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Methods in Molecular Biology (Clifton,... 2024In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass...
In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass spectrometry (UPLC-MS/MS) using deuterated internal standards. This assay detects the presence of nine barbiturate drugs-amobarbital, barbital, butalbital, butabarbital, mephobarbital, secobarbital, pentobarbital, phenobarbital, and thiopental. This protocol describes two LC separation methods-first LC method (2.2 min/sample) is intended to be used as a first step of the analysis that does not separate amobarbital and pentobarbital, and a second, longer (2.7 min/sample) LC method is intended to be used only for samples which have a peak in the amobarbital/pentobarbital retention time on the shorter LC method. Since the frequency at which amobarbital and pentobarbital are observed in clinical populations is low, the shorter LC method helps gain efficiency in a high-volume laboratory environment. Additional features of this protocol that help in efficiency gain are automated extraction using Hamilton™ liquid handling system and algorithmic data review using Ascent™ software.
Topics: Pentobarbital; Amobarbital; Chromatography, Liquid; Tandem Mass Spectrometry; Barbiturates
PubMed: 38036812
DOI: 10.1007/978-1-0716-3541-4_8 -
Journal of Obstetric, Gynecologic, and... Mar 2022To compare glucose concentrations in three sections of individual tubes and among tubes of commercial oral glucose gels commonly used to treat neonatal hypoglycemia in...
OBJECTIVE
To compare glucose concentrations in three sections of individual tubes and among tubes of commercial oral glucose gels commonly used to treat neonatal hypoglycemia in the United States (Glutose 15 [Perrigo, Minneapolis, MN] and Insta-Glucose [Valeant Pharmaceuticals North America LLC, Bridgewater, NJ]).
DESIGN
A quantitative laboratory study.
METHODS
We measured glucose concentrations in aliquots taken from the top, middle, and bottom sections of three different lots and in whole tubes from different lots of Glutose 15 and Insta-Glucose. We measured the glucose content in the gel using hexokinase and glucose-6-phosphate dehydrogenase enzymes on the Siemens ADVIA 1800 analyzer (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY).
RESULTS
The percent difference observed among the three sections of the Glutose 15 tubes was 12.3% to 53.8%. The difference among the three sections of the Insta-Glucose tubes was 40.7% to 79.6%. The concentration of glucose gel is labeled as 40%, but the actual concentration in aliquots of Glutose 15 ranged from 39.64% to 70.96%. The actual concentration in aliquots of Insta-Glucose ranged from 16.45% to 27.47%. The difference in the concentration of glucose among three lots of whole tubes of Glutose 15 was 1.6%, and the difference in concentration among three lots of whole tubes of Insta-Glucose was 8.8%. In Glutose 15, the concentration ranged from 48.3% to 49.1%, and Insta-Glucose, the concentration ranged from 17.2% to 18.8%.
CONCLUSION
Glucose was not uniformly distributed within tubes of Glutose 15 and Insta-Glucose, and this may account for variable results on the efficacy of oral glucose gel as a treatment for neonatal hypoglycemia.
Topics: Blood Glucose; Gels; Glucose; Humans; Hypoglycemia; Infant, Newborn; Infant, Newborn, Diseases
PubMed: 34919803
DOI: 10.1016/j.jogn.2021.11.001 -
Biology of Reproduction Jan 2022Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm...
Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.
Topics: Animals; Cryopreservation; Male; Metabolome; Phenotype; Semen; Semen Analysis; Semen Preservation; Spermatozoa; Sus scrofa; Temperature; Time Factors
PubMed: 34725678
DOI: 10.1093/biolre/ioab200 -
Microorganisms Oct 2022Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the...
Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect underlines the lack of a good strategy for the cultivation and isolation of Aspergillus spp. In order to develop a complete analytical method to detect Aspergillus in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At <72 h, all test systems showed comparable sensitivity, about 1−2 conidia. However, the subculture analysis showed a suboptimal recovery for the methods, despite the positive growth and the visualization of the “Aspergillus balls” in the culture media. To investigate this issue, we studied three different subculture approaches: (i) the use of a sterile subculture unit, (ii) the use of a sterile subculture unit and the collection of a larger aliquot (100 µL), following vigorous agitation of the vials, and (iii) to decapsulate the bottle, withdrawing and centrifuging the sample, and aliquot the pellet onto SDA plates. Our results showed that only the third procedure recovered Aspergillus from all positive culture bottles. This work confirmed that our strategy is a valid and faster method to culture and isolate Aspergillus spp. from blood culture bottles.
PubMed: 36296302
DOI: 10.3390/microorganisms10102026 -
General and Comparative Endocrinology Aug 2024Fecal samples are a non-invasive and relatively accessible matrix for investigating physiological processes in resident killer whale (Orcinus orca) populations. The high...
Fecal samples are a non-invasive and relatively accessible matrix for investigating physiological processes in resident killer whale (Orcinus orca) populations. The high lipid content of the diet (primarily salmonids) leads to lower density fecal material and slower dispersion, facilitating sample collection. As fecal discharge is relatively infrequent and the volume of sample is variable, maximizing analytical options is an important consideration. Here we present an extraction methodology to measure hormones and lipid content from the same fecal aliquot. Lipid extractions are commonly conducted using chloroform and methanol from Folch or Bligh and Dyer (B&D), while alcohol is the primary solvent for hormone extraction. We evaluated the possibility of using the methanol layer from lipid extractions to assess fecal steroid hormone levels. Folch and B&D methanol residues were assayed form metabolites of progesterone (PMs) and corticosterone (GCs), and results were compared to aliquots extracted in 70 % ethanol. Hormone concentrations measured in the methanol layer from Folch and B&D extractions were 55 % to 79 % lower than concentrations in 70 % ethanol. We developed mathematical corrections, using linear regression models fitted to Folch or B&D methanol vs 70 % ethanol hormone concentrations (p < 0.01). Fecal concentrations of PMs and GCs from methanol extractions were biologically validated and are significantly higher in confirmed pregnant females compared to non-pregnant individuals (p < 0.05). This study demonstrates that lipid extraction protocols may be used for the analysis of multiple biomarkers, maximizing the use of small-volume samples.
Topics: Animals; Female; Corticosterone; Feces; Lipids; Progesterone; Whale, Killer
PubMed: 38705419
DOI: 10.1016/j.ygcen.2024.114544 -
European Journal of Pediatrics Mar 2023Manually performed double-volume exchange transfusion (DVET) is tedious, error-prone, and may incur the risk of embolism. We aimed to develop a device that automates the...
UNLABELLED
Manually performed double-volume exchange transfusion (DVET) is tedious, error-prone, and may incur the risk of embolism. We aimed to develop a device that automates the DVET procedure performed through the umbilical venous route. We evaluated changes in blood passing through the device during DVET. We developed an electro-mechanical device with accessories (tubing and valve assembly) to perform a complete DVET. It comprises two syringes driven by a common pump that moves back and forth to withdraw aliquots of the patient's blood and infuse equal volumes of donor blood. In tandem, it draws donor blood from a blood bank bag and pushes the patient blood drawn from the previous cycle into a waste bag, respectively. One-way duckbill valves and a two-way pinch valve ensure the separation of the donor and patient blood. A sensor detects bubbles and clots. A dashboard displays set and measured parameters. We tested the accuracy of the delivered flow rate and volume, electrical safety, embolus detection, and changes in hematological and biochemical values. The delivered flow and volume were within 5% of the set parameters. All electrical safety parameters were within normal limits. The sensor consistently detected microbubbles and clots. There were no clinically significant differences in laboratory parameters between samples drawn directly from the blood bank bag and drawn from the exit port at 80, 100, 120, and 160 s with a fixed aliquot volume.
CONCLUSIONS
Our prototype of a novel device can safely automate a DVET. Further trials of this device are warranted.
WHAT IS KNOWN
• Double volume exchange transfusion is often performed manually, but this is time-consuming and error-prone. • Previous attempts at automation were not widely adopted because they involved inserting two catheters and did not have mechanisms to prevent embolism.
WHAT IS NEW
• This novel device fully automates double volume exchange transfusions through a single-lumen umbilical venous catheter. • It prevents air and clot embolism and has a screen for input and output parameters and alarms.
Topics: Humans; Infant, Newborn; Blood Transfusion; Umbilical Cord; Embolism
PubMed: 36625935
DOI: 10.1007/s00431-022-04791-3 -
Veterinary Dermatology Jun 2021Serum testing for allergen-specific immunoglobulin (Ig)E is commonly employed to identify allergens used for allergen-specific immunotherapy in dogs, yet the reliability...
BACKGROUND
Serum testing for allergen-specific immunoglobulin (Ig)E is commonly employed to identify allergens used for allergen-specific immunotherapy in dogs, yet the reliability of results has been a matter of debate.
OBJECTIVE
The aim of this study was to evaluate the reproducibility of serum tests for environmental allergen-specific IgE in three European laboratories.
ANIMALS/METHODS
Serum was obtained from 33 client-owned dogs diagnosed with atopic dermatitis, divided into three aliquots and sent to the laboratories under different names. Two aliquots were sent simultaneously to one of the laboratories on the first day; the third sample was then sent to the same laboratory on the subsequent day. The laboratory for each patient was chosen according to a predetermined randomization list. The agreement between different samples from the same dog for each of the laboratories was calculated with a Cohen's Kappa test. Spearman's rank coefficients (r ) as well as the coefficients of variation (CV) additionally were calculated.
RESULTS
The intra- and interassay agreements for laboratories A, B and C were 0.79 and 0.75, 0.92 and 0.90, and 0.90 and 0.85, respectively. The CVs were 18.92% and 22.95%, 14.43% and 18.79%, and 15.38% and 18.75% (respectively) and the r 0.73 and 0.68, 0.95 and 0.92, and 0.82 and 0.74 (respectively).
CONCLUSION AND CLINICAL RELEVANCE
The differences in reproducibility between laboratories complicate test interpretation and underline the importance of interpreting results of serum testing for allergen-specific IgE in the context of the patient's clinical history.
Topics: Allergens; Animals; Dog Diseases; Dogs; Europe; Immunoglobulin E; Reproducibility of Results
PubMed: 33686751
DOI: 10.1111/vde.12942 -
Metabolites Apr 2023Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs,... (Review)
Review
Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry (MS), matrix-associated laser desorption/ionization-MS, and liquid chromatography-electrospray ionization-MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.
PubMed: 37110222
DOI: 10.3390/metabo13040564 -
The Journal of Prosthetic Dentistry Jul 2023How the build orientation of a 3-dimensionally (3D) printed denture affects microbial adhesion is unclear.
STATEMENT OF PROBLEM
How the build orientation of a 3-dimensionally (3D) printed denture affects microbial adhesion is unclear.
PURPOSE
The purpose of this in vitro study was to compare the adherence of Streptococcus spp. and Candida spp. on 3D-printed denture bases prepared at different build orientations with conventional heat-polymerized resin.
MATERIAL AND METHODS
Resin specimens (n=5) with standardized 28.3 mm surface area were 3D printed at 0 and 60 degrees, and heat-polymerized (3DP-0, 3DP-60, and HP, respectively). The specimens were placed in a Nordini artificial mouth (NAM) model and exposed to 2 mL of clarified whole saliva to create a pellicle-coated substratum. Suspensions of Streptococcus mitis and Streptococcus sanguinis, Candida albicans and Candida glabrata, and a mixed species, each at 10 cfu/mL were pumped separately into the model for 24 hours to promote microbial adhesion. The resin specimens were then removed, placed in fresh media, and sonicated to dislodge attached microbes. Each suspension (100 μL) was aliquoted and spread on agar plates for colony counting. The resin specimens were also examined under a scanning electron microscope. The interaction between types of specimen and groups of microbes was examined with 2-way ANOVA and then further analysis with Tukey honest significant test and Kruskal-Wallis post hoc tests (α=.05).
RESULTS
A significant interaction was observed between the 3DP-0, 3DP-60, and HP specimen types and the groups of microbes adhering to the corresponding denture resin specimens (P<.05). The difference was statistically significant among 3DP-0, 3DP-60, and HP specimens (P<.05). The adherence of candida was 3.98-times lower on the 3DP-0 than that of HP (P<.05). However, adherence of the mixed-species microbes and streptococci on the 3DP-60 were 1.75 times and 2-fold higher, respectively (P<.05). The scanning electron micrographs showed that 3DP-0 exhibited the lowest microbial adherence compared with HP and 3DP-60.
CONCLUSIONS
Adherence affinity of denture base resin is affected by the build orientation rather than by the group of different microbes. Three-dimensionally printed denture base resin fabricated at a 0-degree build orientation exhibited low affinity for microbial adhesion. Three-dimensionally printed dentures may reduce microbial adhesion when printed at a 0-degree build orientation.
Topics: Denture Bases; Surface Properties; Candida; Candida albicans; Hot Temperature; Materials Testing
PubMed: 37210224
DOI: 10.1016/j.prosdent.2023.04.017 -
Veterinary Sciences May 2021The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study...
The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates from stallions and donkeys were used. After semen collection and dilution, an aliquot was placed on the slide and introduced in the Trumorph device. Then observation was made with a 40x objective and negative phase-contrast microscope. Another aliquot was stained using eosin-nigrosine stain and viewed using 100× magnification. Well-formed sperm were observed, and different abnormalities were identified using Trumorph. The use of eosin-nigrosin staining method and Trumorph led to the same results and both techniques can be used for stallion and donkey sperm morphological analysis. However, considering the fact that Trumorph uses living sperm helps prevent sperm cell alteration during sample preparation. Therefore, Trumorph can be a good alternative to the conventional staining method, which provides a quick test on live sperm.
PubMed: 34066550
DOI: 10.3390/vetsci8050079