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PubMed2002

Problems related to rapid methods for erythrocyte sedimentation rate test and their solution.

The erythrocyte sedimentation rate (ESR) test is one of the most common and traditional laboratory tests in the world. It reflects both plasma concentration of acute-phase proteins of large molecular size and anemia (International Council for Standardization in Hematology, 1993). The ESR test method is easy to perform and inexpensive. Therefore, it is used today as a routine test worldwide. However, the ESR has some demerits, in requiring a large volume of sodium citrate or ethylenediamine tetraacetic acid (EDTA) blood and at least 2-hour testing time. When ESR is tested manually, incorrect results are obtained if the ESR test tube is not stood strait-up and vertically (Dobashi et al, 1994). Reading error for the meniscus line and surrounding temperature at the testing site cause inaccuracy (Manley, 1957). The 2-hour testing time is not practical for modernized automated laboratories. Its test procedures present the risk of infection from contact with pathogen-bearing blood (Imafuku and Yoshida, 2001). In this context, several kinds of simple, rapid and safe methods have been developed. Of these new systems, we selected 4 and evaluated their performance. This paper reports critical reviews of such devices.

Blood Sedimentation↗

PubMed1994

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine.

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology Robot and BenchMate Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid-liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate performed buffer and organic solvent addition, liquid-liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2-100 ng ml-1 in plasma and 0.1-20 micrograms ml-1 in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.

Chromatography, Liquid↗

PubMed1978

Role of veterinarians in modern food hygiene.

Veterinary services and veterinary education and training must keep pace with the constantly changing patterns of agriculture and food processing. Changes in methods of animal production are associated with many problems of food processing and food quality. Veterinary supervision of the animal feed industry and of meat and distribution is essential. Quality testing of meat, milk, and eggs requires the introduction of suitable routine sampling systems, laboratory procedures, and complex evaluation procedures.Food hygiene problems have changed in recent years not only as a result of new methods of animal production, but also because of changes in food processing technology and in the presentation of food to the consumer, increased environmental pollution, increased international trade, and increased tourist travel.Food hygienists must adopt an active and progressive policy and change the scope of food control from a purely negative measure into a positive force working towards improved food quality and the avoidance of losses during production. A modern food hygiene programme should cover all stages of production, processing, and distribution of food and also other ingredients, additives and the water used for production and processing. Veterinarians should also be involved in the registration and licensing of enterprises and this should take into account the premises, the procedures to be used, new techniques in animal husbandry, machines and equipment, etc.In order to facilitate the microbiological analysis of foodstuffs, new mechanized or automated laboratory methods are required, and consideration must be given to adequate sampling techniques.

Animal Feed↗

PubMed1995

Sperm morphology assessment: historical review in relation to fertility.

Careful analysis of sperm morphology has always been an important part of a routine semen examination. However, the usefulness of sperm morphology assessment as a predictor of a man's fertilizing potential has often been challenged due to different classification systems, various slide preparation techniques and inconsistency of analyses within and between laboratories. Automated sperm morphology analysis instruments may overcome the subjective nature of visual assessments of sperm morphology, but the technical problems are numerous and the validity of these instruments has still to be proven. Having reviewed the literature, it seems clear that there is general agreement concerning the clinical relevance and predictive value of this single semen parameter in vivo and in vitro. Nevertheless, even in cases of severe teratozoospermia, fertilization may be possible. Studies on the acrosome reaction are very promising for patients with severe sperm morphology abnormalities that do not have major effects on the fertilizing potential. Most promising is the development of intracytoplasmic sperm injection (ICSI) as the treatment of first choice in cases of severe teratozoospermia with failed fertilization in vitro. Normal fertilization and pregnancy rates can be obtained with ICSI in the presence of extreme teratozoospermia, suggesting that sperm morphology may be important in spermatozoa-zona binding, penetration and spermatozoon-oocyte fusion but fails to be of any predictive value once the spermatozoon reaches the cytoplasm of the oocyte. To conclude, accurate and strict sperm morphology assessment is very useful in evaluating a patient's fertilizing potential, with the main advantage that the methods used to examine this parameter are easy to learn on equipment found in most laboratories.

Classification↗

PubMedDate not supplied

Automated noninvasive determination of mixed venous pCO2.

The determination of mixed venous pCO2 is desirable for assessing the metabolic and respiratory status of a patient. A totally automated, laboratory computer-controlled noninvasive system has been developed to determine mixed venous pCO2 by an equilibrium rebreathing method or by an exponential compartmental analysis for cases in which equilibrium is not achieved. A gas mixture is charged to a 2-liter anesthesia bag contained in a thermostatically controlled chamber used to maintain the temperature at 37 degrees C. This feature improves upon past rebreathing methods and eliminates water vapor as a variable in gas composition measurement. This bag is connected to a rebreathing circuit controlled by a minicomputer. The subject breathes from a mouthpiece attached to a two-way valve and rebreathes the gas mixture for a period of 30 seconds. Inspirate and expirate hoses are placed in the rebreathing bag to ensure a more uniform gas distribution than is generally found in rebreathing systems. Exchange of CO2 takes place between lungs and rebreathing bag, and the concentration of CO2 is continuously monitored by a mass spectrometer. After a period of time, the concentration of CO2 in the rebreathing bag, the alveoli, and the mixed venous blood come into equilibrium, demonstrated by a plateau on the record of CO2 concentration vs. time. Compartmental analysis predicts the mixed venous pCO2 even if an equilibrium is not established. This feature is a significant benefit of this new method, eliminating problems associated with establishing an equilibrium, such as gas mixture volume adjustment, recirculation, and poor ventilation. The predicted value agrees with the equilibrium valve for cases in which equilibrium is reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Automation↗

PubMed2001

Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures.

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.

Animals↗

PubMed1988

Determination of cyanide and cyanogenic compounds in biological systems.

A survey of methods for the qualitative and quantitative determination of cyanide and cyanogenic compounds is presented. Particular attention is paid to determination in complex matrices. Chromatographic methods able to separate mixtures of closely related structures, such as glycosides of enantiomeric hydroxynitriles or lipids with slightly differing fatty acid spectra, are included, as are highly selective methods of detection, such as enzymic post-column cleavage combined with electrochemical detection as used in high-performance liquid chromatography. Details of thin-layer chromatography, including methods for detection, are given for both straight-phase and reverse-phase systems. The survey includes simple field methods as well as automated laboratory methods for the determination of 'free', 'bound' and 'total' cyanide, for example in processed food products from Manihot esculenta Cranz. Sources of enzymes are listed and attention is given to problems of sample storage and preparation. References are given to review articles which include data from methods such as ultraviolet, infrared and proton and carbon-13 nuclear magnetic resonance spectroscopies.

Chemistry Techniques, Analytical↗

PubMed1980

Chemical and hematological screening in patients with malignant and non-malignant conditions.

The value of multiphasic automated laboratory tests in differentiating stages of malignancy and in detecting recurrence of cancer was studied. Chemical SMA-12 tests (including calcium, phosphate, glucose, BUN, uric acid, cholesterol, total protein, albumin, total bilirubin, alkaline phosphatase (AP), lactic dehydrogenase (LDH) and SGOT) and hematological SMA-4 tests (including hematocrit, hemoglobin, WBC and RBC counts) were performed in 1,578 patients. Baseline data obtained from 582 patients with benign, and 996 patients with malignant, lesions were subdivided according to age, sex, primary site, and extent of cancer. With advancing stages of cancer, there was a significant increase of AP and LDH, but decrease of albumin and hemoglobin. Among patients who had sequential tests, there was a significant decrease of uric acid and LDH with control of cancer, and increase of AP with development of distant dissemination.

Adult↗

PubMed2000

Hydrophilicity of Polar and Apolar Domains of Amphiphiles.

The hydrophilicity of polar and apolar domains of various amphiphiles was systematically estimated for their homologues and analogues by measuring the molar adiabatic compressibility of an aqueous solution at infinite dilution. The homologues of protic alkyl H(CH(2))(n)-, perfluoroalkyl F(CF(2))(n)-, and alkylphenyl H(CH(2))(n)(C(6)H(5))- groups (n=0-10) were chosen to represent apolar hydrophobic domains. The polar hydrophilic domains tested were -SO(4)Na, -SO(3)Na, -COONH(4), -N(CH(3))(3)Br, N(C(m)H(2m+1))(4)Br (m=1-5), and -NH(CH(2))(n)SO(3) (n=3, 4) groups. Also tested were the tetraphenyl ionic compounds (C(6)H(5))(4)MX (M=B/X=Na, M=P/X=Cl, M=As/X=Cl) to study the effect of the ionic sign of the core atom across the tetraphenyl apolar shell, the polyethylene glycols H(OCH(2)CH(2))(m)OH (m=1-4) to study the role of apolar -CH(2)- units in the hydrophilic oxyethylene group, and the zwitterionic dimethylaminoalkylsulfonate (CH(3))(2)NH(CH(2))(n)SO(3) homologues to study the effect of intramolecular salt formation on the hydrophilicity of the zwitterion. The adiabatic compressibility of the solution was calculated from measurement of the sound velocity and density of solutions. The introduction of laboratory automation and the numerical control of the system improved the accuracies and efficiencies of the measurements a great deal. The range of the temperature scan was 0-40 degrees C with an effective accuracy of +/-0.001 degrees C and the concentration was automatically scanned down to far below the cmc of the surfactant. The hydrophilicity of various polar and apolar substances was estimated as the decrease of molar adiabatic compressibility of the aqueous solution with increased concentration of their homologues and analogues. The hydrophobic hydration of nonpolar substances was found to be very small at room temperature and was barely detected above 40 degrees C; however, it became large as the temperature was lowered and attained a maximum at 0 degrees C. The cationic charge of quaternary ammonium N(+)(C(n)H(2n+1))(4) was found to enhance the hydrophobic hydration of methylene groups located at a distance of 4 to 6 Å from the core nitrogen atom, while the terminal negative charge of the anionic surfactant R-SO(4)(-), R-SO(3)(-), or R-COO(-) was found to decrease the hydrophobic hydration of -CH(2)- units within the same range. The hydrophilicity of quaternary ammonium and the tetraphenyl ions should be synergistically given by both hydrophobic and ionic hydrations. The hydrophilicity of the perfluoromethylene unit -CF(2)- was found to have a value comparable to that of the protic methylene unit -CH(2)-. The hydrophobic hydration seems to offer a good measure of the hydrophilicity of apolar substances; however, it does not necessarily represent the "hydrophobicity" of the apolar segment when the "surface activity" of the amphiphile is concerned. Copyright 2000 Academic Press.

Journal Article↗

PubMed2002

Potassium channels: gene family, therapeutic relevance, high-throughput screening technologies and drug discovery.

Existing drugs that modulate ion channels represent a key class of pharmaceutical agents across many therapeutic areas and there is considerable further potential for potassium channel drug discovery. Potassium channels represent the largest and most diverse sub-group of ion channels and they play a central role in regulating the membrane potential of cells. Recent advances in genomics have greatly added to the number of these potential drug targets, but selecting a suitable potassium channel for drug discovery research is a key step. In particular, the potential therapeutic relevance of a potassium channel should be taken into account when selecting a target for screening. Potassium channel drug discovery is being driven by a need to identify lead compounds that can provide tractable starting points for medicinal chemistry. Furthermore, advances in laboratory automation have brought significant opportunities to increase screening throughput for potassium channel assays, but careful assay configuration to model drug-target interactions in a physiological manner is an essential consideration. Several potassium channel screening platforms are described in this review in order to provide some insight into the variety of formats available for screening, together with some of their inherent advantages and limitations. Particular emphasis is placed on the mechanistic basis of drug-target interaction and those aspects of structure/function that are of prime importance in potassium channel drug discovery.

Animals↗

PubMed2006

High throughput screening in drug discovery.

Drug discovery is a highly complex and multidisciplinary process which goal is to identify new antitumoral drugs. The screening attrition rate in the current drug discovery protocols suggests that one marketable drug emerges from approximately one million screened compounds. This leads to pressure to screen larger libraries in order to continue the pipeline and to the development of High Throughput Screening. HTS is only a name for specific developments in laboratory automation to collect a large amount of experimental data in a relatively short time. HTS can test hundreds of thousands of compounds per day, however, if fewer compounds could be tested without compromising the probability of success, the cost and time would be greatly reduced. To that end, new developments in large-scale cell biology and compound library design in silico have evolved to obtain data with higher predictability of clinical efficacy.

Animals↗

PubMed1978

Expanding indications for early parathyroidectomy in the elderly female.

In a study of fifty consecutive patients with hyperparathyroidism we have noted: (1) Automated laboratory studies have increased the incidence of diagnosed hyperparathyroidism, especially in the elderly. (2) Bone abnormalities and mental changes are the most frequent symptoms in this older population. (3) Natural history studies have produced some delay in surgical treatment, accentuating mental aberrations and bone-associated deficits. Especially in the elderly, the benefits in personality change, feeling of well-being, and lessened bone deterioration warrant early operation.

Aged↗

PubMed1978

[Autonomic determination of proteins from urine by centrifugal and continuous flow analyses (author's transl)].

A precipitating reagent comprising partially neutralized trichloroacetic acid with magnesium sulfate, sodium dodecyl sulfate and polyethylene glycol is used for automatic determination of proteins from urine by centrifugal analysis and continuous flow analysis. We compare and discuss the results of both methods, their limits of validity and incorporation in an automated laboratory.

Albuminuria↗

PubMed1992

Validation of the simultaneous determination of methylprednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure was developed that accurately quantitates methylprednisolone (MP) and methylprednisolone acetate (MPA) in human plasma over the range 2.00-50.0 ng/ml. The internal standard, fluorometholone, was added to an aliquot of sodium fluoride-potassium oxalate-derived plasma. Samples were prewashed with hexane and extracted twice with methylene chloride. The extracts were dried with anhydrous sodium sulfate, centrifuged, and the organic layer separated and dried under nitrogen. The samples were reconstituted in mobile phase and washed an additional time with hexane before 100 microliters were injected onto a Beckman/Altex Ultrasphere ODS column with ultraviolet absorbance detection at 254 nm. Composition of the mobile phase was acetonitrile-water-glacial acetic acid (33:62:5, v/v/v). Calibration curves were obtained by unweighted, linear regression of peak-height ratios of MP (or MPA)/internal standard versus theoretical concentrations of MP or MPA using a Hewlett-Packard 3357 Laboratory Automation System. Extraction efficiencies for MP and MPA over the linear range were 86.4 and 84.7%, respectively. This method was successfully implemented for the analysis of specimens generated from a single-dose bioavailability and safety study for a new formulation of Depo-Medrol sterile aqueous suspension.

Chromatography, High Pressure Liquid↗

PubMed1988

Laboratory Information Management Systems--part II. Implementation.

In this, the second of two articles on Laboratory Information Management Systems (LIMS), the stages of the acquisition of a system are discussed. First, the laboratory automation strategy is developed leading to the writing of the requirements specification sent to prospective suppliers. The next step, in conjunction with the chosen supplier, is to write the functional and systems specifications from which the LIMS will be tailored. Once installed the LIMS must be validated and in the event of hardware or software changes, should undergo partial or full re-validation. The education and training of users, and operational considerations are presented before concluding with possible developments of LIMS in the future.

Journal Article↗

PubMed2005

Towards a nucleic acid-based diagnosis in clinical parasitology and mycology.

BACKGROUND: Multiple in-house polymerase chain reaction (PCR) assays for the diagnosis of parasitic and fungal diseases have been reported. Encouraging results have been published to anticipate or improve the diagnosis. However, the absence of standardized methods has led to discrepant results. As a consequence, these tests are not recognized as consensual diagnostic criteria. METHODS: The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, the quantitative results provided by these techniques allow to compare rapidly the efficiency of primers, probes, and DNA extraction. Therefore, one can expect a more consensual method to implement comparisons between laboratories. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, the meaning of detecting nucleic acids in a given clinical sample still needs to be defined. This requires well-designed studies with clinical consensual criteria and PCR techniques that are as similar as possible. CONCLUSIONS: The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for decision-making.

Animals↗

PubMed2006

Head to head comparison of N-terminal pro-B-type natriuretic peptide and B-type natriuretic peptide in patients with/without left ventricular systolic dysfunction.

BACKGROUND: Human pro-B-type natriuretic peptide is cleaved into the active B-type natriuretic peptide (BNP) and the inactive fragment NT-proBNP. It is unclear if, similar to BNP, NT-proBNP can be used as a marker of impaired left ventricular (LV) ejection fraction (EF). This study evaluated the analytical performance of both assays to detect LV systolic dysfunction. METHODS: In 72 patients with various degrees of left ventricular systolic dysfunction (LVSD), blood analysis for BNP and NT-proBNP was performed prior to cardiac catheterization, using a point-of-care analyzer (Biosite) and a fully automated laboratory analyzer (Roche-Elecsys), respectively. The within-run and between-run imprecision for BNP and NT-proBNP was calculated. RESULTS: Both markers were able to detect impaired LV EF with the largest area under the receiver-operating-characteristic curve for NT-proBNP (NT-proBNP: 0.851 (0.747-0.924); BNP: 0.803 (0.692-0.887) 95% confidence interval; P = 0.07). A significant correlation was observed between BNP and NT-proBNP (r = 0.9; P < 0.0001). Estimating the within-run imprecision, the coefficient of variance for BNP was 3.14% (n = 20, mean 316 ng/L) to 3.32% (n = 20, mean 820 ng/L) and for NT-proBNP 0.9% (n = 20, mean 4390.8 ng/L) to 1.4% (n = 20, mean 225 ng/L). The between-run imprecision for NT-proBNP ranged between 2.1% (n = 20, mean 224.6 ng/L) and 2% (n = 20, mean 4391 ng/L). Optimal discriminator values for BNP and NT-proBNP were 139 ng/L and 358 ng/L, respectively. However, adjusting the BNP cut-off value to 54 ng/L improved the negative predictive value and sensitivity of the assay. CONCLUSION: Similar to BNP, NT-proBNP is a promising marker in identifying LVSD. Although both assays are reliable and have good analytical performance, their diagnostic cut-off value is dynamic and population-dependent. The slightly wider detection range and the more stable structure of NT-proBNP compared to the BNP assay suggest that NT-proBNP could play an additional role in the evaluation of patients with LV systolic dysfunction.

Adult↗

PubMed2005

Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets.

Forward Pharmacology seeks to identify small or large molecules that modulate a normal or abnormal biological process in living cells or whole organisms and historically has been responsible for the discovery of many clinically used drugs. Forward Pharmacology approaches have become particularly attractive because advances in combinatorial chemistry and laboratory automation have made it possible to generate and interrogate large compound collections in a short period of time. Because many drug discovery efforts are now directed against specific biochemical targets, however, the utility of Forward Pharmacology is limited by the fact that assays to investigate compounds in biological systems are often phenotypic rather than target specific. We discuss here a novel strategy to discover target-based small molecules in intact cells using contemporary Forward Pharmacology in cells with specific genetic manipulations. The method, which we have termed "chemical complementation", is defined as the ability of small molecules to reverse a genetically induced phenotypic change in intact cells. Chemical complementation represents an extension of the commonly used genetic complementation approach, where cDNA libraries are used to investigate the function of genes based on their ability to rescue a specific genetic defect. We present examples of how chemical complementation has been used to identify and credential cell-active, small molecule inhibitors of 2 dual-specificity phosphatases, Cdc25A and MKP-3, which heretofore have eluded small molecule drug discovery efforts.

Animals↗