Concordance of Capillary Electrophoresis and Conventional Gel Electrophoresis in Two Different Groups of Patients with.

BACKGROUND: Serum and urinary protein electrophoresis play an important role in the identification of monoclonal gammopathy. Recently, capillary electrophoresis (CE) has been adapted in many clinical laboratories because of several advantages such as short turnaround time, automation, and high reproducibility. However, there have been unsolved concerns for the concordance between conventional gel and automated capillary electrophoresis methods for protein separation in clinical specimens. In this study, we investigated the diagnostic performance of both methods for detecting monoclonal (M) protein. METHODS: From February 2012 to August 2015, a total of 3,013 CE tests were performed in our hospital. Among these cases, we reconfirmed results of CE (Capillary 2, Sebia, Lysse, France) with those of conventional agarose gel electrophoresis (GE) (Hydragel 4IF, Sebia, Lisses, France) in 28 specimens from 24 patients with newly diagnosed monoclonal gammopathy (group 1). In addition, 22 cases from 15 patients with previously diagnosed monoclonal gammopathy presenting indeterminate or suspicious results on CE (group 2) were also reconfirmed with GE. RESULTS: We compared the results between the two electrophoresis methods in two different groups of patients with newly diagnosed discrete monoclonal peaks vs. pre-existing monoclonal gammopathy with obscure results in follow-up courses. In group 1, agreement rate was 100% (28/28) and there was no discrepant result between these two electrophoresis methods. In contrast, group 2 showed 86.4% (19/22) agreement rate and 0.67 Cohen's kappa value (95% confidence interval, 0.51 - 1.02). CONCLUSIONS: According to our results, both electrophoresis methods can be used with the same level of assurance at the time of initial diagnosis for monoclonal gammopathy. However, in patients with previously diagnosed monoclonal gammopathy in follow-up course after appropriate treatments, discordant results can be observed due to the reduced amount of M proteins. Therefore, we suggest that some ambiguous cases with very small amounts of M components require a combination of both CE and GE methods for accurate interpretation to confirm the presence of M proteins.

The soft tissue cover of the mandibular condyle. Differentiation in histological forms and age-related changes of aggrecan- and versican-like proteoglycans.

Age-related changes of the composition of the extracellular matrix of the soft tissue cover of the mandibular condyle (STC), especially of the large proteoglycans, have been investigated. Proteoglycans were extracted from the STC of neonatal, juvenile and adult domestic pigs, fractionated by density gradient centrifugation and analyzed by electrophoresis/Western blotting. Experiments revealed firstly that a large CS/KS proteoglycan (aggrecan) is an essential constituent of the STC at all ages. This proteoglycan is required for nutrition of avascular tissues, and age-related changes in its average size and substitution with KS (keratan sulfate) may be a response to altered functional loading and tissue architecture of the STC. Secondly it was shown that a large CS/DS (chondroitin sulfate/dermatan sulfate) proteoglycan characterized by a doublet of core proteins at 200 and 250 kDa, thereby resembling perlecan, is present in the tissue of adults, but not of neonates and juveniles. Thirdly a large CS/DS proteoglycan characterized by core proteins at 350, 450 and 550 kDa, thereby resembling versican, was present in juveniles. It was detectable only weakly in neonates and not in adults. Results of core protein analysis were confirmed by results of agarose gel electrophoresis/Western blotting of the undigested proteoglycans isolated directly from the tissue extracts. Versican is believed to destabilize cell-matrix interactions required for cell proliferation and differentiation. In this context, presence of versican-like proteoglycans in the STC of growing individuals and its disappearance in adults appears to be related to the growth potential of the mandibular condyle.

[The electrophoretic properties of gels of high-resolution MS-4 agarose for separating DNA fragments in molecular genetic analysis]. / Issledovanie élektroforeticheskikh svoistv gelei vysokorazreshaiushchei agarozy MS-4 dlia razdeleniia fragmentov DNK v molekuliarno-geneticheskom analize.

The Hispanlab firm (Spain) offers a new series of Molecular Screen analytical agarose, including MS-4 agarose intended for separation of small fragments of DNA. This low-endosmotic agarose attracts attention as a probable alternative to well-known FMC Bio Products special agarose (USA) due to its useful properties. We characterized a gel-electrophoretic system based on MS-4 agarose (and its correspondence to its passport) and tested its fitness for standard molecular genetic expert investigations. The following parameters were assessed: mechanical properties of gel, its transparency, degree of background fluorescence upon ethidium bromide staining, resolving power, and position distortions in the electrophoregram. Important features of MS-4 agarose gels were detected, not typical of other agarose systems, which should be borne in mind during investigations with this agarose.

New method for isolating and quantifying intermediate and beta-very-low-density lipoprotein cholesterol.

We describe a new method, useful to clinical laboratories, for assessing intermediate density (IDL) or beta-very-low-density (beta-VLDL) lipoprotein cholesterol. The technique involves selective precipitation properties of the qualitative Wieland and Seidel post-electrophoretic method that immobilizes IDL and beta-VLDL in the beta-zone of an agarose slide (Clin Chem 1973;19:1139-41). In our method, we separate low-density lipoprotein (LDL) in a second electrophoretic step, in which LDL moves toward the anode, and then quantify the cholesterol of the above lipoproteins remaining in the precipitate band at the beta-zone. Replicate within-run precision (CV) of 15 aliquots of a sera pool was 10.1%. The correlation with sequential ultracentrifugation of 30 samples was r = 0.96 (P less than 0.001). Serum reference values for 30 normal individuals are 57 +/- 7.0 mg/L. Seven phenotype III hyperlipoproteinemic patients had the highest concentrations of IDL or beta-VLDL cholesterol in serum, 1620 +/- 346 mg/L.

New types of large-pore polyacrylamide-agarose mixed-bed matrices for DNA electrophoresis: pore size estimation from Ferguson plots of DNA fragments.

The average pore size value of gels containing polyacrylamide, covalently linked to agarose, was found to be 30% higher than the value of a regular N,N'-methylenebisacrylamide (Bis) cross-linked gel of the same %T. By increasing the agarose concentration (10% of the total amount of polyacrylamide), gels containing low amounts of acrylamide (1.5-2%) are reproducibly obtained; their pore sizes are 130% larger than the pore sizes of a 4%T, 3.3%C polyacrylamide gel. In general, mixed-bed matrices were found to be more elastic and mechanically stronger than classical polyacrylamide gels since an agarose-induced gelation process takes place during their polymerization.

Separation, real-time migration monitoring and selective zone retrieval using a computer controlled system for automated analysis.

High throughput routine analysis of dsDNA fragments or molecular weight determination of proteins via electrophoresis still require significant efforts to obtain results of high reproducibility and accuracy. This paper analyses the use of a fully automated multi-purpose real-time gel electrophoresis system in these applications and evaluates the benefits of this new concept for routine and research. By comparing currently used systems with this new approach, it also addresses the analytical use of information resulting from real-time dynamic migration monitoring via fluorescence photometry over commonly obtained results from post-run fixation, visualization and evaluation at a single time of the separation. The simultaneous separation of components in multi-gel systems, pre-concentration of sample components, and the ability to perform in-gel assays for biological activity are discussed on basis of routine gel separations of restriction enzyme digested DNA fragments, native and denaturing protein separations and enzyme activity determination. Interactive, selective retrieval of separated components in the nano- to microgram range is carried out for real-time isolation of proteins or dsDNA fragments. Results are compared to blotting in respect to ease of use and transfer efficiency and for immediate availability of macromolecules for sequencing or mass spectroscopy. The Windows-based operating software is critically reviewed for functionality, user-friendliness, graphical data representation and GLP compliance for LIMS oriented forensic or certified laboratories. A statistical evaluation of lane-to-lane and gel-to-gel reproducibility of mobility data, quantification and molecular weight determination concludes the paper.

Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol.

Lipoprotein(a) [(Lp(a)], a low-density lipoprotein (LDL)-like particle, contains in addition to LDL a specific protein component, apolipoprotein(a) [apo(a)]. Conventionally, Lp(a) has been measured by immunological methods that distinguish between Lp(a) and LDL by dealing with apo(a) as an antigen. We describe a new method to determine Lp(a) on the basis of its cholesterol content. Very-low-density lipoproteins were removed from serum by preparative ultracentrifugation at a density of 1.006 kg/L. The infranate was subjected to agarose gel electrophoresis to separate Lp(a) and LDL. Lp(a) cholesterol was then determined by direct enzymatic staining for cholesterol. On electrophoresis of the > 1.006 kg/L (bottom) fraction, Lp(a) migrates to the pre-beta position, regardless of the genetic apo(a) isoform. The interassay CVs of Lp(a) cholesterol determinations ranged from 6.9% to 11.5%, and the results correlated well with the Lp(a) concentrations measured by immunonephelometry (r = 0.937). There was an inverse relation between the molecular mass of the genetically determined apo(a) isoforms and Lp(a) cholesterol concentrations. Patients with angiographically proven coronary artery disease (CAD) had significantly more Lp(a) cholesterol than healthy controls did. The ratio of Lp(a) cholesterol to immunologically determined Lp(a) tended to be lower in CAD patients, suggesting that Lp(a) particles contained less cholesterol than apo(a). In addition, the new method allows determination of LDL cholesterol without contamination by Lp(a).

Automatic analysis of agarose gel images.

MOTIVATION: Automatic tools to speed up routine biological processes are very much sought after in bio-medical research. Much repetitive work in molecular biology, such as allele calling in genetic analysis, can be made semi-automatic or task specific automatic by using existing techniques from computer science and signal processing. Computerized analysis is reproducible and avoids various forms of human error. Semi-automatic techniques with an interactive check on the results speed up the analysis and reduce the error. RESULTS: We have successfully implemented an image processing software package to automatically analyze agarose gel images of polymorphic DNA markers. We have obtained up to 90% accuracy for the classification of alleles in good quality images and up to 70% accuracy in average quality images. These results are obtained within a few seconds. Even after subsequent interactive checking to increase the accuracy of allele classification to 100%, the overall speed with which the data can be processed is greatly increased, compared to manual allele classification. AVAILABILITY: The IDL source code of the software is available on request from jonathan.flint@well.ox.ac.uk

[Creatine kinase BB activity in the serum and bronchial aspirate of preterm newborns with respiratory distress syndrome]. / Actividad de la isoenzima CKBB en suero y broncoaspirado de recién nacidos pretérmino con síndrome de distrés respiratorio.

OBJECTIVE: The aim of this study was to test the utility of serum creatine kinase (CK) isoenzyme determinations as a marker of tissue injury in preterm newborns with respiratory distress syndrome (RDS). PATIENTS AND METHODS: Two groups of neonates were studied, 26 suffering from RDS who required mechanical ventilation and 20 healthy newborns with gestational ages, hours of life and birth weights similar to the first group. The activity of CK and its isoenzymes was determined in the bronchial aspirate and serum samples that were obtained before and 24 hours after exogenous surfactant therapy. The isoenzymes were separated by electrophoresis on agarose gel and their activity expressed as a percentage of the total CK. Total proteins were quantified in the bronchial aspirate and CK enzymatic activity expressed in U/mg of protein x 10-3. RESULTS: The CK-BB isoenzyme was significantly increased (p < 0.001) in the serum of infants with RDS compared with the control group. In the bronchial aspirate, the isoenzymatic study showed that the CK-BB isoenzyme represented 98-100% of the total enzymatic CK activity. CONCLUSIONS: The study shows significant differences in the CK isoenzyme patterns of neonates with RDS compared to controls. An increase in serum levels of the CK-BB isoenzyme could be an effective marker of tissue injury in lung disease in the newborn.

Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.

Many bis-intercalating dyes used for fluorescence detection of DNA in electrophoresis have been reported to give band-splitting and band-broadening, which results in poor resolution and a decreased detection sensitivity. We have studied the dimeric dye YOYO-1, and to some extent also TOTO-1 and EthD-1, and found that in complex with DNA these dyes give rise to two components with different electrophoretic mobilities. Electrophoresis experiments and spectroscopic measurements on the two components show that they differ in that the DNA molecules have different amounts of dye bound. Our results exclude that the extra bands are caused by intermolecular cross-linking. Incubation of the samples for increasing times before electrophoresis makes the bands move closer and closer to each other as the dye molecules become more homogeneously distributed among the DNA molecules. Finally, the two bands merge into one at an intermediate position. This equilibration process is extremely slow at room temperature (days), and is therefore not a practical method to eliminate band-splitting in routine analysis. However, we find that if the temperature is raised to 50 degrees C, the dye-DNA complexes equilibrate completely in only 2 h.

Chemiluminographic detection of von Willebrand factor multimeric composition.

Diagnosis of von Willebrand's disease (vWD) requires quantitation of von Willebrand factor (vWF) in plasma plus qualitative assessment of the vWF multimers according to molecular size ranges. Characterization of vWF multimeric size distributions is typically done using sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) followed by immunoblotting in the gel with radiolabeled antibody against vWF and autoradiographic exposure. We applied a western blot technique to vWF multimeric analysis. It included SDS-AGE, electroblotting onto a membrane, and chemiluminescent detection using rabbit anti-human vWF as primary antibody and goat anti-rabbit IgG as secondary antibody conjugated with horseradish peroxidase. Using this method, 18 to 20 vWF multimers were regularly resolved in normal plasma with exposure times of 2 to 4 sec compared to 4 hr or longer by autoradiography. Sensitivity of detection was at least 4-fold enhanced by chemiluminescence compared to radiolabel. Specificity of the assay was confirmed by analysis of plasma samples known to be deficient to different degrees in the larger vWF multimers. The chemiluminographic assay for vWF multimers is superior to the autoradiographic one because it is more sensitive, avoids use of radioactivity, and has shorter total assay time (under 2 days versus five radiolabel).

Principios y aplicaciones de la reacción en cadena de la polimerasa / Principle and application of the chain reaction of the polymerase

El descubrimiento de la reacción en cadena de la polimerasa (PCR), técnica para la amplificación de ácidos nucleicos, ha tenido un enorme impacto sobre áreas diversas, tanto de la investigación básica como de la investigación clínica. Desde su descubrimiento en 1985, los informes sobre una gran variedad de aplicaciones de la PCR han recibido mucha atención en la literatura médica y científica. Esta tecnología ha demostrado tener una gran aplicabilidad para el diagnóstico de enfermedades humanas, incluyendo áreas tan diversas como enfermedades infecciosas, desórdenes genéticos y cáncer. Este artículo presenta una amplia revisión de los principios de la PCR, que incluyen conceptos genéricos que deben manejarse cuando se use o se diseñe un ensayo basado en la PCR. También se discute la aplicación de tales ensayos para el diagnóstico de desórdenes genéticos y para el estudio de enfermedades infecciosas. En los últimos 10 años ha aumentado, de manera notable, la aplicación de herramientas de la biología molecular para el diagnóstico de las enfermedades humanas. Los ensayos con sondas de ADN están, ahora, disponibles comercialmente para la detección e identificación de una gran variedad de patógenos humanos, así como para el diagnóstico de desórdenes genéticos humanos. Una manifestación del rápido crecimiento de la tecnología del ADN, ha sido el desarrollo de técnicas para amplificar secuencias específicas de ácidos nucleicos. En la literatura, han sido descritos muchos métodos, que se enumeran en la tabla i. Una comparación de estos métodos ha sido el tema de una revisión frecuente. Entre ellos se destacan la reacción en cadena de la polimerasa, como la de mayor impacto, tanto como herramienta de investigación como de diagnóstico. El objetivo de este artículo es presentar una breve revisión de los principios y aplicaciones de la amplificación de PCR, que incluyen una discusión de la selección correcta de las secuencias en blanco, el diseño de los "primer" y los medios necesarios para llevar a cabo la técnica

Marcadores pronósticos de carcinoma mamario: experiencia del hospital de oncología / Breast carcinoma prognostic cancer markers: experience in the oncology hospital

Se investigó la presencia de marcadores pronósticos en el citosol de 323 neoplasias de glándula mamaria. En 107 de éstas se cuantificaron las concentraciones de alfa-1-intitripsina y se observó que fueron más bajas en los carcinomas de pacientes con recurrencia tumoral, después de la mastectomía, en comparación con aquellos de pacientes sin recurrencia tumoral (p<0.01). Las pacientes cuyas neoplasias tuvieron valores más altos de alfa-1-antitripsina y no presentaron recurrencia tumoral mostraron una mayor probabilidad se supervivencia en cinco años comparadas con las que tuvieron valores más bajos de alfa-1-antitripsina en el carcinoma y presentaron recurrencia tumoral (p<0.0001). Aparentemente la presencia de concentraciones bajas de alfa-1-antitripsina en el carcinoma mamario, es un indicador de mal pronóstico en estas pacientes. Por otro lado, en 126 carcinomas de mama se investigó el receptor estrogénico, el grado de diferenciación y la recurrencia tumoral y se observó que en las neoplasias grado de diferenciación II con receptor estrogénico negativo, el porcentaje de pacientes con recurrencia tumoral, después de la mastectomía, fue tres veces más alto que en los casos receptor estrogénico positivo. Estos resultadosa sugieren que se puede predecir la presencia de recurrencia tumoral en las pacientes con neoplasias grado de diferenciación II y receptor estrogénico negativo.