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Scandinavian Journal of Clinical and... Jul 2022The body of literature varies significantly regarding serum and urine osmolality stability. Therefore, our aim was to investigate the stability of serum and urine...
OBJECTIVES
The body of literature varies significantly regarding serum and urine osmolality stability. Therefore, our aim was to investigate the stability of serum and urine osmolality at different temperatures (room temperature (RT) 4-8 °C, -20 °C) and time conditions (8 h, 24 h, 1 month).
METHODS
The stability study was conducted following the CRESS guidelines, including 40 serum and urine samples. Samples were aliquoted into three aliquots and stored as follows: primary tube stored at RT for 8 h; two capped aliquots stored at 4-8 °C for 8 h and 24 h; one aliquot stored at -20 °C for 1 month. To minimize imprecision error, serum and urine osmolality were measured by the freezing point depression method in triplicate on OSMOMAT 3000 (Gonotech, Germany) analyzer. Percentage difference (PD%) against baseline measurement was calculated. Deviations were assessed against a reference change value of 5.0%.
RESULTS
The PD% for serum and urine osmolality was below 2.0% for all time/temperature conditions. For serum samples: primary tube after 8 h at RT PD% (95% CI) = 0.0% (-0.3, 0.2%); 8 h at 4-8 °C PD% (95% CI) = -0.4% (-0.7, 0.0%); 24 h at 4-8 °C PD% (95% CI) = -0.7% (-0.7, -0.6%); 1 month at -20 °C PD% (95% CI) = -2.1% (-2.4, -1.5%). For urine samples: after 8 h at RT PD% (95% CI) =0.6% (0.2, 0.9%); 8 h at 4-8 °C PD% (95% CI) = -0.2% (-0.5, 0.1%); 24 h at 4-8 °C PD% (95% CI) = -0.2% (-0.5, 0.0%); 1 month at -20 °C PD% (95% CI) = -2.0% (-3.0, -1.0%).
CONCLUSIONS
Changes in osmolality for tested conditions for serum and urine samples, were within acceptance criteria. Reflex and add-on osmolality testing can be performed within the same day in samples kept at RT for 8 h in primary tube and within 24 h, in aliquoted refrigerated samples, without compromising the reliability of test results. For longer storage, samples should be kept at -20 °C.
Topics: Humans; Osmolar Concentration; Reproducibility of Results; Serum; Specimen Handling; Temperature; Time Factors; Urine
PubMed: 35654415
DOI: 10.1080/00365513.2022.2079094 -
Theriogenology Dec 2022Buffalo spermatozoa are vulnerable to cryo-injuries due to inherent deficiency of endogenous antioxidants, high polyunsaturated fatty acids (PUFA) content in plasma...
Buffalo spermatozoa are vulnerable to cryo-injuries due to inherent deficiency of endogenous antioxidants, high polyunsaturated fatty acids (PUFA) content in plasma membrane and low cholesterol/phospholipid (C/P) ratio. Humanin is a potent cytoprotective agent that protects the cells against oxidative stress and apoptosis. The present study was designed to establish the presence of Humanin in buffalo and effect of Humanin supplementation on freezability of buffalo spermatozoa. Indirect immunofluorescence test revealed presence of Humanin in ejaculated and epididymal spermatozoa, and, elongated spermatids and interstitial space in the testicular tissue section. Humanin levels in seminal plasma were significantly and positively correlated with sperm concentration and individual progressive motility (IPM) in good (n = 22; IPM >70%) and poor (n = 10; IPM <50%) quality ejaculates. For supplementation studies, a total of 24 ejaculates (IPM ≥70%) were collected and each ejaculate was then divided into four aliquots. First aliquot was diluted with egg yolk-tris-glycerol (EYTG) extender without Humanin and served as control group (Group I). Rest three aliquots were diluted with extender containing 2 (Group II), 5 (Group III) and 10 μM Humanin (Group IV), respectively. Semen was cryopreserved using standard protocol and evaluated at pre-freeze for lipid peroxidation (LPO) and post-thaw stages for spermatozoa kinematics, LPO, mitochondrial membrane potential (MMP), capacitation, apoptotic status and DNA integrity. The treatment group that showed best results (5 μM) was compared with control group for in vitro fertility assessment by homologous zona binding assay. The LPO levels were lower (p < 0.05) in 5 and 10 μM Humanin supplemented group. The MMP and DNA integrity were higher (p < 0.05) in 5 μM group than other groups. F-pattern was higher (p < 0.05) and B-pattern was lower (p < 0.05) in 5 and 10 μM Humanin supplemented groups. Lower apoptotic and higher viable spermatozoa (p < 0.05) were observed in 5 μM Humanin group. The mean number of spermatozoa bound to zona pellucida was higher (p < 0.05) in 5 μM Humanin treated group than the control group. The study established the presence of Humanin in buffalo spermatozoa and seminal plasma for very first time and concluded that Humanin supplementation at 5 μM concentration improves the freezability and in vitro fertility of buffalo spermatozoa.
Topics: Male; Animals; Buffaloes; Semen Preservation; Cryoprotective Agents; Sperm Motility; Semen; Spermatozoa; Cryopreservation; Intracellular Signaling Peptides and Proteins; Semen Analysis; Bison; DNA
PubMed: 36183493
DOI: 10.1016/j.theriogenology.2022.09.013 -
Antioxidants (Basel, Switzerland) Sep 2022This study monitored the chemical and biochemical composition of bovine seminal plasma (SP). Freshly ejaculated semen ( = 20) was aliquoted into two parts. The first...
This study monitored the chemical and biochemical composition of bovine seminal plasma (SP). Freshly ejaculated semen ( = 20) was aliquoted into two parts. The first aliquot was immediately assessed to determine the sperm motion parameters. Another motility measurement was performed following an hour-long co-incubation of spermatozoa with SP at 6 °C. The other aliquot was processed to obtain the SP. Seminal plasma underwent the analyses of chemical composition and quantification of selected proteins, lipids and RedOx markers. Determined concentrations of observed parameters served as input data to correlation analyses where associations between micro and macro elements and RedOx markers were observed. Significant correlations of total oxidant status were found with the content of Cu and Mg. Further significant correlations of glutathione peroxidase were detected in relation to Fe and Hg. Furthermore, associations of chemical elements and RedOx markers and spermatozoa quality parameters were monitored. The most notable correlations indicate beneficial effects of seminal Fe on motility and Mg on velocity and viability of spermatozoa. On the contrary, negative correlations were registered between Zn and sperm velocity and seminal cholesterol content and motility. Our findings imply that seminal plasma has a prospective to be developed as the potential biomarker of bull reproductive health.
PubMed: 36139870
DOI: 10.3390/antiox11091796 -
Lab on a Chip Nov 2019Centrifugal microfluidics allows for miniaturization, automation and parallelization of laboratory workflows. The fact that centrifugal forces are always directed...
Centrifugal microfluidics allows for miniaturization, automation and parallelization of laboratory workflows. The fact that centrifugal forces are always directed radially outwards has been considered a main drawback for the implementation of complex workflows leading to the requirement of additional actuation forces for pumping, valving and switching. In this work, we review and discuss the combination of centrifugal with pneumatic forces which enables transport of even complex liquids in any direction on centrifugal systems, provides actuation for valving and switching, offers alternatives for mixing and enables accurate and precise metering and aliquoting. In addition, pneumatics can be employed for timing to carry out any of the above listed unit operations in a sequential and cascaded manner. Firstly, different methods to generate pneumatic pressures are discussed. Then, unit operations and applications that employ pneumatics are reviewed. Finally, a tutorial section discusses two examples to provide insight into the design process. The first tutorial explains a comparatively simple implementation of a pneumatic siphon valve and provides a workflow to derive optimum design parameters. The second tutorial discusses cascaded pneumatic operations consisting of temperature change rate actuated valving and subsequent pneumatic pumping. In conclusion, combining pneumatic actuation with centrifugal microfluidics allows for the design of robust fluidic networks with simple fluidic structures that are implemented in a monolithic fashion. No coatings are required and the overall demands on manufacturing are comparatively low. We see the combination of centrifugal forces with pneumatic actuation as a key enabling technology to facilitate compact and robust automation of biochemical analysis.
PubMed: 31596297
DOI: 10.1039/c9lc00441f -
PloS One 2023Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO2) have recently been adopted for the lethal control of a range...
Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO2) have recently been adopted for the lethal control of a range of invasive carnivores and mustelids. Determining the relative hazard of these compounds to non-target bird species is an important component of ecological risks evaluation. Problematically, some potential non-target bird species may be as small as 10 g in body mass, thus placing limitations on blood volumes that can be routinely sampled. Accordingly, we developed methods to quantify markers of increasing methaemoglobinaemia at their point of collection that required only 5 μL of whole blood. A 3 μL blood aliquot is pipetted into a plastic micro-cuvette and placed in a custom made holder optically coupled to the Ocean Optics spectrometer, enabling absorbance for oxyhaemoglobin (HbO: 575 nm) and MetHb (630 nm) to be determined. Haemoglobin (HbFe2+), packed cell volume (PCV) and lactate (LAC) data were generated from the remaining 2 μL aliquot apportioned to biosensor strips for the Cera-Check® and Lactate Scout® point-of-care devices. After oral doses of PAPP, a methaemoglobinaemia absorbance index (MAI = absorbance 575 nm-absorbance 630 nm) was strongly and significantly associated with dose-dependent declines in HbFe2+ in 9 bird species. Quantifying dose-dependent responses to MetHb-forming agents at the point of sample collection avoids analytical and storage artifacts arising from sample degradation that appears to be a much greater problem in avian blood compared to mammalian blood.
Topics: Animals; Methemoglobinemia; Methemoglobin; Hemoglobins; Propiophenones; Mustelidae
PubMed: 36928076
DOI: 10.1371/journal.pone.0282820 -
Annals of Clinical Biochemistry Jul 2022The Royal College of Pathologists of Australasia Quality Assurance Programs runs a Quality Assurance Program for the assessment of synovial fluid crystals. It provides...
BACKGROUND
The Royal College of Pathologists of Australasia Quality Assurance Programs runs a Quality Assurance Program for the assessment of synovial fluid crystals. It provides aliquots of synovial fluid to various laboratories. The quality of specimens can deteriorate prior to being examined. We aimed to assess whether the addition of dimethyl sulfoxide (DMSO) to synovial fluid specimens helps maintain cellular morphology.
METHODS
Synovial fluid specimens were obtained from 15 patients. Each specimen was aliquoted into 24 samples, with half having DMSO added at a concentration of 10%. For each specimen, six samples containing DMSO and six samples not containing DMSO were stored at-80°C and room temperature. Samples from each group were examined at 1, 2, 3, 6, 7 and 8 weeks. : For each specimen, the final remaining aliquoted samples containing DMSO and not containing DMSO, which were stored at-80°C, were directly compared. A system for grading cellular morphology and assessing for artefacts and cellular clumping was applied by two independent assessors.
RESULTS
A significant difference was found between samples containing DMSO and not containing DMSO which were stored at -80°C ( = .000), in favour of those containing DMSO. Regarding the combined findings of Assessors 1 and 2 and the grading of cellular morphology, a significant difference was found according to "groups" ( = .000), in favour of those containing DMSO and stored at-80°C.
CONCLUSIONS
DMSO contributes to the maintenance of cellular morphology in synovial fluid when stored in frozen conditions.
Topics: Cryopreservation; Dimethyl Sulfoxide; Freezing; Humans; Synovial Fluid
PubMed: 35044280
DOI: 10.1177/00045632221076349 -
PloS One 2023The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the...
The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.
Topics: Models, Statistical; Nucleic Acids; Point-of-Care Systems; Uncertainty
PubMed: 37186607
DOI: 10.1371/journal.pone.0285784 -
Analytical Chemistry Oct 2018A T790M secondary mutation in epidermal-growth-factor receptor (EGFR) is the most well-established EGFR-tyrosine-kinase-inhibitor (TKI) resistance marker in...
A T790M secondary mutation in epidermal-growth-factor receptor (EGFR) is the most well-established EGFR-tyrosine-kinase-inhibitor (TKI) resistance marker in non-small-cell lung cancer (NSCLC). The current methods to rapidly and accurately detect T790M in clinical practice are not satisfactory because of several obstacles, including the unavailability of tumor-tissue rebiopsies and the low DNA copy number of T790M in circulating tumor DNA (ctDNA). Here, we develop library-aliquot-based droplet digital PCR (LAB-ddPCR) to increase detection sensitivity without affecting accuracy. This new LAB-ddPCR method is performed using aliquots of the ctDNA precapture next-generation-sequencing (NGS) library, in which the isolated ctDNA was amplified and enriched. We show that the LAB-ddPCR can precisely distinguish between T790M wild-type and mutation alleles without introducing extra false-positive signals. In a cohort of 70 post-TKI NSCLC patients, the LAB-ddPCR identified 41 T790M-positive cases (sensitivity 58.57%), but ddPCR only detected T790M in 27 cases (sensitivity 38.57%). Taking the ARMS-PCR result from matched tumor rebiopsies into consideration, the LAB-ddPCR method is better than ddPCR. In conclusion, the LAB-ddPCR ctDNA test offers a feasible and flexible option for the rapid and accurate detection of the T790M secondary mutation, which is helpful in dynamically monitoring drug response and disease progression throughout the therapeutic regimen.
Topics: Carcinoma, Non-Small-Cell Lung; Circulating Tumor DNA; DNA Mutational Analysis; ErbB Receptors; Gene Library; Humans; Limit of Detection; Lung Neoplasms; Mutation; Polymerase Chain Reaction
PubMed: 30156405
DOI: 10.1021/acs.analchem.8b01776 -
Biomarker Insights 2022Biobanks have been supporting longitudinal prospective and retrospective studies by providing standardized services for the acquisition, transport, processing, storage,...
BACKGROUND
Biobanks have been supporting longitudinal prospective and retrospective studies by providing standardized services for the acquisition, transport, processing, storage, and distribution of high-quality biological material and associated data. Here, we describe how the Dog Aging Project (DAP), a large-scale longitudinal study of the domestic dog () with translational applications for humans, developed a biobank of canine biospecimens and associated data.
DESIGN AND METHODS
This was accomplished by working with the Cornell Veterinary Biobank, the first biobank in the world to receive accreditation to ISO 20387:2018-General Requirements for Biobanking. The biobank research team was involved in the early collection stages of the DAP, contributing to the development of appropriate workflows and processing fit-for-purpose biospecimens. In support of a dynamic strategy for real-time adjustment of processes, a pilot phase was implemented to develop, test, and optimize the biospecimen workflows, followed by an early phase of collection, processing, and banking of specimens from DAP participants.
RESULTS
During the pilot and early phases of collection, the DAP Biobank stored 164 aliquots of whole blood, 273 aliquots of peripheral blood mononuclear cells, 130 aliquots of plasma, and 70 aliquots of serum, and extracted high molecular weight genomic DNA suitable for whole-genome sequencing from 109 whole blood specimens. These specimens, along with their associated preanalytical data, have been made available for distribution to researchers.
CONCLUSION
We discuss the challenges and opportunities encountered during the implementation of the DAP Biobank, along with novel strategies for promoting biobanking sustainability such as partnering with a DAP quality assurance manager and a DAP marketing and communication specialist and developing a pilot grant structure to fund small innovative research projects.
PubMed: 36468152
DOI: 10.1177/11772719221137217 -
SLAS Technology Oct 2021The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates (cells, magnetic beads, silica beads, and...
The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates (cells, magnetic beads, silica beads, and microcarrier beads) for automated assays. SpinVessels are well suited for aliquoting on robotic liquid handlers and with robotic reagent dispensers, as well as manually. The SpinVessel system combines two critical features: (1) special internal side fins and projections in the bottom of the vessels and (2) an instrument that quickly spins the vessels and repeatedly reverses the spin direction. This rapid reversing motion sends multiple pulses of fluid up the side walls of the SpinVessel, creating a circular radial flow pattern. We tested five different particulates and six different SpinVessels with volume capacities varying from 50 mL to 1200 mL. SpinVessels are compatible with either single-, 8-, 12-, 96-, or 384-channel pipettors or with siphon tubing on robotic reagent dispensers. Experiments have demonstrated high viability of cells and undamaged morphology of microcarrier beads even after hours of constant agitation. The uniformity of aliquots collected at various vertical depths and horizontally across the SpinVessels demonstrated that cells, magnetic beads, and silica beads were uniformly suspended throughout the height and breadth of the SpinVessels, and uniformity of samples was consistent from the beginning to the end of the aliquoting procedure. Only 5 min of mixing is required to resuspend settled particulates. This novel mixing methodology has many applications in laboratory automation where particulate aliquot uniformity and/or particulate integrity are important to automating assays.
Topics: Biological Assay; Robotics; Silicon Dioxide
PubMed: 33955786
DOI: 10.1177/24726303211008864