Development and validation of a low cost blood filtration element separating plasma from undiluted whole blood.
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ABSTRACT:
SUBMITTER: Homsy A
PROVIDER: S-EPMC3365324 | biostudies-other | 2012 Mar
REPOSITORIES: biostudies-other
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Project description:BackgroundWhole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7-8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS.MethodsWhole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02-1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples.ResultsAfter applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×).ConclusionsThe novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.
| S-EPMC8848818 | biostudies-literature
Project description:BackgroundAnalytical methods to measure trace and toxic elements are essential to evaluate exposure and nutritional status. A ten-element panel was developed and validated for clinical testing in whole blood. Retrospective data analysis was conducted on patient samples performed at ARUP Laboratories.MethodsA method was developed and validated to quantify ten elements in whole blood by ICP-MS. Fifty microliters of sample were extracted with 950 μL of diluent containing 1 % ammonium hydroxide, 0.1 % Triton X-100, 1.75 % EDTA along with spiked internal standards. Four calibrators were used for each element and prepared in goat blood to match the patient specimen matrix. Samples were analyzed with an Agilent 7700 ICP-MS with a Cetac MVX 7100 μL Workstation autosampler.ResultsThe assay was linear for all elements with inter- and intra-assay imprecision less than or equal to 11% CV at the low end of the analytical measurement range (AMR) and less than or equal to 4% CV at the upper end of the AMR for all elements. Accuracy was checked with a minimum of 40 repeat patient samples, proficiency testing samples, and matrix-matched spikes. The linear slopes for the ten elements ranged from 0.94 to 1.03 with intercepts below the AMR and R2 ranging from 0.97 to 1.00.ConclusionsThe multi-element panel was developed to analyze ten elements in whole blood to unify the sample preparation and increase batch run efficiency. The improved analytical method utilized matrix-matched calibrators for accurate quantification to meet regulatory requirements. The assay was validated according to guidelines for CLIA-certified clinical laboratories and was suitable for clinical testing to assess nutritional status and toxic exposure.
| S-EPMC9803809 | biostudies-literature
Project description:Blood plasma separation is a prerequisite in numerous biomedical assays involving low abundance plasma-borne biomarkers and thus is the fundamental step before many bioanalytical steps. High-capacity refrigerated centrifuges, which have the advantage of handling large volumes of blood samples, are widely utilized, but they are bulky, non-transportable, and prohibitively expensive for low-resource settings, with prices starting at $1,500. On the other hand, there are low-cost commercial and open-source micro-centrifuges available, but they are incapable of handling typical clinical amounts of blood samples (2-10mL). There is currently no low-cost CE marked centrifuge that can process large volumes of clinical blood samples on the market. As a solution, we customised the rotor of a commercially available low-cost micro-centrifuge (~$125) using 3D printing to enable centrifugation of large clinical blood samples in resource poor-settings. Our custom adaptor ($15) can hold two 9 mL S-Monovette tubes and achieve the same separation performance (yield, cell count, hemolysis, albumin levels) as the control benchtop refrigerated centrifuge, and even outperformed the control in platelet separation by at least four times. This low-cost open-source centrifugation system capable of processing clinical blood tubes could be valuable to low-resource settings where centrifugation is required immediately after blood withdrawal for further testing.
| S-EPMC9269434 | biostudies-literature
Project description:The levels of trace elements in whole blood and plasma have been widely used for assessing nutritional status and monitoring exposure and can vary widely in populations from different geographical regions. In this study, whole blood samples (n = 120) and plasma samples (n = 120) were obtained from healthy donors attending the Red Cross Blood Bank (Queensland Red Cross Blood Service), which provided information for age and sex. There were 71 males (age range: 19-73 years) and 49 females (age range: 18-72 years) for whole blood samples, and 59 males (age range: 19-81 years) and 61 females (age range: 19-73 years) for plasma samples. The main aim of the study was to provide information on blood reference levels of 21 trace elements (Ag, Al, As, Bi, Br, Cd, Co, Cr, Cu, Hg, I, Mn, Mo, Ni, Pb, Sb, Se, Tl, U, V, Zn) in Queensland. The study also aimed to assess differences in trace element blood levels between males and females and the effect of age. The trace element levels in blood samples were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and the standard reference materials of Seronorm (Trace Elements Whole Blood) and UTAK (Trace Elements Serum) were used for quality control and assurance. The study found wide variations of trace element levels in whole blood and plasma, and generally the levels were comparable to other countries. No detectable levels were found for Bi, Cr, U and V in whole blood, but V levels were found in plasma samples. There were significant differences between males and females for whole blood Cu (p < 0.001), I (p = 0.009), Tl (p = 0.016) and Zn (p = 0.016). Significant differences were also found for plasma Cu (p < 0.001) and Se (p = 0.003) between males and females. There were trends of increased levels of blood Pb, Se and Zn with age. The study has provided further information on a wide range of trace elements in blood as reference levels for Queensland and Australia which are currently lacking.
| S-EPMC7967308 | biostudies-literature
Project description:ObjectivesThe simulation of microlaryngeal skills is rarely seen in surgical training, but it is particularly important in phonomicrosurgery. This study described and validated the laryngeal surgical simulator through surgical training.MethodsA simple and low-cost simulator was developed for the fixation of the suspension laryngoscope and porcine larynges. Twenty participants with work skills and experience did preparation before training, and performed suture and carbon dioxide (CO2) laser cordectomy for simulator evaluation. The results were proposed by the aspects of time taken for each procedure, the global rating scale, a procedure-specific assessment, and a post-simulation questionnaire.ResultsAll participants completed the preparation within 9 minutes and reached the conclusion that the microlaryngeal surgical simulator was helpful in improving their surgical skills. The performance of experts was superior to that of novices in both suture and CO2 laser cordectomy.ConclusionThis simulator could be easily assembled and was successfully validated by microlaryngeal surgical training both subjectively and objectively. It may be helpful to clinicians in microlaryngeal skills.
| S-EPMC7010492 | biostudies-literature
Project description:Several studies have demonstrated that training with a laparoscopic simulator improves laparoscopic technical skills. We describe how to build a homemade, low-cost laparoscopic training simulator (LABOT) and its validation as a training instrument. First, sixty surgeons filled out a survey characterized by 12 closed-answer questions about realism, ergonomics, and usefulness for surgical training (global scores ranged from 1-very insufficient to 5-very good). The results of the questionnaires showed a mean (±SD) rating score of 4.18 ± 0.65 for all users. Then, 15 students (group S) and 15 residents (group R) completed 3 different tasks (T1, T2, T3), which were repeated twice to evaluate the execution time and the number of users' procedural errors. For T1, the R group had a lower mean execution time and a lower rate of procedural errors than the S group; for T2, the R and S groups had a similar mean execution time, but the R group had a lower rate of errors; and for T3, the R and S groups had a similar mean execution time and rate of errors. On a second attempt, all the participants tended to improve their results in doing these surgical tasks; nevertheless, after subgroup analysis of the T1 results, the S group had a better improvement of both parameters. Our laparoscopic simulator is simple to build, low-cost, easy to use, and seems to be a suitable resource for improving laparoscopic skills. In the future, further studies should evaluate the potential of this laparoscopic box on long-term surgical training with more complex tasks and simulation attempts.
| S-EPMC6981870 | biostudies-literature
Project description:BackgroundOne major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol.MethodsWe designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory.ResultsThe sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89).ConclusionsThese approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.
| S-EPMC8280604 | biostudies-literature
Project description:A method for analysis of toxicologically important arsenic species in blood plasma and whole blood by selective hydride generation with cryotrapping (HG-CT) coupled either to atomic absorption spectrometry (AAS) with a quartz multiatomizer or to inductively coupled plasma mass spectrometry (ICPMS) has been validated. Sample preparation, which involved only 5 times dilution with addition of Triton X-100, Antifoam B, and l-cysteine, suppressed excessive foaming in a hydride generator. Calibration slopes for whole blood and blood plasma spiked with arsenate, monomethylarsonate, and dimethylarsinate at 0.25-1 μg L-1 As and 0.025-0.1 μg L-1 As for AAS and ICPMS detection, respectively, did not differ from slopes in aqueous solutions. HG-CT-AAS was used to analyze samples with elevated levels of arsenic species-blood plasma from patients treated with arsenic trioxide for acute promyelocytic leukemia and whole blood from mice fed an arsenic-containing diet. A good agreement between results of the direct analysis and analysis after mild digestion in phosphoric acid proved the good efficiency of the direct HG-CT procedure for the arsenic species in these types of biological samples. In the next step, plasma and whole blood from healthy donors that were spiked with the plasma from leukemia patients at levels of 0.15-0.4 μg L-1 As were analyzed by direct HG-CT-ICPMS. Good recoveries for all species even at these low levels (88-104%) were obtained. Limits of detection in blood and plasma were 0.014 μg L-1 for inorganic arsenic and below 0.002 μg L-1 As for methylated arsenic species. Thus, the ultrasensitive direct HG-CT-ICPMS method is uniquely suited for analyses of blood plasma and whole blood from individuals at low exposure levels.
| S-EPMC6611167 | biostudies-literature
Project description:Trace elements and minerals are compounds that are essential for the support of a variety of biological functions and play an important role in the formation of and the defense against oxidative stress. Here we describe a technique, allowing sequential detection of the trace elements (K, Zn, Se, Cu, Mn, Fe, Mg) in serum and whole blood by an ICP-MS method using single work-up, which is a simple, quick and robust method for the sequential measurement and quantification of the trace elements Sodium (Na), Potassium (K), Calcium (Ca), Zinc (Zn), Selenium (Se), Copper (Cu), Iron (Fe), Manganese (Mn) and Magnesium (Mg) in whole blood as well as Copper (Cu), Selenium (Se), Zinc (Zn), Iron (Fe), Magnesium (Mg), Manganese (Mn), Chromium (Cr), Nickel (Ni), Gold (Au) and Lithium (Li) in human serum. For analysis, only 100 μl of serum or whole blood is sufficient, which make this method suitable for detecting trace element deficiency or excess in newborns and infants. All samples were processed and analyzed by ICP-MS (Agilent Technologies). The accuracy, precision, linearity and the limit of quantification (LOQ), Limit of Blank (LOB) and the limit of detection (LOD) of the method were assessed. Recovery rates were between 80-130% for most of the analyzed elements; repeatabilities (Cv %) calculated were below 15% for most of the measured elements. The validity of the proposed methodology was assessed by analyzing a certified human serum and whole blood material with known concentrations for all elements; the method described is ready for routine use in biomonitoring studies.
| S-EPMC7239469 | biostudies-literature
Project description:The functional biomechanics of the lumbar spine have been better understood by finite element method (FEM) simulations. However, there are still areas where the behavior of soft tissues can be better modeled or described in a different way. The purpose of this research is to develop and validate a lumbar spine section intended for biomechanical research. A FE model of the 50th percentile adult male (AM) Total Human Model for Safety (THUMS) v6.1 was used to implement the modifications. The main modifications were to apply orthotropic material properties and nonlinear stress-strain behavior for ligaments, hyperelastic material properties for annulus fibrosus and nucleus pulposus, and the specific content of collagenous fibers in the annulus fibrosus ground substance. Additionally, a separation of the nucleus pulposus from surrounding bones and tissues was implemented. The FE model was subjected to different loading modes, in which intervertebral rotations and disc pressures were calculated. Loading modes contained different forces and moments acting on the lumbar section: axial forces (compression and tension), shear forces, pure moments, and combined loading modes of axial forces and pure moments. The obtained ranges of motion from the modified numerical model agreed with experimental data for all loading modes. Moreover, intradiscal pressure validation for the modified model presented a good agreement with the data available from the literature. This study demonstrated the modifications of the THUMS v6.1 model and validated the obtained numerical results with existing literature in the sub-injurious range. By applying the proposed changes, it is possible to better model the behavior of the human lumbar section under various loads and moments.
| S-EPMC10424670 | biostudies-literature