated during pregnancy. Detection of a sudden decrease in blood progesterone product levels in the final days of pregnancy is considered an objective way of predicting impending parturition. Point-of-care (POC) tests eliminate the cost involved in transporting samples to an external laboratory and provide an almost instant result, facilitating decision-making for animal monitoring and management. This proof-of-concept study aims to investigate the ability of the AgPlus POC immunoassay system to measure 4-pregnen-3,20-dione in pregnant elephant serum samples and adapt the method for detection of the preparturient progesterone decrease. Frozen serum samples of two pregnant elephants (N = 82) and fresh serum samples of one pregnant elephant (N = 10) were analyzed using both the POC method and a radioimmunoassay in a reference laboratory. Statistical analysis of the data showed that there was no significant difference between the two methods for detection of the progesterone drop, indicating that the POC method can be considered appropriate for use in elephant parturition prediction. Refinement of the methodology, an increase of sample size, and temporal tandem radioimmunoassay would be required to further validate this method for use in elephant reproductive management.