Proteome identification of common immunological proteins of two nematode parasites.

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

Biochemical profile of renal amyloidosis in a Latin American cohort.

BACKGROUND: Two-thirds of patients with immunoglobulin light chain (AL) amyloidosis have renal involvement. The biochemical profile of kidney damage is poorly described. METHODS: A cross-sectional study was conducted involving patients diagnosed with AL amyloidosis and renal involvement between January 1, 2010, and April 30, 2022 at the Hospital Italiano de Buenos Aires. Participants were retrospectively identified from the Institutional Amyloidosis Registry. Patients diagnosed with AL amyloidosis and evidence of renal involvement were included. Individuals with other types of amyloidosis were excluded. The selection process involved a thorough review of medical records and registry data to ensure accurate identification and inclusion of eligible participants. RESULTS: Seventy-seven patients were included. At diagnosis, 90% of the subjects had proteinuria, with a median of 4.3 g/24 h, 61% had renal failure, and 47% presented nephrotic syndrome. Semi-automated urinary electrophoresis revealed 55% with non-selective and 21% with moderately selective glomerular proteinuria. Urine immunofixation indicated 64% with lambda monoclonal free light chains and 12% with kappa. Serum immunofixation demonstrated 48% with lambda monoclonal type and 25% with lambda IgG. At the time of diagnosis of AL amyloidosis, the median age was 66 years (IQR 53-72) and 49% were men. In addition to kidney involvement, other organs were also affected: heart in 53%, gastrointestinal system in 19%, peripheral nervous system in 16%, and liver in 16% of patients. CONCLUSION: Our study provides a biochemical profile in renal amyloidosis due to immunoglobulin light chains in a Latin American population. Proteinuria emerged as the most common finding in this cohort with frequent multiorgan involvement.

Comparative analysis of electrophoresis and interferometric optical detection method for molecular weight determination of proteins.

Analytical detection methods play a pivotal role in scientific research, enabling the identification and quantification of specific analytes in various disciplines. This scientific report aims to compare two very different methodologies for determining the Molecular Mass (MM, also known as Molecular Weight, MW) of proteins: electrophoresis gel and the Interferometric Optical Detection Method (IODM). For this purpose, several proteins with different MM were selected. The electrophoresis technique was employed to validate the structure and MM of different parts or fragments of the Matrix Metallopeptidase 9 antibody (anti-MMP9), antibody against S100 calcium binding protein A6 (anti-S100A6) and Cystatin S4 antibody (anti-CST4) by examining the presence of bands with expected sizes. The IODM was applied to study the above-mentioned proteins (part of the antibodies) together with the protein G, as a reference to correlate the MM and protein sizes with the measured signal. We report the evidence of IODM as a competitive analytical approach for the determination of the MM of proteins for the first time. This innovative method allows for accurate MM determination using minimal sample volumes and concentrations, employing a simple experimental procedure that eliminates the requirement for protein denaturation.

[Development of a Novel Male Reproductive Toxicity Assessment Method to Predict Male-mediated Effects on the Next Generation].

Less than 10% of the candidate drug compounds are associated with male reproductive toxicity. Genetic and/or epigenetic information on sperm may be crucial for fetal development. Therefore, developmental toxicity, such as paternally transmitted birth defects, is possible if genetic abnormalities in the male germ line persist and accumulate in the sperm during spermatogenesis. First, this study provides an overview of chemical and male reproductive toxicity, which may lead to developmental toxicity from the perspective of male reproduction. Second, we demonstrate methods for evaluating male reproductive toxicity to anticipate male-mediated developmental toxicity. We developed a novel staining technique for evaluating sperm quality, as well as a noninvasive imaging analysis of male reproductive toxicity. The former is a mammalian male germ cell-specific staining method using reactive blue 2 dye (RB2), as previously confirmed in human sperm, and a method for detecting the early-stage DNA fragmentation in a single nucleus from mouse spermatozoa using single-cell pulsed-field gel electrophoresis. The latter is a new, ready-to-use, and compact magnetic resonance imaging (MRI) platform utilizing a high-field permanent magnet to evaluate male reproductive toxicity. The histopathological analysis supported the suitability of the MRI platform. The present study, for the first time, revealed a rapid, noninvasive evaluation of male reproductive toxicity in vivo using compact MRI. These novel toxicity assessments can help predict male-mediated developmental toxicity, contributing to accelerated drug discovery and drug repositioning.

Serum chemistry, thyroxine concentration, and blood cell morphology of wild, young Blackchin Guitarfish Glaucostegus cemiculus sampled on the eastern Mediterranean coastline.

OBJECTIVE: The Blackchin Guitarfish Glaucostegus cemiculus is endemic to the Mediterranean Sea and is critically endangered, but relevant routine laboratory data are unavailable. Our objectives were to determine the packed cell volume (PCV), comprehensive serum chemistry analytes, and serum total thyroxine (sTT4) concentration; compare serum albumin and serum globulin concentrations as measured by two different methods; and describe the blood cell morphology of healthy, free-ranging Blackchin Guitarfish. METHODS: Wild Blackchin Guitarfish were captured using a seine net. Blood samples for serum chemistry and hematological analyses were obtained and measured using routine laboratory methods. The fish were tagged and released. RESULT: This study included 43 Blackchin Guitarfish (17 males and 26 females) that were younger than 6 months as estimated based on total length and body weight. The median PCV (n = 23) was 22% (minimum-maximum [min-max] = 15-25%). Median sTT4 (n = 10) measured by chemiluminescence immunoassay was 7.86 nmol/L (min-max = 7.52-9.57 nmol/L). The study included a comprehensive, 25-analyte serum chemistry analysis (e.g., serum iron and unbound and total iron-binding capacity) and a morphological description of all blood cells. Serum electrophoresis (SEP; n = 13) yielded a consistent serum albumin-migrating protein fraction and four globulin fractions. Serum electrophoretograms corroborating these results are presented. CONCLUSION: In Blackchin Guitarfish, the serum albumin-migrating fraction measured by SEP combined with serum total protein concentration yields a much higher albumin concentration compared to that measured by bromocresol green spectrophotometry. The true identity of this albumin-migrating fraction remains to be identified. The analytes' calculated 2.5-97.5% interpercentile intervals should be considered as reference intervals applying to Blackchin Guitarfish of similar age but should be applied cautiously to adult fish.

Comparison of multiplex PCR capillary electrophoresis assay and PCR-reverse dot blot assay for human papillomavirus DNA genotyping detection in cervical cancer tissue specimens.

Background: The study aimed to evaluate the positivity rates and genotype distribution of the multiplex PCR capillary electrophoresis (MPCE) and PCR-Reverse Dot Blot (PCR-RDB) assays for human papillomavirus (HPV) detection in cervical cancer tissue specimens, and to explore their detection principles and applications in large-scale population screening. Methods: The MPCE and PCR-RDB assays were performed separately on 425 diagnosed cervical cancer tissue specimens. Subsequently, the results of both assays were compared based on the HPV infection positivity rates and genotype distribution. Results: The overall positive rates of HPV genotypes for the MPCE and PCR-RDB assays were 97.9% and 92.9%, respectively. A p-value < 0.001 indicated a statistically significance difference in consistency between the two assays. The kappa value was 0.390, indicating that the consistency between both assays was fair. HPV16 was the most common single-genotype infection type, with infection rates detected via MPCE and PCR-RDB assays being 75.7% and 68.3%, respectively. In the age group >50 years, the HPV multiple-type infection rate detected via MPCE assay was significantly higher than that detected by the PCR-RDB assay, with a statistically significant difference (p = 0.002). Conclusion: To reduce the false-negative rate and improve screening efficiency, the MPCE assay, which targets the oncogenic gene E6/E7 segments, can be extended to the general female population for the early detection, diagnosis, and treatment of cervical cancer.

Comprehensive Study of Total Nitrogen Content and Microfluidic Profiles in Additive-Enriched Plant-Based Drinks.

The growing consumption of plant-based milk substitutes raises important questions about their composition. The various additives used by manufacturers, including those employed as flavor enhancers, protein additives, and stabilizers, may contain both protein and non-protein nitrogen components. In our study, we examined not only popular milk alternatives but also other milk substitutes made from specific plants. We present a reproducible and rapid method for the simultaneous qualitative and quantitative determination of the total nitrogen content in milk alternatives, focusing on applicability. Using the microchip gel electrophoretic method, we determined that the total nitrogen content differed from the protein content indicated on the packaging. Our results, along with statistical evaluations, supported the hypothesis that different brands of products, derived from the same plant source, resulted in different microfluidic profiles, likely due to the presence of additives. As expected, the microfluidic profiles of additive-free products differed from those of fortified products made from the same plant-based milk replacer. Total nitrogen content provides crucial information for individuals with kidney disease, as is essential to reduce the burden on the kidneys to slow deterioration, alleviate symptoms and avoid complications.

[Familial bisalbuminemia]. / Familiäre Bisalbuminämie.

Bisalbuminemia is characterized by two albumin peaks in the electrophoresis of serum. There are different forms of bisalbuminemia: inherited and acquired. The acquired form is mainly transitory, whereas the familial form is permanent. The frequency of bisalbuminemia in the general population has been reported to be between 0.0003 and 0.01%. This paper presents a case of familial bisalbuminemia as well as the family tree-to the extent obtainable. A married couple, in which the husband had bisalbuminemia, had seven children and 18 grandchildren. Bisalbuminemia was also found in two children and in two grandchildren.

Advances in Hemoglobinopathies and Thalassemia Evaluation.

Hemoglobin (Hb) disorders are among the most prevalent inherited diseases. Despite a limited number of involved genes, these conditions represent a broad clinical and prognostic spectrum. The menu of laboratory tests is extensive. From widely available modalities, for example, complete blood count to rather sophisticated molecular technologies, the investigation of Hb disorders recapitulates an increasing complexity of laboratory workup in other medical fields. This review highlights a current state of biochemical and molecular investigation of Hb disorders and offers a glimpse on technologies that are yet to be fully embraced in clinical practice.

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Short-term impact of vitamin K2 supplementation on biochemical parameters and lipoprotein fractions.

This study explored the short-term effects of vitamin K2 (VK2) supplementation on biochemical parameters (vitamin D, vitamin E, vitamin A, alkaline phosphatase, calcium, phosphorus (P), magnesium, metallothionein, triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and lipoprotein fractions (albumin, HDL, very low-density lipoprotein (VLDL), LDL, and chylomicrons). A short-term experiment (24 h, six probands) was performed to track changes in VK2 levels after a single-dose intake (360 µg/day). Liquid chromatography-tandem mass spectrometry was used to monitor vitamin K levels (menaquinone-4 (MK-4), menaquinone-7 (MK-7), and vitamin K1 [VK1]) with a limit of detection of 1.9 pg/mL for VK1 and 3.8 pg/mL for the two forms of VK2. Results showed that MK-7 levels significantly increased within 2-6 h post-administration and then gradually declined. MK-4 levels were initially low, showing a slight increase, whereas VK1 levels rose initially and then decreased. Biochemical analyses indicated no significant changes in sodium, chloride, potassium, calcium, magnesium, albumin, or total protein levels. A transient increase in P was observed, peaking at 12 h before returning to baseline. Agarose gel electrophoresis of lipoprotein fractions revealed distinct chylomicron bands and variations in VLDL and HDL mobility, influenced by dietary lipids and VK2 supplementation. These findings suggest effective absorption and metabolism of MK-7 with potential implications for bone metabolism and cardiovascular health.

Assessing and Evaluating HbS Concentration in Asymptomatic Sickle Cell Patients and Patients With Sickle Cell Crisis Through High-Performance Liquid Chromatography (HPLC) in a Tertiary Hospital in Central India.

Background Sickle cell disease (SCD) is a significant health concern, particularly due to the variability in disease severity and frequency of crisis episodes among patients. Accurate assessment of HbS concentrations is crucial for understanding the disease's progression and severity. This study aimed to assess and evaluate HbS concentrations in sickle cell patients and those experiencing sickle cell crisis using high-performance liquid chromatography (HPLC). The objectives included screening individuals for SCD, diagnosing the disease using Hb electrophoresis, estimating HbS concentration via HPLC, and comparing HbS concentration values between sickle cell patients and those in crisis. Methods An analytical study design was employed at Jawaharlal Nehru Medical College, Sawangi, Wardha, Maharashtra, involving 80 participants diagnosed with SCD. Data collection included clinical assessments, routine sickling tests, Hb electrophoresis, and HPLC for HbS concentration measurement. Descriptive and inferential statistics were utilized for data analysis, including chi-square tests, Mann-Whitney U tests, and regression analyses. Results Significant differences in HbS concentrations were observed between different patient groups. Individuals with the SS pattern exhibited higher HbS levels than those with the AS pattern (p = 0.001). Non-crisis patients had significantly higher mean HbS concentrations than crisis patients (p = 0.001). A moderate positive correlation (0.476, p = 0.001) was found between HbS concentrations and clinical outcomes. No significant differences in HbS concentrations were noted based on sex or age group. Longitudinal analysis revealed a significant increase in HbS levels over time (p = 0.001). Conclusion The study underscores the importance of HbS concentration measurement in understanding the severity and progression of SCD. HPLC proves to be a valuable tool in accurately estimating HbS levels, aiding in better clinical management of the disease.

A multiplex microbial profiling system for the identification of the source of body fluid and skin samples.

Determining the source of body fluids is crucial in forensic investigations, as it provides valuable information about suspects and the nature of the crime. Microbial markers that trace the source of tissues and body fluids based on site specificity and temporal stability are often used effectively for this purpose. In this study, a multiplex system comprising seven microbial markers (Finegoldia magna, Corynebacterium tuberculostearicum, Cutibacterium acnes, Haemophilus parainfluenzae, Streptococcus oralis, Prevotella melaninogenica and Faecalibacterium prausnitzii) was developed to distinguish between skin, saliva, and feces samples. Based on these markers, the system produces electropherograms that are specific for each sample type. We collected 492 samples from six different skin sites (palm, antecubital crease, inguinal crease, cheek, upper back, and toe web space), the buccal mucosa, and stool were collected to further test the system. Beta diversity analysis revealed distinct clustering among the three sample groups. Additionally, skin microenvironment cluster analysis was used to identify skin sites accurately. This analysis classified skin samples into four distinct microenvironments: dry, moist, oily, and foot. Finally, we established a machine learning prediction model based on random forest regression to identify the skin microenvironment, achieving an overall prediction accuracy of 79 %. The multiplex system developed in this study accurately identifies the sources of body fluids, and the skin microenvironment. These findings offer new insights into the application of microbial markers in forensic science.

Exploring the manganese-dependent interaction between a transcription factor and its corresponding DNA: insights from gas-phase electrophoresis on a nES GEMMA instrument.

Manganese ion homeostasis is vital for bacteria and is achieved via manganese-dependent transcription factors. Manganese mediation of transcription factor attachment to the corresponding oligonucleotide sequences can be investigated, e.g. via electrophoretic mobility shift assays (EMSA). Formation of specific biocomplexes leads to differences in the migration pattern upon gel electrophoresis. Focusing on electrophoresis in the gas-phase, applying a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) also known as nES differential mobility analyzer (nES DMA), and on transcription factors (MntR proteins) from Bacillus subtilis and Mycobacterium tuberculosis, we took interest in the gas-phase electrophoresis of the corresponding biospecific complexes. We compared nES GEMMA, separating analytes in the nanometer regime (a few to several hundred nm in diameter) in the gas-phase in their native state according to particle size, to EMSA data. Indeed we were able to demonstrate manganese-mediated attachment of MntR to target genomic sequences with both analytical techniques. Despite some inherent pitfalls of the nES GEMMA method like analyte/instrument surface interactions, we were able to detect the target complexes. Moreover, we were able to calculate the molecular weight (MW) of the obtained species by application of a correlation function based on nES GEMMA obtained data. As gas-phase electrophoresis also offers the possibility of offline hyphenation to orthogonal analysis techniques, we are confident that nES GEMMA measurements are not just complementary to EMSA, but will offer the possibility of further in-depth characterization of biocomplexes in the future.

Miniaturized nanoelectrospray interface for coupling capillary electrophoresis with mass spectrometry detection.

A miniaturized electrospray interface consisting of a microfluidic nanosprayer and nanospray module is reported in the presented short communication. The nanosprayer was fabricated using silicon (Si) technology suitable for cost-efficient high-volume mass production. The nanospray module enabled the positioning of the nanosprayer in front of a mass spectrometry entrance and its coupling with capillary electrophoresis based on the liquid junction principle. A case study of top-down and bottom-up proteomic analyses of intact cytochrome c and its tryptic digest demonstrates the practical applicability of the developed interface.

Characterization and Techno-Functional Properties of High Protein Walnut Flour from an Oil by-Product.

A high protein walnut flour (HPWF) was obtained by defatting walnut flour (WF), which is a by-product of the oil industry. The objective of this study was the chemical and techno-functional characterization of HPWF. Composition, amino acid content, protein secondary structure, protein solubility and thermal transitions were measured. Besides, the techno-functional properties, emulsion activity and stability, and water holding and oil absorption capacities, of HPWF were evaluated. Also, the molecular mass of proteins under denaturing conditions and the microstructure of HPWF were evaluated by electrophoresis and confocal scanning laser microscopy, respectively. HPWF had 55.4% protein content and 21.5% total dietary fibre. In terms of HPWF amino acid composition, the limiting amino acids were the sulphurated cysteine and methionine. By FTIR analysis, the main secondary structures were ß-sheet (49%) followed by α-helix (24%); both structures are considered to be ordered. Likewise, HPWF soluble proteins increased at basic pH and HPWF proteins were separated in 11 bands with molecular masses ranging from 97 kDa to 18 kDa by electrophoresis. With respect to techno-functional properties, HPWF presented good emulsion activity (51%) and high thermal emulsion stability (46%). In addition, HPWF retained 571% and 242% of water and oil by weight, respectively. Finally, the micrograph showed the predominance of protein structures and fibre fragments, and the presence of few lipids mostly trapped. These results showed that HPWF is an interesting source of plant-based proteins and walnut flour can be used to obtain high protein ingredients from non-traditional sources.

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis.

BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

An enhanced and rapid method for von Willebrand factor multimer analysis for mechanical circulatory device testing.

BACKGROUND: Von Willebrand factor (VWF) is a critical glycoprotein in hemostasis and is an important factor in diagnosing bleeding disorders. Albeit the analysis of VWF is often compromised by inconsistent methodologies and challenges quantifying multimeric size. Current VWF multimer analysis methods are costly, time-consuming, and often inconsistent; thus, demanding skilled professionals. This study aimed to streamline and optimize the VWF multimer analysis technique, making it more efficient and reproducible, particularly for identifying or predicting mechanical circulatory support (MCS) induced bleeding disorders. METHODS: Blood samples from healthy volunteers were exposed to high shear forces via a Medtronic HeartWare ventricular assist device. VWF multimers were analyzed using vertical-gel agarose electrophoresis and Western blotting. Differences in VWF distribution were determined using densitometry, and two methods of densitometric analysis were compared: proprietary software against open-source software. RESULTS: Using the developed method: (i) protocol duration was accelerated from three days (in classical methods) to ~ eight hours; (ii) the resolution of the high molecular weight (HMW) VWF multimers were substantially improved; and (iii) densitometric analysis tools were validated. Additionally, the densitometry analysis using two software types showed a strong correlation between results, with the proprietary software reporting slightly higher HMW VWF percentages. CONCLUSION: This methodology is recommended for affordable, accurate, and reproducible VWF multimer evaluations during MCS use and testing. Further research comparing this method with semi-automated methods would provide additional insight and improve inter-laboratory comparisons.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits.

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5' flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3' region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Incidental detection of hereditary bisalbuminemia in a patient with positive DAT coombs: A case-based review.

Bisalbuminemia is a rare, typically benign condition marked by the presence of a bifid albumin band on serum protein electrophoresis. It can either be inherited due to a point mutation or acquired in association with various medical conditions, most commonly diabetes mellitus. Bisalbuminuria, the presence of bifid albumin in urine, may or may not accompany bisalbuminemia. Both conditions are often discovered incidentally during screening for monoclonal gammopathy. Bisalbuminemia and related variants provide insights into albumin's genetic diversity and functional roles, influencing clinical diagnostics and research in human genetics. Understanding these variants aids in distinguishing benign conditions from potential disease states, guiding appropriate clinical management. In this case-based review, we present a case of hereditary bisalbuminemia identified unexpectedly during an investigation of a positive Direct Antiglobulin Test Coombs in an adult female patient. This review aims to highlight the key features of bisalbuminemia, a rare condition that should be recognized by clinicians.