Additional Centrifugation Step Research Articles

Highly efficient diluent-free solvent extraction of uranium using mixtures of protonated trioctylamine and quaternary ammonium salts. Comparative Life Cycle Assessment with the conventional extractant

A more environmentally friendly solvent extraction process was formulated for uranium extraction using diluent-free extractants based on trialkylammonium compounds. The effect of ammonium cations and anions toward extraction efficiency, viscosity and phase separation was also evaluated. These systems were shown to be highly efficient and simple to implement as they rely on the same chemical components as those applied in the actual industrial process. It has also been demonstrated that using such alternatives allows not only to prevent third phase formation and subsequent extractant degradation, but also reduces the evaporation loss of solvent by a factor of 20. Among the systems investigated, the pure [TOAH]2[SO4] was selected as the simplest and most efficient. A comparative Life Cycle Assessment (LCA) was also conducted between this system and the conventional solvent to compare more objectively their sustainability. Results based on four environmental indicators show that the new system is 18 times less impactful. Contrary to expectations, this study also showed that the pure (diluent-free) extractant system needs to be at least 2 times more efficient in terms of metal extraction to become significantly more sustainable. Other main effects contributing to the LCA are derived from: (i) the production of chemicals, which is much more harmful for extractants than for aliphatic diluents, (ii) the loss of Volatile Organic Compounds (VOC) to the environment, and (iii) to a lesser extent, the additional centrifugation step that may be required to separate more viscous systems.

Magnetic activated carbon: A promising approach for the removal of methylene blue from wastewater

The contamination of water bodies by synthetic dyes, namely methylene blue (MB), is a significant environmental concern due to their contrary effects on aquatic ecosystems. This study proposes a novel approach by integrating magnetic properties into activated carbon to improve the removal efficiency of methylene blue and simplifies the separation process. The synthesis of magnetic activated carbon involved the impregnation of activated carbon (Rice husk as a precursor) with iron oxide nanoparticles ((Fe3O4-RAC), which imparted magnetic properties to the carbon matrix. The characterization studies were performed to perceive the morphology of the adsorbent. The adsorption tests were examined under various factors such as contact time, initial MB concentration, adsorbent dosage and pH. The data revealed that the equilibrium adsorption of methylene blue onto Fe3O4-RAC was achieved within 60 min, with a maximal adsorption strength of 32.25 mg g−1. The Langmuir model (R2 = 0.997) demonstrated the best fit, suggesting monolayer adsorption behaviour. Furthermore, the adsorption kinetics tailed pseudo-second-order (R2 = 0.995), indicating chemisorption as the rate-determining step. The results indicated a positive correlation between temperature and MB adsorption, signifying the endothermic nature. The magnetic properties of Fe3O4-RAC facilitated the separation of the adsorbent from the treated solution using an external magnetic field, eliminating the need for additional filtration or centrifugation steps. The regeneration of Fe3O4-RAC was also investigated, and it was found that the composite could be efficiently regenerated using ethanol as a desorption agent, allowing for repeated use. In conclusion, magnetic activated carbon demonstrated promising potential as an efficient and reusable adsorbent for the removal of methylene blue from wastewater.

Isolation of extracellular vesicles from human plasma samples: The importance of controls.

Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. We performed a comparative study to evaluate the effect ofEV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. Appropriate controlsare required when adding any additives during EV isolationas even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Composition and Techno-functional Properties of Grape Seed Flour Protein Extracts

Defatted grape seed flour (DGSF), a byproduct of the grape seed oil production, was investigated as a potential source of protein-enriched food ingredients with functional properties. The operation conditions of the alkaline extraction followed by the acid precipitation method were studied by response surface methodology, aiming to maximize the protein extraction yield. These conditions were temperature 50 °C, solvent/meal ratio 1:15, pH 11, and 150 min extraction time in two sequential extraction cycles, achieving a product with 32.63 ± 0.07% (w/w) d.m. of protein content. A second extract with 38.17 ± 1.34% (w/w) d.m. of protein was obtained under the same conditions, but using an additional centrifugation step before the acid precipitation. The amino acid profile, solubility, water and oil retention capacity, and emulsifying and foaming properties of the DGSF and the obtained extracts were compared. Both extracts could be used as food ingredients with different nutritional and technological purposes.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

Composition of Brazil Nut (Bertholletia excels HBK), Its Beverage and By-Products: A Healthy Food and Potential Source of Ingredients.

The consumption of plant-based beverages is a growing trend and, consequently, the search for alternative plant sources, the improvement of beverage quality and the use of their by-products, acquire great interest. Thus, the purpose of this work was to characterize the composition (nutrients, phytochemicals and antioxidant activity) of the Brazil nut (BN), its whole beverage (WBM), water-soluble beverage (BM-S), and its by-products of the beverage production: cake, sediment fraction (BM-D), and fat fraction (BM-F). In this study, advanced methodologies for the analysis of the components were employed to assess HPLC-ESI-QTOF (phenolic compounds), GC (fatty acids), and MALDI-TOF/TOF (proteins and peptides). The production of WBM was based on a hot water extraction process, and the production of BM-S includes an additional centrifugation step. The BN showed an interesting nutritional quality and outstanding content of unsaturated fatty acids. The investigation found the following in the composition of the BN: phenolic compounds (mainly flavan-3-ols as Catechin (and glycosides or derivatives), Epicatechin (and glycosides or derivatives), Quercetin and Myricetin-3-O-rhamnoside, hydroxybenzoic acids as Gallic acid (and derivatives), 4-hydroxybenzoic acid, ellagic acid, Vanillic acid, p-Coumaric acid and Ferulic acid, bioactive minor lipid components (β-Sitosterol, γ-Tocopherol, α-Tocopherol and squalene), and a high level of selenium. In beverages, WBM had a higher lipid content than BM-S, a factor that influenced the energy characteristics and the content of bioactive minor lipid components. The level of phenolic compounds and selenium were outstanding in both beverages. Hydrothermal processing can promote some lipolysis, with an increase in free fatty acids and monoglycerides content. In by-products, the BM-F stood out due to its bioactive minor lipid components, the BM-D showed a highlight in protein and mineral contents, and the cake retained important nutrients and phytochemicals from the BN. In general, the BN and its beverages are healthy foods, and its by-products could be used to obtain healthy ingredients with appreciable biological activities (such as antioxidant activity).

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Development and evaluation of an automated multi-channel multiplug filtration cleanup device for pesticide residue analysis on mulberry leaves and processed tea.

An automated multi-channel multiplug filtration cleanup (m-PFC) device was designed and developed. m-PFC columns were suitably installed in the device. The cycle times, speed and nitrogen pressure parameters of the m-PFC column were optimized. The device was utilized to analyze the 82 pesticide residues in fresh mulberry leaves and processed tea with GC-MS/MS detection. Method validation was performed on 82 pesticide residues in fresh mulberry leaves and processed tea at spiked levels of 0.01, 0.05 and 0.5 mg kg−1. The fortified recoveries of 82 pesticides were 72–115% and the relative standard deviations were 1–15%, except for diniconazole and clodinafop-propargyl in mulberry leaves. The automated multi-channel m-PFC device was successfully applied to detect the pesticide residues in fresh mulberry leaves and processed tea samples. With comparison to the conventional QuEChERS method, the current method using this device did not need additional vortex or centrifugation steps, and could process 48–64 samples in about one hour. The automated m-PFC method saved labor and improved the precision and was shown to be efficient and practical in pesticide residue analysis.

The challenges of developing and optimising an assay to measure 25-hydroxyvitamin D in saliva

Accurately detecting vitamin D deficiency (defined as concentration in blood of 25-hydroxyvitamin D (25(OH)D), <20 ng/mL) is important for both clinicians and researchers. Drawing blood may be difficult in some populations, such as infants and children. We thus explored the development of a method to measure 25(OH)D concentrations in saliva, using a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay. Using 25(OH)D3 standards spiked into synthetic saliva, we generated a standard curve with high correlation (r = 0.999, Pearson’s); the intra-assay and inter-assay variation were ≤3.2% and ≤13.2% (CV%), respectively. Passive collection of saliva via drooling into glass or polypropylene tubes yielded higher levels of 25(OH)D3 than chewing on a synthetic swab. Chewing gum for at least 4 min reduced saliva levels of 25(OH)D3. Differences in the levels of 25(OH)D3 in saliva between the passive drooling and stimulated swab-chewing methods were normalised by adjusting for measured levels of vitamin D binding protein in saliva. Freezing samples immediately, or after 24 h of refrigeration did not affect 25(OH)D3 levels. When saliva levels of 25(OH)D3 were averaged from samples collected daily for three consecutive days, for which an additional centrifugation step was performed after samples were defrosted (to remove mucin), there was a positive (but non-significant) correlation between 25(OH)D3 levels in saliva and serum (r = 0.57, p = 0.24, Pearson’s) with significant correlations (r ≥ 0.88, p < 0.05) observed after further adjusting for saliva flow rate. The time of day of the collection made little difference to 25(OH)D3 levels measured in saliva. In conclusion, we have developed an LC–MS/MS assay that accurately measures saliva 25(OH)D3 levels, which correlated with serum levels. However, for a measurement that correlates with serum 25(OH)D it may be necessary to average results from saliva collected on three consecutive days, and adjust for differences in saliva flow rate. This would increase costs, and combined with the processing requirements for samples, could limit the applicability of this assay to large cohort and field studies.

Loop-mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species.

Invasive Aedes mosquito species (Diptera: Culicidae) are of public health concern in Europe because they are either recognized or potential vectors of pathogens. Loop-mediated isothermal amplification (LAMP) is a rapid and simple method for amplifying DNA with high specificity and efficiency, with the technique having potential for application in the field, including in high-throughput format. Specific LAMP assays based on rDNA internal transcribed spacers 1 or 2 sequences, considering intraspecies variability at these loci, were developed for Aedes aegypti, Aedes albopictus, Aedes japonicus, Aedes koreicus and the indigenous Aedes geniculatus. No such assays could be developed for Aedes atropalpus and Aedes triseriatus because both loci were too short to serve as target. The assays rely on the clearly visible colour change from violet to sky blue after successful amplification. Sensitivity of egg detection was confirmed with ratios of up to one mosquito egg in 99 other eggs. Simple sample preparation of adults or eggs by mechanical homogenization in water required an additional heat treatment or centrifugation step to avoid non-specific colour changes. Thus, further technical improvements are needed to render these assays truly field-applicable, which would greatly facilitate surveillance of these invasive mosquito species and allow for prompt implementation of control measures.

Enhancement of Progenitor Cells by Two-Step Centrifugation of Emulsified Lipoaspirates.

Adipose-derived stem cells, endothelial progenitor cells, and soluble factors jointly contribute to the regenerative effect of fat grafts. Nanofat grafting emulsifies the lipoaspirate and increases the progenitor cell yield. In the present study, the authors evaluated their extended nanofat grafting method that includes two additional centrifugation steps and results in a lipoaspirate of low volume that they termed "lipoconcentrate." Furthermore, the authors investigated the oily fractions after centrifugation for their regenerative potential. Lipoaspirates of 20 healthy patients were processed by emulsification and/or centrifugation. Six groups were created: native (not emulsified) fat, 1× centrifuged native fat, 2× centrifuged native fat, nanofat (emulsified), 1× centrifuged nanofat, and lipoconcentrate (i.e., 2× centrifuged nanofat). The oily phases after the centrifugation steps were collected. Progenitor cells and basic fibroblast growth factor, insulin-like growth factor 1, matrix metalloproteinase-9, platelet-derived growth factor-BB, and vascular endothelial growth factor-A levels were measured by flow cytometry and immunoassays. Lipoconcentrate contained significantly higher numbers of adipose-derived stem cells and endothelial progenitor cells per gram compared with all other fractions. No difference of all five soluble factors between groups was found. The oily phases after centrifugation showed no or very few adipose-derived stem cells and endothelial progenitor cells, and no or very low levels of soluble factors. Centrifugation of emulsified lipoaspirates increases the progenitor cell count in the lipoaspirate. The oily phase after centrifugation of lipoaspirates may be disposable because of the minuscule content of progenitor cells and soluble factors.

An Efficient Method for Purifying High-Quality RNA from the Hepatopancreas of the Pacific AbaloneHaliotis discus hannai

RNA isolation from the hepatopancreas of adult abalone is challenging, as indicated by the variable results of reverse transcription-polymerase chain reaction (RT-PCR) amplification. In this study, three RNA isolation methods were tested to evaluate their usefulness in RNA isolation from various abalone tissues. Unlike the conventional method (method I), methods II and III included additional centrifugation and lithium chloride precipitation steps, respectively. RNA quality assessment based on the RT-PCR amplification of ribosomal protein L3 (RPL3) showed little difference in the quality of RNA isolated from muscle, regardless of the isolation method used. While RNA isolated from the hepatopancreas using methods I and II resulted in lower levels of RPL3 amplification, method III was found to be a more effective method of RNA extraction from the hepatopancreas, as indicated by a 30–60-fold increase in the RPL3 level. Competitive inhibition experiments using mixtures of RNA prepared by each method were performed to investigate which RT-PCR step was inhibited by materials present in the RNA preparation. An almost complete absence of RPL3 amplification was observed in RT-PCR with a cDNA template prepared from a mixture of RNAs containing a small amount of hepatopancreatic RNA isolated using method I. A drastic decrease in the incorporation of a fluorescence-labeled nucleotide into cDNA was also evident in the reverse transcription containing hepatopancreatic RNA isolated using method I. These results indicate a distinct inhibition of cDNA synthesis by materials present in the hepatopancreatic RNA preparation isolated using method I, but not present when method III is used. A competitive inhibition experiment was performed using mixtures of cDNA prepared from RNA isolated by each method to investigate whether the materials used in the preparation inhibited the polymerase chain reaction (PCR) step. The level of RPL3 amplification was proportional to the amount of cDNA prepared from hepatopancreatic RNA isolated using method III. The comparable degree of amplification regardless of the amount of cDNA prepared from RNA isolated using method I, together with the low cDNA synthesis in the latter, indicated that it had little, if any, inhibitory effect on the PCR step. This study reports an RNA isolation method that is applicable to the hepatopancreas and other abalone tissues with similar efficacy.

Molecularly imprinted magnetic nanoparticles for the micro solid-phase extraction of thiabendazole from citrus samples.

The preparation of molecularly imprinted core-shell magnetic nanoparticles and their subsequent use in the solid-phase extraction of thiabendazole from citrus sample extracts is described. Molecularly imprinted core-shell magnetic nanoparticles were prepared by the precipitation copolymerization of the imprinting polymerization mixture on the surface of vinyl-modified silica magnetic nanoparticles and were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The obtained molecularly imprinted core-shell magnetic nanoparticles exhibited a high selectivity for thiabendazole and were easily collected and separated by an external magnetic field without additional centrifugation or filtration steps. Under optimum conditions, a magnetic molecularly imprinted solid-phase extraction method was developed allowing the extraction of thiabendazole from citrus sample extracts and final determination by high-performance liquid chromatography with fluorescence detection. The detection limit was 0.2mg/kg, far lower than the maximum residue limit established within the European Union for thiabendazole in citrus samples.

Development and optimization of an in vitro cultivation protocol allows for isolation of Borrelia miyamotoi from patients with hard tick-borne relapsing fever

ObjectivesBorrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients. MethodsModified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro. Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF. ResultsDipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 μg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g, suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF. ConclusionsDirect culture from K2-EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers.

Molecularly imprinted core-shell magnetic nanoparticles for selective extraction of triazines in soils.

In this work, a propazine-imprinted polymer was synthesized on the surface of modified magnetic nanoparticles to be used in the solid-phase extraction of triazines in soil samples. The effect of different solvents on the selective extraction of target analytes was assessed to establish the optimum rebinding conditions. The obtained magnetic molecularly imprinted particles exhibited high selectivity for triazines and were easily collected and separated by an external magnetic field without additional centrifugation or filtration steps. Under optimum conditions, a magnetic molecularly imprinted solid-phase extraction method was developed allowing the extraction of several triazines (desisopropylatrazine, desethylatrazine, simazine, atrazine, and propazine) from soil samples and their subsequent final determination by high-performance liquid chromatography with diode-array detection. Recoveries for the triazines studied were within the range 5.4% to 40.6%, with relative standard deviations lower than 7.0% (n=3). The detection limits were within 0.1 to 3ngg-1 , depending upon the triazine and the type of soil used.

Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

Influence of the separation procedure on the properties of magnetic nanoparticles: Gaining in vitro stability and T1–T2 magnetic resonance imaging performance

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) coated with polyacrylic acid (PAA) were synthesized by a hydrothermal method in gram-scale quantity and extensively characterized. Only the nanoparticles subjected to an additional centrifugation step showed narrow size distribution, high polymeric coverage, and ideal superparamagnetism. In addition to improved physico-chemical properties, these nanoparticles feature high stability in vitro as well as dual T1–T2 performance as contrast agents (CAs) for magnetic resonance imaging (MRI), highlighting the importance of the additional separation step in obtaining material with the desired properties.

Increased count, motility, and total motile sperm cells collected across three consecutive ejaculations within 24 h of oocyte retrieval: implications for management of men presenting with low numbers of motile sperm for assisted reproduction.

The purpose of this study was to quantitate changes in seminal volume, sperm count, motility, qualitative forward progression, and total motile sperm cells per ejaculate, across three consecutive ejaculates collected from individuals within 24 h preceding an IVF cycle. Men presenting with oligoasthenozoospermia or asthenozoospemia attempted three ejaculates within 24 h preceding IVF. Ejaculate 1 was produced the afternoon prior to oocyte retrieval, and ejaculates 2 and 3 were produced the morning of oocyte retrieval with 2-3 h between collections. Ejaculates 1 and 2 were extended 1:1 v/v with room temperature rTYBS. Test tubes were placed into a beaker of room temperature water, then placed at 4 °C for gradual cooling. Ejaculate 3 was not extended, but pooled with ejaculates 1 and 2 and processed for intracytoplasmic sperm injection (ICSI). Out of 109 oocyte retrievals, 28 men were asked to attempt multiple consecutive ejaculations. Among this population, 25/28 (89.3 %) were successful, and 3/28 men (10.7 %) could only produce two ejaculates. Mean volumes for ejaculates 1, 2, and 3 were significantly different from each other (p < 0.01); the volume decreased for each ejaculate. Mean sperm counts, motility, qualitative forward progression, and total motile cells per ejaculate for the ejaculates1, 2, and 3 demonstrated the following: ejaculates 2 and 3 were not significantly different, but counts, motility, and total motile sperm were improved over ejaculate 1 (p < 0.01). Pooling three consecutive ejaculates within 24 h increased the numbers of available motile sperm in this population by 8-fold compared to the first ejaculate alone, facilitating avoidance of sperm cryopreservation and additional centrifugation steps that could affect sperm viability and/or function.

The Utility of Assessing the Gross Appearance of Fine Needle Aspiration Specimens for a Better Yield on Cell Blocks

Introduction: Cell blocks (CBs) with adequate cellularity are important for diagnosis and ancillary studies. Cytology samples from breast cancer, primary or metastatic, are unique in that they must be fixed in 10% neutral buffered formalin (NBF) to comply with the ASCO/CAP guidelines for evaluation of estrogen and progesterone receptors (ER, PR) and Her2 neu (Her2) by immunohistochemistry (IHC). The aim of this study was to determine whether the addition of 95% ethyl alcohol (ETOH) into NBF and an additional centrifugation step would improve cellularity of CBs without compromising ER/PR/Her2 IHC or FISH. Materials and Methods: 18 breast cancer specimens, >1 cm in size and known ER+ and Her2(3+) status were selected. Scrapings from each case were performed at the time of grossing and placed in conical tubes all with 10% NBF labeled 1, 2 and 3, and processed as seen in Figure 1. Histological processing followed standard cycle for CBs with adequate fixation time. CBs were evaluated for cellularity, stained for ER and Her2, and scored. Known Her2(3+) cases were submitted for FISH. Results: The CBs with the ETOH/centrifugation step showed better cell aggregation. 17/18 CBs (94%) had similar or improved cellularity. There was a 93% concordance of ER and 100% concordance of Her2 between CBs and the biopsy/resection. Her2 FISH testing in CBs following the new method were more successful in showing amplification (5/6) compared with the old method (1/6). FISH failed due to lack of cells. Conclusion: Adding ETOH/centrifugation step into the processing provides the same or better cellularity in the majority of cases while complying with the ASCO/CAP guidelines. Better cellular aggregation in the slide helps screening, FISH analysis and possibly molecular testing. Staining pattern of ER and Her2 in CBs prepared with the addition of ETOH has high concordance rate with stains performed on paired biopsy/ resection. Figure 1