Detection of Hemoglobin Constant Spring: A Comparison of Capillary Electrophoresis Versus High-Performance Liquid Chromatography.

INTRODUCTION: In Malaysia, Hemoglobin Constant Spring (Hb CS) is the most common non-deletional α-thalassemia, caused by a mutation at the termination codon of the α2-globin gene (TAA>CAA). Detection typically involves identifying an abnormal peak at zone 2 on capillary electrophoresis (CE) or a small peak at the C-window on high-performance liquid chromatography (HPLC), indicative of Hb CS. OBJECTIVE: This study aimed to investigate the correlation between HPLC and CE in detecting Hb CS, evaluating their respective diagnostic accuracies and limitations. METHODS: A cross-sectional study was conducted at Hospital Sultanah Nur Zahirah involving secondary school students (Form 4) from Terengganu who participated in a thalassemia screening program conducted by the Ministry of Health (MOH) from January 2019 to December 2022. Blood samples from subjects showing a positive peak in zone 2 of CE and a small peak at the C-window of HPLC were selected. Molecular studies of these samples were performed to confirm the presence of Hb CS. For the statistical analysis, the Pearson correlation coefficient was employed to assess the relationship between CE and HPLC results. RESULTS: Hb CS was confirmed in all samples by molecular studies, revealing 92.3% heterozygous, 7.2% compound heterozygous, and 0.5% homozygous cases. CE detected 92.3% of heterozygous Hb CS cases, while HPLC detected only 48.2%. For compound heterozygous Hb CS, CE detected 100%, whereas HPLC detected 89.3%. Both homozygous cases were detected by CE and HPLC. The Pearson correlation coefficient test showed a significant linear relationship (p<0.001) between CE's zone 2 peak values and HPLC's C-window peaks (n=389).  Conclusion: These findings highlight the efficacy of CE as a reliable method for Hb CS detection, suggesting its potential superiority over traditional HPLC in clinical settings.

Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions.

Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 min but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low limit of detection (LOD) and limit of quantification (LOQ) (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in antibody drugs.

Comment on "Cellular aggregation dictates universal spreading behaviour of a whole-blood drop on a paper strip".

Laha et al. studied the diffusive behavior of a whole-blood drop on filter paper using the generalized capillary bundle model. However, some model parameters should be further refined to accurately reflect the physics involved in this diffusion process. Moreover, citations are missing for some key equations. Addressing these aspects will improve the model applicability to this application and benefit readers in accessing more accurate and detailed information.

Interactions of electrophoretically silent hemoglobin Hekinan II [HBA1:c.84G>T] with various forms of α-thalassemias and other hemoglobinopathies: novel insights into the molecular and hematological characteristics and genetic origins.

To determine the molecular basis, genotype - phenotype relationship, and genetic origin of Hemoglobin (Hb) Hekinan associated with several forms of α-thalassemia and other hemoglobinopathies for a better understanding of its diverse clinical phenotypes. Seventeen participants with suspected abnormal Hb were studied. Hb analysis was performed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutational and α-haplotypic and structural analyses were conducted, and the effects of mutations on globin-chain stability were determined. All participants harbored Hb Hekinan II (HBA1:c.84 G>T) co-inherited with another α-globin gene anomaly. Three novel genotypes, (ααHekinan/αCSα), (ααHekinan/αCSα,ßA/ßE), and (ααHekinan/αCSα,ßE/ßE), were characterized. Despite being co-inherited with both α- and ß-Hb variants Hb Hekinan II led to minimal changes in erythrocyte parameters, suggesting a non-pathological nature. HPLC but not CE revealed a distinct small shoulder-like Hb pattern. Thai Hb Hekinan II was strongly associated with haplotype [+ - S + - - -] and the possibility of four different haplotypes, while two Burmese Hb Hekinan II were associated with haplotypes [± - S + - + -] and [± - S + - - -]. The novel genotypes identified provide a fresh perspective on Hb Hekinan II diversity. HPLC has superior identification capabilities for samples of Hb Hekinan II co-inherited with α-thalassemia. Thai and Burmese Hb Hekinan II have diverse origins.

Advancements in Multiple-Step On-Line Preconcentration Techniques for Enhanced Sensitivity in Capillary Electrophoresis.

Multiple-step on-line preconcentration, a combination of at least two stacking techniques has been developed to increase the sensitivity in capillary electrophoresis (CE) for analytes in various samples. It is usually conducted sequentially, or in some cases, synergistically, where different stacking modes occur simultaneously. Multiple-step techniques allow simultaneous preconcentration and separation of various kinds of analytes in different complex samples in a single CE run. This review aims to provide recent advances in multiple-step on-line preconcentration techniques in CE. We critically review technical papers published for the last 7 years up until July 2024, subsequently organized according to the combination of the main stacking techniques, that is, field amplification, large volume sample stacking, transient isotachophoresis, micelle to solvent or micelle to cyclodextrin stacking, and others. The procedures, fundamental mechanism, analytical figures of merits achieved, and their feasibility for complicated sample matrices are reviewed.

The impact of managed aquifer recharge on the fate and transport of pesticides in agricultural soils.

Groundwater aquifers worldwide experience unsustainable depletion, compounded by population growth, economic development, and climate forcing. Managed aquifer recharge provides one tool to alleviate flood risk and replenish groundwater. However, concerns grow that intentional flooding of farmland for groundwater recharge, a practice known as Ag-MAR, may increase the leaching of pesticides and other chemicals into groundwater. This study employs a physically based unsaturated flow model to determine the fate and transport of residues of four pesticide in three vadose zone profiles characterized by differing fractions of sand (41 %, 61 %, and 84 %) in California's Central Valley. Here, we show that the complex heterogeneity of alternating coarse and fine-grain hydrogeologic units controls the transit times of pesticides and their adsorption and degradation rates. Unsaturated zones that contain a higher fraction of sand are more prone to support preferential flow, higher recharge rates (+8 %), and faster (42 %) water flow and pesticide transport, more flooding-induced pesticide leaching (about 22 %), as well as more salt leaching correlating with increased risks of groundwater contamination. Interestingly, considering preferential flow predicted higher degradation and retention rates despite shorter travel times, attributed to the trapping of pesticides in immobile zones where they degrade more effectively. The findings underscore the importance of considering soil texture and structure in Ag-MAR practices to minimize environmental risks while enhancing groundwater recharge. The study also highlights that selecting less mobile pesticides can reduce leaching risks in sandy areas.

Nitroglycerin challenge identifies microcirculatory target for improved resuscitation in patients with circulatory shock.

BACKGROUND: Circulatory shock and multi-organ failure remain major contributors to morbidity and mortality in critically ill patients and are associated with insufficient oxygen availability in the tissue. Intrinsic mechanisms to improve tissue perfusion, such as up-regulation of functional capillary density (FCD) and red blood cell velocity (RBCv), have been identified as maneuvers to improve oxygen extraction by the tissues; however, their role in circulatory shock and potential use as resuscitation targets remains unknown. To fill this gap, we examined the baseline and maximum recruitable FCD and RBCv in response to a topical nitroglycerin stimulus (FCDNG, RBCvNG) in patients with and without circulatory shock to test whether this may be a method to identify the presence and magnitude of a microcirculatory reserve capacity important for identifying a resuscitation target. METHODS: Sublingual handheld vital microscopy was performed after initial resuscitation in mechanically ventilated patients consecutively admitted to a tertiary medical ICU. FCD and RBCv were quantified using an automated computer vision algorithm (MicroTools). Patients with circulatory shock were retrospectively identified via standardized hemodynamic and clinical criteria and compared to patients without circulatory shock. RESULTS: 54 patients (57 ± 14y, BMI 26.3 ± 4.9 kg/m2, SAPS 56 ± 19, 65% male) were included, 13 of whom presented with circulatory shock. Both groups had similar cardiac index, mean arterial pressure, RBCv, and RBCvNG. Heart rate (p < 0.001), central venous pressure (p = 0.02), lactate (p < 0.001), capillary refill time (p < 0.01), and Mottling score (p < 0.001) were higher in circulatory shock after initial resuscitation, while FCD and FCDNG were 10% lower (16.9 ± 4.2 and 18.9 ± 3.2, p < 0.01; 19.3 ± 3.1 and 21.3 ± 2.9, p = 0.03). Nitroglycerin response was similar in both groups, and circulatory shock patients reached FCDNG similar to baseline FCD found in patients without shock. CONCLUSION: Critically ill patients suffering from circulatory shock were found to present with a lower sublingual FCD. The preserved nitroglycerin response suggests a dysfunction of intrinsic regulation mechanisms to increase the microcirculatory oxygen extraction capacity associated with circulatory shock and identifies a potential resuscitation target. These differences in microcirculatory hemodynamic function between patients with and without circulatory shock were not reflected in blood pressure or cardiac index.

Sex- and age-based reference intervals for capillary complete blood count parameters among urban preschoolers in southeast China based on a large community population.

Background and aim: Pediatricians commonly use the complete blood count (CBC) of capillary blood to evaluate health status, guide diagnoses, and determine treatment strategies. This study aimed to establish sex- and age-specific reference intervals (RIs) for 23 capillary CBC parameters for urban preschoolers in Fuzhou, Southeast China. Materials and methods: Capillary blood CBC data of 18,369 healthy preschoolers who underwent annual physical examinations at Fujian Maternity and Child Health Hospital between January 01, 2022, and November 31, 2023, were analyzed retrospectively. To fully validate the new RIs, the data of all apparently healthy children within the same age cohort at the same institution were comprehensively analyzed in December 2023. The new RIs were assessed by comparing them with the RIs currently used in laboratories and those obtained from different regions, sample types, or methodologies. Results: Dynamic temporal changes that differ between males and females were observed in the blood system of 3-7-year-old children in this region. The new sex- and age-specific RIs for capillary CBC parameters were feasible to guide clinical decision-making in the local region. Conclusions: Our findings demonstrated the importance of establishing sex- and age-specific RIs for each region and underscored the necessity of continuous adjustment of clinical Rls based on statistical rules and clinical responses.

Superficial Vessel Density and Determinants of Macular Microvasculature in Healthy Chinese Subjects.

BACKGROUND: The purpose of this study was to investigate the vessel density (VD) of the macular superficial capillary plexus (SCP) and its associated factors in healthy subjects. METHODS: This prospective, cross-sectional study enrolled healthy Chinese volunteers. The optical coherence tomography angiography (OCTA) was used to measure the superficial VD in macula. A generalized estimation equation (GEE) model was used to analyze the ocular and systemic associated factors. RESULTS: A total of 516 eyes of 262 healthy subjects were included (mean age 38.59±21.03 years). The total VD of macular SCP was 16.85±2.28 mm- 1. The VD in the inner ring and outer ring were significantly higher than that in the central ring, with the density being highest in nasal quadrant and lowest in the superior quadrant. After adjusting the ocular and systemic factors, age (ß=-0.0085, P=0.0122) and SSI (ß=1.3261, P <0.001) were significantly associated with total VD of the macular SCP. CONCLUSIONS: Among the healthy Chinese subjects included in the study, the mean total VD of macular SCP was 16.85 ± 2.28mm-1. The macular superficial capillary density was positively associated with age and SSI. Further studies are still required to better understand the change of macular VD in aging and other pathological conditions.

Capillary regression leads to sustained local hypoperfusion by inducing constriction of upstream transitional vessels.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a feature of many neurological disorders and injuries and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ~59% of the injuries resulted in regression of the capillary segment 7 to 14 d following injury, and the remaining repaired to reestablish blood flow within 7 d. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days postinjury in both awake and anesthetized mice. The degree of vasomotor dynamics was chronically attenuated in the ACT zone consequently reducing blood flow in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how focal capillary injury and regression can impair the microvascular sensory web and contribute to cerebral hypoperfusion.

Quantitative expression of LNAPL pollutant concentrations in capillary zone by coupling multiple environmental factors based on random forest algorithm.

The capillary zone plays a crucial role in migration and transformation of pollutants. Light nonaqueous liquids (LNAPLs) have become the main organic pollutant in soil and groundwater environments. However, few studies have focused on the concentration distribution characteristics and quantitative expression of LNAPL pollutants within capillary zone. In this study, we conducted a sandbox-migration experiment using diesel oil as a typical LNAPL pollutant, with the capillary zone of silty sand as the research object. The variation characteristics of LNAPL pollutants (total petroleum hydrocarbon) concentration and environmental factors (moisture content, electrical conductivity, pH, and oxidationreduction potential) were essentially consistent at different locations with the same height. These characteristics differed within range of 10.0-50.0 cm and above 60.0 cm from groundwater. A model for quantitative expression of concentrations was constructed by coupling multiple environmental factors of 968 sets-7744 data via random forest algorithm. The goodness of fit (R2) for both training and test sets was greater than 0.90, and the mean absolute percentage error (MAPE) was less than 16.00 %. The absolute values of relative errors in predicting concentrations at characteristic points were less than 15.00 %. The constructed model can accurately and quantitatively express and predict concentrations in capillary zone.

Patient-centric microsampling for abrocitinib pharmacokinetics: multiple-analytes assay bridging using Tasso device.

Aim: The feasibility of using Tasso devices (Tasso-SST® and Tasso+) collecting capillary blood samples for measuring abrocitinib and its metabolites were evaluated, and assay concordance established between capillary and venous blood samplings.Methods: Capillary serum and venous plasma concentrations were measured using their respective qualified and validated assays. Concentration and exposure comparisons were conducted for abrocitinib and its metabolites (M1, M2 and M4) to establish assay concordance.Results: The correlation coefficient between capillary serum and venous plasma concentrations were >0.98 for all four analytes from three separate assays, and PK parameters (AUClast and Cmax) were compared and met bioequivalence criteria.Conclusion: These results demonstrate the feasibility of patient-centric microsampling device, such as Tasso, in future abrocitinib pediatric study.

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Intra- and inter-day variations in oral metabolites from mouth-rinsed water determined using capillary electrophoresis-mass spectrometry metabolomics.

BACKGROUND AND AIMS: Collecting clinical samples without inconveniencing participants is desirable. The profile of metabolites in mouth-rinsed water is similar to that in saliva. However, the intra- and inter-day variations in unstimulated or stimulated saliva metabolites from mouth-rinsed water have yet to be clarified. Thus, we aimed to fill this research gap using capillary electrophoresis-mass spectrometry metabolomics. MATERIALS AND METHODS: We collected mouth-rinsed water from 15 healthy participants at 9:00, 11:30, 14:00, and 16:30 daily for 3 days. In total, 509 metabolite concentrations from 180 samples were obtained using capillary electrophoresis time-of-flight mass spectrometry. Variations in each metabolite were evaluated using the Wilcoxon signed-rank test to determine at which time/day significant differences occurred after removing metabolites without significant changes using the Friedman test. RESULTS: Of 167 frequently detected metabolites, 100 exhibited intra-day variations, and none exhibited inter-day variations. Intra-day variations were classified into four patterns, and the intra-day variation in each metabolite was assessed. The variations may reflect elapsed time after meals, oral cleaning, or circadian rhythms. CONCLUSION: This study could serve as a reference for improving the design of future clinical trials and the accuracy of metabolome analysis of mouth-rinsed water samples collected at different dates and times.

Acute macular neuroretinopathy following COVID-19 infection: Three case reports.

BACKGROUND: During the Coronavirus disease 2019 (COVID-19) pandemic, a notable increase in acute macular neuroretinopathy (AMN) cases was observed. This study aimed to investigate the potential association between AMN and COVID-19 by examining 3 cases in China. CASE SUMMARY: The first case involved a 30-year-old man who presented with progressive vision loss following a COVID-19 infection. Optical coherence tomography (OCT) and near-infrared imaging identified hallmark AMN lesions, hyperreflective disruptions within the outer plexiform layer, and hyporeflective anomalies in the ellipsoid zone, leading to an AMN diagnosis. Despite partial visual recovery, OCT angiography (OCTA) revealed persistent microvascular changes, specifically a decreased vascular density in the deep capillary plexus. The second case was a 24-year-old woman who experienced blurred vision and exhibited bilateral cotton-wool spots on fundus examination post-COVID-19. Imaging confirmed the presence of AMN along with paracentral acute middle maculopathy (PAMM). Follow-up OCTA found a progressive reduction in vascular density, indicating ongoing microvascular compromise. The third case was a 28-year-old woman who reported sensations of occlusion in her right eye following a COVID-19 infection. Imaging confirmed both AMN and PAMM, revealing similar decreases of microvascular density on OCTA despite a significant improvement in visual acuity. We noted that all 3 patients had received the COVID-19 vaccine prior to the appearance of symptoms. CONCLUSION: The findings highlight the diagnostic utility of advanced ocular imaging in detecting AMN in COVID-19 patients and the importance of comprehensive eye examinations.

Chiral-induced covalent organic framework as novel chiral stationary phase for chiral separation using open-tubular capillary electrochromatography.

As a novel class of chiral stationary phase (CPS) material, chiral covalent organic frameworks (CCOFs) have already shown great promise in open-tubular capillary electrochromatography (OT-CEC) for chiral separation. The synthesis methods of CCOFs used in OT-CEC mainly include bottom-up, post modification and chiral induction. The CCOFs synthesized by bottom-up and post modification strategies already have lots of applications in capillary electrochromatography, however, the chiral-induced synthesized via an asymmetric catalytic strategy has not yet been reported for using as the chiral stationary phase (CPS) in OT-CEC or even in chromatographic separation. Herein, the chiral-induced COF (Λ)-TpPa-1 was synthesized by asymmetric catalytic synthesis and coated on the inner surface of a capillary by an in-situ growth strategy as the CPS for chiral drug separation. The baseline separation of six enantiomers was achieved within 14 min, with a high-resolution (Rs) range from 1.85 to 6.75. Moreover, the resolution and migration time of the capillary keep stable within 160 runs, showing its superior stability and repeatability. This research provides a new idea for the development and application of novel CPS materials in the field of capillary electrochromatography separation, also shows the new application of chiral induced COFs. Furthermore, the chiral-induced CCOFs can be easily applied to other chromatographic separation fields, exhibiting its extensive application value in chiral analysis separation.

Hydrogen-bonded organic frameworks as stationary phase for open-tubular capillary electrochromatography.

BACKGROUND: Capillary electrochromatography (CEC) stationary phases have always been the focus of attention. The selection of excellent stationary phases are the key to realize separate of different compounds. Hydrogen-bonded organic frameworks (HOFs) are porous materials connected by hydrogen bonds between molecules, which have the advantages of renewable, high specific surface area and mild synthesis conditions. At present, HOFs are used in gas adsorption and storage, catalysis and drug delivery. Because of its unique advantages, HOFs have a bright future as CEC stationary phases. RESULTS: Using melamine (MA) and 1,3,6,8-tetra (4-carboxylphenyl)pyrene (H4TBAPy) as reaction monomers, a HOFs named MA/PFC-1 was synthesized by solvent evaporation at room temperature. The inner wall of the capillary column was coated with MA/PFC-1 by chemical bonding. Sulfonamides were used as the target analytes. The effects of pH, phosphate buffer solution concentration, organic additive content and applied voltage on sulfonamides separation were investigated. The MA/PFC-1-coated capillary column had good resolution (>1.5) and reproducibility. The intra-day, inter-day, column-to-column, and inter-batch precision of the retention times were 0.03%-0.09%, 0.04%-0.09%, 0.03%-0.14% and 0.06%-0.09%, respectively. The intra-day, inter-day, column-to-column, and inter-batch precision of the peak areas were 0.11%-0.25%, 0.13%-0.20%, 0.12%-0.15% and 0.08%-0.15%, respectively. The MA/PFC-1-coated capillary column was run 150 consecutive times, and the results showed no noticeable change, which proved that this method had good stability. SIGNIFICANCE: This work applied HOFs to CEC. The results show the that MA/PFC-1-coated capillary column has good separation performance. The MA/PFC-1-coated capillary column has been successfully applied to the determination of sulfamethoxazole in tablets, which has practical application value. To open up the application of HOFs in CEC and provide a new idea for developing new CEC stationary phases.

5q14.3 Microdeletion Syndrome With Simultaneous Involvement of MEF2C and RASA1. Clinical Case and Review of the Literature.

5q14.3 microdeletion syndrome is a rare condition involving multiple genes such as MEF2C and RASA1 and is potentially classified as a neurocutaneous syndrome. Deletion of the MEF2C gene accounts for the majority of clinical manifestations, including global developmental delay, intellectual disability, seizures, and behavioral disorders. RASA1 deletion is linked to capillary malformations with arteriovenous malformations (CM-AVM). Until now, only 17 cases have been described with deletions of both genes. We present the first case described in Spain with the microdeletion in the 5q14.3 cytoband simultaneously affecting both MEF2C and RASA1, exhibiting the typical manifestations of this entity, and review the published cases to date.

Soft and Deformable Thermoresponsive Hollow Rod-Shaped Microgels.

Depending on their aspect ratio, rod-shaped particles exhibit a much richer 2D and 3D phase behavior than their spherical counterparts, with additional nematic and smectic phases accompanied by defined orientational ordering. While the phase diagram of colloidal hard rods is extensively explored, little is known about the influence of softness in such systems, partly due to the absence of appropriate model systems. Additionally, investigating higher volume fractions for long rods is usually complicated because non-equilibrium dynamical arrest is likely to precede the formation of more defined states. This has motivated us to develop micrometric rod-like microgels with limited sedimentation that can respond to temperature and reversibly reorganize into defined phases via annealing and seeding procedures. A detailed procedure is presented for synthesizing rod-shaped hollow poly(N-isopropylacrylamide) microgels using micrometric silica rods as sacrificial templates. Their morphological characterization is conducted through a combination of microscopy and light scattering techniques, evidencing the unconstrained swelling of rod-shaped hollow microgels compared to core-shell microgel rods. Different aspects of their assembly in dispersion and at interfaces are further tested to illustrate the opportunities and challenges offered by such systems that combine softness, anisotropy, and thermoresponsivity.