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PubMed1992

Accuracy and precision of a robotic sample processor.

The accuracy and precision of the Packard Probe 1000, a computer-driven robotic sample processor, were determined using NCCLS Guideline 18-P, employing a rigorous gravimetric procedure. For volumes of 25, 100, and 200 muL, inaccuracy ranged from -0.5 to +0.6% (saline) and -0.8 to +0.7% (human serum), and imprecision from 0.1 to 0.6% (saline) and 0.02 to 0.8% (human serum), in the dip and sip mode of pipetting. For the same volumes of saline in the dispense-through pipetting mode, inaccuracy ranged from -0.1 to -0.9% and imprecision from 0.1 to 0.5%. To obtain this level of performance, the instrument's operating parameters were changed from the factory default settings. This automated liquid-handling system is capable of impressive accuracy and precision, but it must be calibrated in the same manner as any other volumetric device. Performance varies with the volume, type of liquid, and mode of pipetting. Inaccuracy of less than or equal to 1.0% and imprecision of less than =1.0% can be attained along with rapid, walkaway pipetting of calibrators, controls, and large numbers of patient samples.

Chemistry, Clinical↗

PubMedDate not supplied

Microalbuminuria in diabetes mellitus: efficacy of a new screening method in comparison with timed overnight urine collection.

Diabetic nephropathy and end-stage renal failure are still a major cause of mortality amongst patients with diabetes mellitus (DM). In this study, we evaluated the Clinitek-Microalbumin (CM) screening test strip for the detection of microalbuminuria (MA) in a random morning spot urine in comparison with the quantitative assessment of albuminuria in the timed overnight urine collection ("gold standard"). One hundred thirty-four children, adolescents, and young adults with insulin-dependent DM Type 1 were studied at 222 outpatient visits. Because of urinary tract infection and/or haematuria, the data of 13 visits were excluded. Finally, 165 timed overnight urine were collected in the remaining 209 visits (79% sample per visit rate). Ten (6.1%) patients presented MA of > or =15 microg/min. In comparison however, 200 spot urine could be screened (96% sample/visit rate) yielding a significant increase in compliance and screening rate (P<.001, McNemar test). Furthermore, at 156 occasions, the gold standard and CM could be directly compared. The sensitivity and the specificity for CM in the spot urine (cut-off > or =30 mg albumin/l) were 0.89 [95% confidence interval (CI) 0.56-0.99] and 0.73 (CI 0.66-0.80), respectively. The positive and negative predictive value were 0.17 (CI 0.08-0.30) and 0.99 (CI 0.95-1.00), respectively. Considering CM albumin-to-creatinine ratio, the results were poorer than with the albumin concentration alone. Using CM instead of quantitative assessment of albuminuria is not cost-effective (35 US dollars versus 60 US dollars/patient/year). In conclusion, to exclude MA, the CM used in the random spot urine is reliable and easy to handle, but positive screening results of > or =30 mg albumin/l must be confirmed by analyses in the timed overnight collected urine. Although the screening compliance is improved, in terms of analysing random morning spot urine for MA, we cannot recommend CM in a paediatric diabetic outpatient setting because the specificity is far too low.

Adolescent↗

PubMed1997

Practice guideline for examination of the placenta: developed by the Placental Pathology Practice Guideline Development Task Force of the College of American Pathologists.

The Placental Pathology Practice Guideline Development Task Force, a multidisciplinary group, has prepared this guideline to assist those involved with placental examination. It provides recommendations related to indications and methods for placental examination as well as sample worksheets. An algorithm for the handling of placentas summarizes the recommendations of the guideline. A summary of specific findings of placental examination together with their pathogenesis and clinical associations is also provided. Recommendations related to reporting with sample reporting formats are included. The guideline is intended as an educational tool, and its use should be guided by the individual circumstances and care setting of specific cases.

Humans↗

PubMed1998

Effects of various handling and storage conditions on stability of Treponema pallidum DNA in cerebrospinal fluid.

Treponema pallidum DNA from even small numbers of organisms was detectable in cerebrospinal fluid (CSF) stored at room temperature or at 4 degrees C for several hours and in CSF subjected to three freeze-thaw cycles. These results suggest that negative PCR results for T. pallidum from patients diagnosed with T. pallidum invasion of the central nervous system are probably not due to the loss of target DNA prior to testing.

Blotting, Southern↗

PubMed2005

Prognostic factors and reporting of prostate carcinoma in radical prostatectomy and pelvic lymphadenectomy specimens.

This paper, based on the activity of the Morphology-Based Prognostic Factors Committee of the 2004 World Health Organization-sponsored International Consultation, describes various methods of handling radical prostatectomy specimens for both routine clinical use and research purposes. The correlation between radical prostatectomy findings and postoperative failure is discussed in detail. This includes issues relating to pelvic lymph node involvement, detected both at the time of frozen section and in permanent sections. Issues of seminal vesicle invasion, including its definition, routes of invasion and relationship to prognosis, are covered in detail. The definition, terminology and incidence of extra-prostatic extension are elucidated, along with its prognostic significance relating to location and extent. Margins of resection are covered in terms of their definition, the etiology, incidence and sites of positive margins, the use of frozen sections to assess the margins and the relationship between margin positivity and prognosis. Issues relating to grade within the radical prostatectomy specimen are covered in depth, including novel ways of reporting Gleason grade and the concept of tertiary Gleason patterns. Tumor volume, tumor location, vascular invasion and perineural invasion are the final variables discussed relating to the prognosis of radical prostatectomy specimens. The use of multivariate analysis to predict progression is discussed, together with proposed modifications to the TNM system. Finally, biomarkers to predict progression following radical prostatectomy are described, including DNA ploidy, microvessel density, Ki-67, neuroendocrine differentiation, p53, p21, p27, Bcl-2, Her-2/neu, E-cadherin, CD44, retinoblastoma proteins, apoptotic index, androgen receptor status, expression of prostate-specific antigen and prostatic-specific acid phosphatase and nuclear morphometry.

Biomarkers, Tumor↗

PubMed1975

Multiple vascular and gastric cannulation of swine for studies of gastrointestinal, liver, and peripheral tissue metabolism.

The implantation of multiple chronic indwelling vascular cannulas and of a gastric cannula into swine was described. Technics for housing, care, and handling of the animals pre- and post-surgically were established to reduce the incidence of infection and to allow collection of urine and feces in metabolic studies. This regimen has been used in 20 gilts to study absorption, tissue distribution, metabolism, and excretion of nutrients. On the average, the cannulas remained patent for 4 wk.

Anesthesia, Inhalation↗

PubMed2001

Automated high-performance liquid chromatographic method for the determination of guanidinoacetic acid in dried blood spots: a tool for early diagnosis of guanidinoacetate methyltransferase deficiency.

A new automated method for the assay of guanidinoacetic acid (GAA) in dried blood spot (DBS) on filter paper is reported. The method, based on reversed-phase (RP)-HPLC, precolumn derivatisation with benzoin and fluorescence detection, has shown good precision and sensitivity and requires only minimal sample handling. The validity of the method was demonstrated by analysing the neonatal blood spot of a patient affected by guanidinoacetate methyltransferase (GAMT) deficiency. GAA concentration was found to be nearly 12-fold higher than the mean control value. We propose this method as an inexpensive and widely applicable tool for the diagnosis of GAMT deficiency.

Automation↗

PubMed2002

Anticoagulant effects on plasma coenzyme Q(10) estimated by HPLC with coulometric detection.

BACKGROUND: The proportion of reduced coenzyme Q(10) (Q(10)H(2)) in total coenzyme Q(10) (TQ(10)), referred to as the Q(10)H(2):TQ(10) ratio, may be used as a possible marker of in vivo oxidative stress. However, the ranges for Q(10)H(2):TQ(10) ratio from previous reports are quite variable. Sample handling and preparation appear to have a profound effect on the stability of Q(10)H(2). METHODS: Paired tests were used to estimate TQ(10), Q(10)H(2), oxidized coenzyme Q(10) (Q(10)), and Q(10)H(2):TQ(10) ratio in patient samples collected in vacutainers containing heparin or EDTA. Sample tubes were immediately placed on ice and promptly centrifuged. After harvesting plasma, 100 microl of plasma was extracted with 1-propanol and centrifuged. The supernatant was injected directly into a high-performance liquid chromatography (HPLC) system. RESULTS: Significantly higher values (p=0.0015) of TQ(10), Q(10)H(2), and Q(10)H(2):TQ(10) ratio were noted in heparinized plasma as compared to EDTA plasma; Q(10) concentrations were lower in heparinized plasma. When vacutainers containing specimen were opened and kept refrigerated, the Q(10)H(2):TQ(10) ratios in heparinized samples were stable over 7 h with variation <3%. Blood Q(10)H(2) in closed heparin vacutainers kept refrigerated was stable up to 24 h. CONCLUSIONS: Our results indicate that the heparinized plasma is superior to the EDTA plasma in all measurements for coenzyme Q(10).

Anticoagulants↗

PubMed2002

[A system for biomaterial handling and sample pretreatment in conditions of weightlessness].

To perform laboratory biochemical, immunologic and microbiological analyses and special pretreatment and stabilization of biosamples immediately on the space station, system Plasma-03 was designed and manufactured and associated procedure for sample collection and pretreatment was developed. Main elements of the system are a refrigerator/thermostat, centrifuge and returnable container, and a set of accessories and expendables. The system meets the requirements to the equipment to be employed in crew health monitoring and biochemical experiments on board the International space station.

Biochemical Phenomena↗

PubMed2005

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory.

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.

Animals↗

PubMed1992

A simple method for handling grids.

A simple method for handling electron microscope grids is described here. While assuring their identification and safety, this method provides improved handling, temporary storage, and identification of grids bearing ultra-thin sections, and in addition, provides a novel method for preparing bulk samples. Grids are attached at their edges to the weakly adhesive surface of a "Post-it" note pad which sits in a petri dish. The grids are safely immobilized on the pad, classified based on their location, and identified by convenient pad notation. Grid manipulation and identification are simplified using this device, which is easily assembled from readily available and inexpensive materials.

Microscopy, Electron↗

PubMed2003

Evaluation of the clinical usefulness of isolation of fetal DNA from the maternal circulation.

OBJECTIVE: To assess the reliability of isolating free fetal DNA from maternal usefulness. DESIGN: Fetal DNA was isolated from plasma or serum that was either collected prospectively or from archived samples collected for the purposes of second trimester screening. METHODS: Prospective samples were collected from patients undergoing prenatal diagnostic procedures (n = 24). A second group of samples from Rhesus negative women (n = 28) were assayed in which blood had originally been collected for maternal triple serum screening. DNA was extracted from all samples and assayed for the presence of the beta-globin gene, sex-determing region Y (SRY) gene and Rh gene. All DNA sample handling and extraction was carried out by a single operator, and polymerase chain reaction (PCR) was carried out using previously published PCR primers and appropriate controls. The accuracy of results was assessed relative to the karyotype in the case of the SRY gene or cord blood phenotype in the case of the Rh gene. RESULTS: The SRY PCR results were compared to fetal cell karyotypes obtained from invasive diagnostic testing, 21 out of the 24 samples were correctly 'sexed'. The RhD PCR results were compared to fetal cord blood samples at the time of delivery, and showed both false positive and false negative results. Two RhD negative babies were genotyped as RhD positive, despite repeat analysis. CONCLUSION: It is possible to isolate fetal DNA from maternal serum. It is a potentially clinically useful technique in our laboratory and can be used to detect male fetuses, and Rh negative fetuses. To be useful in clinical practice, it is necessary to safeguard against contamination at the time of sample handling, and to use the optimal range of primers available to cover the polymorphisms present within the RhD gene. Although not robust enough yet to be used with diagnostic certainty in our hands, immense improvements in technique, probes and real-time PCR equipment make this type of diagnosis a reality in the near future.

Chromosomes, Human, Y↗

PubMed2005

Influence of pre-treatment step on PAHs analyses in contaminated soils.

There is no specific standardization for the pre-treatment of a soil sample for PAHs analyses. The ISO/FDIS 14507 method for soil quality gives a guidance for sample pre-treatment before measurement of organic contaminants in soil. It refers to cryogenic crushing which is not so easy to carry out. So, analysts lead to develop in house methods which can be very different from a laboratory to another and can involve deviations in the pollution quantification. This study has explored different ways of drying, grinding and sieving in order to determine the influence of the soil sample handling on the measured pollutant concentration. It appears that the influence of pre-treatment depends on the soil nature and on the PAHs distribution. As a conclusion, it is advised "light" drying ways like air drying or drying at 40 degrees C in a drying oven instead of more "drastic" ways (freeze-drying, cryogenic crushing) which can induce some losses. Crushing and sieving are also recommended for more reproducible measures.

Environmental Monitoring↗

PubMed1997

Micro-extraction of commonly abused benzodiazepines for urinary screening by liquid chromatography.

We have developed a micro-extraction procedure for the analysis of seven commonly prescribed benzodiazepines (chlordiazepoxide, diazepam, lorazepam, nitrazepam, nordiazepam, oxazepam, and temazepam) in urine using liquid chromatography. The method is reliable and sensitive, uses small volumes (100 microL) of urine and is suitable for the detection and quantification of low concentrations of benzodiazepines. The micro-extraction procedure allowed rapid sample processing, which is important for routine sample handling. The limit of detection for the seven benzodiazepines ranged from 0.10-0.71 mg/L and recovery of the different benzodiazepines was good, ranging from 70-105%. Between- and within-assay coefficients of variation ranged from 6.3% to 13.8%, and 2% to 3.5%, respectively. Chlordiazepoxide chromatographed poorly (between assay coefficient of variation 35.4%, within-assay 7%), and we set the cut-off value for this compound at 5.0 mg/L.

Benzodiazepines↗

PubMed2005

[Ecotoxicological bioassays on aquatic sediments: experimental problems of exposure matrices].

In this review a discussion on some factors influencing the exposure matrices which, in turn, influences the reliability of ecotoxicological bioassays on aquatic sediments, has been carried out. These factors include the variability induced on sediments by the sampling, storage, handling, and preparative operations. The exposure matrices-sediments in toto, interstitial water and elutriate, can be deeply modified by these actions, which alter the chemicals bioavailability and, therefore, the bioassay meaning. In order to obtain reproducible and scientifically valid data, to be used in the ecological risk assessment, all these factors need to be considered and kept under control.

Biological Assay↗

PubMed1976

The salivary secretions in health and disease.

The potential usefulness of analysis of the salivary secretions in diagnosis and prognosis is beginning to be explored in depth. The preliminary work already undertaken indicates that modern methods applied to this secretion may provide information that is different from that obtained in other body fluids. Saliva is collected at the point of its manufacture and, therefore, is unaffected by collection or storage in the body. It is the product both of protein synthesis within the glands and of most of the known water and electrolyte exchange mechanisms. Salivary composition is affected by both autonomic and hormonal stimuli. As the specific influence of each of these factors is better understood, studies of this fluid will provide important clues to the understanding of disease and the evaluation of therapy. There are few places in the body where it is possible directly, utilizing a non-invasive technique, to examine the product of a large number of important biological processes. It is obvious that careful handling of collection and analytic techniques are essential if these secretions are to be utilized. Future investigations in clinical situations should take full advantage of the strong base of knowledge of the physiology of these glands. Development of this field depends on careful clinical investigations designed to make full use of our current knowledge.

Amylases↗

PubMed2001

Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection.

Microfabrication techniques have become increasingly popular in the development of the next generation of DNA analysis systems. While significant progress has been reported by many researchers, complete microfabricated integrated DNA analysis devices are still in the earliest stages of development. Most miniaturized analysis systems have incorporated noncross-linked polymer solutions as the separation medium of choice and the operation of these systems necessitates the use of high electric fields and long separation lengths. In this paper, we present two techniques that may help alleviate this problem and accelerate the development of the so-called 'lab-on-a-chip' systems. We present the use of photodefinable polyacrylamide gels as a sieving medium for DNA electrophoresis. These gels offer the significant advantages of faster curing times, locally controlled gel interface, and simpler handling over chemically polymerized gels. We also introduce an electrode-defined sample compaction and injection technique. This technique helps achieve sample compaction without migration into the gel and offers significant control over the size and application of the sample plug. The use of these technologies for double-stranded DNA separations in microfabricated separation systems is demonstrated.

Acrylic Resins↗

PubMed2003

Sources of sampling variation in saliva cortisol in dogs.

The main advantage of collecting saliva cortisol as opposed to plasma cortisol is that it is non-invasive and therefore it is now widely used in stress measurement studies on farm animals and dogs. Although a plasma cortisol response to handling associated with blood collection generally occurs at 3 min from the commencement of handling, there is no information in the literature on the time course of the response of salivary cortisol concentration to handling. The aims of these experiments were to (1). determine if there is a response to up to 4 min handling that affects cortisol concentration in saliva and (2). determine the main causes of variation in saliva cortisol in dogs over time. In experiment 1, saliva was collected from six Kelpies at 0 min then 2, 3 or 4 min after the commencement of restraint. There was no handling effect found in up to 4 min sampling time. In experiment 2, saliva was collected from six Labrador Retrievers five times in 2 h (14:00-16:00), three days a week for four weeks. Some of the sources of variation in saliva cortisol over time included between dog variation that varied over a period of days and variation between occasions that affected the group of dogs as a whole.

Animals↗