Identification and quantification of high fluorescence-stained lymphocytes as antibody synthesizing/secreting cells using the automated routine hematology analyzer XE-2100.

OBJECTIVES: The aim of this study was to classify and quantify the high fluorescence lymphocytes area (HFL-count) from the SYSMEX XE-2100 leucocyte differential channel as antibody-synthesizing or -secreting cells (ASC, plasma cells or lymphoplasmacytoid cells) in reactive diseases. To unequivocally identify the HFL cells, all possibly eligible cell populations have been investigated: activated B-lymphocytes, activated T-lymphocytes, large granular lymphocytes (LGL), activated monocytes, and immature granulocytes. METHODS: In total, 85 patients were analyzed on the XE-2100 and compared with the automated image analysis system Cellavision Diffmaster 96 based on artificial neural network and immunophenotyping method with the BD FACSCalibur. RESULTS: Reproducibility tests for HFL demonstrated a mean coefficient of variation of 13.9% for very low results and 1.5% for high results. The linearity data showed a good correlation (R(2) = 0.99) between expected and measured HFL. The comparison with possibly eligible cell populations showed no significant correlation between activated monocytes and immature granulocytes, with most immature granulocytes (promyelocyte I or II), natural killer cells or LGLs, activated T-lymphocytes, and sub-T-lymphocytes populations. However, for activated B-lymphocytes an excellent significant correlation with the peripheral blood smear, and the immunophenotyping method has been found with R(2) = 0.900, P < 0.001 and R(2) = 0.897, P < 0.001, respectively. The slope of 1.1 and intercept of minus 5 cells/microL of the regression equation between HFL-count and ASC (smear) do indicate an excellent quantification of the HFL-count, as well. CONCLUSION: The fully automated SYSMEX XE-2100 HFL-count identifies and quantifies the ASC cells (activated B-lymphocytes) with high precision and reliability in patients without hematology system diseases, thus providing a potential screening and monitoring tool for any patient with suspected infection. Additional studies are required to comprehend in more detail the full clinical utility of an HFL (ASC) count as a potential diagnostic indicator of inflammation, infection, or sepsis.

Evaluation of antioxidative properties of Geranium macrorrhizum and Potentilla fruticosa extracts in Dutch style fermented sausages.

Antioxidative properties of Geranium macrorrhizum, Potentilla fruticosa and Rosmarinus officinalis (as a reference) extracts were evaluated in Dutch style fermented sausages. Extracts were incorporated into sausages during preparation. The sausages were subsequently fermented, tested and compared to a standard spices mix, traditionally used for the production of such sausages. Formation of the primary oxidation products - peroxides, and secondary - TBARS and hexanal was monitored. The polar extracts from Potentilla showed some antioxidant activity, especially in combination with ascorbate, however the activity was low compared to the standard spices mix. Polar extracts from Geranium showed only negligible antioxidant activity.

The Effects of Ex Vivo Administration of Granulocyte-Macrophage Colony-Stimulating Factor and Endotoxin on Cytokine Release of Whole Blood Are Determined by Priming Conditions.

BACKGROUND: Lipopolysaccharide- (LPS-) induced tumour necrosis factor alpha (TNFα) secretion in critically ill patients can be considered as a measure of immune responsiveness. It can be enhanced by granulocyte-macrophage colony stimulating factor (GM-CSF). We investigated the effect of GM-CSF on ex vivo stimulated cytokine production using various preincubation regimens in healthy donors and patients with sepsis. RESULTS: The maxima for the stimuli occurred 3 hours after stimulation. In donors, there was an increase (p < 0.001) of LPS-induced TNFα levels following incubation with GM-CSF. The simultaneous incubation with GM-CSF and LPS caused an inhibition of TNFα production (p < 0.001). Postincubation with GM-CSF did not yield any difference. In patients, preincubation with GM-CSF yielded an enhanced ex vivo TNFα-response when TNFα levels were low. Patients with increased TNFα concentrations did not show a GM-CSF stimulation effect. The GM-CSF preincubation yielded an increase of IL-8 production in patients and donors. CONCLUSIONS: This study demonstrates the immune-modulating properties of GM-CSF depending on the absence or presence of LPS or systemic TNFα. The timing of GM-CSF administration may be relevant for the modulation of the immune system in sepsis. The lack of stimulation in patients with high TNFα may represent endotoxin tolerance.

Biofabrication of reinforced 3D-scaffolds using two-component hydrogels.

Progress in biofabrication technologies is mainly hampered by the limited number of suitable hydrogels that can act as bioinks. Here, we present a new bioink for 3D-printing, capable of forming large, highly defined constructs. Hydrogel formulations consisted of a thermoresponsive polymer mixed with a poly(ethylene glycol) (PEG) or a hyaluronic acid (HA) cross-linker with a total polymer concentration of 11.3 and 9.1 wt% respectively. These polymer solutions were partially cross-linked before plotting by a chemoselective reaction called oxo-ester mediated native chemical ligation, yielding printable formulations. Deposition on a heated plate of 37 °C resulted in the stabilization of the construct due to the thermosensitive nature of the hydrogel. Subsequently, further chemical cross-linking of the hydrogel precursors proceeded after extrusion to form mechanically stable hydrogels that exhibited a storage modulus of 9 kPa after 3 hours. Flow and elastic properties of the polymer solutions and hydrogels were analyzed under similar conditions to those used during the 3D-printing process. These experiments showed the ability to extrude the hydrogels, as well as their rapid recovery after applied shear forces. Hydrogels were printed in grid-like structures, hollow cones and a model representing a femoral condyle, with a porosity of 48 ± 2%. Furthermore, an N-hydroxysuccinimide functionalized thermoplastic poly-ε-caprolactone (PCL) derivative was successfully synthesized and 3D-printed. We demonstrated that covalent grafting of the developed hydrogel to the thermoplastic reinforced network resulted in improved mechanical properties and yielded high construct integrity. Reinforced constructs also containing hyaluronic acid showed high cell viability of chondrocytes, underlining their potential for further use in regenerative medicine applications.

Automation and validation of a rapid method to assess neutrophil and monocyte activation by routine fluorescence flow cytometry in vitro.

The aim of the present study was to design an automated-gating hematology fluorescence flow cytometry methodology permitting the assessment of neutrophil and monocyte activation in EDTA-anticoagulated whole blood based on cell granularity, lipid membrane components, cell shape and volume, and total cell nucleic acid (NA) compounds. For particularly monitoring the proper functioning of patients' innate immune system as the first line defense against microbial invaders, the suitable test system should be rapid, simple, reliable by yielding reproducible results. It must be validated against established methods, and it must prove to work in selected clinical settings, e.g. in intensive care unit (ICU) environments. The adaptation of a routine hematology cell analyser utilizing fluorescence flow cytometry resulted in a potentially useful system for all requirements. It proved to detect in real-time and in a reliable and reproducible way the main cellular response reactions of neutrophils and monocytes during externally stimulated immune defense. Validation was successful when comparing it to established methods. The quantified activation effects were dose dependent from the applied activating agents. Cellular response kinetics could be measured and described and showed to be in line with the prevailing cell response models. Upon applying the test method to a healthy population of volunteers and a first cohort of ICU patients with and without evident immune depression, the test revealed excellent cellular responses to external activating cytotoxic stimuli (lipopolysaccharide; LPS) for the control group, slightly weaker response from ICU patients without immune depression and no response from patients with evident immune depression.We conclude that routine hematology fluorescence flow cytometry can accurately and reproducibly measure different activation steps of monocytes and polymorphonuclear neutrophilic granulocytes to defined external stimuli. This may potentially be applied as a STAT (Latin statim = immediately) and routine screening and surveillance method for inflammatory diseases.

Static headspace gas chromatography of acetaldehyde in aqueous foods and polythene terephthalate.

Polythene terephthalate (PET) is frequently used as a packaging material for mineral water and other non-alcoholic beverages. PET contains detectable amounts of acetaldehyde, which is able to migrate to its packed product. An automated headspace gas chromatographic method for the determination of acetaldehyde has been developed and was used for the quantification of acetaldehyde in aqueous food products and their PET packages. A cold trap, mounted in the GC oven, between the auto sampler and analytical column was introduced as a new application. The detection limit of acetaldehyde was found to be 3 ng/ml with a standard deviation of 3%. The contents of acetaldehyde found in carbonated mineral water and lemonade ranged between 11 ng/ml and 7447 ng/ml, while the contents of acetaldehyde in the PET packages ranged from 1.1 microgram/g to 3.8 micrograms/g.

Development and validation of analytical methods for monomeric and oligomeric migrants from nylon 12 packaging materials.

Analytical methods for the determination of laurolactam--the monomer of nylon 12--as well as the cyclic dimer and trimer were established. High performance liquid chromatography using ultraviolet (HPLC-UV) and mass spectrometric detection (HPLC-MS) were both found suitable to identify and quantify monomer, cyclic dimer and trimer well below the specific migration limit (SML) of laurolactam, being 5 mg/kg of food (simulant). Gas chromatography with flame ionisation detection (GC-FID) showed to be an appropriate method for the detection of only laurolactam in aqueous and fatty food simulants. Food simulants could be analysed directly by all three methods, or after a change of solvents. For olive oil, a method for sample clean-up by size-exclusion chromatography (SEC) was established.

Alternative fatty food simulants and diffusion kinetics of nylon 12 food packaging.

The migration of laurolactam and cyclic di- and trimer of nylon 12 was assessed using three different films and five food simulants (olive oil, isooctane, 95% ethanol, 50% ethanol, water). Substitute test conditions for migration into olive oil according to European Union Directive EC/97/48 were applied using 95% ethanol and isooctane. Results showed that 95% ethanol overestimated while isooctane underestimated the respective migration into olive oil. Water was the best olive oil substitute, as migration of laurolactam into water and olive oil using the same temperature gave similar results. Additionally, diffusion kinetics of laurolactam were investigated by migration kinetic studies using isooctane and olive oil. Diffusion coefficients determined with isooctane were significantly higher than those found using olive oil. It was proved that isooctane had an interaction and olive oil was inert to the polymer. The diffusion conductance parameter, A(p), for nylon 12 determined using olive oil ranged from 0.3 to 0.6.

Combined GC and sniffing port analysis of volatile compounds in rubber rings mounted on beer bottles.

For product development reasons two types (A and B) of rubber rings, used on clasps of swing top beer bottles, were investigated for the presence of volatile compounds, which could affect the taste/odour of the packed beer due to (vapour phase) migration. Samples were incubated under different conditions and, after dynamic headspace sampling, analysed by combined GC and sniffing port analysis. Compounds were also identified by GC-MS. The main differences were located in the presence of isobutene isomers in Ring Type A, which were absent in Ring Type B. These compounds were described mainly as 'toy-like' and 'rubber'. Therefore the risk for off-flavour development is expected to be diminished by using Ring Type B.

Sensory analysis of polystyrene packaging material taint in cocoa powder for drinks and chocolate flakes.

Polystyrene packaging material taint was sensorily evaluated in cocoa powder for drinks and chocolate flakes using short-cut signal detection measures on differences between control and test samples and on recognition of styrene. No differences were observed in cocoa powder for drinks and plain chocolate flakes treated with 0.5 dm2 polystyrene of 1 mm thickness. However, differences were detected in milk chocolate flakes and plain chocolate flakes, which were in contact with a larger area or thicker polystyrene packaging material. The latter results were confirmed by the styrene recognition test, so polystyrene is a potential source of off-flavour for chocolate products. The amount of residual styrene in the polystyrene used was about 320 ppm, while the amounts of styrene ranged from 7 to 132 ppb in cocoa drinks and from 414 to 1447 ppb in chocolate flakes.

Taste interaction of styrene/ethylbenzene mixtures in an oil-in-water emulsion.

Styrene and ethylbenzene are present in detectable amounts in polystyrene packaging materials and migration of these compounds may cause a taint of the packed food. The effect of mixtures of styrene and ethylbenzene on the detection threshold concentration in a 5% oil-in-water emulsion was investigated with the constant stimulus differences test. The detection threshold concentrations of the individual compounds in the emulsion were found to be 1.0 ppm for styrene and 3.0 ppm for ethylbenzene. Linear regression of the percentages of correct answers and the logarithm of the sum of the concentrations of both compounds in the mixtures expressed as fractions of their threshold concentration gave the following relationship: % of correct answers = 42.5 x log (sum of threshold fractions) +74.8 (r = 0.87). Calculation of the sum of threshold fractions at the 75% point of correct answers gave a value of 1.0, which clearly indicates that the effect of mixing of styrene and ethylbenzene is additive at sub-threshold levels in a 5% oil-in-water emulsion.

Influence of flavour absorption by food-packaging materials (low-density polyethylene, polycarbonate and polyethylene terephthalate) on taste perception of a model solution and orange juice.

The influence of flavour absorption by low-density polyethylene (LDPE), polycarbonate (PC) and polyethylene terephthalate (PET) on taste perception of a model solution containing seven flavour compounds and orange juice in glass bottles was studied with and without pieces of the respective plastic films after dark storage at 20 degrees C. Owing to absorption, the amount of flavour compounds in the model solution exposed to LDPE decreased substantially. From the model flavour solution valencene was almost completely absorbed by LDPE, followed to a lesser extent by decanal, hexyl acetate, octanal and nonanone. Less flavour compounds were absorbed from the model solution by PC and PET. In contrast to LDPE, valencene was absorbed in the lowest amounts and decanal in the highest. Limonene was readily absorbed from orange juice by LDPE, while myrcene, valencene, pinene and decanal were absorbed in smaller quantities. Only three flavour compounds were absorbed from orange juice by PC and PET in very small amounts: limonene, myrcene and decanal. Although the flavour content between controls and polymer-treated samples differed substantially, the loss of flavour compounds due to absorption by LDPE, PC and PET did not influence taste perception of a model solution and orange juice significantly up to 29 days of dark storage at 20 degrees C as determined by triangular taste panel tests.

Modelling the effect of oil/fat content in food systems on flavour absorption by LLDPE.

One of the phenomena in food-packaging interactions is flavour absorption. Absorption of flavour compounds from food products into food-packaging materials can result in loss of flavour compounds or an unbalance in the flavour profile changing a product's quality. The food matrix influences the amounts of absorbed flavour compounds; the presence of oil or fat especially determines the ability to absorb flavour compounds from the food to the package. On the other hand, the polarity of the flavour compound itself is a characteristic that also influences the level of absorption into synthetic polymers. A model based on the effect of the polarity (logP) of flavour compounds and on their partitioning coefficients between the food (matrix) and the packaging material is described. The model can be used for predicting absorption of flavour compounds from foods into LLDPE. However, an attempt to apply the proposed model on real foods shows serious limitations of the model for (very) low fat products. Predictive values deviate from the measured values, probably due to other interaction phenomena, e.g. with proteins. Predictive and measured values from a product with a substantial amount of fat match much better, suggesting that the model is valid for products having a substantial amount of (free) fat.

Feasibility study for the development of certified reference materials for specific migration testing. Part 1: initial migrant concentration and specific migration.

The paper describes a project with the main objective of developing the know how to produce certified reference materials (CRMs) for specific migration testing. Certification parameters discussed are the initial concentration of the migrant in the polymer (C(P),0) and the specific migration into a food simulant under certain temperature/time conditions. Sixteen preliminary candidate CRMs were defined and produced. The most important polymers (low- and high-density polyethylene (LDPE and HDPE), polypropylene (PP), polystyrene (PS), polyethylene terephtalate (PET), plasticized polyvinyl chloride (PVC), rigid PVC, polyamides (PA)) and additives as well as monomers representing different physicochemical properties as target substances for migration were chosen. The stability and homogeneity of the migrants in the materials were tested and methods for the determination of the certification parameters were developed and validated. > From the 16 materials produced, the six most suitable CRM candidates (LDPE//Irganox 1076/Irgafos 168, LDPE//1,4-diphenyl-1,3-butadiene (DPBD), HDPE//Chimassorb 81/Uvitex OB, PP homo//Irganox 1076/Irgafos 168, HIPS, 1% mineral oil//styrene, PA 6//caprolactam) were selected. The feasibility of CRM production for the six candidate materials was demonstrated and a trial certification exercise was performed with participation of all four partner laboratories. All six materials showed suitable properties for future production as certified reference materials.

The food matrix of spinach is a limiting factor in determining the bioavailability of beta-carotene and to a lesser extent of lutein in humans.

Carotenoid bioavailability depends, amongst other factors, on the food matrix and on the type and extent of processing. To examine the effect of variously processed spinach products and of dietary fiber on serum carotenoid concentrations, subjects received, over a 3-wk period, a control diet (n = 10) or a control diet supplemented with carotenoids or one of four spinach products (n = 12 per group): whole leaf spinach with an almost intact food matrix, minced spinach with the matrix partially disrupted, enzymatically liquefied spinach in which the matrix was further disrupted and the liquefied spinach to which dietary fiber (10 g/kg wet weight) was added. Consumption of spinach significantly increased serum concentrations of all-trans-beta-carotene, cis-beta-carotene, (and consequently total beta-carotene), lutein, alpha-carotene and retinol and decreased the serum concentration of lycopene. Serum total beta-carotene responses (changes in serum concentrations from the start to the end of the intervention period) differed significantly between the whole leaf and liquefied spinach groups and between the minced and liquefied spinach groups. The lutein response did not differ among spinach groups. Addition of dietary fiber to the liquefied spinach had no effect on serum carotenoid responses. The relative bioavailability as compared to bioavailability of the carotenoid supplement for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber for beta-carotene was 5.1, 6.4, 9.5 and 9.3%, respectively, and for lutein 45, 52, 55 and 54%, respectively. We conclude that the bioavailability of lutein from spinach was higher than that of beta-carotene and that enzymatic disruption of the matrix (cell wall structure) enhanced the bioavailability of beta-carotene from whole leaf and minced spinach, but had no effect on lutein bioavailability.

Influence of flavour absorption on oxygen permeation through LDPE, PP, PC and PET plastics food packaging.

The effect of flavour absorption on the oxygen permeability of low-density polyethylene (LDPE), polypropylene (PP), polycarbonate (PC) and polyethylene terephthalate (PET) was studied using an isostatic continuous flow system. Polymer samples were exposed to a model solution containing limonene, hexyl acetate, nonanone and decanal at 40 degrees C. After exposure, one part of each sample was analysed for absorbed flavour compounds using a Large Volume Injection GC Ultrasonic 'in vial' extraction method, and from the other part, oxygen permeability was measured in a permeation cell at 25 degrees C. After 8 h of exposure, LDPE and PP samples showed a significant linear (R2 = 0.82 and 0.99) increase in oxygen permeability of 21 and 130%, respectively. Owing to swelling of the polymer samples resulting from flavour absorption, the structure of the polymeric network changed (i.e. opened) and consequently increased oxygen permeability. The oxygen permeability of exposed PC showed a significant linear (R2 = 0.78) decrease of 11% after 21 days. PC obviously did not swell like LDPE or PP. Therefore, it was suggested that absorbed flavour compounds occupied or blocked 'microcavities' through which normally oxygen is transported. Absorption of flavour compounds by PET did not affect the oxygen permeability of PET significantly.

beta-carotene does not change markers of enzymatic and nonenzymatic antioxidant activity in human blood.

In vitamin A-replete populations, increased concentrations of serum carotenoids have been associated with a decreased risk of degenerative diseases. The mechanism of action of carotenoids in determining antioxidant activity is largely unknown. The aim of the study was to examine the effect of carotenoid supplementation and spinach intake on erythrocyte enzyme antioxidant activities, serum or plasma nonenzymatic antioxidant concentrations, and concentrations of oxidatively damaged amino acids in plasma. Subjects received for 3 wk a basic diet (n = 10), a basic diet with a carotenoid supplement (n = 12) or with a spinach product (n = 12 per group), i.e., whole-leaf, minced, liquefied or liquefied spinach plus added dietary fiber. After 3 wk of dietary intervention, changes in serum or plasma concentrations of ascorbic acid, alpha-tocopherol, FRAP (ferric reducing ability of plasma) and uric acid and erythrocyte enzyme activities were assessed, and differences among experimental groups were tested. Consumption of spinach resulted in greater (P < 0.01) erythrocyte glutathione reductase activity and lower (P < 0.05) erythrocyte catalase activity and serum alpha-tocopherol concentration compared with the control group. Consumption of the carotenoid supplement led to lower alpha-tocopherol responses (P = 0.02) compared with the basic diet only. Our data suggest that the short-term changes in erythrocyte glutathione reductase activity and serum alpha-tocopherol concentration can be attributed to an increased carotenoid (lutein and zeaxanthin) intake, but beta-carotene is unlikely to be a causative factor. Lower erythrocyte catalase activity after intervention with spinach products may be related to other constituents in spinach such as flavonoids.

Antioxidant properties of differently processed spinach products.

The effect of variously processed spinach products (whole-leaf, minced and enzymatically liquefied spinach) on lipid oxidation was determined. In an autoxidative methyl linoleate (MeLo) system the inhibition of hydroperoxide formation, measured by HPLC after three days of oxidation, was in descending order: whole-leaf > liquefied > minced spinach. The inhibition of formation of thiobarbituric acid-reactive substances (TBARS) and hexanal by spinach was determined in cooked meatballs with added spinach after two days of storage at 4 degrees C. The formation of TBARS was inhibited by liquefied spinach at 200 g/kg meat; all other spinach products tested at 100 and 200 g/kg were pro-oxidative. The formation of hexanal was inhibited by both minced and liquefied spinach at 100 and 200 g/kg meat. The variously processed spinach products behaved differently when tested for their antioxidant activity (MeLo) or oxidative stability (meatballs). We conclude that the effect of spinach products on lipid oxidation is affected by processing.