[Examples of accreditation of serum and urinary proteins electrophoresis]. / Expériences d'accréditation des électrophorèses sériques et urinaires.

Serum proteins and urinary proteins electrophoresis are useful biological tests. They are often prescribed for the screening of monoclonal gammopathys and also during follow-up for treatment response. This test can be accredited according to standard NF ISO 15189 since laboratories use analysers and adapted reagents, but there are numerous protocols for the method validation. This paper present the results of a survey proposed in 2019 to biologists by CNBH and SFBC. The aim of this survey is to give biologists a choice among several protocols that have been positively evaluated by COFRAC.

Double inner standard plot model of an electrophoresis titration chip for a portable and green assay of protein content in milk.

High portability and environmental safety ("green") are two of the most important objectives pursued by microfluidic methods. However, there remain many challenges for the design of portable and visual microfluidic methods (e.g., chip electrophoresis) due to use of a cumbersome pump, power supply and detector. Herein, a facile double inner standard plot (DISP) model of electrophoresis titration (ET) was proposed for portable and visual assay of proteins in test milk samples without use of a pump, power supply or detector based on a moving reaction boundary (MRB) chip. The DISP-ET model predicted that: (i) by setting the upper limit (UL) and lower limit (LL) of double inner standard milk protein contents, points U and L were, respectively, achieved in the relationship D = -aC + b (D: MRB motion distance; C: protein content); and (ii) the two points divided both the C-axis and D-axis into "poor", "eligible" and "superior" rulers scaled for quantitative assay of test samples. To demonstrate the model of DISP-ET, an original portable device (120 mm × 78 mm × 30 mm, 341 g) was designed, which had a chip (25 mm × 25 mm × 4 mm) of three channels (15 mm × 200 µm × 80 µm), platinum electrodes, a lithium cell and touch screen. A series of experiments were undertaken based on the developed portable device. The relevant experiments demonstrated systemically the validity of the DISP-ET model, theory and method. In particular, the experiments clearly showed the advantages of the DISP-ET chip: portability, visuality, green use, rapidity, and flexibility for real-life use. Finally, the device was applied for a portable and visual assay of fresh milk from a cow on a dairy farm. The DISP-ET model opens a window for designing portable and visual quantitative methods of food-safety control and clinical diagnoses.

Comparison of two different electrophoretic methods in studying the genetic diversity among plantains (Musa spp.) using ISSR markers.

Inter simple sequence repeat markers were employed for the genotyping of 16 plantain ecotypes. Two different electrophoretic systems namely conventional gel electrophoresis (CVGE) and fully automated high-resolution CGE were used to evaluate the genetic diversity. Comparative analysis indicated that all parameters related to marker informativeness were higher in CGE except polymorphic information content. But genetic diversity parameters like effective number of alleles, Nei's gene diversity (1973) and Shannon's information index showed higher values (1.52 ± 0.12, 0.34 ± 0.05 and 0.52 ± 0.05, respectively) in CVGE as against CGE (1.29 ± 0.04, 0.22 ± 0.02 and 0.38 ± 0.03, respectively) system. The unweighed pair group method with arithmetic averages was used to obtain the dendrogram for both analyses. The results of dendrogram and principal component analysis were found to be consistent in both systems except for some minor disagreements. The clone-specific bands could be used in the identification and development of SCAR markers. Inter simple sequence repeat markers used in this study provided sufficient polymorphism and reproducible banding pattern for evaluating the genetic diversity of different plantain ecotypes. Lack of accuracy and consistency of the CVGE warrants the employment of high-throughput CGE for diversity analysis as it provided better separation of bands with higher resolution.

Iron stains on paper. Can electrophoretic removal become an effective alternative to chemical cleaning?

Research in restoration and conservation is directed vs. more sustainable working materials, methods and technologies. Electrophoretic removal, from porous material, of undesired stains due to charged species is theoretically an interesting alternative to chemical cleaning methods, but the lack of specific and comprehensive research work leads to controversial opinions about the efficiency and the needed harmfulness for the treated objects. In this work paper, samples with artificial rust stains were subjected to electrophoretic cleaning treatments in mineral water as electrolyte. Treatments were carried out either in a bath by complete sample immersion between the distanced electrodes or by sample wetting and sandwiching between the electrodes. Evaluation of cleaning efficiency and treatment effects was based on colour change measurements (image analysis of scanned paper samples before and after the treatment and by colorimetric data via spectrophotometric measurements), investigation of morphological changes by SEM observations and folding endurance measurements.

A Comparison of Protein Stability in Prefillable Syringes Made of Glass and Plastic.

The development of protein therapeutics requires stabilization of these labile molecules during shipment and storage. Biologics, particularly monoclonal antibodies, are frequently packaged at high concentration in prefillable syringes traditionally made of glass. However, some biologics are unstable in glass due to sensitivity to silicone oil, tungsten, glue, or metal ions. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), with a Flurotec-laminated piston, have none of these issues. This study compared the stability of several proteins including biotherapeutics when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when subjected to mild agitation by end-over-end rotation at room temperature. At each time point, proteins were analyzed by several techniques including turbidity, size exclusion high-performance liquid chromatography, reversed phase high-performance liquid chromatography, ion-exchange chromatography, electrophoresis, and light scattering to monitor changes in aggregation and degradation. The results show that proteins have comparable stability when stored in glass syringes or in syringes made of CZ sterilized by E-beam or autoclave. In addition, proteins stressed by agitation were generally more stable and aggregated less in syringes made of CZ than in ones made of glass.LAY ABSTRACT: Biotherapeutic protein drugs such as monoclonal antibodies are frequently packaged at high concentration in prefillable syringes, which allows the drug to be directly administered by the patient or caregiver. Protein drugs, or biologics, can be unstable, and may aggregate, particularly when shaken. These aggregates can be immunogenic, stimulating the body's immune system to produce antibodies that can reduce the drug's efficacy. Although prefillable syringes are traditionally made of glass, some biologics are unstable in glass syringes due to the presence of substances used in their manufacture, including silicone oil, which is necessary for lubricity. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), have none of these issues. This study compared the stability of several biotherapeutic proteins when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when mildly agitated at room temperature. Proteins were analyzed by several techniques to detect changes in aggregation and degradation. The results show that biotherapeutic proteins have similar stability whether stored in syringes made of glass or CZ. In addition, proteins subjected to agitation were generally more stable and aggregated less in CZ syringes than in glass syringes.

Radiolabeling, quality control, and biological characterization of 177 Lu-labeled kanamycin.

Kanamycin is an antibiotic, isolated from Streptomyces kanamyceticus, which is used to treat serious bacterial infections. The fact that the present radioligand 99m Tc-kanamycin used for diagnosis is short-lived, raised a need to label and study kanamycin with one of the most important beta (ß) radiation emitting isotope 177 Lu. Labeling yield of 177 Lu-kanamycin was confirmed by different chromatography techniques such as paper chromatography, TLC, HPLC. Several experiments were performed to optimize labeling with changing reaction conditions such as pH, temperature, amount of ligand, and reaction time. In vitro stability analysis was performed incubation with human serum. Electrophoresis analysis was also conducted to determine the charge on 177 Lu-kanamycin. The biodistribution and scintigraphy were performed in normal mice and rabbit, respectively, at different time intervals of postinjection. 177 Lu-kanamycin was prepared with very high yield (~100%), with excellent stability in vivo and in vitro (>99% 6 hr postprep.), at pH 7. Maximum labeling was achieved at less reaction time (15 min), with maximum conjugation of the ligand (12.5 mg) with 177 Lu. Electrophoresis analysis showed net neutral charge. The radioligand showed rapid clearance from body in biodistribution and scintigraphy studies. The preparation 177 Lu-kanamycin could be used as a radio-pharmaceutical for infection imaging purpose, especially when transporting the radioligand to long-range distances.

Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples.

The current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The detection limit of ELISA is about 250 pg/ml. However, to quantify analytes that are related to various stages of tumors including early detection requires detecting well below the current limit of the ELISA test. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are <100 pg/ml and the prostate specific antigen level of the early stage of prostate cancer is about 1 ng/ml. Further, it has been reported that there are significantly less than 1pg/mL of analytes in the early stage of tumors. Therefore, depending on the tumor type and the stage of the tumors, it is required to quantify various levels of analytes ranging from ng/ml to pg/ml. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes (

IFM (Intergroupe francophone du myélome) recommendations for uniform interpretation of serum and urine protein electrophoresis in multiple myeloma diagnosis and follow-up.

Serum and urine proteins electrophoresis take a major place in multiple myeloma management, at time of diagnosis, during follow-up for treatment response evaluation and also in detection of relapse. The Intergroupe francophone du myélome (IFM) suggests recommendations to clinicians and biologists, to perform and interpret these biochemical analysis, with the objective of harmonizing practices between laboratories and improving patients' follow-up.

Serum-free light-chain analysis in diagnosis and management of multiple myeloma and related conditions.

The introduction of the serum-free light-chain (S-FLC) assay has been a breakthrough in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The first method, proposed in 2001, quantifies serum-free light-chains using polyclonal antibodies. More recently, assays based on monoclonal antibodies have entered into clinical practice. S-FLC measurement plays a central role in the screening for multiple myeloma and related conditions, in association with electrophoretic techniques. Analysis of S-FLC is essential in assessing the risk of progression of precursor diseases to overt plasma cell dyscrasias. It is also useful for risk stratification in solitary plasmacytoma and AL amyloidosis. The S-FLC measurement is part of the new diagnostic criteria for multiple myeloma, and provides a marker to follow changes in clonal substructure over time. Finally, the evaluation of S-FLC is fundamental for assessing the response to treatment in monoclonal light chain diseases.

The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissue.

RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investigative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue.

Sources of variation analysis and derivation of reference intervals for ALP, LDH, and amylase isozymes using sera from the Asian multicenter study on reference values.

BACKGROUND: Sources of variation (SV) of ALP, LDH, and amylase isozymes were explored. METHODS: We analyzed 3511 sera from well-defined healthy individuals recruited during the 2009 Asian project for derivation of common reference intervals (RIs). Up-to-date electrophoresis auto-analyzer and reagents were employed for high resolution and reproducibility. SVs including sex, age, body mass index (BMI), ABO blood groups, and levels of drinking, smoking, and exercise were analyzed by multiple regression analysis. RIs were determined by parametric methods after refining healthy individuals by use of latent reference values exclusion method. RESULTS: Age-related changes in ALP2-3 were different in females: ALP2, linear increase from 20-64y; ALP3, lowering until 45 y and rising steeply thereafter. ALP2 increased with BMI especially in females. ALP5 was barely detectable except in blood-types O and B. Age-related increases in LDH1-LDH3 were noted in females, whereas BMI-related increases were found only for LDH2-LDH5 in both sexes. Pancreatic amylase showed age-related increase in females and was slightly higher in blood-type O. RIs for absolute and relative activities of each isozyme were derived in consideration of sex and age. CONCLUSIONS: Investigation of these isozymes revealed various age-, BMI-, and blood-type-related changes that are all relevant in clinical interpretation of enzyme test results.

On benchmark problems, challenges, and competitions in electrokinetics-A review.

In this critical review, we comment on the absence of widely shared benchmark problems and relevant challenges or even attractive competitions in the field of electrokinetics. We argue that in some other scientific domains that are, similarly as electrokinetics, strongly multidisciplinary, the existence of these tools is very beneficial because it stimulates the discussion about what constitutes the bottleneck of further progress, allows easier exploitation of results provided by other scientific and engineering disciplines, and, last but not least, makes the research domain attractive and visible to a broader public, including students. The goal of this review is to provoke some discussion that might perhaps lead to compensating for these shortcomings.

Comparison of Hevylite™ IgA and IgG assay with conventional techniques for the diagnosis and follow-up of plasma cell dyscrasia.

BACKGROUND: Heavy/light chain assay allows the characterization and quantification of immunoglobulin light chains bound to heavy chains for each Ig'k and Ig'λ immunoglobulin class, discriminating between the involved/uninvolved isotypes in plasma cell dyscrasia. The Ig'k/Ig'λ ratio (heavy/light chain ratio) enables to monitor the trend of monoclonal component during therapy and disease evolution. OBJECTIVE: In this study, we evaluate the impact of the heavy/light chain assay in monitoring multiple myeloma patients in comparison with conventional techniques. METHODS: Serum samples of 28 patients with IgG or IgA monoclonal component were collected for a mean of 109 days and analyzed. The heavy/light chain assay was compared with classical immunoglobulin quantification (Ig'Tot), serum immunofixation electrophoresis, serum protein electrophoresis, and serum-free light chains quantification. Serum samples from 30 healthy patients were used as control (polyclonal). RESULTS: Heavy/light chain ratio and serum immunofixation electrophoresis were comparable in 86% of the cases, and free light chain ratio and heavy/light chain ratio in 71.8%. Heavy/light chain assay and Ig'Tot measurements showed a concentration-dependent agreement in monoclonal patients. The heavy/light chain assay was able to quantify the monoclonal component migrating in SPE ß region: this occurred in 10% of our IgG and 50% of our IgA patients. CONCLUSIONS: The concordance scores indicate that heavy/light chain and Ig'Tot assays show differences at high monoclonal component values. The heavy/light chain ratio, serum immunofixation electrophoresis, and free light chain ratio showed partial concordance. Our study confirmed that, in the context of heavy/light chain assay, heavy/light chain Ig'k and Ig'λ absolute values and heavy/light chain ratio are both important tools to monitor the presence of monoclonal component that are difficult to be identified in SPE.

Preparing size markers for gel electrophoresis.

Here we present two simple methods for preparing radiolabeled size markers for gel electrophoresis. The first procedure describes the generation of an RNA marker ladder by the alkaline hydrolysis of (32)P 5'- or 3'-end-labeled RNA. The second procedure describes the labeling of DNA fragments produced by digestion of pBR322 with the restriction enzyme MspI.

Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand.

BACKGROUND: Although protein electrophoresis of serum (SPEP) and urine (UPEP) specimens is a well-established laboratory technique, the reporting of results using this important method varies considerably between laboratories. The Australasian Association of Clinical Biochemists recognized a need to adopt a standardized approach to reporting SPEP and UPEP by clinical laboratories. METHODS: A Working Party considered available data including published literature and clinical studies, together with expert opinion in order to establish optimal reporting practices. A position paper was produced, which was subsequently revised through a consensus process involving scientists and pathologists with expertise in the field throughout Australia and New Zealand. RESULTS: Recommendations for standardized reporting of protein electrophoresis have been produced. These cover analytical requirements: detection systems; serum protein and albumin quantification; fractionation into alpha-1, alpha-2, beta and gamma fractions; paraprotein quantification; urine Bence Jones protein quantification; paraprotein characterization; and laboratory performance, expertise and staffing. The recommendations also include general interpretive commenting and commenting for specimens with paraproteins and small bands together with illustrative examples of reports. CONCLUSIONS: Recommendations are provided for standardized reporting of protein electrophoresis in Australia and New Zealand. It is expected that such standardized reporting formats will reduce both variation between laboratories and the risk of misinterpretation of results.

Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis.

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 10 10(3) kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30-2400 kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10 min using only 6 pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.

Homozygous bisalbuminemia or homozygous mutant albumin?

INTRODUCTION: Bisalbuminemia is a genetic condition in which an albumin variant is found in serum in addition to normal albumin. METHODS AND MATERIALS: Serum protein electrophoresis using the Sebia HYDRASYS electrophoresis system was performed on an 84 year old male. RESULTS: Serum protein electrophoresis showed a single albumin band migrating faster than normal albumin. CONCLUSION: The presence of a homozygous albumin variant band, albumin Naskapi, is noted.