Polyethylene Glycol Precipitation Research Articles

Detection of SARS-CoV-2 in wastewater as an earlier predictor of COVID-19 epidemic peaks in Venezuela

Wastewater-based epidemiological surveillance has proven to be a useful and cost-effective tool for detecting COVID-19 outbreaks. Here, our objective was to evaluate its potential as an early warning system in Venezuela by detecting SARS-CoV-2 RNA in wastewater and its correlation with reported cases of COVID-19. Viral RNA was concentrated from wastewater collected at various sites in Caracas (northern Venezuela), from September 2021 to July 2023, using the polyethylene glycol (PEG) precipitation method. Viral quantification was performed by RT-qPCR targeting the N1 and ORF1ab genes. A significant association (p < 0.05) was found between viral load in wastewater and reported cases of COVID-19 up to six days after sampling. During the whole study, two populated areas of the city were persistent hotspots of viral infection. The L452R mutation, suggestive of the presence of the Delta variant, was identified in the only sample where a complete genomic sequence could be obtained. Significant differences (p < 0.05) between the physicochemical conditions of the wastewater samples positive and negative for the virus were found. Our results support proof of concept that wastewater surveillance can serve as an early warning system for SARS-CoV-2 outbreaks, complementing public health surveillance in those regions where COVID-19 is currently underreported.

Enhancing Gene Delivery in NB-4 Cells: Overcoming Transduction and Selection Challenges

Efficient gene transduction and cell viability are critical factors in genetic manipulation for research and therapeutic purposes. In this study, we explored the challenges associated with transducing the NB-4 cell line, a well-established model for acute promyelocytic leukemia (APL), using lentiviral vectors. While the initial transduction efficiency in NB-4 cells reached approximately 30%, we observed a significant decrease in cell viability, a phenomenon not observed in other acute leukemia cell lines such as THP-1 cells. We identified that this toxicity could be mitigated by purifying viral particles through ultracentrifugation or polyethylene glycol (PEG) precipitation, indicating that toxic substances, potentially secondary metabolites released by HEK293, could be responsible for the cell death. Nevertheless, cell selection by puromycin was still ineffective; crucially, we discovered that the human phosphoglycerate kinase (hPGK) promoter, commonly used in the PLKO1 vector, may become silenced in NB-4 cells, preventing effective selection with puromycin. By replacing the hPGK promoter with the elongation factor-1 alpha (EF1α) promoter, we successfully achieved high transduction efficiency and robust selection, demonstrating the potential for this modified vector system to facilitate genetic studies in APL models. These findings provide important insights into optimizing gene transduction protocols not only for NB-4 cells but also for other challenging cell lines, offering a refined approach for gene delivery and selection in cell models.

Comparative Efficiency of Polyethylene Glycol, Ammonium Sulphate, Methanol Precipitation, and Ultrafiltration Techniques for the Down Streaming of Viral Antigen

Background: Foot and mouth disease (FMD) is ubiquitous worldwide but endemic in many countries of Africa, Asia, South America, and the Middle East. Many reasons contribute to the incidence of viral diseases even in vaccinated animals. These reasons include low antigenic payload, low PD50, improper formulation, unstable vaccine containing antigen, and genetically different from field strain. Among these, the most important one is the low antigenic load per dose of the vaccine. Vaccine failure is mainly due to the direct use of virus suspension in the vaccine without the concentration of viral antigen. Another reason to concentrate the antigen is small volume storage in the vaccine bank. These issues are mostly concerned with developing countries like Pakistan which lack antigen concentration technology. The concentration of the virus is a major milestone to be achieved for the production of an effective vaccine as well as for the diagnostic tool.Methods: Different techniques including precipitation with polyethylene glycol, ammonium sulfate, methanol, and filtration through an ultra-filter membrane were used for the concentration of viral suspension. Antigen quantification in terms of µg/ml was determined through size exclusion chromatography by using Sephacryl S-300 as a stationary phase.Results: Percentage recovery of FMDV calculated through analysis of chromatograms found 77.80%, 59.75%, 32.50%, and 13.83% for polyethylene glycol, ammonium sulfate, ultra-filtration, and methanol treated samples respectively.Conclusion: Classical polyethylene glycol precipitation showed a maximum percentage recovery of foot and mouth disease virus as compared to other concentration methods.Keywords: Foot and mouth disease virus (FMDV); Concentration Methods; Polyethylene Glycol (PEG); Size Exclusion Chromatography (SEC); Sephacryl S-300

A curious case of a negative control line on a dengue duo assay due to interference - A case report.

An elderly patient who presented with 1week of fever and respiratory symptoms was tested for dengue infection with an Abbott Bioline™ Dengue Duo immunochromatographic assay. Unexpectedly, the control line of the dengue non-structural protein 1 (NS1) component was absent, necessitating result invalidation. It remained absent when the test was repeated on the SD Biosensor Standard™ Q Dengue Duo, but present on the Wells Bio careUSTM Dengue Combo and Asan Easy Test® Dengue DUO assays, suggesting potential interference. Dilution, polyethylene glycol (PEG) precipitation and centrifugation with a 100kDa filter were performed to reduce/remove the potential interferent. Sera from other patients that showed a control line, and a test line that was either positive or negative for NS1, were used as controls. Upon dilution with negative control serum, a faint control line emerged. PEG precipitation resulted in disappearance of control and test lines in the positive control. Filtration led to emergence of the control line for the patient's serum but caused the test line for the positive control serum to disappear. Overall, investigations suggested the presence of a high molecular weight (>100kDa) substance which interferes with chicken IgY-anti-chicken IgY binding at the control line of affected assays. Our results highlight two important points: firstly, some commonly used laboratory procedures (e.g. PEG or filtration) may inadvertently remove the target biomarker (e.g. multimeric NS1) and should be interpreted with appropriate controls. Secondly, alternative kits that use a different antigen-antibody combination for the control line can be considered when similar patients are encountered in future.

A-071 Macroprolactin Screening in Patients with Hyperprolactinemia: Comparison Between Different Criteria for Defining Macroprolactinemia

Abstract Background Prolactin (PRL) exists in different forms in human serum. The predominant form is monomeric PRL, the other forms include dimeric PRL and polymeric PRL, also known as macroprolactin, a biologically inactive form. Macroprolactin can be measured by almost all immunoassays for PRL. This may lead to misdiagnosis and mismanagement of patients. Polyethylene glycol (PEG) precipitation is the most widely used method for detecting macroprolactin, generally employing a cutoff of 40% to distinguish macroprolactinemia from true hyperprolactinemia. This approach lacks specificity, so the reported result may be misinterpreted in cases in which excess macroprolactin occurs simultaneously with supraphysiological concentrations of monomeric prolactin. Nowadays, it is recommended to report results as post-PEG PRL using method-specific post-PEG reference intervals, classifying as macroprolactinemia (false or pseudohyperprolactinemia) those individuals with post- PEG PRL within post-PEG reference intervals and true hyperprolactinemia above the upper limit of the post-PEG reference interval. The objective of this study was to evaluate the prevalence of macroprolactinemia in patients with hyperprolactinemia using two different criteria, a cutoff &amp;lt; 40% in PEG precipitation and a post- PEG PRL within post-PEG assay specific reference range. Methods We analyzed blood samples requesting serum macroprolactin from patients admitted to a private reference clinical laboratory in Rio de Janeiro, Brazil, from 01/01/2019 to 12/31/2023. Anonymized data on laboratory tests was available from a database of the local Laboratory Information System. All patients included had PRL levels above the reference range, males &amp;gt;15.2 ng/mL, females &amp;gt;23.3 ng/mL (ECLIA, Modular, Roche). Results 9423 patients, 80.1% women (W) were evaluated. Men (M) were statistically significantly older than women (p&amp;lt;0.001) (45.6; SD=15.0 vs. 38.9; SD=11.5). Prolactin median levels were significantly higher in women (p&amp;lt;0.001) than men (37.4ng/mL, IQR=26 vs. 23.8ng/mL, IQR=17.1). Macroprolactinemia detection was greater (p&amp;lt;0.001) by post-peg PRL concentration criteria (9.7% - 10.1%W vs. 8.4%M), than by &amp;lt; 40% PRL recovery criteria (5.5% - 5.7%W vs. 4.8%M). Dividing patients by prolactin levels (&amp;lt;50.0; 50.0 to &amp;lt;100.0; 100.0 to &amp;lt;150.0, &amp;gt;150.0 ng/mL, we found macroprolactinemia in 5.2% (5.2%W vs. 5.2%M), 7.7% (8.2%W vs. 2.4%M), 2.8% (2.8%W vs. 2.5%M) and 1.6% (1.3%W vs. 2.6%M) - &amp;lt;40% PRL recovery criteria; and 12.1% (12.8%W vs. 9.8%M), 4.4% (4.8%W vs. 0.6%M), 0.8% (0.8%W vs. 0%M), 0.0% (0%W vs. 0%M) - post-PEG PRL concentration criteria. By the last criteria, no patient with total PRL more than 150 ng/mL had macroprolactinemia. Conclusions The criterion for defining macroprolactinemia has a big impact on the study results and needs to be considered in data interpretation and comparison with other studies. PEG precipitation is an easy and fast screening method for macroprolactin. Its main purpose should be determining whether the bioactive monomeric prolactin concentration is increased which is why the post-PEG PRL with corresponding reference interval is the most suitable way of reporting results.

A-125 Difference Between the Results of Vitros® Dry Biochemistry and Wet Biochemistry with Kinetic Method in Diagnosing the Comorbidity of Lung Adenocarcinoma and Aacroamylasemia: Case Report

Abstract Background Amylase (AMY) is a biomarker that comes mainly from the pancreas and salivary glands. Macroamylasemia is a condition often accompanied by malignant tumors and clinical features that include: (i) recurrent abdominal pain, no abnormality in the salivary glands, serum amylase (AMY) level ≥2 to 3 times higher than the normal value and poor patient response to treatments for pancreatitis; (ii) normal serum creatinine and urea levels, increased serum AMY level, and urine AMY level within the normal range or slightly lower. Because macroamylasemia may cause abnormally increased serum AMY levels for a long period of time, misdiagnosis of pancreatitis and delayed diagnosis/treatment of the disease may occur. We report a case of lung adenocarcinoma with macroamylasemia that was co-diagnosed using wet chemistry with kinetic method and dry chemistry to measure AMY. Methods A 47-year-old male patient was previously diagnosed with “acute pancreatitis and lung infection” after initial admission to a local hospital with paroxysmal cough and abdominal pain without obvious cause. To further diagnose the patient, we determined the blood AMY level by using the wet chemistry with kinetic method and the Vitros® dry chemistry test, in addition to conducting a physical exam, abdominal and chest computed tomography (CT), and other lab tests. Two methods, calculation of amylase/creatinine clearance ratio (Cam/Ccr ratio) and protein precipitation by polyethylene glycol (PEG), were also performed to validate assumed interference by serum macroamylase (MAMS) in the wet chemistry method compared to the dry chemistry method, which has technology to capture large molecules and minimize the impact of interferents. MAMS is a high-molecular-weight AMY complex that is synthesized by normal serum AMY bound with macromolecular substances (eg, immunoglobulins and carbohydrates). Results The final diagnosis was a stage IIB cT3N0M0 adenocarcinoma of the upper lobe of the right lung and macroamylasemia. Symptoms and radiologic findings were atypical in terms of pancreatitis. Serum AMY was 2,178U/L when measured with the wet chemistry with kinetic method. When the specimen was re-tested with Vitros dry chemistry, the AMY level was 369 U/L. After precipitation with 25% PEG 6000 solution, the blood AMY level from wet chemistry decreased from 2395.1 U/L to 246.7 U/L, yielding a precipitation rate of 89.7%. Cam/Ccr ratio was calculated as 0.23% (&amp;lt;1%). The calculated PEG precipitation rate and Cam/Ccr ratio from this study were consistent with the ranges considered for macroamylasemia, which are ≥73% and Cam/Ccr ratio &amp;lt;1%, respectively. Conclusions High serum AMY levels do not necessarily indicate pancreatitis or parotitis; wet chemistry results can be impacted by the presence of serum macroamylase. Instead of relying solely on blood AMY in diagnosing acute pancreatitis, clinicians should also consider the correlations of blood AMY with urine AMY and blood lipase. In patients with prolonged blood AMY elevation but normal lipase and urine AMY, re-testing of AMY with a more interference-resistant methodology (e.g. Vitros dry chemistry) may be requested. Accordingly, Cam/Ccr ratio calculation and PEG precipitation can be performed to validate the diagnosis of macroamylasemia.

Macro-FSH is a rare cause of inappropriately high FSH concentrations.

Assessment of hormone concentrations can be subjected to laboratory pitfalls. Macro-hormones are hormone-autoantibody complexes which are cleared slowly from circulation and cause a false elevation in hormones' concentrations. Macro-prolactin and macro-thyroid-stimulating hormone (TSH) are most frequently encountered while macro-follicle-stimulating hormone (FSH) has been rarely reported. We describe the case of a 30-year-old woman who had a gynaecological consultation due to failure in achieving pregnancy after 8 months of unprotected intercourse. She had regular menses, did not complain of climacteric symptoms and her medical history was unremarkable. Antral follicle count and anti-mullerian hormone concentrations were normal, and regular ovulation was documented. Unexpectedly, high early follicular phase FSH concentrations were confirmed on two occasions (57 and 51 IU/L), raising the suspicion of primary ovarian insufficiency. After excluding Turner's syndrome and autoimmune oophoritis, a laboratory artifact was hypothesized. Following polyethylene glycol precipitation, FSH levels dropped from 41.1 IU/L to 6.54 IU/L (recovery 16%) and the presence of macro-FSH was concluded. Laboratory interference can lead to misdiagnosis and unnecessary treatments. A laboratory artifact should be suspected when inconsistency exists between clinical presentation and laboratory results. Only five other cases of macro-FSH have been reported to date. Although macro-hormones generally have low biological activity and do not require treatment, the role of anti-FSH antibodies has been hypothesized in primary ovarian insufficiency and in vitro fertilization failure. Hormone quantification is a cornerstone in the diagnostic workup of endocrine disorders, but it can be subjected to laboratory interferences which can lead to unnecessary investigations and inappropriate treatments. A laboratory artifact should be suspected when a discrepancy is observed between clinical presentation and laboratory results, when extremely unusual analyte concentrations are observed and when inconsistent results are obtained by different analytical methods. Macro-hormones are hormone-autoantibody complexes which are cleared slowly from circulation and cause a false elevation in hormone concentrations. Macro-prolactin and macro-TSH are most frequently encountered, while macro-FSH has been rarely reported. Macro-hormones can be detected by polyethylene glycol precipitation, gel filtration chromatography, or by using protein G or protein A columns. Although macro-hormones generally have low biological activity and do not require treatment, the role of anti-FSH antibodies has been hypothesized in primary ovarian insufficiency and in vitro fertilization failure.

Optimizing Conditions of Polyethylene Glycol Precipitation for Exosomes Isolation From MSCs Culture Media for Regenerative Treatment.

Mesenchymal stem cell (MSC)-derived exosomes, as a cell-free alternative to MSCs, offer enhanced safety and significant potential in regenerative medicine. However, isolating these exosomes poses a challenge, complicating their broader application. Commonly used methods like ultracentrifugation (UC) and tangential flow filtration are often impractical due to the requirement for costly instruments and ultrafiltration membranes. Additionally, the high cost of commercial kits limits their use in processing large sample volumes. Polyethylene glycol (PEG) precipitation offers a more convenient and cost-effective alternative, but there is a critical need for optimized and standardized isolation protocols using PEG precipitation across different cell types and fluids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: UC and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method (about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.

Wastewater Surveillance of SARS-CoV-2: A Comparison of Two Concentration Methods.

Wastewater surveillance is crucial for the epidemiological monitoring of SARS-CoV-2. Various concentration techniques, such as skimmed milk flocculation (SMF) and polyethylene glycol (PEG) precipitation, are employed to isolate the virus effectively. This study aims to compare these two methods and determine the one with the superior recovery rates. From February to December 2021, 24-h wastewater samples were collected from the Ioannina Wastewater Treatment Plant's inlet and processed using both techniques. Subsequent viral genome isolation and a real-time RT-qPCR detection of SARS-CoV-2 were performed. The quantitative analysis demonstrated a higher detection sensitivity with a PEG-based concentration than SMF. Moreover, when the samples were positive by both methods, PEG consistently yielded higher viral loads. These findings underscore the need for further research into concentration methodologies and the development of precise protocols to enhance epidemiological surveillance through wastewater analysis.

In vitro antimicrobial and antibiofilm activity of phage cocktail against Mammaliicoccus sciuri, a causative agent of bovine mastitis.

In this research paper the in vitro antimicrobial and antibiofilm activity of phage cocktail against the coagulase negative Mammaliicoccus sciuri was investigated. Three M. sciuri isolates obtained from clinical bovine mastitis samples were characterized and identified by 16S rRNA gene sequencing. Bacteriophages with lytic activity against M. sciuri isolates were isolated from dairy farm effluents. Two typical phages were isolated using standard enrichment and plaque assay techniques, purified by polyethylene glycol precipitation, and morphologically characterized based on shape and size using transmission electron microscopy. This was followed by determination of host range using spot tests and stability to varying temperature, pH and UV treatment. The phage cocktail suppressed bacterial activity within 30 min of exposure. Crystal violet assay showed that the tested phages and their cocktail significantly reduced the biofilm biomass of all three M. sciuri strains compared to the untreated control in vitro within 24 h with a single dosing. Transmission electron micrography of the purified phage particle revealed an icosahedral head and a rigid contractile tail, characteristic of the class Caudoviricetes. The findings open new avenues in phage-based antimicrobial approaches for controlling contagious and teat skin opportunistic bacteria causing bovine mastitis.

Prevalence of hepatitis A and E viruses in wastewater in Asian countries

Hepatitis A and E viruses (HAV and HEV, respectively) remain a significant global health concern despite advancements in healthcare and vaccination programs. Regular monitoring and vaccine efficacy of HAV are still lacking in different countries. This study aimed to investigate HAV and HEV prevalence in developed, developing, and least-developed Asian countries using wastewater as a surveillance tool. A total of 232 untreated wastewater samples were collected from six wastewater treatment plants, a sewage treatment plant, or an open drainage in six countries [Nepal (n = 51), Indonesia (n = 37), Thailand (n = 30), Vietnam (n = 27), the Philippines (n = 17), and Japan (n = 70)] between April and October 2022. Viruses in wastewater were concentrated by simple centrifugation or polyethylene glycol precipitation method, followed by viral RNA extraction and reverse transcription-quantitative polymerase chain reaction. HAV and HEV RNA were detected in the samples from Nepal (51 % for HAV and 2 % for HEV), Thailand (3 % for both viruses), and Japan (1 % for HAV and 24 % for HEV). Only HAV RNA was found in 11 % of the samples in Indonesia, whereas only HEV RNA was detected in Vietnam and the Philippines, with a positive ratio of 15 % and 12 %, respectively. These results highlighted the geographic variability in HAV and HEV prevalence, underscoring the need for localized public health strategies to address specific viral hepatitis challenges in each country.

Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

BackgroundBacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics.ResultsUsing a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature.ConclusionThis study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Interference by vitamin B12-macrocomplexes: towards an effective detection and correct interpretation of hypo- and hypervitaminemia

Abstract Objectives The presence of macro-B12 is a cause of factual hypervitaminemia B12. Precipitation with polyethylene glycol (PEG) is a simple method of removing high-molecular-weight biomolecules. However, “free” molecule may co-precipitate. This fact imposes the need to establish new reference values for the post-precipitation result. The main aims of the study were to (Baeza I, Corchón A, Pérez JM, Remacha AF, Rocha H, Rodríguez-Portugal E, et al. Basic Aspects of vitamin B12. In: Communication Committee of the Spanish Society of Laboratory Medicine (SEQCML). Clinical significance of vitamin B12 deficiency. Beyond megaloblastic anemia; 2022. ISBN: 978-84-89975-60-6): establish post-PEG reference intervals (RIs) for total vitamin B12 (Iqtidar N, Chaudary MN. Misdiagnosed vitamin B12 deficiency a challenge to be confronted by use of modern screening markers. J Pak Med Assoc 2012;62:1223–9); compare the two bibliographic criteria for defining the presence of macro-B12 (Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood 2017;129:2603–11); evaluate the joint usefulness of percentage post-PEG vitamin B12 recovery and post-PEG RIs to inform the real vitamin B12 status; and (Carmel R. Diagnosis and management of clinical and subclinical cobalamin deficiencies: why controversies persist in the age of sensitive metabolic testing. Biochimie 2013;95:1047–55), propose interpretative comments for ease of interpretation. Methods Prospective study in which 488 serum samples from “healthy” individuals were included and analyzed to determine recovery and RIs following PEG precipitation. Subsequently, a retrospective analysis was carried out in order to evaluate the joint usefulness of both definitions for a suspicion of macro-B12. A total of 297 cases were included. Results Recovery and post-PEG RIs determined on an Alinity i platform ranged from 60 to 107 % and from 118 to 506 pmol/L, respectively. McNemar’s test revealed statistically significant differences between both criteria for estimating interference. However, both methodologies showed good agreement. In 27 cases, the presence of macro-B12 coexisted with true hypervitaminemia B12. No case of coexistence of macro-B12 with vitamin B12 deficiency was detected in our series. Conclusions Laboratory reports should include total vitamin B12 concentration, recovery and post-PEG vitamin B12 concentration with their adjusted RIs to better assess the body vitamin status.

The interference of anti-TSH autoantibody on clinical TSH detection.

It is well known that macro-thyroid-stimulating hormone (macro-TSH) could interfere with the detection of TSH. The anti-TSH autoantibody is an essential component of macro-TSH. However, the epidemiological characteristics and the clinical interference of the anti-TSH autoantibody are unclear. In this study, the radioimmunoprecipitation technique was used to detect the anti-TSH autoantibody. Platforms with different detection mechanisms were applied to measure the TSH in patients with the anti-TSH autoantibody. Polyethylene glycol (PEG) precipitation was used to determine the immunoassay interference. The prevalence of the anti-TSH autoantibody in patients with mild subclinical hypothyroidism (SCH) and autoimmune thyroiditis, but normal thyroid function, was 4.78%. All 10 patients with anti-TSH antibodies had autoimmune diseases, with five of them having significant clinical test interference. The appearance of the anti-TSH antibody is not associated with thyroid autoantibodies. The presence of the anti-TSH autoantibody can interfere with the detection of TSH and can affect clinical diagnosis and treatment.

Rare coincidence: Macro-thyroid-stimulating hormone and multiple manufacturer-specific interferences in thyroid hormone immunoassays.

Immunoassays are widely used for laboratory assessment of endocrine functions including thyroid hormones. While usually adequate for patient evaluation, they are known to potentially suffer from interference from a variety of factors. We report the case of a 44year-old male patient without clinical symptoms of thyroid disease who presented for specialist evaluation after pathological thyroid function tests prompted a transferal by his primary care practitioner. Thyroid function tests showed discrepant results across immunoassays and platforms of different manufacturers. Polyethylene glycol precipitation prompted the diagnosis of macro-thyroid-stimulating hormone, while heterophilic and non-specific antibody blocking reagents proved ineffective in eliminating the interference in thyroid-stimulating hormone, free triiodothyronine and free thyroxine measurements. Further assessment ruled out a diagnosis of familial dysalbuminemic hyperthyroxinemia, leaving an exclusion diagnosis of manufacturer-specific interference in free triiodothyronine and free thyroxine assays due to unknown factors. Both clinicians and laboratory specialists must be aware of potential interference in immunoassays which otherwise might be misleading, potentially triggering unnecessary (invasive) follow-up procedures or therapeutic interventions. Close communication is required for successful troubleshooting. To our knowledge, no other case of both macro-thyroid-stimulating hormone and manufacturer-specific interference in a single patient has been documented thus far.

Comparative analyses of SARS-CoV-2 RNA concentrations in Detroit wastewater quantified with CDC N1, N2, and SC2 assays reveal optimal target for predicting COVID-19 cases

To monitor COVID-19 through wastewater surveillance, global researchers dedicated significant endeavors and resources to develop and implement diverse RT-qPCR or RT-ddPCR assays targeting different genes of SARS-CoV-2. Effective wastewater surveillance hinges on the appropriate selection of the most suitable assay, especially for resource-constrained regions where scant technical and socioeconomic resources restrict the options for testing with multiple assays. Further research is imperative to evaluate the existing assays through comprehensive comparative analyses. Such analyses are crucial for health agencies and wastewater surveillance practitioners in the selection of appropriate methods for monitoring COVID-19. In this study, untreated wastewater samples were collected weekly from the Detroit wastewater treatment plant, Michigan, USA, between January and December 2023. Polyethylene glycol precipitation (PEG) was applied to concentrate the samples followed by RNA extraction and RT-ddPCR. Three assays including N1, N2 (US CDC Real-Time Reverse Transcription PCR Panel for Detection of SARS-CoV-2), and SC2 assay (US CDC Influenza SARS-CoV-2 Multiplex Assay) were implemented to detect SARS-CoV-2 in wastewater. The limit of blank and limit of detection for the three assays were experimentally determined. SARS-CoV-2 RNA concentrations were evaluated and compared through three statistical approaches, including Pearson and Spearman's rank correlations, Dynamic Time Warping, and vector autoregressive models. N1 and N2 demonstrated the highest correlation and most similar time series patterns. Conversely, N2 and SC2 assay demonstrated the lowest correlation and least similar time series patterns. N2 was identified as the optimal target to predict COVID-19 cases. This study presents a rigorous effort in evaluating and comparing SARS-CoV-2 RNA concentrations quantified with N1, N2, and SC2 assays and their interrelations and correlations with clinical cases. This study provides valuable insights into identifying the optimal target for monitoring COVID-19 through wastewater surveillance.

A simple method to overcome paraproteinemic interferences in chemistry and immunoassays.

Interferences on chemistry and immunoassay results due to paraproteinemia may lead to erroneous diagnoses and treatment. Such interferences are difficult to recognize and even more difficult to deal with. This report describes 1 such case where multiple measurands were affected and how the interferant was overcome. Paraproteins present in an immunoglobulin (Ig)G-lambda multiple myeloma specimen interfered with results of total bilirubin, direct bilirubin, inorganic phosphate, iron, ferritin, and total thyroxine measured on 3 platforms: AU5800, Alinity ci, and cobas pure. Repeat testing upon dilution with normal saline or deproteinization by polyethylene glycol precipitation gave unsatisfactory results on some or all the affected measurands. Repeat testing after dilution of the interferant serum with a healthy serum corrected the anomalous results for all the affected measurands. Dilution of paraproteinemic serum with a healthy serum of known concentrations appears to be the most suitable method to negate the effects of paraproteinemic interferences.

Experimental Study of Interference in Calcitonin Testing: A Case Report and Literature Review.

Calcitonin (CT) is a sensitive serum marker of medullary thyroid carcinoma usually detected via immunoassays; however, its levels are easily disturbed by several endogenous factors. The study aimed to discuss a case of suspected interference resulting in aberrant CT values and review previous reports of CT interference. A female patient visited our clinic with a physical ultrasound examination showing a slightly enlarged thyroid gland with small nodules. She had elevated CT levels, inconsistent with the clinical presentation and other findings. We evaluated the results by retesting using the same platform, platform validation, multiplex dilution, Polyethylene Glycol (PEG) precipitation, heterophilic blocking tubes, and RET gene analysis. Retesting CT using the same platform confirmed the high value obtained. However, serial dilution of the sample produced nonlinear results, suggesting some interference. While PEG precipitation did not significantly reduce the CT level, incubating the sample in HBTs normalized the CT value, indicating interference from heterophilic antibodies. Gene sequencing revealed no RET mutations. In cases where elevated CT levels are inconsistent with clinical presentations and other findings, the laboratory technicians should communicate with clinicians, analyze the reasons for the inconsistent results, and use different methods to verify the results. Accurate testing provides realistic and reliable data for doctors and patients and helps to avoid unnecessary procedures.

Interference With VIP to Distinguish Between Real and False VIPoma: National Study From the French Endocrine Tumors Group.

Vasoactive intestinal peptide (VIP)-secreting tumors (VIPomas) are digestive neuroendocrine tumors in which the hormonal secretion is life-threatening. Biological confirmation is obtained by demonstrating an elevation in plasma VIP, usually using radioimmunoassay (RIA). In some cases, analytical interference is suspected. We developed 3 different techniques to detect interference in VIP RIA. Three techniques were used: RIA after Sephadex column chromatography separation, RIA after polyethylene glycol precipitation, and 125I-labeled VIP binding test. We included patients with suspicion of false positive VIP (FPV) elevation. We then compared results with those of a group of "real," proven VIPoma (RV). A total of 15 patients with FPV elevation and 9 RV patients were included. Interference was detected in all FPV patients vs none in RV. Clinical and biochemical parameters did not differ between FPV and RV patients, but VIP concentration in RIA was significantly higher in FPV patients than in RV patients (228 pmol/L vs 66 pmol/L, P = .038). Using a 125I-labeled VIP binding test, median proportion of radioactivity in the pellet was significantly higher in FPV than in RV patients (53% vs 13%, P < .0001). A 20.5% threshold presented excellent performances (sensitivity 100% [79.6-100], specificity 100% [70.1-100]). We developed 3 different laboratory techniques to reveal interference in RIA VIP assays. The diagnostic performance of all 3 was excellent. These techniques must be employed in cases of discordance between VIP elevation and clinical presentation.