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Effects of storage and handling conditions on concentrations of individual carotenoids, retinol, and tocopherol in plasma.

We investigated the effects of storage and handling on measured values for carotenoids, retinol, and tocopherol in plasma. We found no significant differences in the concentrations of these analytes measured in plasma samples that were frozen immediately after separation as compared with replicate samples maintained at room temperature in the dark for 24 h. Analytes were stable in solvents for at least 18 h at 23 degrees C after extraction. Purging samples with nitrogen gas before freezing had no detectable beneficial effects. All analytes were stable in plasma stored at -70 degrees C for at least 28 months or at -20 degrees C for five months. By 15 months the concentrations of carotenoids were significantly less (P less than 0.05) in plasma stored at -20 degrees C than in plasma stored at -70 degrees C, while retinol and tocopherol concentrations were not significantly different. Concomitant with the decrease in carotenoids was the appearance of unidentified peaks in the ultraviolet. Adding ascorbic acid or butylated hydroxytoluene antioxidants to the precipitating solvent did not alter the losses of carotenoids or alter the appearance of unidentified peaks. Under appropriate conditions, plasma carotenoids, retinol, and tocopherol are stable for more than two years.

Ascorbic Acid↗

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem.

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.

Acrosome↗

To freeze or not to freeze. A comparison of methods for the handling of breast biopsies with no palpable abnormality.

Recently there have been several editorials suggesting that breast biopsies with small or nonpalpable lesions should not be subjected to frozen section examination. In this study, the approach of routinely performing frozen sections on all breast biopsies was compared to the recommended approach of performing frozen sections only on cases with gross lesions < 1.0 cm. From July 1, 1992 through June 30, 1993, there were 444 breast biopsies submitted. Nearly all of these (98%) had frozen sections performed. Among these cases, there were 14 false-negative frozen section diagnoses equating to a frozen section sensitivity of 84%, and a frozen section false-negative rate of 3.3%. From July 1, 1994 through June 30, 1995, there were 601 breast biopsies submitted. Of these cases, 310 contained a gross lesion > 1.0 cm and were subjected to frozen section examination. The remaining 291 cases contained either no distinct lesion or a gross lesion < 1.0 cm. These cases were not frozen. Among the 310 cases that were frozen, there were 3 false-negative frozen section diagnoses equating to a frozen section sensitivity of 96%, and a frozen section false-negative rate of 1.0%. These findings indicate that careful gross examination combined with selective freezing of only cases containing a gross lesion > 1.0 cm improves frozen section accuracy. This study provides statistical support for previous recommendations that frozen section examination should be limited to cases with distinct gross lesions > 1.0 cm.

Biopsy↗

Laboratory handling of Helicobacter pylori critically influences the results of in-vitro metronidazole resistance determination.

In-vitro metronidazole resistance rates of Helicobacter pylori determined by Etest are high, and the predictive value of metronidazole resistance is low. It was hypothesised that altered laboratory methods could reduce the overestimation of resistance and improve the predictive value of the Etest. Pre-treatment isolates (n = 150) of H. pylori from 150 patients were investigated by Etest with incubation for 72 h. Treatment with metronidazole, tetracycline and bismuth for 10 days failed to eradicate H. pylori in 23 patients. After isolate storage for 3 years, resistance determination results by agar dilution and Etest, with incubation for 72 and 31 h, were compared. The rate of metronidazole resistance was reduced significantly during storage, and instability of resistance was associated significantly with treatment outcome. Isolates that retained in-vitro resistance had significantly (p 0.008) higher treatment failure rates (n = 13; 42%) than isolates that lost resistance (n = 3; 9%). The reproducibility achieved by dual testing with agar dilution and Etest was 41% and 70% for +/- 1 and +/- 2 log2 dilutions, respectively, after incubation for 72 h, and 85% and 92%, respectively, after incubation for 31 h. Thus, the predictive value was improved from 25% to 50% by the altered laboratory conditions (p 0.04). MIC values of 2-8 mg/L signified an intermediate risk of treatment failure.

Adult↗

Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin.

Detected levels of IL-6, TNF-alpha and leptin may be affected by methods of storage, anticoagulant or repeated freezing-thawing. Blood samples from 22 healthy subjects were: (i) allowed to stand for 1, 2, 4 or 6 h prior to, or after separation, before freezing at -70 degrees C; (ii) taken into tubes with lithium heparin, sodium citrate, EDTA or no anticoagulant, separated and frozen; and (iii) separated, and plasma repeatedly freeze-thawed for up to six cycles prior to assay. Leptin was assayed by RIA, and IL-6 and TNF-alpha by high-sensitivity ELISA. (i) IL-6 and TNF-alpha levels were not altered significantly in separated samples but IL-6 declined by mean (SEM) 14.3% (3.7%) and TNF-alphaincreased by 9.6% (2.3%) in samples left unseparated for 4 h (P=0.003 and 0.002, respectively). Leptin remained unchanged. (ii) Serum and EDTA-plasma samples gave comparable results for all three cytokines, but levels in the other anticoagulant samples were highly variable. (iii) IL-6 and leptin levels were not altered by up to 6 cycles of freeze-thawing, but TNF-alpha increased by 17.0% (3.7%) after 3 cycles. Concentrations of these molecules are significantly altered by storage conditions, therefore they need to be standardized for epidemiological and clinical studies, and between-study comparisons of levels may not be reliable.

Adult↗

High turbulence liquid chromatography online extraction and tandem mass spectrometry for the simultaneous determination of suberoylanilide hydroxamic acid and its two metabolites in human serum.

A reliable and sensitive method incorporating high turbulence liquid chromatography (HTLC) online extraction with tandem mass spectrometry (MS/MS), for simultaneous determination of suberoylanilide hydroxamic acid (SAHA) and its two metabolites, SAHA-glucuronide (M1) and 4-anilino-4-oxobutanoic acid (M2), in human serum, has been developed to support clinical studies. The HTLC technology significantly reduces the time required for sample clean-up since sample extraction and analysis are performed online. Clinical samples, internal standards (IS) and buffer are transferred into 96-well plates using a robotic liquid handling system. A 20 microL aliquot of prepared sample is directly injected into the HTLC/LC-MS/MS system where the matrix is rapidly washed away to waste and the analytes are retained on the narrow-bore extraction column (0.5 x 50 mm), using an aqueous mobile phase at 1.5 mL/min. Analytes are then eluted from the extraction column and transferred to the analytical column using a gradient mobile phase prior to detection by MS/MS. Interference with determination of SAHA from in-source dissociation of M1 is eliminated by the chromatographic separation. The resolution of SAHA and M1 did not change for more than 1500 serum sample injections by applying an acid wash (15% acetic acid) on the extraction column. The linear calibration ranges for SAHA, M1, and M2 are 2-500, 5-2000, and 10-2000 ng/mL, respectively. Assay intraday validation was conducted using five calibration curves prepared in five lots of human control serum. The precision expressed as relative standard deviation (RSD) is less than 6.8% and accuracy is 94.6-102.9% of nominal values for all three analytes. Assay specificity, freeze/thaw stability, storage stability, and matrix effects were also assessed.

Anti-Inflammatory Agents, Non-Steroidal↗

Optimal preparation techniques for human saphenous vein grafts.

Endothelial damage during preparation of saphenous vein grafts is an important cause of early and late graft failure. To determine optimal preparation techniques for human saphenous veins (SVs), we made a scanning electron microscopic comparison of the effects of handling techniques, immersion media, and distention pressures on human SV morphology. Segments of SVs from 30 patients were divided into seven groups of five veins each. The effects of both blood and saline solution immersion at 4 degrees and 28 degrees C, 100 and 300 mm Hg distention pressures, and trauma from surgical instrumentation were examined. All groups of veins were fixed by perfusion with glutaraldehyde at 100 mm Hg to simulate arterial pressure and were examined with a scanning electron microscope. SVs immersed in warm saline solution showed massive endothelial cell loss, whereas SVs immersed in warm blood showed only moderate endothelial damage. Cold blood and saline immersion fully preserved endothelium; however, cold saline immersion produced mural edema. Distention to 300 mm Hg with saline solution produced severe endothelial damage and edema, an effect lessened by blood distention. Vascular clamping destroyed endothelium and fractured the intima and media. Marked luminal stenoses were caused by four of seven side branch ties that appeared normal externally. We conclude that human SVs are preserved best by a "no touch" harvesting technique in which manipulation is minimized, side branch ties are placed away from the SV wall, veins are immersed in cold blood, and distention above 100 mm Hg is avoided.

Blood↗

Pharmacokinetic study of sisomicin in humans.

Detailed analyses of the pharmacokinetics of sisomicin administered at doses of 25, 50 and 100 mg intravenously and intramuscularly to healthy volunteers established that the drug is handled by a two-compartment open model system with a disposition (elimination) half-life of 2.6 hr. The kinetic estimates over this dose range are linear and independent of dose and were verified by a 60-min infusion experiment in which dose and the maximum serum concentration achieved (5 microgram/ml) were predicted correctly. Sisomicin was rapidly distributed to the tissue compartment, and equilibrium between the central and the tissue compartment was established by 30 min after dosing. Renal clearance (55 ml/min) of sisomicin was about 30% less than total body clearance (78 ml/min). Total urinary excretion of sisomicin during a 24-hr period following drug administration was about 70% of the dose. The disposition kinetics of sisomicin following intramuscular administration are similar to those obtained following rapid intravenous administration. Intramuscular bioavailability of sisomicin for the doses of 25, 50, and 100 mg was greater than 95%. Based on these results, various initial loading infusion doses and maintenance infusion rates were calculated to provide specific desired peak and steady-state serum sisomicin concentrations rapidly. The purpose was not to expose patients to potentially toxic high peak concentrations of drug while maintaining these concentrations during the current therapeutic dosing intervals of 8 to 12 hr.

Biological Availability↗

Effects of duration of capture and sample handling on critical care blood analytes in free-ranging bottlenose dolphins.

OBJECTIVE: To determine effects of duration of capture and sample-handling procedures on blood analytes in free-ranging bottlenose dolphins. DESIGN: Cross-sectional study. ANIMALS: 154 free-ranging bottlenose dolphins of various ages and both sexes. PROCEDURES: Blood samples were drawn from each dolphin within 10 minutes of capture and before release and analyzed by use of a portable analyzer with a single-use 8-analyte disposable cartridge. Analyte values were compared according to duration between sample acquisition and analysis (time to run [TTR]) and duration between net encirclement and sample acquisition (time to bleed [TTB]). RESULTS: Neither TTB nor TTR significantly affected sodium or chloride concentration. Potassium concentration was not significantly affected by TTR, whereas the effect of TTB was significant. Glucose, total CO(2), HCO(3), Hct, and base excess of extracellular fluid values were significantly affected by TTR. Increased TTB resulted in significantly increased total CO(2), HCO(3), and base excess when TTR was kept within 10 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: The effect of TTB on certain acid-base and electrolyte values was readily measured in free-ranging bottlenose dolphins, and such values may provide a reference range for those variables.

Animals↗

Quantitative whole-body autoradiography: recommendations for the standardization of the method.

The objective of this article is to identify the best conditions for preparing, handling, and exposing radioactive sections by using the Fujix BAS 2000 system for quantitative radioluminography. Regarding the influence of thickness of sections, thicker sections may allow shorter exposure times due to the increased radioactivity, but on the other hand they take more time for the freeze-drying process, resolution will be reduced, and the recovery of radioactivity will be lower due to increased self-absorption particularly in organs like bones or teeth. The pretreatment of the sections should depend on the method of exposure. Powdering with talcum is the most recommendable method when vacuum packaging the imaging plates and sections. Spraying with Nobecutan is recommended when using the cartridge method. Particularly for low concentrations, the vacuum-contact method should be the method of choice. To avoid a flare effect, the geometrical arrangement of the sections on the imaging plate (IP) should always be vertical to the scanning direction of the laser. An exposure time longer than 10 days is not recommended and the time between the end of exposure and start of scanning should be as short as possible. In order to reduce the background signal, it is necessary to expose the IPs in a shielding box in a cold environment. No positive chemographic effects of sections were found.

Animals↗

Handling of environmental and biological samples via pre-column technologies.

Sample handling is still a weak point in chromatography and in analytical chemistry in general. One consideration is the automation potential of new procedures. Solid-liquid extraction techniques in combination with pre-column technology are particularly promising in this regards. The construction and geometry of pre-columns both for conventional and narrow-bore HPLC are of major importance, since band broadening should be kept at a minimum for an optimal functioning of the analytical system. The various operations that can be carried out with such a pre-column are trace-enrichment, clean-up of the sample which depends on the type of adsorbents used in the precolumn, i.e., polar or apolar materials, ion exchangers or metal covered surfaces, etc., protection of the analytical column, field sampling and storage of samples and as a substrate for on-column chemical derivatizations. These various operations are demonstrated with practical examples from the fields of environmental and biological analysis. The selectivity can be further enhanced by coupling pre-column technology with selective detection models such as diode array UV, electrochemical or fluorescence detection. This enables the construction of optimal and integrated analysis systems which are fully automated and microprocessor controlled. They can also be made compatible with miniaturized LC-technology.

Autoanalysis↗

Manure sampling for nutrient analysis: variability and sampling efficacy.

Reliable estimation of nutrient concentrations is required to manage animal manure for protecting waters while sustaining crop production. This study was conducted to investigate sample variability and reliable nutrient analysis for several manure types and handling systems. Serial samples were collected from dairy, swine, and broiler poultry operations while manure was being loaded onto hauler tanks or spreaders for field application. Samples were analyzed for total solids (TS), total nitrogen (N), ammoniacal nitrogen (NH4-N), total phosphorus (P), and potassium (K). The least number of samples needed for reliable testing of total N and P, defined as +/- 10% of the experimental means with 99% probability, was obtained for each farm using a computer-intensive random resampling technique. Sample variability within farms, expressed as the coefficient of variation (CV), was mostly 6 to 8% for farms that used agitation of manure storages but several times higher (20-30%) on farms where no agitation was applied during the sampling period. Results from the random resampling procedure indicated that for farms that used agitation, three to five samples were adequate for a representative composite for reliable testing of total N and P; whereas for farms without agitation, at least 40 samples would be required. Data also suggest that using book values for manure nutrient estimations could be problematic because the discrepancies between book standards and measured farm data varied widely from a small amount to several fold.

Agriculture↗

Effects of sample handling temperatures on bovine skim milk progesterone concentrations.

The effect of incubation of whole milk at various temperatures and times on the amount of progesterone (nmol/l) in the skim milk fraction was determined. For this study, milk samples were obtained from 10 pregnant Holstein cows. The whole milk samples were incubated at 37 degrees C (near normal body temperature of the cow) for 4 h and the initial skim milk progesterone concentration was determined. After that, the experiment was carried out in two main steps: (I) The test tubes containing the whole milk were divided into 4 groups and incubated at different temperatures (0, 4, 20 and 37 degrees C). Samples were removed at 30, 60, 90 and 120 min. (II) After 120 min of incubation at different temperatures, the remaining test tubes were returned to the water bath at 37 degrees C for another 30, 60 and 90 min. The initial average of skim milk progesterone concentrations after incubating the whole milk at 37 degrees C for 4 h was 11.0 +/- 4.4 nmol/l. When the whole milk was incubated at 0 degrees C, the skim milk progesterone concentration increased (P < 0.05) to 14.6 nmol/l at 30 min and reached 16.2 nmol/l at 60 min of incubation. At 4 degrees C incubation temperature, skim milk progesterone increased significantly (P < 0.05) to 15.3 nmol/l and reached 16.9 nmol/l after 90 min. When the whole milk was left at 20 degrees C, the initial skim milk progesterone values decreased to 9.5 nmol/l after 30 min incubation and no further decreases were found even if the whole milk was returned to 37 degrees C for 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Optimizing oriented planar-supported lipid samples for solid-state protein NMR.

Sample orientation relative to the static magnetic field of an NMR spectrometer allows study of membrane proteins in the lipid bilayer setting. The straightforward preparation and handling of extremely thin mica substrates with consistent surface properties has prompted us to examine oriented phospholipid bilayer and hexagonal phases on mica. The spectral characteristics of oriented lipid samples formed on mica are as good as or better than those on glass. Nine solvents with varying dielectric constants were used to cast lipid films or for vesicle spreading; film characteristics were then compared, and static solid-state 31P-NMR was used to characterize the degree of orientation of the hydrated lipid species. Lipids with four headgroup chemistries were tested: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Solvent affected orientation of POPG, DOPA, and DOPE, but not POPC. Film characteristics varied with solvent, with ramifications for producing homogeneous oriented lipid samples. POPC was used to optimize the amount of lipid per substrate and compare hydration methods. POPG did not orient reproducibly, whereas POPG-POPC mixtures did. DOPA showed 1-2 oriented states depending upon hydration level and deposition method. DOPE formed an oriented hexagonal phase that underwent a reversible temperature-induced phase transition to the oriented bilayer phase.

Aluminum Silicates↗

Earlier initiation of antibiotic treatment for severe infections after interventions to improve the organization and specific guidelines in the emergency department.

OBJECTIVE: To examine whether combined interventions improve the timely administration of antibiotic therapy and acquisition of material for culture from patients admitted to the emergency department with a serious infectious disease. METHODS: Guidelines and educational programs were developed to facilitate timely antibiotic administration: guidelines on handling patients with serious infections and on ordering immediate treatment, guidelines on obtaining culture samples, lectures to medical and nursing staff, improvement of availability of antibiotics in the emergency department, and removal of financial restraints on stocking and ordering of antibiotics. Fifty consecutive patients were evaluated after this series of interventions and compared with the results in 50 patients evaluated before the interventions. The interval from presentation to the emergency department until the administration of antibiotics, number of samples taken for microbiological investigations, and number of patients receiving a first dose of antibiotic at routinely scheduled drug distribution rounds were evaluated. RESULTS: The median time to the initial dose of antibiotics administered decreased from 5.0 hours to 3.2 hours (P = .04). The number of blood cultures obtained did not change. The percentage of sputum cultures obtained increased from 28% to 50%, and the percentage of urine cultures obtained increased from 50% to 100%. The percentage of patients whose first dose of antibiotic was delayed until a routinely scheduled drug distribution round decreased from 54% to 32% (P = .03). CONCLUSIONS: Combined interventions to expedite diagnostic and therapeutic actions through directed clinical practice guidelines and organizational measures are successful. This may lead to a substantial quality improvement in the process of care.

Aged↗

Airborne molds and actinomycetes in the work environment of farmer's lung patients in Finland.

Occurrence of molds and actinomycetes in the breathing zone of farmers during the handling of hay, straw, or grain was studied with the use of an Andersen sampler on 35 farms in Finland. On 24 farms there was a person with recently diagnosed farmer's lung disease, and on 11 farms people were free of the disease. The total spore concentration and the concentrations of the spores of Thermoactinomyces (T) vulgaris, Micropolyspora (M) faeni, and Aspergillus (A) umbrosus were statistically significantly higher on the farms of patients with farmer's lung than on the disease-free farms. The mean proportions of the spores of thermotolerant and thermophilic microbes were greater on the farms of farmer's lung patients than on the reference farms. T vulgaris was the predominant actinomycete species. Both T vulgaris and A umbrosus were found on all farms of farmer's lung patients, but M faeni on only about half of such farms. The findings match the results of previous microbiological analyses of Finnish moldy hay and serological analyses of Finnish farmer's lung patients. It seems that T vulgaris, not M faeni, may be the main causative agent of farmer's lung in Finland. The possible etiologic role of A umbrosus requires further investigation. Because the farmers often failed to identify the moldiness of the plant material in contrast to researchers, it might be possible, through training, to improve farmers' ability to identify moldiness.

Air Microbiology↗

Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates.

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

Bacteriological Techniques↗

Human-parathormone assay for use in dogs: validation, sample handling studies, and parathyroid function testing.

Ten commercially available parathormone (PTH) assays were competitively validated, using dilutional parallelism, intra-assay and interassay coefficients of variation, and sensitivity and measured responses of 2 dogs to calcium and EDTA infusions. A 2-site immunoradiometric assay for intact human-PTH was superior to the others for estimating canine-PTH, met the criteria for validity, and was further investigated. A series of sample-handling studies was performed. Serum and plasma samples stored at 24 C lost 15% (n = 5; P less than 0.05) of PTH between 2 and 24 hours. This did not occur at 6 C. The mean PTH concentration of sera from blood samples clotted at 24 C was 6% (P less than 0.05) higher than equivalent EDTA samples. Serum samples stored at 6 and 37 C deteriorated 35% and 100% (n = 5; P less than 0.05), respectively, after 1 week, whereas samples stored at -20 and -70 C for 4 weeks did not deteriorate. There was no significant deterioration of PTH in samples frozen (-40 C) and thawed up to 7 times (n = 5). Parathyroid function testing was investigated by use of 2-hour infusions of disodium EDTA (25 mg/kg/h), 10-minute infusions of calcium gluconate (3 mg of elemental calcium/kg/10 min), and physiologic saline controls (n = 8). Renal function was monitored before and after EDTA infusion by exogenous creatinine clearance. Infusion of disodium EDTA increased mean PTH concentration from 67 (time 0) to 317 and 235 pg/ml at 90 and 180 minutes, respectively (P less than 0.001). Infusion of calcium gluconate decreased mean PTH concentration from 84 (time 0) to 14 and 12 pg/ml at 15 and 60 minutes, respectively (P less than 0.005). There were no observable side effects of the infusions in normal conscious dogs and no differences in exogenous creatinine clearance after EDTA infusion.

Animals↗