Determining the Frequency of Macroamylasemia in Patients with Hyperamylasemia using PEG Precipitation Method

Abstract Objective: The aim of this study was to compare the quality and efficiency of exosomes extracted from knee joint effusion by different methods, laying a foundation for further research on exosomes from knee joint effusion. Methods:To separate and extract joint exosomes by 8% polyethylene glycol (PEG) precipitation method, ultracentrifugation method (UC) and ultrafiltration with exclusion chromatography (SECF). Transmission electron microscopy (TEM) and nanoparticle tracing technology (NTA) were used to detect particle morphology and particle size, and Western Blot (WB) was used to detect marker proteins of granule surface (CD9, CD63, Flotillin-1 and calnexin).Results: Three methods separated vesicle-like round particles from joint effusion successfully. The results of TEM show that the particles obtained by the three extraction methods are round or oval vesicles, the plasma membrane is intact, the size is different, and the diameter is distributed between 30 and 150 nm. Compared with SECF group, PEG group had more background particle impurities. and the broken vesicle fragments can be seen in the UC group; The results of NTA show that the main peaks of the three groups of particles are between 100-120nm, and the particle concentration is greater than 1×1010/ml; The results of WB show that the protein expressions of CD9, CD63 and Flotillin-1 in the suspension extracted by the three methods were higher, the expression of calnexin protein in the PEG group was higher than the UC group and SECF group.Conclusion: The three extraction methods can extract the exosomes of joint effusion successfully. The quality of exosomes obtained by the SECF method is relatively high, while the PEG precipitation method contains a small amount of impurity particles. UC method does not guarantee the integrity of exosomes. In summary, we recommend the SECF method to extract and isolate joint effusion exudates-derived exosomes when further studying the role of exosomes in the pathogenesis of knee osteoarthritis.

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Macroprolactinemia: Diagnostic, Clinical, and Pathogenic Significance

Clinical and Developmental Immunology ◽

10.1155/2012/167132 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 38

Author(s):

Akira Shimatsu ◽

Naoki Hattori

Keyword(s):

Middle Age ◽

Molecular Form ◽

Protein A ◽

Gel Filtration ◽

Cross Reactivity ◽

Precipitation Method ◽

Binding Studies ◽

Rat Pituitary ◽

Peg Precipitation Method

Macroprolactinemia is characterized by a large molecular mass of PRL (macroprolactin) as the main molecular form of PRL in sera, the frequent elevation of serum PRL (hyperprolactinemia), and the lack of symptoms. Macroprolactin is largely a complex of PRL with immunoglobulin G (IgG), especially anti-PRL autoantibodies. The prevalence of macroprolactinemia is 10–25% in patients with hyperprolactinemia and 3.7% in general population. There is no gender difference and a long-term followup demonstrates that macroprolactinemia develops before middle age and is likely a chronic condition. Polyethylene-glycol- (PEG-) precipitation method is widely used for screening macroprolactinemia, and gel filtration chromatography, protein A/G column, andI125-PRL binding studies are performed to confirm and clarify its nature. The cross-reactivity of macroprolactin varies widely according to the immunoassay systems. The epitope on PRL molecule recognized by the autoantibodies is located close to the binding site for PRL receptors, which may explain that macroprolactin has a lower biological activity. Hyperprolactinemia frequently seen in macroprolactinemic patients is due to the delayed clearance of autoantibody-bound PRL. When rats are immunized with rat pituitary PRL, anti-PRL autoantibodies are produced and hyperprolactinemia develops, mimicking macroprolactinemia in humans. Screening of macroprolactinemia is important for the differential diagnosis of hyperprolactinemia to avoid unnecessary examinations and treatments.

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Comparison of Some Recent Methods for the Differentiation of Elevated Serum Amylase and the Detection of Macroamylasaemia

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456328902600508 ◽

1989 ◽

Vol 26 (5) ◽

pp. 422-426 ◽

Cited By ~ 6

Author(s):

Armand Van Deun ◽

Christa Cobbaert ◽

Angeline Van Orshoven ◽

Georges Claeys ◽

Willy Lissens

Keyword(s):

Wheat Germ ◽

Serum Amylase ◽

Amylase Activity ◽

Elevated Serum ◽

Precipitation Method ◽

Serum Samples ◽

Agarose Electrophoresis ◽

Pancreatic Isoamylase ◽

Peg Precipitation Method ◽

A Minor

A pancreatic isoamylase method (Pancreatic Alpha-Amylase EPS, Boehringer) that uses monoclonal antibodies showed almost complete immunoinhibition of salivary (S) amylase activity with only a minor decrease of pancreatic (P) amylase activity. The method displayed good sensitivity and linearity. The correlations of P-amylase activities determined by this technique with a wheat-germ inhibition method and with agarose electrophoresis followed by densitometric scanning were excellent. However, both the wheat-germ and monoclonal inhibition methods failed to detect macroamylasaemia. To recognise macroamylases we used the PEG precipitation method and confirmed the results with agarose electrophoresis. Of 161 serum samples with elevated amylase activities, only four out of five with macroamylasaemia were detected by the PEG precipitation method. No false positives were demonstrated. After PEG precipitation of 28 samples, P-amylase determinations were performed on the supernatants. Again, four out of five with macroamylasaemia were recognised. We consider P-amylase measurement and, when macroamylasaemia is suspected, the combined use of the PEG precipitation method and P-amylase or total amylase determination to be the most practical way to differentiate between elevated serum amylase levels.

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Extraction And Quality Evaluation of Exosomes From Joint Effusion

10.21203/rs.3.rs-48470/v1 ◽

2020 ◽

Author(s):

Anmin Ruan ◽

Pu Chen ◽

Jun Zhou ◽

Xiaozhe Zhang ◽

Yufeng Ma ◽

...

Keyword(s):

Knee Joint ◽

Particle Morphology ◽

Extraction Methods ◽

Joint Effusion ◽

Precipitation Method ◽

Transmission Electron ◽

Marker Proteins ◽

Exclusion Chromatography ◽

Peg Precipitation Method

Abstract Objective: The aim of this study was to compare the quality and efficiency of exosomes extracted from knee joint effusion by different methods, laying a foundation for further research on exosomes from knee joint effusion. Methods: Separate and extract joint exosomes by 8% polyethylene glycol (PEG) precipitation method, ultracentrifugation method (UC) and ultrafiltration with exclusion chromatography (SECF). Transmission electron microscopy (TEM) and nanoparticle tracing technology (NTA) were used to detect particle morphology and particle size, and Western Blot (WB) was used to detect granule protein surface marker proteins (CD9, CD63, Flotillin-1 and calnexin).Results: All three methods successfully separated vesicle-like round particles from joint effusion. TEM results show that the particles obtained by the three extraction methods are round or oval vesicles, the plasma membrane is intact, the size is different, and the diameter is distributed between 30 and 150 nm. Compared with SECF group, PEG group had more background particle impurities. and the broken vesicle fragments can be seen in the UC group; NTA results show that the main peaks of the three groups of particles are between 100-120nm, and the particle concentration is greater than 1×1010/ml; WB results show that the expressions of CD9, CD63 and Flotillin-1 protein in the suspension extracted by the three methods were all higher, the expression of calnexin protein was higher in the PEG group than the UC group and SECF group.Conclusion: The three extraction methods can successfully extract the exosomes of joint effusion. The quality of exosomes obtained by the SECF method is relatively high, while the PEG precipitation method contains a small amount of impurity particles. UC method does not guarantee the integrity of exosomes. In summary, when further studying the role of exosomes in the pathogenesis of knee osteoarthritis, we recommend the SECF method to extract and isolate joint exudates.

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Laboratory detection of macro-aspartate aminotransferase: Case report and evaluation of the PEG-precipitation method

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2012.03.004 ◽

2012 ◽

Vol 45 (9) ◽

pp. 691-693 ◽

Cited By ~ 6

Author(s):

Lisbeth Patteet ◽

Marc Simoens ◽

Marian Piqueur ◽

Annick Wauters

Keyword(s):

Case Report ◽

Aspartate Aminotransferase ◽

Precipitation Method ◽

Peg Precipitation Method

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Optimizing laboratory defined macroprolactin algorithm

Biochemia Medica ◽

10.11613/bm.2019.020706 ◽

2019 ◽

Vol 29 (2) ◽

pp. 346-351

Author(s):

Milica Šostarić ◽

Adriana Bokulić ◽

Domagoj Marijančević ◽

Ivana Zec

Keyword(s):

Reference Interval ◽

Repeated Measurements ◽

Precipitation Method ◽

Upper Limit ◽

Significant Difference ◽

Before And After ◽

Absolute Bias ◽

The Difference ◽

Peg Precipitation Method

Introduction: Macroprolactinaemia is a well-known analytical problem in diagnostics of hyperprolactinaemia usually detected with polyethylene glycol (PEG) precipitation method. Since there is no harmonization in macroprolactin detection and reporting results, this study proposes and evaluates the usefulness of in-house developed algorithm. The aims were to determine the most suitable way of reporting results after PEG treatment and the possibilities of rationalizing the precipitation procedure. Materials and methods: This is a retrospective study based on extracted data for 1136 patients. Prolactin concentrations were measured before and after PEG precipitation on Roche cobas e601. Macroprolactinaemia was defined by percentage recovery and post-PEG prolactin concentrations. Results: Prevalence of macroprolactinaemia using recovery criteria of ≤ 40%, ≤ 60%, and post-PEG prolactin concentrations was 3.3%, 8.8% and 7.8%, respectively. Raising the cut-off value from the upper limit of the manufacturer’s reference interval to 32.9 μg/L does not drastically change detected macroprolactinaemia with recovery criteria. Post-PEG prolactin concentrations showed more than half of the patients with macroprolactinaemia would be overlooked. Regardless of the criteria, a cut-off of 47.0 μg/L would miss most of the macroprolactinaemic patients. Repeated recovery measurements of follow-up patients showed there is a significant difference with mean absolute bias of 9%. Conclusions: Post-PEG prolactin concentration with corresponding reference interval is the most suitable way of reporting results. All samples with prolactin concentration above the upper limit of the manufacturer’s reference interval should be submitted to PEG precipitation. Follow-up period could be prolonged since the difference between the recoveries of repeated measurements is not clinically significant.

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Extraction And Quality Evaluation of Exosomes From Joint Effusion

10.21203/rs.3.rs-49025/v1 ◽

2020 ◽

Author(s):

Anmin Ruan ◽

Yueshan Yin ◽

Pu Chen ◽

Jun Zhou ◽

Xiaozhe Zhang ◽

...

Keyword(s):

Knee Joint ◽

Particle Morphology ◽

Extraction Methods ◽

Joint Effusion ◽

Precipitation Method ◽

Transmission Electron ◽

Marker Proteins ◽

Exclusion Chromatography ◽

Peg Precipitation Method

Abstract Objective: The aim of this study was to compare the quality and efficiency of exosomes extracted from knee joint effusion by different methods, laying a foundation for further research on exosomes from knee joint effusion. Methods: Separate and extract joint exosomes by 8% polyethylene glycol (PEG) precipitation method, ultracentrifugation method (UC) and ultrafiltration with exclusion chromatography (SECF). Transmission electron microscopy (TEM) and nanoparticle tracing technology (NTA) were used to detect particle morphology and particle size, and Western Blot (WB) was used to detect granule protein surface marker proteins (CD9, CD63, Flotillin-1 and calnexin).Results: All three methods successfully separated vesicle-like round particles from joint effusion. TEM results show that the particles obtained by the three extraction methods are round or oval vesicles, the plasma membrane is intact, the size is different, and the diameter is distributed between 30 and 150 nm. Compared with SECF group, PEG group had more background particle impurities. and the broken vesicle fragments can be seen in the UC group; NTA results show that the main peaks of the three groups of particles are between 100-120nm, and the particle concentration is greater than 1×1010/ml; WB results show that the expressions of CD9, CD63 and Flotillin-1 protein in the suspension extracted by the three methods were all higher, the expression of calnexin protein was higher in the PEG group than the UC group and SECF group.Conclusion: The three extraction methods can successfully extract the exosomes of joint effusion. The quality of exosomes obtained by the SECF method is relatively high, while the PEG precipitation method contains a small amount of impurity particles. UC method does not guarantee the integrity of exosomes. In summary, when further studying the role of exosomes in the pathogenesis of knee osteoarthritis, we recommend the SECF method to extract and isolate joint exudates.

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MS2 Virus-like Particle as an Efficient Drug Delivery Tool: Optimizing the Synthesis and Purification Protocol

10.21203/rs.3.rs-667960/v1 ◽

2021 ◽

Author(s):

Khadijeh Hashemi ◽

Mohammad Mahdi Ghahramani Seno ◽

Mohammad Reza Ahmadian ◽

Bizhan Malaekeh-Nikouei ◽

Mohammad Reza Bassami ◽

...

Keyword(s):

Drug Delivery ◽

Precipitation Method ◽

Rna Sequences ◽

Bacteriophage Ms2 ◽

Virus Like Particle ◽

Effects Of Ph ◽

Peg Precipitation Method ◽

The Stability ◽

Purification Protocol

Abstract Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated.In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30nm and suitable homogeneity with minimum 12-month stability at 4◦C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 hours in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.

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