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A representative microbial sampling method for large commercial containers of raw beef based on purge.

The purge from beef combos ( a boxed collection of beef trimmings) was tested as a means of representatively sampling the microbial content of this raw product. In the first experiment, purge was sampled from model beef combos that had been inoculated with bovine feces.. Data from this experiment indicated a strong correlation (r = 0.94) between the total aerobic bacteria counts derived from the purge samples of a model beef combo and the total aerobic bacteria present in a rinse sample of the entire model beef combo. In a second experiment, two 500-g meat pieces were inoculated with an antibiotic-resistant Escherichia coli O157:H7 and place at various levels within a 75-cm meat column. The marked bacteria were retrievable from the purge of the meat column after 24 h, showing that bacteria are carried downward into the purge. During the third part of the study, 90 beef combos (approximately 900 kg beef/combo) were randomly selected at the receiving dock of a commercial grinding facility and sampled using both purge and concurrently used 11-g core samples. Purge samples from these combos recovered significantly greater numbers of mesophilic and psychrotrophic aerobic bacteria, coliforms, and E. coli than core samples from the same combos. Additionally, coliforms and E. coli were recoverable from 100% and 80%, respectively, of the purge samples taken, whereas core samples were only able to recover 60% and 40%, respectively, from the same combos. These findings indicate that a purge sample from a beef combo is a more efficacious sampling method for determining the general bacterial profile and identifying the presence of coliforms and E. coli than randomly taken core samples.

Animals↗

Sampling plans for the determination of aflatoxin B1 in large shipments of animal feedstuffs.

Incremental samples (50, 100, and 500 g) were systematically collected from large shipments of copra meal pellets, copra cake, and palm kernel cake to study the distribution of aflatoxin B1 and evaluate adherence of distribution to the model, CV(2)is (EQ) = A + B/Mis (where CVis = coefficient of variation of the true concentration of aflatoxin B1 within the incremental samples; Mis = mass of the incremental samples; and A and B are constants). Also evaluated was the distribution of aflatoxin B1 among 1 kg composite samples, produced both by random combination of existing incremental samples and by collection of 1 kg composite samples (composed of 10 x 100 g increments) from additional batches of copra meal pellets and cottonseed cake. The efficiency of selected sample preparation (grinding and subdivision) procedures was compared, culminating in the development and description of a variety of sampling plans. The coefficient of variation (CV) among incremental samples varied from 0 to 38%, and was independent of incremental sample size. No significant difference (F-test, 5% significance level) was found between the efficacy of 4 sample preparation methods when these methods were applied to the commodities described above. Various sampling plans were evaluated with estimated CVs from 4.0 to 12.5%, for the aflatoxin B1 content of the composite samples.

Aflatoxin B1↗

Testing green coffee for ochratoxin A, part III: performance of ochratoxin A sampling plan.

Green coffee shipments are often inspected for ochratoxin A (OTA) and classified into good or bad categories depending on whether the OTA estimates are above or below a defined regulatory limit. Because of the uncertainty associated with the sampling, sample preparation, and analytical steps of an OTA test procedure, some shipments of green coffee will be misclassified. The misclassification of lots leads to some good lots being rejected (sellers' risk) and some bad lots being accepted (buyers' risk) by an OTA sampling plan. Reducing the uncertainty of an OTA test procedure and using an accept/reject limit less than the regulatory limit can reduce the magnitude of one or both risks. The uncertainty of the OTA test procedure is most effectively reduced by increasing sample size (or increasing the number of samples analyzed), because the sampling step is the largest source of uncertainty in the OTA test procedure. The effects of increasing sample size and changing the sample accept/reject limit relative to the regulatory limit on the performance of OTA sampling plans for green coffee were investigated. For a given accept/reject limit of 5 microg/kg, increasing sample size increased the percentage of lots accepted at concentrations below the regulatory limit and increased the percentage of lots rejected at concentrations above the regulatory limit. As a result, increasing sample size reduced both the number of good lots rejected (sellers' risk) and the number of bad lots accepted (buyers' risk). For a given sample size (1 kg), decreasing the sample accept/reject limit from 5 to 2 microg/kg relative to a fixed regulatory limit of 5 microg/kg decreased the percentage of lots accepted and increased the percentage of lots rejected at all OTA concentrations. As a result, decreasing the accept/reject limit below the regulatory limit increased the number of good lots rejected (sellers' risk), but decreased the number of bad lots accepted (buyers' risk).

Carcinogens↗

Regression-based reference limits: determination of sufficient sample size.

Regression analysis is the method of choice for the production of covariate-dependent reference limits. There are currently no recommendations on what sample size should be used when regression-based reference limits and confidence intervals are calculated. In this study we used Monte Carlo simulation to study a reference sample group of 374 age-dependent hemoglobin values. From this sample, 5000 random subsamples, with replacement, were constructed with 10-220 observations per sample. Regression analysis was used to estimate age-dependent 95% reference intervals for hemoglobin concentrations and erythrocyte counts. The maximum difference between mean values of the root mean square error and original values for hemoglobin was 0.05 g/L when the sample size was > or = 60. The parameter estimators and width of reference intervals changed negligibly from the values calculated from the original sample regardless of what sample size was used. SDs and CVs for these factors changed rapidly up to a sample size of 30; after that changes were smaller. The largest and smallest absolute differences in root mean square error and width of reference interval between sample values and values calculated from the original sample were also evaluated. As expected, differences were largest in small sample sizes, and as sample size increased differences decreased. To obtain appropriate reference limits and confidence intervals, we propose the following scheme: (a) check whether the assumptions of regression analysis can be fulfilled with/without transformation of data; (b) check that the value of v, which describes how the covariate value is situated in relation to both the mean value and the spread of the covariate values, does not exceed 0.1 at minimum and maximum covariate positions; and (c) if steps 1 and 2 can be accepted, the reference limits with confidence intervals can be produced by regression analysis, and the minimum acceptable sample size will be approximately 70.

Child, Preschool↗

A highly sensitive and quantitative telomerase activity assay with pancreatic juice is useful for diagnosis of pancreatic carcinoma without problems due to polymerase chain reaction inhibitors: analysis of 100 samples of pancreatic juice from consecutive patients.

BACKGROUND: Early detection of pancreatic carcinoma is difficult even with current diagnostic tools. Novel biomarkers and detection techniques are urgently needed. Telomerase activity is a promising diagnostic marker. However, the conventional telomeric repeat amplification protocol (TRAP) assay is not suitable for clinical application because of its complexity, time-consuming nature, and the effects of polymerase chain reaction (PCR) inhibitors in samples leading to difficulties in quantification. METHODS: The authors used a hybridization protection assay in combination with TRAP (TRAP/HPA) to investigate the effects of PCR inhibitors in pancreatic juice on quantification of telomerase activity. They analyzed 117 consecutive samples of pancreatic juice to determine the feasibility of TRAP/HPA for diagnosis of pancreatic carcinoma. RESULTS: The authors found that TRAP/HPA was 1000-fold more sensitive than the conventional TRAP assay, and that the effects of PCR inhibitors could be avoided by diluting samples. In a large analysis of pancreatic juice samples with TRAP/HPA, 17 samples were excluded from the final analysis because of insufficient follow-up periods or inadequate treatment of the samples. Relative telomerase activity (RTA) in samples from patients with pancreatic carcinoma was significantly higher in comparison to samples from patients with pancreatitis and 13 (61.9%) of 21 samples from patients with pancreatic carcinoma showed high RTA (> 4 U). Meanwhile, high RTAs were observed in 4 of 35 (11.4%) samples from patients with intraductal papillary mucinous tumor and in 1 of 40 samples (2.5%) fom patients without malignant disease. CONCLUSIONS: TRAP/HPA accurately evaluated weak telomerase activity in pancreatic juice samples without the problem due to PCR inhibitors. This large analysis of nonselected pancreatic juice samples suggested that TRAP/HPA is a promising approach for the diagnosis of pancreatic carcinoma.

Adult↗

Classification of human tumors using gene expression profiles obtained after microarray analysis of fine-needle aspiration biopsy samples.

BACKGROUND: Gene expression profiling using gene-discovery, high-density microarray technologies is a powerful tool. One potential application is the development of tumor classifiers that predict the site of origin. For this technology to be relevant, however, it must be applicable to tumor biopsy samples, which most often are fine-needle aspiration biopsy (FNAB) samples. METHODS: Surgically resected tumors were sampled by FNAB using different gauge needles. A portion of the excised tumor was also collected. RNA samples were extracted using standard techniques and the quality and quantity of the RNA samples were measured for each sample. Thirteen representative FNAB samples and two representative tissue samples were submitted for microarray analysis and then subjected to a tumor classifier. RESULTS: Fourteen of 18 samples analyzed for quantity and quality of RNA yielded an adequate amount of RNA (> 1 microg total RNA). Tumor type contributed to the RNA yield because one of the four inadequate samples was retrieved from a patient with lobular carcinoma of the breast and the other three samples were retrieved from patients with retroperitoneal sarcomas. Of the 13 samples submitted for microarray analysis, 9 were classified correctly as to tumor type using a tissue-based tumor classifier. CONCLUSIONS: The authors demonstrated that FNABs reproducibly obtained an adequate amount of RNA for microarray analysis when a standardized collection procedure was used. Furthermore, the samples generated interpretable gene expression profiles that could be matched accurately with a tumor classifier established on tissue specimens. The current study showed that FNAB produced adequate material for microarray analysis when utilizing a standardized collection procedure.

Biopsy, Fine-Needle↗

Applying sample survey methods to clinical trials data.

This paper outlines the utility of statistical methods for sample surveys in analysing clinical trials data. Sample survey statisticians face a variety of complex data analysis issues deriving from the use of multi-stage probability sampling from finite populations. One such issue is that of clustering of observations at the various stages of sampling. Survey data analysis approaches developed to accommodate clustering in the sample design have more general application to clinical studies in which repeated measures structures are encountered. Situations where these methods are of interest include multi-visit studies where responses are observed at two or more time points for each patient, multi-period cross-over studies, and epidemiological studies for repeated occurrences of adverse events or illnesses. We describe statistical procedures for fitting multiple regression models to sample survey data that are more effective for repeated measures studies with complicated data structures than the more traditional approaches of multivariate repeated measures analysis. In this setting, one can specify a primary sampling unit within which repeated measures have intraclass correlation. This intraclass correlation is taken into account by sample survey regression methods through robust estimates of the standard errors of the regression coefficients. Regression estimates are obtained from model fitting estimation equations which ignore the correlation structure of the data (that is, computing procedures which assume that all observational units are independent or are from simple random samples). The analytic approach is straightforward to apply with logistic models for dichotomous data, proportional odds models for ordinal data, and linear models for continuously scaled data, and results are interpretable in terms of population average parameters. Through the features summarized here, the sample survey regression methods have many similarities to the broader family of methods based on generalized estimating equations (GEE). Sample survey methods for the analysis of time-to-event data have more recently been developed and implemented in the context of finite probability sampling. Given the importance of survival endpoints in late phase studies for drug development, these methods have clear utility in the area of clinical trials data analysis. A brief overview of methods for sample survey data analysis is first provided, followed by motivation for applying these methods to clinical trials data. Examples drawn from three clinical studies are provided to illustrate survey methods for logistic regression, proportional odds regression and proportional hazards regression. Potential problems with the proposed methods and ways of addressing them are discussed.

Clinical Trials as Topic↗

Demonstration of a longitudinal concentration gradient along scala tympani by sequential sampling of perilymph from the cochlear apex.

Local applications of drugs to the inner ear are increasingly being used to treat patients' inner ear disorders. Knowledge of the pharmacokinetics of drugs in the inner ear fluids is essential for a scientific basis for such treatments. When auditory function is of primary interest, the drug's kinetics in scala tympani (ST) must be established. Measurement of drug levels in ST is technically difficult because of the known contamination of perilymph samples taken from the basal cochlear turn with cerebrospinal fluid (CSF). Recently, we reported a technique in which perilymph was sampled from the cochlear apex to minimize the influence of CSF contamination (J. Neurosci. Methods, doi: 10.1016/j.jneumeth.2005.10.008 ). This technique has now been extended by taking smaller fluid samples sequentially from the cochlear apex, which can be used to quantify drug gradients along ST. The sampling and analysis methods were evaluated using an ionic marker, trimethylphenylammonium (TMPA), that was applied to the round window membrane. After loading perilymph with TMPA, 10 1-muL samples were taken from the cochlear apex. The TMPA content of the samples was consistent with the first sample containing perilymph from apical regions and the fourth or fifth sample containing perilymph from the basal turn. TMPA concentration decreased in subsequent samples, as they increasingly contained CSF that had passed through ST. Sample concentration curves were interpreted quantitatively by simulation of the experiment with a finite element model and by an automated curve-fitting method by which the apical-basal gradient was estimated. The study demonstrates that sequential apical sampling provides drug gradient data for ST perilymph while avoiding the major distortions of sample composition associated with basal turn sampling. The method can be used for any substance for which a sensitive assay is available and is therefore of high relevance for the development of preclinical and clinical strategies for local drug delivery to the inner ear.

Animals↗

Rates of detection of Salmonella and Campylobacter in meats in response to the sample size and the infection level of each species.

Pork, beef and chicken meat samples were collected from slaughter houses, poultry-processing plants and meat shops. Rates of incidence of Salmonella spp., Campylobacter jejuni and C. coli with respect to the sample size were compared and the most probable number for these species were determined. Salmonella spp. were detected in 69 (24.1%) of 286 chicken meat samples, in three (3.2%) of 94 pork samples, and in one (1.9%) of 52 beef samples. With chicken meat, the rates of detection were: 19.9% in 25-g, 15.7% in 10-g, and 12.2% in 1-g samples. The populations in most probable numbers, that gave positive results in 31 (20.8%) of 149 samples, ranged from 30 to 10(4) per 100 g, the majority (93.5%) being between 30 and 10(3) per 100 g. C. jejuni and C. coli were detected in 106 (67.9%) of 156 chicken meat samples, in two (2.1%) of 94 pork samples, and none of 52 beef samples. The results obtained with different sample sizes of chicken were compared. Positive rates were 55.8%, 39.7%, 27.6% in 10 g, 1 g, and 0.1 g, respectively. The most probable numbers in 107 (68.6%) positives out of 156 chicken samples examined ranged from 30 to 10(6) per 100 g: 46 (29.5%) contained between 10(2) and 10(3) per 100 g, 22 (14.1%) between 10(3) and 10(4) per 100 g, and the other 19 samples (12.2%) between 10(4) and 10(5) per 100 g.

Animals↗

Antibodies against nematodes in serum, milk and bulk milk samples as possible estimators of infection in dairy cows.

Antibodies against Ostertagia spp., Cooperia spp. and Dictyocaulus viviparus were assessed in serum and individual milk samples of cows and in bulk milk samples from the refrigerating tank. Titres in milk samples were related to serum titers, but also influenced by milk yield, lactation stage and age of the cow. Correlation coefficients between serum and individual milk samples were highly significant, and varied from 0.42 to 0.56. Correlations between herd means of serum titres and herd means of individual milk titres varied from 0.41 to 0.61, and those between herd means of serum titres and means of bulk milk samples from 0.43 to 0.67. Finally, the correlations between herd means of individual milk samples and means of bulk milk samples varied from 0.52 to 0.82. This variation was dependent upon worm species and whether or not correction for milk yield was applied. The discrimination of herds was evaluated by the between/within herd ratio of variances. F-values for all types of samples were highly significant, and at least as high for milk samples as for serum samples. If, as has been shown for serum samples, a positive relationship exists between the milk yield response to anthelmintic treatment and the mean herd titre of milk samples or bulk milk samples, assessment of these titres could offer a rational basis for treatment of dairy cows.

Age Factors↗

Evaluation of an improved blood-conserving POCT sampling system.

OBJECTIVE: To evaluate a modified point-of-care (POCT) testing i-STAT analyzing cartridge that connects directly to the sampling port of a blood-conserving sampling line. DESIGN AND METHODS: In an in vitro setup, blood samples were drawn from a blood-conserving sampling line connected to a miniature cardiopulmonary bypass (CPB) system. Blood collection from the sampling port was either performed with a syringe necessitating subsequent sample loading on a standard i-STAT cartridge (conventional procedure) or with a modified i-STAT sampling cartridge allowing blood flow from the sampling port directly into the cartridge (modified procedure). The loaded cartridges were subsequently inserted into the i-STAT Portable Clinical Analyzer for sample analysis. Multiple parameters such as blood gases, electrolytes, hematocrit, and glucose were measured. A series of 30 paired measurements was performed. Corresponding series of values were compared using linear regression analysis and Bland-Altman bias analysis (P < 0.05). RESULTS: Twenty-five complete measurement series consisting of 12 parameters (pH, pCO(2), pO(2), SO(2), base excess, bicarbonate concentration, sodium, potassium, ionized calcium, hemoglobin concentration, hematocrit, glucose) were evaluated. Linear regression analysis between the two sampling methods tested demonstrated an excellent correlation for all parameters (Pearson correlation coefficients: 0.859-0.999). Bias and precision between corresponding series showed clinically acceptable performance levels for all parameters. CONCLUSIONS: The modified i-STAT sampling cartridge allows reliable diagnostic blood sampling directly from a blood-conserving sampling line. The technique presented is also applicable to other POCT systems, thus reducing diagnostic blood loss because of the minimal amount of blood required for analysis.

Blood Chemical Analysis↗

Interference and blood sample preparation for a pyruvate enzymatic assay.

BACKGROUND: To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis. METHOD: A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4 degrees C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated. RESULTS: When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4 degrees C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P < 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4 degrees C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin. CONCLUSIONS: For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay.

Adult↗

An evaluation of sampling- and culturing methods in the Norwegian action plan against Campylobacter in broilers.

The Norwegian Action Plan against Campylobacter in broilers was implemented in May 2001 with the objective of reducing human exposure to Campylobacter through Norwegian broilers. From each flock, samples collected at the farm about one week prior to slaughter, and then again at the slaughter plant, are examined for the presence of Campylobacter. All farmers with positive flocks are followed up with bio-security advice. Sampling of broiler products at retail level is also included in the Action Plan. The aim of this study was to evaluate the existing sampling and culturing methods of the Norwegian Action Plan against Campylobacter in broilers. The material collected was pooled faecal samples, pooled cloacae samples and caecae samples from individuals. The highest number of positives, from culturing of the pooled faecal samples, the pooled cloacae swabs and the caecae swabs from individuals, were obtained at incubation temperature 41.5 degrees C. When comparing the results at incubation temperature 37 and 41.5 degrees C, the faecal samples from the farms demonstrated a high concordance, with a kappa value of 0.88. The results from culturing cloacae swabs and caecae samples from slaughter plant level at two temperatures did not agree very well with a kappa value of 0.21 and moderate value of 0.57, respectively, but were both disconcordant at a level of 0.05. Modelling farm level data indicated that if increasing the number of pooled samples per flock from two (in existing regime) to three, the flock sensitivity increases from 89% to 95%. Modelling of slaughter plant data indicated that three pooled cloacae swabs are needed to identify 90% of the positive flocks. The results from the modelling of caecae data indicated that samples from seven individuals are sufficient to identify 90% of the positive flocks and caecae samples could thus be an alternative to cloacae sampling at slaughter plant level.

Animals↗

Effect of pooling bovine fecal samples on the sensitivity of detection of E. coli O157:H7.

To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.

Animals↗

Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization.

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially "sorted" samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes. STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome-specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples. RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the X/Y ratio for sorted samples. CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples.

Analysis of Variance↗

Stability of PO2, PCO2, and pH in fresh blood samples stored in a plastic syringe with low heparin in relation to various blood-gas and hematological parameters.

OBJECTIVE: To assess the stability of PO2, PCO2 and pH in fresh individual blood samples drawn in a plastic syringe with low heparin and stored at 22 degrees C or on ice in relation to various biochemical and hematological parameters of the samples. METHODS: PO2, PCO2, and pH were measured at determined times in samples kept at 22 degrees C or on ice. The magnitude of change in blood-gas parameters over time for each group of samples was determined. The change in PO2 was examined according to its initial value, the oxygen content (CaO2), the hemoglobin concentration, the leukocyte and platelet counts of each sample. RESULTS: The changes in PO2, PCO2, and pH over time were about three times lower in samples stored on ice than in the ones at 22 degrees C. Nevertheless, rapid increases in PO2 were observed for samples stored on ice with an initial PO2 between 50 and 250 mm Hg. Samples with PO2 over 250 mmHg exhibited a decrease in PO2 over time. No correlation was observed between the change in PO2 and the oxygen content, the hemoglobin concentration or the leukocyte and platelet counts for both groups of samples stored at 22 degrees C or on ice. CONCLUSION: PO2 in samples with an initial PO2 between 50 and 250 mm Hg stored on ice should be analyzed within 30 min. However, PCO2 and pH didn't exhibit clinically significant changes within 60 min under similar conditions. The magnitude of change in PO2 for samples kept on ice was dependent on their hemoglobin capacity for buffering oxygen, which is in converse relation with the oxygen saturation level of hemoglobin (SatHbO2). It is believed that the alteration in PO2 for samples stored on ice is produced predominantly by diffusion of oxygen through the plastic wall of the syringe, whereas it would be mainly of metabolic origin in samples at room temperature.

Blood Gas Analysis↗

Within-sample variation of fecal glucocorticoid measurements.

Fecal glucocorticoid metabolite analysis is a useful tool for monitoring adrenocortical activity in captive and free-ranging wildlife. Glucocorticoid metabolites may not be evenly distributed within fecal samples and this variability could affect the interpretation of glucocorticoid metabolite measurements. Furthermore, the precision (i.e., repeatability of measurements) of fecal glucocorticoid measurements from well-mixed samples is unknown. We collected fresh white-tailed deer (Odocoileus virginianus) feces at various times pre- and post-adrenocorticotropin injection to provide samples with low (<75 ng/g), medium (75-90 ng/g), and high (>90 ng/g) glucocorticoid concentrations in case variability differs in samples of dissimilar hormone metabolite concentrations. We compared two sampling methods (selection of three pellet groups [one from each end of the fecal mass and one from the center] versus sampling three small portions of the thoroughly mixed fecal mass) to estimate within-sample variation of glucocorticoid metabolites. Glucocorticoid metabolite measures from pellet groups were higher than fecal glucocorticoid measures from mixed samples in the low (F=3.10; df = 1,56; P=0.08) and medium concentration (F=7.28; df = 1,50; P=0.01) groups. Fecal glucocorticoid metabolite estimates from mixed samples were less variable than glucocorticoid metabolite measures using pellet groups from the same fecal mass, although these differences were not statistically significant (low group: F=0.59; df = 1,38; P=0.45; medium group: F=0.13; df = 1,34; P=0.72; high group: F=2.30; df = 1,28; P=0.14). The mean coefficient of variation was <10% across all treatment groups and sampling methods. However, a power analysis indicated the mixed sub-sample technique requires fewer samples to detect statistically significant differences than pellet groups. Our results suggest that glucocorticoid metabolites may not be evenly distributed in white-tailed deer feces. Consequently, using only a few pellets from a fecal mass may bias assay interpretation. We suggest researchers homogenize the entire fecal mass before removing a sub-sample of fecal material for analysis. Also, other sources of variation must be considered when interpreting results of fecal glucocorticoid studies.

Animals↗

Evaluation of a synthetic tripeptide as antigen for detection of IgM and IgG antibodies to Trypanosoma cruzi in serum samples from patients with Chagas disease or viral diseases.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.

Acute Disease↗