Capillary Electrophoresis Research Articles

Pressurized liquid extraction of sulfite for quantitation by capillary zone electrophoresis.

Sulfite is an additive used in shrimp processing to prevent discoloration. However, sulfite can cause health issues for sensitive consumers, making its monitoring necessary. Determining sulfite concentrations is complex because the Monier-Williams reference method is laborious and has low analytical throughput. Additionally, new techniques are needed to stabilize sulfite during the extraction process because this analyte undergoes rapid oxidation. A new method involving extraction and derivatization of sulfite with formaldehyde through automated pressurized liquid extraction (PLE), followed by quantitation by capillary zone electrophoresis with diode-array detector in indirect mode, was developed and optimized using multivariate planning. The PLE procedure was compared to another solid-liquid extraction method. The new method successfully stabilized and extracted sulfite from shrimp in few steps with adequate precision (CV<3.8%), producing extracts that were stable for 10 days. Recovery was satisfactory (97%-99%), and the limits of detection (4.6mgkg-1) and quantitation (15.4mgkg-1) were suitable for the intended purpose.

Electroextraction of Large Volume Samples Using Paper Points Coupled With Hollow Fiber Membranes: Study of Parameters and Strategies to Enhance Analytical Performance.

Electroextraction (EE) encompasses a range of sample preparation methods whose effectiveness, selectivity, and efficiency are significantly influenced by the physical-chemical characteristics of analytes, samples, and instrumental conditions. This article explores, for the first time, various strategies aimed at enhancing the extraction efficiency of a recent approach of EE utilizing a paper point (PP) combined with a hollow fiber (HF) (abbreviated as PP-HF-EE) to extract various cationic and anionic model compounds from water samples. The study also explores, experimentally, the impact of agitation, organic filter composition, PP diameter, and PP brand on extraction performance, and proves that all these factors are quite important, especially when digital image analysis is utilized for determination. Furthermore, this work demonstrates the ease and feasibility of simultaneously extracting cations and anions using PP-HF-EE and proposes a straightforward method to enhance analyte concentration on the vertex of the PP through a base-to-vertex focusing. Lastly, it is demonstrated, using capillary electrophoresis coupled to a UV-Vis detector, that for PP-HF-EE, the extraction efficiency and pre-concentration factor are less dependent on other parameters when multiple PPs per sample are utilized, with signal enhancement values of up to 111 and 339 for nortriptyline and haloperidol, respectively. All the findings and strategies presented herein constitute significant contributions that can facilitate future research in method development, particularly in the utilization of PP-HF-EE and similar EE approaches.

Urinary proteomic signature of mineralocorticoid receptor antagonism by spironolactone: evidence from the HOMAGE trial

Abstract Background Heart failure (HF) is characterised by collagen deposition. Urinary proteomic profiling (UPP) detects many sequenced peptides, for &amp;gt;70% derived from collagens. Purpose UPP and serum fibrosis markers were analysed together in patients at risk of HF with as objective to generate insights into the antifibrotic action of spironolactone. Methods In the open-label HOMAGE trial, patients were randomised to usual therapy combined or not with spironolactone 25-50 mg/d and followed for 9 months (n=290; 23.8% women; median age: 73 years). UPP was done by capillary electrophoresis coupled with mass spectrometry; 1498 urinary peptides with detectable signal in ≥30% of patients were analysed. Serum markers of COL1A1 synthesis (PICP and PICP/CITP) were measured. After rank normalisation of the biomarker distributions, between-group differences in their changes were assessed by multivariable-adjusted models. Correlations between the changes in urinary peptides and in serum PICP and PICP/CITP were compared between groups using Fisher Z transform. Results Among all the urinary peptides, only the changes in collagen fragments remained significantly different (p&amp;lt;0.05) between randomisation groups after accounting for baseline levels, covariables and multiple testing. Compared to the control group, spironolactone reduced 16 of 27 collagen-derived urinary peptides. From baseline to 9 months, serum PICP and the PICP/CITP ratio decreased from 79.0 to 75.4 µg/L and from 21.3 to 18.3, respectively (p≤0.0129), reflecting decreased COL1A1 synthesis. Spironolactone did not affect the correlations between changes in urinary COL1A1 fragments and the serum fibrosis markers. Conclusions Spironolactone downregulated urinary collagen-derived peptides, probably by shrinking the body-wide pool of collagens. Spironolactone did not affect the interaction between urinary and serum fibrosis markers, suggesting that collagen scaffolding is maintained, thereby explaining why some urinary collagen fragments increased. Combining urinary and serum fibrosis biomarkers opens new avenues for discovery of antifibrotic drugs and refines insight in the action of antifibrotic drugs.Figure 1Figure 2

OSTEO18, a novel urinary proteomic signature, associated with osteoporosis in heart transplant recipients

Abstract Background Immunosuppressive treatment in heart transplant (HTx) recipient causes osteoporosis. The urinary proteomic profile (UPP) includes peptide fragments derived from the bone extracellular matrix. Purpose Study aims were to develop and validate a multidimensional UPP biomarker for osteoporosis in HTx patients from single sequenced urinary peptides identifying the parent proteins. Methods A single-center HTx cohort was analyzed. Urine samples were measured by capillary electrophoresis coupled with mass spectrometry. Cases with osteoporosis and matching controls were randomly selected from all available 389 patients. In derivation case-control dataset, 1576 sequenced peptides detectable in ≥30% of patients. Applying statistical analysis on these, an 18-peptide multidimensional osteoporosis UPP biomarker (OSTEO18) was generated by support vector modeling. The 2 replication datasets included 118 and 94 patients. For further validation, the whole cohort was analyzed. Statistical methods included logistic regression and receiver operating characteristic curve (ROC) analysis. Results In derivation dataset, the AUC, sensitivity and specificity of OSTEO18 were 0.83 (95% CI: 0.76-0.90), 74.3% and 87.1%, respectively. In replication datasets, results were confirmatory. In the whole cohort (154 osteoporotic patients [39.6%]), the ORs for osteoporosis increased (p &amp;lt; 0.0001) across OSTEO18 quartiles from 0.39 (95% CI: 0.25-0.61) to 3.14 (2.08-4.75). With full adjustment for known osteoporosis risk factors, OSTEO18 improved AUC from 0.708 to 0.786 (p = 0.0003) for OSTEO18 categorized (optimized threshold: 0.095) and to 0.784 (p = 0.0004) for OSTEO18 as continuously distributed classifier. Conclusion OSTEO18 is a clinically meaningful novel biomarker indicative of osteoporosis in HTx recipients and is being certified as in-vitro diagnostic.

Method Validation for the Determination of Ciprofloxacin in Lucilia sericata Larvae via Capillary Electrophoresis Combined With Mass Spectrometry.

Substances derived from insects can serve therapeutic functions due to their diverse biological properties. This article focuses on the species Lucilia sericata and the benefits of larval therapy in patients who, due to hospitalization, have developed pressure ulcers and other difficult-to-heal wounds. Larval therapy, also known as maggot debridement therapy, employs sterile fly larvae to treat chronic, non-healing wounds by enzymatically degrading necrotic tissue and decreasing bacterial colonization. The larvae are applied to the wound for a period of 48-72h, during which they effectively clean the wound and stimulate tissue regeneration. This therapeutic approach is particularly efficacious for recalcitrant wounds, such as diabetic foot ulcers and pressure sores, which have not responded to conventional treatments. Larvae may also constitute an alternative material in entomotoxicological studies to detect substances ingested at not only toxic but also therapeutic doses. The present work describes a method for assaying ciprofloxacin in L. sericata larvae using capillary electrophoresis coupled to mass spectrometry. The developed method features high sensitivity with a limit of quantification of 100±0.018ng/mL, as well as accuracy and precision estimated within 87%-103% and 1%-4%, respectively. An application of a simple and fast precipitation of proteins procedure for sample cleaning resulted in a highly satisfactory recovery of the analyte (90%-104%). The method was linear in a range of 100-1000ng/mL with a determination coefficient higher than 0.9973. The method was used to determine ciprofloxacin in larval homogenate after antibiotic administration to the patient at a dose of 500mg twice daily per os during application of the larvae dressing. Ciprofloxacin was shown to distribute from the patient's circulation to the larvae at a concentration of 150ng/mL (750ng/g).

Novel Applications of CE-ICP-MS/MS: Monitoring of Antiaging GHK-Cu Cosmetic Component Encapsulation in Liposomes.

The hyphenation of the separation technique with the high-sensitive mass spectrometry detection is one of the driving forces of modern analysis enabling measurements in complex matrices. In particular, capillary electrophoresis coupled to inductively coupled plasma tandem mass spectrometry allows for speciation analysis of selected analytes with a superior resolution. The mild, physiological-friendly conditions of this separation technique offer the unique advantage of analyzing chemical entities in their intact form, which has been successfully exploited in various areas. Herein, we report the pioneering application of such a hyphenated technique in the cosmetic field to investigate the encapsulation of copper tripeptide complex (GHK-Cu) in liposomes. By monitoring copper and phosphorus signals, the formation of liposomes via a simple ethanol injection method was confirmed, and the concentration of GHK-Cu in the liposomes was assessed. The application of coupling of capillary electrophoresis with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) in cosmetic studies could lead to the development of diverse liposomal formulations with preferential properties and expand their accessibility.

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Comparison of Two Different Integration Methods for Quantifying Monoclonal Proteins on Agarose Gel and Capillary Zone Electrophoresis Instruments

Assessment of diagnostic accuracy of Gazelle: A point-of-care testing device for screening β-thalassemia trait

Background &amp; objectives Timely detection of population with β-thalassemia trait (BTT) followed by genetic counselling is an advocated method of preventing the birth of a child with β-thalassemia major. In this study we aim to assess the diagnostic accuracy of Gazelle, a point-of-care (POC) testing device, in screening for BTT in hospital laboratory setting. Methods Standards for Reporting Diagnostic Accuracy (STARD) guidelines were followed in developing study design, recruiting study participants and sample size calculation for the current research. A consecutive sample of 446 participants was recruited for this study and was tested for the reference test Gazelle as well as the gold standard cation exchange high performance liquid chromatography (CE-HPLC). Low serum ferritin levels are known to interfere with the production of HbA2 and in turn lead to false negative results. Hence, the study population was divided into two categories with respect to a cut off value of 15ng/dl of serum ferritin and the results were analyzed. Results Overall diagnostic accuracy of Gazelle for detecting BTT was found to be 95.3 per cent with a confidence interval (CI) of (92.9 - 97.1) and the sensitivity was 94.5 per cent with a 95% CI of (84.8 - 98.8). When analyzed by the serum ferritin level the diagnostic accuracy was found to be 94.7 per cent (91.1% - 97.1%) and 95.7 per cent (91.8% - 98.1%) for participants with serum ferritin level as &gt; 15 ng/ml and &lt; 15 ng/ml, respectively. Interpretation &amp; conclusions This study found Gazelle to be a good screening tool for β-thalassemia trait with high sensitivity, specificity and accuracy. However, it is recommended that the final confirmation of the diagnosis done by a diagnostic test like HPLC or Capillary Zone Electrophoresis (CZE).

Multimass Analysis of Adeno-Associated Virus Vectors by Orbitrap-Based Charge Detection Mass Spectrometry.

Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids. Analytical methods have been established for evaluating CQAs, such as analytical ultracentrifugation, capillary gel electrophoresis with laser-induced fluorescence detection, and capillary gel electrophoresis using sodium dodecyl sulfate with UV detection. Here, we present a multimass analysis of AAV vectors using orbitrap-based charge detection mass spectrometry (CDMS), a single-ion mass spectrometry. Orbitrap-based CDMS facilitates the quantitative evaluation of the genome packaging status based on the mass distribution of empty and full particles. Additionally, we established a novel method to analyze the encapsidated genome directly without pretreatment, such as protein digestion or heat treatment, and to estimate the stoichiometric variation of VP for the capsid based on the mass distribution constituted by the single peak corresponding to AAV particles. Orbitrap-based CDMS is a distinctive method that allows multiple mass characterizations of AAV vectors with a small sample volume of 20 μL for 1013 cp/mL in a short time (30 min), and it holds the potential to become a new standard method in the assessment of CQAs for AAV vectors.

Critical analysis of analytical techniques developed for statins in biological fluids, environmental and fermentation samples.

Statins are the most prescribed drug for regulating the high cholesterol level in the blood, which can lead to severe complications, such as cardiovascular diseases and other health complications. A wide range of analytical techniques have been employed for the quantification of statins from various origins, including fermentation derived (lovastatin, pravastatin, and compactin), semi-synthetic (simvastatin), and synthetic (atorvastatin, rosuvastatin, and fluvastatin) routes. The presence of more than one structural form and structural analogue generated in the biosynthesis pathway, as well as reaction intermediates and macromolecules in the clinical sample, complicates the quantification of statins. Furthermore, significant concentrations of statins in environmental samples pose serious health and ecology hazards, and estimating statins in those diluted samples is extremely difficult. On the other hand, the: cost, accurate estimation of the desired one from other structural forms, sample complexity, time, limits of detection and quantification, were major criteria distinguishing the usability of each technique. As a result, the current manuscript focuses on analytical techniques such as molecular spectroscopy (normal and derivatives UV-Visible spectrophotometer), chromatography (TLC, HP-TLC, HPLC, GC, swing column, micellar, and supercritical fluid), mass spectroscopy (HPLC-MS/MS and GC-MS/MS), sequential flow injection, capillary electrophoresis, and cyclic voltammetry, as well as their: optimal operating conditions, limits of detection and quantification, advancements, and limitations. Furthermore, various online and offline sample preparations (precipitation, solid phase extraction, liquid-liquid extraction, and micellar extraction) have been highlighted as an essential pretreatment technique to avoid the interference caused by structural analogues and other macromolecules. The greener and more sustainable concepts used in analytical approaches for the quantification statins are also highlighted with current advancements.

Molecular and haematological characterisation of haemolytic anaemia associated with biallelic KLF1 mutations: a case series

AimsKrüppel-like factor 1 (KLF1) is an erythroid-specific transcription factor playing an important role in erythropoiesis and haemoglobin (Hb) switching. Biallelic KLF1 mutations can cause haemolytic anaemia with thalassaemia-like syndromes but...

Inverse Shifting-PCR Modified by Capillary Electrophoresis for Detecting F8 int22h and int1h Inversions in Severe Hemophilia A Patients and Probable Carriers.

Globally, intron 22 inversions (Inv22s) of the factor VIII gene (F8) are the most frequent pathogenic variants and account for 45-50% of severe hemophilia A (SHA) cases, while intron 1 inversion (Inv1) explains 1-5% of SHA cases. The detection of both inversions by an inverse shifting-polymerase chain reaction (IS-PCR) is the first choice worldwide for the diagnosis of patients and carriers of SHA. To improve its sensitivity and reproducibility in the visualization of PCR products, we approached the IS-PCR with fluorescent capillary electrophoresis instead of agarose electrophoresis. Based on the original protocol, we modified two primers by 5'-end labeling with FAM™ fluorescent dye for the detection of the PCR products by capillary electrophoresis. Additionally, the "fast enzymes" BclI and T4-Ligase were incorporated for work saving in the genomic digestion and ligation reactions, respectively. Once we accomplished the standardization and verified the reproducibility of the modified IS-PCR method, we applied it for the diagnosis of a cohort of SHA patients and carriers. The modified IS-PCR by fluorescent capillary electrophoresis for PCR product detection is more sensitive than agarose electrophoresis. The method was also improved by using the new rapid enzymes to save time in the whole process.

Separation of tolperisone and its degradation products by a dual cyclodextrin capillary electrophoresis system to study their potential role in allergic events

Tolperisone is a centrally acting muscle relaxant that has been used for the treatment of post-stroke spasticity and low back pain. Recently, the safety of tolperisone pharmaceutical products has been reassessed due to growing concerns over allergic adverse events. Reactive degradants of tolperisone may be responsible for these hypersensitivity reactions. By forming adducts with proteins, they may act as haptens that could evoke allergic reactions. The objective of this study was to examine the presence of these degradants in tolperisone pharmaceutical products and to assess their reactivity to elucidate their possible role in the pro-allergic effect of tolperisone. For this purpose, capillary electrophoresis UV detection (CE-UV) method was developed and validated for the quantification of degradants. A dual cyclodextrin system was applied to achieve the appropriate migration order enabling the analysis of 2-methyl-1-(4-methylphenyl)prop-2-en-1-one (MMP) and 1-(4-methylphenyl)propan-1-one (MMPO) in the presence of high concentrations of tolperisone. MMP was identified as the main degradant in forced degradation tests of the active pharmaceutical ingredient. Differences in MMP content of tolperisone products by different manufacturers have also been found, highlighting the role of formulation in their stability. High reactivity of MMP was demonstrated as rapid and almost complete adduct formation with cysteine was found. This degradant thus might be responsible for the allergic adverse effects of tolperisone even when it is present in trace amounts in tablets by readily reacting with proteins in vivo.

High-Efficiency Enzyme Assay and Screening of Enzyme-Inhibiting Nanomaterials Using Capillary Electrophoresis with Hierarchically Porous Metal-Organic Framework-Based Immobilized Enzyme Microreactor.

Enzyme-inhibiting nanomaterials have significant potential for regulating enzyme activity. However, a universal and efficient method for systematically screening and evaluating the inhibitory effects of various nanomaterials on drug target enzymes has not been established. While the integrated technique of immobilized enzyme microreactor (IMER) with capillary electrophoresis (CE) serves as an effective tool for enzyme analysis, it still faces challenges such as low enzyme loadability, unsatisfactory stability, and limited applicability. Herein, hierarchical porous metal-organic frameworks (HP-MOFs) were explored as high-performance enzyme immobilization carriers and stationary phases to develop a novel HP-MOFs-based IMER-CE microanalysis system for efficient online enzyme assay and systematic screening of enzyme-inhibiting nanomaterials. As a proof-of-concept demonstration, the model enzyme xanthine oxidase (XOD) was immobilized on a HP-UiO-66-NH2 coated capillary, serving as an efficient and durable IMER for screening potential XOD-inhibiting nanomaterials. The hierarchically micro- and mesoporous structure and superior enzyme loadability of as-prepared HP-UiO-66-NH2-IMER was intensively characterized, followed by systematic evaluation of the separation performance of HP-UiO-66-NH2 coated column and the enzyme kinetics of the immobilized XOD. Compared to the microporous UiO-66-NH2-IMER, the HP-UiO-66-NH2-IMER-CE system showed significant improvements in enzyme loading, maximum reaction rate, repeatability, and long-term stability. Furthermore, the established method was effectively employed to screen the XOD inhibitory activity of various nanomaterials, revealing that graphene oxide, single wall carbon nanotube and three other nanomaterials exhibited inhibitory potentials. The HP-MOFs-based microanalysis system can be easily expanded by modifying the types of immobilized enzymes and holds the potential to accelerate the identification and rational design of effective enzyme-inhibiting nanomaterials.

Altered spike IgG Fc N-linked glycans are associated with hyperinflammatory state in adult COVID-19 and Multisystem Inflammatory Syndrome in Children

Abstract Severe COVID-19 and multisystem inflammatory syndrome (MIS-C) are characterized by excessive inflammatory cytokines/chemokines. In adults, disease severity is associated with SARS-CoV-2-specific IgG Fc afucosylation, which induces pro-inflammatory cytokine secretion from innate immune cells. This study aimed to define spike IgG Fc glycosylation following SARS-CoV-2 infection in adults and children and following SARS-CoV-2 vaccination in adults and the relationships between glycan modifications and cytokines/chemokines. We analyzed longitudinal (n=146) and cross-sectional (n=49) serum/plasma samples from adult and pediatric COVID-19 patients, MIS-C patients, adult vaccinees, and adult and pediatric controls. We developed methods for characterizing bulk and spike IgG Fc glycosylation by capillary electrophoresis (CE) and measured levels of ten inflammatory cytokines/chemokines by multiplexed ELISA. Spike IgG were more afucosylated than bulk IgG during acute adult COVID-19 and MIS-C. We observed an opposite trend following vaccination, but it was not significant. Spike IgG were more galactosylated and sialylated and less bisected than bulk IgG during adult COVID-19, with similar trends observed during pediatric COVID-19/MIS-C and following SARS-CoV-2 vaccination. Spike IgG glycosylation changed with time following adult COVID-19 or vaccination. Afucosylated spike IgG exhibited inverse and positive correlations with inflammatory markers in MIS-C and following vaccination, respectively; galactosylated and sialylated spike IgG inversely correlated with pro-inflammatory cytokines in adult COVID-19 and MIS-C; and bisected spike IgG positively correlated with inflammatory cytokines/chemokines in multiple groups. We identified previously undescribed relationships between spike IgG glycan modifications and inflammatory cytokines/chemokines that expand our understanding of IgG glycosylation changes that may impact COVID-19 and MIS-C immunopathology.

Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing

During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R2 > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.

Case Report: Hemoglobin Hasharon with Concurrent Alpha Thalassemia in a Patient with Maple Syrup Urine Disease

Abstract Hemoglobin Hasharon (Hb Hasharon) is an unstable hemoglobin variant stemming from a mutation in the alpha globin gene. This mutation involves the replacement of aspartic acid with histidine at position 47 of the alpha globin chain. Initially identified in 1967 in Israel, Hb Hasharon has since been sporadically reported in various populations, including in Italy, Greece, Germany, and among Latin Americans of Mediterranean descent. Only three English articles have reported several cases of the combined alpha thalassemia deletion and Hasharon mutation, all between 1978-1980. Notably, there has been no prior report of Hb Hasharon with concurrent alpha thalassemia in patients with maple syrup urine disease (MSUD). We hereby present the first case of Hb Hasharon with alpha thalassemia in a patient diagnosed with MSUD. This patient, a 16-month-old female, received a prenatal diagnosis of MSUD through amniocentesis, confirming homozygosity for a known pathogenic variant in DBT (c.75_76delAT, p.C26wfsX2). Prompt initiation of formula feeding has effectively controlled her MSUD. The patient was referred for further evaluation due to hemoglobin newborn screen showing hemoglobin A, Hb F and “variant hemoglobin”. Her parents denied any family history of hemoglobinopathy. Her complete blood count was predominantly normal except for mild microcytosis, with a mean corpuscular volume of 69.1 fl (normal range 70.0-86.0 fl). The patient didn’t exhibit neonatal jaundice or any other signs or symptoms of hemolysis. The reticulocyte count was also within normal limits. The hemoglobin electrophoresis results revealed a complex pattern, with 28.8% of an unknown hemoglobin between zone 4 and 5 on capillary zone electrophoresis (CZE) as well as a possible A2 variant in zone 1. High-performance liquid chromatography (HPLC) indicated a 29.1% peak at a retention time of 4.72 minutes, along with several smaller peaks, that were indicative of the degradation of the unstable unknown hemoglobin. Although the zones and retention time aligned with Hb Hasharon, the percentage differed from the expected range of 19-20% in CZE and 18-21% in HPLC for this variant, prompting the team to order alpha and beta globin sequencing tests. Beta globin gene complete sequencing yielded negative results, while the sequencing of alpha globin gene revealed that the patient carries one copy of the -alpha3.7 alpha-globin deletion and one copy of the c.142G&amp;gt;C (p.Asp48His) variant in the alpha2-globin (HBA2) gene, resulting in an -alpha/alpha2alpha1 genotype with Hb Hasharon. While Hb Hasharon is generally not clinically significant, its co-occurrence with alpha-thalassemia can lead to hemolytic anemia. In our patient, clinic and chemical parameters were predominantly within normal ranges, except for mild microcytosis. The potential interaction between MSUD and Hb Hasharon with concurrent alpha thalassemia remains unknown. Continued patient monitoring will enable us to evaluate whether these conditions may influence each other’s clinical and biochemical profiles.

Reemergence of Hemoglobin Yukuhashi

Abstract The abnormal hemoglobin, Hb Yukuhashi, identified in Japan in 1963, exhibits slower migration compared to Hb A and is associated with target cells on peripheral smear. A similar hemoglobin, Hb Dhofar, is found in an Omani tribesman and is defined as a substitution of arginine for proline at the 58th amino acid position of the beta-globin chain. Despite both Yukuhashi and Dhofar seemingly arising from the same exonic point mutation, variations in expression were clarified through beta-globin gene amplification. Hb Dhofar, unlike Yukuhashi, was linked to a splice-site mutation at codon 29, explaining its reduced expression. Despite these hemoglobins’ structural alterations, their functional properties on a molecular level, including oxygen affinity and heme-heme interaction, remained largely unaffected. While mutations at codon 58 have been observed alongside other mutations in Hb Dhofar and Hb C-Ziguinchor, this case represents the first sequencing and report of a beta-globin gene mutation solely at codon 58, as only Yukuhashi’s protein coding region had been sequenced, not the whole gene. “Hemoglobin D” was detected on newborn screening of a male child with no other significant medical history or physical abnormalities. Capillary Electrophoresis and Cation-Exchange HPLC showed abnormal hemoglobin fractions, with 20.1% in the S Zone and 25.7% in the D-window, respectively. This led to a presumptive diagnosis of Hemoglobin Dhofar trait; however, discrepancies in hematologic parameters and relative hemoglobin levels were noted. Genetic testing of the Beta Globin gene confirmed a C to G substitution at codon 58 but lacked the characteristic codon 29 mutations associated with Hemoglobin Dhofar. The patient, was discovered to possess a mutation at codon 58 on the beta globin gene, a mutation not previously reported in isolation. In contrast to previous studies on Hb Dhofar, where variant Hb levels were lower and accompanied by abnormal RBC indices indicative of beta thalassemia trait, this case exhibited higher variant Hb levels with normal RBC indices, suggestive of a typical beta globin variant. The mutation at codon 58 did not significantly alter intermolecular interactions of the globin which may mean the variant is clinically asymptomatic in nature. Continued follow-up is necessary to confirm this suspicion, although initial assessments at 3 and 6 months of age showed normal RBC parameters. This case represents the first instance of an isolated mutation at codon 58 of the beta globin gene, emphasizing the need to consider this mutation in interpreting hemoglobinopathy screening results.

SNaPshot minisequencing for SARS CoV 2 Variant Genotyping

Abstract Introduction/Objective The SNaPshot Multiplex System (Thermo Fisher) can investigate up to fifty SNP markers simultaneously by using PCR amplification and subsequent dideoxy single base extension of an unlabeled primer and capillary electrophoresis. After electrophoresis and fluorescence detection, the alleles of a single marker appear as different colored peaks at roughly the same size in the electropherogram plot. The size of the different allele peaks vary slightly due to differences in molecular weight of the dyes. We propose a new set of primers and probes to use this system to differentiate variants of SARs Cov 2 variants. Methods/Case Report SNAPSHOT analysis was performed using the primers designed internally and analysis was done of up to 7 mutation hot spots, which were 417, 4511, 478, 484, 501, 614, and 681. By implementing the algorithm of systems and methods herein with the traces depicted in Figures, true positive and true negative outcomes for the different COVID variants can be performed, based on the highlighted peaks, which are not necessarily the most intense peaks. 10 Positive controls achieved from in stro transcript plasmid was tested then 10 viral RNA obtained through BEI Resources, NIAID was tested after mixed into 30 pooled negative PCR-swab specimens. Genmapper was used for analysis of results. 40 Korean patient samples were tested as well. Results (if a Case Study enter NA) Both In vitro transcript and derived specimens of wild type, alpha, beta, delta, gamma, kappa, omicron, and pirola showed expected results in 78/80 of the specimens. For example,delta B.1.617.2 variant showed 1 = A (green) at Primer 452R-F, 2 = G (blue): Primer 484Q-F, and 3 = A (green) at Primer 614-F. Of note, hetero type was noted in a few of the patient strains but none of the BEI strains and this may be due to a transfromation of the virus and worth investigation. Conclusion The SNaPshot® Multiplex System is a accurate system that may be applied to multiple applications including viral strain anylsis and here we can show how it may be effectively used for SARS-Cov-2 gentotyping. Using this technique, it is possible to analyze more than 50 SNPs distributed throughout the genome in a single multiplex reaction, making it an advantage when compared with traditional sequencing reaction. This is a important study that shows that this technique may be used in multiple field applictions.

A Case for Co-oximetry: Methemoglobinemia secondary to a Rare Hemoglobin Variant

Abstract Introduction/Objective The presence of dyshemoglobins and some inherited hemoglobin (Hb) variants can cause over or underestimation of oxygen saturation (O2sat) when using a pulse oximeter (ox). These noninvasive medical devices rely on two wavelengths, a pulsing arterial bed, and light absorption properties of Hb to measure the difference between deoxy- and oxy-Hb. In contrast, a co-oximeter (co-ox) requires arterial blood. By using at least four wavelengths, co-ox measures the functional and fractional O2sat to detect all forms of Hb, including those that do not bind oxygen (O2), such as methemoglobin (metHb). Methemoglobinemia may be congenital or acquired, resulting from exposure to certain toxins, metabolic disorders, enzymatic deficiencies such as cytochrome b5 reductase deficiency, or hemoglobinopathies. Methods/Case Report A healthy, adolescent male undergoing a sports physical at his pediatrician’s office was hypoxic by pulse ox and sent to the emergency department (ED) for evaluation. On exam, he had cold, blue tinged fingertips and lips but was otherwise asymptomatic. His O2sat was 88-90% on room air with minimal improvement with 6L of supplemental O2. Co-ox revealed a metHb level &amp;gt; 20%. The father also mentioned that the mother had frequent ED visits for hypoxia. Capillary Hb electrophoresis showed peaks in the HbA, HbF, and HbA2 zones of 78.3%, 17.9%, and 3.6%, respectively. All CBC parameters were within the reference intervals. The large peak in the HbF zone would suggest hereditary persistence of fetal hemoglobin (HPFH); however, the clinical picture was not consistent. Further reference laboratory investigation determined that the patient was heterozygous for Hb Tübingen (HbTüb), a beta globin chain variant resulting from a point mutation at position 106. By mass spectrometry, the variant quantified as 38% of the total hemoglobin, indicating underestimation by electrophoretic techniques. Results (if a Case Study enter NA) NA Conclusion This case presents an essentially asymptomatic patient with hypoxia and elevated methemoglobin levels. Electrophoretic hemoglobinopathy and CBC workup were clinically inconsistent with HPFH and the presence of an aberrant hemoglobin migrating in the HbF must be considered in the differential diagnosis. The manifestation of HbTüb as methemoglobinemia in this context adds complexity to the diagnostic puzzle. Further laboratory exploration into the underlying mechanisms driving this unique presentation is crucial for a comprehensive understanding and tailored management approach.