Polyethylene Glycol Precipitation Research Articles

Macroprolactinemia in patients with hyperprolactinemia: an experience from a single tertiary center.

Macroprolactinemia frequently causes misdiagnosis, unnecessary investigation and inappropriate treatment in hyperprolactinemic patients. Aim of this study is to investigate the prevalence and clinical characteristics of Thai patients with macroprolactinemia. We performed a cross-sectional study in 56 hyperprolactinemic patients (51 women and 5 men) whose sera were subsequently tested for the presence of macroprolactin. Recovery of less than 40% of serum prolactin after polyethylene glycol (PEG) precipitation was indicative of macroprolactinemia. Our study revealed 19.64% (11/56) of patients with hyperprolactinemia were found to have a preponderance of macroprolactin. All patients with macroprolactinemia were women, of which eight of them were initially diagnosed as idiopathic hyperprolactinemia and mistreated with dopamine agonist medications. Interestingly, neuroradiological abnormalities were reported in three patients with macroprolactinemia, 2 cases with prolactinoma and one case with stalk effect hyperprolactinemia. In conclusion, nearly one-fifth of our patients with hyperprolactinemia have macroprolactinemia. This finding suggests that the diagnostic algorithm of all patients with hyperprolactinemia should include the PEG precipitation test as the initial step.Domain: Endocrinology

Optimizing Extraction of Proteins from Carcinus Maenus After Removal of Low‐Molecular Weight Chitins

Crab shells as waste products are presently of economic interest only for the value of the chitin polymer present in their shells, which may be used to formulate foods or pharmaceutical products. Other materials in the crab shells, including protein, are discarded as waste. Crab shells have a large protein content A key question to examine is if there are bioactive proteins and/or peptides that have health benefits which could be recovered and utilized as a value added fraction from the waste stream. For example proteins isolated from crab shells have been found to contain antimicrobial and moderate antitumor agents (Sawssen, 2015.)In this work, the procedures used for removal of low molecular weight (LMW) chitin were followed (Ye, 2016). After removal of LMW chitin, the acetic acid extraction of proteins from crab shells was optimized using 50%, 25% and 12% acetic acid. One of the challenges associated with the extraction of proteins from crab shells is the concomitant demineralization of the shells which results in a high calcium carbonate solution in solution. In this project, a variety of different methods for separating the protein from the acidic solution and the calcium carbonate were utilized including dialysis, polyethylene glycol precipitation strategies.These findings will be of value to the community of researchers investigating either bioactive materials in the shells of marine organisms.

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications.

Macroprolactinemia Detection by Magnetically Assisted Polyethylene Glycol Precipitation: Potential for Automation.

Macroprolactin is an immunoglobulin-prolactin complex that is not bioactive in vivo but the prolactin component remains immunoreactive. The complex is a universal source of interference in prolactin immunoassays and commonly results in misdiagnosis of hyperprolactinemia with consequent clinical mismanagement of patients. Removal of macroprolactin by precipitation with polyethylene glycol (PEG) is an effective technique for identifying such patients but unfortunately not universally employed due to the manual nature of the procedure. We developed a modified PEG precipitation technique using magnetic nanoparticles that we termed Magnetically Assisted PEG Precipitation (MAPP). This procedure was verified against an established PEG precipitation procedure. The MAPP procedure we developed was robust, reproducible, and affords the potential for automation of macroprolactin screening in clinical laboratories. Comparisons of prolactin levels obtained following MAPP in sera from patients with either true hyperprolactinemia or macroprolactinemia generated results comparable to that of conventional PEG precipitation. The MAPP technique yields results comparable to those of traditional PEG precipitation. Elimination of the need for centrifugation affords the possibility of automation and hence more widespread adoption of routine PEG screening by clinical laboratories.

Detection and Characterization of Hepatitis E Virus Genotype 3 in Wastewater and Urban Surface Waters in Germany

In highly populated areas, environmental surveillance of wastewater and surface waters is a key factor to control the circulation of viruses and risks for public health. Hepatitis E virus (HEV) genotype 3 is considered as an emerging pathogen in industrialized countries. Therefore, this study was carried out to determine the prevalence of HEV in environmental waters in urban and suburban regions in Germany. HEV was monitored in water samples using quantitative RT-PCR (RT-qPCR) and nested RT-PCR without or with virus concentration via polyethylene glycol precipitation or ultracentrifugation. By RT-qPCR, 84–100% of influent samples of wastewater treatment plants were positive for HEV RNA. Genotypes HEV-3c and 3f were identified in wastewater, with HEV-3c being the most prevalent genotype. These data correlate with subtypes identified earlier in patients from the same area. Comparison of wastewater influent and effluent samples revealed a reduction of HEV RNA of about 1 log10 during passage through wastewater treatment plants. In addition, combined sewer overflows (CSOs) after heavy rainfalls were shown to release HEV RNA into surface waters. About 75% of urban river samples taken during these CSO events were positive for HEV RNA by RT-qPCR. In contrast, under normal weather conditions, only around 30% of river samples and 15% of samples from a bathing water located at an urban river were positive for HEV. Median concentrations of HEV RNA of all tested samples at this bathing water were below the limit of detection.

Persistent elevation of aspartate aminotransferase in a child after incomplete Kawasaki disease: a case report and literature review

BackgroundInterpretation of abnormalities in liver function tests, especially in asymptomatic children, is a common problem faced by clinicians. Isolated elevation of aspartate aminotransferase may further puzzle physicians. Macro-aspartate aminotransferase (AST) results from complexes AST produces with other plasma components, such as immunoglobulin. To our knowledge, this is the first report on a case of macro-AST-associated incomplete Kawasaki disease (KD). It is to make physicians aware of this benign condition and help to prevent extensive, unnecessary investigations and invasive workups.Case presentationA 16-month old boy with a 7-day history of fever was admitted to our pediatric ward for pyrexia workup. After complete investigations, KD was confirmed by a pediatric rheumatologist. During his admission and serial follow-up tests, an isolated AST elevation was noted. Comprehensive tests were performed and using the polyethylene glycol (PEG) precipitation method, macro-AST was confirmed. The patient has been followed up for 3 years, and so far, the benign nature of this condition has been confirmed.ConclusionClinicians should consider testing for macro-AST when elevated AST is the only abnormal lab finding. Although an uncommon finding, macro-AST may be seen in both children and adults. There are many reasons for this phenomenon, including resolved acute hepatitis or in some cases, inflammatory bowel disease, hepatic malignancy, monoclonal gammapathy, celiac disease, or KD; however, it may be observed in asymptomatic healthy children as well. Using the PEG precipitation method, a definitive diagnosis can be made. In none of these conditions does macro-AST have any prognostic significance. An appreciation of macro-AST may prevent the need for more invasive investigations to which patients may be unnecessarily subjected. It is important to recognize this condition as benign and assure patients that no specific treatment is required.

Discrepancy between Cardiac Troponin Assays Due to Endogenous Antibodies

Despite well-described analytical effects of autoantibodies against cardiac troponin (cTn) I on experimental assays, no study has systematically examined their impact on cTn assays in clinical use. We determined the effects of endogenous antibodies on 5 different cTnI assays and a cTnT assay. cTn was measured by 6 methods: Siemens hs-cTnI Centaur, Siemens hs-cTnI Vista, Abbott hs-cTnI Architect, Beckman hs-cTnI Access, Beckman cTnI Access, and Roche hs-cTnT Elecsys. Measurements were repeated on 5 assays (all except Siemens hs-cTnI Vista) following immunoglobulin depletion by incubation with protein A. Low recovery of cTnI (<40%) following immunoglobulin depletion was considered positive for macro-cTnI. Protein A findings were validated by gel filtration chromatography and polyethylene glycol precipitation. In a sample of 223 specimens selected from a community laboratory that uses the Siemens hs-cTnI Centaur assay and from which cTn was requested, 76% of samples demonstrated increased cTnI (median, 88 ng/L; interquartile range, 62-204 ng/L). Macro-cTnI was observed in 123 (55%) of the 223 specimens. Comparisons of cTnI assays markedly improved once patients with macro-cTnI were removed. Passing-Bablok regression analysis between hs-cTnI assays demonstrated different slopes for patients with and without macro-cTnI. In patients with macro-cTnI, 89 (72%) showed no effect on the recovery of cTnT, whereas 34 (28%) had reduced recovery of cTnT. The proportion of results above the manufacturers' 99th percentile varied with the cTn assay and macro-cTnI status. We suggest that the observed discrepancy between hs-cTnI assays may be attributed in part to the presence of macro-cTnI.

The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production

Abstract Actinomycetes have been identified as one of the most diverse groups of microorganisms that play a vital role in the production of enzymes and nutraceuticals. In addition, prior studies on the wild type strain of Kitasatospora sp. emphasized on its ability to exhibit high β-mannanase activity. This study aimed to purify, characterize and evaluate the potential of this strain in the production of mannooligosaccharides using mannan polymer. The enzyme was produced by submerged fermentation of a medium contains locus bean gum as a carbon source. The crude mannanase was subjected to polyethylene glycol precipitation and ion exchange chromatography. The completion of the purification process was confirmed by SDS-PAGE and purified of enzyme characterization were investigated, analysis hydrolysis product was conducted by TLC. The enzymes exhibited the activity of 37.0 U/mL. A purification factor of 1.4-fold was achieved with specific activity of 6.3 U/mg. An increase of activity was recorded from 15.0 U/mL and 4.4 U/mL to 19.3 U/mL and 6.3 U/mL. In addition, the total protein decreased from 338.5 mg/mL to 45.7 mg/mL. The purified β-mannanase has the molecular weight was approximately 37.0 kDa with optimal activity at pH 6.0 and 60 °C and relatively stability at a pH variety of 6.0–9.0, retaining > 90% activity. This product was capable of hydrolysing various mannan polymers (porang potato, palm sugar fruit, coconut cake, palm cernel cake) and other commercial mannan (LBG, β-mannan, konjac, ivory nut), subsequently producing various sizes of mannoligosaccharides and mannose potential for food and feed industry.

Efficient Transduction of T-Lymphocytes by Lentiviral Particles in Oncoimmunological Studies

Aim. To compare different methods of lentivirus concentration in order to select the best way of providing high-level transduction for generating laboratory CAR-T cells. Materials &amp; Methods. Concentration of lentiviral supernatant was carried out by 4 methods: ultrafiltration, ultracentrifugation, polyethylene glycol (PEG), water-soluble non-ionic polymer, precipitation method, and ion-exchange chromatography. Functional viral titer was determined by mCherry reporter protein expression in the transduced HeLa cell line as well as by rapid immunochromatographic (IC) tests. Physical titer was determined by ELISA. Transduction efficiency of healthy donor’s T-lymphocytes was assessed by flow cytometry with respect to signal intensity of reporter protein FusionRed. Functional activity of generated anti-CD19 CAR-T was evaluated by microscopy after co-cultivation with CD19-HeLa cell line as well as subsequent cytokine testing. Results. Lentivirus purification and concentration by ultrafiltration provided the greatest number of transduced cells, i.e. 84.7 %. Methods of ultracentrifugation, PEG precipitation, and ion-exchange chromatography yielded 56.08 %, 74.22 %, and 21.05 % of T-cell transduction, respectively. Results of rapid IC tests were comparable (г = 0.91) with cell line titer data. The mean T-cell transduction efficiency was 59.55 % ± 2.94 %, and its maximum reached 76.26 %. Conclusion. The focus was laid on optimization of CAR-T cell production during the generation of lentiviral vectors and their purification. Ultrafiltration was selected as the best method of lentiviral supernatant concentration to efficiently transduce T-lymphocytes and to generate functional CAR-T cell population.

Makroamilazemija kao razlog hiperamilazemije u klinički nejasnim stanjima - prikaz slučaja

Macroamylasemia is a rare condition characterized by elevated serum amylase levels due to the existence of macromolecules - complex amylase that cannot be secreted normally by the kidneys due to their size. It is a benign condition that is usually free of marked clinical symptoms and signs. The prevalence of macroamylasemia in the population is between 1 and 2%. The main clinical significance of macromylasemia is that it is often a diagnostic problem because it requires differentiation of this condition from other causes of hyperamylasemia. Therefore, it is rational to avoid unnecessary diagnostics and treatment, which burden both the patient and the health system. Macromalasemia as a diagnosis should be considered in any patient with elevated serum amylase activity in whom serum lipase and urine amylase levels are normal. Laboratory confirmation of the diagnosis of amylasemia rests on tests: electrophoresis or polyethylene glycol precipitation test. This paper presents a patient who has been routinely treated clinically due to non-specific gastrointestinal problems. During laboratory treatment, the patient's serum was found to have elevated amylase values. Additional diagnostics did not identify organic pancreatic diseases or other diseases that may be related to hyperamylasemia, and by calculating the ratio of renal amylase clearance to creatinine clearance of less than 1%, we conclude that it is most likely a macroamylasemia. Specific treatment was not required. The patient comes for a check-up once every 6 months.

Assessment of macroprolactinemia by the polyethylene glycol precipitation method

Introduction. Macroprolactinemia is one of the common causes of hyperprolactinemia, especially in cases where standard routine commercial immunometric assays are used for prolactin level measurement. Two forms of prolactin, inactive dimeric prolactin (50 - 60 kDa) and a low-activity tetramer (molecular weight above 150 kDa), contribute to this condition. The relatively low incidence of symptoms in macroprolactinemia patients has necessitated a relatively simple method to detect large, biologically inactive prolactin molecules, such as polyethylene glycol precipitation method. The aim of this study was to compare the precipitation method using polyethylene glycol dissolved in phosphate buffered saline in relation to polyethylene glycol dissolved in water. Material and Methods. This study included 82 patients who visited the Center of Laboratory Medicine, Clinical center of Vojvodina, to determine serum prolactin levels. The obtained serum samples were divided into two aliquots. The first aliquot was frozen and stored at - 20?C and the other was immediately analyzed. Aqueous solution was added to one aliquot and polyethylene glycol Merck 6000 to the other. All serum and supernatant samples were analyzed using the automated Abbott Architect i2000sr immunoassay. Recovery values were calculated as the ratio of double values of free prolactin and total prolactin concentration, expressed as a percentage. Results. The prolactin concentrations and calculated recovery values were lowest in fresh supernatant prolactin treated with phosphate buffered saline. Statistical analysis showed no significant difference in the calculated recovery values obtained by precipitation of fresh and frozen sera using polyethylene glycol dissolved in phosphate buffered saline (p = 0.893). Conclusion. Precipitation using polyethylene glycol dissolved in phosphate buffered saline is a more reliable method for laboratory detection of macroprolactinemia compared to polyethylene glycol dissolved in water.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY)

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Different Diets Change the Expression of Bovine Serum Extracellular Vesicle-miRNAs.

Simple SummaryStudies over the last decade have shown that cells can communicate with neighboring or distant cells through complex packets stuffed with selected proteins, lipids, and nucleic acids, called extracellular vesicles. The wrapped macromolecules are miRNAs, which play a central role in mediating the signal communication of creatural patho/physiological systems. Extracellular vesicle-miRNAs vary among species and different body fluids, such as milk, urine, saliva, cerebrospinal fluid and blood, providing general and individual characters of the vesicles. Cow’s milk is significant in the supply of human nutrition. Therefore, the extracellular vesicle-related physiological process of dairy cows should be of concern. This study clarified the miRNA profiling of bovine serum and found their potential influence on immunity. Moreover, we found that different diets could affect miRNA expression. The results implied that people could implement effective dietary strategies to intervene in the physiological state of animals.Cells can communicate with neighboring or distant cells using extracellular vesicles (EVs), mainly attributed to their containing miRNAs. Given that diets can change host circulatory miRNA profiling, and EVs are the major miRNA carriers in serum, we hypothesized that different diets could change bovine circulating EV-miRNA expression. We partly replaced alfalfa hay with whole cotton seed and soybean hull in the feed formula of the tested cows. Blood EVs were isolated using a polyethylene glycol precipitation kit. Particle size analysis revealed exosomes were dominant in bovine serum EVs. Small RNAs were enriched in bovine serum EVs, including miRNAs, snRNAs, tiRNAs, Cis-regulatory elements, piRNAs, etc. In total, 359 types of Bos taurus miRNAs were identified by Solexa sequencing. Each cow in the control group contained about 244 types of serum EV-miRNAs, compared to 246 types in the tested group. There were 15 immune-related miRNAs in the top 20 serum EV-miRNAs, accounting for about 80% of the total. Seven differently expressed known miRNAs were detected in responding to different diets. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed differently expressed miRNAs were related to hormone signal pathways and protein metabolism. Bovine serum EVs are abundant with miRNAs, most of which are immune-related. Different diets eventually change the miRNA profiling of bovine serum EVs.

Detection by Flow Cytometry of Anti-DNA Autoantibodies and Circulating DNA Immune Complexes in Lupus Erythematosus

A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.

Manufacturing of plasma-derived C1-inhibitor concentrate for treatment of patients with hereditary angioedema.

Background: Replacement therapy with plasma-derived C1-inhibitor (C1-INH) has been used for decades to treat patients with hereditary angioedema (HAE) with C1-INH deficiency. Objective: This article reviewed the rationale for using C1-INH replacement therapy in patients with HAE and the process of manufacturing plasma-derived C1-INH. Methods: The manufacture of C1-INH is an involved and carefully monitored process that includes screening and selection of prospective donors, the collection of source plasma, and purification with dedicated pathogen reduction steps. Donor eligibility is determined by restrictive criteria established and monitored by regulatory agencies as well as voluntary standards implemented by plasma collection centers that exceed government regulations. Individual and pooled donations are tested for transfusion-transmissible infections, including hepatitis B virus, hepatitis C virus, human immunodeficiency virus, parvovirus B19, and hepatitis A virus, by using enzyme-linked immunosorbent assays or nucleic acid amplification technologies. Frozen plasma that is cleared for manufacturing undergoes controlled thawing and centrifugation, and the resulting supernatant (i.e., cryoprecipitate-depleted plasma) is used to manufacture several plasma-derived therapies, including C1-INH. In addition to chromatography steps, the manufacturing process consists of dedicated and effective pathogen reduction steps, including pasteurization, hydrophobic interaction chromatography or polyethylene glycol precipitation, and virus filtration. Manufacturers continuously monitor the safety profile of C1-INH products by robust pharmacovigilance processes that enable systematic collection and evaluation of all suspected adverse drug reaction reports as well as evaluation of safety information from all other sources. Results and Conclusion: These procedures used in donor screening, donation and manufacturing pool testing, manufacturing, and pharmacovigilance ensure that plasma-derived C1-INH products have the safety, quality, identity, potency, and purity that is necessary to provide the intended therapeutic effect.

Mid-manufacturing storage: Antibody stability after chromatography and precipitation based capture steps.

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, −20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re‐converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at −20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.

Macro vitamin B12: an underestimated threat.

Background The correct identification of the macro-B12 interference (macroforms) is paramount to avoid potential erroneous clinical decisions. Our objectives were to determine whether immunoassays are affected by the presence of macro-B12 and to validate a polyethylene glycol (PEG) precipitation procedure to detect it. Methods Sixty-two serum samples obtained from healthy volunteers were analyzed to determine recovery and reference intervals (RIs) following PEG precipitation. Thereafter, 50 serum samples with very high levels of B12 (>1476 pmol/L) were randomly selected to search for macro-B12 interferences. Serum samples obtained from healthy volunteers and related PEG aliquots were analyzed on a Cobas® immunoassay. Patients' samples were analyzed on both Cobas® and Architect® immunoassays. Finally, samples suspected to contain macro-B12 were analyzed by size-exclusion chromatography (SEC) to confirm the presence of macro-B12. Results Recovery and post-PEG RIs determined on a Cobas 8000® in healthy volunteers ranged from 68.3% to 108.4% and from 122.1 to 514.4 pmol/L, respectively. Fifteen samples (30%) were found to show macro-B12 while using the recovery criteria, and nine samples (18%) while using the post-PEG RI. The other immunoassay ran on the Architect i2000® was also affected by the presence of macro-B12. Size-exclusion chromatography studies confirmed the presence of macro-B12 (immunoglobulin-B12 complexes). Conclusions The prevalence of macro-B12 in elevated B12 samples is high. We suggest to systematically screen for the presence of macro-B12 with PEG precipitation procedure in samples with elevated B12 levels to avoid potential misdiagnosis or harmful clinical consequences.

Evaluation of percentage recovery together with modified reference range in hyperprolactinemia

Abstract Objective Macroprolactinemia is an important cause of hyperprolactinemia. The aim of this study was to examine the added value of the consideration of modified reference range in determination of macroprolactinemia and true hyperprolactinemia. Materials and methods Three hundred and ninety patients with high and 131 with normal prolactin (PRL) levels were included in this study. PRL had been analyzed before and after polyethylene glycol precipitation (post-PEG PRL). Recovery percentage (R%) &lt;40% and &gt;60% had been reported as macroprolactinemia and true hyperprolactinemia, respectively. Post-PEG PRL levels were evaluated according to the modified reference range obtained from those of the normoprolactinemic subjects. Results According to the R% criterion; macroprolactinemia had been detected in 24.9% and true hyperprolactinemia in 67.4% of hyperprolactinemic patients. When the data were evaluated considering the post-PEG PRL levels according to the modified reference range; 13 (13.4%) of the 97 macroprolactinemia reports would be considered as true hyperprolactinemia and 6 (2.3%) of the 263 true hyperprolactinemia reports would be changed as macroprolactinemia. Conclusion Discrimination capacity of R% criterion for true hyperprolactinemia and macroprolactinemia is limited, and we suggest that, in accordance with R% criterion, laboratory reports should include the post-PEG PRL levels along with the modified reference range.

Production of adeno-associated virus vectors for in vitro and in vivo applications

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010–1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Circulating immune complexes levels correlate with the progression of canine leishmaniosis in naturally infected dogs

Dogs are the main domestic reservoir of Leishmania infantum, and in cases of uncontrolled infection, a strong humoral immune response is elicited, which is inefficient against the parasites. Previous studies have suggested that an adequate antigen/antibody ratio, with a moderate prevalence of antigens with respect to the antibodies, could result in the formation of circulating immune complexes (CIC) in canine leishmaniosis (CanL). Deposition of these complexes in tissues has been associated with vasculitis, uveitis, arthritis, dermatitis and especially glomerulonephritis and renal failure. However, little is known about the relationship between the presence of CIC and disease progression. The aim of this study was to evaluate serum CIC level and its correlation with disease severity in infected dogs with different stages of disease and non-infected animals as a control. A total of 60 dogs were included in the study, classified according to the proposed LeishVet classification criteria: healthy non-infected (n = 13); healthy infected (n = 12); sick stage I (n = 9); sick stage II (n = 17); sick stage III (n = 8); and sick stage IV (n = 1). CIC were isolated from serum samples using a modified polyethylene glycol precipitation method, and their levels measured by ELISA and bicinchoninic acid protein assay. A nanoparticle tracking analysis was performed to investigate the relationship between the molecular size distribution of the CIC and disease progression. In conclusion, the results confirmed a positive association between CIC levels, their molecular size and disease progression that suggests a potential use of CIC as biomarkers of CanL.