Unpreserved Samples Research Articles

The possible influence of micro-organisms and putrefaction in the production of GHB in post-mortem biological fluid

In recent years, the post-mortem production of the drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids (e.g. blood and urine) has caused various interpretative problems for toxicologists. Previously, other researchers have shown certain microbial species ( Pseudomonas spp. and Clostridium aminobutyricum) possess the necessary enzymes to convert GABA to GHB. A preliminary investigation involving putrefied post-mortem blood indicated there was no observed relationship between “endogenous” GHB concentrations and concentrations of common putrefactive markers (tryptamine and phenyl-2-ethylamine). Microbiological analysis identified the presence of various micro-organisms: Clostridia spp., Escherichia coli, Proteus vulgaris, Enterococcus faecalis and Aeromonoas spp. Equine plasma, human blood and urine samples were inoculated with these and an additional micro-organism ( Pseudomonas aeruginosa) and incubated at 22 °C for 1 month. Following comparison with control samples and pre-inoculation concentrations, the data indicated an apparent production of GHB in unpreserved P. aeruginosa inoculated blood (2.3 mg/l). All other fluoride-preserved and unpreserved samples (including controls) had GHB concentrations <1 mg/l. Although this concentration is lower than is typically associated with “endogenous” post-mortem GHB concentrations, this paper proposes a potential microbial production of GHB with time.

α1-Microglobulin Is Stable in Human Urine ex Vivo

Increased concentrations of urinary α1-microglobulin may imply proximal tubular damage (1). α1-Microglobulin has generally been considered to be stable in human urine (1)(2). Tencer et al. (2) observed good stability in 10 urine samples stored at room temperature for 7 days, at 4 °C for 30 days, and at −20 °C for 6 months. In contrast, Donaldson et al. (3) noted significant losses of α1-microglobulin in urine stored at −20 °C and that this problem was exacerbated in more acidic (pH <6.0) urines; they recommend that urine should be neutralized on receipt. The manufacturers of our assay recommend that urines be assayed fresh or stored at 4 °C for a period of less than 1 week and warn against freezing samples. Urine samples are often stored before batch analysis. To clarify the appropriate storage conditions for urinary α1-microglobulin, we studied stability under standardized conditions. Random unpreserved urine samples were collected from 19 patients at a single nephrology clinic, and urinary pH was determined (mean pH 5.87; range, 5.08–6.85). Samples …

Analysis of cocaine, benzoylecgonine, ecogonine methyl ester, and ecgonine by high-pressure liquid chromatography-API mass spectrometry and application to a short-term degradation study of cocaine in plasma.

A method for the determination of cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and ecgonine (ECG) in plasma by liquid chromatography-mass spectrometry (LC-MS-MS) was developed. The analytes were isolated from human plasma by subsequent solid-phase extraction and were separated on a Zorbax Eclipse XDB-C8 narrow-bore column using an ammonium acetate buffer/acetonitrile/methanol gradient. A Turbolonspray source was used for ionization. The analytes were characterized by their particular molecular ion and several fragments. Multiple reaction monitoring (MRM) was used for isolation and quantitation. The assay was rapid, highly sensitive, and reliable. The method was applied to monitor the in vitro degradation of cocaine in plasma. Fresh unpreserved and preserved (0.25% KF) plasma samples were spiked with 1,000 ng cocaine/mL. Aliquots of both series were stored at 4 degrees C and 20 degrees C and were analyzed at selected storage times of up to 15 days. In all samples, degradation of cocaine that was dependent on storage time and temperature and on sample preservation could be observed. The formation of BE did not occur to a significant extent (< 12%, referred to the initial concentration of COC), and its concentration was slightly higher in preserved compared with unpreserved plasma at both storage temperatures chosen. EME was formed in considerably higher amounts compared to BE. As already observed for COC, its formation and degradation were dependent on storage time, temperature, and preservation. EME is suggested to be the major source of ECG, which was detectable in all samples after 1-2 days of storage. Although the degradation of COC was shown to be highly dynamic in nature, the sum of all hydrolysis products of COC accounted for the initial COC concentration at any particular time of storage. Therefore, production of hitherto unknown degradation products of COC seems unlikely. Moreover, the common transformation product of BE and EME appeared to be stable, and ECG is suggested as a promising postcollection artifact.

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNA later, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO ( rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNA later is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples.

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

Cryopreservation of Artificial Cartilage: Viability and Functional Examination after Thawing

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 × 10⁶/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4°C followed by a 24-hour storage at –80°C. After that, the tissue units were transferred into liquid nitrogen (–196°C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.

Simultaneous GC-MS Analysis of meta- and para-Hydroxybenzoylecgonine and Norbenzoylecgonine: A Secondary Method to Corroborate Cocaine Ingestion Using Nonhydrolytic Metabolites

Positive benzoylecgonine (BZE) urinalysis results are sometimes challenged in legal and administrative proceedings on the grounds that the presence of BZE is due to the addition of cocaine to the urine sample with subsequent in vitro hydrolysis to BZE. Consequently, counsel for the respondent or defendant may move that an ecgonine methyl ester (EME) analysis be preformed because EME is presumed to be solely an in vivo cocaine metabolite. For these reasons, a sensitive and rapid gas chromatographic-mass spectrometric procedure was developed for the simultaneous analysis of m-hydroxybenzoylecgonine (m-OHBZE), p-hydroxybenzoylecgonine (p-OHBZE), and N-desmethyl benzoyl ecgonine (norBZE), all of which are cocaine metabolites believed to arise exclusively via in vivo metabolism. Analysis of human urine specimens previously reported positive for BZE using GC-MS at the Department of Defense cutoff of 100 ng/mL demonstrated that at least one of the three metabolites was present in 79 of the 82 specimens studied (96.3%). Thus, the simultaneous analysis of r-OHBZE, p-OHBZE, and norBZE could be used to substantiate that the presence of BZE in urine specimens is the result of cocaine ingestion. Additionally, the premise that EME is a "true" in vivo cocaine metabolite was investigated by assessing the stability of cocaine in unpreserved urine samples at several pHs ranging from 5.0 to 9.0.

Differentiation of Entamoeba histolytica and Entamoeba dispar cysts using polymerase chain reaction on DNA isolated from faeces with spin columns.

Since Entamoeba histolytica and Entamoeba dispar were formally recognized as two different species at the World Health Organization (WHO)/Pan American Health Organization (PAHO)/United Nations Educational, Scientific and Cultural Organization (UNESCO) meeting in Mexico City in 1997, the specific differentiation of the two morphologically identical species would seem relevant in clinical diagnosis. Several polymerase chain reaction (PCR)-based methods have been described and used successfully, but methods for DNA isolation from cysts in stool samples are time-consuming and problematic due to inhibitory factors in faeces. The use of the slightly modified QIAamp tissue method (Qiagen, Germany) for DNA isolation was evaluated in 657 unpreserved faecal samples from cases of suspected Entamoeba histolytica/Entamoeba dispar infection. In only 1.7% of the cases was PCR hampered by inhibitors present in the faeces. The DNA isolation procedure was found to be rapid, simple and one that could easily be implemented in a routine diagnostic setting. In 98.8% of Entamoeba histolytica/Entamoeba dispar cyst-positive faecal samples, the true identity of the cysts could be determined using PCR specific for Entamoeba histolytica and Entamoeba dispar, respectively.

Evaluation of degradation of urinary catecholamines and metanephrines and deconjugation of their sulfoconjugates using stability-indicating reversed-phase ion-pair HPLC with electrochemical detection

The possibility of the deconjugation of urinary catecholamine sulfates (CA-S) and metanephrine sulfates (MN-S) during storage was studied using a reversed-phase ion-pair HPLC method with electrochemical detection. Stability profiles of catecholamines (norepinephrine (NE), epinephrine (E) and dopamine (DA)) and metanephrines (normetanephrine (NM) and metanephrine (MN)) in preserved and unpreserved pooled urine and aqueous samples stored at 10 and 30°C over 8 days were compared. Results showed that these compounds exhibited stable or declining profiles in aqueous samples but fluctuating profiles in pooled urine samples. It was concluded that deconjugation of CA-S and MN-S occurred in both preserved and unpreserved urine samples. Therefore, at 10 and 30°C, levels of detected urinary catecholamines and metanephrines could be affected by both the degradation of the free amines and deconjugation of the CA-S and MN-S. Based on these findings, another batch of unpreserved urine and aqueous samples were prepared and stored at −80°C. Under this condition, all compounds were found to be stable with less than 10% variations in both unpreserved urine and aqueous samples for at least 22 days. These indicated that urine samples could be stored unpreserved at −80°C for at least 3 weeks without significant degradation of the free amines or deconjugation of their sulfoconjugates.

Effects of water sample preservation and storage on nitrogen and phosphorus determinations: implications for the use of automated sampling equipment

The effects of freezing, acidification, refrigeration and extended storage without refrigeration on measured concentrations of forms of nitrogen and phosphorus were assessed in water samples taken from polluted and unpolluted streams in dry and wet weather. Emphasis was placed on likely changes associated with the use of automatic samplers in field conditions. The response of measured nutrient concentrations to different preservation methods and to extended storage without preservation varied widely between samples. For polluted streams where nutrient concentrations were high (ammoniacal nitrogen >0.1 mg l −1; oxidised nitrogen, total Kjeldahl nitrogen, total and filterable phosphorus >1 mg l −1), there was generally little proportional change in nutrients other than ammoniacal nitrogen after up to 6 d of storage without preservation. Where nutrient concentrations were low, up to 90% of ammoniacal nitrogen, 50% of oxidised nitrogen, 84% of total Kjeldahl nitrogen and 67% of total phosphorus was lost after 6 d. If samples cannot be retrieved immediately, automatic samplers should be refrigerated where possible. Acidification is a suitable alternative for preservation of nitrogen. Samples for filterable phosphorus should be filtered as soon as possible after collection. In some circumstances, unpreserved samples may provide an acceptable level of accuracy for evaluation of broad temporal trends or estimation of nutrient loadings.

Automated Screening Method for Determining Optimum Preservative Systems for Personal and Home Care Products

A procedure was designed to determine the minimum preservative level (MPL) for personal and home care products. A highly preserved sample and an unpreserved sample were combined at different concentrations within a 96-well microtiter plate by using an autodilutor. A unique tip design made it possible to accurately deliver viscous test materials that cannot be dispensed using vacuum- or fluid-filled systems. After inoculation, the sample was evaluated at a specified time interval for the presence of surviving bacteria, yeast, and mold. The lowest concentration of preservative with no microbial growth is the recommended level of preservative for the product. Because sample turbidity may interfere with determination of the endpoint, a colorimetric endpoint was used to indicate growth of microorganisms and to differentiate product from growth. The predicted levels were tested with a modified Cosmetic, Toiletry, and Fragrance Association method. The method successfully predicted effective preservative levels in many personal and home care products with a broad range of viscosities.

Use of Biopsy Samples of Humpback Whale (Megaptera novaeangliae) Skin for Stable Isotope (δ13C) Determination

Previous work has shown that stable isotope indicators taken from the muscle tissue of dead, stranded cetaceans can be used to assess diet. Recent advances in remote biopsy techniques have provided a means to collect skin and blubber tissues from live animals. This study examines the potential of biopsy samples taken from humpback whales (Megaptera novaeangliae) for isotopic assessments of diet by 1) determining if isotopic differences exist between the two tissue types obtained in a biopsy (skin and blubber) and the traditional source for isotopic analysis, muscle tissue, 2) examining the effects of two different lipid extraction techniques on the removal of the preservative dimethyl sulfphoxide (DMSO) from tissues, and 3) assessing procedural reproducibility for automated isotopic analysis of skin derived from biopsy samples. Results demonstrate that carbon isotopic values ( δ 13C) of muscle were not significantly different from those of skin (Scheffe, p = 0.4985; δ 13C = -19.1‰ ± 0.7 and -19.5‰ ± 0.5 for muscle and skin respectively; mean ± SD). The values for blubber ( δ 13C = -23.7‰ ± 0.2) were significantly lower than those of muscle or skin (Scheffe, p = 0.0001). This result was consistent with previous studies indicating that the δ 13C of lipids is typically lighter than those tissues with which it is associated. The analysis also indicates that samples preserved in DMSO have significantly lower δ 13C than unpreserved samples (paired t-test, p = 0.010). Two methods of lipid extraction, sonication and Soxhlet ext rac t ion, were successful in removing DMSO from samples . The procedural reproducibility for δ 13C was 0.1‰. In summary, skin tissue yielded from biopsy samples may be used in isotopic assessments of diet. The use of biopsies as a source allows the technique to be used in longitudinal, non-lethal sampling.

Commentary on the Subsampling Procedures Used for Rapid Bioassessments

Commentary on the Subsampling Procedures Used for Rapid Bioassessments

Effects of Sampling Area and Subsampling Procedure on Comparisons of Taxa Richness among Streams

the biotic integrity of communities. In all cases, we make the implicit and sometimes brash assumption that we can really measure the number of taxa in a community. Although measuring taxa richness might appear straightforward, accurate measurement has been extraordinarily difficult; and despite years of effort, no universally accepted methods for its measurement have emerged. The essential problem is that we can never completely census a taxonomic assemblage or entire community; we rely instead on estimates that describe some portion of the real taxa richness of an assemblage. The problem of knowing what percent of the taxa present have been collected is exacerbated when investigators fail to explicitly define their universe of interest (i.e., the spatial bounds of the community or communities in question). Comparisons of taxa richness among studies that used different sampling and subsampling methods are especially difficult and should be viewed skeptically. The difficulty of obtaining accurate measurements of richness is due to the collector's curve

Urinary cortisol analysis for monitoring adrenal activity in elephants

AbstractCortisol was measured in dichloromethane‐extracted elephant urine using an 125I solid‐phase radioimmunoassay (RIA). The cortisol RIA was validated by demonstrating 1) parallelism between dilutions of pooled urinary extracts and the standard curve, 2) significant recovery of exogenous cortisol added to elephant urine, and 3) a relationship between changes in peripheral and urinary cortisol after an adrenocorticotropin hormone (ACTH) challenge. One African (Loxodonta africana) and one Asian (Elephas maximus) elephant were given three injections of ACTH (1.25 mg) at 2 h intervals. Serum cortisol increased four‐ to eightfold within 30 min after the first injection and peaked (nine‐ to twelvefold increase) after the second injection. Serum concentrations began to decline 2–3 h after the last injection but were still approximately fourfold higher than baseline at the end of the collection period (hour 8). In the urine, cortisol concentrations were increased in the first sample postinjection (1.5–4 h) and peaked twenty‐ to fortyfold by ∼6 h. Urinary cortisol remained elevated at 8 h, but returned to baseline the following morning. Analysis of high performance liquid chromatography fractions of extracted urine revealed that immunoactivity was associated with free cortisol (∼90% of total immunoactivity) and a more polar, unidentified metabolite. A method for preserving urine was developed to allow storing unfrozen samples. One pool of urine from each of one African and two Asian elephants was divided into aliquots, placed in tubes containing absolute ethanol (10%), sodium azide (0.1%) or distilled water (control), and frozen after 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 weeks of storage at ∼25°C. In unpreserved samples, cortisol concentrations were reduced 46% by 2 weeks and 95% by 24 weeks. In contrast, ethanol‐ and sodium azide‐preserved samples retained 100 and 95% of cortisol immunoactivity through 8 weeks and 93 and 85% of activity through 12 weeks, respectively. We infer from these data that changes in urinary cortisol excretion in the elephant reflect fluctuations in adrenal activity and may be a useful indicator of stress. Additionally, urine samples can be collected and stored unfrozen for at least 2 months before any appreciable loss in cortisol immunoactivity occurs, a finding potentially useful to field application of this technique. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America .

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

Summary An elisa containing lipoarabinomannan (lam) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to lam elisa testing immediately, and after 1 year of storage at −70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in lam elisa results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk lam elisa and was 0.15 for serum lam elisa, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to lam elisa testing. Results were compared, using the area under the receiver operating characteristic curves. The milk lam elisa had specificity (± 95% confidence limits) of 87 ± 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 ± 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 ± 0.03 for the milk elisa and 0.75 ± 0.02 for the serum elisa. In this population of cattle, the milk lam elisa had comparable accuracy to serum lam elisa, although the milk lam elisa was slightly less reproducible (higher coefficient of variation).

Efficacy of 1% Sodium Fluoride as a Preservative in Urine Samples Containing Glucose and Candida albicans

Whether urine samples used in forensic science DUI testing can be compromised by endogenous ethanol production is a recurrent and yet unresolved issue. This study first assessed unpreserved urine samples that were collected, processed, and analyzed repeatedly over 13 to 41 days using a standard gas chromatographic procedure for ethanol analysis. Despite extensive microbial growth, ethanol was not detected in any test sample. The extent of ethanol production in samples supplemented with glucose, Candida albicans, or both was determined to evaluate the potential for ethanol production in urine samples associated with pathological conditions such as urinary tract yeast infections and diabetes mellitus. Ethanol production under each of the above treatment conditions was assessed in the presence and absence of 1% sodium fluoride as a microbial suppressant. Mean ethanol concentrations were determined for unpreserved samples containing urine only (0.003 +/- 0.005 g%), urine plus yeast (0.006 +/- 0.009 g%) and urine plus glucose (0.067 +/- 0.070 g%). Unpreserved samples supplemented with both yeast and glucose attained mean ethanol concentrations of 0.164 +/- 0.057 g% (P < 0.01). Ethanol could not be detected in any corresponding duplicate samples, which were preserved with 1% sodium fluoride. A lack of ethanol production in any of the unpreserved urine samples indicates that false DUI convictions due to endogenous ethanol production are very unlikely. And while endogenous ethanol production is possible in the presence of both glucose and contaminating C. albicans, 1% sodium fluoride completely eliminated microbial fermentation.

Dissolved organic carbon methods: a critical review

According to the most recent high-temperature catalytic oxidation (HTCO) analyses, the dissolved organic carbon content of seawater can be divided into material oxidizable, and hence measurable, by the standard wet chemical and photochemical methods, and material which is not oxidizable by either chemical or photochemical methods, but can be measured by HTCO methods. Both of these categories contain materials which are biologically extremely labile, disappearing within hours or days in unpreserved samples. A major problem in all methods is preservation of this labile material. As all of the methods are routinely performed, the amount of the labile material included in the measurement is unknown, and may vary between total inclusion, for samples analyzed as soon as they are drawn, to total exclusion, for samples stored at room temperature and analyzed some weeks later. This fraction may make up a third to a half of the total dissolved organic carbon (DOC). A less labile, chemically resistant fraction can be measured as the difference between HTCO and wet oxidation results for seawater below the oxygen minimum layer. This material must be available to some extent to biological, but not to chemical degradation. This requirement sets limits on the possible composition of the material. The most pressing needs for future work in this field are reliable preservation methods, which will allow measurement of the labile materials, suitable primary standards, to permit calibration of analytical systems, and water sufficiently free of organic carbon to be usable in determining instrumental blanks.

High-temperature combustion analysis of dissolved organic carbon produced in phytoplankton cultures

Abstract A high-temperature catalytic oxidation (HTCO) unit was constructed for the measurement of dissolved organic carbon (DOC) in seawater. This unit, which used a 5% Pt catalyst, consistently measured a greater amount of DOC than did a wet oxidation technique using UV light. In medium in which algal cultures had been grown, as much as half of the DOC measured by HTCO disappeared in a day in unpreserved samples. Of the preservation methods tested, addition of mercuric chloride after filtration appeared to be the most satisfactory. In algal cultures, the rates of production and disappearance of DOC were dependent on the species of algae present and their physiological state.

Salivary testosterone measurements: Collecting, storing, and mailing saliva samples

Salivary testosterone measurements can be especially useful in field studies, but reliable ways of collecting and handling samples need to be established. Using cotton dental rolls to collect saliva leads to inflated testosterone scores. Sugarfree gum can be used satisfactorily to stimulate saliva among both male and female subjects. Leaving unpreserved saliva samples at room temperature for 2 weeks or mailing them unrefrigerated is satisfactory for male subjects but leads to inflated scores for female subjects.