Capillary Electrophoresis Assay Research Articles

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, β-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

Long-term HIF-1α stabilization reduces respiration, promotes mitophagy, and results in retinal cell death

Ocular hypertension during glaucoma can lead to hypoxia, activation of the HIF transcription factors, and a metabolic shift toward glycolysis. This study aims to test whether chronic HIF activation and the attendant metabolic reprogramming can initiate glaucoma-associated pathology independently of ocular hypertension. HIF-1α stabilization was induced in mice for 2 and 4 weeks by inhibiting prolyl hydroxylases using the small molecule Roxadustat. HIF-1α stabilization and the expression of its downstream bioenergetic targets were investigated in the retina by immunofluorescence, capillary electrophoresis, and biochemical enzyme activity assays. Roxadustat dosing resulted in significant stabilization of HIF-1α in the retina by 4 weeks, and upregulation in glycolysis-associated proteins (GLUT3, PDK-1) and enzyme activity in both neurons and glia. Accordingly, succinate dehydrogenase, mitochondrial marker MTCO1, and citrate synthase activity were significantly decreased at 4 weeks, while mitophagy was significantly increased. TUNEL assay showed significant apoptosis of cells in the retina, and PERG amplitude was significantly decreased with 4 weeks of HIF-1α stabilization. A significant increase in AMPK activation and glial hypertrophy, concomitant with decreases in retinal ganglion cell function and inner retina cell death suggests that chronic HIF-1α stabilization alone is detrimental to retina metabolic homeostasis and cellular survival.

AGREE, hexagonal and whiteness assessment approaches for evaluating two novel analytical methods; capillary zone electrophoresis and spectrophotometric assays for simultaneous determination of pantoprazole, chlordiazepoxide, and clidinium bromide ternary mixtures

Going Green through pharmaceutical analysis is not a luxury anymore. Maintaining sustainability and eco-friendliness is targeted as urgently as the validation of the newly developed procedures is. Although no firm guidelines have been implemented yet, the AGREE, Hexagon, and Whiteness “RGB 12” models confirm and fairly arbitrate the method greenness approval. In this consensus, two novel methods; Capillary Zone Electrophoresis-Diode Array Detection and Double Divisor Ratio Spectra Derivative Spectrophotometric assays are developed and fully validated for the simultaneous determination of Clidinium bromide, Chlordiazepoxide, and Pantoprazole ternary polypill. Linearity ranges of 1–100 μg/mL for the three drugs were achieved using CZE, meanwhile, 2.5–50, 1–30, and 5–80 μg/mL for Clidinium bromide, Chlordiazepoxide, and Pantoprazole, respectively were achieved using Spectrophotometry. Both methods have been validated as per International Council for Harmonization guidelines and applied for three drugs’ estimation in laboratory-made pills. Greenness and sustainability appraisals of the proposed procedures were evaluated by the three former models versus the reported HPLC and HPTLC methods for the cited mixture. Suggested methods declared higher green attributes than previous ones.

Diagnostic performance of glycated haemoglobin (HbA1c) for diabetes mellitus in dogs.

The objective of this study was to determine the diagnostic performance of glycated haemoglobin (HbA1c) for canine diabetes mellitus (DM) and compare it with that of serum fructosamine. Aliquots of blood samples collected for diagnostic purposes from adult dogs were used. HbA1c was measured using a previously validated capillary electrophoresis assay. The dogs were allocated into four groups: (1) DM; (2) hyperadrenocorticism (HAC); (3) long-term corticosteroid therapy (CST); and (4) various chronic diseases (VD). In total, 88 dogs were included as follows: DM (n=11), HAC (n=10), CST (n=14), and VD (n=53). Fructosamine was measured in all four groups as follows: DM (n=6), HAC (n=7), CST (n=9), and VD (n=42). Median (range) serum glucose concentration was higher (P<0.001) in the DM group (22.8mmol/L; range, 15.6-29.3mmol/L) compared to HAC (5.9mmol/L; range, 4.2-6.8mmol/L), CST (5.6mmol/L; range, 4.3-23.3mmol/L), and VD (5.5mmol/L; range, 4.1-9.4mmol/L) groups. Mean (±standard deviation) HbA1c was higher (P<0.001) in the DM group (6.3%±1.5%) compared to HAC (1.9%±0.5%), CST (1.7%±0.5%), and VD (1.9%±0.5%) groups. All diabetic dogs and none of the other dogs had HbA1c levels above the cut-off value for DM (3.3%), indicating an accuracy of 100% in diagnosing DM. Significant differences (P<0.01) were observed in median fructosamine between the DM group (389 μmol/L; range, 348-865 μmol/L) and the HAC (306 μmol/L; range, 167-348 μmol/L) and the VD (316 μmol/L; range, 189-500 μmol/L) groups. Fructosamine had an accuracy of 84.4% for the diagnosis of DM. When used for the diagnosis of canine DM, HbA1c measured with this specific assay had excellent diagnostic accuracy and was superior to serum fructosamine.

Validation of Urinary Thiocyanate as a Robust Biomarker of Active Tobacco Smoking in the Prospective Urban and Rural Epidemiological Study.

Tobacco smoking is a leading preventable cause of premature death globally. Urinary thiocyanate is a biomarker of cyanide exposure from tobacco smoke; however, few studies have evaluated its utility in diverse populations of smokers. We examined the associations between urinary thiocyanate and self-reported never and current smokers among 1000 participants from 14 countries in the Prospective Urban and Rural Epidemiological study. We analyzed urinary thiocyanate in light and heavy smokers as compared to never-smokers from high- (HICs), middle- (MICs), and low-income countries (LICs) using a validated capillary electrophoresis method in conjunction with standardized questionnaires. The median urinary thiocyanate concentration was 31 μM, which ranged from 8.6 μM to 52 μM for never-smokers (n = 335) and current smokers (n = 660), respectively. Urinary thiocyanate was correlated with daily cigarette consumption (r = 0.621) and total nicotine equivalents (r = 0.514). Thiocyanate also displayed a better dose-response than urinary cotinine. A moderate association of urinary thiocyanate was found in biochemically verified never-smokers (r ~0.38) because of intake of vegetables, fruits, and dairy. Receiver-operating characteristic curves established cutoff values for urinary thiocyanate to differentiate current from never-smokers with an optimal threshold of 23.9 μM (Area Under the Curve or AUC = 0.861), which lowered progressively from HICs, MICs, and LICs. Elevated thiocyanate was evident in current smokers from high-income countries likely reflecting differences in smoking topography and greater toxicant burden. Background urinary thiocyanate in never-smokers was associated with goitrogenic food intake that obscured detection of secondhand smoke exposure. Urinary thiocyanate is a sensitive biomarker of active tobacco smoking relative to cotinine that can be measured by an inexpensive capillary electrophoresis assay. Regional cutoff values are demonstrated to improve discrimination of smoking status in developing countries because of differences in smoking habits and cigarette products consumed, as well as intake of goitrogenic foods. Urinary thiocyanate may allow for more reliable estimates of the hazards of tobacco smoking between countries with varying socioeconomic development as compared to self-reports.

Comparing Data Generated Using Multiparametric Flow Cytometry, Capillary Electrophoresis, and Next-Generation Sequencing in a Cohort of B-Cell Lymphoproliferative Disorder Samples

Background: Determining whether suspected lymphoid proliferation disorder (LPD) samples are benign or malignant remains a significant challenge for the hematopathologist. For the vast majority of LPDs, the current gold standard includes assessment of clinical characteristics, cytomorphology, and immunophenotyping by flow cytometry; however, 10-15% of LPDs require further resolution by molecular methods, such as PCR-based capillary electrophoresis or next-generation sequencing, to rule out clonality based on V-D-J rearrangements in the immunoglobulin heavy-chain (IGH) gene. Invivoscribe (San Diego, CA) has developed multiple complementary methodologies to evaluate LPDs; including multiparametric flow cytometry (MFC), capillary electrophoresis (CE), and next-generation sequencing (NGS). To compare performance between these three methods, a cohort of 21 clinical samples were tested in a pilot study. Method: The CAP/CLIA Hematolymphoid Screening assay, a 10-color flow panel, was used to identify all major types of hematopoietic neoplasia according to the 2006 Bethesda Consensus and 2017 WHO Classification. For molecular testing, DNA was extracted from peripheral blood (PB), then PCR-based assays were used to determine presence or absence of clonality. The Research Use Only (RUO) molecular assays were designed for CE (ABI 3500xL) and NGS (MiSeqTM) detection, utilizing framework multiplex PCR master mixes targeting the conserved framework regions FR1, FR2, or FR3 within the VH and the JH regions of the IGH gene. Twenty-one (21) de-identified PB samples were collected from informed consensus donors (LabPMM, San Diego, CA) or from residual samples (Lahey Hospital, Burlington, MA) and tested under an IRB approved protocol. An aliquot of each PB sample was used for MFC evaluation, then the remaining DNA was extracted and tested using the CE and NGS methods. Test results were reported as "present abnormal" or "normal" for MFC and "clonal" or "non-clonal" for molecular assays based on pre-defined criteria. Results: The MFC assay identified 12 out of 21 (57%) samples as "present abnormal" and 9 out of 21 (43%) samples as "normal." Both molecular assays (CE and NGS) reported 12 out of 21 (57%) samples as "clonal" and 9 out of 21 (43%) samples as "non-clonal." The results between CE and NGS assays were 100% in agreement for clonality detection. Comparisons between the CE and MFC assays again resulted in 100% agreement; all 12 "present abnormal" samples by MFC were reported as "clonal" by CE, while all 9 "normal" samples by MFC were "non-clonal" by CE. In addition to presence of clonality, the NGS method was able to detect the prevalence of a specific clonal V-D-J rearrangement sequence (expressed as % reads), which ranged from 10% to 99% in the 12 clonal samples. This pilot study demonstrated excellent concordance (21/21) between these 3 methods (MFC, CE and NGS). Accordingly, MFC, CE and NGS are all suitable methods for the evaluation and stratification of B-cell LPD samples. The combined use of all 3 methods provides a comprehensive assessment of clonality: including surface markers identified by MFC, DNA rearrangement lengths from CE, and the specific DNA rearrangement sequences from NGS. In addition, the NGS method allows for tracking of these clonal sequences in relation to disease status. Conclusion: This pilot study demonstrated 100% concordance between 3 different methods (MFC, CE, and NGS) when evaluating samples from LPD subjects. Depending on laboratory capabilities and the type of information desired, any one of these technologies can provide appropriate results when studying clonality. The alignment between MFC and NGS allows the combined use of both methods to provide rapid diagnosis via the MFC service and identification of specific biomarkers for subsequent tracking of these clonal populations in clinical studies.

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line.

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing.

Bayesian latent class analysis of the characteristics of diagnostic tests to assess the passive immunity transfer status in neonatal Belgian Blue beef calves

The aim of the current study was to assess the diagnostic characteristics of radial immunodiffusion (RID), capillary electrophoresis (CE) and digital brix refractometry (Bx) for the diagnosis of failure of passive transfer (FPT) of immunity in neonatal Belgian Blue beef calves in the absence of a gold standard using a Bayesian latent class model. Belgian blue beef calves (n = 202) from a large farm in the south of Belgium were blood-sampled at 48–72 h of age and tested for FPT. The true prevalence of FPT in this population of calves was 34.5 % (95 % BCI: 26.1–44.3) using a FPT cut-off point of 10 g IgG/L. This true prevalence increased to 66.3 (95 % BCI: 56.9–74.8) and 88.9 % (95 % BCI: 83.1–93.2) at FPT cut-off points of respectively 18 and 25 g IgG/L serum. The Bland-Altman plot comparing the RID and CE methods, revealed that the serum IgG concentrations obtained by RID were on average 2.25 (95 % CI 1.62–2.88) g/L higher than those measured by CE. Optimal cut-off values for CE, corresponding to the FPT values as measured by RID of 10, 18, and 25 g IgG/L serum, were respectively 10, 15, and 20 g IgG/L. The overall diagnostic accuracy of the three diagnostic tests was comparable at the FPT cut-off point of 10 g IgG/L serum (i.e. 85 %). At higher cut-off points for FPT, the RID and CE assays presumably performed better that the Bx method. In conclusion, we demonstrated that: (1) the CE method is a good alternative for the RID assay, the latter having important constraints when considering its practicality, and (2) the Bx method is a cheap and user-friendly indirect method to evaluate the FPT in new-born Belgian Blue beef calves.

The Prevalence and Risk Factors Associated with Iodine Deficiency in Canadian Adults.

Iodine is a trace micronutrient that is critical for normal thyroid function and human health. Inadequate dietary intake is associated with cognitive impairment, infertility, growth retardation and iodine deficiency disorders in affected populations. Herein, we examined the prevalence of iodine deficiency in adults (median age of 61 years) based on the analysis of 24 h urine samples collected from 800 participants in four clinical sites across Canada in the Prospective Urban and Rural Epidemiological (PURE) study. Urinary iodide together with thiocyanate and nitrate were measured using a validated capillary electrophoresis assay. Protective/risk factors associated with iodine deficiency were identified using a binary logistic regression model, whereas daily urinary iodine concentration (24 h UIC, μg/L) and urinary iodine excretion (24 h UIE, μg/day) were compared using complementary statistical methods with covariate adjustments. Overall, our Canadian adult cohort had adequate iodine status as reflected by a median UIC of 111 μg/L with 11.9% of the population <50 μg/L categorized as having moderate to severe iodine deficiency. Iodine adequacy was also evident with a median 24 h UIE of 226 μg/day as a more robust metric of iodine status with an estimated average requirement (EAR) of 7.1% (< 95 μg/day) and a tolerable upper level (UL) of 1.8% (≥1100 μg/day) based on Canadian dietary reference intake values. Participants taking iodine supplements (OR = 0.18; p = 6.35 × 10−5), had greater 24 h urine volume (OR = 0.69; p = 4.07 × 10−4), excreted higher daily urinary sodium (OR = 0.71; p = 3.03 × 10−5), and/or were prescribed thyroxine (OR = 0.33; p = 1.20 × 10−2) had lower risk for iodine deficiency. Self-reported intake of dairy products was most strongly associated with iodine status (r = 0.24; p = 2.38 × 10−9) after excluding for iodine supplementation and T4 use. Participants residing in Quebec City (OR = 2.58; p = 1.74 × 10−4) and Vancouver (OR = 2.54; p = 3.57 × 10−4) were more susceptible to iodine deficiency than Hamilton or Ottawa. Also, greater exposure to abundant iodine uptake inhibitors from tobacco smoking and intake of specific goitrogenic foods corresponded to elevated urinary thiocyanate and nitrate, which were found for residents from Quebec City as compared to other clinical sites. Recent public health policies that advocate for salt restriction and lower dairy intake may inadvertently reduce iodine nutrition of Canadians, and further exacerbate regional variations in iodine deficiency risk.

Chimerism monitoring using biallelic single nucleotide or insertion/deletion polymorphisms: How many markers to screen?

Background/aimsChimerism monitoring by means of high-throughput sequencing or quantitative PCR of biallelic single nucleotide and insertion/deletion polymorphisms has shown potential for improved patient care when compared to the gold standard capillary electrophoresis assays. When designing chimerism assays the number of markers to screen needs consideration: it determines the informativity rate and accuracy of the assay, but screening too many markers increases the assay’s cost and complexity. The minimal number of biallelic markers to screen is currently unstudied. Materials/methodsA simulation framework accounting for marker minor allele frequencies, the number of markers screened, marker allelic constellations and donor-recipient relatedness was constructed. The framework was validated through analysis of 324 clinical samples. ResultsEmpirical clinical data confirm the validity of the simulation framework. With guidelines suggesting to monitor at least three informative markers, we demonstrate that, for optimized assays, at least 40 biallelic markers need to be screened to achieve enough informative markers in over 99% of cases. We propose and discuss several assay optimization strategies. ConclusionCurrently used chimerism assays often screen too little or too many markers, leaving room for optimization. Through support of the simulation framework here introduced and validated, more informative, cost-effective chimerism assays can be designed.

Phenotypic Expression of Known and Novel Hemoglobin A2-Variants, Hemoglobin A2-Mae Phrik [Delta 52(D3) Asp > Gly, HBD:c.158A > G], Associated with Hemoglobin E [Beta 26(B8) Glu > Lys, HBB:c.79G > A] in Thailand

The interactions of δ-globin variants with α- and β-thalassemia or other hemoglobinopathies cause complex thalassemic syndromes and potential diagnostic problems. Understanding the molecular basis and phenotypic expression is crucial. Four unrelated Thai subjects with second hemoglobin (Hb) A2 fractions were studied. A standard automated cell counter was used to acquire initial hematological data. Hb analysis was carried out by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assays. Globin gene mutations and haplotype were identified by appropriate DNA analysis. An allele-specific polymerase chain reaction method was developed to provide a simple molecular diagnostic test. Hb analysis revealed a Hb A2 variant in all cases. DNA analysis of the δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu > Lys] variant in combination with Hb E in three cases. Analysis of the remaining case identified a novel δ-Hb variant, namely Hb A2-Mae Phrik [δ52(D3)GAT > GGT; Asp > Gly], found in association with Hb E and α+-thalassemia, indicative of the as yet undescribed combination of triple heterozygosity of globin gene defects. An allele-specific PCR-based assay was successfully developed to identify this variant. The β-haplotype of the Hb A2 Mae-Phrik allele was strongly associated with haplotype [+ − − − − ± +]. This study advanced our understanding of the phenotypic expression of known and novel δ-Hb variants coinherited with other globin gene defects, routinely causing problems with diagnosis. Therefore, knowledge and recognition of this Hb variant and molecular assessments are crucial to improving diagnosis.

The effect of age and sex on glycated hemoglobin in dogs.

We investigated the effect of age and sex on canine glycated hemoglobin (HbA1c) using a validated capillary electrophoresis assay. Aliquots of EDTA blood samples collected for routine health checks were used. HbA1c was measured using the Capillarys 2 flex-piercing system (Sebia). We included 58 clinically and hematologically healthy, normoglycemic dogs (29 males, 29 females), allocated to 3 age groups: young (14 dogs <1-y-old), adult (31 dogs 1-7.9-y-old), and senior (13 dogs ≥8-y-old). The mean (± SD) HbA1c was not significantly different (p = 0.428) between the age groups (young: 1.68 ± 0.54%; adult: 1.59 ± 0.41%; senior: 1.80 ± 0.57%). The HbA1c was not significantly correlated with age (rho = 0.144, p = 0.280). The median (range) HbA1c was not significantly different (p = 0.391) between male [1.7% (0.5-2.5%)] and female [1.5% (1.0-2.7%)] dogs. Age and sex do not appear to affect canine HbA1c; however, a study of geriatric dogs would be needed to fully exclude an effect of age on HbA1c.

Next Generation Sequencing (NGS) Based IGH and TCR Clonality Assays Provide Excellent Specificity and Sensitivity for Routine Clonal Characterization and Monitoring of Lymphoproliferative Disorders

Background:IGH and TCR gamma clonality testing is an important component in the diagnosis of lymphoproliferative disorders. Capillary electrophoresis (CE) of multiplexed PCR products is currently considered the gold standard method of analysis. Although robust, simple and highly reproducible, it does not provide full characterization of clonal sequences and lacks sensitivity and specificity to track clones in subsequent samples. Here we describe our clinical implementation of an NGS based assay for routine assessment of lymphoproliferative disorders and outline the clinical utility for initial clonal characterization and minimal residual disease monitoring through patient specific clone tracking.Methods:Blood, bone marrow and FFPE tissue samples submitted for routine clonality assessment during Jan 2016 to March 2017 were identified. DNA was extracted and tested using both standard CE and LymphoTrack® IGH + TRG - MiSeq assays (InvivoScribe). Results were analyzed using an in-house developed software (LymphoClone) as well as proprietary InvivoScribe software. The lymphoma classification at the time of initial diagnosis, disease status and results of ancillary studies, including morphologic assessment, immunohistochemical analysis, flow cytometry (FC) and molecular test results were recorded for each case.Results:A total of 716 samples were analyzed, including 534 samples at the time of initial diagnosis (Dx) for clonal characterization and 182 follow up samples for disease monitoring. The case distribution is outlined in table 1. Sensitivity for initial clonal characterization was superior for the NGS assays, allowing clonal detection down to 1% tumor content, compared to 5-10% for the CE assays. Clonal rearrangement was detected in 94% of Dx cases by NGS (502/534) vs 89% CE (475/534). For monitoring samples with a previously characterized clone, NGS was equivalent to FC for detection of plasma cell neoplasms (PCN) but showed distinct advantages in disease monitoring for B-ALL, B and T cell lymphomas. Among 182 monitoring samples, NGS testing allowed residual disease detection in 25 (14%) more cases (137/182 vs 112/182) compared to flow cytometry. In all cases, residual disease was confirmed by an ancillary method (qPCR for a molecular marker) or a subsequent sample with higher disease level. The sensitivity for MRD detection for NGS has been established as 1x10-5. Sensitivity for FC ranged between 1x10-4 to 1x10-5 (1x10-4 for T and B-ALL and 1x10-5 for PCN and B cell lymphomas). Higher sensitivities may be attained by NGS with increasing DNA input. The NGS assay provided additional information on distinct clonal tumor population behavior, including suppression and/or re-emergence of subpopulations following treatment. In the current study, turnaround time (TAT) was higher for NGS vs FC, 9 days vs 1 day, respectively.Conclusions:Routine assessment of clonality by NGS methods is feasible and provides significant improvement over existing CE clonality assays, enabling comprehensive clonal characterization and disease monitoring with high sensitivity which is similar and often superior than FC. Based on current technology and protocols, TAT is longer for NGS compared to both CE and FC assays, which could limit its use as a stand-alone test in select clinical settings. Further protocol optimization is in progress to reduce this time to ~7 days. The NGS assays further define behaviors of clonal tumor populations and should therefore be considered in the routine assessment of lymphoid malignancies in conjunction with FC and other ancillary molecular tests. [Display omitted] DisclosuresArcila:Raindance Tecnologies: Honoraria; Invivoscribe: Honoraria; Archer: Honoraria. Park:Amgen: Consultancy. Dogan:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peer Review Institute: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche Pharmaceuticals: Consultancy; Celgene: Consultancy.

Validation of a Next-Generation Sequencing–Based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing

Assessment of T-cell receptor γ gene (TRG) rearrangements is an importants consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard of such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment size-based methods, given that they generate results with specific clonotype sequence information and allow for more accurate quantitation. The present study evaluated various test parameters and performance characteristics of a commercially available NGS-based TRG gene-rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%. The concordance rates were 88% to 95% in the Vγ1-8, Vγ10, and Vγ11 gene families, but lower in the Vγ9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing clonotypes of the same fragment size. This clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay.

Diagnosing a Challenging Case of Primary Amyloidosis using Capillary Electrophoresis

Amyloid Light chain (AL) amyloidosis is characterised by deposition of intact free light chains or their fragments in extracellular space. Here, authors describe the journey of a diagnostically challenging patient who presented with features of nephrotic syndrome and was finally diagnosed with AL amyloidosis. A 45-year-old female presented to the Outpatient Department (OPD) with gradually progressive generalised body swelling. On examination, hepatomegaly, cardiomegaly and macroglossia were observed. Renal biopsy, capillary serum protein electrophoresis, immunotyping and serum free light chain assay were performed along with routine blood and urine investigations to detect the presence of monoclonal protein. Bone marrow biopsy was conducted for confirmation of diagnosis. Proteinuria with hypoalbuminaemia 1.8 g/dL was detected during routine investigations. Renal biopsy showed presence of amyloid deposits in glomerular mesangium and walls of medium sized blood vessels which tested positive for Immunoglobulin (Ig) G, IgA, kappa and lambda chains on immune florescence. Serum protein capillary electrophoresis findings demonstrated increase in beta 2 fraction and distortion in gamma region. Immuno typing showed presence of monoclonal IgA heavy chains and lambda light chains. Bone marrow biopsy confirmed presence of plasma cell dyscrasia. Based on these findings authors concluded that capillary gel electrophoresis is more sensitive method than agarose gel electrophoresis in detecting beta migrating monoclonal proteins.

The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission

Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans—one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and l-citrulline (co-product of NO synthesis from l-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.

α-Thalassemia Intermedia Results from Interactions of Unstable Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] with the α-Thalassemia-1 [– –SEA (Southeast Asian)] Deletion in Thailand

The clinical consequences of many abnormal hemoglobins (Hbs) interacting with α- or β-thalassemia (α- or β-thal) or other hemoglobinopathies have not been described. We evaluated a 75-year-old Thai woman and her 45-year-old daughter. Hematological data was obtained on an automated cell counter. Hemoglobin (Hb) analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) assays. Mutations and globin haplotypes were identified by appropriated DNA techniques. The proband presented with moderate anemia and inclusion bodies in most of the red blood cells (RBCs), while altered RBC parameters were absent in her daughter. Hemoglobin analysis showed an abnormal Hb peak only in the proband. DNA analysis identified a G>T substitution at codon 31 of the α1-globin gene, corresponding to Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] in both subjects. The α-thal-1 [– –SEA (Southeast Asian)] deletion was also identified in the proband, but not in her daughter. These mutations could be identified using newly developed allele-specific polymerase chain reaction (ASPCR) assays. The α haplotypic analysis demonstrated the Thai Hb Prato allele was associated with haplotype [+ – S + – + –] [the S represents the inter ζ hypervariable region (HVR)]. The combination of the unstable Hb Prato with α-thal-1 result in α-thal intermedia (α-TI) phenotypes. A simple DNA method is essential for detection, and a haplotypic α-globin gene cluster are presented.

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16.

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α2β2 and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)‐4‐{[(S)‐4‐benzyl‐2‐oxo‐1,3‐oxazolidin‐3‐yl]carbonyl}‐10‐(3,4,5‐trimethoxyphenyl)‐4,5,10,10a‐tetrahydrofuro[3,4‐b]carbazole‐1,3(3aH)‐dione ((+)‐3 a)) was prepared in an one‐pot, three‐component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide (9) and N‐methylimide (10) substructure. The enantiomer (−)‐3 a (K i=4.9 μM) of the lead compound (+)‐3 a (K i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein‐protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)‐3 a did not show an increased enzyme inhibition of the CK2α2β2 holoenzyme, the CK2α subunit or the mutated CK2α′ C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)‐9 a (K i=3.6 μM) and the N‐methylimide (+)‐10 a (K i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)‐12, with a carboxy moiety in the 4‐position, displays the highest CK2α/CK2β interaction inhibition (K i=1.8 μM) of this series of compounds.

Food Authentication: Identification and Quantitation of Different Tuber Species via Capillary Gel Electrophoresis and Real-Time PCR.

Truffles are hypogeous fungi mainly found in Europe and Asia. Due to their special aroma and taste, some truffle species are sold on the international market at an extremely high price. Among the economically relevant species, the white Alba truffle (Tuber magnatum) and the black Périgord truffle (T. melanosporum) are the most appreciated species. The fruiting bodies of the Asian black truffle are morphologically very similar to T. melanosporum, and those of the Bianchetto truffle (T. albidum Pico) are similar to T. magnatum, but are of little economic value. Highly valued species are adulterated with cheaper ones, especially. Because of this problem, the aim of this study was the development of methods for detecting possible admixtures to protect consumers from fraud. This study is based on seven different truffle species (117 fruiting bodies) from different growing regions. Additionally, selected truffle products were included. Using this material, a real-time PCR (polymerase chain reaction) assay allowing the detection and quantitation of Asian black truffles in T. melanosporum up to 0.5% was developed. In addition, a capillary gel electrophoresis assay was designed, which allows the identification and quantitation of different species. The methods can be used to ensure the integrity of truffle products.

A Simple Approach to Analyze Sugar Nectar Composition in Flowers Using Capillary Electrophoresis and Enzymatic Assays

Despite the obvious importance of nectar, the composition of this solution remains surprisingly understudied to most of the flowers. Here we describe a simple, low-cost, and reliable methodology to analyze the three main sugars present in 210 nectar samples of Christmas Bellflowers using capillary electrophoresis (CE) and enzymatic assays. CE separation (-16 kV) was made using an electrolyte containing 36 mM NaOH pH 12, 15 mM sorbic acid, 0.5 mM hexadecyl trimethylammonium bromide (CTAB), and indirect UV detection. 72.9% of analyzed nectar samples were sucrose dominant; 13.3% sucrose rich only and 13.8% were fructose rich. The concentrations of fructose and glucose present in most of the samples were very similar, while the amount of sucrose varied considerably. The average amounts of fructose, glucose, sucrose and total sugar were 0.22, 0.21, 0.34 and 0.8 M, respectively. The nectar sample shows a pH average of 5. α-Glucosidase activity was observed in 65% of the samples with an average activity of 0.013 µU µL-1 providing a possible explanation for the similar concentrations of fructose and glucose in the samples. Under our knowledge, this is the first article that shows the analysis of sugars in nectar flowers using CE.