Polyethylene Glycol Precipitation Research Articles

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids

BackgroundInduced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.MethodsDifferentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.ResultsWe observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.ConclusionOur data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Macroprolactin over time: Is there any point in rechecking it in people with a persistently elevated serum prolactin?

Macroprolactinemia may influence the interpretation of serum prolactin levels-a recognised phenomenon since 1981. The degree of macroprolactinaemia over time is less well described. We determined how macroprolactin status (based on polyethylene glycol (PEG) precipitation) varied by analysing serial measurements in hyperprolactinaemic individuals over a period of 9 years. Results from 1810 individuals were included. All serum total prolactin results (measured using Roche Cobas 8000 analyser) were extracted from the laboratory information system for the period 1 January 2012 to 1 April 2021, along with relevant patient demographic/test data. Samples with a macroprolactin screening test performed (on samples with prolactin > 700 miu/L) were included in the main analysis. During the study period, 2782 macroprolactin checks were performed (12.5% of all prolactin tests) in 1810 individuals (599 males/2183 females, median-age: 35,interquartilerange: 25-47, range: 16-93 years). Multiple macroprolactin checks were carried out on 465 patients (1437 measurements) with 94 patients (141 measurements) screening positive (<60% recovery). Only 19 patients (18 female) had at least one result above and one below the 60% screening cut-off, with 10 of these patients having results close to the 60% cut-off; in 9 patients, results were clearly different between repeat samples. In seven cases, the adjusted monomeric prolactin showed a potentially clinically significant difference. In this study, only 19/465 patients appeared to change macroprolactin status based on a 60% PEG recovery cut-off. The majority of these 19 patients were on antipsychotic/antidepressant medication(s) or had a prolactinoma; in only 7did monomeric prolactin change significantly. This suggests that once macroprolactin status has been determined, clinical decision making is rarely affected by repeating it.

Anti-HBsAg IgY polyclonal antibodies potential as capture antibody for HBsAg Detection Kit development

Hepatitis B, affecting about 296 million globally, is a significant concern, with Indonesia ranking second in Southeast Asia for case numbers. The disease's latent initial phase, devoid of early symptoms upon hepatitis B virus (HBV) infection, highlights the demand for precise diagnostics. This research aimed to develop anti-HBsAg polyclonal antibodies (pAb) for application as capture agents within a sandwich enzyme-linked immunosorbent assay (ELISA). Chicken egg-derived IgY antibodies have advantages over mammalian ones due to simpler extraction and higher yield. In a study involving 21-week-old chickens, four intramuscular injections of 500 µg HBsAg antigen in Freund's adjuvant were administered at two-week intervals. Eggs as IgY source were collected daily and then IgY was isolated from eggyolk using polyethylene glycol (PEG) precipitation. The Bradford method was used to measure the total protein concentration, while the existence of IgY and pAb specific IgY against Anti-HBsAg was verified through SDS-PAGE and sandwich ELISA using HRP as a reporter, respectively. The resulting SDS-PAGE showed two distinct IgY bands: a 68 kDa heavy chain and a 23 kDa light chain. Using these anti-HBsAg IgY antibodies as capturing agents, the slightly elevation of IgY pAb against HBsAg level has been identified within the second week following the initial immunization. Subsequently, from the third to the eighth week, antibody levels escalated significantly, ranging from 2 to 13-fold higher than those observed in the second week. These findings suggest the potential use of IgY pAb as effective capture antibodies in sandwich ELISA for HBsAg antigen detection.

Concentration strategies for spiked and naturally present biomarkers in non-invasively collected first-void urine

BackgroundFirst-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation.MethodsFVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured.ResultsThe findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed.ConclusionsWith this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.

Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

The cases of tracing the source of patients infected with Omicron variant of SARS-CoV-2 based on wastewater-based epidemiology in Shenzhen

Wastewater-based epidemiology (WBE) is an emerging discipline, which has been applied to drug abuse tracking and infectious disease pathogen surveillance. During the COVID-19 epidemic, WBE has been applied to monitor the epidemic trend and SARS-CoV-2 variants etc. In order to detect hidden COVID-19 cases and prevent transmission in the community, wastewater surveillance system for monitoring SARS-CoV-2 RNA was developed in Shenzhen. The sewage sampling sites were set up in key places such as the port areas, urban villages and residential communities of Futian, Nanshan, Luohu and Yantian districts. From July 26 to November 30, 2022, a total of 369 sewage sampling sites were set up, covering 1.93 million people. Continuous sampling was carried out for 3 hours in the peak period of water use every day. Sewage virus enrichment and SARS-CoV-2 nucleic acid detection were carried out by polyethylene glycol precipitation method and RT-qPCR, and a positive water sample disposal process was molded. This article aims to introduce the case of source tracing of COVID-19 infected patients based on urban sewage in Shenzhen. The sewage monitoring of Honghu water treatment plant in Luohu District played an early warning role, and the source of infection was traced. In the disposal of positive water samples in Futian South Road, Futian District, the important experience of monitoring point layout was obtained. In the sewage monitoring of Nanshan village, Nanshan District, the existence of occult infection was revealed. Sharing the experience of tracing the source of COVID-19 patients to avoid the spread of COVID-19 in the community based on wastewater surveillance of SARS-CoV-2 RNA in Shenzhen, and summarizing the advantages and application prospects of sewage surveillance can provide new ideas for monitoring emerging or re-emerging pathogens that are known to exhibit gastrointestinal excretion in the future.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.

Heterophilic Interference of Rheumatoid Factor in TSH Immunometric Assay: A Cross-Sectional Observational Study.

Considering the inherent vulnerability of immunoassays for heterophilic interference and the potential of Rheumatoid Factor (RF) to act as a heterophile-like antibody, we conducted this study to investigate if RF leads to any such heterophilic interference in seropositive rheumatoid arthritis (RA) patients. The study was done on the TSH assay as it is a noncompetitive, double antibody sandwich assay, which is known to be vulnerable to heterophilic interference. In this cross-sectional observational study, eighty-four consecutive newly diagnosed RF-positive RA patients underwent TSH, Free T4, and anti-TPO estimation using the chemiluminescence technique (CLIA) on Siemens Immulite 1000 platform. The samples were screened for TSH interference using four methods: 1) analysis on a different platform, 2) assessment of linearity using doubling dilutions, 3) polyethylene glycol (PEG) precipitation, and 4) addition of a commercial blocker. Ten samples had a loss of linearity on serial dilution, indicating potential interference. After heterophile blocker treatment, five cases exhibited interference. One patient had diagnostic interpretation discordance on the second platform. No sample on PEG precipitation suggested the influence of antibodies. It is worth noting that even in cases where interference was suspected, the clinical interpretation was largely unaffected by the correction of TSH values based on mean dilution or measurement after heterophile blocker treatment. RF can cause heterophilic interference in TSH immunoassays used commercially. However, in most cases, this interference does not affect clinical decision-making.

Isolation of Phage WO Particles from Wolbachia-Infected Arthropods.

Nearly all arthropod-associated Wolbachia contain intact and/or genomic remnants of phage WO, temperate bacteriophages that facilitate horizontal gene transfer, genomic rearrangement of the bacterial chromosome, and symbiotic interactions between Wolbachia and their arthropod hosts. Integrated prophage WO genomes produce active, lytic particles; but the lack of a cell-free culturing system for Wolbachia render them difficult to purify and study. This chapter describes polyethylene glycol (PEG) precipitation of phage particles from Wolbachia-infected arthropods, followed by confirmation of phage WO isolation and purification using electron microscopy and PCR.

From stable to stable: how does stress influence the first line of immune defense in sport horses

Stressful factors, including physical exercise, can influence the body’s defense mechanisms and can be involved in the etiology and evolution of systemic diseases. The aim of this study was to investigate the influence of various stressors to which sport horses are commonly subjected on the innate immune system. For this, four experimental protocols were designed including different animals, namely: (a) resting horses subjected to everyday medium level exercise (n = 18, average age = 7 years, range: 6 to 11 years), (b) medium course transportation, where the distance of one-way trips represented 270 km (n = 8, average age = 7 years, range: 6 to 9 years), (c) a long course, with a 561 km one-way distance (n = 5, average age = 8.2, range: 7 to 11 years) and (d) pre- and post-competition investigations (n = 9, average age = 7 years, range: 6 to 11). Blood was sampled on heparine (50 UI/ml) before and after the workout or transportation. The phagocytic activity of leukocytes was measured by a carbon particle inclusion test, along with total immunoglobulin/opsonin levels (Ig) (24% Zn sulphate precipitation test) and circulating immune complexes (4.2% polyethylene glycol precipitation test). The results were statistically interpreted by use of Micrososft Excel program (Student’s t test). The various types of stress differently influenced the equine athletes. While a moderate daily exercise did not substantially influence the humoral and cellular effectors of innate immunity, transportation exerted stronger beneficial effects based on the distance to the competition site. As a fast acting antimicrobial mechanism, the phagocytosis underwent the most pronounced changes, positively influenced mainly by the short course transportation. While the transportation stress enhanced phagocytosis, the competition decreased the potential of the cells to react, without significantly influencing their numbers. The experiment suggested that management measures, such as age-tailored exercise and maternal care and diet could help in alleviating stress from stable to stable, during the competition season, thus improving animal welfare and allowing a better health forecast.

HUMORAL IMMUNITY IN PATIENTS WITH MULTIPLE SCLEROSIS

Introduction. More than 1 million people in the world suffer from multiple sclerosis (MS). The etiology of MS remains not fully understood. The main role in the pathogenesis of MS belongs to the combination of chronic inflammation and neurodegenerative processes, in the genesis of which autoimmune processes play a significant role. Progress in studying the mechanisms of the pathological process of demyelinating diseases has been achieved mainly thanks to immunological research. One of the manifestations of activation of humoral immunity in MS is increased synthesis of immunoglobulins in brain tissue and cerebrospinal fluid&#x0D; The aim of the study. To study the state of humoral immunity in multiple sclerosis patients with different types of disease course&#x0D; Research methods. 68 patients with a verified diagnosis of multiple sclerosis (MS) based on the MacDonald et al criteria aged 19 to 65 years (37 women, 29 men) were examined. The primary progressive type of the course was established in 19 patients, the secondary-progressive - in 3, the remitting-relapsing type of the course - in 26 patients. The control group consisted of 80 practically healthy male volunteers aged 25-45 years.&#x0D; The concentration of immunoglobulins A, G, M in blood serum was determined by the Mancini method (1965). The content of circulating immune complexes (CIC) in blood serum was determined by the method of precipitation in polyethylene glycol.&#x0D; Results and discussion. The highest content of Ig A was observed in the group of patients with primary progressive form of MS, which was 3.7 times higher than normal values and 1.2 times higher than in the general group of MS patients. In the group of patients with the secondary progressive form of MS, the content of Ig A was 3.1 times higher than in the control group and 1.2 times lower than in the group with the primary progressive form. In the group of patients with relapsing-remitting form of MS, there was an increase in the level of Ig A three times relative to the normal indicator, and a decrease (by 1.2 times) relative to the level in the group with a primary progressive form of MS.&#x0D; The content of Ig M in the blood serum of patients with MS exceeded the norm by 5 times. In the group with the primary progressive form of MS, the level of Ig M was 3.5 times higher than the level in the control group and 1.5 times lower than the level in the group of patients with MS. In patients with secondary progressive form of MS, the content of Ig M was 2.6 times higher than the norm, but 1.3 times lower than the level in the group with primary progressive form of MS and 1.9 times lower than in the group of patients with MS. In patients with the relapsing-remitting form of MS, the level of Ig M was: 4.6 times higher than the level in the control group and 1.7 times higher than the content in patients with the secondary-progressive form of MS, and also 1.3 times higher, than in the group with the primary progressive form.&#x0D; In all groups of patients with MS with different types of the course of the disease, the most pronounced increase in the level of Ig G was noted.&#x0D; The highest level of CIC was recorded in the general group of patients with MS and in the group with secondary progressive form of MS.&#x0D; Conclusions. An imbalance of the humoral link of immunity was observed in patients with multiple sclerosis. A probable increase in the concentration of Ig A in the blood serum of patients with multiple sclerosis indicates the presence of infection of the mucous membranes, which is especially pronounced in patients with the primary progressive form of multiple sclerosis. A significant increase in the content of Ig G in the blood serum of patients with multiple sclerosis indicates a chronic course of the process, which is especially evident in the relapsing-relapsing form of multiple sclerosis. An increased concentration of IgM in the blood serum of patients with multiple sclerosis may indicate an exacerbation of the course of chronic inflammation, especially in the case of relapsing-remitting multiple sclerosis. The detected fluctuations of the average content of CIC in the blood serum of patients with multiple sclerosis did not go beyond the physiological norm.

EcoHIV-Infected Mice Show No Signs of Platelet Activation.

Platelets express several surface receptors that could interact with different viruses. To understand the mechanisms of HIV-1's interaction with platelets, we chose the EcoHIV model. While EcoHIV is an established model for neuroAIDS, its effects on platelets are ill-defined. Our results indicate that EcoHIV behaves differently from HIV-1 and is cleared from circulation after 48 h post-infection. The EcoHIV course of infection resembles an HIV-1 infection under the effects of combined antiretroviral therapy (cART) since infected mice stayed immunocompetent and the virus was readily detected in the spleen. EcoHIV-infected mice failed to become thrombocytopenic and showed no signs of platelet activation. One explanation is that mouse platelets lack the EcoHIV receptor, murine Cationic Amino acid Transporter-1 (mCAT-1). No mCAT-1 was detected on their surface, nor was any mCAT-1 mRNA detected. Thus, mouse platelets would not bind or become activated by EcoHIV. However, impure virus preparations, generated by Polyethylene Glycol (PEG) precipitation, do activate platelets, suggesting that nonspecific PEG-precipitates may contain other platelet activators (e.g., histones and cell debris). Our data do not support the concept that platelets, through general surface proteins such as DC-SIGN or CLEC-2, have a wide recognition for different viruses and suggest that direct platelet/pathogen interactions are receptor/ligand specific.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Reporting of Hyperprolactinaemia Post-polyethylene Glycol (PEG) Precipitation at Hospital Tengku Ampuan Rahimah Klang, Malaysia

Introduction: Macroprolactinaemia is defined as hyperprolactinaemia due to excess macroprolactin in the presence of normal monomeric prolactin. Failure to identify macroprolactinaemia may result in patients being subjected to unnecessary investigations and inappropriate treatment for hyperprolactinaemia. In our centre, screening for macroprolactinaemia is currently performed at the request of the treating physician. The study thus aimed to determine the frequency of macroprolactinaemia in samples with serum prolactin ≥700 mIU/L in Hospital Tengku Ampuan Rahimah (HTAR) and to determine the presence of true hyperprolactinaemia in these cases. Methods: A cross-sectional study among hyperprolactinaemic subjects in HTAR, using serum specimens received by the laboratory for measurement of prolactin between October 2018 and September 2019. Samples with prolactin ≥700 mIU/L were screened for macroprolactinaemia using the polyethylene glycol (PEG) precipitation technique. Macroprolactinaemia was present when the percentage recovery of prolactin post-PEG was &lt;40%. Assay-specific post-PEG monomeric prolactin levels were also reported, with levels above the upper limit of reference intervals indicated the presence of true hyperprolactinaemia. Results: A total of 101 samples were subjected to PEG precipitation. Macroprolactinaemia was found in four (4%) samples, whilst eight (7.9%) were categorised as indeterminate (percentage recovery of 40-60%). The remaining 89 (88.1%) samples had a percentage recovery &gt;60%, hence considered negative for macroprolactinaemia. All four samples with macroprolactinaemia also had raised monomeric prolactin levels indicating the co-existence of macroprolactinaemia in subjects with true hyperprolactinaemia. Similarly, in the indeterminate group, all eight had raised monomeric prolactin levels. Conclusion: In one-year period in HTAR, macroprolactinaemia was detected in 4% of subjects with prolactin ≥700 mIU/L, all of whom also had raised monomeric prolactin levels. The common occurrence of both macroprolactinaemia and true hyperprolactinaemia warrants the reporting of both percentage recovery and monomeric prolactin levels post-PEG.

Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serum.

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.

Prolactin Monomeric Polyethylene Glycol Measurement Method and Study of Reference Value Verification

Purpose: Prolactin in the blood is separated into three types, and over 90% of prolactin presents as a double monomer (23 KDa). Rarely, it can exist in the size of big prolactin (150 KDa), which is called macroprolactin and is known as an autoantibody complex. When macroprolactin accounts for more than 60% of prolactin in the blood, it is called macroprolactinemia. The presence of such macroprolactin was first reported in a patient with hyperprolactinemia but without typical symptoms. Macroprolactinemia is emerging as an important cause of idiopathic hyperprolactinemia. The polyethylene glycol (PEG) precipitation method using the property of precipitating large-molecular-weight proteins is simple and recently has been widely used as a screening test. The results are in good agreement with the results of gel chromatography. The purpose of this study was to confirm the measurement method and reference value verification of monomeric prolactin in blood prolactin using the PEG precipitation method. Materials and Methods: For 40 examinees who visited the Gangnam Center of Seoul National University Hospital in 2021, the prolactin level was verified using radioimmunoassay (RIA). For macroprolactinemia PEG precipitation method, 25% PEG (molecular weight 6000kDa) solution and serum were mixed in equal amounts in a test tube, then left at room temperature for 20 minutes and centrifuged at 4℃ for 30 minutes (1500g). The prolactin level was measured in the supernatant. Results : After confirming that more than 90% of the 40 tested samples within the reference range &lt;25 ng/mL, the same value as the reference value for prolactin was applied. Since the concentration of monomeric prolactin in serum from which macroprolactin has been removed from blood is diluted 1:1 with PEG, our laboratory is currently reporting the result by multiplying the result by a dilution factor of 2. Conclusion: Radioimmunoassay using PEG precipitation method using the property of precipitating large molecular weight proteins is simple and effective for quantitative measurement of monomeric prolactin in blood prolactin.

Macro-Vitamin B12 as Cause of Falsely Elevated Cobalamin Levels.

High blood concentrations of vitamin B12 are often caused by over-supplementation. However, there are instances in which augmented vitamin B12 levels are seen in the absence of supplements. Macro-vitamin B12 is an underrated cause of supra-physiological cobalamin plasma levels. A 70-year-old man was referred to an ambulatory internal medicine centre because of high vitamin B12 levels yet he denied taking supplements. An X-ray showed a tumour in the right upper lobe of the lung, which triggered further examinations. An MRI scan of the brain came back normal as well as a CT scan of the abdomen, and colonoscopy. The pulmonologist requested a PET-CT scan, which showed an isolated 18-FDG uptake in the area of the lung mass that was detected earlier. The patient underwent surgery with adjuvant cis-platinum and gemcitabine and is still making good progress. The vitamin B12 levels persisted after successful treatment of lung adenocarcinoma; determination of vitamin B12 after PEG (polyethylene glycol) precipitation showed normal concentrations. A high vitamin B12 plasma concentration in the absence of vitamin supplementation can be a daunting diagnostic problem for the internist, as there are several possible underlying causes. In this case the diagnosis of lung carcinoma was made, the patient was treated appropriately, yet this pathology had no correlation with the cobalamin levels. A high vitamin B12 concentration can be the impetus of thorough medical inquiries. Internists should be careful not to forget macro-vitamin B12 as a possible source of falsely elevated vitamin B12 values. When encountering an otherwise unexplainable B12 hypervitaminosis, the diagnosis of macro-vitamin B12 should be taken into account to avoid unnecessary extensive medical examinations.PEG precipitation can distinguish between a 'real' high vitamin B12 and macro-vitamin B12.A high vitamin B12 concentration is no guarantee for adequate cobalamin storage. In case of suspicion, a vitamin B12 measurement after PEG precipitation should be considered.

Effect of wastewater collection and concentration methods on assessment of viral diversity

Monitoring of potentially pathogenic human viruses in wastewater is of crucial importance to understand disease trends in communities, predict potential outbreaks, and boost preparedness and response by public health departments. High throughput metagenomic sequencing opens an opportunity to expand the capabilities of wastewater surveillance. However, there are major bottlenecks in the metagenomic enabled wastewater surveillance, including the complexities in selecting appropriate sampling and concentration/virus enrichment methods as well as in bioinformatic analysis of complex samples with low human virus concentrations. To evaluate the abilities of two commonly used sampling and concentration methods in virus identification, virus communities concentrated with Virus Adsorption-Elution (VIRADEL) and PolyEthylene Glycol (PEG) precipitation were compared for three interceptor sites. Results indicated that more viral reads were obtained by the VIRADEL concentration method, with 2.84 ± 0.57 % viral reads in the sample. For samples concentrated with PEG, the average proportion of viral reads in the sample was 0.63 ± 0.19 %. In all wastewater samples, bacteriophage affiliated with the families Siphoviridae, Myoviridae and Podoviridae were found to be the abundant populations. Comparison against a custom Swiss-Prot human virus database indicated that the relatively abundant human viruses (average proportions in human virus community greater than 1.00 %) in samples concentrated with the VIRADEL method were Orthopoxvirus, Rhadinovirus, Parapoxvirus, Varicellovirus, Hepatovirus, Simplexvirus, Molluscipoxvirus, Parechovirus, Lymphocryptovirus, and Spumavirus. In samples concentrated with the PEG method, fewer human viruses were found to be relatively abundant. These were Orthopoxvirus, Rhadinovirus, Varicellovirus, Simplexvirus, Molluscipoxvirus, Lymphocryptovirus, and Betacoronavirus. Contigs of Betacoronavirus, which contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were identified in VIRADEL and PEG samples. Our study demonstrates the feasibility of using metagenomics in wastewater surveillance as a first screening tool and the need for selecting the appropriate virus concentration methods and optimizing bioinformatic approaches in analyzing metagenomic data of wastewater samples.

Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

SAT616 Differentiating Resistant Prolactinoma From Macroprolactinemia

Abstract Disclosure: S. Lacount: None. P. Belany: None. Introduction: Prolactinomas are the most common secretory tumor of the pituitary gland. Hyperprolactinemia can present as hypogonadism, infertility, galactorrhea, or it may remain asymptomatic. In asymptomatic hyperprolactinemia, macroprolactinemia and other secondary causes should be considered and ruled out. Prolactinomas treated with dopamine agonists typically respond well, with decreasing tumor size and prolactin level reduction. We present a case of a prolactinoma not responding biochemically or anatomically to maximum doses of dopamine agonists, concerning for resistant prolactinoma versus macroprolactinemia. Clinical Case: A 62 y.o. male with PMH of HTN, HLD and CAD who presented with fatigue and headaches in 2009. Initial work-up showed an elevated prolactin level to 178 ng/mL (reference range 2.0-18.0 ng/mL). MRI pituitary showed a 1.8 x 1.9 x 1.5 cm mass but without optic chiasmal compression. Patient refused medications and opted to pursue endoscopic endonasal approach (EEA), which was completed in March 2009. Pathology and hormonal immunostains confirmed prolactinoma. Due to persistently elevated prolactin levels following EEA, cabergoline was started. Follow-up MRI Pituitary in November 2012 showed stable heterogeneous residual tissue without tumor recurrence. Despite escalating cabergoline doses (up to 8 mg weekly), his prolactin levels remained slightly elevated (50 ng/mL) after surgery with unchanged symptoms from presentation. Due to the association of cabergoline and cardiac valve disease development, his dose was decreased back down to 0.5 mg twice weekly. Given his persistent asymptomatic hyperprolactinemia that was nonresponsive to high-dose cabergoline, there was consideration for macroprolactinemia causing a falsely high level. Screening polyethylene glycol (PEG) precipitation test in November 2022 was normal. MRI pituitary in December 2022 showed a progressive sellar lesion with increased size and cavernous sinus invasion near the carotid artery. Patient was referred to neurosurgery in January 2023 for further evaluation of potential resistant prolactinoma. Due to the MRI findings and persistently elevated prolactin levels, he will undergo repeat EEA in May 2023 for treatment of resistant prolactinoma. Conclusion: Dopamine agonists are very effective in treating prolactinomas. In asymptomatic hyperprolactinemia, PEG precipitation is an inexpensive test to screen for macroprolactinemia. Once macroprolactinemia and other secondary causes are ruled out, resistant prolactinoma can be considered. Resistant prolactinoma is defined by failure to achieve normoprolactinemia and at least 50% reduction in tumor size on maximum-tolerated therapy. Risk factors for resistant prolactinoma include larger tumor volume and male gender. Treatment options include different or increased dopamine agonist doses, radiotherapy and resection. Presentation: Saturday, June 17, 2023