High-resolution Electrophoresis Research Articles

BRCA1 and BRCA2 whole cDNA analysis in unsolved hereditary breast/ovarian cancer patients

Germline pathogenic variants in BRCA1 and BRCA2 genes (BRCA1/2) explain an important fraction of hereditary breast/ovarian cancer (HBOC) cases. Genetic testing generally involves examining coding regions and exon/intron boundaries, thus the frequency of deleterious variants in non-coding regions is unknown. Here we analysed BRCA1/2 whole cDNA in a large cohort of 320 unsolved high-risk HBOC cases in order to identify potential splicing alterations explained by variants in BRCA1/2 deep intronic regions. Whole RNA splicing profiles were analysed by RT-PCR using Sanger sequencing or high-resolution electrophoresis in a QIAxcel instrument.Known predominant BRCA1/2 alternative splicing events were detected, together with two novel events BRCA1 ▼21 and BRCA2 Δ18q_27p. BRCA2 exon 3 skipping was detected in one patient (male) affected with breast cancer, caused by a known Portuguese founder mutation (c.156_157insAluYa5). An altered BRCA2 splicing pattern was detected in three patients, consisting in the up-regulation of ▼20A, Δ22 and ▼20A+Δ22 transcripts. In silico analysis and semi-quantitative data identified the polymorphism BRCA2 c.8755–66T>C as a potential modifier of Δ22 levels.Our findings suggest that mRNA alterations in BRCA1/2 caused by deep intronic variants are rare in Spanish population. However, RNA analysis complements DNA-based strategies allowing the identification of alterations that could go undetected by conventional testing.

Abstract 724: Application of oncoMonitor™ ctDNA tracking technology for monitoring of therapy and early detection of recurrence in metastatic colorectal cancer

Abstract Introduction: Liquid biopsy based on detection of circulating tumor DNA (circulating-tumor DNA, ctDNA) is finding new applications in clinical management of solid cancers. From the initial use as an alternative to classical tissue biopsy for investigation of molecuar profiles in prediction or resistance detection of targeted therapies the approach is now applied to monitoring of minimal residual disease (MRD). Patients with metastatic colorectal cancer undergoing liver resection are subject to standard post-operative surveillance based mainly on periodic CT or PET/CT imaging (complemented eventually by MR imaging). Some reports have previously demonstrated benefit from performing a ctDNA detection complimentary to standard imaging to improve follow-up efficiency. Methods: In this report we demonstrate use of our oncoMonitor™ ctDNA technology, based on detection of somatic mutations derived from tumor in patient plasma. Unlike various approaches targeting oncogenic hotspot mutants, this technology can trace virtually any somatic mutation found in any gene (e.g. a tumor supressor). The relatively simple approach is based on rapid scanning of 100 to 140bp target amplicons for presence of mutations revealed based on differential melting by high-resolution denaturing capillary electrophoresis (DCE). The detection sensitivity is 0.1% MAF. Patients: In our concordance study during which a total of 41 patients with metatatic colorectal cancer were followed for 3 years from metastasectomy. oncoMonitor™ tast was applied to evaluate ctDNA along with standard CT or PET/CT imaging and tumor markers (CEA/CA19-9). Results: A total of 23 recurrences were clinically confirmed during surveillance. All recurrences were detected by oncoMonitor™ (23/23, 100%), while at the same time only 17 instances were detected by CT or PET/CT imaging (17/23, 73.9%) and 16 by tumor markers CEA/CA19-9 (16/23, 69.6%). There were 2 patients after R0 resection with persistent ctDNA positivity who had recurrence within 6 months from surgery. Conclusions: oncoMonitor™ technology is suitable for quick and sensitive detection of ctDNA in plasma of patients with metatatic colorectal cancer after liver resection. Evaluation of ctDNA presence improves efficiency of post-operative surveillance. Supported by Czech Ministry of Health project no. 17-31909A. Citation Format: Marek Minarik, Barbora Belsanova, Renata Ptackova, Tereza Halkova, Filip Pazdirek, Jiri Pudil, Miroslav Levy, Jaromir Simsa, Jiri Hoch, Miroslav Ryska, Lubos Petruzelka, Lucie Benesova. Application of oncoMonitor™ ctDNA tracking technology for monitoring of therapy and early detection of recurrence in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 724.

Standardization of 1- and 2-D protein profiling of Haloxylon species: Basis of proteome analysis of haloxerophytes

One and two dimensional electrophoresis is a powerful technique for separation of proteins from complex biological sources. These are effective techniques of gene expression analysis at translational level especially for the species of which genome is not yet sequenced. High resolution electrophoresis separation of proteins from complex biological systems is crucial in protein science. Three protein extraction methods, such as Phenol, Multi detergent and TCA acetone were analyzed in the present investigation to find out appropriate protein extraction method amenable to one dimensional (1-D) and two dimensional (2-D) protein profiling of Haloxylonrecurvumand H. salicornicum. Plant species selected for the present investigation are major components of saline and arid ecosystem and valuable source of food additives, nutritional ingredients, herbal medicines and fuel in the region. Phenolic components present in these plants are major hindrances for extraction of pure proteins. High concentrations of good quality proteins represented by clear, intense and more bands/spots in 1-D and 2-D gels were extracted with Phenol extraction method in comparison to TCA acetone and Multi detergent methods. This is possibly thefirst report on extraction and purification of proteins amenable to 1-D and 2-D proteome profiling of Haloxylon species of Indian arid zone, and forms a basis for further comparative proteome analysis of these two contrasting species with respect to salinity and drought tolerant ability for prospects.

CRISPR/Cas9 system for disruption of biochemical pathway for sterol synthesis in Artemisia annua L.

WHO recommends artemisinin-based combination therapy for curing malaria which is still a health problem in Indonesia. Artemisia annua L. is the primary source for artemisinin comes from subtropical China and India. Some introduction materials are grown in Indonesia, but they produce low concentration of artemisinin. Artemisinin synthesis uses the isoprenoid pathway, in which farnesyl diphospate (FDP) serves as the main precursor. FDP is the main precursor for sterol synthesis as well. The gene controls sterol biosynthesis is Squalene synthase (SQS). So then SQS is considered a competitive gene for artemisinin biosynthesis. CRISPR/cas9 is the most advanced system for genome editing, and can be used to induce a targeted-mutation. The aim of this research was to elevate Artemisinin content through disruption of the biochemical pathway of sterol synthesis using CRISPR/Cas9. Materials and methods used were as follows. Artemisia CRISPR/Cas9 seeds were processed and grown in selection medium and then soil. Seventy-seven Artemisia kanamycin resistant lines were produced. Fifty-two (of 77) lines were confirmed to have T-DNA by PCR with SQS-Fn and SQS-Rn primers, produced about 710 bp DNA fragment. By using SQS-Fs and SQS-Rs primers, those produced shorter DNA fragments (about 470 bp). A high-resolution electrophoresis QIAxcel was applied to identify for any nucleotide difference occured in PCR product of lines. Results showed that 44 lines (84.61%) have varied sizes with one to three nucleotides differences compared to control (plasmid, 468 bp). By assuming this analysis was right, it may be said that mutations has occured in SQS gene of Artemisia lines due to the CRISPR/Cas9.

Selective screening for detection of mucopolysaccharidoses in Malaysia; A two-year study (2014–2016)

IntroductionMucopolysaccharidoses (MPS) are a group of inherited disorders caused by the deficiency of a specific lysosomal enzyme involved in glycosaminoglycans (GAGs) degradation. This enzyme deficiency leads to accumulation of GAGs in the lysosomes, resulting in organ dysfunction and enlargement. We aimed to detect cases of MPS in patients with suggestive signs and symptoms. MethodsThis was a 2-year cross-sectional study conducted during June 2014 to May 2016. Urine and whole blood samples were taken from high-risk MPS patients. All urine samples were analysed for GAGs and characterised by high resolution electrophoresis (HRE). Whole blood was collected in ethylenediaminetetraacetic acid (EDTA) tube and analysed for specific enzymes based on the clinical history and HRE findings. ResultsFrom the 60 samples tested, 15 were positive for MPS; (Type I = 1), (Type II = 4), (Type IIIA = 3), (Type IVA = 1), (Type VI = 6). The overall prevalence of MPS among high-risk Malaysian patients was 26% (95% CI 14.72% to 37.86%). One patient had mucolipidosis. The mean age of patients when diagnosed was 5 years old. Patients with MPS were more likely to present with hepatosplenomegaly compared to other symptoms (OR = 0.974, p < .05). ConclusionOne in 4 high-risk patients was diagnosed with MPS being MPS type VI the most common among Malaysian patients. Hepatosplenomegaly was the most common symptom. Patients with suspected MPS should be screened by urinary GAGs analysis and diagnosis confirmed by enzyme activity analysis.

Heat dissipation in slab gel electrophoresis: The effect of embedded TiO2 nanoparticles on the thermal profiles

Despite the fast development of novel and high-resolution electrophoresis techniques such as capillary-based methods and microfluidic devices, the slab gel electrophoresis is still a popular method for the separation of biomolecules in medicine and biology. It is a low cost and simple method and offers high throughput. However, this technique is limited to low voltages leading to slow separations. Producing the heat during the electrophoresis known as Joule heating inevitably leads to a rise in the gel temperature. For the first time, this work offers a whole gel temperature measurement by using a thermal camera which presents accurate temperature profiles in the gel with a resolution of more than 10 pixel/mm2 and a precision of 0.1 °C. Titania, TiO2, nanoparticles (NPs) were embedded into the polyacrylamide (PA) gel to improve the electrophoretic separation of proteins. By embedding 0.025% w/v TiO2 NPs, heat dissipation increases by 16.5% at applied voltage of 200 V compared with that of PA gel with no embedded TiO2 NPs. The thermal images showed that the composite gel was 2.5 °C in average cooler than PA gel after 15 min of electrophoresis run at 200 V. The maximum separation voltage increased by 30 V in the composite PA/TiO2 gel compared with the pure PA gel. Moreover, the average number of theoretical plates over the 10 protein peaks, as a criterion of separation performance, increased by about 63% at 180 V when TiO2 NPs were included into the gel.

Infrared Spectroscopy of Blood Serum from Patients with Multiple Myeloma

A combination of high-resolution agarose gel protein electrophoresis, refractometric analysis, and infrared spectroscopy was applied to investigate the blood serum in patients with multiple myeloma (MM). Analysis of the FTIR spectra of the serum obtained from MM patients and healthy donors revealed a decreased amount of α-helixes and increased content of β-sheets in serum proteins from MM patients compared to healthy donors. These findings suggest an interaction between serum proteins and stabilization of the resulting complexes, accompanied by the formation of associated β-sheets.

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.

ABSTRACTThe in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.

A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences

HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis.

A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future.

Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.

Improved densitometric quantification of β-region paraproteins with high-resolution gel electrophoresis

Improved densitometric quantification of β-region paraproteins with high-resolution gel electrophoresis

Typing of Four Cases of Monoclonal Gammopathy: A Revival of Immunosubstraction Role

Monoclonal gammopathy of undetermined significance (MGUS) is characterized by increased production of an immunoglobuling (Ig) from a clone of plasma cells and is a pre-malignant disorders in subjects older than 50 years. The prevalence of MGUS in Caucasian population is still not determined. MGUS is characterized by the presence of a monoclonal-protein(M-protein) (IgG and IgA) lower than 30 g/L, bone marrow plasma cell percentage lower than 10%, and absence of clinical signs related to multiple myeloma (MM). MGUS can be responsible for damage to organs through the production of toxic M proteins that may have autoantibody activity or deposit pathologically in the organ tissues. Many techniques are available for the characterization of M-proteins. These techniques can involve different expenses, skills, labor time, and sensitivity in detecting monoclonal proteins also at low-level. Detection of M-proteins needs of assays based on high-resolution electrophoresis and im-munofixation (or immunosubtraction). We show suggestive clinical cases where the subjects involved had not an apparent disease but they showed an interesting pattern in electrophoresis. All cases were investigated by capillary’s electrophoresis and immunofixation to confirm or not the clinical suspect, and then if the immunofixation is not exhaustive, additionally immunosubstraction is done. However in some cases, the interpretation of the peaks is not so easy. Clinical and scientific data provided evidences that immunofixaction technique can fail the identification of monoclonal components. In that cases, we opted for the immunosubtraction method as a third level test, in that cases when immunofixation failed the identification of a monoclonal protein.

Multiplexed set of 10 microsatellite markers for identification of potato varieties

Genetic identification of potato varieties is a demanded instrument for development of new cultivars registration system, protection of plant breeders’ rights, and variety homogeneity control. The most perspective approach for distinction and identification of varieties continues to remain the use of short tandem repeats. STR amplification with the subsequent high resolution electrophoresis allows such a unique characteristic of a variety to be obtained as the DNA profile. A large scale of samples requires the creation of a robust and time-saving technique based on fragment sizing. We selected 10 polymorphic STR loci of potato: STI0032, STG0016, STI0001, STI0004, STM1104, STM5127, STI0030, STI0033, STI0014, STM5114 and designed a multiplex panel for potato DNA profiling. Fluorescent labelling of primers and size distinction of amplicones allowed us to use one tube for PCR and capillary electrophoresis. We also modified the CTAB-protocol for DNA extraction from tubers and other parts of potato plants, the PCR mix recipe and the amplification protocol for good results. Using Genetic Analyzer allows the length of alleles to be defined with an accuracy of one nucleotide and digitized genetic profiles to be developed. We created a unique DNA profile for each of 40 varieties and 23 breeding lines from Russia and other countries and evaluated the homogeneity of 8 varieties. The proposed technique оf potato DNA profiling allows a large number of samples to be rapidly analyzed in the 96-well plate format.

Validation of an electrophoretic method to detect albuminuria in cats.

Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were <10%, percentage recovery was between 97% and 109% with a high linearity (r = 0.99). HRE allowed the visualisation of a faint band of albumin and a diffused band between alpha and beta zones in healthy cats, while profiles from diseased cats were variable. Albumin (mg/dl) and urine albumin:creatinine ratio (UAC) were significantly ( P <0.05) different between healthy and diseased cats. After ROC analysis, UAC values of 0.035 and 0.074 had a high sensitivity and high specificity, respectively, to classify proteinuria and identify borderline proteinuric cats. Moreover, a UAC of 0.017 had a high sensitivity in distinguishing between healthy and diseased cats. However, UAC was not able to distinguish between renal (CKD) and non-renal diseases (LUTD/OTHER), probably owing to the pathophysiology of CKD in cats, which is characterised by low-grade proteinuria and less glomerular involvement than in dogs. Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.

Comparison of FXII contact activation with or without proteolytic cleavage

Event Abstract Back to Event Comparison of FXII contact activation with or without proteolytic cleavage Yuan Yan1, Lichong Xu1 and Christopher A. Siedlecki1 1 The Pennsylvania State University Hershey Medical Center, Department of Surgery and Bioengineering, United States Introduction: Surface-induced plasma coagulation is potentiated by contact activation of the blood zymogen factor XII (FXII, Hageman factor). Recent studies revealed that FXII surface activation exhibited different characteristics in neat-buffer and in plasma[1]. aFXIIa and βFXIIa are well known products of FXII contact activation via enzymatic cleavage in the presence of plasma proteins such as prekallikrein, plasmin, and trypsin[2]. Activation products of FXII in buffer (FXII autoactivation) are yet to be fully understood. This study compared the products of FXII activation with or without adding prekallikrein (PK) with regard to activated FXII composition, procoagulant and amidolytic activities. Materials and Methods: Activators used were glass particles (425-600 µm, Sigma Aldrich) in either cleaned or silanized form. 500 µL of 30 µg/ml FXII in PBS was brought into contact with 100 mg of different activators with or without added PK for 1 hr on a rotating hematology mixer. The products present in the supernatant were then assessed by high-resolution electrophoresis (Agilent Protein 230 kit) for molecular weight analysis, a plasma coagulation time assay for procoagulant yield, and a chromogenic assay with Pefa-5963 for amidolytic yield. Results and Discussion: Electrophoretic analyses of FXII activation products in neat-buffer with or without PK. In the absence of PK, the products of FXII activation showed only one band in the size of 95-98 kDa regardless of surface activator, indicating that no proteolytic cleavage takes place in FXII autoactivation. Instead, these FXII-derived products appear to be of the same molecular weight as initial FXII. Note that they present as~ 20% higher molecular weight as observed by automated electrophoresis due to the glycosylation of FXII[3]. In the presence of PK, FXII-derived fragments with smaller molecular weight of about 33 kDa were observed. This matches the size of shared catalytic domain by both aFXIIa and βFXIIa. Yields of FXII-derived products by different activators are shown in Table 1 according to the concentrations calculated by electrophoresis software. Functional analyses of FXII activation products in buffer with or without PK (Figure 1). FXII autoactivation takes place at all surface types, not only at anionic, as demonstrated by measurable procoagulant and amidolytic activities. However, procoagulant yields from OTS and APTES were much lower than that of Glass, suggesting activators of varying surface energy induce different FXII conformers displaying remarkably different procoagulant activity. The presence of PK (or proteolytic cleavage) amplified both procoagulant and amidolytic yields from all surface types in a manner roughly proportional to the amount of βFXIIa and total FXIIa, respectively. Conclusion: FXII contact activation in buffer does not produce proteolytic fragments. It is likely that FXII undergoes non-enzymatic conformational changes in response to surface contact, thus showing different procoagulant and amidolytic activities depending on surface energy. The presence of prekallikrein amplifies both activities, likely as the result of subsequent enzymatic cleavage of FXII into aFXIIa and βFXIIa.

Typing Candida Species Using Microsatellite Length Polymorphism and Multilocus Sequence Typing.

To gain more insight into the epidemiological relationships between isolates of Candida spp. obtained from various origins, several molecular typing techniques have been developed. Two methods have emerged in the 2000s as soon as enough knowledge of the Candida spp. genomes was available to choose adequate loci and primers, namely microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST). To contrast with previous PCR-based methods, specific amplifications with stringent conditions easily reproducible are the basis of MLP and MLST. MLST relies on Sanger sequencing to detect single-nucleotide polymorphisms within housekeeping genes. MLP needs a first in silico step to select tandemly repeated stretches of two to five nucleotides. One of the two primers used to amplify a microsatellite locus is labeled and fragment sizing is automatically performed using high-resolution electrophoresis platforms. MLST provides results easily comparable between laboratories and active MLST schemes are publicly available for the main Candida species. For comparative studies, MLP needs standards to compensate for the electrophoretic variations depending on the platforms used. Both methods can help us gain insight into the genetic relatedness of fungal isolates, both with advantages and drawbacks, and the choice of one method rather than the other depends on the task in question.

Old but Still Relevant: High Resolution Electrophoresis and Immunofixation in Multiple Myeloma.

High resolution electrophoresis (HRE) and immunofixation (IFX) of serum and urine are integral to the diagnostic work-up of multiple myeloma. Unusual electrophoresis patterns are common and may be misinterpreted. Though primarily the responsibility of the hematopathologist, clinicians who are responsible for managing myelomas may benefit from knowledge of these. In this review article we intend to discuss the patterns and importance of electrophoresis in present day scenario. Patterns of HRE and IFX seen in our laboratory over the past 15years were studied. Monoclonal proteins are seen on HRE as sharply defined bands, sometimes two, lying from γ- to α-globulin regions on a background of normal, increased or decreased polyclonal γ-globulins, showing HRE to be a rapid and dependable method of detecting M-protein in serum or urine. Immunofixation complements HRE and due to its greater sensitivity, is able to pick up small or light chain bands, not apparent on electrophoresis, including biclonal disease even when electrophoresis shows only one M-band. Special features liable to misinterpretation are discussed. Familiarity with the interpretation of the varied patterns seen in health and disease is essential for providing dependable laboratory support in the management of multiple myeloma.

Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response

Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison with healthy controls (HCs; n=16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p=0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination.

Urinary Glycosaminoglycan Electrophoresis With Optimized Keratan Sulfate Separation Using Peltier System for the Screening of Mucopolysaccharidoses

The purpose of this communication is to indicate a simple and rapid method with a small volume of urine sample to detect urine glycosaminoglycan (GAG) and serve as a screening procedure for mucopolysaccharidoses (MPSs). Total GAG measurement for patients with MPS disorders is considered to be the first step in diagnosis of those heterogeneous group of lysosomal storage disorders presenting clinical phenotype. In this study, modified 9-dimethylmethylene blue method is used for total GAG measurement. Following GAG quantitation, the procedure described here allows GAG isolation from a very a small volume of urine sample and subjected to high-resolution GAG electrophoresis, which can be easily performed in routine clinical diagnostic laboratories. Glycosaminoglycan precipitation is a modified method based on total GAG concentration in the urine. For optimized isolation of total GAG for electrophoresis, instead of considering the urine creatinine concentration, 300 μg/mL GAG containing urine is considered to be the target concentration for the best precipitation with 1000 μL cetylpyridinium chloride (CPC)/citrate buffer. Glycosaminoglycan concentration-based precipitation of urine with CP Ca llows the laboratory to be able to work with as mall volume of urine sample by keeping the precipitating ratio with CPC constant for samples that contain GAG less than 300 μg/mL. Based on the effect of cold buffer using low voltage, GAGs high-resolution electrophoresis banding patterns described here enable a clear separation of keratan sulfate from chondroitin sulfate as well as dermatan sulfate (DS1 and DS2) and heparan sulfate. By this procedure, GAG patterns are more clear, easily identified, and provide a guide for the enzyme analysis deficient in the MPS disorders.