Laboratory Assays for Epstein-Barr Virus-Related Disease

PubMed Central

Gulley, Margaret L.; Tang, Weihua

2008-01-01

Epstein-Barr virus (EBV) infects various cell types in a wide spectrum of benign and malignant diseases. Laboratory tests for EBV have improved and are increasingly used in diagnosis, prognosis, prediction, and prevention of diseases ranging from infectious mononucleosis to selected subtypes of lymphoma, sarcoma, and carcinoma. Indeed, the presence of EBV is among the most effective tumor markers supporting clinical management of cancer patients. In biopsies, localization of EBER transcripts by in situ hybridization remains the gold standard for identifying latent infection. Other RNA- and protein-based assays detect lytic viral replication and can distinguish carcinoma-derived from lymphocyte-derived EBV in saliva or nasopharyngeal brushings. Analysis of blood using EBV viral load and serology reflects disease status and risk of progression. This review summarizes prior research in the context of basic virologic principles to provide a rational strategy for applying and interpreting EBV tests in various clinical settings. Such assays have been incorporated into standard clinical practice in selected settings such as diagnosis of primary infection and management of patients with immune dysfunction or nasopharyngeal carcinoma. As novel therapies are developed that target virus-infected cells or overcome the adverse effects of infection, laboratory testing becomes even more critical for determining when intervention is appropriate and the extent to which it has succeeded. PMID:18556771

Missed detection of significant positive and negative shifts in gentamicin assay: implications for routine laboratory quality practices.

PubMed

Koerbin, Gus; Liu, Jiakai; Eigenstetter, Alex; Tan, Chin Hon; Badrick, Tony; Loh, Tze Ping

2018-02-15

A product recall was issued for the Roche/Hitachi Cobas Gentamicin II assays on 25 th May 2016 in Australia, after a 15 - 20% positive analytical shift was discovered. Laboratories were advised to employ the Thermo Fisher Gentamicin assay as an alternative. Following the reintroduction of the revised assay on 12 th September 2016, a second reagent recall was made on 20 th March 2017 after the discovery of a 20% negative analytical shift due to erroneous instrument adjustment factor. The practices of an index laboratory were examined to determine how the analytical shifts evaded detection by routine internal quality control (IQC) and external quality assurance (EQA) systems. The ability of the patient result-based approaches, including moving average (MovAvg) and moving sum of outliers (MovSO) approaches in detecting these shifts were examined. Internal quality control data of the index laboratory were acceptable prior to the product recall. The practice of adjusting IQC target following a change in assay method resulted in the missed negative shift when the revised Roche assay was reintroduced. While the EQA data of the Roche subgroup showed clear negative bias relative to other laboratory methods, the results were considered as possible 'matrix effect'. The MovAvg method detected the positive shift before the product recall. The MovSO did not detect the negative shift in the index laboratory but did so in another laboratory 5 days before the second product recall. There are gaps in current laboratory quality practices that leave room for analytical errors to evade detection.

Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.

PubMed

Mondal, Dinesh; Ghosh, Prakash; Khan, Md Anik Ashfaq; Hossain, Faria; Böhlken-Fascher, Susanne; Matlashewski, Greg; Kroeger, Axel; Olliaro, Piero; Abd El Wahed, Ahmed

2016-05-13

Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD. A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh. The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent. The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.

Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity.

PubMed

Sommer, Jurg M; Buyue, Yang; Bardan, Sara; Peters, Robert T; Jiang, Haiyan; Kamphaus, George D; Gray, Elaine; Pierce, Glenn F

2014-11-01

Due to variability in the one-stage clotting assay, the performance of new factor IX (FIX) products should be assessed in this assay. The objective of this field study was to evaluate the accuracy of measuring recombinant FIX Fc fusion protein (rFIXFc) activity in clinical haemostasis laboratories using the one-stage clotting assay. Human haemophilic donor plasma was spiked with rFIXFc or BeneFIX® at 0.80, 0.20, or 0.05 IU/ml based on label potency. Laboratories tested blinded samples using their routine one-stage assay and in-house FIX plasma standard. The mean spike recoveries for BeneFIX (n=30 laboratories) were 121 %, 144 %, and 168 % of expected at nominal 0.80, 0.20, and 0.05 IU/ml concentrations, respectively. Corresponding rFIXFc spike recoveries were 88 %, 107 %, and 132 % of expected, respectively. All BeneFIX concentrations were consistently overestimated by most laboratories. rFIXFc activity was reagent-dependent; ellagic acid and silica gave higher values than kaolin, which underestimated rFIXFc. BeneFIX demonstrated significantly reduced chromogenic assay activity relative to one-stage assay results and nominal activity, while rFIXFc activity was close to nominal activity at three concentrations with better dilution linearity than the typical one-stage assay. In conclusion, laboratory- and reagent-specific assay variabilities were revealed, with progressively higher variability at lower FIX concentrations. Non-parallelism against the FIX plasma standard was observed in all one-stage assays with rFIXFc and BeneFIX, leading to significant overestimation of FIX activity at lower levels and generally high inter-laboratory variability. Compared to the accuracy currently achieved in clinical laboratories when measuring other rFIX products, most laboratories measured rFIXFc activity with acceptable accuracy and reliability using routine one-stage assay methods and commercially available plasma standards.

Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

1975-01-01

A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

Measurements for 8 common analytes in native sera identify inadequate standardization among 6 routine laboratory assays.

PubMed

Stepman, Hedwig C M; Tiikkainen, Ulla; Stöckl, Dietmar; Vesper, Hubert W; Edwards, Selvin H; Laitinen, Harri; Pelanti, Jonna; Thienpont, Linda M

2014-06-01

External quality assessment (EQA) with commutable samples is essential for assessing the quality of assays performed by laboratories, particularly when the emphasis is on their standardization status and interchangeability of results. We used a panel of 20 fresh-frozen single-donation serum samples to assess assays for the measurement of creatinine, glucose, phosphate, uric acid, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. The commercial random access platforms included: Abbott Architect, Beckman Coulter AU, Ortho Vitros, Roche Cobas, Siemens Advia, and Thermo Scientific Konelab. The assessment was done at the peer group level and by comparison against the all-method trimmed mean or reference method values, where available. The considered quality indicators were intraassay imprecision, combined imprecision (including sample-matrix interference), bias, and total error. Fail/pass decisions were based on limits reflecting state-of-the-art performance, but also limits related to biological variation. Most assays showed excellent peer performance attributes, except for HDL- and LDL cholesterol. Cases in which individual assays had biases exceeding the used limits were the Siemens Advia creatinine (-4.2%), Ortho Vitros phosphate (8.9%), Beckman Coulter AU triglycerides (5.4%), and Thermo Scientific Konelab uric acid (6.4%), which lead to considerable interassay discrepancies. Additionally, large laboratory effects were observed that caused interlaboratory differences of >30%. The design of the EQA study was well suited for monitoring different quality attributes of assays performed in daily laboratory practice. There is a need for improvement, even for simple clinical chemistry analytes. In particular, the interchangeability of results remains jeopardized both by assay standardization issues and individual laboratory effects. © 2014 The American Association for Clinical Chemistry.

Mars Science Laboratory thermal control architecture

NASA Technical Reports Server (NTRS)

Bhandari, Pradeep; Birur, Gajanana; Pauken, Michael; Paris, Anthony; Novak, Keith; Prina, Mauro; Ramirez, Brenda; Bame, David

2005-01-01

The Mars Science Laboratory (MSL) mission to land a large rover on Mars is being planned for launch in 2009. This paper will describe the basic architecture of the thermal control system, the challenges and the methods used to overcome them by the use of an innovative architecture to maximize the use of heritage from past projects while meeting the requirements for the design.

Rapid laboratory investigation of the thermal properties of planetary analogues by using the EXTASE thermal probe.

NASA Astrophysics Data System (ADS)

Nadalini, R.; Extase Team

The thermal properties of the constituent materials of the upper meters of planets and planetary bodies are of extreme interest. During the design and the verification of various planetary missions, the need to model and test appropriate simulants in laboratory is often raised. To verify the thermal properties of deployed laboratory simulants, the EXTASE thermal probe is a fast, precise, and easy-to-use tool. EXTASE is a thermal profile probe, able to measure the temperature and inject heat into the selected material at 16 different locations along its 45cm long slender cylindrical body. It has been developed following the experience of MUPUS, with the purpose of observing such properties on Earth, in situ and in a short time. We have used EXTASE, under laboratory cold and standard conditions, on several sand mixtures, soils, granular and compact ices, under vacuum and at normal pressure levels, to collect a great number of time- and depth-dependent temperature curves that represent the thermal dynamical response of the material. At the same time, two independent models have been developed to verify the experimental results by reaching the same results with a simulation of the same process. The models, analytical and numerical, which account for all material parameters (conductivity, density, capacity), have been developed and fine tuned until their results are superposed to the experimental curves, thus allowing the determination of the distinct thermal properties. In addition, a test campaign is under planning to use EXTASE to determine, rapidly and efficiently, the thermal properties of various regolith simulants to be used in the simulation of planetary subsurface processes.

Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international inter-laboratory scoring exercise.

PubMed

Bolognesi, Claudia; Knasmueller, Siegfried; Nersesyan, Armen; Roggieri, Paola; Ceppi, Marcello; Bruzzone, Marco; Blaszczyk, Ewa; Mielzynska-Svach, Danuta; Milic, Mirta; Bonassi, Stefano; Benedetti, Danieli; Da Silva, Juliana; Toledo, Raphael; Salvadori, Daisy Maria Fávero; Groot de Restrepo, Helena; Filipic, Metka; Hercog, Klara; Aktas, Ayça; Burgaz, Sema; Kundi, Michael; Grummt, Tamara; Thomas, Philip; Hor, Maryam; Escudero-Fung, Maria; Holland, Nina; Fenech, Michael

2017-03-01

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions

Laboratory Procedures in Thermal Expansion and Viscosity of Liquids

ERIC Educational Resources Information Center

Dawson, Paul Dow

1974-01-01

Describes the laboratory procedures for the measurement of thermal expansion and viscosity of liquids. These experiments require inexpensive equipment and are suitable for secondary school physical science classes. (JR)

Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

PubMed Central

Crowther, Gregory J.; Napuli, Alberto J.; Thomas, Andrew P.; Chung, Diana J.; Kovzun, Kuzma V.; Leibly, David J.; Castaneda, Lisa J.; Bhandari, Janhavi; Damman, Christopher J.; Hui, Raymond; Hol, Wim G. J.; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Zhang, Zhongsheng; Fan, Erkang; Van Voorhis, Wesley C.

2010-01-01

In the last decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. Here we present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. We conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. PMID:19470714

Agreement between allergen-specific IgE assays and ensuing immunotherapy recommendations from four commercial laboratories in the USA

PubMed Central

Plant, Jon D; Neradelik, Moni B; Polissar, Nayak L; Fadok, Valerie A; Scott, Brian A

2014-01-01

Background Canine allergen-specific IgE assays in the USA are not subjected to an independent laboratory reliability monitoring programme. Hypothesis/Objectives The aim of this study was to evaluate the agreement of diagnostic results and treatment recommendations of four serum IgE assays commercially available in the USA. Methods Replicate serum samples from 10 atopic dogs were submitted to each of four laboratories for allergen-specific IgE assays (ACTT®, VARL Liquid Gold, ALLERCEPT® and Greer® Aller-g-complete®). The interlaboratory agreement of standard, regional panels and ensuing treatment recommendations were analysed with the kappa statistic (κ) to account for agreement that might occur merely by chance. Six comparisons of pairs of laboratories and overall agreement among laboratories were analysed for ungrouped allergens (as tested) and also with allergens grouped according to reported cross-reactivity and taxonomy. Results The overall chance-corrected agreement of the positive/negative test results for ungrouped and grouped allergens was slight (κ = 0.14 and 0.13, respectively). Subset analysis of the laboratory pair with the highest level of diagnostic agreement (κ = 0.36) found slight agreement (κ = 0.13) for ungrouped plants and fungi, but substantial agreement (κ = 0.71) for ungrouped mites. The overall agreement of the treatment recommendations was slight (κ = 0.11). Altogether, 85.1% of ungrouped allergen treatment recommendations were unique to one laboratory or another. Conclusions and clinical importance Our study indicated that the choice of IgE assay may have a major influence on the positive/negative results and ensuing treatment recommendations. PMID:24461034

Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

ERIC Educational Resources Information Center

Mascotti, David P.; Waner, Mark J.

2010-01-01

A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

Betrixaban: Impact on Routine and Specific Coagulation Assays-A Practical Laboratory Guide.

PubMed

Siriez, Romain; Evrard, Jonathan; Dogné, Jean-Michel; Pochet, Lionel; Gheldof, Damien; Chatelain, Bernard; Mullier, François; Douxfils, Jonathan

2018-06-11

 Betrixaban is a novel direct oral factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in adult patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anti-coagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced.  The aim of this study is to determine which coagulation assay(s) should be used to assess the impact of betrixaban on haemostasis and provide laboratory guidance for their interpretation.  Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/mL in platelet-poor plasma. Different reagents from several manufacturers were tested and the impact of betrixaban on pro-thrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russel viper venom time (dRVV-T), chromogenic anti-Xa assays, thrombin generation assay (TGA), and a large panel of haemostasis diagnostic tests has been assessed.  A concentration-dependent prolongation of aPTT, PT and dRVV-T is observed. The sensitivity mainly depends on the reagent. Chromogenic anti-Xa assays show high sensitivity depending on the reagent and/or the methodology. These assays applicable for other direct factor Xa inhibitors have to be adapted to obtain a relevant range of measurement. TGA may also be attractive to assess the anti-coagulant activity of betrixaban.  Adapted chromogenic anti-Xa assays are the most appropriate assays to estimate the concentration of betrixaban. Betrixaban significantly affects several haemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests. Schattauer GmbH Stuttgart.

Thermal-Structures and Materials Testing Laboratory

NASA Technical Reports Server (NTRS)

Teate, Anthony A.

1997-01-01

Since its inception and successful implementation in 1997 at James Madison University, the Thermal Structures and Materials Testing Laboratory (T-SaMTL) funded by the NASA Langley Research Center is evolving into one of the University's premier and exemplary efforts to increase minority representation in the sciences and mathematics. Serving ten (10) students and faculty directly and almost fifty (50) students indirectly, T-SAMTL, through its recruitment efforts, workshops, mentoring program, tutorial services and its research and computational laboratories has marked the completion of the first year with support from NASA totaling $ 100,000. Beginning as an innovative academic research and mentoring program for underrepresented minority science and mathematics students, the program now boasts a constituency which consists of 50% graduating seniors in the spring of 1998 with 50% planning to go to graduate school. The program's intent is to increase the number of underrepresented minorities who receive doctoral degrees in the sciences by initiating an academically enriched research program aimed at strengthening the academic and self actualization skills of undergraduate students with the potential to pursue doctoral study in the sciences. The program provides financial assistance, academic enrichment, and professional and personal development support for minority students who demonstrate the potential and strong desire to pursue careers in the sciences and mathematics. James Madison University was awarded the first $100,000, in April 1997, by The NASA Langley Research Center for establishment and support of its Thermal Structures and Materials Testing

Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I.

PubMed

Gustafson, A-L; Stedman, D B; Ball, J; Hillegass, J M; Flood, A; Zhang, C X; Panzica-Kelly, J; Cao, J; Coburn, A; Enright, B P; Tornesi, M B; Hetheridge, M; Augustine-Rauch, K A

2012-04-01

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60-70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish. Copyright © 2011 Elsevier Inc. All rights reserved.

Agreement between allergen-specific IgE assays and ensuing immunotherapy recommendations from four commercial laboratories in the USA.

PubMed

Plant, Jon D; Neradelik, Moni B; Polissar, Nayak L; Fadok, Valerie A; Scott, Brian A

2014-02-01

Canine allergen-specific IgE assays in the USA are not subjected to an independent laboratory reliability monitoring programme. The aim of this study was to evaluate the agreement of diagnostic results and treatment recommendations of four serum IgE assays commercially available in the USA. Replicate serum samples from 10 atopic dogs were submitted to each of four laboratories for allergen-specific IgE assays (ACTT(®) , VARL Liquid Gold, ALLERCEPT(®) and Greer(®) Aller-g-complete(®) ). The interlaboratory agreement of standard, regional panels and ensuing treatment recommendations were analysed with the kappa statistic (κ) to account for agreement that might occur merely by chance. Six comparisons of pairs of laboratories and overall agreement among laboratories were analysed for ungrouped allergens (as tested) and also with allergens grouped according to reported cross-reactivity and taxonomy. The overall chance-corrected agreement of the positive/negative test results for ungrouped and grouped allergens was slight (κ = 0.14 and 0.13, respectively). Subset analysis of the laboratory pair with the highest level of diagnostic agreement (κ = 0.36) found slight agreement (κ = 0.13) for ungrouped plants and fungi, but substantial agreement (κ = 0.71) for ungrouped mites. The overall agreement of the treatment recommendations was slight (κ = 0.11). Altogether, 85.1% of ungrouped allergen treatment recommendations were unique to one laboratory or another. Our study indicated that the choice of IgE assay may have a major influence on the positive/negative results and ensuing treatment recommendations. © 2014 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the ESVD and the ACVD.

Applicability of the DPPH assay for evaluating the antioxidant capacity of food additives - inter-laboratory evaluation study -.

PubMed

Shimamura, Tomoko; Sumikura, Yoshihiro; Yamazaki, Takeshi; Tada, Atsuko; Kashiwagi, Takehiro; Ishikawa, Hiroya; Matsui, Toshiro; Sugimoto, Naoki; Akiyama, Hiroshi; Ukeda, Hiroyuki

2014-01-01

An inter-laboratory evaluation study was conducted in order to evaluate the antioxidant capacity of food additives by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Four antioxidants used as existing food additives (i.e., tea extract, grape seed extract, enju extract, and d-α-tocopherol) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were used as analytical samples, and 14 laboratories participated in this study. The repeatability relative standard deviation (RSD(r)) of the IC50 of Trolox, four antioxidants, and the Trolox equivalent antioxidant capacity (TEAC) were 1.8-2.2%, 2.2-2.9%, and 2.1-2.5%, respectively. Thus, the proposed DPPH assay showed good performance within the same laboratory. The reproducibility relative standard deviation (RSD(R)) of IC50 of Trolox, four antioxidants, and TEAC were 4.0-7.9%, 6.0-11%, and 3.7-9.3%, respectively. The RSD(R)/RSD(r) values of TEAC were lower than, or nearly equal to, those of IC50 of the four antioxidants, suggesting that the use of TEAC was effective for reducing the variance among the laboratories. These results showed that the proposed DPPH assay could be used as a standard method to evaluate the antioxidant capacity of food additives.

Analytic Validation of Immunohistochemical Assays: A Comparison of Laboratory Practices Before and After Introduction of an Evidence-Based Guideline.

PubMed

Fitzgibbons, Patrick L; Goldsmith, Jeffrey D; Souers, Rhona J; Fatheree, Lisa A; Volmar, Keith E; Stuart, Lauren N; Nowak, Jan A; Astles, J Rex; Nakhleh, Raouf E

2017-09-01

- Laboratories must demonstrate analytic validity before any test can be used clinically, but studies have shown inconsistent practices in immunohistochemical assay validation. - To assess changes in immunohistochemistry analytic validation practices after publication of an evidence-based laboratory practice guideline. - A survey on current immunohistochemistry assay validation practices and on the awareness and adoption of a recently published guideline was sent to subscribers enrolled in one of 3 relevant College of American Pathologists proficiency testing programs and to additional nonsubscribing laboratories that perform immunohistochemical testing. The results were compared with an earlier survey of validation practices. - Analysis was based on responses from 1085 laboratories that perform immunohistochemical staining. Of 1057 responses, 65.4% (691) were aware of the guideline recommendations before this survey was sent and 79.9% (550 of 688) of those have already adopted some or all of the recommendations. Compared with the 2010 survey, a significant number of laboratories now have written validation procedures for both predictive and nonpredictive marker assays and specifications for the minimum numbers of cases needed for validation. There was also significant improvement in compliance with validation requirements, with 99% (100 of 102) having validated their most recently introduced predictive marker assay, compared with 74.9% (326 of 435) in 2010. The difficulty in finding validation cases for rare antigens and resource limitations were cited as the biggest challenges in implementing the guideline. - Dissemination of the 2014 evidence-based guideline validation practices had a positive impact on laboratory performance; some or all of the recommendations have been adopted by nearly 80% of respondents.

Can a Point-of-Care Troponin I Assay be as Good as a Central Laboratory Assay? A MIDAS Investigation

PubMed Central

Diercks, Deborah; Birkhahn, Robert; Singer, Adam J.; Hollander, Judd E.; Nowak, Richard; Safdar, Basmah; Miller, Chadwick D.; Peberdy, Mary; Counselman, Francis; Chandra, Abhinav; Kosowsky, Joshua; Neuenschwander, James; Schrock, Jon; Lee-Lewandrowski, Elizabeth; Arnold, William; Nagurney, John

2016-01-01

Background We aimed to compare the diagnostic accuracy of the Alere Triage Cardio3 Tropinin I (TnI) assay (Alere, Inc., USA) and the PathFast cTnI-II (Mitsubishi Chemical Medience Corporation, Japan) against the central laboratory assay Singulex Erenna TnI assay (Singulex, USA). Methods Using the Markers in the Diagnosis of Acute Coronary Syndromes (MIDAS) study population, we evaluated the ability of three different assays to identify patients with acute myocardial infarction (AMI). The MIDAS dataset, described elsewhere, is a prospective multicenter dataset of emergency department (ED) patients with suspected acute coronary syndrome (ACS) and a planned objective myocardial perfusion evaluation. Myocardial infarction (MI) was diagnosed by central adjudication. Results The C-statistic with 95% confidence intervals (CI) for diagnosing MI by using a common population (n=241) was 0.95 (0.91-0.99), 0.95 (0.91-0.99), and 0.93 (0.89-0.97) for the Triage, Singulex, and PathFast assays, respectively. Of samples with detectable troponin, the absolute values had high Pearson (RP) and Spearman (RS) correlations and were RP =0.94 and RS=0.94 for Triage vs Singulex, RP =0.93 and RS=0.85 for Triage vs PathFast, and RP =0.89 and RS=0.73 for PathFast vs Singulex. Conclusions In a single comparative population of ED patients with suspected ACS, the Triage Cardio3 TnI, PathFast, and Singulex TnI assays provided similar diagnostic performance for MI. PMID:27374704

Can a Point-of-Care Troponin I Assay be as Good as a Central Laboratory Assay? A MIDAS Investigation.

PubMed

Peacock, W Frank; Diercks, Deborah; Birkhahn, Robert; Singer, Adam J; Hollander, Judd E; Nowak, Richard; Safdar, Basmah; Miller, Chadwick D; Peberdy, Mary; Counselman, Francis; Chandra, Abhinav; Kosowsky, Joshua; Neuenschwander, James; Schrock, Jon; Lee-Lewandrowski, Elizabeth; Arnold, William; Nagurney, John

2016-09-01

We aimed to compare the diagnostic accuracy of the Alere Triage Cardio3 Tropinin I (TnI) assay (Alere, Inc., USA) and the PathFast cTnI-II (Mitsubishi Chemical Medience Corporation, Japan) against the central laboratory assay Singulex Erenna TnI assay (Singulex, USA). Using the Markers in the Diagnosis of Acute Coronary Syndromes (MIDAS) study population, we evaluated the ability of three different assays to identify patients with acute myocardial infarction (AMI). The MIDAS dataset, described elsewhere, is a prospective multicenter dataset of emergency department (ED) patients with suspected acute coronary syndrome (ACS) and a planned objective myocardial perfusion evaluation. Myocardial infarction (MI) was diagnosed by central adjudication. The C-statistic with 95% confidence intervals (CI) for diagnosing MI by using a common population (n=241) was 0.95 (0.91-0.99), 0.95 (0.91-0.99), and 0.93 (0.89-0.97) for the Triage, Singulex, and PathFast assays, respectively. Of samples with detectable troponin, the absolute values had high Pearson (R(P)) and Spearman (R(S)) correlations and were R(P)=0.94 and R(S)=0.94 for Triage vs Singulex, R(P)=0.93 and R(S)=0.85 for Triage vs PathFast, and R(P)=0.89 and R(S)=0.73 for PathFast vs Singulex. In a single comparative population of ED patients with suspected ACS, the Triage Cardio3 TnI, PathFast, and Singulex TnI assays provided similar diagnostic performance for MI.

Determination of serum hCG levels by radioreactor assay in the clinical laboratory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyko, W.L.

1979-09-01

The radioreceptorassay (RRA) has been used for measuring human chorionic gonadotropin (hCG) in sera from 751 individuals. The RRA is shown to be sensitive (98%) and specific (99.8%) in detecting hCG in a wide variety of conditions, including normal pregnancy and threatened or missed abortions. As a rapid qualitative or semiquantitative assay for hCG, the RRA is a valuable adjunct in the laboratory to less sensitive tests for hCG. Variation among different quantitative assays for hCG is examined, and it is concluded that the same assay system should be used for monitoring hCG levels in a single individual over amore » period of time in order to avoid inconsistent results. Application of the quantitative RRA for hCG in detecting the midcycle luteinizing hormone surge in infertillity is also presented.« less

Differential sensitivity of von Willebrand factor (VWF) 'activity' assays to large and small VWF molecular weight forms: a cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays.

PubMed

Favaloro, E J; Bonar, R; Chapman, K; Meiring, M; Funk Adcock, D

2012-06-01

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura. © 2012 International Society on Thrombosis and Haemostasis.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

PubMed

Reus, Astrid A; Reisinger, Kerstin; Downs, Thomas R; Carr, Gregory J; Zeller, Andreas; Corvi, Raffaella; Krul, Cyrille A M; Pfuhler, Stefan

2013-11-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a

Comet assay in reconstructed 3D human epidermal skin models—investigation of intra- and inter-laboratory reproducibility with coded chemicals

PubMed Central

Pfuhler, Stefan

2013-01-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a

Laboratories | NREL

Science.gov Websites

| Z A Accelerated Exposure Testing Laboratory Advanced Optical Materials Laboratory Advanced Thermal Laboratory Structural Testing Laboratory Surface Analysis Laboratory Systems Performance Laboratory T Thermal Storage Materials Laboratory Thermal Storage Process and Components Laboratory Thin-Film Deposition

Protein–ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grøftehauge, Morten K., E-mail: m.k.groftehauge@durham.ac.uk; Hajizadeh, Nelly R.; Swann, Marcus J.

2015-01-01

The biophysical characterization of protein–ligand interactions in solution using techniques such as thermal shift assay, or on surfaces using, for example, dual polarization interferometry, plays an increasingly important role in complementing crystal structure determinations. Over the last decades, a wide range of biophysical techniques investigating protein–ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmonmore » resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.« less

New Laboratory Observations of Thermal Pressurization Weakening

NASA Astrophysics Data System (ADS)

Badt, N.; Tullis, T. E.; Hirth, G.

2017-12-01

Dynamic frictional weakening due to pore fluid thermal pressurization has been studied under elevated confining pressure in the laboratory, using a rotary-shear apparatus having a sample with independent pore pressure and confining pressure systems. Thermal pressurization is directly controlled by the permeability of the rocks, not only for the initiation of high-speed frictional weakening but also for a subsequent sequence of high-speed sliding events. First, the permeability is evaluated at different effective pressures using a method where the pore pressure drop and the flow-through rate are compared using Darcy's Law as well as a pore fluid oscillation method, the latter method also permitting measurement of the storage capacity. Then, the samples undergo a series of high-speed frictional sliding segments at a velocity of 2.5 mm/s, under an applied confining pressure and normal stress of 45 MPa and 50 MPa, respectively, and an initial pore pressure of 25 MPa. Finally the rock permeability and storage capacity are measured again to assess the evolution of the rock's pore fluid properties. For samples with a permeability of 10-20 m2 thermal pressurization promotes a 40% decrease in strength. However, after a sequence of three high-speed sliding events, the magnitude of weakening diminishes progressively from 40% to 15%. The weakening events coincide with dilation of the sliding interface. Moreover, the decrease in the weakening degree with progressive fast-slip events suggest that the hydraulic diffusivity may increase locally near the sliding interface during thermal pressurization-enhanced slip. This could result from stress- or thermally-induced damage to the host rock, which would perhaps increase both permeability and storage capacity, and so possibly decrease the susceptibility of dynamic weakening due to thermal pressurization in subsequent high-speed sliding events.

An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format.

PubMed

Bergsdorf, Christian; Fiez-Vandal, Cédric; Sykes, David A; Bernet, Pascal; Aussenac, Sonia; Charlton, Steven J; Schopfer, Ulrich; Ottl, Johannes; Duckely, Myriam

2016-03-01

Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners. © 2015 Society for Laboratory Automation and Screening.

Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators.

PubMed

Onoue, Satomi; Hosoi, Kazuhiro; Toda, Tsuguto; Takagi, Hironori; Osaki, Naoto; Matsumoto, Yasuhiro; Kawakami, Satoru; Wakuri, Shinobu; Iwase, Yumiko; Yamamoto, Toshinobu; Nakamura, Kazuichi; Ohno, Yasuo; Kojima, Hajime

2014-06-01

A previous multi-center validation study demonstrated high transferability and reliability of reactive oxygen species (ROS) assay for photosafety evaluation. The present validation study was undertaken to verify further the applicability of different solar simulators and assay performance. In 7 participating laboratories, 2 standards and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals, were assessed by the ROS assay using two different solar simulators (Atlas Suntest CPS series, 3 labs; and Seric SXL-2500V2, 4 labs). Irradiation conditions could be optimized using quinine and sulisobenzone as positive and negative standards to offer consistent assay outcomes. In both solar simulators, the intra- and inter-day precisions (coefficient of variation; CV) for quinine were found to be below 10%. The inter-laboratory CV for quinine averaged 15.4% (Atlas Suntest CPS) and 13.2% (Seric SXL-2500V2) for singlet oxygen and 17.0% (Atlas Suntest CPS) and 7.1% (Seric SXL-2500V2) for superoxide, suggesting high inter-laboratory reproducibility even though different solar simulators were employed for the ROS assay. In the ROS assay on 42 coded chemicals, some chemicals (ca. 19-29%) were unevaluable because of limited solubility and spectral interference. Although several false positives appeared with positive predictivity of ca. 76-92% (Atlas Suntest CPS) and ca. 75-84% (Seric SXL-2500V2), there were no false negative predictions in both solar simulators. A multi-center validation study on the ROS assay demonstrated satisfactory transferability, accuracy, precision, and predictivity, as well as the availability of other solar simulators. Copyright © 2013 Elsevier Ltd. All rights reserved.

International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

PubMed Central

Engelhardt, Othmar G.; Wood, John; Heath, Alan; Katz, Jacqueline M.; Peiris, Malik; Hoschler, Katja; Hungnes, Olav; Zhang, Wenqing; Van Kerkhove, Maria D.

2015-01-01

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future. PMID:26108286

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

NASA Astrophysics Data System (ADS)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

Femtomole-Scale High-Throughput Screening of Protein Ligands with Droplet-Based Thermal Shift Assay.

PubMed

Liu, Wen-Wen; Zhu, Ying; Fang, Qun

2017-06-20

There is a great demand to measure protein-ligand interactions in rapid and low cost way. Here, we developed a microfluidic droplet-based thermal shift assay (dTSA) system for high-throughput screening of small-molecule protein ligands. The system is composed of a nanoliter droplet array chip, a microfluidic droplet robot, and a real-time fluorescence detection system. Total 324 assays could be performed in parallel in a single chip with an 18 × 18 droplet array. The consumption of dTSA for each protein or ligand sample was only 5 nL (femtomole scale), which is significantly reduced by over 3 orders of magnitude compared with those in 96- or 384-well plate-based systems. We also observed the implementation of TSA in nanoliter droplet format could substantially improve assay precision with relative standard deviation (RSD) of 0.2% (n = 50), which can be ascribed to the enhanced thermal conduction in small volume reactors. The dTSA system was optimized by studying the effect of droplet volumes, as well as protein and fluorescent dye (SYPRO Orange) concentrations. To demonstrate its potential in drug discovery, we applied the dTSA system in screening inhibitors of human thrombin with a commercial library containing 100 different small molecule compounds, and two inhibitors were successfully identified and confirmed.

Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

PubMed

Hasan, Mohammad R; Tan, Rusung; Al-Rawahi, Ghada; Thomas, Eva; Tilley, Peter

2016-08-01

Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

USGS Publications Warehouse

Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; ,; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.

2013-01-01

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

BOW SHOCK FRAGMENTATION DRIVEN BY A THERMAL INSTABILITY IN LABORATORY ASTROPHYSICS EXPERIMENTS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki-Vidal, F.; Lebedev, S. V.; Pickworth, L. A.

The role of radiative cooling during the evolution of a bow shock was studied in laboratory-astrophysics experiments that are scalable to bow shocks present in jets from young stellar objects. The laboratory bow shock is formed during the collision of two counterstreaming, supersonic plasma jets produced by an opposing pair of radial foil Z-pinches driven by the current pulse from the MAGPIE pulsed-power generator. The jets have different flow velocities in the laboratory frame, and the experiments are driven over many times the characteristic cooling timescale. The initially smooth bow shock rapidly develops small-scale nonuniformities over temporal and spatial scalesmore » that are consistent with a thermal instability triggered by strong radiative cooling in the shock. The growth of these perturbations eventually results in a global fragmentation of the bow shock front. The formation of a thermal instability is supported by analysis of the plasma cooling function calculated for the experimental conditions with the radiative packages ABAKO/RAPCAL.« less

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus.

PubMed

Tignon, Marylène; Gallardo, Carmina; Iscaro, Carmen; Hutet, Evelyne; Van der Stede, Yves; Kolbasov, Denis; De Mia, Gian Mario; Le Potier, Marie-Frédérique; Bishop, Richard P; Arias, Marisa; Koenen, Frank

2011-12-01

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV

A Thermal Management of Electronics Course and Laboratory for Undergraduates

ERIC Educational Resources Information Center

Okamoto, Nicole; Hsu, Tai-Ran; Bash, Cullen E.

2009-01-01

A novel thermal management of electronics course with an associated laboratory has been developed for mechanical, electrical, and computer engineering students. The lecture topics, term project, computer modeling project, and six associated experiments that were built from scratch are described. Over half of the course lectures as well as all lab…

Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories

PubMed Central

Levine, Gail L.; Sambor, Anna; Carter, Donald K.; Sato, Alicia; Kopycinski, Jakub; Hayes, Peter; Hahn, Bridget; Birungi, Josephine; Tarragona-Fiol, Tony; Wan, Hong; Randles, Mark; Cooper, Andrew Raxworthy; Ssemaganda, Aloysius; Clark, Lorna; Kaleebu, Pontiano; Self, Steven G.; Koup, Richard; Wood, Blake; McElrath, M. Juliana; Cox, Josephine H.; Hural, John; Gilmour, Jill

2010-01-01

Background The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates. Methods We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study. Findings Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study. Interpretation The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of

Method of assaying uranium with prompt fission and thermal neutron borehole logging adjusted by borehole physical characteristics

DOEpatents

Barnard, Ralston W.; Jensen, Dal H.

1982-01-01

Uranium formations are assayed by prompt fission neutron logging techniques. The uranium in the formation is proportional to the ratio of epithermal counts to thermal or eqithermal dieaway. Various calibration factors enhance the accuracy of the measurement.

Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice.

PubMed

Schmidt, Katja; Butt, Julia; Mauter, Petra; Vogel, Klaus; Erles-Kemna, Andrea; Pawlita, Michael; Nicklas, Werner

2017-01-01

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.

Performance of the TB-LAMP diagnostic assay in reference laboratories: Results from a multicentre study.

PubMed

Pham, Thu Hang; Peter, Jonathan; Mello, Fernanda C Q; Parraga, Tommy; Lan, Nguyen Thi Ngoc; Nabeta, Pamela; Valli, Eloise; Caceres, Tatiana; Dheda, Keertan; Dorman, Susan E; Hillemann, Doris; Gray, Christen M; Perkins, Mark D

2018-03-01

To evaluate the diagnostic performance of TB-LAMP, a manual molecular tuberculosis (TB) detection method, and provide comparison to the Xpert MTB/RIF assay. In a large multicentre study, two sputum samples were collected from participants with TB symptoms in reference laboratories in Peru, South Africa, Brazil, and Vietnam. Each sample was tested with TB-LAMP. The reference standard consisted of four direct smears, four cultures, and clinical and radiological findings. Individuals negative on conventional tests were followed up after 8 weeks. The Xpert MTB/RIF assay was performed on fresh or frozen samples as a molecular test comparison. A total of 1036 adults with suspected TB were enrolled. Among 375 culture-confirmed TB cases with 750 sputum samples, TB-LAMP detected 75.6% (95% confidence interval (CI) 71.8-79.4%), including 97.9% (95% CI 96.4-99.4%) of smear-positive TB samples and 46.6% (95% CI 40.6-52.7%) of smear-negative TB samples. Specificity in 477 culture-negative participants not treated for TB (954 sputum samples) was 98.7% (95% CI 97.9-99.6%). TB-LAMP test results were indeterminate in 0.3% of cases. TB-LAMP detects nearly all smear-positive and half of smear-negative TB cases and has a high specificity when performed in reference laboratories. Performance was similar to the Xpert MTB/RIF assay. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

PubMed

Pine, P S; Boedigheimer, M; Rosenzweig, B A; Turpaz, Y; He, Y D; Delenstarr, G; Ganter, B; Jarnagin, K; Jones, W D; Reid, L H; Thompson, K L

2008-11-01

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

PubMed

Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

2012-05-01

We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

Night Vision Laboratory Static Performance Model for Thermal Viewing Systems

DTIC Science & Technology

1975-04-01

Research and Development Technical Report f ECOM-� • i’.__1’=• =•NIGHT VISION LABORATORY STATIC PERFORMANCE MODEL 1 S1=• : FOR THERMAL VIEWING...resolvable temperature Infrared imaging Minimum detectable temperature1.Detection and recognition performance Night visi,-)n Noise equivalent temperature...modulation transfer function (MTF). The noise charactcristics are specified by the noise equivalent temper- ature difference (NE AT), The next sections

Using ATP-driven bioluminescence assay to monitor microbial safety in a contemporary human cadaver laboratory.

PubMed

Benninger, Brion; Maier, Thomas

2015-03-01

The objective of this study was to utilize a cost-effective method for assessing the levels of bacterial, yeast, and mold activity during a human dissection laboratory course. Nowadays, compliance with safety regulations is policed by institutions at higher standards than ever before. Fear of acquiring an unknown infection is one of the top concerns of professional healthcare students, and it provokes anti-laboratory anxiety. Human cadavers are not routinely tested for bacteria and viruses prior to embalming. Human anatomy dissecting rooms that house embalmed cadavers are normally cleaned after the dissected cadavers have been removed. There is no evidence that investigators have ever assessed bacterial and fungal activities using adenosine triphosphate (ATP)-driven bioluminescence assays. A literature search was conducted on texts, journals, and websites regarding bacterial, yeast, and mold activities in an active cadaver laboratory. Midway into a clinical anatomy course, ATP bioluminescence assays were used to swab various sites within the dissection room, including entrance and exiting door handles, water taps, cadaver tables, counter tops, imaging material, X-ray box switches, and the cadaver surfaces. The results demonstrated very low activities on cadaver tables, washing up areas, and exiting door handles. There was low activity on counter tops and X-ray boxes. There was medium activity on the entrance door handles. These findings suggest an inexpensive and accurate method for monitoring safety compliance and microbial activity. Students can feel confident and safe in the environment in which they work. © 2014 Wiley Periodicals, Inc.

Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

PubMed

Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L

2015-01-01

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

Thermal infrared remote sensing and Kirchhoff's law: 1. Laboratory measurements

NASA Technical Reports Server (NTRS)

Salisbury, J. W.; Wald, A.; Daria, D. M.

1993-01-01

Kirchoff's Law, as originally conceived, applies only to samples in thermal equilibrium with their surroundings. Most laboratory measurements of emissivity only approach this condition and it never applies in remote sensing applications. In particular, the background is often much cooler than the radiating sample, and this has led to a long controversy about the applicability of Kirchhoff's Law under such conditions. It has also led to field and laboratory measurement techniques that use some form of the 'emissivity box' approach, which surrounds the sample with a background as close as possible to the sample temperature. In our experiments, we have heated soil samples in air on a hot plate in the laboratory to a much higher temperature than the room temperature background. Spectral emissivity was measured, except the known emissivities of both the primary and secondary Christiansen features were used, instead of assuming an emissivity of unity at these wavelengths. The results from this investigation are discussed in brief.

CPTAC Assay Portal: a repository of targeted proteomic assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2014-06-27

To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigatorsmore » to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.« less

Adapting High-Throughput Screening Methods and Assays for Biocontainment Laboratories

PubMed Central

Tigabu, Bersabeh; White, E. Lucile; Bostwick, Robert; Tower, Nichole; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.; Noah, James W.

2015-01-01

Abstract High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories. PMID:25710545

Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

ERIC Educational Resources Information Center

Hicks, Katherine A.

2016-01-01

Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation 1

PubMed Central

Hardy, R. W. F.; Holsten, R. D.; Jackson, E. K.; Burns, R. C.

1968-01-01

The methodology, characteristics and application of the sensitive C2H2-C2H4 assay for N2 fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N2ase-catalyzed reduction of C2H2 to C2H4, gas chromatographic isolation of C2H2 and C2H4, and quantitative measurement with a H2-flame analyzer. As little as 1 μμmole C2H4 can be detected, providing a sensitivity 103-fold greater than is possible with 15N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C2H2-reducing activity in the field. Applications to field samples included an evaluation of N2 fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg N2 fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N2 fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N2 fixing activity of soybeans. The validity of measuring N2 fixation in terms of C2H2 reduction was established through extensive comparisons of these activities using defined systems, including purified N2ase preparations and pure cultures of N2-fixing bacteria. With this assay it now becomes possible and practicable to conduct comprehensive surveys of N2 fixation, to make detailed comparisons among different N2-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N2 fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N2

NCL-01: Nanomedicine Drug Release Study in Human Plasma Using Stable Isotope Tracer Ultrafiltration Assay (SITUA)  | Frederick National Laboratory for Cancer Research

Cancer.gov

The Nanotechnology Characterization Laboratory will evaluate drug release from a nanoparticulate formulation in vitro in human plasma, using a novel stable isotope tracer ultrafiltration assay (SITUA) developed at the laboratory. The SITUA is a metho

University Physics Students' Ideas of Thermal Radiation Expressed in Open Laboratory Activities Using Infrared Cameras

ERIC Educational Resources Information Center

Haglund, Jesper; Melander, Emil; Weiszflog, Matthias; Andersson, Staffan

2017-01-01

Background: University physics students were engaged in open-ended thermodynamics laboratory activities with a focus on understanding a chosen phenomenon or the principle of laboratory apparatus, such as thermal radiation and a heat pump. Students had access to handheld infrared (IR) cameras for their investigations. Purpose: The purpose of the…

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

PubMed Central

Zemtsova, Galina E.; Montgomery, Merrill; Levin, Michael L.

2015-01-01

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays. PMID:25607846

A laboratory-scale pretreatment and hydrolysis assay for determination of reactivity in cellulosic biomass feedstocks.

PubMed

Wolfrum, Edward J; Ness, Ryan M; Nagle, Nicholas J; Peterson, Darren J; Scarlata, Christopher J

2013-11-14

The rapid determination of the release of structural sugars from biomass feedstocks is an important enabling technology for the development of cellulosic biofuels. An assay that is used to determine sugar release for large numbers of samples must be robust, rapid, and easy to perform, and must use modest amounts of the samples to be tested.In this work we present a laboratory-scale combined pretreatment and saccharification assay that can be used as a biomass feedstock screening tool. The assay uses a commercially available automated solvent extraction system for pretreatment followed by a small-scale enzymatic hydrolysis step. The assay allows multiple samples to be screened simultaneously, and uses only ~3 g of biomass per sample. If the composition of the biomass sample is known, the results of the assay can be expressed as reactivity (fraction of structural carbohydrate present in the biomass sample released as monomeric sugars). We first present pretreatment and enzymatic hydrolysis experiments on a set of representative biomass feedstock samples (corn stover, poplar, sorghum, switchgrass) in order to put the assay in context, and then show the results of the assay applied to approximately 150 different feedstock samples covering 5 different materials. From the compositional analysis data we identify a positive correlation between lignin and structural carbohydrates, and from the reactivity data we identify a negative correlation between both carbohydrate and lignin content and total reactivity. The negative correlation between lignin content and total reactivity suggests that lignin may interfere with sugar release, or that more mature samples (with higher structural sugars) may have more recalcitrant lignin. The assay presented in this work provides a robust and straightforward method to measure the sugar release after pretreatment and saccharification that can be used as a biomass feedstock screening tool. We demonstrated the utility of the assay by

Toward a microfluidic-based rapid amylase assay system.

PubMed

Holmes, Richard J; Summersgil, Philip; Ryan, Timothy; Brown, Bernard J Treves; Mockbil, Amal; Grieve, Bruce D; Fielden, Peter R

2009-08-01

This article describes work into a prototype system for the assay of amylase, using microfludic technologies. The new system has a significantly shorter cycle time than the current laboratory methods, which generally use microtitre plates, yet is capable of generating significantly superior results. As such, we have shown that sensitivity is enhanced by a factor of 10 in the standard assay trials, and by a factor of 2 in the real-sample lab trials. In both assays, the use of a microreactor system reduced the reaction time by a factor of 6.2, from 20 min incubation to 3.2 min. Basing the conclusion on the Megazyme Cerealpha Standard Method, and using the Cerealpha units as a measure of assay efficiency, the typical response for the microfluidic assay was shown to be 1.0 x 10(-3) CU/mL (standard deviation [SD] 2.5 x 10(-4) CU/mL), compared to 2.56 x 10(-4) CU/mL (SD 5.94 x 10(-5) CU/mL) for the standard macroassay. It is believed that this improvement in the reaction schematics is due to the inherent advantages of microfluidic devices such as superior mixing, higher thermal efficiency, and enhanced reaction kinetics.

Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment

ERIC Educational Resources Information Center

Johnson, R. Jeremy; Savas, Christopher J.; Kartje, Zachary; Hoops, Geoffrey C.

2014-01-01

Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal…

Method of assaying uranium with prompt fission and thermal neutron borehole logging adjusted by borehole physical characteristics. [Patient application

DOEpatents

Barnard, R.W.; Jensen, D.H.

1980-11-05

Uranium formations are assayed by prompt fission neutron logging techniques. The uranium in the formation is proportional to the ratio of epithermal counts to thermal or epithermal dieaway. Various calibration factors enhance the accuracy of the measurement.

The transient divided bar method for laboratory measurements of thermal properties

NASA Astrophysics Data System (ADS)

Bording, Thue S.; Nielsen, Søren B.; Balling, Niels

2016-12-01

Accurate information on thermal conductivity and thermal diffusivity of materials is of central importance in relation to geoscience and engineering problems involving the transfer of heat. Several methods, including the classical divided bar technique, are available for laboratory measurements of thermal conductivity, but much fewer for thermal diffusivity. We have generalized the divided bar technique to the transient case in which thermal conductivity, volumetric heat capacity and thereby also thermal diffusivity are measured simultaneously. As the density of samples is easily determined independently, specific heat capacity can also be determined. The finite element formulation provides a flexible forward solution for heat transfer across the bar, and thermal properties are estimated by inverse Monte Carlo modelling. This methodology enables a proper quantification of experimental uncertainties on measured thermal properties and information on their origin. The developed methodology was applied to various materials, including a standard ceramic material and different rock samples, and measuring results were compared with results applying traditional steady-state divided bar and an independent line-source method. All measurements show highly consistent results and with excellent reproducibility and high accuracy. For conductivity the obtained uncertainty is typically 1-3 per cent, and for diffusivity uncertainty may be reduced to about 3-5 per cent. The main uncertainty originates from the presence of thermal contact resistance associated with the internal interfaces in the bar. These are not resolved during inversion and it is imperative that they are minimized. The proposed procedure is simple and may quite easily be implemented to the many steady-state divided bar systems in operation. A thermally controlled bath, as applied here, may not be needed. Simpler systems, such as applying temperature-controlled water directly from a tap, may also be applied.

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources

USGS Publications Warehouse

Sinigalliano, Christopher D.; Ervin, Jared S.; Van De Werfhorst, Laurie C.; Badgley, Brian D.; Ballestée, Elisenda; Bartkowiaka, Jakob; Boehm, Alexandria B.; Byappanahalli, Muruleedhara N.; Goodwin, Kelly D.; Gourmelon, Michèle; Griffith, John; Holden, Patricia A.; Jay, Jenny; Layton, Blythe; Lee, Cheonghoon; Lee, Jiyoung; Meijer, Wim G.; Noble, Rachel; Raith, Meredith; Ryu, Hodon; Sadowsky, Michael J.; Schriewer, Alexander; Wang, Dan; Wanless, David; Whitman, Richard; Wuertz, Stefan; Santo Domingo, Jorge W.

2013-01-01

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR® Green qPCR method, and two TaqMan® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.

Transitioning cytomegalovirus viral load testing from a laboratory developed test to the cobas® CMV quantitative nucleic acid assay.

PubMed

Payne, Michael; Merrick, Linda; Lawson, Tanya; Ritchie, Gordon; Lowe, Christopher

2018-04-16

Commutability between human cytomegalovirus (CMV) viral load assays (VLA) is poor, despite the development of a WHO CMV International Standard (CMV IS). We evaluated a new CMV VLA, cobas ® CMV, as compared to our current laboratory developed CMV VLA (LDT), for clinical use. Both the LDT and cobas ® CMV were run in parallel for 109 patient samples. In addition, 104 replicates, over 8 dilutions, of the CMV IS were tested. Conversion factors and correlation between the two assays were calculated. The correlation coefficient between the LDT and cobas ® CMV was 0.91 for patient samples. The Bland-Altman graph displayed a systematic bias of +0.31 log 10 for the cobas ® CMV as compared to the LDT. The bias was greater for lower CMV viral loads. This increase in CMV viral loads was not seen with testing of the CMV IS dilutions by both the LDT and cobas ® CMV. CMV VLA inter-assay commutability continues to be an issue when switching CMV testing platforms and requires communication between the laboratory and clinicians during the transition period to prevent misinterpretation of results. © 2018 Wiley Periodicals, Inc.

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

PubMed Central

2012-01-01

Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either

Thermal Performance of the Mars Science Laboratory Rover During Mars Surface Operations

NASA Technical Reports Server (NTRS)

Novak, Keith S.; Kempenaar, Joshua E.; Liu, Yuanming; Bhandari, Pradeep; Lee, Chern-Jiin

2013-01-01

On November 26, 2011, NASA launched a large (900 kg) rover as part of the Mars Science Laboratory (MSL) mission to Mars. Eight months later, on August 5, 2012, the MSL rover (Curiosity) successfully touched down on the surface of Mars. As of the writing of this paper, the rover had completed over 200 Sols of Mars surface operations in the Gale Crater landing site (4.5 deg S latitude). This paper describes the thermal performance of the MSL Rover during the early part of its two Earth-0.year (670 Sols) prime surface mission. Curiosity landed in Gale Crater during early Spring (Ls=151) in the Southern Hemisphere of Mars. This paper discusses the thermal performance of the rover from landing day (Sol 0) through Summer Solstice (Sol 197) and out to Sol 204. The rover surface thermal design performance was very close to pre-landing predictions. The very successful thermal design allowed a high level of operational power dissipation immediately after landing without overheating and required a minimal amount of survival heating. Early morning operations of cameras and actuators were aided by successful heating activities. MSL rover surface operations thermal experiences are discussed in this paper. Conclusions about the rover surface operations thermal performance are also presented.

Thermal Performance of the Mars Science Laboratory Rover During Mars Surface Operations

NASA Technical Reports Server (NTRS)

Novak, Keith S.; Kempenaar, Joshua E.; Liu, Yuanming; Bhandari, Pradeep; Lee, Chern-Jiin

2013-01-01

On November 26, 2011, NASA launched a large (900 kg) rover as part of the Mars Science Laboratory (MSL) mission to Mars. Eight months later, on August 5, 2012, the MSL rover (Curiosity) successfully touched down on the surface of Mars. As of the writing of this paper, the rover had completed over 200 Sols of Mars surface operations in the Gale Crater landing site (4.5 degrees South latitude). This paper describes the thermal performance of the MSL Rover during the early part of its two Earth-0.year (670 Sols) prime surface mission. Curiosity landed in Gale Crater during early Spring (Solar longitude=151) in the Southern Hemisphere of Mars. This paper discusses the thermal performance of the rover from landing day (Sol 0) through Summer Solstice (Sol 197) and out to Sol 204. The rover surface thermal design performance was very close to pre-landing predictions. The very successful thermal design allowed a high level of operational power dissipation immediately after landing without overheating and required a minimal amount of survival heating. Early morning operations of cameras and actuators were aided by successful heating activities. MSL rover surface operations thermal experiences are discussed in this paper. Conclusions about the rover surface operations thermal performance are also presented.

Thermal Analysis of a Disposable, Instrument-Free DNA Amplification Lab-on-a-Chip Platform.

PubMed

Pardy, Tamás; Rang, Toomas; Tulp, Indrek

2018-06-04

Novel second-generation rapid diagnostics based on nucleic acid amplification tests (NAAT) offer performance metrics on par with clinical laboratories in detecting infectious diseases at the point of care. The diagnostic assay is typically performed within a Lab-on-a-Chip (LoC) component with integrated temperature regulation. However, constraints on device dimensions, cost and power supply inherent with the device format apply to temperature regulation as well. Thermal analysis on simplified thermal models for the device can help overcome these barriers by speeding up thermal optimization. In this work, we perform experimental thermal analysis on the simplified thermal model for our instrument-free, single-use LoC NAAT platform. The system is evaluated further by finite element modelling. Steady-state as well as transient thermal analysis are performed to evaluate the performance of a self-regulating polymer resin heating element in the proposed device geometry. Reaction volumes in the target temperature range of the amplification reaction are estimated in the simulated model to assess compliance with assay requirements. Using the proposed methodology, we demonstrated our NAAT device concept capable of performing loop-mediated isothermal amplification in the 20⁻25 °C ambient temperature range with 32 min total assay time.

New Laboratory Technique to Determine Thermal Conductivity of Complex Regolith Simulants Under High Vacuum

NASA Astrophysics Data System (ADS)

Ryan, A. J.; Christensen, P. R.

2016-12-01

Laboratory measurements have been necessary to interpret thermal data of planetary surfaces for decades. We present a novel radiometric laboratory method to determine temperature-dependent thermal conductivity of complex regolith simulants under high vacuum and across a wide range of temperatures. Here, we present our laboratory method, strategy, and initial results. This method relies on radiometric temperature measurements instead of contact measurements, eliminating the need to disturb the sample with thermal probes. We intend to determine the conductivity of grains that are up to 2 cm in diameter and to parameterize the effects of angularity, sorting, layering, composition, and cementation. These results will support the efforts of the OSIRIS-REx team in selecting a site on asteroid Bennu that is safe and meets grain size requirements for sampling. Our system consists of a cryostat vacuum chamber with an internal liquid nitrogen dewar. A granular sample is contained in a cylindrical cup that is 4 cm in diameter and 1 to 6 cm deep. The surface of the sample is exposed to vacuum and is surrounded by a black liquid nitrogen cold shroud. Once the system has equilibrated at 80 K, the base of the sample cup is rapidly heated to 450 K. An infrared camera observes the sample from above to monitor its temperature change over time. We have built a time-dependent finite element model of the experiment in COMSOL Multiphysics. Boundary temperature conditions and all known material properties (including surface emissivities) are included to replicate the experiment as closely as possible. The Optimization module in COMSOL is specifically designed for parameter estimation. Sample thermal conductivity is assumed to be a quadratic or cubic polynomial function of temperature. We thus use gradient-based optimization methods in COMSOL to vary the polynomial coefficients in an effort to reduce the least squares error between the measured and modeled sample surface temperature.

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

PubMed

Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

2017-08-17

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen.

PubMed

Wang, Jianning; O'Keefe, Joseph; Orr, Della; Loth, Leo; Banks, Malcolm; Wakeley, Philip; West, Donna; Card, Roderick; Ibata, Georgina; Van Maanen, Kees; Thoren, Peter; Isaksson, Mats; Kerkhofs, Pierre

2008-01-01

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.

A Laboratory to Demonstrate the Effect of Thermal History on Semicrystalline Polymers Using Rapid Scanning Rate Differential Scanning Calorimetry

ERIC Educational Resources Information Center

Badrinarayanan, Prashanth; Kessler, Michael R.

2010-01-01

A detailed understanding of the effect of thermal history on the thermal properties of semicrystalline polymers is essential for materials scientists and engineers. In this article, we describe a materials science laboratory to demonstrate the effect of parameters such as heating rate and isothermal annealing conditions on the thermal behavior of…

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Assay to Characterize Histopathologic Changes Following Thermal Injury

PubMed Central

Lee, Ji Min; Park, Ji Hyun; Kim, Bo Young

2018-01-01

Background Despite the wide application of lasers and radiofrequency (RF) surgery in dermatology, it is difficult to find studies showing the extent of damage dependent on cell death. Objective We evaluated histopathologic changes following in vivo thermal damage generated by CO2 laser, 1,444 nm long-pulsed neodymium:yttrium-aluminum-garnet (LP Nd:YAG) laser and RF emitting electrosurgical unit. Methods Thermal damage was induced by the above instruments on ventral skin of rat. Specimens were stained with hematoxylin and eosin, along with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, to highlight the degree of irreversible cellular injury. Results The volume of vaporization was largest with the CO2 laser. Area of cell death area identified by TUNEL assay, when arranged from widest to narrowest, was 1,444 nm LP Nd:YAG laser, CO2 laser, and RF emitting electrosurgical unit. Conclusion This histopathologic evaluation of the acute characterization of injury across devices may be advantageous for attaining better treatment outcomes. PMID:29386831

CaTs Lab (CHAOS and Thermal Sciences Laboratory)

NASA Technical Reports Server (NTRS)

Teate, Anthony A.

2002-01-01

The CHAOS and Thermal Sciences Laboratory (CaTs) at James Madison University evolved into a noteworthy effort to increase minority representation in the sciences and mathematics. Serving ten students and faculty directly, and nearly 50 students indirectly, CaTs, through recruitment efforts, workshops, mentoring programs, tutorial services and research and computational laboratories, fulfilled its intent to initiate an academically enriched research program aimed at strengthening the academic and self-actualization skills of undergraduate students with potential to pursue doctoral study in the sciences. The stated goal of the program was to increase by 5% the number of enrolled mathematics and science students into the program. Success far exceeded the program goals by producing 100% graduation rate of all supported recipients during its tenure, with 30% of the students subsequently in pursuit of graduate degrees. Student retention in the program exceeded 90% and faculty participation exceeded the three members involved in mentoring and tutoring, gaining multi-disciplinary support. Aggressive marketing of the program resulted in several paid summer internships and commitments from NASA and an ongoing relationship with CHROME, a nationally recognized organization which focuses on developing minority students in the sciences and mathematics. Success of the program was only limited by the limited fiscal resources at NASA which resulted in phasing out of the program.

Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

PubMed

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano; Bastien, Patrick

2015-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

PubMed Central

Filisetti, Denis; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Pelloux, Hervé; Touafek, Fériel; Varlet-Marie, Emmanuelle; Yera, Hélène; Candolfi, Ermano

2014-01-01

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents. PMID:25339393

Advanced Thermal Batteries.

DTIC Science & Technology

1981-06-01

ADVANCED THERMAL BATTERIES NATIONAL UNION ELECTRIC CORPORATION ADVANCE SCIENCE DIVISION 1201 E. BELL STREET BLXXMINGTON, ILLINOIS 61701 JUNE 1981...December 1978 in: " Advanced Thermal Batteries " AFAPL-TR-78-114 Air Force Aero Propulsion Laboratory Air Force Wright Aeronautical Laboratories Air Force...March 1980 in: " Advanced Thermal Batteries " AFAPL-TR-80-2017 Air Force Aero Propulsion Laboratory Air Force Wright Aeronautical Laboratories Air Force

Pathology consultation on anticoagulation monitoring: factor X-related assays.

PubMed

Wool, Geoffrey D; Lu, Chuanyi M

2013-11-01

To review various anticoagulation therapies and related laboratory monitoring issues, with a focus on factor X-related chromogenic assays. A case-based approach is used to review pertinent published literatures and product inserts of anticoagulation drugs and to look back on clinical use of factor X-related chromogenic assays. The number of anticoagulants available to clinicians has increased greatly in the past decade. Whether and how these anticoagulants should be monitored are areas of uncertainty for clinicians, which can lead to misuse of laboratory assays and suboptimal patient management. Factor X-related assays are of particular concern because of the similar and often confusing test names. Based on a common clinical case scenario and literature review regarding anticoagulant monitoring, an up-to-date discussion and review of the various factor X-related assays are provided, focusing on the differences in test designs and clinical utilities between the chromogenic anti-Xa and chromogenic factor X activity assays. Anticoagulation therapy and related laboratory monitoring are rapidly evolving areas of clinical practices. A good knowledge of relevant laboratory assays and their clinical applications is necessary to help optimize patient care.

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

PubMed

Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard

2017-12-01

therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be easily used in any coagulation laboratory, is performed as a single test, and is quantitative. This assay has a high robustness, is accurate and presents a good intra- (<3%) and inter-assay (<5%) variability. It contributes solving most of the laboratory issues faced when testing factor V-Leiden. Quantitation of Factor V-L could contribute to a better assessment of thrombotic risk in affected patients, as this complication is first associated to and caused by the presence of a defined amount of FVa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Designing Experiments on Thermal Interactions by Secondary-School Students in a Simulated Laboratory Environment

ERIC Educational Resources Information Center

Lefkos, Ioannis; Psillos, Dimitris; Hatzikraniotis, Euripides

2011-01-01

Background and purpose: The aim of this study was to explore the effect of investigative activities with manipulations in a virtual laboratory on students' ability to design experiments. Sample: Fourteen students in a lower secondary school in Greece attended a teaching sequence on thermal phenomena based on the use of information and…

Evaluation of the Roche prototype 454 HIV-1 ultradeep sequencing drug resistance assay in a routine diagnostic laboratory.

PubMed

Garcia-Diaz, A; Guerrero-Ramos, A; McCormick, A L; Macartney, M; Conibear, T; Johnson, M A; Haque, T; Webster, D P

2013-10-01

Studies have shown that low-frequency resistance mutations can influence treatment outcome. However, the lack of a standardized high-throughput assay has precluded their detection in clinical settings. To evaluate the performance of the Roche prototype 454 UDS HIV-1 drug resistance assay (UDS assay) in a routine diagnostic laboratory. 50 plasma samples, previously characterized by population sequencing and that had shown ≥1 resistance associated mutation (RAM), were retrospectively tested by the UDS assay, including 18 B and 32 non-B subtypes; viral loads between 114-1,806,407 cp/ml; drug-naive (n=27) and drug-experienced (n=23) individuals. The UDS assay was successful for 37/50 (74%) samples. It detected all RAMs found by population sequencing at frequencies above 20%. In addition, 39 low-frequency RAMs were exclusively detected by the UDS assay at frequencies below 20% in both drug-naïve (19/26, 73%) and drug-experienced (9/18, 50%) individuals. UDS results would lead to changes from susceptible to resistant to efavirenz (EFV) in one drug-naive individual with suboptimal response to an EFV-containing regimen and from susceptible to resistance to lamivudine (3TC) in one drug naïve subject who subsequently failed a 3TC-containing regimen and in a treatment experienced subject who had failed a 3TC-containing regimen. The UDS assay performed well across a wide range of subtypes and viral loads; it showed perfect agreement with population sequencing for all RAMs analyzed. In addition, the UDS assay detected additional mutations at frequencies below 20% which correlate with patients' treatment history and had in some cases important prognostic implications. Copyright © 2013 Elsevier B.V. All rights reserved.

A bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings.

PubMed

Sarkar, Susmita; Tang, Xinli L; Das, Dipankar; Spencer, John S; Lowary, Todd L; Suresh, Mavanur R

2012-01-01

The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.

For geological investigations with airborne thermal infrared multispectral images: Transfer of calibration from laboratory spectrometer to TIMS as alternative for removing atmospheric effects

NASA Technical Reports Server (NTRS)

Edgett, Kenneth S.; Anderson, Donald L.

1995-01-01

This paper describes an empirical method to correct TIMS (Thermal Infrared Multispectral Scanner) data for atmospheric effects by transferring calibration from a laboratory thermal emission spectrometer to the TIMS multispectral image. The method does so by comparing the laboratory spectra of samples gathered in the field with TIMS 6-point spectra for pixels at the location of field sampling sites. The transference of calibration also makes it possible to use spectra from the laboratory as endmembers in unmixing studies of TIMS data.

Adaptation of the ORAC assay to the common laboratory equipment and subsequent application to antioxidant plastic films.

PubMed

Bentayeb, K; Vera, P; Rubio, C; Nerin, C

2009-06-01

The oxygen radical absorbance capacity (ORAC) method has been adapted to the instrumental laboratory and optimized for the determination of the antioxidant capacity of a novel active packaging. As the ORAC assay requires the monitorization of a reaction at controlled temperature by means of the fluorescence signal decrease over time, specific instrumental is usually necessary. In this work, a common liquid chromatographic device has been adapted to perform the ORAC assay, leaving it accessible to any laboratory. Using this adaptation, five different essential oils have been determined resulting in the following antioxidant order: clove (2.66 g Trolox per gram of essential oil), oregano (2.25), cinnamon (1.93), rosemary (1.66), and ginger (1.47). After incorporating the essential oils to the film, its antioxidant capacity has also been checked and related to the concentration of essential oil as well as the thickness of the active film. The results point out that for the same amount of essential oil incorporated measured as grams per square meter, thicker films have more antioxidant capacity than the thinner and more concentrated ones. Furthermore, the antioxidant capacity found in the films was always higher than expected taking into account the amount of essential oil incorporated. Some likely explanations have been proposed, leading to the improvement of the antioxidant film under development.

Pumped Fluid Loop Heat Rejection and Recovery Systems for Thermal Control of the Mars Science Laboratory

NASA Technical Reports Server (NTRS)

Bhandari, Pradeep; Birur, Gajanana; Prina, Mauro; Ramirez, Brenda; Paris, Anthony; Novak, Keith; Pauken, Michael

2006-01-01

This viewgraph presentation reviews the heat rejection and heat recovery system for thermal control of the Mars Science Laboratory (MSL). The MSL mission will use mechanically pumped fluid loop based architecture for thermal control of the spacecraft and rover. The architecture is designed to harness waste heat from an Multi Mission Radioisotope Thermo-electric Generator (MMRTG) during Mars surface operations for thermal control during cold conditions and also reject heat during the cruise aspect of the mission. There are several test that are being conducted that will insure the safety of this concept. This architecture can be used during any future interplanetary missions utilizing radioisotope power systems for power generation.

Low Temperature (<100K) Regolith Thermal Conductivity - Preliminary Laboratory Data

NASA Astrophysics Data System (ADS)

Siegler, M.; Zhong, F.; Woods-Robinson, R.; Paige, D. A.

2016-12-01

The Diviner Lunar Radiometer, aboard the Lunar Reconnaissance Orbiter, has shown materials with in the polar cold traps of the Moon to have thermal inertias at least 1 order of magnitude than the rest of the lunar surface. This detection was unexpected, but has a potentially straight-forward explanation in solid state theory (see companion Woods-Robinson et. al. abstract). Thermal conductivity, λ, of a solid should be directly proportional to the specific heat capacity, cp, phonon mean-free path, l, and phonon velocity, v, as: λ(T)=cplvAs temperature decreases, cp also decreases, while l increases. Phonon velocity, v, is generally thought to be constant with temperature. Therefore, thermal conductivity, λ, as a function temperature, T, can be thought of as a battle between cp and l. In crystalline materials, the increase of l with decreasing T generally dominates. However, in polycrystalline materials, like are found on most planetary surfaces, the growth of l (which is fundimantally a measurement of likelihood of phonon scattering) is limited by phonon scattering off of individual grains and subgrain boundaries. In these cases, cpdominates, causing thermal conductivity to plummet at low (<100K for silicate materials) temperatures. Therefore, thermal conductivity as a function of temperature should be inherently related to crystallinity of a given material. In regolith, this solid state drop in material thermal conductivity of polycrystalline materials will act on top of a well understood, but difficult to predict, physical bottleneck of heat transfer at grain contact points. This leads to λ on the order of 10-3 Wm-1K-1 in lunar regolith. Preliminary models predict thermal conductivities on the order 10-5 to 10-4 Wm-1K-1are likely at temperatures below 50K for materials dominated by small crystals (amorphous materials such as glass). Here we report on preliminary laboratory measurements of regolith and regolith simulants down to 15K and 10-7 torr. These results

Protein-ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI).

PubMed

Grøftehauge, Morten K; Hajizadeh, Nelly R; Swann, Marcus J; Pohl, Ehmke

2015-01-01

Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.

Tritium assay of Li/sub 2/O in the LBM/LOTUS experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quanci, J.; Azam, S.; Bertone, P.

1986-11-01

The Lithium Blanket Module (LBM) is an assembly of over 20,000 cylindrical lithium oxide pellets in an array representative of a limited-coverage breeding zone for a toroidal fusion device. A principal objective of the LBM program is to test the ability of advanced neutronics coding to model the tritium breeding characteristics of a fusion device blanket. The LBM has been irradiated at the Ecole Polytechnique Federale de Lausanne (EPFL) LOTUS facility with a 14 MeV point-neutron source. Princeton Plasma Physics Laboratory (PPPL) and EPFL assayed the tritium bred in lithium oxide diagnostic samples placed at various positions in the LBM.more » PPPL employed a thermal extraction technique while EPFL used a dissolution method. The results for the assay are reported and compared to MCNP Monte Carlo neutronics calculations for the LBM/LOTUS system.« less

Field Measurements Indicate Unexpected, Serious Underestimation of Mussel Heart Rates and Thermal Tolerance by Laboratory Studies

PubMed Central

Tagliarolo, Morgana; McQuaid, Christopher D.

2016-01-01

Attempts to predict the response of species to long-term environmental change are generally based on extrapolations from laboratory experiments that inevitably simplify the complex interacting effects that occur in the field. We recorded heart rates of two genetic lineages of the brown mussel Perna perna over a full tidal cycle in-situ at two different sites in order to evaluate the cardiac responses of the two genetic lineages present on the South African coast to temperature and the immersion/emersion cycle. “Robomussel” temperature loggers were used to monitor thermal conditions at the two sites over one year. Comparison with live animals showed that robomussels provided a good estimate of mussel body temperatures. A significant difference in estimated body temperatures was observed between the sites and the results showed that, under natural conditions, temperatures regularly approach or exceed the thermal limits of P. perna identified in the laboratory. The two P. perna lineages showed similar tidal and diel patterns of heart rate, with higher cardiac activity during daytime immersion and minimal values during daytime emersion. Comparison of the heart rates measured in the field with data previously measured in the laboratory indicates that laboratory results seriously underestimate heart rate activity, by as much as 75%, especially during immersion. Unexpectedly, field estimates of body temperatures indicated an ability to tolerate temperatures considered lethal on the basis of laboratory measurements. This suggests that the interaction of abiotic conditions in the field does not necessarily raise vulnerability to high temperatures. PMID:26840775

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round.

PubMed

Ehling, G; Hecht, M; Heusener, A; Huesler, J; Gamer, A O; van Loveren, H; Maurer, Th; Riecke, K; Ullmann, L; Ulrich, P; Vandebriel, R; Vohr, H-W

2005-08-15

The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC(3) values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.

CO2 Insulation for Thermal Control of the Mars Science Laboratory

NASA Technical Reports Server (NTRS)

Bhandari, Pradeep; Karlmann, Paul; Anderson, Kevin; Novak, Keith

2011-01-01

The National Aeronautics and Space Administration (NASA) is sending a large (>850 kg) rover as part of the Mars Science Laboratory (MSL) mission to Mars in 2011. The rover's primary power source is a Multi-Mission Radioisotope Thermoelectric Generator (MMRTG) that generates roughly 2000 W of heat, which is converted to approximately 110 W of electrical power for use by the rover electronics, science instruments, and mechanism-actuators. The large rover size and extreme thermal environments (cold and hot) for which the rover is designed for led to a sophisticated thermal control system to keep it within allowable temperature limits. The pre-existing Martian atmosphere of low thermal conductivity CO2 gas (8 Torr) is used to thermally protect the rover and its components from the extremely cold Martian environment (temperatures as low as -130 deg C). Conventional vacuum based insulation like Multi Layer Insulation (MLI) is not effective in a gaseous atmosphere, so engineered gaps between the warm rover internal components and the cold rover external structure were employed to implement this thermal isolation. Large gaps would lead to more thermal isolation, but would also require more of the precious volume available within the rover. Therefore, a balance of the degree of thermal isolation achieved vs. the volume of rover utilized is required to reach an acceptable design. The temperature differences between the controlled components and the rover structure vary from location to location so each gap has to be evaluated on a case-by-case basis to arrive at an optimal thickness. For every configuration and temperature difference, there is a critical thickness below which the heat transfer mechanism is dominated by simple gaseous thermal conduction. For larger gaps, the mechanism is dominated by natural convection. In general, convection leads to a poorer level of thermal isolation as compared to conduction. All these considerations play important roles in the

Multiplex tandem mass spectrometry assay for newborn screening of X-linked adrenoleukodystrophy, biotinidase deficiency, and galactosemia with flexibility to assay other enzyme assays and biomarkers.

PubMed

Hong, Xinying; Kumar, Arun Babu; Ronald Scott, C; Gelb, Michael H

2018-03-29

All States screen for biotinidase deficiency and galactosemia, and X-linked adrenoleukodystrophy (X-ALD) has recently been added to the Recommended Uniform Screening Panel (RUSP).We sought to consolidate these tests by combining them into a single multiplex tandem mass spectrometry assay as well as to improve the current protocol for newborn screening of galactosemia.A 3 mm punch of a dried blood spot (DBS) was extracted with organic solvent for analysis of the C26:0-lysophosphatidylcholine biomarker for X-ALD.An additional punch was used to assay galactose-1-phosphate uridyltransferase (GALT) and biotinidase.All assays were combined for a single injection for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (2.3 min per sample).The GALT LC-MS/MS assay does not give a false positive for galactosemia if glucose-6-phosphate dehydrogenase is deficient.The multiplex assay shows acceptable reproducibility and provides for rapid analysis of X-ALD, biotinidase deficiency, and galactosemia.The throughput and ease of sample preparation are acceptable for newborn screening laboratories.We also show that the LC-MS/MS assay is expandable to include several other diseases including Pompe and Hurler diseases (enzymatic activities and biomarkers).Because of consolidation of assays, less manpower is needed compared to running individual assays on separate platforms.The flexibility of the LC-MS/MS platform allows each newborn screening laboratory to analyze the set of diseases offered in their panel. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein–ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI)

PubMed Central

Grøftehauge, Morten K.; Hajizadeh, Nelly R.; Swann, Marcus J.; Pohl, Ehmke

2015-01-01

Over the last decades, a wide range of biophysical techniques investigating protein–ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography. PMID:25615858

Evaluation of the Xpert MTB/RIF assay for diagnosis of tuberculosis and rifampin resistance in county-level laboratories in Hunan province, China.

PubMed

Hu, Peilei; Bai, Liqiong; Liu, Fengping; Ou, Xichao; Zhang, Zhiying; Yi, Songlin; Chen, Zhongnan; Gong, Daofang; Liu, Binbin; Guo, Jingwei; Tan, Yunhong

2014-01-01

The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis (TB) countries. However, there were little data obtained by validation or demonstration study of the assay in China. In this study, the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province, China. Consecutive patients with suspected pulmonary tuberculosis (PTB) and suspicion for multidrug-resistant tuberculosis (MDR-TB) were enrolled. For each patient suspected to have PTB, three sputum specimens (one spot sputum, one night sputum, and one morning sputum) were collected and each sputum was tested with smear microscopy, Löwenstein-Jensen (LJ) culture, and Xpert MTB/RIF test. For comparison across subgroups and testing methods, 95% confidence intervals were calculated. All analyses were done with SPSS 16.0, and P < 0.05 was regarded as significant. For case detection, the sensitivity of Xpert MTB/RIF was 100% for smear- and culture-positive TB and 88.6% for smear-negative and culture-positive TB; the overall sensitivity was 94.5% for all culture-positive patients. The specificity was 99.8%. The sensitivity of Xpert MTB/RIF assay was 22.0% in clinical TB patients and the specificity reached 100.0% in the group of patients who are infected with nontuberculous mycobacteria. For the detection of rifampin resistance, the sensitivity of MTB/RIF RIF-resistance detection was 92.9%, and the specificity was 98.7%. Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests, 20 (76.9%) patients were infected by MDR-TB. The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance, which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China. Further

Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2016-02-12

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative tomore » other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.« less

Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

PubMed

Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

2016-01-01

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

The help of simulation codes in designing waste assay systems using neutron measurement methods: Application to the alpha low level waste assay system PROMETHEE 6

NASA Astrophysics Data System (ADS)

Mariani, A.; Passard, C.; Jallu, F.; Toubon, H.

2003-11-01

The design of a specific nuclear assay system for a dedicated application begins with a phase of development, which relies on information from the literature or on knowledge resulting from experience, and on specific experimental verifications. The latter ones may require experimental devices which can be restricting in terms of deadline, cost and safety. One way generally chosen to bypass these difficulties is to use simulation codes to study particular aspects. This paper deals with the potentialities offered by the simulation in the case of a passive-active neutron (PAN) assay system for alpha low level waste characterization; this system has been carried out at the Nuclear Measurements Development Laboratory of the French Atomic Energy Commission. Due to the high number of parameters to be taken into account for its development, this is a particularly sophisticated example. Since the PAN assay system, called PROMETHEE (prompt epithermal and thermal interrogation experiment), must have a detection efficiency of more than 20% and preserve a high level of modularity for various applications, an improved version has been studied using the MCNP4 (Monte Carlo N-Particle) transport code. Parameters such as the dimensions of the assay system, of the cavity and of the detection blocks, and the thicknesses of the nuclear materials of neutronic interest have been optimised. Therefore, the number of necessary experiments was reduced.

Colorimetric micro-assay for accelerated screening of mould inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2013-01-01

Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

PubMed

Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

2017-10-01

A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories

PubMed Central

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; Matteis, Giovanna De; Zacomo, Silvia Di; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-01-01

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory. A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays. The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples. In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F. PMID:28427233

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

PubMed

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; De Matteis, Giovanna; Di Zacomo, Silvia; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-05-16

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Application of statistical process control to qualitative molecular diagnostic assays.

PubMed

O'Brien, Cathal P; Finn, Stephen P

2014-01-01

Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

International Technology Transfer of a GCLP-Compliant HIV-1 Neutralizing Antibody Assay for Human Clinical Trials

PubMed Central

Todd, Christopher A.; Greene, Kelli M.; Montefiori, David C.; Sarzotti-Kelsoe, Marcella

2012-01-01

The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody – Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials. PMID:22303476

International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials.

PubMed

Ozaki, Daniel A; Gao, Hongmei; Todd, Christopher A; Greene, Kelli M; Montefiori, David C; Sarzotti-Kelsoe, Marcella

2012-01-01

The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody-Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials.

Solar thermal power storage applications lead laboratory overview

NASA Technical Reports Server (NTRS)

Radosevich, L. G.

1980-01-01

The implementation of the applications elements of the thermal energy storage for Solar Thermal Applications program is described. The program includes the accelerated development of thermal storage technologies matched to solar thermal power system requirements and scheduled milestones. The program concentrates on storage development in the FY80 to 85 time period with emphasis on the more near-term solar thermal power system application.

An Undergraduate Laboratory Experiment that Utilizes a Glass Fiber Filter Assay to Determine the Steroid Specificity and Equilibrium Binding Properties of Glucocorticoid Receptors.

ERIC Educational Resources Information Center

John, Nancy J.; Firestone, Gary L.

1987-01-01

Describes two complementary laboratory exercises that use the glass fiber assay to assess receptor specificity and hormone binding affinity in rat liver cytoplasmic extracts. Details the methods, materials and protocol of the experiments. Discusses the basic concepts illustrated and the feasibility of using the experiments at the undergraduate…

Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents.

PubMed

O'Hara, Denise M; Theobald, Valerie; Egan, Adrienne Clements; Usansky, Joel; Krishna, Murli; TerWee, Julie; Maia, Mauricio; Spriggs, Frank P; Kenney, John; Safavi, Afshin; Keefe, Jeannine

2012-06-01

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

Critical Value Reporting at Egyptian Laboratories.

PubMed

Mosallam, Rasha; Ibrahim, Samaa Zenhom

2015-06-12

To examine critical value reporting policies and practices and to identify critical value ranges for selected common laboratory assays at inpatient division of laboratories of Alexandria hospitals. A cross-sectional descriptive study design was used. Subjects were from inpatient division of all laboratories of Alexandria hospitals (40 laboratories). Data were collected using a questionnaire composed of 4 sections. The first section explored hospital and laboratory characteristics. The second section assessed policies and procedures of critical value reporting. The third section explored the reporting process. The fourth section explored critical value ranges for selected common laboratory assays. Written procedure for reporting of critical values was present in 77.5% of laboratories and a comprehensive list of critical values in 72.55%. For laboratories having a critical value list, the number of tests in the list ranged from 7 to 40. Three-fifths of laboratories had a policy for assessing the timeliness of reporting and 3 quarters stated that the laboratory policy requires feedback (60.0% and 75.0%, respectively). The hospital laboratory physician was responsible for critical value reporting followed by the laboratory technician (75.0% and 50.0%, respectively). The call is received mainly by nurses and physicians ordering the test (67.5% and 55.0%, respectively) and the channel of reporting is mainly the telephone or through sending test report to the ward (67.5% and 50.0%, respectively). Wireless technologies are used in reporting in only 10.0% of hospitals. The cutoff limits for reporting different assays showed considerable interlaboratory variation. Critical value policies and practices showed interinstitutional variation with deficiencies in some reporting practices. Selection of critical assays for notification and setting the limits of notification exhibited wide variation as well.

Laboratory heterogeneity of the lupus anticoagulant: a multicentre study using different clotting assays on a panel of 78 samples. Hemostasis Committee of the "Société Française de Biologie Clinique".

PubMed

1992-05-15

The laboratory heterogeneity of the lupus anticoagulant (LA) was investigated in a multicentre study using a panel of 78 plasma samples diagnosed as containing a LA. Consecutive samples were collected by 12 participants using various screening tests, and sent to 7 laboratories which performed one or more clotting assays among the following: activated partial thromboplastin time (APTT), dilute Russell viper venom time, kaolin clotting time (KCT), dilute tissue thromboplastin time (dTTI) and a platelet neutralization test. For APTT and dTTI, 10 versions of these tests including standard and mixing procedures were carried out. They varied by reagents, phospholipid concentration or methodology. Cut-off times were determined for each test by comparing the results of the panel to those of a control population. When the data of all clotting assays were pooled, 70 of the 78 selected plasmas were considered to contain LA, 15 of them having a low-titer inhibitor. Sensitivity, defined as the proportion of positive results among LA-containing plasmas, varied from 62 to 100% and was positively related to responsiveness (defined as the mean ratio of clotting time to cut-off time). Laboratory heterogeneity of LA-containing plasma was illustrated by a star symbol plot analysis. Different populations of samples, with LA preferentially recognized by one assay (or group of assays) irrespective of the overall sensitivity of this assay, were identified. Multiple component analysis demonstrated the heterogeneity of low-titer inhibitors, which complicates their recognition in routine laboratory investigation.

Photon-induced Processing of Interstellar Ices in the Laboratory. Focus on Their Non-thermal Desorption.

NASA Astrophysics Data System (ADS)

Martin-Domenech, Rafael; Munoz Caro, Guillermo; Cruz-Diaz, Gustavo A.; Oberg, Karin I.

2018-06-01

Some of the processes that take place in the interstellar medium (ISM)can be simulated in laboratories on Earth under astrophysically relevant conditions. For example, the energetic processing of the ice mantles that accrete on top of dust grains in the coldest regions of the ISM, leading to the production of new species and their desorption to the gas phase. In particular, observation of complex organic molecules (COMs) in cold interstellar environments stress the need for not only a solid state formation but also for non-thermal desorption mechanisms that can account for the observed abundances in regions where thermal desorption is inhibited. Laboratory Astrophysics can be used to test different non-thermal desorption processes and extract yields than can be extrapolated to the astrophysical scenario with theoretical models. 0th generation COMs like CH3OH and H2CO can be formed at very low temperatures. In this talk, we present laboratory simulations of the UV photoprocessing of a binary ice mixture composed by water (the main component of astrophysical ices) and methane. Formation of CO, CO2, CH3OH and H2CO was confirmed by IR spectroscopy and subsequent TPD. At the same time, photodesorption of CO and H2CO was detected by means of a Quadrupole Mass Spectrometer, with yields on the order of 10-4 and 10-5 molecules per incident photon, respectively. In general, photodesorption can take place through a direct mechanism, where the absorbing molecule (or its photofragments) are desorbed; or through an indirect mechanism where the absorbed energy is transferred to a surface molecule which is the one finally desorbing. In the case of photoproducts, the evolution of the photodesorption yield gives information on the photodesorption mechanism: a constant photodesorption yield is observed when the photoproducts are desorbed right after their formation; while an increasing yield is measured when the photoproducts are desorbed later after energy transfer from another

Detection of Enterovirus D68 in Canadian Laboratories

PubMed Central

Hatchette, Todd F.; Drews, Steven J.; Grudeski, Elsie; Booth, Tim; Martineau, Christine; Dust, Kerry; Garceau, Richard; Gubbay, Jonathan; Karnauchow, Tim; Krajden, Mel; Levett, Paul N.; Mazzulli, Tony; McDonald, Ryan R.; McNabb, Alan; Mubareka, Samira; Needle, Robert; Petrich, Astrid; Richardson, Susan; Rutherford, Candy; Smieja, Marek; Tellier, Raymond; Tipples, Graham

2015-01-01

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68. PMID:25740765

Prediction of In-Space Durability of Protected Polymers Based on Ground Laboratory Thermal Energy Atomic Oxygen

NASA Technical Reports Server (NTRS)

Banks, Bruce A.; deGroh, Kim K.; Rutledge, Sharon; DiFilippo, Frank J.

1996-01-01

The probability of atomic oxygen reacting with polymeric materials is orders of magnitude lower at thermal energies (greater than O.1 eV) than at orbital impact energies (4.5 eV). As a result, absolute atomic oxygen fluxes at thermal energies must be orders of magnitude higher than orbital energy fluxes, to produce the same effective fluxes (or same oxidation rates) for polymers. These differences can cause highly pessimistic durability predictions for protected polymers and polymers which develop protective metal oxide surfaces as a result of oxidation if one does not make suitable calibrations. A comparison was conducted of undercut cavities below defect sites in protected polyimide Kapton samples flown on the Long Duration Exposure Facility (LDEF) with similar samples exposed in thermal energy oxygen plasma. The results of this comparison were used to quantify predicted material loss in space based on material loss in ground laboratory thermal energy plasma testing. A microindent hardness comparison of surface oxidation of a silicone flown on the Environmental Oxygen Interaction with Materials-III (EOIM-III) experiment with samples exposed in thermal energy plasmas was similarly used to calibrate the rate of oxidation of silicone in space relative to samples in thermal energy plasmas exposed to polyimide Kapton effective fluences.

Microfluidic cartridges for DNA purification and genotyping processed in standard laboratory instruments

NASA Astrophysics Data System (ADS)

Focke, Maximilian; Mark, Daniel; Stumpf, Fabian; Müller, Martina; Roth, Günter; Zengerle, Roland; von Stetten, Felix

2011-06-01

Two microfluidic cartridges intended for upgrading standard laboratory instruments with automated liquid handling capability by use of centrifugal forces are presented. The first microfluidic cartridge enables purification of DNA from human whole blood and is operated in a standard laboratory centrifuge. The second microfluidic catridge enables genotyping of pathogens by geometrically multiplexed real-time PCR. It is operated in a slightly modified off-the-shelf thermal cycler. Both solutions aim at smart and cost-efficient ways to automate work flows in laboratories. The DNA purification cartridge automates all liquid handling steps starting from a lysed blood sample to PCR ready DNA. The cartridge contains two manually crushable glass ampoules with liquid reagents. The DNA yield extracted from a 32 μl blood sample is 192 +/- 30 ng which corresponds to 53 +/- 8% of a reference extraction. The genotyping cartridge is applied to analyse isolates of the multi-resistant Staphyloccus aureus (MRSA) by real-time PCR. The wells contain pre-stored dry reagents such as primers and probes. Evaluation of the system with 44 genotyping assays showed a 100% specificity and agreement with the reference assays in standard tubes. The lower limit of detection was well below 10 copies of DNA per reaction.

Comparison of Established and Emerging Biodosimetry Assays

PubMed Central

Rothkamm, K.; Beinke, C.; Romm, H.; Badie, C.; Balagurunathan, Y.; Barnard, S.; Bernard, N.; Boulay-Greene, H.; Brengues, M.; De Amicis, A.; De Sanctis, S.; Greither, R.; Herodin, F.; Jones, A.; Kabacik, S.; Knie, T.; Kulka, U.; Lista, F.; Martigne, P.; Missel, A.; Moquet, J.; Oestreicher, U.; Peinnequin, A.; Poyot, T.; Roessler, U.; Scherthan, H.; Terbrueggen, B.; Thierens, H.; Valente, M.; Vral, A.; Zenhausern, F.; Meineke, V.; Braselmann, H.; Abend, M.

2014-01-01

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools. PMID:23862692

Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

PubMed

Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

2016-12-01

Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Laboratory evaluations on thermal debonding of ceramic brackets.

PubMed

Sernetz, F; Kraut, J

1991-01-01

The purpose of this laboratory study was to define the working parameters and physiological safety and efficacy of the Dentaurum Ceramic Debonding Unit. Extracted mandibular incisors were utilized because of their low thermal mass and low heat sensitivity. The teeth were embedded in plastic and placed on a turning force measuring apparatus. An electrothermal element was placed in the pulp chamber (filled with a conducting paste). The thermoelement temperature was registered on y-t recorder as was the turning momentum required to remove the ceramic brackets with the Dentaurum Ceramic Debonding Unit. Ceramic brackets from GAC (Allure III), Unitek (Transcend) and Dentaurum (Fascination) using one and two component adhesives (Monolok, Concise), were tested. Scanning electron microscopic views taken after debonding showed predictable (and favorable) adhesive failure at the bracket base/resin interface. No enamel damage was demonstrated. All brackets were removable under three seconds with a clinically reproducible turning force of 85-100 Nmm allowing for intrapulpal temperature increases under the 5 degrees C biocompatible threshold. The Dentaurum Ceramic Debonding Unit provided a safe, reliable, efficient modality of removing ceramic brackets while maintaining a physiologically acceptable rise in pulpal temperature without damage to tooth enamel or pulpal tissue.

When is it time to get married? Or when should the assay user and the assay developer collaborate?

PubMed Central

Swan, S H; Lasley, B L

1991-01-01

Hormone assays are being developed in the laboratory to detect specific molecular markers in nonclinical populations. Epidemiology is increasingly using these assays to improve the precision with which disease processes and exposures can be defined. This growing body of molecular epidemiology requires a high degree of cooperation between the assay developer and the assay user. We draw on our experience in using a sensitive hormone assay for the detection of early pregnancy via urinary human chorionic gonadotropin to illustrate these points. We conclude that this collaborative effort, in addition to making this study possible, has provided unexpected rewards. PMID:1954925

Analytical and Clinical Performance Evaluation of the Abbott Architect PIVKA Assay.

PubMed

Ko, Dae-Hyun; Hyun, Jungwon; Kim, Hyun Soo; Park, Min-Jeong; Kim, Jae-Seok; Park, Ji-Young; Shin, Dong Hoon; Cho, Hyoun Chan

2018-01-01

Protein induced by vitamin K absence (PIVKA) is measured using various assays and is used to help diagnose hepatocellular carcinoma. The present study evaluated the analytical and clinical performances of the recently released Abbott Architect PIVKA assay. Precision, linearity, and correlation tests were performed in accordance with the Clinical Laboratory Standardization Institute guidelines. Sample type suitability was assessed using serum and plasma samples from the same patients, and the reference interval was established using sera from 204 healthy individuals. The assay had coefficients of variation of 3.2-3.5% and intra-laboratory variation of 3.6-5.5%. Linearity was confirmed across the entire measurable range. The Architect PIVKA assay was comparable to the Lumipulse PIVKA assay, and the plasma and serum samples provided similar results. The lower reference limit was 13.0 mAU/mL and the upper reference limit was 37.4 mAU/mL. The ability of the Architect PIVKA assay to detect hepatocellular carcinoma was comparable to that of the alpha-fetoprotein test and the Lumipulse PIVKA assay. The Architect PIVKA assay provides excellent analytical and clinical performance, is simple for clinical laboratories to adopt, and has improved sample type suitability that could broaden the assay's utility. © 2018 by the Association of Clinical Scientists, Inc.

Managing the Mars Science Laboratory Thermal Vacuum Test for Safety and Success

NASA Technical Reports Server (NTRS)

Evans, Jordan P.

2010-01-01

The Mars Science Laboratory is a NASA/JPL mission to send the next generation of rover to Mars. Originally slated for launch in 2009, development problems led to a delay in the project until the next launch opportunity in 2011. Amidst the delay process, the Launch/Cruise Solar Thermal Vacuum Test was undertaken as risk reduction for the project. With varying maturity and capabilities of the flight and ground systems, undertaking the test in a safe manner presented many challenges. This paper describes the technical and management challenges and the actions undertaken that led to the ultimate safe and successful execution of the test.

Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

PubMed

Jacobson, R H; Downing, D R; Lynch, T J

1982-11-15

A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

Optical Characterization Laboratory | Energy Systems Integration Facility |

Science.gov Websites

Laboratory offers the following capabilities. Solar Thermal Calibration The Optical Characterization collectors for solar thermal energy generation to enable the study of increasingly stable (less intermittent Characterization Laboratory's environmental characterization hub offers high-temperature/humidity thermal chambers

Teaching about citric acid cycle using plant mitochondrial preparations: Some assays for use in laboratory courses*.

PubMed

Vicente, Joaquim A F; Gomes-Santos, Carina S S; Sousa, Ana Paula M; Madeira, Vítor M C

2005-03-01

Potato tubers and turnip roots were used to prepare purified mitochondria for laboratory practical work in the teaching of the citric acid cycle (TCA cycle). Plant mitochondria are particularly advantageous over the animal fractions to demonstrate the TCA cycle enzymatic steps, by using simple techniques to measure O(2) consumption and transmembrane potential (ΔΨ). The several TCA cycle intermediates induce specific enzyme activities, which can be identified by respiratory parameters. Such a strategy is also used to evidence properties of the TCA cycle enzymes: ADP stimulation of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase; activation by citrate of downstream oxidation steps, e.g. succinate dehydrogenase; and regulation of the activity of isocitrate dehydrogenase by citrate action on the citrate/isocitrate carrier. Furthermore, it has been demonstrated that, in the absence of exogenous Mg(2+) , isocitrate-dependent respiration favors the alternative oxidase pathway, as judged by changes of the ADP/O elicited by the inhibitor n-propyl galate. These are some examples of assays related with TCA cycle intermediates we can use in laboratory courses. Copyright © 2005 International Union of Biochemistry and Molecular Biology, Inc.

From Concept-to-Flight: An Active Active Fluid Loop Based Thermal Control System for Mars Science Laboratory Rover

NASA Technical Reports Server (NTRS)

Birur, Gajanana C.; Bhandari, Pradeep; Bame, David; Karlmann, Paul; Mastropietro, A. J.; Liu, Yuanming; Miller, Jennifer; Pauken, Michael; Lyra, Jacqueline

2012-01-01

The Mars Science Laboratory (MSL) rover, Curiosity, which was launched on November 26, 2011, incorporates a novel active thermal control system to keep the sensitive electronics and science instruments at safe operating and survival temperatures. While the diurnal temperature variations on the Mars surface range from -120 C to +30 C, the sensitive equipment are kept within -40 C to +50 C. The active thermal control system is based on a single-phase mechanically pumped fluid loop (MPFL) system which removes or recovers excess waste heat and manages it to maintain the sensitive equipment inside the rover at safe temperatures. This paper will describe the entire process of developing this active thermal control system for the MSL rover from concept to flight implementation. The development of the rover thermal control system during its architecture, design, fabrication, integration, testing, and launch is described.

A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

ERIC Educational Resources Information Center

Harding, Ethelynda E.; Kimsey, R. Scott

1998-01-01

Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results.

PubMed

Zantek, N D; Hsu, P; Refaai, M A; Ledford-Kraemer, M; Meijer, P; Van Cott, E M

2013-06-01

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or <40 U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73 U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20 U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0 U/dL). For specimens with FVII >50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant. © 2013 Blackwell Publishing Ltd.

Detection of enterovirus D68 in Canadian laboratories.

PubMed

Hatchette, Todd F; Drews, Steven J; Grudeski, Elsie; Booth, Tim; Martineau, Christine; Dust, Kerry; Garceau, Richard; Gubbay, Jonathan; Karnauchow, Tim; Krajden, Mel; Levett, Paul N; Mazzulli, Tony; McDonald, Ryan R; McNabb, Alan; Mubareka, Samira; Needle, Robert; Petrich, Astrid; Richardson, Susan; Rutherford, Candy; Smieja, Marek; Tellier, Raymond; Tipples, Graham; LeBlanc, Jason J

2015-05-01

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Thermal biology, torpor and behaviour in sugar gliders: a laboratory-field comparison.

PubMed

Geiser, Fritz; Holloway, Joanne C; Körtner, Gerhard

2007-07-01

Most studies on animal physiology and behaviour are conducted in captivity without verification that data are representative of free-ranging animals. We provide the first quantitative comparison of daily torpor, thermal biology and activity patterns, conducted on two groups of sugar gliders (Petaurus breviceps, Marsupialia) exposed to similar thermal conditions, one in captivity and the other in the field. Our study shows that activity in captive gliders in an outdoor aviary is restricted to the night and largely unaffected by weather, whereas free-ranging gliders omit foraging on cold/wet nights and may also forage in the afternoon. Torpor occurrence in gliders was significantly lower in captivity (8.4% after food deprivation; 1.1% for all observations) than in the field (25.9%), mean torpor bout duration was shorter in captivity (6.9 h) than in the field (13.1 h), and mean body temperatures during torpor were higher in captivity (25.3 degrees C) than in the field (19.6 degrees C). Moreover, normothermic body temperature as a function of air temperature differed between captive and free-ranging gliders, with a >3 degrees C difference at low air temperatures. Our comparison shows that activity patterns, thermal physiology, use of torpor and patterns of torpor may differ substantially between the laboratory and field, and provides further evidence that functional and behavioural data on captive individuals may not necessarily be representative of those living in the wild.

Local lymph node assay: how testing laboratories apply OECD TG 429 for REACH purposes.

PubMed

Rovida, Costanza

2011-01-01

The Local Lymph Node Assay (LLNA) is the official method for assessing the allergic contact dermatitis potential of chemicals for the purposes of REACH regulation. The LLNA went through a validation process that allowed the delineation of a robust protocol for performing new tests. The OECD accepted this method in 2002 and published OECD TG 429. The European Chemical Agency (ECHA) recently published data that were submitted in the registration dossiers of chemicals. This database was analysed to determine how testing laboratories apply OECD TG 429. This analysis comes after a detailed analysis of four full study reports that were also prepared for REACH purposes. Although the majority of the tests are fully compliant with OECD TG 429, some showed major deviations, and a number of others used more animals than necessary. This suggests that in vivo tests need to be planned more carefully and consciously to obtain meaningful results with the minimum animal number necessary.

Assessing the relative importance of environmental effects, carry-over effects and species differences in thermal stress resistance: a comparison of Drosophilids across field and laboratory generations.

PubMed

Schiffer, Michele; Hangartner, Sandra; Hoffmann, Ary A

2013-10-15

There is increasing interest in comparing species of related organisms for their susceptibility to thermal extremes in order to evaluate potential vulnerability to climate change. Comparisons are typically undertaken on individuals collected from the field with or without a period of acclimation. However, this approach does not allow the potential contributions of environmental and carry-over effects across generations to be separated from inherent species differences in susceptibility. To assess the importance of these different sources of variation, we here considered heat and cold resistance in Drosophilid species from tropical and temperate sites in the field and across two laboratory generations. Resistance in field-collected individuals tended to be lower when compared with F1 and F2 laboratory generations, and species differences in field flies were only weakly correlated to differences established under controlled rearing conditions, unlike in F1-F2 comparisons. This reflected large environmental effects on resistance associated with different sites and conditions experienced within sites. For the 8 h cold recovery assay there was no strong evidence of carry-over effects, whereas for the heat knockdown and 2 h cold recovery assays there was some evidence for such effects. However, for heat these were species specific in direction. Variance components for inherent species differences were substantial for resistance to heat and 8 h cold stress, but small for 2 h cold stress, though this may be a reflection of the species being considered in the comparisons. These findings highlight that inherent differences among species are difficult to characterise accurately without controlling for environmental sources of variation and carry-over effects. Moreover, they also emphasise the complex nature of carry-over effects that vary depending on the nature of stress traits and the species being evaluated.

Serotype determination of Salmonella by xTAG assay.

PubMed

Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

2017-10-01

Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

PubMed

Baumeister, Mark A; Zhang, Nan; Beas, Hilda; Brooks, Jesse R; Canchola, Jesse A; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) ("Versant Assay") currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.

Problems with GH assays and strategies toward standardization.

PubMed

Bidlingmaier, Martin

2008-12-01

Disorders affecting GH secretion--either GH deficiency or GH excess (acromegaly)--are biochemically defined through peak or nadir concentrations of human GH in response to dynamic tests. Immunoassays employing polyclonal or monoclonal antibodies are routinely used for the analysis of GH concentrations, and many different assays are available on the market today. Unfortunately, the actual value reported for the GH concentration in a specific patient's sample to a large extent depends on the assay method used by the respective laboratory. Variability between assay results exceeds 200%, limiting the applicability of consensus guidelines in clinical practice. Reasons for the heterogeneity in GH assay results include the heterogeneity of the analyte itself, the availability of different preparations for calibration, and the interference from matrix components such as GH-binding protein. Furthermore, the reporting of results in mass units or international units together with the application of variable conversion factors led to confusion. International collaborations proposed measures to improve the comparability of assay results, recommending the use of a single, recombinant calibrator for all assays and reporting only in mass units as first steps. However, because of the differences in epitope specificity of antibodies used in different assays, method-specific cut-off levels for dynamic tests might remain necessary to correctly interpret and compare results from different laboratories.

A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

PubMed Central

Baumeister, Mark A.; Zhang, Nan; Beas, Hilda; Brooks, Jesse R.; Canchola, Jesse A.; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R.

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements. PMID:22479381

Evaluating 6 ricin field detection assays.

PubMed

Slotved, Hans-Christian; Sparding, Nadja; Tanassi, Julia Tanas; Steenhard, Nina R; Heegaard, Niels H H

2014-01-01

This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

PubMed

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

Integrated Summary Report: Validation of Two Binding Assays ...

EPA Pesticide Factsheets

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 89

Thermal comfort of aeroplane seats: influence of different seat materials and the use of laboratory test methods.

PubMed

Bartels, Volkmar T

2003-07-01

This study determined the influence of different cover and cushion materials on the thermal comfort of aeroplane seats. Different materials as well as ready made seats were investigated by the physiological laboratory test methods Skin Model and seat comfort tester. Additionally, seat trials with human test subjects were performed in a climatic chamber. Results show that a fabric cover produces a considerably higher sweat transport than leather. A three-dimensional knitted spacer fabric turns out to be the better cushion alternative in comparison to a moulded foam pad. Results from the physiological laboratory test methods nicely correspond to the seat trials with human test subjects.

Mussel micronucleus cytome assay.

PubMed

Bolognesi, Claudia; Fenech, Michael

2012-05-17

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

PubMed

Stellrecht, K A; Espino, A A; Maceira, V P; Nattanmai, S M; Butt, S A; Wroblewski, D; Hannett, G E; Musser, K A

2014-05-01

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.

PubMed

Cheng, Fang; Wu, Jiajie; Zhang, Jin; Pan, Aihu; Quan, Sheng; Zhang, Dabing; Kim, HaeYeong; Li, Xiang; Zhou, Shan; Yang, Litao

2016-05-15

Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Manual versus automated γ-H2AX foci analysis across five European laboratories: can this assay be used for rapid biodosimetry in a large scale radiation accident?

PubMed

Rothkamm, Kai; Barnard, Stephen; Ainsbury, Elizabeth A; Al-Hafidh, Jenna; Barquinero, Joan-Francesc; Lindholm, Carita; Moquet, Jayne; Perälä, Marjo; Roch-Lefèvre, Sandrine; Scherthan, Harry; Thierens, Hubert; Vral, Anne; Vandersickel, Veerle

2013-08-30

The identification of severely exposed individuals and reassurance of the 'worried well' are of prime importance for initial triage following a large scale radiation accident. We aim to develop the γ-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo γ-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate γ-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2-4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the γ-H2AX assay. We conclude that the γ-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

21 CFR 226.58 - Laboratory controls.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Laboratory controls. Laboratory controls shall include the establishment of adequate specifications and test... establishment of master records containing appropriate specifications and a description of the test procedures... necessary laboratory test procedures to check such specifications. (c) Assays which shall be made of...

Electrochemical Assay of Gold-Plating Solutions

NASA Technical Reports Server (NTRS)

Chiodo, R.

1982-01-01

Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

Profile of Instrumentation Laboratory's HemosIL® AcuStar HIT-Ab(PF4-H) assay for diagnosis of heparin-induced thrombocytopenia.

PubMed

Nagler, Michael; Cuker, Adam

2017-05-01

Immunoassays play an essential role in the diagnosis of heparin-induced thrombocytopenia (HIT). The objective of this article is to review HemosIL® AcuStar HIT-Ab(PF4-H) (Instrumentation Laboratory, Bedford, MA, USA), a new chemiluminescent immunoassay for HIT. Areas covered: The authors searched the published literature for evaluation studies of HemosIL® AcuStar HIT-Ab(PF4-H) and sought information from the manufacturer. In this paper, the authors discuss the analytical principle and technical aspects of the assay; describe its diagnostic performance in validation studies; report on its reproducibility, cost-effectiveness, and regulatory status; and discuss the implications of the assay on clinical practice and means of integrating it in diagnostic pathways. HemosIL® AcuStar HIT-Ab(PF4-H) is compared with other rapid assays and widely used enzyme-linked immunoassays for the diagnosis of HIT. Expert commentary: HemosIL® AcuStar HIT-Ab(PF4-H) is automatable, can be performed 24 h per day, offers a rapid turnaround time, and appears to have favorable diagnostic accuracy, particularly at thresholds above that listed in the label. These advantages could lead to improved patient outcomes through rapid provision of results at the point of care, enhancing the accuracy of initial diagnosis.

Laboratory Testing Protocols for Heparin-Induced Thrombocytopenia (HIT) Testing.

PubMed

Lau, Kun Kan Edwin; Mohammed, Soma; Pasalic, Leonardo; Favaloro, Emmanuel J

2017-01-01

Heparin-induced thrombocytopenia (HIT) represents a significant high morbidity complication of heparin therapy. The clinicopathological diagnosis of HIT remains challenging for many reasons; thus, laboratory testing represents an important component of an accurate diagnosis. Although there are many assays available to assess HIT, these essentially fall into two categories-(a) immunological assays, and (b) functional assays. The current chapter presents protocols for several HIT assays, being those that are most commonly performed in laboratory practice and have the widest geographic distribution. These comprise a manual lateral flow-based system (STiC), a fully automated latex immunoturbidimetric assay, a fully automated chemiluminescent assay (CLIA), light transmission aggregation (LTA), and whole blood aggregation (Multiplate).

Comparison of Estrogen Receptor Assay Results from Pathology Reports with Results from Central Laboratory Testing: Implications for Population-Based Studies of Breast Cancer

PubMed Central

Collins, LC; Marotti, J; Baer, HJ; Deitz, AC; Colditz, GA; Tamimi, RM

2014-01-01

Population-based studies of women with breast cancer commonly utilize information culled from pathology reports rather than central pathology review. The reliability of this information, particularly with regard to tumor biomarker results, is of concern. To address this, we evaluated the concordance between estrogen receptor (ER) results as determined from the original pathology reports and ER results obtained on the same specimens following testing in a single laboratory. Tissue microarrays (TMAs) were constructed from paraffin blocks of 3,167 breast cancers that developed in women enrolled in the Nurses’ Health Study. ER immunostains were performed on all TMA sections in single run. Results of ER immunostains performed on the TMA sections were compared with ER assay results abstracted from pathology reports. Among 1,851 cases of invasive breast cancer in which both ER results from pathology reports and central ER test results were available, the reported ER status and the ER status as determined from immunostains on TMAs were in agreement in 1,651 cases (87.3 %; kappa value 0.64, p<0.0001). When the comparison was restricted to ER assays originally performed by immunohistochemistry, the agreement rate increased to 92.3% (kappa value 0.78, p<0.0001). These results provide a framework for the accuracy of ER results abstracted from clinical records. Further, they suggest that utilizing ER assay results from pathology reports is a reasonable, albeit imperfect, alternative to central laboratory ER testing for large, population-based studies of patients with breast cancer. PMID:18230800

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

PubMed

Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E

2014-11-28

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

Laboratory assays evaluate the influence of physical guidelines on subterranean termite (Isoptera: Rhinotermitidae) tunneling, bait discovery, and consumption.

PubMed

Swoboda, L E; Miller, D M

2004-08-01

Laboratory assays were conducted to determine whether physical guidelines could direct subterranean termite foraging behavior. Several materials (wood, plastics, and wood thermoplastic composites) were evaluated for their potential to serve as termite guidelines. Termite tunneling along the different types of guidelines was measured. The proportion of baits discovered when connected by a guideline was compared with the proportion of unconnected baits discovered. Termite consumption of baits also was quantified. Assay results indicated that the termites did not respond to all guideline materials in the same way. Termites built significantly longer tunnels along wood guidelines than they did along any of the plastic guidelines tested. However, tunnel length along the wood and the wood thermoplastic composites was not significantly different. The probability of two baits being discovered when they were connected by wood guidelines was significantly greater than when the baits were connected by plastic guidelines or left unconnected (no guideline). Pairs of baits connected by wood thermoplastic composites were also significantly more likely to be discovered than unconnected baits. Bait consumption was not enhanced by the presence of the guidelines. It is likely that guidelines made of wood competed with the baits as a termite food resource.

Impact of the New Abbott mPLUS Feature on Clinical Laboratory Efficiencies of Abbott RealTime Assays for Detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae

PubMed Central

Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin

2013-01-01

Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents. PMID:24088850

Impact of the New Abbott mPLUS feature on clinical laboratory efficiencies of abbott RealTime assays for detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae.

PubMed

Lucic, Danijela; Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin

2013-12-01

Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents.

Thermal instabilities in a soft and complex lithosphere: laboratory experiments and numerical simulations

NASA Astrophysics Data System (ADS)

Massmeyer, A.; Davaille, A. B.; Rolf, T.; Tackley, P. J.; Di Giuseppe, E.

2012-12-01

The upwelling of hot material in the lithosphere remains far from understood. This is due to the complexity of the mechanical behaviour of lithospheric material, which presents solid as well as viscous properties. Mushroom-shaped less viscous plumes or more viscous finger-shaped diapirs, depending on the viscosity ratio between the rising and the matrix materials, are known to migrate through ductile, quasi-newtonian lithosphere; while dikes fracture and propagate through a solid matrix. But what happens in between these two end-members? To answer this question, we perform a combined study of laboratory experiments and numerical simulations on the development of thermal plumes in aqueous solutions of Carbopol, a polymer gel suspension forming a continous network of micrometric sponges. This fluid is shear thinning and presents a yield-stress, whereby flow occurs only if the local stress exceeds a critical value. Below this value, the fluid acts as an elastic solid. Our experimental setup consists of a localized heat-source, placed in the center of a squared plexiglas tank. At t=0, a constant thermal power is applied locally to the fluid. For the numerical simulations, we replace the rigid plastic regions by an extremely viscous fluid, and therefore neglect the elastic contribution to the local stress. We systematically studied the influence of the rheological parameters, as well as the supplied heat. Depending on the Yield number Y0, which compares the thermally-induced stress to the yield stress, three different regims are observed. For low Y0, no convection develops; while for intermediate values, a small-scale convection cell appears and remains confined around the heater. For high Y0, thermal instabilities rise through the tank. Their morphology differs from the mushroom-shape typically encountered in newtonian fluids. Combined temperature and velocity field measurements show that a plug flow develops within the plume thermal anomaly, therefore producing a

Implementation of Good Clinical Laboratory Practice (GCLP) guidelines within the External Quality Assurance Program Oversight Laboratory (EQAPOL).

PubMed

Todd, Christopher A; Sanchez, Ana M; Garcia, Ambrosia; Denny, Thomas N; Sarzotti-Kelsoe, Marcella

2014-07-01

The EQAPOL contract was awarded to Duke University to develop and manage global proficiency testing programs for flow cytometry-, ELISpot-, and Luminex bead-based assays (cytokine analytes), as well as create a genetically diverse panel of HIV-1 viral cultures to be made available to National Institutes of Health (NIH) researchers. As a part of this contract, EQAPOL was required to operate under Good Clinical Laboratory Practices (GCLP) that are traditionally used for laboratories conducting endpoint assays for human clinical trials. EQAPOL adapted these guidelines to the management of proficiency testing programs while simultaneously incorporating aspects of ISO/IEC 17043 which are specifically designed for external proficiency management. Over the first two years of the contract, the EQAPOL Oversight Laboratories received training, developed standard operating procedures and quality management practices, implemented strict quality control procedures for equipment, reagents, and documentation, and received audits from the EQAPOL Central Quality Assurance Unit. GCLP programs, such as EQAPOL, strengthen a laboratory's ability to perform critical assays and provide quality assessments of future potential vaccines. © 2013.

Against the proposition: for the diagnosis of viral infections, commercial assays provide more reliable results than do in-house assays.

PubMed

James, Vivienne

2008-01-01

There are no differences inherent in the design of commercial or in-house assays and their early development is similar. The same principles apply and it is on the same criteria of accuracy, reproducibility and clinical relevance of results that all assays are judged. However, if there is sufficient uptake of a commercial assay, its strengths and any flaws soon become apparent and it will only be the best commercial assays that remain in the market. For the in-house assays it is through comparability studies and external quality assessment (EQA) schemes that the best can be demonstrated, albeit this information is only accessible initially to the EQA provider and the laboratories using the assays. The EQA results described here support my supposition that, for the diagnosis of viral infections, commercial assays do not provide more reliable results than do in-house assays.

International network for comparison of HIV neutralization assays: the NeutNet report.

PubMed

Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

2009-01-01

Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing

Thermal Properties of G-348 Graphite

DOE Office of Scientific and Technical Information (OSTI.GOV)

McEligot, Donald; Swank, W. David; Cottle, David L.

2016-05-01

Fundamental measurements have been obtained in the INL Graphite Characterization Laboratory to deduce the temperature dependence of thermal conductivity for G-348 isotropic graphite, which has been used by City College of New York in thermal experiments related to gas-cooled nuclear reactors. Measurements of thermal diffusivity, mass, volume and thermal expansion were converted to thermal conductivity in accordance with ASTM Standard Practice C781-08. Data are tabulated and a preliminary correlation for the thermal conductivity is presented as a function of temperature from laboratory temperature to 1000C.

Thermal Properties of G-348 Graphite

DOE Office of Scientific and Technical Information (OSTI.GOV)

McEligot, Donald M.; Swank, W. David; Cottle, David L.

Fundamental measurements have been obtained in the INL Graphite Characterization Laboratory to deduce the temperature dependence of thermal conductivity for G-348 isotropic graphite, which has been used by City College of New York in thermal experiments related to gas-cooled nuclear reactors. Measurements of thermal diffusivity, mass, volume and thermal expansion were converted to thermal conductivity in accordance with ASTM Standard Practice C781-08 (R-2014). Data are tabulated and a preliminary correlation for the thermal conductivity is presented as a function of temperature from laboratory temperature to 1000C.

Interpreting the Laboratory Reports for Vit D.

PubMed

Sarmah, Devajit; Sharma, Booloo

2014-09-01

Importance for Vit D estimation has increased in the recent years due to its link to various diseases. Measurements of Vit D by different diagnostic laboratories is however not uniform. There is variation pertaining to assay methodology and also variation in the measurement of different metabolites of Vit D. There are also various confounders which influence Vit D assays and which in most instances are overlooked. Also a matter of concern regarding Vit D assays is the lack of assay standardisation. These factors contribute to the variation in the reports generated by the diagnostic laboratories. Therefore interpretation of Vit D reports needs proper understanding of these interfering factors and further reports need to be correlated substantially with the clinical findings.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Accelerated Colorimetric Micro-assay for Screening Mold Inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2014-01-01

Rapid quantitative laboratory test methods are needed to screen potential antifungal agents. Existing laboratory test methods are relatively time consuming, may require specialized test equipment and rely on subjective visual ratings. A quantitative, colorimetric micro-assay has been developed that uses XTT tetrazolium salt to metabolically assess mold spore...

Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies.

PubMed

Kitchen, Steve; Tiefenbacher, Stefan; Gosselin, Robert

2017-04-01

The advent of modified factor VIII (FVIII) and factor IX (FIX) molecules with extended half-lives (EHLs) compared with native FVIII and FIX represents a major advance in the field of hemophilia care, with the potential to reduce the frequency of prophylactic injections and/or to increase the trough level prior to subsequent injections. Monitoring treatment through laboratory assays will be an important part of ensuring patient safety, including any tailoring of prophylaxis. Several approaches have been used to extend half-lives, including PEGylation, and fusion to albumin or immunoglobulin. Some of these modifications affect factor assays as routinely performed in hemophilia centers; so, laboratories will need to use FVIII and FIX assays which have been shown to be suitable on a product-by-product basis. For some products, there are marked differences between results obtained using one-stage or chromogenic assays and results obtained using different reagents in the one-stage assay. The laboratory should use an assay in which the recovery of the product closely aligns with the assay used by the pharmaceutical company to assign potency to the product, so that the units reported by the laboratory agree with those used to demonstrate efficacy of the product during clinical trials. Reported assay differences in relation to several of the EHL FVIII and FIX molecules will be reviewed in this article. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Ground Laboratory Soft X-Ray Durability Evaluation of Aluminized Teflon FEP Thermal Control Insulation

NASA Technical Reports Server (NTRS)

Banks, Bruce A.; deGroh, Kim K.; Stueber, Thomas J.; Sechkar, Edward A.

1998-01-01

Metallized Teflon fluorinated ethylene propylene (FEP) thermal control insulation is mechanically degraded if exposed to a sufficient fluence of soft x-ray radiation. Soft x-ray photons (4-8 A in wavelength or 1.55 - 3.2 keV) emitted during solar flares have been proposed as a cause of mechanical properties degradation of aluminized Teflon FEP thermal control insulation on the Hubble Space Telescope (HST). Such degradation can be characterized by a reduction in elongation-to-failure of the Teflon FER Ground laboratory soft x-ray exposure tests of aluminized Teflon FEP were conducted to assess the degree of elongation degradation which would occur as a result of exposure to soft x-rays in the range of 3-10 keV. Tests results indicate that soft x-ray exposure in the 3-10 keV range, at mission fluence levels, does not alone cause the observed reduction in elongation of flight retrieved samples. The soft x-ray exposure facility design, mechanical properties degradation results and implications will be presented.

Thermal behavior of an asphalt pavement in the laboratory and in the parking lot.

PubMed

Martinkauppi, J B; Mäkiranta, A; Kiijärvi, J; Hiltunen, E

2015-01-01

The urban, constructed areas are full of buildings and different kinds of pavements and have a noticeable lack of trees and flora. These areas are accumulating the heat from the Sun, people, vehicles, and constructions. One interesting heat collector is the asphalt pavement. How does the heat transfer to different layers under the pavement or does it? What are the temperatures under the pavement in Finland where the winter can be pretty hard? How can those temperatures be measured accurately? These are the main questions this paper gives the preliminary answers to. First the thermal behavior of asphalt and the layers beneath are researched in the laboratory and then the measurement field is bored and dug in the parking in the Western coast of Finland, 63°5'45'' N. Distributed temperature sensing method was found to be a good choice for temperature measurements. Thermal behavior of pavement has been monitored in different layers and the preliminary results have been published here. The goal of this research is to assess the applicability of asphalt pavements for heat energy collection.

Audit of Use and Overuse of Serum Protein Immunofixation Electrophoresis and Serum Free Light Chain Assay in Tertiary Health Care: A Case for Algorithmic Testing to Optimize Laboratory Utilization.

PubMed

Heaton, Christopher; Vyas, Shikhar G; Singh, Gurmukh

2016-04-01

Overuse of laboratory tests is a persistent issue. We examined the use and overuse of serum immunofixation electrophoresis and serum free light chain assays to develop an algorithm for optimizing utilization. A retrospective review of all tests, for investigation of monoclonal gammopathies, for all patients who had any of these tests done from April 24, 2014, through July 25, 2014, was carried out. The test orders were categorized as warranted or not warranted according to criteria presented in the article. A total of 237 patients were tested, and their historical records included 1,503 episodes of testing for one or more of serum protein electrophoresis, serum immunofixation electrophoresis, and serum free light chain assays. Only 46% of the serum immunofixation and 42% serum free light chain assays were warranted. Proper utilization, at our institution alone, would have obviated $64,182.95/year in health care costs, reduced laboratory cost of reagent alone by $26,436.04/year, and put $21,904.92/year of part B reimbursement at risk. Fewer than half of the serum immunofixation and serum free light chain assays added value. The proposed algorithm for testing should improve utilization. Risk to part B billing may be a disincentive to reducing test utilization. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Glucose-6-phosphate dehydrogenase laboratory assay: How, when, and why?

PubMed

Minucci, Angelo; Giardina, Bruno; Zuppi, Cecilia; Capoluongo, Ettore

2009-01-01

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD-deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow-chart for the complete diagnostic approach of G6PD-deficiency.

Differences in serum thyroglobulin measurements by 3 commercial immunoradiometric assay kits and laboratory standardization using Certified Reference Material 457 (CRM-457).

PubMed

Lee, Ji In; Kim, Ji Young; Choi, Joon Young; Kim, Hee Kyung; Jang, Hye Won; Hur, Kyu Yeon; Kim, Jae Hyeon; Kim, Kwang-Won; Chung, Jae Hoon; Kim, Sun Wook

2010-09-01

Serum thyroglobulin (Tg) is essential in the follow-up of patients with differentiated thyroid carcinoma (DTC). However, interchangeability and standardization between Tg assays have not yet been achieved, even with the development of an international Tg standard (Certified Reference Material 457 [CRM-457]). Serum Tg from 30 DTC patients and serially diluted CRM-457 were measured using 3 different immunoradiometric assays (IRMA-1, IRMA-2, IRMA-3). The intraclass correlation coefficient (ICC) method was used to describe the concordance of each IRMA to CRM-457. The serum Tg measured by 3 different IRMAs correlated well (r > .85, p < .0001), but clinically relevant discrepancies were found in 13.3% of patients. IRMA-3, which claims to be standardized to CRM-457, showed the best ICC (p(1) = .98) for the CRM-457. Hospitals caring for patients with DTC should either set their own cutoffs for IRMAs for Tg based on their patient pools, or adopt IRMAs standardized to CRM-457 and calibrate their laboratory using CRM-457.

Computer simulation of thermal and fluid systems for MIUS integration and subsystems test /MIST/ laboratory. [Modular Integrated Utility System

NASA Technical Reports Server (NTRS)

Rochelle, W. C.; Liu, D. K.; Nunnery, W. J., Jr.; Brandli, A. E.

1975-01-01

This paper describes the application of the SINDA (systems improved numerical differencing analyzer) computer program to simulate the operation of the NASA/JSC MIUS integration and subsystems test (MIST) laboratory. The MIST laboratory is designed to test the integration capability of the following subsystems of a modular integrated utility system (MIUS): (1) electric power generation, (2) space heating and cooling, (3) solid waste disposal, (4) potable water supply, and (5) waste water treatment. The SINDA/MIST computer model is designed to simulate the response of these subsystems to externally impressed loads. The computer model determines the amount of recovered waste heat from the prime mover exhaust, water jacket and oil/aftercooler and from the incinerator. This recovered waste heat is used in the model to heat potable water, for space heating, absorption air conditioning, waste water sterilization, and to provide for thermal storage. The details of the thermal and fluid simulation of MIST including the system configuration, modes of operation modeled, SINDA model characteristics and the results of several analyses are described.

Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

PubMed

Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

2015-11-01

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. Copyright © 2015 Elsevier B.V. All rights

Design and Preliminary Thermal Performance of the Mars Science Laboratory Rover Heat Exchangers

NASA Technical Reports Server (NTRS)

Mastropietro, A. J.; Beatty, John; Kelly, Frank; Birur, Gajanana; Bhandari, Pradeep; Pauken, Michael; Illsley, Peter; Liu, Yuanming; Bame, David; Miller, Jennifer

2010-01-01

The challenging range of proposed landing sites for the Mars Science Laboratory Rover requires a rover thermal management system that is capable of keeping temperatures controlled across a wide variety of environmental conditions. On the Martian surface where temperatures can be as cold as -123 degrees Centigrade and as warm as 38 degrees Centigrade, the Rover relies upon a Mechanically Pumped Fluid Loop (MPFL) and external radiators to maintain the temperature of sensitive electronics and science instruments within a -40 degrees Centigrade to 50 degrees Centigrade range. The MPFL also manages significant waste heat generated from the Rover power source, known as the Multi Mission Radioisotope Thermoelectric Generator (MMRTG). The MMRTG produces 110 Watts of electrical power while generating waste heat equivalent to approximately 2000 Watts. Two similar Heat Exchanger (HX) assemblies were designed to both acquire the heat from the MMRTG and radiate waste heat from the onboard electronics to the surrounding Martian environment. Heat acquisition is accomplished on the interior surface of each HX while heat rejection is accomplished on the exterior surface of each HX. Since these two surfaces need to be at very different temperatures in order for the MPFL to perform efficiently, they need to be thermally isolated from one another. The HXs were therefore designed for high in-plane thermal conductivity and extremely low through-thickness thermal conductivity by using aerogel as an insulator inside composite honeycomb sandwich panels. A complex assembly of hand welded and uniquely bent aluminum tubes are bonded onto the HX panels and were specifically designed to be easily mated and demated to the rest of the Rover Heat Recovery and Rejection System (RHRS) in order to ease the integration effort. During the cruise phase to Mars, the HX assemblies serve the additional function of transferring heat from the Rover MPFL to the separate Cruise Stage MPFL so that heat

Guidelines on Good Clinical Laboratory Practice

PubMed Central

Ezzelle, J.; Rodriguez-Chavez, I. R.; Darden, J. M.; Stirewalt, M.; Kunwar, N.; Hitchcock, R.; Walter, T.; D’Souza, M. P.

2008-01-01

A set of Good Clinical Laboratory Practice (GCLP) standards that embraces both the research and clinical aspects of GLP were developed utilizing a variety of collected regulatory and guidance material. We describe eleven core elements that constitute the GCLP standards with the objective of filling a gap for laboratory guidance, based on IND sponsor requirements, for conducting laboratory testing using specimens from human clinical trials. These GCLP standards provide guidance on implementing GLP requirements that are critical for laboratory operations, such as performance of protocol-mandated safety assays, peripheral blood mononuclear cell processing and immunological or endpoint assays from biological interventions on IND-registered clinical trials. The expectation is that compliance with the GCLP standards, monitored annually by external audits, will allow research and development laboratories to maintain data integrity and to provide immunogenicity, safety, and product efficacy data that is repeatable, reliable, auditable and that can be easily reconstructed in a research setting. PMID:18037599

The narrow therapeutic window of glycated hemoglobin and assay variability.

PubMed

Hosseini, S S; Bibler, I; Charles, M A

1999-12-01

Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

PubMed Central

Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

2009-01-01

Background Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Methods Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. Findings PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. Conclusions The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting

Comparing ImmunoCard with two EIA assays for Clostridium difficile toxins.

PubMed

Chan, Edward L; Seales, Diane; Drum, Hong

2009-01-01

To compare three Clostridium difficile EIA kits for the detection of C. difficile toxins from clinical specimens. A total of 287 fresh and stored stool specimens were tested using all three assays. Stools with discrepant results were sent to a reference laboratory for tissue cytotoxin assay. Trinity Medical Center, a community hospital with network hospitals. Patients with diarrhea submitted stools for detection of C. difficile toxins. Of the 287 stool specimens, 116 were positive and 171 negative for C. difficile toxins. The sensitivity, specificity, and positive and negative predictive values of Meridian EIA assay were 99.1, 97.7, 96.6, and 99.4%; ImmunoCard were 100, 98.2, 97.5, and 100%; BioStar OIA assay were 94, 98.8, 98.2, and 96% respectively. ImmunoCardprovides the best sensitivity (100%) for C. difficile toxins A and B detection. The BioStar OIA rapid test missed seven positive stool specimens possibly due to failure to detect toxin B. ImmunoCard has slightly higher predictive values, shorter turnaround time and greater convenience compared to the Meridian EIA Assay. ImmunoCard may be cost effective not only in smaller laboratories, but also in high volume laboratories, when used on a STAT basis or single request.

The EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations.

PubMed

Roy, Shambhu; Kulkarni, Rohan; Hewitt, Nicola J; Aardema, Marilyn J

2016-07-01

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline 487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48h and 72h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMN assay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermally-Induced Chemistry and the Jovian Icy Satellites: A Laboratory Study of the Formation of Sulfur Oxyanions

NASA Technical Reports Server (NTRS)

Loeffler, Mark J.; Hudson, Reggie L.

2011-01-01

Laboratory experiments have demonstrated that magnetospheric radiation in the Jovian system drives reaction chemistry in ices at temperatures relevant to Europa and other icy satellites. Here we present new results on thermally-induced reactions at 50-100 K in solid H2O-SO2 mixtures, reactions that take place without the need for a high-radiation environment. We find that H2O and SO2 react to produce sulfur Oxyanions, such as bisulfite, that as much as 30% of the SO2 can be consumed through this reaction, and that the products remain in the ice when the temperature is lowered, indicating that these reactions are irreversible. Our results suggest that thermally-induced reactions can alter the chemistry at temperatures relevant to the icy satellites in the Jovian system.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

PubMed

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

PubMed Central

Ramage, Jason G.; Prentice, Kristin W.; DePalma, Lindsay; Venkateswaran, Kodumudi S.; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R.; Sharma, Shashi; Estacio, Peter L.; Stanker, Larry; Hodge, David R.

2016-01-01

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert® test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 106 spores/mL (ca. 1.5 × 105 spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

PubMed

Islam, Rafiq; Briscoe, Chad; Bower, Joseph; Cape, Stephanie; Arnold, Mark; Hayes, Roger; Warren, Mark; Karnik, Shane; Stouffer, Bruce; Xiao, Yi Qun; van der Strate, Barry; Sikkema, Daniel; Fang, Xinping; Tudoroniu, Ariana; Tayyem, Rabab; Brant, Ashley; Spriggs, Franklin; Barry, Colin; Khan, Masood; Keyhani, Anahita; Zimmer, Jennifer; Caturla, Maria Cruz; Couerbe, Philippe; Khadang, Ardeshir; Bourdage, James; Datin, Jim; Zemo, Jennifer; Hughes, Nicola; Fatmi, Saadya; Sheldon, Curtis; Fountain, Scott; Satterwhite, Christina; Colletti, Kelly; Vija, Jenifer; Yu, Mathilde; Stamatopoulos, John; Lin, Jenny; Wilfahrt, Jim; Dinan, Andrew; Ohorodnik, Susan; Hulse, James; Patel, Vimal; Garofolo, Wei; Savoie, Natasha; Brown, Michael; Papac, Damon; Buonarati, Mike; Hristopoulos, George; Beaver, Chris; Boudreau, Nadine; Williard, Clark; Liu, Yansheng; Ray, Gene; Warrino, Dominic; Xu, Allan; Green, Rachel; Hayward-Sewell, Joanne; Marcelletti, John; Sanchez, Christina; Kennedy, Michael; Charles, Jessica St; Bouhajib, Mohammed; Nehls, Corey; Tabler, Edward; Tu, Jing; Joyce, Philip; Iordachescu, Adriana; DuBey, Ira; Lindsay, John; Yamashita, Jim; Wells, Edward

2018-04-01

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

PubMed

Pillet, Sylvie; Verhoeven, Paul O; Epercieux, Amélie; Bourlet, Thomas; Pozzetto, Bruno

2015-06-01

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Thermoreception and nociception of the skin: a classic paper of Bessou and Perl and analyses of thermal sensitivity during a student laboratory exercise.

PubMed

Kuhtz-Buschbeck, Johann P; Andresen, Wiebke; Göbel, Stephan; Gilster, René; Stick, Carsten

2010-06-01

About four decades ago, Perl and collaborators were the first ones who unambiguously identified specifically nociceptive neurons in the periphery. In their classic work, they recorded action potentials from single C-fibers of a cutaneous nerve in cats while applying carefully graded stimuli to the skin (Bessou P, Perl ER. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J Neurophysiol 32: 1025-1043, 1969). They discovered polymodal nociceptors, which responded to mechanical, thermal, and chemical stimuli in the noxious range, and differentiated them from low-threshold thermoreceptors. Their classic findings form the basis of the present method that undergraduate medical students experience during laboratory exercises of sensory physiology, namely, quantitative testing of the thermal detection and pain thresholds. This diagnostic method examines the function of thin afferent nerve fibers. We collected data from nearly 300 students that showed that 1) women are more sensitive to thermal detection and thermal pain at the thenar than men, 2) habituation shifts thermal pain thresholds during repetititve testing, 3) the cold pain threshold is rather variable and lower when tested after heat pain than in the reverse case (order effect), and 4) ratings of pain intensity on a visual analog scale are correlated with the threshold temperature for heat pain but not for cold pain. Median group results could be reproduced in a retest. Quantitative sensory testing of thermal thresholds is feasible and instructive in the setting of a laboratory exercise and is appreciated by the students as a relevant and interesting technique.

13C Nuclear magnetic resonance studies of kerogen from Cretaceous black shales thermally altered by basaltic intrusions and laboratory simulations

USGS Publications Warehouse

Dennis, L.W.; Maciel, G.E.; Hatcher, P.G.; Simoneit, B.R.T.

1982-01-01

Cretaceous black shales from DSDP Leg 41, Site 368 in the Eastern Atlantic Ocean were thermally altered during the Miocene by an intrusive basalt. The sediments overlying and underlying the intrusive body were subjected to high temperatures (up to ~ 500??C) and, as a result, their kerogen was significantly altered. The extent of this alteration has been determined by examination by means of 13C nuclear magnetic resonance, using cross polarization/magic-angle spinning (CP/MAS). Results indicate that the kerogen becomes progressively more aromatic in the vicinity of the intrusive body. Laboratory heating experiments, simulating the thermal effects of the basaltic intrusion, produced similar results on unaltered shale from the drill core. The 13C CP/MAS results appear to provide a good measure of thermal alteration. ?? 1982.

Laboratory compliance with the American Society of Clinical Oncology/college of American Pathologists guidelines for human epidermal growth factor receptor 2 testing: a College of American Pathologists survey of 757 laboratories.

PubMed

Nakhleh, Raouf E; Grimm, Erin E; Idowu, Michael O; Souers, Rhona J; Fitzgibbons, Patrick L

2010-05-01

To ensure quality human epidermal growth receptor 2 (HER2) testing in breast cancer, the American Society of Clinical Oncology/College of American Pathologists guidelines were introduced with expected compliance by 2008. To assess the effect these guidelines have had on pathology laboratories and their ability to address key components. In late 2008, a survey was distributed with the HER2 immunohistochemistry (IHC) proficiency testing program. It included questions regarding pathology practice characteristics and assay validation using fluorescence in situ hybridization or another IHC laboratory assay and assessed pathologist HER2 scoring competency. Of the 907 surveys sent, 757 (83.5%) were returned. The median laboratory accessioned 15 000 cases and performed 190 HER2 tests annually. Quantitative computer image analysis was used by 33% of laboratories. In-house fluorescence in situ hybridization was performed in 23% of laboratories, and 60% of laboratories addressed the 6- to 48-hour tissue fixation requirement by embedding tissue on the weekend. HER2 testing was performed on the initial biopsy in 40%, on the resection specimen in 6%, and on either in 56% of laboratories. Testing was validated with only fluorescence in situ hybridization in 47% of laboratories, whereas 10% of laboratories used another IHC assay only; 13% used both assays, and 12% and 15% of laboratories had not validated their assays or chose "not applicable" on the survey question, respectively. The 90% concordance rate with fluorescence in situ hybridization results was achieved by 88% of laboratories for IHC-negative findings and by 81% of laboratories for IHC-positive cases. The 90% concordance rate for laboratories using another IHC assay was achieved by 80% for negative findings and 75% for positive cases. About 91% of laboratories had a pathologist competency assessment program. This survey demonstrates the extent and characteristics of HER2 testing. Although some American Society of

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay

DTIC Science & Technology

2015-12-01

several commercially available assays from VecTOR Test Systems Inc. for malaria, West Nile virus, Rift Valley fever virus, dengue , chikungunya, and...Sabin AB. 1955. Recent advances in our knowledge of dengue and sandfly fever. Am J Trop Med Hyg 4:198–207. Sather GE. 1970. Catalogue of arthropod

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

PubMed Central

Morales-Soto, Nydia; Anyan, Morgen E.; Mattingly, Anne E.; Madukoma, Chinedu S.; Harvey, Cameron W.; Alber, Mark; Déziel, Eric; Kearns, Daniel B.; Shrout, Joshua D.

2015-01-01

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment. PMID:25938934

Thermal Behavior of an Asphalt Pavement in the Laboratory and in the Parking Lot

PubMed Central

Martinkauppi, J. B.; Mäkiranta, A.; Kiijärvi, J.; Hiltunen, E.

2015-01-01

The urban, constructed areas are full of buildings and different kinds of pavements and have a noticeable lack of trees and flora. These areas are accumulating the heat from the Sun, people, vehicles, and constructions. One interesting heat collector is the asphalt pavement. How does the heat transfer to different layers under the pavement or does it? What are the temperatures under the pavement in Finland where the winter can be pretty hard? How can those temperatures be measured accurately? These are the main questions this paper gives the preliminary answers to. First the thermal behavior of asphalt and the layers beneath are researched in the laboratory and then the measurement field is bored and dug in the parking in the Western coast of Finland, 63°5′45′′ N. Distributed temperature sensing method was found to be a good choice for temperature measurements. Thermal behavior of pavement has been monitored in different layers and the preliminary results have been published here. The goal of this research is to assess the applicability of asphalt pavements for heat energy collection. PMID:25861679

Dr. Johney Green Jr. - Associate Laboratory Director for Mechanical and

Science.gov Websites

Thermal Engineering Sciences | NREL Dr. Johney Green Jr. - Associate Laboratory Director for Mechanical and Thermal Engineering Sciences Dr. Johney Green Jr. - Associate Laboratory Director for Mechanical and Thermal Engineering Sciences A photo of Johney Green In his role, Johney Green oversees early

Assay optimisation and technology transfer for multi-site immuno-monitoring in vaccine trials

PubMed Central

Harris, Stephanie A.; Satti, Iman; Bryan, Donna; Walker, K. Barry; Dockrell, Hazel M.; McShane, Helen; Ho, Mei Mei

2017-01-01

Cellular immunological assays are important tools for the monitoring of responses to T-cell-inducing vaccine candidates. As these bioassays are often technically complex and require considerable experience, careful technology transfer between laboratories is critical if high quality, reproducible data that allows comparison between sites, is to be generated. The aim of this study, funded by the European Union Framework Program 7-funded TRANSVAC project, was to optimise Standard Operating Procedures and the technology transfer process to maximise the reproducibility of three bioassays for interferon-gamma responses: enzyme-linked immunosorbent assay (ELISA), ex-vivo enzyme-linked immunospot and intracellular cytokine staining. We found that the initial variability in results generated across three different laboratories reduced following a combination of Standard Operating Procedure harmonisation and the undertaking of side-by-side training sessions in which assay operators performed each assay in the presence of an assay ‘lead’ operator. Mean inter-site coefficients of variance reduced following this training session when compared with the pre-training values, most notably for the ELISA assay. There was a trend for increased inter-site variability at lower response magnitudes for the ELISA and intracellular cytokine staining assays. In conclusion, we recommend that on-site operator training is an essential component of the assay technology transfer process and combined with harmonised Standard Operating Procedures will improve the quality, reproducibility and comparability of data produced across different laboratories. These data may be helpful in ongoing discussions of the potential risk/benefit of centralised immunological assay strategies for large clinical trials versus decentralised units. PMID:29020010

Multiparameter behavioral profiling reveals distinct thermal response regimes in Caenorhabditis elegans

PubMed Central

2012-01-01

Background Responding to noxious stimuli by invoking an appropriate escape response is critical for survival of an organism. The sensations of small and large changes in temperature in most organisms have been studied separately in the context of thermotaxis and nociception, respectively. Here we use the nematode C. elegans to address the neurogenetic basis of responses to thermal stimuli over a broad range of intensities. Results C. elegans responds to aversive temperature by eliciting a stereotypical behavioral sequence. Upon sensation of the noxious stimulus, it moves backwards, turns and resumes forward movement in a new direction. In order to study the response of C. elegans to a broad range of noxious thermal stimuli, we developed a novel assay that allows simultaneous characterization of multiple aspects of escape behavior elicited by thermal pulses of increasing amplitudes. We exposed the laboratory strain N2, as well as 47 strains with defects in various aspects of nervous system function, to thermal pulses ranging from ΔT = 0.4°C to 9.1°C and recorded the resulting behavioral profiles. Conclusions Through analysis of the multidimensional behavioral profiles, we found that the combinations of molecules shaping avoidance responses to a given thermal pulse are unique. At different intensities of aversive thermal stimuli, these distinct combinations of molecules converge onto qualitatively similar stereotyped behavioral sequences. PMID:23114012

Enzyme-Linked Antibodies: A Laboratory Introduction to the ELISA Assay

NASA Astrophysics Data System (ADS)

Anderson, Gretchen L.; McNellis, Leo A.

1998-10-01

A fast and economical laboratory exercise is presented that qualitatively demonstrates the power of enzyme-linked antibodies to detect a specific antigen. Although ELISAs are commonly used in disease diagnosis in clinical settings, this application uses biotin, covalently attached to albumin, to take advantage of readily available reagents and avoids problems associated with potentially pathogenic antigens. The laboratory exercise is suitable for high school or freshman level biochemistry courses, and can be completed within two hours.

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays.

PubMed

Hau, Jean Christophe; Fontana, Patrizia; Zimmermann, Catherine; De Pover, Alain; Erdmann, Dirk; Chène, Patrick

2011-06-01

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

NASA Dryden Flight Loads Laboratory

NASA Technical Reports Server (NTRS)

Horn, Tom

2008-01-01

This viewgraph presentation reviews the work of the Dryden Flight Loads Laboratory. The capabilities and research interests of the lab are: Structural, thermal, & dynamic analysis; Structural, thermal, & dynamic ground-test techniques; Advanced structural instrumentation; and Flight test support.

Development and implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells.

PubMed

Todd, Christopher A; Greene, Kelli M; Yu, Xuesong; Ozaki, Daniel A; Gao, Hongmei; Huang, Yunda; Wang, Maggie; Li, Gary; Brown, Ronald; Wood, Blake; D'Souza, M Patricia; Gilbert, Peter; Montefiori, David C; Sarzotti-Kelsoe, Marcella

2012-01-31

Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents. A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass-fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms. Copyright © 2011 Elsevier B.V. All rights reserved.

The Pitfalls of Companion Diagnostics: Evaluation of Discordant EGFR Mutation Results from a Clinical Laboratory and a Central Laboratory.

PubMed

Turner, Scott A; Peterson, Jason D; Pettus, Jason R; de Abreu, Francine B; Amos, Christopher I; Dragnev, Konstantin H; Tsongalis, Gregory J

2016-05-01

Accurate identification of somatic mutations in formalin-fixed, paraffin-embedded tumor tissue is required for enrollment into clinical trials for many novel targeted therapeutics, including trials requiring EGFR mutation status in non-small-cell lung carcinomas. Central clinical trial laboratories contracted to perform this analysis typically rely on US Food and Drug Administration-approved targeted assays to identify these mutations. We present two cases in which central laboratories inaccurately reported EGFR mutation status because of improper identification and isolation of tumor material and failure to accurately report assay limitations, resulting in enrollment denial. Such cases highlight the need for increased awareness by clinical trials of the limitation of these US Food and Drug Administration-approved assays and the necessity for a mechanism to reevaluate discordant results by alternative laboratory-developed procedures, including clinical next-generation sequencing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Ground Laboratory Soft X-Ray Durability Evaluation of Aluminized Teflon FEP Thermal Control Insulation. Revised

NASA Technical Reports Server (NTRS)

Banks, Bruce A.; deGroh, Kim K.; Stueber, Thomas J.; Sechkar, Edward A.; Hall, Rachelle L.

1998-01-01

Metallized Teflon fluorinated ethylene propylene (FEP) thermal control insulation is mechanically degraded if exposed to a sufficient fluence of soft x-ray radiation. Soft x-ray photons (4-8 A in wavelength or 1.55 - 3.2 keV) emitted during solar flares have been proposed as a cause of mechanical properties degradation of aluminized Teflon FEP thermal control insulation on the Hubble Space Telescope (HST). Such degradation can be characterized by a reduction in elongation-to-failure of the Teflon FEP. Ground laboratory soft x-ray exposure tests of aluminized Teflon FEP were conducted to assess the degree of elongation degradation which would occur as a result of exposure to soft x-rays in the range of 3-10 keV. Tests results indicate that soft x-ray exposure in the 3-10 keV range, at mission fluence levels, does not alone cause the observed reduction in elongation of flight retrieved samples. The soft x-ray exposure facility design, mechanical properties degradation results and implications will be presented.

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

PubMed

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths. We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings

PubMed Central

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Background Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. Methodology/Principal Findings In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the “archaic” method of hand-transferring PCR tubes between water baths. Conclusions/Significance We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach

Bioanalytical method transfer considerations of chromatographic-based assays.

PubMed

Williard, Clark V

2016-07-01

Bioanalysis is an important part of the modern drug development process. The business practice of outsourcing and transferring bioanalytical methods from laboratory to laboratory has increasingly become a crucial strategy for successful and efficient delivery of therapies to the market. This chapter discusses important considerations when transferring various types of chromatographic-based assays in today's pharmaceutical research and development environment.

Designing experiments on thermal interactions by secondary-school students in a simulated laboratory environment

NASA Astrophysics Data System (ADS)

Lefkos, Ioannis; Psillos, Dimitris; Hatzikraniotis, Euripides

2011-07-01

Background and purpose: The aim of this study was to explore the effect of investigative activities with manipulations in a virtual laboratory on students' ability to design experiments. Sample Fourteen students in a lower secondary school in Greece attended a teaching sequence on thermal phenomena based on the use of information and communication technology, and specifically of the simulated virtual laboratory 'ThermoLab'. Design and methods A pre-post comparison was applied. Students' design of experiments was rated in eight dimensions; namely, hypothesis forming and verification, selection of variables, initial conditions, device settings, materials and devices used, process and phenomena description. A three-level ranking scheme was employed for the evaluation of students' answers in each dimension. Results A Wilcoxon signed-rank test revealed a statistically significant difference between the students' pre- and post-test scores. Additional analysis by comparing the pre- and post-test scores using the Hake gain showed high gains in all but one dimension, which suggests that this improvement was almost inclusive. Conclusions We consider that our findings support the statement that there was an improvement in students' ability to design experiments.

HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT

EPA Science Inventory

The rapid and sensitive detection of organophosphate insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelati...

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round.

PubMed

Ehling, G; Hecht, M; Heusener, A; Huesler, J; Gamer, A O; van Loveren, H; Maurer, Th; Riecke, K; Ullmann, L; Ulrich, P; Vandebriel, R; Vohr, H-W

2005-08-15

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8--10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A--C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.

Protein immobilization techniques for microfluidic assays

PubMed Central

Kim, Dohyun; Herr, Amy E.

2013-01-01

Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

Quantitative comparison of DNA methylation assays for biomarker development and clinical applications.

PubMed

2016-07-01

DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

Quality Assurance Specifications for Planetary Protection Assays

NASA Astrophysics Data System (ADS)

Baker, Amy

As the European Space Agency planetary protection (PP) activities move forward to support the ExoMars and other planetary missions, it will become necessary to increase staffing of labo-ratories that provide analyses for these programs. Standardization of procedures, a comprehen-sive quality assurance program, and unilateral training of personnel will be necessary to ensure that the planetary protection goals and schedules are met. The PP Quality Assurance/Quality Control (QAQC) program is designed to regulate and monitor procedures performed by labora-tory personnel to ensure that all work meets data quality objectives through the assembly and launch process. Because personnel time is at a premium and sampling schedules are often de-pendent on engineering schedules, it is necessary to have flexible staffing to support all sampling requirements. The most productive approach to having a competent and flexible work force is to establish well defined laboratory procedures and training programs that clearly address the needs of the program and the work force. The quality assurance specification for planetary protection assays has to ensure that labora-tories and associated personnel can demonstrate the competence to perform assays according to the applicable standard AD4. Detailed subjects included in the presentation are as follows: • field and laboratory control criteria • data reporting • personnel training requirements and certification • laboratory audit criteria. Based upon RD2 for primary and secondary validation and RD3 for data quality objectives, the QAQC will provide traceable quality assurance safeguards by providing structured laboratory requirements for guidelines and oversight including training and technical updates, standardized documentation, standardized QA/QC checks, data review and data archiving.

Policies and practices in haemostasis testing among laboratories in Croatia: a survey on behalf of a Working Group for Laboratory Coagulation of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

PubMed

Bronić, Ana; Herak, Desiree Coen; Margetić, Sandra; Milić, Marija

2017-02-15

The objective of this survey was to assess current policies and practice in haemostasis testing among both hospital and outpatient laboratories in Republic of Croatia. A questionnaire with seventy questions divided into nine sections was created in May 2015. Participants were asked about their practice related to test request form, sample collection, prothrombin time (PT) and activated partial thromboplastin time assays, other individual haemostasis assays, point-of-care testing (POCT), reporting of coagulation tests results and quality assurance of procedures, the personnel and other laboratory resources, as well as on issues related to education and implementation of additional coagulation assays in their laboratory. The survey was administered and data were collected between June and September 2015. A total survey response rate was 104/170 (61.2%). Most respondents were faced with incomplete information on prescribed therapy and diagnosis on the test request or inappropriate samples withdrawn on distant locations, but also do not have protocols for handling samples with high haematocrit values. Reporting of PT-INR and D-dimer results was different between laboratories. Although almost all laboratories developed a critical value reporting system, reporting a value to general practitioners is still a problem. Result on coagulation POCT testing showed that not all devices were supervised by laboratories, which is not in compliance with Croatian Chamber of Medical Biochemistry acts. Obtained results highlighted areas that need improvement and different practice patterns in particular field of haemostasis testing among laboratories. A harmonization of the overall process of haemostasis testing at national level should be considered and undertaken.

Temperament in bullheads: do laboratory and field explorative behaviour variables correlate?

NASA Astrophysics Data System (ADS)

Kobler, Alexander; Engelen, Brecht; Knaepkens, Guy; Eens, Marcel

2009-10-01

The relevance of temperament traits for life history strategy or productivity is increasingly acknowledged. Temperament traits are often either observed in captivity or in the wild, but studies combining both observations are very rare. We examine whether exploratory behaviour in the bullhead ( Cottus perifretum), assayed under laboratory conditions, predicts this behaviour under field conditions. Forty-three PIT-tagged individuals were first assayed for exploration of a novel environment in the aquarium and then released into an unfamiliar stream stretch, where they were later relocated using a mobile antenna. Explorative behaviour assayed in the laboratory was significantly positively related to the exploration in the field, thus predicting distance moved in the field release. Both in the laboratory and in the field, explorative behaviour was not related to individual body length. When bullheads that did not leave the refuge in the aquarium (laboratory assay) and, therefore, did not explore the new environment were excluded from the analysis, the correlation between laboratory and field explorative behaviour variables became weaker. However, overall, our results illustrate that exploration rate of bullheads in isolated single-individual experiments can be used to predict this behaviour in the natural ecosystem.

Accelerated stability assay (ASA) for colloidal systems.

PubMed

Chong, Josephine Y T; Mulet, Xavier; Boyd, Ben J; Drummond, Calum J

2014-05-12

Assessment of the stability of colloidal systems, in particular lyotropic liquid crystalline dispersions, such as cubosomes and hexosomes, is typically performed qualitatively or with limited throughput on specialized instruments. Here, an accelerated stability assay for colloidal particles has been developed in 384-well plates with standard laboratory equipment. These protocols enable quantitative assessments of colloidal stability. To demonstrate the applicability of the assay, several steric stabilizers for cubic phase nanostructured particles (cubosomes) have been compared to the current "gold standard" Pluronic F127.

FluxCTTX: A LIMS-based tool for management and analysis of cytotoxicity assays data

PubMed Central

2015-01-01

Background Cytotoxicity assays have been used by researchers to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds or screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical. These assays may be used as an alternative to animal experimentation and are becoming increasingly important in modern laboratories. However, the execution of these assays in large scale and different laboratories requires, among other things, the management of protocols, reagents, cell lines used as well as the data produced, which can be a challenge. The management of all this information is greatly improved by the utilization of computational tools to save time and guarantee quality. However, a tool that performs this task designed specifically for cytotoxicity assays is not yet available. Results In this work, we have used a workflow based LIMS -- the Flux system -- and the Together Workflow Editor as a framework to develop FluxCTTX, a tool for management of data from cytotoxicity assays performed at different laboratories. The main work is the development of a workflow, which represents all stages of the assay and has been developed and uploaded in Flux. This workflow models the activities of cytotoxicity assays performed as described in the OECD 129 Guidance Document. Conclusions FluxCTTX presents a solution for the management of the data produced by cytotoxicity assays performed at Interlaboratory comparisons. Its adoption will contribute to guarantee the quality of activities in the process of cytotoxicity tests and enforce the use of Good Laboratory Practices (GLP). Furthermore, the workflow developed is complete and can be adapted to other contexts and different tests for management of other types of data. PMID:26696462

Laboratory assessment of novel oral anticoagulants: method suitability and variability between coagulation laboratories.

PubMed

Helin, Tuukka A; Pakkanen, Anja; Lassila, Riitta; Joutsi-Korhonen, Lotta

2013-05-01

Laboratory tests to assess novel oral anticoagulants (NOACs) are under evaluation. Routine monitoring is unnecessary, but under special circumstances bioactivity assessment becomes crucial. We analyzed the effects of NOACs on coagulation tests and the availability of specific assays at different laboratories. Plasma samples spiked with dabigatran (Dabi; 120 and 300 μg/L) or rivaroxaban (Riva; 60, 146, and 305 μg/L) were sent to 115 and 38 European laboratories, respectively. International normalized ratio (INR) and activated partial thromboplastin time (APTT) were analyzed for all samples; thrombin time (TT) was analyzed specifically for Dabi and calibrated anti-activated factor X (anti-Xa) activity for Riva. We compared the results with patient samples. Results of Dabi samples were reported by 73 laboratories (13 INR and 9 APTT reagents) and Riva samples by 22 laboratories (5 INR and 4 APTT reagents). Both NOACs increased INR values; the increase was modest, albeit larger, for Dabi, with higher CV, especially with Quick (vs Owren) methods. Both NOACs dose-dependently prolonged the APTT. Again, the prolongation and CVs were larger for Dabi. The INR and APTT results varied reagent-dependently (P < 0.005), with less prolongation in patient samples. TT results (Dabi) and calibrated anti-Xa results (Riva) were reported by only 11 and 8 laboratories, respectively. The screening tests INR and APTT are suboptimal in assessing NOACs, having high reagent dependence and low sensitivity and specificity. They may provide information, if laboratories recognize their limitations. The variation will likely increase and the sensitivity differ in clinical samples. Specific assays measure NOACs accurately; however, few laboratories applied them. © 2013 American Association for Clinical Chemistry.

Transferability and within- and between-laboratory reproducibilities of EpiSensA for predicting skin sensitization potential in vitro: A ring study in three laboratories.

PubMed

Mizumachi, Hideyuki; Sakuma, Megumi; Ikezumi, Mayu; Saito, Kazutoshi; Takeyoshi, Midori; Imai, Noriyasu; Okutomi, Hiroko; Umetsu, Asami; Motohashi, Hiroko; Watanabe, Mika; Miyazawa, Masaaki

2018-05-03

The epidermal sensitization assay (EpiSensA) is an in vitro skin sensitization test method based on gene expression of four markers related to the induction of skin sensitization; the assay uses commercially available reconstructed human epidermis. EpiSensA has exhibited an accuracy of 90% for 72 chemicals, including lipophilic chemicals and pre-/pro-haptens, when compared with the results of the murine local lymph node assay. In this work, a ring study was performed by one lead and two naive laboratories to evaluate the transferability, as well as within- and between-laboratory reproducibilities, of EpiSensA. Three non-coded chemicals (two lipophilic sensitizers and one non-sensitizer) were tested for the assessment of transferability and 10 coded chemicals (seven sensitizers and three non-sensitizers, including four lipophilic chemicals) were tested for the assessment of reproducibility. In the transferability phase, the non-coded chemicals (two sensitizers and one non-sensitizer) were correctly classified at the two naive laboratories, indicating that the EpiSensA protocol was transferred successfully. For the within-laboratory reproducibility, the data generated with three coded chemicals tested in three independent experiments in each laboratory gave consistent predictions within laboratories. For the between-laboratory reproducibility, 9 of the 10 coded chemicals tested once in each laboratory provided consistent predictions among the three laboratories. These results suggested that EpiSensA has good transferability, as well as within- and between-laboratory reproducibility. Copyright © 2018 John Wiley & Sons, Ltd.

Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2'-deoxyuridine incorporation.

PubMed

Kojima, Hajime; Takeyoshi, Masahiro; Sozu, Takashi; Awogi, Takumi; Arima, Kazunori; Idehara, Kenji; Ikarashi, Yoshiaki; Kanazawa, Yukiko; Maki, Eiji; Omori, Takashi; Yuasa, Atsuko; Yoshimura, Isao

2011-01-01

The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2'-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.

Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments.

PubMed

Akbulut, D; Grant, K A; McLauchlin, J

2005-09-01

An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.

Clinical performance of the LCx HCV RNA quantitative assay.

PubMed

Bertuzis, Rasa; Hardie, Alison; Hottentraeger, Barbara; Izopet, Jacques; Jilg, Wolfgang; Kaesdorf, Barbara; Leckie, Gregor; Leete, Jean; Perrin, Luc; Qiu, Chunfu; Ran, Iris; Schneider, George; Simmonds, Peter; Robinson, John

2005-02-01

This study was conducted to assess the performance of the Abbott laboratories LCx HCV RNA Quantitative Assay (LCx assay) in the clinical setting. Four clinical laboratories measured LCx assay precision, specificity, and linearity. In addition, a method comparison was conducted between the LCx assay and the Roche HCV Amplicor Monitor, version 2.0 (Roche Monitor 2.0) and the Bayer VERSANT HCV RNA 3.0 Assay (Bayer bDNA 3.0) quantitative assays. For precision, the observed LCx assay intra-assay standard deviation (S.D.) was 0.060-0.117 log IU/ml, the inter-assay S.D. was 0.083-0.133 log IU/ml, the inter-lot S.D. was 0.105-0.177 log IU/ml, the inter-site S.D. was 0.099-0.190 log IU/ml, and the total S.D. was 0.113-0.190 log IU/ml. The specificity of the LCx assay was 99.4% (542/545; 95% CI, 98.4-99.9%). For linearity, the mean pooled LCx assay results were linear (r=0.994) over the range of the panel (2.54-5.15 log IU/ml). A method comparison demonstrated a correlation coefficient of 0.881 between the LCx assay and Roche Monitor 2.0, 0.872 between the LCx assay and Bayer bDNA 3.0, and 0.870 between Roche Monitor 2.0 and Bayer bDNA 3.0. The mean LCx assay result was 0.04 log IU/ml (95% CI, -0.08, 0.01) lower than the mean Roche Monitor 2.0 result, but 0.57 log IU/ml (95% CI, 0.53, 0.61) higher than the mean Bayer bDNA 3.0 result. The mean Roche Monitor 2.0 result was 0.60 log IU/ml (95% CI, 0.56, 0.65) higher than the mean Bayer bDNA 3.0 result. The LCx assay quantitated genotypes 1-4 with statistical equivalency. The vast majority (98.9%, 278/281) of paired LCx assay-Roche Monitor 2.0 specimen results were within 1 log IU/ml. Similarly, 86.6% (240/277) of paired LCx assay and Bayer bDNA 3.0 specimen results were within 1 log, as were 85.6% (237/277) of paired Roche Monitor 2.0 and Bayer specimen results. These data demonstrate that the LCx assay may be used for quantitation of HCV RNA in HCV-infected individuals.

Benzodiazepine Synthesis and Rapid Toxicity Assay

ERIC Educational Resources Information Center

Fletcher, James T.; Boriraj, Grit

2010-01-01

A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

Evaluation of the technical performance of novel holotranscobalamin (holoTC) assays in a multicenter European demonstration project.

PubMed

Morkbak, Anne L; Heimdal, Randi M; Emmens, Kathleen; Molloy, Anne; Hvas, Anne-Mette; Schneede, Joern; Clarke, Robert; Scott, John M; Ueland, Per M; Nexo, Ebba

2005-01-01

A commercially available holotranscobalamin (holo-TC) radioimmunoassay (RIA) (Axis-Shield, Dundee, Scotland) was evaluated in four laboratories and compared with a holoTC ELISA run in one laboratory. The performance of the holoTC RIA assay was comparable in three of the four participating laboratories. The results from these three laboratories, involving at least 20 initial runs of "low", "medium" and "high" serum-based controls (mean holoTC concentrations 34, 60 and 110 pmol/L, respectively) yielded an intra-laboratory imprecision of 6-10%. No systematic inter-laboratory deviations were observed on runs involving 72 patient samples (holoTC concentration range 10-160 pmol/L). A fourth laboratory demonstrated higher assay imprecision for control samples and systematic deviation of results for the patient samples. Measurement of holoTC by ELISA showed an imprecision of 4-5%, and slightly higher mean values for the controls (mean holoTC concentrations 40, 70 and 114 pmol/L, respectively). Comparable results were obtained for the patient samples. The long-term intra-laboratory imprecision was 12% for the holoTC RIA and 6% for the ELISA. In conclusion, it would be prudent to check the calibration and precision prior to starting to use these holoTC assays in research or clinical practice. The results obtained using the holoTC RIA were similar to those obtained using the holoTC ELISA assay.

21 CFR 225.58 - Laboratory controls.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory controls. 225.58 Section 225.58 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory controls. (a) The periodic assay of medicated feeds for drug components provides a measure of...

21 CFR 225.58 - Laboratory controls.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory controls. 225.58 Section 225.58 Food...: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Product Quality Control § 225.58 Laboratory controls. (a) The periodic assay of medicated feeds for drug components provides a measure of...

21 CFR 225.58 - Laboratory controls.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory controls. 225.58 Section 225.58 Food...: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Product Quality Control § 225.58 Laboratory controls. (a) The periodic assay of medicated feeds for drug components provides a measure of...

21 CFR 225.58 - Laboratory controls.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory controls. 225.58 Section 225.58 Food...: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Product Quality Control § 225.58 Laboratory controls. (a) The periodic assay of medicated feeds for drug components provides a measure of...

The laboratory diagnosis of testosterone deficiency.

PubMed

Paduch, Darius A; Brannigan, Robert E; Fuchs, Eugene F; Kim, Edward D; Marmar, Joel L; Sandlow, Jay I

2014-05-01

The evaluation and treatment of hypogonadal men has become an important part of urologic practice. Fatigue, loss of libido, and erectile dysfunction are commonly reported, but nonspecific symptoms and laboratory verification of low testosterone (T) are an important part of evaluation in addition to a detailed history and physical examination. Significant intraindividual fluctuations in serum T levels, biologic variation of T action on end organs, the wide range of T levels in human serum samples, and technical limitations of currently available assays have led to poor reliability of T measurements in the clinical laboratory setting. There is no universally accepted threshold of T concentration that distinguishes eugonadal from hypogonadal men; thus, laboratory results have to be interpreted in the appropriate clinical setting. This review focuses on clinical, biological, and technological challenges that affect serum T measurements to educate clinicians regarding technological advances and limitations of the currently available laboratory methods to diagnose hypogonadism. A collaborative effort led by the American Urological Association between practicing clinicians, patient advocacy groups, government regulatory agencies, industry, and professional societies is underway to provide optimized assay platforms and evidence-based normal assay ranges to guide clinical decision making. Until such standardization is commonplace in clinical laboratories, the decision to treat should be based on the presence of signs and symptoms in addition to serum T measurements. Rigid interpretation of T ranges should not dictate clinical decision making or define coverage of treatment by third party payers. Copyright © 2014 Elsevier Inc. All rights reserved.

Ink dating using thermal desorption and gas chromatography/mass spectrometry: comparison of results obtained in two laboratories.

PubMed

Koenig, Agnès; Bügler, Jürgen; Kirsch, Dieter; Köhler, Fritz; Weyermann, Céline

2015-01-01

An ink dating method based on solvent analysis was recently developed using thermal desorption followed by gas chromatography/mass spectrometry (GC/MS) and is currently implemented in several forensic laboratories. The main aims of this work were to implement this method in a new laboratory to evaluate whether results were comparable at three levels: (i) validation criteria, (ii) aging curves, and (iii) results interpretation. While the results were indeed comparable in terms of validation, the method proved to be very sensitive to maintenances. Moreover, the aging curves were influenced by ink composition, as well as storage conditions (particularly when the samples were not stored in "normal" room conditions). Finally, as current interpretation models showed limitations, an alternative model based on slope calculation was proposed. However, in the future, a probabilistic approach may represent a better solution to deal with ink sample inhomogeneity. © 2014 American Academy of Forensic Science.

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

PubMed

Park, Douglas L; Coates, Scott; Brewer, Vickery A; Garber, Eric A E; Abouzied, Mohamed; Johnson, Kurt; Ritter, Bruce; McKenzie, Deborah

2005-01-01

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.

Simulating thermal stress features on hot planetary surfaces in vacuum at high temperature facility in the PEL laboratory

NASA Astrophysics Data System (ADS)

Maturilli, A.; Ferrari, S.; Helbert, J.; D'Incecco, P.; D'Amore, M.

2011-12-01

In the Planetary Emissivity Laboratory (PEL) at the Institute for Planetary Research of the German Aerospace Center (DLR) in Berlin, we set-up a simulation chamber for the spectroscopic investigation of minerals separates under Mercurial conditions. The chamber can be evacuated to 10-4 bar and the target samples heated to 700 K within few minutes, thanks to the innovative inductive heating system. While developing the protocol for the high temperature spectroscopy measurements we discovered interesting "morphologies" on the sample surfaces. The powders are poured into stainless steel cups of 50 mm internal diameter, 8 mm height and 3 mm depth, having a 5 mm thick base (thus leaving 3 mm free space for the minerals), and rim 1 mm thick. We selected several minerals of interest for Mercurial surface composition and for each of them we analyzed various grain size separates, to study the influence of grain dimensions to the process of thermal stressing. We observed that for the smaller grain size separate (0-25 μm) the thermal stress mainly induces large depressions and fractures, while on larger grain sizes (125-250 μm) small depressions and a cratered surface. Our current working hypothesis is that these features are mainly caused by thermal stress induced by a radiatively quickly cooling surface layer covering the much hotter bulk material. Further investigation is ongoing to understand the processes better. The observed morphologies exhibit surprising similarities to features observed at planetary scale size for example on Mercury and even on Venus. Especially the high resolution images provided currently from MESSENGER'S Mercury Dual Imaging System (MDIS) instrument has revealed plains dominated by polygonal fractures whose origin still have to be determined. Our laboratory analogue studies might in the future provide some insight into the processes creating those features

Thermal protective uniforms and hoods: impact of design modifications and water content on burn prevention in New York City firefighters: laboratory and field results

PubMed Central

Prezant, D; Malley, K; Barker, R; Guerth, C; Kelly, K

2001-01-01

Objectives—To determine (1) the effectiveness of hoods in reducing head burns, (2) the impact of clothes worn under the protective outer uniform (modern = long sleeve shirt and long pants; modified modern = short sleeve T-shirt and short pants) on burns, and (3) whether water content (dry, damp or saturated) affects the level of thermal protection. Setting—Fire Department of the City of New York (FDNY). Methods—Laboratory tests (fully dressed manikin) evaluated the different uniform and water conditions when exposed to an average 24 cal/cm2 heat flux, approximately 2250°F air temperature. FDNY field results compared (1) head burns during winters wearing the hood to winters without hood and (2) upper and lower extremity burns during summers wearing traditional, modern, and modified modern uniforms. Results—Laboratory tests showed that thermal protection was: (1) dramatically improved by the hood with protection increasing as water content increased and (2) not significantly different between modern and modified modern uniforms, regardless of water content. FDNY field results confirmed these tests showing (1) significant decreases in neck burns (by 54%), ear burns (by 60%), and head burn totals (by 46%) wearing the hood and (2) no significant differences in upper or lower extremity burns wearing modern compared with modified modern uniforms. Conclusions—Based on combined laboratory and field results, we strongly recommend the use of modern thermal protective hoods and the modified modern uniform. PMID:11565971

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

PubMed Central

Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

2014-01-01

The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

Policies and practices in haemostasis testing among laboratories in Croatia: a survey on behalf of a Working Group for Laboratory Coagulation of the Croatian Society of Medical Biochemistry and Laboratory Medicine

PubMed Central

Bronić, Ana; Herak, Desiree Coen; Margetić, Sandra; Milić, Marija

2017-01-01

Introduction The objective of this survey was to assess current policies and practice in haemostasis testing among both hospital and outpatient laboratories in Republic of Croatia. Materials and methods A questionnaire with seventy questions divided into nine sections was created in May 2015. Participants were asked about their practice related to test request form, sample collection, prothrombin time (PT) and activated partial thromboplastin time assays, other individual haemostasis assays, point-of-care testing (POCT), reporting of coagulation tests results and quality assurance of procedures, the personnel and other laboratory resources, as well as on issues related to education and implementation of additional coagulation assays in their laboratory. The survey was administered and data were collected between June and September 2015. Results A total survey response rate was 104/170 (61.2%). Most respondents were faced with incomplete information on prescribed therapy and diagnosis on the test request or inappropriate samples withdrawn on distant locations, but also do not have protocols for handling samples with high haematocrit values. Reporting of PT-INR and D-dimer results was different between laboratories. Although almost all laboratories developed a critical value reporting system, reporting a value to general practitioners is still a problem. Result on coagulation POCT testing showed that not all devices were supervised by laboratories, which is not in compliance with Croatian Chamber of Medical Biochemistry acts. Conclusion Obtained results highlighted areas that need improvement and different practice patterns in particular field of haemostasis testing among laboratories. A harmonization of the overall process of haemostasis testing at national level should be considered and undertaken. PMID:28392741

The level of laboratory testing required for diagnosis or exclusion of a platelet function disorder using platelet aggregation and secretion assays.

PubMed

Mezzano, Diego; Quiroga, Teresa; Pereira, Jaime

2009-03-01

The major advances from research on platelet molecular and cell biology, physiology, and pathophysiology over the past decades have not been adequately translated to clinical laboratory diagnosis. Hereditary platelet function disorders (PFDs) are at least as prevalent in the general population as von Willebrand disease (VWD) although PFDs tend not be as well recognized or evaluated. Clinical mucous and skin bleeding in patients with PFDs is prototypic of primary hemostasis disorders, and the bleeding pattern is not distinguishable from that of other primary hemostasis disorders such as VWD. However, different treatment needs, between these discrete disorders, make a precise diagnosis mandatory. Currently, clinicians receive reliable laboratory reports when testing patients with severe PFDs, such as Glanzmann thrombasthenia and Bernard-Soulier syndrome, due to the distinctive laboratory defects that these disorders present, together with the availability of differential diagnostic tests. This is not the case for the majority of PFDs generically classified as "platelet secretion disorders," which are a heterogeneous group of "mild bleeding disorders," for which there are not universally accepted diagnostic criteria. An important reason for robust diagnostic tests is the high proportion (more than 50% in some reports) of patients with unequivocal bleeding who have no precise diagnosis established after a complete laboratory workup. It is paradoxical that the current "gold standard" test for PFD diagnosis, light transmission aggregometry (LTA), has not been standardized after more than four decades of worldwide clinical use. This review describes current diagnostic assays for PFD in a clinical hemostasis laboratory, relating these with current knowledge on platelet function and pathophysiology. Special emphasis will be given to LTA and platelet secretion tests, as well as to the reasons why sensitive tests are needed to explore the lesser known participation of

Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

PubMed

Kitchen, S; Kershaw, G; Tiefenbacher, S

2016-07-01

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. © 2016 John Wiley & Sons Ltd.

Engineering of a miniaturized, robotic clinical laboratory.

PubMed

Nourse, Marilyn B; Engel, Kate; Anekal, Samartha G; Bailey, Jocelyn A; Bhatta, Pradeep; Bhave, Devayani P; Chandrasekaran, Shekar; Chen, Yutao; Chow, Steven; Das, Ushati; Galil, Erez; Gong, Xinwei; Gessert, Steven F; Ha, Kevin D; Hu, Ran; Hyland, Laura; Jammalamadaka, Arvind; Jayasurya, Karthik; Kemp, Timothy M; Kim, Andrew N; Lee, Lucie S; Liu, Yang Lily; Nguyen, Alphonso; O'Leary, Jared; Pangarkar, Chinmay H; Patel, Paul J; Quon, Ken; Ramachandran, Pradeep L; Rappaport, Amy R; Roy, Joy; Sapida, Jerald F; Sergeev, Nikolay V; Shee, Chandan; Shenoy, Renuka; Sivaraman, Sharada; Sosa-Padilla, Bernardo; Tran, Lorraine; Trent, Amanda; Waggoner, Thomas C; Wodziak, Dariusz; Yuan, Amy; Zhao, Peter; Young, Daniel L; Robertson, Channing R; Holmes, Elizabeth A

2018-01-01

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

Evaluation of the Abbott ARCHITECT Toxo IgM assay.

PubMed

Sickinger, Eva; Braun, Hans-Bertram; Praast, Gerald; Stieler, Myriam; Gundlach, Cordelia; Birkenbach, Claudia; Prostko, John; Palafox, Mary Ann; Frias, Edwin; Hsu, Stephen; Matias, Matthew; Pucci, Dominick; Hausmann, Michael; Sagel, Ulrich; Smith, Darwin

2009-07-01

Development of the ARCHITECT Toxo IgM assay has been done to assist the clinician in acute Toxoplasma gondii infection detection, especially in pregnant women. Its use, in conjunction with ARCHITECT Toxo IgG and Toxo Avidity assays, will provide an array of assays particularly useful in the monitoring of pregnant females to determine the risk of maternal transmission of the parasite. Specificity results from 2 testing sites, using populations of pregnant females, hospital patients, and blood donors, demonstrated that the assay has an overall resolved relative specificity of 99.89% (confidence interval, 99.68-99.98%). Relative specificity for pregnant female specimens was 99.95% (n = 2031). Excellent seroconversion sensitivity was observed for the ARCHITECT Toxo IgM assay, which was similar to the Abbott AxSYM Toxo IgM assay (Abbott Laboratories, Abbott Park, IL). In more than 90% of the panels tested, the 1st bleed detected in the serial bleeds was the same for both assays.

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

Advances in Developing HIV-1 Viral Load Assays for Resource-Limited Settings

PubMed Central

Wang, ShuQi; Xu, Feng; Demirci, Utkan

2010-01-01

Commercial HIV-1 RNA viral load assays have been routinely used in developed countries to monitor antiretroviral treatment (ART). However, these assays require expensive equipment and reagents, well-trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. Inexpensive alternatives such as the Ultrasensitive p24 assay, the Reverse Transcriptase (RT) assay and in-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) have been developed. However, they are still time-consuming, technologically complex and inappropriate for decentralized laboratories as point-of-care (POC) tests. Recent advances in microfluidics and nanotechnology offer new strategies to develop low-cost, rapid, robust and simple HIV-1 viral load monitoring systems. We review state-of-the-art technologies used for HIV-1 viral load monitoring in both developed and developing settings. Emerging approaches based on microfluidics and nanotechnology, which have potential to be integrated into POC HIV-1 viral load assays, are also discussed. PMID:20600784

Electric Motor Thermal Management R&D; NREL (National Renewable Energy Laboratory)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennion, Kevin

2015-06-09

Thermal constraints place significant limitations on how electric motors ultimately perform. Without the ability to remove heat, the motor cannot operate without sacrificing performance, efficiency, and reliability. Finite element analysis and computational fluid dynamics modeling approaches are being increasingly utilized in the design and analysis of electric motors. As the models become more sophisticated, it is important to have detailed and accurate knowledge of both the passive thermal performance and the active cooling performance. In this work, we provide an overview of research characterizing both passive and active thermal elements related to electric motor thermal management. To better characterize themore » passive thermal performance, work is being performed to measure motor material thermal properties and thermal contact resistances. The active cooling performance of automatic transmission fluid (ATF) jets is also being measured to better understand the heat transfer coefficients of ATF impinging on motor copper windings.« less

CLSI-based transference and verification of CALIPER pediatric reference intervals for 29 Ortho VITROS 5600 chemistry assays.

PubMed

Higgins, Victoria; Truong, Dorothy; Woroch, Amy; Chan, Man Khun; Tahmasebi, Houman; Adeli, Khosrow

2018-03-01

Evidence-based reference intervals (RIs) are essential to accurately interpret pediatric laboratory test results. To fill gaps in pediatric RIs, the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) project developed an age- and sex-specific pediatric RI database based on healthy pediatric subjects. Originally established for Abbott ARCHITECT assays, CALIPER RIs were transferred to assays on Beckman, Roche, Siemens, and Ortho analytical platforms. This study provides transferred reference intervals for 29 biochemical assays for the Ortho VITROS 5600 Chemistry System (Ortho). Based on Clinical Laboratory Standards Institute (CLSI) guidelines, a method comparison analysis was performed by measuring approximately 200 patient serum samples using Abbott and Ortho assays. The equation of the line of best fit was calculated and the appropriateness of the linear model was assessed. This equation was used to transfer RIs from Abbott to Ortho assays. Transferred RIs were verified using 84 healthy pediatric serum samples from the CALIPER cohort. RIs for most chemistry analytes successfully transferred from Abbott to Ortho assays. Calcium and CO 2 did not meet statistical criteria for transference (r 2 <0.70). Of the 32 transferred reference intervals, 29 successfully verified with approximately 90% of results from reference samples falling within transferred confidence limits. Transferred RIs for total bilirubin, magnesium, and LDH did not meet verification criteria and are not reported. This study broadens the utility of the CALIPER pediatric RI database to laboratories using Ortho VITROS 5600 biochemical assays. Clinical laboratories should verify CALIPER reference intervals for their specific analytical platform and local population as recommended by CLSI. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish.

PubMed

Van Dolah, Frances M; Leighfield, Tod A; Doucette, Gregory J; Bean, Laurie; Niedzwiadek, Barbara; Rawn, Dorothea F K

2009-01-01

A single-laboratory validation (SLV) study was conducted for the microplate receptor binding assay (RBA) for paralytic shellfish poisoning (PSP) toxins in shellfish. The basis of the assay is the competition between [3H]saxitoxin (STX) and STX in a standard or sample for binding to the voltage dependent sodium channel. A calibration curve is generated by the addition of 0.01-1000 nM STX, which results in the concentration dependent decrease in [3H]STX-receptor complexes formed and serves to quantify STX in unknown samples. This study established the LOQ, linearity, recovery, accuracy, and precision of the assay for determining PSP toxicity in shellfish extracts, as performed by a single analyst on multiple days. The standard curve obtained on 5 independent days resulted in a half-maximal inhibition (IC50) of 2.3 nM STX +/- 0.3 (RSD = 10.8%) with a slope of 0.96 +/- 0.06 (RSD = 6.3%) and a dynamic range of 1.2-10.0 nM. The LOQ was 5.3 microg STX equivalents/100 g shellfish. Linearity, established by quantification of three levels of purified STX (1.5, 3, and 6 nM), yielded an r2 of 0.97. Recovery from mussels spiked with three levels (40, 80, and 120 microg STX/100 g) averaged 121%. Repeatability (RSD(r)), determined on six naturally contaminated shellfish samples on 5 independent days, was 17.7%. A method comparison with the AOAC mouse bioassay yielded r2 = 0.98 (slope = 1.29) in the SLV study. The effects of the extraction method on RBA-based toxicity values were assessed on shellfish extracted for PSP toxins using the AOAC mouse bioassay method (0.1 M HCI) compared to that for the precolumn oxidation HPLC method (0.1% acetic acid). The two extraction methods showed linear correlation (r2 = 0.99), with the HCl extraction method yielding slightly higher toxicity values (slope = 1.23). A similar relationship was observed between HPLC quantification of the HCI- and acetic acid-extracted samples (r2 = 0.98, slope 1.19). The RBA also had excellent linear correlation

Use of a Rapid Ethylene Glycol Assay: a 4-Year Retrospective Study at an Academic Medical Center.

PubMed

Rooney, Sydney L; Ehlers, Alexandra; Morris, Cory; Drees, Denny; Davis, Scott R; Kulhavy, Jeff; Krasowski, Matthew D

2016-06-01

Ethylene glycol (EG) is a common cause of toxic ingestions. Gas chromatography (GC)-based laboratory assays are the gold standard for diagnosing EG intoxication. However, GC requires specialized instrumentation and technical expertise that limits feasibility for many clinical laboratories. The objective of this retrospective study was to determine the utility of incorporating a rapid EG assay for management of cases with suspected EG poisoning. The University of Iowa Hospitals and Clinics core clinical laboratory adapted a veterinary EG assay (Catachem, Inc.) for the Roche Diagnostics cobas 8000 c502 analyzer and incorporated this assay in an osmolal gap-based algorithm for potential toxic alcohol/glycol ingestions. The main limitation is that high concentrations of propylene glycol (PG), while readily identifiable by reaction rate kinetics, can interfere with EG measurement. The clinical laboratory had the ability to perform GC for EG and PG, if needed. A total of 222 rapid EG and 24 EG/PG GC analyses were documented in 106 patient encounters. Of ten confirmed EG ingestions, eight cases were managed entirely with the rapid EG assay. PG interference was evident in 25 samples, leading to 8 GC analyses to rule out the presence of EG. Chart review of cases with negative rapid EG assay results showed no evidence of false negatives. The results of this study highlight the use of incorporating a rapid EG assay for the diagnosis and management of suspected EG toxicity by decreasing the reliance on GC. Future improvements would involve rapid EG assays that completely avoid interference by PG.

[Multicenter evaluation of h-FABP semi-quantitative assay (Cardio Detect) in central laboratory: the point in acute myocardial infarction diagnosis].

PubMed

Lefèvre, G; Fayet, J-M; Graïne, H; Berny, C; Maupas-Schwalm, F; Capolaghi, B; Morin, C

2007-01-01

The diagnostic performance of heart-Fatty Acid Binding Protein (h-FABP) (semi-quantitative CardioDetect test) and cardiac troponin I (TnIc) blood assays were compared in one hundred patients presenting with suspicion of acute coronary syndrome. Final patient diagnosis was "acute myocardial infarction" in 36 cases, "non ST myocardial infarction" in 25 cases and "non ischemic pathologies" in 39 cases. h-FABP results were positive in 26 patients, negative in 57 patients and ambiguous in 17 patients, the latter corresponding to the final diagnosis of "acute myocardial infarction" in 5 cases, "non ST myocardial infarction" in 2 cases and "non ischemic pathologies " in 10 cases. At admission, h-FABP and TnIc exhibiteda sensitivity of 54% an 66%, respectively and a specificity of 86% and 95%, respectively. Positive and negative predictive values were 81% and 64% for h-FABP, respectively and 92% and 75% for cTnI, respectively. h-FABP and cTnI demonstrated a similar diagnostic efficiency if admission delay is less than 4 hours after onset of chest pain (area under ROC curve TnIc = 0.767 +/- 0.091 ; area under ROC curve h-FABP = 0.622 +/- 0.109 ; p = 0.144). On the contrary, cTnI assay demonstrated a better efficiency than h-FABP (p< 0.005) for patients admitted in a delay of 4 to 12 hours after the onset of chest pain. If chosen cTnI cut-off corresponded to the recent consensus definition used for monitoring acute coronary syndrome patients, h-FABP semi-quantitative assay realized within central laboratory did not demonstrated a better diagnostic efficiency than cTnI.

Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

PubMed

Herremans, Tineke; Hogema, Boris M; Nabuurs, Marrigje; Peeters, Marcel; Wegdam-Blans, Marjolijn; Schneeberger, Peter; Nijhuis, Carla; Notermans, Daan W; Galama, Joep; Horrevorts, Anton; van Loo, Inge H M; Vlaminckx, Bart; Zaaijer, Hans L; Koopmans, Marion P; Berkhout, Hanneke; Socolovschi, Cristina; Raoult, Didier; Stenos, John; Nicholson, William; Bijlmer, Henk

2013-01-01

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever. Copyright © 2013 Elsevier Inc. All rights reserved.

The use of laboratory tests in the diagnosis of SLE.

PubMed

Egner, W

2000-06-01

ANA IIF is an effective screening assay in patients with clinical features of SLE and will detect most anti-ssDNA, anti-dsDNA, ENAs, and other autoantibodies. False positives are common. The clinical importance cannot be extrapolated from the ANA titre or pattern, although higher titres (> 1/160) are more likely to be important. HEp-2 cells are the most sensitive substrate for ANA detection, but this must be balanced against an increased incidence of insignificant positivity. ANA positive samples should be subjected to more specific assays for the diagnosis of SLE. A combination of ENA (Ro/La/Sm/RNP) and dsDNA assays will detect most patients with SLE as long as the characteristics of the assays used are well understood. ESR and CRP measurements provide useful additional information. Sjogren's syndrome and MCTD will produce overlapping serology with SLE, and anti-dsDNA titres are sometimes seen in autoimmune hepatitis and rheumatoid arthritis. All results should be reported in the light of the clinical details, by an experienced immunologist. A suggested diagnostic protocol is outlined in fig 1. The type of assay used crucially influences the predictive value of the tests. ELISA technology dominates routine laboratory practice, but tends to produce more false positive and true weak positive results, which may reduce the PPV of the test. This can be minimised by using IgG specific conjugates and careful assay validation. The NPV for SLE [figure: see text] is high for most assays but the PPV varies. Where necessary, laboratories should use crithidia or Farr dsDNA assays to confirm dubious ELISA dsDNA results, and ID/IB to confirm dubious ENA results. For monitoring, a precise, quantitative assay is required. It is unclear whether the detection of IgM or low affinity antibodies has a role here. A combination of anti-dsDNA, C3, C4, CRP, and ESR assays provides the most useful clinical information. Anti-ssDNA assays are likely to be useful, and are potentially more

Interferences from blood collection tube components on clinical chemistry assays

PubMed Central

Bowen, Raffick A.R.; Remaley, Alan T.

2014-01-01

Improper design or use of blood collection devices can adversely affect the accuracy of laboratory test results. Vascular access devices, such as catheters and needles, exert shear forces during blood flow, which creates a predisposition to cell lysis. Components from blood collection tubes, such as stoppers, lubricants, surfactants, and separator gels, can leach into specimens and/or adsorb analytes from a specimen; special tube additives may also alter analyte stability. Because of these interactions with blood specimens, blood collection devices are a potential source of pre-analytical error in laboratory testing. Accurate laboratory testing requires an understanding of the complex interactions between collection devices and blood specimens. Manufacturers, vendors, and clinical laboratorians must consider the pre-analytical challenges in laboratory testing. Although other authors have described the effects of endogenous substances on clinical assay results, the effects/impact of blood collection tube additives and components have not been well systematically described or explained. This review aims to identify and describe blood collection tube additives and their components and the strategies used to minimize their effects on clinical chemistry assays. PMID:24627713

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

PubMed

Barnett, David; Louzao, Raaul; Gambell, Peter; De, Jitakshi; Oldaker, Teri; Hanson, Curtis A

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations. © 2013 International Clinical Cytometry Society.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part II - preanalytical issues.

PubMed

Davis, Bruce H; Dasgupta, Amar; Kussick, Steven; Han, Jin-Yeong; Estrellado, Annalee

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

An interlaboratory transfer of a multi-analyte assay between continents.

PubMed

Georgiou, Alexandra; Dong, Kelly; Hughes, Stephen; Barfield, Matthew

2015-01-01

Alex has worked at GlaxoSmithKline for the past 15 years and currently works within the bioanalytical and toxicokinetic group in the United Kingdom. Alex's role in previous years has been the in-house support of preclinical and clinical bioanalysis, from method development through to sample analysis activities as well as acting as PI for GLP bioanalysis and toxicokinetics. For the past two years, Alex has applied this analytical and regulatory experience to focus on the outsourcing of preclinical bioanalysis, toxicokinetics and clinical bioanalysis, working closely with multiple bioanalytical and in-life CRO partners worldwide. Alex works to support DMPK and Safety Assessment outsourcing activities for GSK across multiple therapeutic areas, from the first GLP study through to late stage clinical PK studies. Transfer and cross-validation of an existing analytical assay between a laboratory providing current analytical support, and a laboratory needed for new or additional support, can present the bioanalyst with numerous challenges. These challenges can be technical or logistical in nature and may prove to be significant when transferring an assay between laboratories in different continents. Part of GlaxoSmithKline's strategy to improve confidence in providing quality data, is to cross-validate between laboratories. If the cross-validation fails predefined acceptance criteria, then a subsequent investigation would follow. This may also prove to be challenging. The importance of thorough planning and good communication throughout assay transfer, cross-validation and any subsequent investigations is illustrated in this case study.

Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

PubMed

Chung, Thomas D Y

2013-01-01

Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis

PubMed Central

An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting

2015-01-01

The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive samples, 704 fresh syphilis-negative samples, 48 preselected potentially interfering samples, and 30 “borderline” samples and was compared head to head with the commercially available Lumipulse G TP-N. In this study, the HISCL anti-TP assay was in perfect agreement with the applied testing algorithms with an overall agreement of 100%, comparable to that of Lumipulse G TP-N (99.63%). The sensitivity and specificity of the HISCL anti-TP assay were 100% (95% confidence interval [CI], 98.42% to 100%) and 100% (95% CI, 99.37% to 100%), respectively. Considering the excellent ease of use and automation, high throughput, and its favorable sensitivity and specificity, the HISCL anti-TP assay may represent a new choice for syphilis screening in high-volume laboratories. PMID:25972403

Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis.

PubMed

An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

2015-07-01

The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive samples, 704 fresh syphilis-negative samples, 48 preselected potentially interfering samples, and 30 "borderline" samples and was compared head to head with the commercially available Lumipulse G TP-N. In this study, the HISCL anti-TP assay was in perfect agreement with the applied testing algorithms with an overall agreement of 100%, comparable to that of Lumipulse G TP-N (99.63%). The sensitivity and specificity of the HISCL anti-TP assay were 100% (95% confidence interval [CI], 98.42% to 100%) and 100% (95% CI, 99.37% to 100%), respectively. Considering the excellent ease of use and automation, high throughput, and its favorable sensitivity and specificity, the HISCL anti-TP assay may represent a new choice for syphilis screening in high-volume laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mars Science Laboratory Entry Capsule Aerothermodynamics and Thermal Protection System

NASA Technical Reports Server (NTRS)

Edquist, Karl T.; Hollis, Brian R.; Dyakonov, Artem A.; Laub, Bernard; Wright, Michael J.; Rivellini, Tomasso P.; Slimko, Eric M.; Willcockson, William H.

2007-01-01

The Mars Science Laboratory (MSL) spacecraft is being designed to carry a large rover (greater than 800 kg) to the surface of Mars using a blunt-body entry capsule as the primary decelerator. The spacecraft is being designed for launch in 2009 and arrival at Mars in 2010. The combination of large mass and diameter with non-zero angle-of-attack for MSL will result in unprecedented convective heating environments caused by turbulence prior to peak heating. Navier-Stokes computations predict a large turbulent heating augmentation for which there are no supporting flight data1 and little ground data for validation. Consequently, an extensive experimental program has been established specifically for MSL to understand the level of turbulent augmentation expected in flight. The experimental data support the prediction of turbulent transition and have also uncovered phenomena that cannot be replicated with available computational methods. The result is that the flight aeroheating environments predictions must include larger uncertainties than are typically used for a Mars entry capsule. Finally, the thermal protection system (TPS) being used for MSL has not been flown at the heat flux, pressure, and shear stress combinations expected in flight, so a test program has been established to obtain conditions relevant to flight. This paper summarizes the aerothermodynamic definition analysis and TPS development, focusing on the challenges that are unique to MSL.

A Curious Year on Mars - Long-Term Thermal Trends for Mars Science Laboratory Rover's First Martian Year

NASA Technical Reports Server (NTRS)

Cucullu, Gordy C., III; Zayas, Daniel; Novak, Keith; Wu, Pat

2014-01-01

By the time of this writing, Curiosity, the Mars Science Laboratory (MSL) Rover, has weathered four seasons in Gale Crater, just south of and approaching the foothills of the 5-km high Aeolis Mons, known as "Mount Sharp," at 4.59 deg south latitude. The mission design included a much broader latitude range of 30 deg north to 30 deg south constraining some of the Rover environmental requirements and operations. To date, Curiosity has relayed over 150 MB of thermal telemetry. Curiosity has relayed over 150 MB of thermal telemetry through four seasons. The trends and idiosyncrasies revealed through four seasons of telemetry from Mars are discussed. The better-characterized thermal environment allows for less conservatism in operational models and increases the amount of science data collection. Examples include: the elimination of overheating concerns for some cameras and the use of the previous sol's temperature telemetry along with the conservative soak temperature curve from the winter thermal model, to produce a custom heating prescription for the upcoming weeks thus increasing operation time and reducing heating times. This paper discusses the lessons learned for Rover operation as well as general idiosyncrasies discovered about the local environment-such as the effect of orientation on subsystem temperature variation, a regular morning and afternoon wind, ground and air microclimates with distinct temperature differences from other terrain, and how the Rover affects the local environment. This paper further documents and explains some of the interesting highs and lows of the temperature telemetry data as well as offers explanations for sudden temperature changes on board the Rover.

46 CFR 160.174-5 - Independent laboratory.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 6 2011-10-01 2011-10-01 false Independent laboratory. 160.174-5 Section 160.174-5...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Thermal Protective Aids § 160.174-5 Independent laboratory. (a... laboratory accepted by the Coast Guard under subpart 159.010 of this chapter. (b) [Reserved] ...

46 CFR 160.174-5 - Independent laboratory.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 6 2010-10-01 2010-10-01 false Independent laboratory. 160.174-5 Section 160.174-5...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Thermal Protective Aids § 160.174-5 Independent laboratory. (a... laboratory accepted by the Coast Guard under subpart 159.010 of this chapter. (b) [Reserved] ...

Evaluation of the DCA Vantage analyzer for HbA 1c assay.

PubMed

Szymezak, Jean; Leroy, Nathalie; Lavalard, Emmanuelle; Gillery, Philippe

2008-01-01

Measurement of HbA 1c is key in monitoring diabetic patients in both laboratories and clinical units, where HbA 1c results are used as part of patient education. We have evaluated the DCA Vantage, a new device for immunological assay of HbA 1c. HbA 1c results obtained were evaluated in terms of precision, linearity, specificity and practicability, and were compared with results obtained by a Variant II HPLC method. The method exhibited intra- and inter-assay coefficients of variation lower than 2.6% and 4.0%, respectively, and good correlation with the comparison HPLC method (r2=0.9776). No interference was noted in the presence of labile HbA 1c or carbamylated hemoglobin. The new device exhibited improved practicability characteristics and allowed better sample identification, better management of quality control routines and greater connectivity possibilities compared to the previous DCA 2000 analyzer. This new analyzer exhibited analytical and practical characteristics very suitable for HbA 1c assay for laboratory or point-of-care use according to good laboratory practice.

Assaying Wnt5A-mediated Invasion in Melanoma Cells

PubMed Central

O'Connell, Michael P.; French, Amanda D.; Leotlela, Poloko D.; Weeraratna, Ashani T.

2009-01-01

Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate and lung cancers. Assays to test motility and invasion include both in vivo assays, and in vitro assays. The former assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require MMP-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment. PMID:19099260

[Investigation of reference intervals of blood gas and acid-base analysis assays in China].

PubMed

Zhang, Lu; Wang, Wei; Wang, Zhiguo

2015-10-01

To investigate and analyze the upper and lower limits and their sources of reference intervals in blood gas and acid-base analysis assays. The data of reference intervals were collected, which come from the first run of 2014 External Quality Assessment (EQA) program in blood gas and acid-base analysis assays performed by National Center for Clinical Laboratories (NCCL). All the abnormal values and errors were eliminated. Data statistics was performed by SPSS 13.0 and Excel 2007 referring to upper and lower limits of reference intervals and sources of 7 blood gas and acid-base analysis assays, i.e. pH value, partial pressure of carbon dioxide (PCO2), partial pressure of oxygen (PO2), Na+, K+, Ca2+ and Cl-. Values were further grouped based on instrument system and the difference between each group were analyzed. There were 225 laboratories submitting the information on the reference intervals they had been using. The three main sources of reference intervals were National Guide to Clinical Laboratory Procedures [37.07% (400/1 079)], instructions of instrument manufactures [31.23% (337/1 079)] and instructions of reagent manufactures [23.26% (251/1 079)]. Approximately 35.1% (79/225) of the laboratories had validated the reference intervals they used. The difference of upper and lower limits in most assays among 7 laboratories was moderate, both minimum and maximum (i.e. the upper limits of pH value was 7.00-7.45, the lower limits of Na+ was 130.00-156.00 mmol/L), and mean and median (i.e. the upper limits of K+ was 5.04 mmol/L and 5.10 mmol/L, the upper limits of PCO2 was 45.65 mmHg and 45.00 mmHg, 1 mmHg = 0.133 kPa), as well as the difference in P2.5 and P97.5 between each instrument system group. It was shown by Kruskal-Wallis method that the P values of upper and lower limits of all the parameters were lower than 0.001, expecting the lower limits of Na+ with P value 0.029. It was shown by Mann-Whitney that the statistic differences were found among instrument

Internal quality assurance in a clinical virology laboratory. II. Internal quality control.

PubMed Central

Gray, J J; Wreghitt, T G; McKee, T A; McIntyre, P; Roth, C E; Smith, D J; Sutehall, G; Higgins, G; Geraghty, R; Whetstone, R

1995-01-01

AIMS--In April 1991 additional quality control procedures were introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. Internal quality control (IQC) samples were gradually included in the serological assays performed in the laboratory and supplemented kit controls and standard sera. METHODS--From April 1991 to December 1993, 2421 IQC procedures were carried out with reference sera. RESULTS--The IQC samples were evaluated according to the Westgard rules. Violations were recorded in 60 of 1808 (3.3%) controls and were highest in the IQC samples of complement fixation tests (25/312 (8%) of controls submitted for complement fixation tests). CONCLUSIONS--The inclusion of IQC samples in the serological assays performed in the laboratory has highlighted batch to batch variation in commercial assays. The setting of acceptable limits for the IQC samples has increased confidence in the validity of assay results. PMID:7730475

Diagnostic performance of serological assays for anti-HBs testing: Results from a quality assessment program.

PubMed

Raven, Stijn; Hautvast, Jeannine; Steenbergen, Jim van; Akkermans, Reinier; Weykamp, Cas; Smits, Francis; Hoebe, Christian; Vossen, Ann

2017-02-01

Post-vaccination testing after hepatitis B vaccination is indispensable to evaluate long-term immunological protection. Using a threshold level of antibodies against hepatitis B surface antigen (anti-HBs) to define serological protection, implies reproducible and valid measurements of different diagnostic assays. In this study we assess the performance of currently used anti-HBs assays. In 2013, 45 laboratories participated in an external quality assessment program using pooled anti-HBs serum samples around the cutoff values 10IU/l and 100IU/l. Laboratories used either Axsym (Abbott Laboratories), Architect (Abbott Laboratories), Access (Beckman-Coulter), ADVIA Centaur anti-HBs2 (Siemens Healthcare Diagnostics), Elecsys, Modular or Cobas (Roche Diagnostics) or Vidas Total Quick (Biomerieux) for anti-HBs titre quantification. We analysed covariance using mixed-model repeated measures. To assess sensitivity/specificity and agreement, a true positive or true negative result was defined as an anti-HBs titre respectively above or below the cutoff value by ≥4 of 6 assays. Different anti-HBs assays were associated with statistically significant (P<0.05) differences in anti-HBs titres in all dilutions. Sensitivity and specificity ranged respectively from 64%-100% and 95%-100%. Agreement between assays around an anti-HBs titre cutoff value of 10IU/l ranged from 93%-100% and was 44% for a cutoff value of 100IU/l. Around a cutoff value of 10IU/l use of the Access assay may result in false-negative results. Concerning the cutoff value of 100IU/l, a sample being classified below or above this cutoff relied heavily on the specific assay used, with both the Architect and the Access resulting in false-negative results. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a pan-rickettsial molecular diagnostic test based on recombinase polymerase amplification assay.

PubMed

Kissenkötter, Jonas; Hansen, Sören; Böhlken-Fascher, Susanne; Ademowo, Olusegun George; Oyinloye, Oladapo Elijah; Bakarey, Adeleye Solomon; Dobler, Gerhard; Tappe, Dennis; Patel, Pranav; Czerny, Claus-Peter; Abd El Wahed, Ahmed

2018-03-01

Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Service impact of a change in HIV-1 viral load quantification assay.

PubMed

Tipple, Craig; Oomeer, Soonita; Dosekun, Olamide; Mackie, Nicola

2014-01-01

Due to discontinuation of the Siemens Versant HIV-1 RNA (bDNA) assay in the UK, our laboratory switched to the Roche Cobas Ampliprep/Taqman HIV-1 viral load (VL) assay (Roche) in April 2013. This assay has a lower cut-off of 20 RNA copies/mL (compared with <50 for the Siemens assay). Our laboratory demonstrated previously that a significant proportion (18%) of patients undetectable using bDNA HIV-1 RNA quantification exhibited low level viraemia (LLV) using the new assay. Local guidelines recommend that patients stable on therapy receive twice-yearly VLs. We evaluated the impact of the introduction of the new assay on our clinical service. A retrospective cohort analysis of treated patients with stable undetectable VL by bDNA (<50 copies/mL) followed by ≥ one low-level (<400 copies/mL) VL with the Roche assay. Demographic data were collected in addition to frequency of VL testing and genotypic resistance assays. Referrals to virtual clinic (VC) were recorded. Patients were identified using laboratory data and information collected from electronic patient records. RESULTS were analyzed with SPSS v18. One hundred and ninety patients were included. 79.5% male; 60.6% homosexual; mean age of 46 years. Duration on stable treatment was 46.35 (std. dev. 38.15) months. Current treatment regimens were 43.3% PI-based; 43.3% NNRTI-based and 13.7% other. Patients were stratified into VL 20-49 copies/mL (n=109); VL 50-199 copies/mL (n=71) and VL 200-399 copies/mL (n=10). In total, there were 471 VLs measured of which 274 were additional as a result of the assay switch. This resulted in six HIV-1 genotype requests and 16 VC discussions (Table 1). Longer duration on HAART was associated with reduced frequency of VL testing. The relative risk of ongoing detectability according to drug class are: PI 1.62 (95% CI 1.18-2.21); NNRTI 0.507 (95% CI 0.30-0.85) and other 1.09 (95% CI 0.48-2.43). Changes in assay can result in difficulties in interpretation of patient results. The assay

An Opposite Pattern to the Conventional Thermal Hypothesis: Temperature-Dependent Variation in Coloration of Adults of Saccharosydne procerus (Homoptera: Delphacidae)

PubMed Central

Yin, Haichen; Shakeel, Muhammad; Kuang, Jing; Li, Jianhong

2015-01-01

Melanism is a common polymorphism in many insect species that also influences immune function. According to the thermal melanin hypothesis, ectothermic individuals from cooler environments have darker cuticles and higher polyphenol oxidase (PO) levels, which represent a better immunocompetence. In this study, the links among environmental temperature, melanism, and PO activity of Saccharosydne procerus (Matsumura) were examined. Most S. procerus have a black spot on their forewings at high temperatures in the field and in the laboratory. In PO activity assay, a positive association between PO level and temperature was found. Our results showed that a diversification of melanism occurred under different temperatures and that melanism in S. procerus presented an opposite pattern to the one proposed by the thermal hypothesis. PMID:26024474

The ODTX System for the Study of Thermal Sensitivity and Thermal Explosion Violence of Energetic Materials

NASA Astrophysics Data System (ADS)

Hsu, Peter; Hust, Gary; Reynolds, John; Springer, Keo; Fried, Larry; Maienschein, Jon

2013-06-01

Incidents caused by fire and combat operations in battlefields can expose energetic materials to unexpected heat that may cause thermal explosion, structural damage and casualty. Some explosives may thermally explode at fairly low temperatures (<100 C) and the violence from thermal explosion may cause a significant damage. Thus it is important to understand the response of energetic materials to thermal insults. The One Dimensional Time to Explosion (ODTX) system at the Lawrence Livermore National Laboratory can measure times to explosion, threshold thermal explosion temperature, and determine kinetic parameters of energetic materials. Samples of different configurations (pressed part, powder, paste, and liquid) can be tested in the system. The ODTX testing can also provide useful data for assessing the thermal explosion violence of energetic materials. In this paper, we will present some recent ODTX experimental data and compare thermal explosion violence of different energetic materials. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

Advanced Thermal Batteries.

DTIC Science & Technology

1980-03-01

A-AOB 13 EREAC BLONTOdLEUEAAVNESENEDV F 1/ ADVANCED THERMAL BATTERIES .(U) MAR 80 D M RYAN F33615-77-C-317 UNCLASSIFIED AFWAL-TR-80-2017...iii - -.... This report is for the second year of work. The first year of work was reported December 1978 in: " Advanced Thermal Batteries " AFAPL-TR-78...work. The first year of work was reported December 1978 in: " Advanced Thermal Batteries " AFAPL-TR-78-114 Air Force Aero Propulsion Laboratory Air

Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

PubMed

Rösner, Stephan; Gehlweiler, Kevin; Küsters, Uta; Kolbert, Mathias; Hübner, Kirsten; Pfennigwerth, Niels; Mack, Dietrich

2018-01-01

Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

PubMed Central

Heyndrickx, Leo; Heath, Alan; Sheik-Khalil, Enas; Alcami, Jose; Bongertz, Vera; Jansson, Marianne; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Ramaswamy, Meghna; Sattentau, Quentin; Tolazzi, Monica; Schuitemaker, Hanneke; Willems, Betty; Wrin, Terri; Fenyö, Eva Maria; Scarlatti, Gabriella

2012-01-01

Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus

International network for comparison of HIV neutralization assays: the NeutNet report II.

PubMed

Heyndrickx, Leo; Heath, Alan; Sheik-Khalil, Enas; Alcami, Jose; Bongertz, Vera; Jansson, Marianne; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Ramaswamy, Meghna; Sattentau, Quentin; Tolazzi, Monica; Schuitemaker, Hanneke; Willems, Betty; Wrin, Terri; Fenyö, Eva Maria; Scarlatti, Gabriella

2012-01-01

Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend

Key learnings from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 in vitro assays.

PubMed

LeBaron, Matthew J; Coady, Katie K; O'Connor, John C; Nabb, Diane L; Markell, Lauren K; Snajdr, Suzanne; Sue Marty, M

2014-02-01

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid. © 2014 Wiley Periodicals, Inc.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part I - rationale and aims.

PubMed

Davis, Bruce H; Wood, Brent; Oldaker, Teri; Barnett, David

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

PubMed

Reisinger, Kerstin; Blatz, Veronika; Brinkmann, Joep; Downs, Thomas R; Fischer, Anja; Henkler, Frank; Hoffmann, Sebastian; Krul, Cyrille; Liebsch, Manfred; Luch, Andreas; Pirow, Ralph; Reus, Astrid A; Schulz, Markus; Pfuhler, Stefan

2018-03-01

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion ® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion ® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will

Identification of six Listeria species by real-time PCR assay.

PubMed

Hage, E; Mpamugo, O; Ohai, C; Sapkota, S; Swift, C; Wooldridge, D; Amar, C F L

2014-06-01

The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories. © 2014 The Society for Applied Microbiology.

Comparison of estrogen receptor results from pathology reports with results from central laboratory testing.

PubMed

Collins, Laura C; Marotti, Jonathan D; Baer, Heather J; Tamimi, Rulla M

2008-02-06

We compared estrogen receptor (ER) assay results abstracted from pathology reports with ER results determined on the same specimens by a central laboratory with an immunohistochemical assay. Paraffin sections were cut from tissue microarrays containing 3093 breast cancer specimens from women enrolled in the Nurses' Health Study, 1851 of which had both pathology reports and tissue available for central laboratory testing. All sections were immunostained for ER at the same time. The original assays were biochemical for 1512 (81.7%) of the 1851 specimens, immunohistochemical for 336 (18.2%), and immunofluorescent for three (0.2%). ER results from pathology reports and repeat central laboratory testing were in agreement for 87.3% of specimens (1615 of the 1851 specimens; kappa statistic = 0.64, P < .001). When the comparison was restricted to the specimens for which the ER assays were originally performed by immunohistochemistry, the agreement rate increased to 92.3% of specimens (310 of the 336 specimens; kappa statistic = 0.78, P < .001). Thus, ER assay results from pathology reports appear to be a reasonable alternative to central laboratory ER testing for large, population-based studies of patients with breast cancer.

Laboratory Diagnosis and Characterization of Fungal Disease in Patients with Cystic Fibrosis (CF): A Survey of Current UK Practice in a Cohort of Clinical Microbiology Laboratories.

PubMed

Boyle, Maeve; Moore, John E; Whitehouse, Joanna L; Bilton, Diana; Downey, Damian G

2018-03-02

There is much uncertainty as to how fungal disease is diagnosed and characterized in patients with cystic fibrosis (CF). A 19-question anonymous electronic questionnaire was developed and distributed to ascertain current practice in clinical microbiology laboratories providing a fungal laboratory service to CF centres in the UK. Analyses of responses identified the following: (1) current UK laboratory practice, in general, follows the current guidelines, but the scope and diversity of what is currently being delivered by laboratories far exceeds what is detailed in the guidelines; (2) there is a lack of standardization of fungal tests amongst laboratories, outside of the current guidelines; (3) both the UK CF Trust Laboratory Standards for Processing Microbiological Samples from People with Cystic Fibrosis and the US Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech) Guidelines 43 Cystic Fibrosis Microbiology need to be updated to reflect both new methodological innovations, as well as better knowledge of fungal disease pathophysiology in CF; (4) there is a need for clinical medicine to decide upon a stratification strategy for the provision of new fungal assays that will add value to the physician in the optimal management of CF patients; (5) there is also a need to rationale what assays should be performed at local laboratory level and those which are best served at National Mycology Reference Laboratory level; and (6) further research is required in developing laboratory assays, which will help ascertain the clinical importance of 'old' fungal pathogens, as well as 'emerging' fungal pathogens.

The comet assay: ready for 30 more years.

PubMed

Møller, Peter

2018-02-24

During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

NETL - Thermogravimetric Analysis Laboratory

ScienceCinema

Richards, George

2018-06-22

Researchers in NETL's Thermal Analysis Laboratory are investigating chemical looping combustion. As a clean and efficient fossil fuel technology, chemical looping combustion controls CO2 emissions and offers a promising alternative to traditional combustion.

Steroid Assays in Paediatric Endocrinology

PubMed Central

2010-01-01

Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation. Conflict of interest:None declared. PMID:21274330

Ejecta patterns of Meteor Crater, Arizona derived from the linear un-mixing of TIMS data and laboratory thermal emission spectra

NASA Technical Reports Server (NTRS)

Ramsey, Michael S.; Christensen, Philip R.

1992-01-01

Accurate interpretation of thermal infrared data depends upon the understanding and removal of complicating effects. These effects may include physical mixing of various mineralogies and particle sizes, atmospheric absorption and emission, surficial coatings, geometry effects, and differential surface temperatures. The focus is the examination of the linear spectral mixing of individual mineral or endmember spectra. Linear addition of spectra, for particles larger than the wavelength, allows for a straight-forward method of deconvolving the observed spectra, predicting a volume percent of each endmember. The 'forward analysis' of linear mixing (comparing the spectra of physical mixtures to numerical mixtures) has received much attention. The reverse approach of un-mixing thermal emission spectra was examined with remotely sensed data, but no laboratory verification exists. Understanding of the effects of spectral mixing on high resolution laboratory spectra allows for the extrapolation to lower resolution, and often more complicated, remotely gathered data. Thermal Infrared Multispectral Scanner (TIMS) data for Meteor Crater, Arizona were acquired in Sep. 1987. The spectral un-mixing of these data gives a unique test of the laboratory results. Meteor Crater (1.2 km in diameter and 180 m deep) is located in north-central Arizona, west of Canyon Diablo. The arid environment, paucity of vegetation, and low relief make the region ideal for remote data acquisition. Within the horizontal sedimentary sequence that forms the upper Colorado Plateau, the oldest unit sampled by the impact crater was the Permian Coconino Sandstone. A thin bed of the Toroweap Formation, also of Permian age, conformably overlays the Coconino. Above the Toroweap lies the Permian Kiabab Limestone which, in turn, is covered by a thin veneer of the Moenkopi Formation. The Moenkopi is Triassic in age and has two distinct sub-units in the vicinity of the crater. The lower Wupatki member is a fine

Estimating thermal performance curves from repeated field observations

USGS Publications Warehouse

Childress, Evan; Letcher, Benjamin H.

2017-01-01

Estimating thermal performance of organisms is critical for understanding population distributions and dynamics and predicting responses to climate change. Typically, performance curves are estimated using laboratory studies to isolate temperature effects, but other abiotic and biotic factors influence temperature-performance relationships in nature reducing these models' predictive ability. We present a model for estimating thermal performance curves from repeated field observations that includes environmental and individual variation. We fit the model in a Bayesian framework using MCMC sampling, which allowed for estimation of unobserved latent growth while propagating uncertainty. Fitting the model to simulated data varying in sampling design and parameter values demonstrated that the parameter estimates were accurate, precise, and unbiased. Fitting the model to individual growth data from wild trout revealed high out-of-sample predictive ability relative to laboratory-derived models, which produced more biased predictions for field performance. The field-based estimates of thermal maxima were lower than those based on laboratory studies. Under warming temperature scenarios, field-derived performance models predicted stronger declines in body size than laboratory-derived models, suggesting that laboratory-based models may underestimate climate change effects. The presented model estimates true, realized field performance, avoiding assumptions required for applying laboratory-based models to field performance, which should improve estimates of performance under climate change and advance thermal ecology.

Efficacy of alkyl dimethyl benzyl ammonium chloride on suppression of Physalospora vaccinii in laboratory assays.

PubMed

Tubajika, K M

2006-10-01

Growth of Physalospora vaccinii on inoculated agar growth medium and cranberries treated with 0.1, 1, 10, 100, and 1,000 ppm of alkyl dimethyl benzyl ammonium chloride (ADBAC) was investigated in the laboratory. In vitro growth assays, the colony diameter, and mycelial dry weight of P. vaccinii was reduced at 1,000 ppm ADBAC. Mild or no reduction of fungal growth and mycelial dry weight was observed at concentrations less than 100 ppm when compared with the nonamended control. Growth of P. vaccinii on inoculated cranberries was inhibited by treatment with 10 and 100 ppm ADBAC. Complete inhibition of fungus growth was also achieved at 1,000 ppm ADBAC. Area under the disease progress curve values in wounded fruits were 75, 77, and 100% at 10, 100, and 1,000 ppm ADBAC, respectively, whereas area under the disease progress curve values in fruits immersed in ADBAC and pathogen were reduced 47 to 100% compared with the untreated fruits used as controls. No P. vaccinii or other fungi were detected on the control fruits inoculated with sterile distilled water. This is the first report on the use of ADBAC to control a field and storage rotting fungus, P. vaccinii. ADBAC is likely to be an important component to any integrated approach for reducing the risks associated with the presence of pathogenic microorganisms in or on foods.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

PubMed Central

Staats, Janet S.; Enzor, Jennifer H.; Sanchez, Ana M.; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N.; Weinhold, Kent J.

2014-01-01

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is “Good”). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. PMID:24968072

Effect of Fungal Competition on Decay Rates in Bicultured Soil Bottle Assays

Treesearch

Grant T. Kirker; Amy Blodgett; Patricia K. Lebow; Carol A. Clausen

2016-01-01

For decades, wood scientists and preservative formulators have employed the monocultured soil bottle assay to test efficacy of wood treatment in the laboratory as a rapid predictor of field performance. This study examines the effects of bicultured soil bottle assays on the decay by common wood decay fungi. Mycelial interactions were noted in early stages of...

Blood collection tubes as medical devices: The potential to affect assays and proposed verification and validation processes for the clinical laboratory.

PubMed

Bowen, Raffick A R; Adcock, Dorothy M

2016-12-01

Blood collection tubes (BCTs) are an often under-recognized variable in the preanalytical phase of clinical laboratory testing. Unfortunately, even the best-designed and manufactured BCTs may not work well in all clinical settings. Clinical laboratories, in collaboration with healthcare providers, should carefully evaluate BCTs prior to putting them into clinical use to determine their limitations and ensure that patients are not placed at risk because of inaccuracies due to poor tube performance. Selection of the best BCTs can be achieved through comparing advertising materials, reviewing the literature, observing the device at a scientific meeting, receiving a demonstration, evaluating the device under simulated conditions, or testing the device with patient samples. Although many publications have discussed method validations, few detail how to perform experiments for tube verification and validation. This article highlights the most common and impactful variables related to BCTs and discusses the validation studies that a typical clinical laboratory should perform when selecting BCTs. We also present a brief review of how in vitro diagnostic devices, particularly BCTs, are regulated in the United States, the European Union, and Canada. The verification and validation of BCTs will help to avoid the economic and human costs associated with incorrect test results, including poor patient care, unnecessary testing, and delays in test results. We urge laboratorians, tube manufacturers, diagnostic companies, and other researchers to take all the necessary steps to protect against the adverse effects of BCT components and their additives on clinical assays. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Miniature silicon electronic biological assay chip and applications for rapid battlefield diagnostics

NASA Astrophysics Data System (ADS)

Cunningham, Brian T.; Regan, Robert A.; Clapp, Christopher; Hildebrant, Eric; Weinberg, Marc S.; Williams, John

1999-07-01

Assessing the medical condition of battlefield personnel requires the development of rapid, portable biological diagnostic assays for a wide variety of antigens and enzymes. Ideally, such an assay would be inexpensive, small, and require no added reagents while maintaining the sensitivity and accuracy of laboratory-based assays. In this work, a microelectromechanical (MEMS) based biological assay sensor is presented which is expected to meet the above requirements. The sensor is a thin silicon membrane resonator (SMR) which registers a decrease in resonant frequency when mass is adsorbed onto its surface. By coating the sensor surface with a monolayer of antibody, for example, we have detected the corresponding antigen with a detection resolution of 0.25 ng/ml in phosphate buffer solution. Micromachining techniques are being used to integrate many (64 elements on the first test chip) identical SMR sensors into a single silicon chip which would be capable of simultaneously performing a wide variety of biomedical assays. The sensors require only a small printed circuit board and 8V power supply to operate and provide a readout. The presentation will describe the operation of the SMR sensor, the fabrication of the sensor array, and initial test results using commercially available animal immunoglobulins in laboratory-prepared test solutions.

Procedures and equipment for staining large numbers of plant root samples for endomycorrhizal assay.

PubMed

Kormanik, P P; Bryan, W C; Schultz, R C

1980-04-01

A simplified method of clearing and staining large numbers of plant roots for vesicular-arbuscular (VA) mycorrhizal assay is presented. Equipment needed for handling multiple samples is described, and two formulations for the different chemical solutions are presented. Because one formulation contains phenol, its use should be limited to basic studies for which adequate laboratory exhaust hoods are available and great clarity of fungal structures is required. The second staining formulation, utilizing lactic acid instead of phenol, is less toxic, requires less elaborate laboratory facilities, and has proven to be completely satisfactory for VA assays.

An in-house assay for BK polyomavirus quantification using the Abbott m2000 RealTime system.

PubMed

Muldrew, Kenneth L; Lovett, Jennie L

2013-11-01

BK polyomavirus (BKPyV) quantification is useful for monitoring renal transplant patient response to therapy. The Abbott m2000 RealTime System employed by some clinical laboratories to perform US Food and Drug Administration-approved assays can also be used to develop in-house assays such as the one presented here. This study aimed to validate an in-house quantitative real-time PCR assay targeting the BKPyV major capsid VP1 gene for assessment of viral load using the Abbott m2000 RealTime System. BKPyV load was measured in 95 urine and plasma samples previously tested for BKPyV by one of three laboratories (46 BKPyV-positive samples consisting of 35 plasma and 11 urine samples; 49 samples negative for BKPyV consisting of 47 plasma and two urine samples). Two additional plasma specimens from the College of American Pathologists proficiency testing survey were also analysed. Precision studies were performed by diluting a high-viral-titre patient sample into BKPyV-negative pooled plasma to create high-positive (6.16 log10 copies ml(-1)) and low-positive (3.16 log10 copies ml(-1)) samples. For precision studies of inter-assay variability, a high-positive (7.0 log10 copies ml(-1)) and a low-positive (3.0 log10 copies ml(-1)) sample were measured in 20 separate runs. The assay's limit of quantification and limit of detection were 2.70 and 2.25 log10 copies ml(-1), respectively. The assay was linear from 2.70 to 9.26 log10 copies ml(-1). Of the 48 known positives, 43 were detected as positive, with three reported by the reference laboratory as values lower than the limit of detection. Two known positives at 3.27 and 3.80 log10 copies ml(-1) tested negative by the m2000 BKPyV assay. Of the 49 known negative samples, 48 were negative by the m2000 BKPyV load assay, with one sample confirmed positive by a reference laboratory. Qualitative analysis prior to discrepancy testing demonstrated a sensitivity of 89.58 % and a specificity of 97.96 %. Precision studies

The role of the independent clinical laboratory in new assay development and commercialization.

PubMed

Ellis, David G

2003-01-01

Most would agree that these are exciting times in the field of laboratory medicine. As the body of scientific knowledge expands and research activities, such as those catalyzed by the sequencing of the human genome, bring us closer to the promise of personalized medicine, the clinical laboratory industry will have increasing opportunities to partner with owners of intellectual property to develop and commercialize new diagnostic tests. The large, independent clinical laboratories are particularly well positioned to commercialize important new tests, with their broad market penetration, infrastructure, and the scale to run esoteric tests cost-effectively.

Identifying thermal breakdown products of thermoplastics.

PubMed

Guillemot, Marianne; Oury, Benoît; Melin, Sandrine

2017-07-01

Polymers processed to produce plastic articles are subjected to temperatures between 150°C and 450°C or more during overheated processing and breakdowns. Heat-based processing of this nature can lead to emission of volatile organic compounds (VOCs) into the thermoplastic processing shop. In this study, laboratory experiments, qualitative and quantitative emissions measurement in thermoplastic factories were carried out. The first step was to identify the compounds released depending on the thermoplastic nature, the temperature and the type of process. Then a thermal degradation protocol that can extrapolate the laboratory results to industry scenarios was developed. The influence of three parameters on released thermal breakdown products was studied: the sample preparation methods-manual cutting, ambient, or cold grinding-the heating rate during thermal degradation-5, 10 20, and 50°C/min-and the decomposition method-thermogravimetric analysis and pyrolysis. Laboratory results were compared to atmospheric measurements taken at 13 companies to validate the protocol and thereby ensure its representativeness of industrial thermal processing. This protocol was applied to most commonly used thermoplastics to determine their thermal breakdown products and their thermal behaviour. Emissions data collected by personal exposure monitoring and sampling at the process emission area show airborne concentrations of detected compounds to be in the range of 0-3 mg/m 3 under normal operating conditions. Laser cutting or purging operations generate higher pollution levels in particular formaldehyde which was found in some cases at a concentration above the workplace exposure limit.

Laboratory tests for identification or exclusion of heparin induced thrombocytopenia: HIT or miss?

PubMed

Favaloro, Emmanuel J

2018-02-01

Heparin induced thrombocytopenia (HIT) is a potentially fatal condition that arises subsequent to formation of antibodies against complexes containing heparin, usually platelet-factor 4-heparin ("anti-PF4-heparin"). Assessment for HIT involves both clinical evaluation and, if indicated, laboratory testing for confirmation or exclusion, typically using an initial immunological assay ("screening"), and only if positive, a secondary functional assay for confirmation. Many different immunological and functional assays have been developed. The most common contemporary immunological assays comprise enzyme-linked immunosorbent assay [ELISA], chemiluminescence, lateral flow, and particle gel techniques. The most common functional assays measure platelet aggregation or platelet activation events (e.g., serotonin release assay; heparin-induced platelet activation (HIPA); flow cytometry). All assays have some sensitivity and specificity to HIT antibodies, but differ in terms of relative sensitivity and specificity for pathological HIT, as well as false negative and false positive error rate. This brief article overviews the different available laboratory methods, as well as providing a suggested approach to diagnosis or exclusion of HIT. © 2017 Wiley Periodicals, Inc.

Thermal performance of a photographic laboratory process: Solar Hot Water System

NASA Technical Reports Server (NTRS)

Walker, J. A.; Jensen, R. N.

1982-01-01

The thermal performance of a solar process hot water system is described. The system was designed to supply 22,000 liters (5,500 gallons) per day of 66 C (150 F) process water for photographic processing. The 328 sq m (3,528 sq. ft.) solar field has supplied 58% of the thermal energy for the system. Techniques used for analyzing various thermal values are given. Load and performance factors and the resulting solar contribution are discussed.

The Case for Laboratory Developed Procedures

PubMed Central

Sabatini, Linda M.; Tsongalis, Gregory J.; Caliendo, Angela M.; Olsen, Randall J.; Ashwood, Edward R.; Bale, Sherri; Benirschke, Robert; Carlow, Dean; Funke, Birgit H.; Grody, Wayne W.; Hayden, Randall T.; Hegde, Madhuri; Lyon, Elaine; Pessin, Melissa; Press, Richard D.; Thomson, Richard B.

2017-01-01

An explosion of knowledge and technology is revolutionizing medicine and patient care. Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures. Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments. A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories. PMID:28815200

Verification of performance specifications for a US Food and Drug Administration-approved molecular microbiology test: Clostridium difficile cytotoxin B using the Becton, Dickinson and Company GeneOhm Cdiff assay.

PubMed

Schlaberg, Robert; Mitchell, Michael J; Taggart, Edward W; She, Rosemary C

2012-01-01

US Food and Drug Administration (FDA)-approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturer's performance standards. We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. Performance data from the verification studies conducted by the laboratory were consistent with the manufacturer's performance standards and the assay was implemented into the laboratory's test menu. Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.

Vehicle Thermal Management Facilities | Transportation Research | NREL

Science.gov Websites

Management Facilities Vehicle Thermal Management Facilities Image of a building with two semi truck evaluation facilities to develop advanced thermal management technologies for vehicles. Vehicle Testing and apparatus. Combined fluid loops bench research apparatus in the Vehicle Thermal Management Laboratory. Photo

Determination of paralytic shellfish toxins in shellfish by receptor binding assay: collaborative study.

PubMed

Van Dolah, Frances M; Fire, Spencer E; Leighfield, Tod A; Mikulski, Christina M; Doucette, Gregory J

2012-01-01

A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic e shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with (3)H saxitoxin (STX) diHCl for binding to voltage-gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (microg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-07-01

Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Laboratory diagnosis of Chlamydia pneumoniae infections

PubMed Central

Peeling, Rosanna W

1995-01-01

Chlamydia pneumoniae is an important cause of respiratory illness. There is a need for accurate and rapid laboratory diagnostic methods that will lead to improved patient care, appropriate use of antimicrobial therapy and a better understanding of the epidemiology of this emerging pathogen. Culture is highly specific but is technically demanding, expensive, has a long turnaround time and its sensitivity is highly dependent on transport conditions. Antigen detection tests such as enzyme immunoassay and direct fluorescent antibody assay, and molecular detection methods such as the polymerase chain reaction assay, may provide a rapid diagnosis without the requirement for stringent transport conditions. The results of these tests should be interpreted with caution until more thorough evaluation is available. Serology remains the method of choice. The limitations of different serological methods for the laboratory diagnosis of C pneumoniae are discussed. PMID:22514397

Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei.

PubMed

Duval, Brea D; Elrod, Mindy G; Gee, Jay E; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R

2014-06-01

Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested. © The American Society of Tropical Medicine and Hygiene.

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

PubMed

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Detection of Zaire ebolavirus in swine: Assay development and optimization.

PubMed

Pickering, B S; Collignon, B; Smith, G; Marszal, P; Kobinger, G; Weingartl, H M

2018-02-01

Ebolaviruses (family Filoviridae, order Mononegavirales) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low-level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7 dpi, 3 days earlier than virus neutralization tests (VNT). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results. © 2017 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of Health and Agriculture, Canadian Food Inspection Agency.

Comparison of the Chiron Quantiplex branched DNA (bDNA) assay and the Abbott Genostics solution hybridization assay for quantification of hepatitis B viral DNA.

PubMed

Kapke, G E; Watson, G; Sheffler, S; Hunt, D; Frederick, C

1997-01-01

Several assays for quantification of DNA have been developed and are currently used in research and clinical laboratories. However, comparison of assay results has been difficult owing to the use of different standards and units of measurements as well as differences between assays in dynamic range and quantification limits. Although a few studies have compared results generated by different assays, there has been no consensus on conversion factors and thorough analysis has been precluded by small sample size and limited dynamic range studied. In this study, we have compared the Chiron branched DNA (bDNA) and Abbott liquid hybridization assays for quantification of hepatitis B virus (HBV) DNA in clinical specimens and have derived conversion factors to facilitate comparison of assay results. Additivity and variance stabilizing (AVAS) regression, a form of non-linear regression analysis, was performed on assay results for specimens from HBV clinical trials. Our results show that there is a strong linear relationship (R2 = 0.96) between log Chiron and log Abbott assay results. Conversion factors derived from regression analyses were found to be non-constant and ranged from 6-40. Analysis of paired assay results below and above each assay's limit of quantification (LOQ) indicated that a significantly (P < 0.01) larger proportion of observations were below the Abbott assay LOQ but above the Chiron assay LOQ, indicating that the Chiron assay is significantly more sensitive than the Abbott assay. Testing of replicate specimens showed that the Chiron assay consistently yielded lower per cent coefficients of variance (% CVs) than the Abbott assay, indicating that the Chiron assay provides superior precision.

David Florida Laboratory Thermal Vacuum Data Processing System

NASA Technical Reports Server (NTRS)

Choueiry, Elie

1994-01-01

During 1991, the Space Simulation Facility conducted a survey to assess the requirements and analyze the merits for purchasing a new thermal vacuum data processing system for its facilities. A new, integrated, cost effective PC-based system was purchased which uses commercial off-the-shelf software for operation and control. This system can be easily reconfigured and allows its users to access a local area network. In addition, it provides superior performance compared to that of the former system which used an outdated mini-computer and peripheral hardware. This paper provides essential background on the old data processing system's features, capabilities, and the performance criteria that drove the genesis of its successor. This paper concludes with a detailed discussion of the thermal vacuum data processing system's components, features, and its important role in supporting our space-simulation environment and our capabilities for spacecraft testing. The new system was tested during the ANIK E spacecraft test, and was fully operational in November 1991.

Inflammation Thread Runs across Medical Laboratory Specialities

PubMed Central

Lung, Thomas; Risch, Lorenz; Risch, Martin; Medina Escobar, Pedro; Bodmer, Thomas

2016-01-01

We work on the assumption that four major specialities or sectors of medical laboratory assays, comprising clinical chemistry, haematology, immunology, and microbiology, embraced by genome sequencing techniques, are routinely in use. Medical laboratory markers for inflammation serve as model: they are allotted to most fields of medical lab assays including genomics. Incessant coding of assays aligns each of them in the long lists of big data. As exemplified with the complement gene family, containing C2, C3, C8A, C8B, CFH, CFI, and ITGB2, heritability patterns/risk factors associated with diseases with genetic glitch of complement components are unfolding. The C4 component serum levels depend on sufficient vitamin D whilst low vitamin D is inversely related to IgG1, IgA, and C3 linking vitamin sufficiency to innate immunity. Whole genome sequencing of microbial organisms may distinguish virulent from nonvirulent and antibiotic resistant from nonresistant varieties of the same species and thus can be listed in personal big data banks including microbiological pathology; the big data warehouse continues to grow. PMID:27493451

Inflammation Thread Runs across Medical Laboratory Specialities.

PubMed

Nydegger, Urs; Lung, Thomas; Risch, Lorenz; Risch, Martin; Medina Escobar, Pedro; Bodmer, Thomas

2016-01-01

We work on the assumption that four major specialities or sectors of medical laboratory assays, comprising clinical chemistry, haematology, immunology, and microbiology, embraced by genome sequencing techniques, are routinely in use. Medical laboratory markers for inflammation serve as model: they are allotted to most fields of medical lab assays including genomics. Incessant coding of assays aligns each of them in the long lists of big data. As exemplified with the complement gene family, containing C2, C3, C8A, C8B, CFH, CFI, and ITGB2, heritability patterns/risk factors associated with diseases with genetic glitch of complement components are unfolding. The C4 component serum levels depend on sufficient vitamin D whilst low vitamin D is inversely related to IgG1, IgA, and C3 linking vitamin sufficiency to innate immunity. Whole genome sequencing of microbial organisms may distinguish virulent from nonvirulent and antibiotic resistant from nonresistant varieties of the same species and thus can be listed in personal big data banks including microbiological pathology; the big data warehouse continues to grow.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

PubMed

Dickey, Robert W; Plakas, Steven M; Jester, Edward L E; El Said, Kathleen R; Johannessen, Jan N; Flewelling, Leanne J; Scott, Paula; Hammond, Dan G; Van Dolah, Frances M; Leighfield, Tod A; Bottein Dachraoui, Marie-Yasmine; Ramsdell, John S; Pierce, Richard H; Henry, Mike S; Poli, Mark A; Walker, Calvin; Kurtz, Jan; Naar, Jerome; Baden, Daniel G; Musser, Steve M; White, Kevin D; Truman, Penelope; Miller, Aaron; Hawryluk, Timothy P; Wekell, Marleen M; Stirling, David; Quilliam, Michael A; Lee, Jung K

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA.

PubMed

Michelin, Birgit D A; Muller, Zsofia; Stelzl, Evelyn; Marth, Egon; Kessler, Harald H

2007-02-01

The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced. In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test. Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated. When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay. The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

Drosophila comet assay: insights, uses, and future perspectives

PubMed Central

Gaivão, Isabel; Sierra, L. María

2014-01-01

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

Gamma neutron assay method and apparatus

DOEpatents

Cole, J.D.; Aryaeinejad, R.; Greenwood, R.C.

1995-01-03

The gamma neutron assay technique is an alternative method to standard safeguards techniques for the identification and assaying of special nuclear materials in a field or laboratory environment, as a tool for dismantlement and destruction of nuclear weapons, and to determine the isotopic ratios for a blend-down program on uranium. It is capable of determining the isotopic ratios of fissionable material from the spontaneous or induced fission of a sample to within approximately 0.5%. This is based upon the prompt coincidence relationships that occur in the fission process and the proton conservation and quasi-conservation of nuclear mass (A) that exists between the two fission fragments. The system is used in both passive (without an external neutron source) and active (with an external neutron source) mode. The apparatus consists of an array of neutron and gamma-ray detectors electronically connected to determine coincident events. The method can also be used to assay radioactive waste which contains fissile material, even in the presence of a high background radiation field. 7 figures.

Gamma neutron assay method and apparatus

DOEpatents

Cole, Jerald D.; Aryaeinejad, Rahmat; Greenwood, Reginald C.

1995-01-01

The gamma neutron assay technique is an alternative method to standard safeguards techniques for the identification and assaying of special nuclear materials in a field or laboratory environment, as a tool for dismantlement and destruction of nuclear weapons, and to determine the isotopic ratios for a blend-down program on uranium. It is capable of determining the isotopic ratios of fissionable material from the spontaneous or induced fission of a sample to within approximately 0.5%. This is based upon the prompt coincidence relationships that occur in the fission process and the proton conservation and quasi-conservation of nuclear mass (A) that exists between the two fission fragments. The system is used in both passive (without an external neutron source and active (with an external neutron source) mode. The apparatus consists of an array of neutron and gamma-ray detectors electronically connected to determine coincident events. The method can also be used to assay radioactive waste which contains fissile material, even in the presence of a high background radiation field.

Physico-Geometrical Kinetics of Solid-State Reactions in an Undergraduate Thermal Analysis Laboratory

ERIC Educational Resources Information Center

Koga, Nobuyoshi; Goshi, Yuri; Yoshikawa, Masahiro; Tatsuoka, Tomoyuki

2014-01-01

An undergraduate kinetic experiment of the thermal decomposition of solids by microscopic observation and thermal analysis was developed by investigating a suitable reaction, applicable techniques of thermal analysis and microscopic observation, and a reliable kinetic calculation method. The thermal decomposition of sodium hydrogen carbonate is…

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

PubMed

Domingo, Cristina; Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

2012-12-01

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

PubMed

Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

2016-02-01

Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. © 2015 Society for Laboratory Automation and Screening.

Integrated Summary Report: Validation of Two Binding Assays Using Human Recombinant Estrogen Receptor Alpha (hrERa)

EPA Science Inventory

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (h...

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

PubMed

Shalaby, Mohamed A; El-Deeb, Ayman; El-Tholoth, Mohamed; Hoffmann, Donata; Czerny, Claus-Peter; Hufert, Frank T; Weidmann, Manfred; Abd El Wahed, Ahmed

2016-11-02

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Evaluation of the VIDAS Anti-HCV Assay for Detection of Hepatitis C Virus Infection.

PubMed

Hyun, Jungwon; Ko, Dae Hyun; Kang, Hee Jung; Whang, Dong Hee; Cha, Young Joo; Kim, Hyun Soo

2016-11-01

Anti-hepatitis C virus antibody (anti-HCV) assays are recommended for screening HCV-infected persons. The VIDAS Anti-HCV Assay (bioMérieux, France), based on the enzyme-linked fluorescence test principle, was recently introduced in Korea. We evaluated the clinical performance of the VIDAS assay. One hundred HCV-positive and 1,002 HCV-negative blood samples confirmed by Architect anti-HCV (Abbott Laboratories, USA) and COBAS TaqMan HCV real-time PCR (Roche Diagnostics, USA) or the Procleix Ultrio Plus Assay (Gen-Probe Incorporated, USA) were obtained from the Human Serum Bank (HSB) and tested by VIDAS. In case of discrepant results, we conducted a recombinant immunoblot assay (RIBA). The agreement rates for known HCV-positive and HCV-negative samples between the VIDAS assay and the HSB testing were 100% (95% confidence interval [CI]: 96.4-100%) and 99.5% (95% CI: 98.8-99.8%), respectively. One of the five discrepant samples was positive for Core 2+ and NS3-2 2+ reactivity, two samples were negative, and the other two were indeterminate regarding NS4 2+ reactivity in RIBA. We observed a significant but weak positive correlation between the titers of VIDAS and Architect assays (r=0.315, P<0.001). The VIDAS anti-HCV assay, developed on the VIDAS automated immunoassay platform based on the ready-to-use, single-sample test concept may be useful in small-to-medium-sized laboratories. It showed good agreement with Architect anti-HCV and COBAS PCR assays and is therefore useful for detection of HCV infection. Weakly test-positive (ambiguous) samples require additional testing by another anti-HCV, RIBA, or HCV RNA assay.

Evaluation of the VIDAS Anti-HCV Assay for Detection of Hepatitis C Virus Infection

PubMed Central

Hyun, Jungwon; Ko, Dae-Hyun; Kang, Hee Jung; Whang, Dong Hee

2016-01-01

Background Anti-hepatitis C virus antibody (anti-HCV) assays are recommended for screening HCV-infected persons. The VIDAS Anti-HCV Assay (bioMérieux, France), based on the enzyme-linked fluorescence test principle, was recently introduced in Korea. We evaluated the clinical performance of the VIDAS assay. Methods One hundred HCV-positive and 1,002 HCV-negative blood samples confirmed by Architect anti-HCV (Abbott Laboratories, USA) and COBAS TaqMan HCV real-time PCR (Roche Diagnostics, USA) or the Procleix Ultrio Plus Assay (Gen-Probe Incorporated, USA) were obtained from the Human Serum Bank (HSB) and tested by VIDAS. In case of discrepant results, we conducted a recombinant immunoblot assay (RIBA). Results The agreement rates for known HCV-positive and HCV-negative samples between the VIDAS assay and the HSB testing were 100% (95% confidence interval [CI]: 96.4-100%) and 99.5% (95% CI: 98.8-99.8%), respectively. One of the five discrepant samples was positive for Core 2+ and NS3-2 2+ reactivity, two samples were negative, and the other two were indeterminate regarding NS4 2+ reactivity in RIBA. We observed a significant but weak positive correlation between the titers of VIDAS and Architect assays (r=0.315, P<0.001). Conclusions The VIDAS anti-HCV assay, developed on the VIDAS automated immunoassay platform based on the ready-to-use, single-sample test concept may be useful in small-to-medium-sized laboratories. It showed good agreement with Architect anti-HCV and COBAS PCR assays and is therefore useful for detection of HCV infection. Weakly test-positive (ambiguous) samples require additional testing by another anti-HCV, RIBA, or HCV RNA assay. PMID:27578508

Design of laboratory experiments to study photoionization fronts driven by thermal sources

DOE PAGES

Drake, R. P.; Hazak, G.; Keiter, P. A.; ...

2016-12-20

This study analyzes the requirements of a photoionization-front experiment that could be driven in the laboratory, using thermal sources to produce the necessary flux of ionizing photons. It reports several associated conclusions. Such experiments will need to employ the largest available facilities, capable of delivering many kJ to MJ of energy to an x-ray source. They will use this source to irradiate a volume of neutral gas, likely of N, on a scale of a few mm to a few cm, increasing with source energy. For a gas pressure of several to ten atmospheres at room temperature, and a sourcemore » temperature near 100 eV, one will be able to drive a photoionization front through a system of tens to hundreds of photon mean free paths. The front should make the familiar transition from the so-called R-Type to D-Type as the radiation flux diminishes with distance. The N is likely to reach the He-like state. Preheating from the energetic photons appears unlikely to become large enough to alter the essential dynamics of the front beyond some layer near the surface. For well-chosen experimental conditions, competing energy transport mechanisms are small.« less

DESIGN OF LABORATORY EXPERIMENTS TO STUDY PHOTOIONIZATION FRONTS DRIVEN BY THERMAL SOURCES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drake, R. P.; Keiter, P. A.; Davis, J. S.

2016-12-20

This paper analyzes the requirements of a photoionization-front experiment that could be driven in the laboratory, using thermal sources to produce the necessary flux of ionizing photons. It reports several associated conclusions. Such experiments will need to employ the largest available facilities, capable of delivering many kJ to MJ of energy to an X-ray source. They will use this source to irradiate a volume of neutral gas, likely of N, on a scale of a few mm to a few cm, increasing with source energy. For a gas pressure of several to ten atmospheres at room temperature, and a sourcemore » temperature near 100 eV, one will be able to drive a photoionization front through a system of tens to hundreds of photon mean free paths. The front should make the familiar transition from the so-called R-Type to D-Type as the radiation flux diminishes with distance. The N is likely to reach the He-like state. Preheating from the energetic photons appears unlikely to become large enough to alter the essential dynamics of the front beyond some layer near the surface. For well-chosen experimental conditions, competing energy transport mechanisms are small.« less

Experimental methods of determining thermal properties of granite

USDA-ARS?s Scientific Manuscript database

Determination of thermal properties of granite using the block method is discussed and compared with other methods. Problems that limit the accuracy of contact method in determining thermal properties of porous media are evaluated. Thermal properties of granite is determined in the laboratory with a...

Assay for Arf GTP-binding Proteins | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Laboratory of Cellular and Molecular Biology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize an antibody-based proteomics assay.

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis

PubMed Central

Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-01-01

ABSTRACT Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. PMID:28202794

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

PubMed

Robert-Gangneux, Florence; Brenier-Pinchart, Marie-Pierre; Yera, Hélène; Belaz, Sorya; Varlet-Marie, Emmanuelle; Bastien, Patrick

2017-05-01

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations ( P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal. Copyright © 2017 American Society for Microbiology.

Fischer Assays of Oil-Shale Drill Cores and Rotary Cuttings from the Greater Green River Basin, Southwestern Wyoming

USGS Publications Warehouse

,

2008-01-01

Chapter 1 of this CD-ROM is a database of digitized Fischer (shale-oil) assays of cores and cuttings from boreholes drilled in the Eocene Green River oil shale deposits in southwestern Wyoming. Assays of samples from some surface sections are also included. Most of the Fischer assay analyses were made by the former U.S. Bureau of Mines (USBM) at its laboratory in Laramie, Wyoming. Other assays, made by institutional or private laboratories, were donated to the U.S. Geological Survey (USGS) and are included in this database as well as Adobe PDF-scanned images of some of the original laboratory assay reports and lithologic logs prepared by USBM geologists. The size of this database is 75.2 megabytes and includes information on 971 core holes and rotary-drilled boreholes and numerous surface sections. Most of these data were released previously by the USBM and the USGS through the National Technical Information Service but are no longer available from that agency. Fischer assays for boreholes in northeastern Utah and northwestern Colorado have been published by the USGS. Additional data include geophysical logs, groundwater data, chemical and X-ray diffraction analyses, and other data. These materials are available for inspection in the office of the USGS Central Energy Resources Team in Lakewood, Colorado. The digitized assays were checked with the original laboratory reports, but some errors likely remain. Other information, such as locations and elevations of core holes and oil and gas tests, were not thoroughly checked. However, owing to the current interest in oil-shale development, it was considered in the public interest to make this preliminary database available at this time. Chapter 2 of this CD-ROM presents oil-yield histograms of samples of cores and cuttings from exploration drill holes in the Eocene Green River Formation in the Great Divide, Green River, and Washakie Basins of southwestern Wyoming. A database was compiled that includes about 47

Thermal Protection Materials Development

NASA Technical Reports Server (NTRS)

Selvaduray, Guna; Cox, Michael

1998-01-01

The main portion of this contract year was spent on the development of materials for high temperature applications. In particular, thermal protection materials were constantly tested and evaluated for thermal shock resistance, high-temperature dimensional stability, and tolerance to hostile environmental effects. The analytical laboratory at the Thermal Protection Materials Branch (TPMB), NASA-Ames played an integral part in the process of materials development of high temperature aerospace applications. The materials development focused mainly on the determination of physical and chemical characteristics of specimens from the various research programs.

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

PubMed Central

McSharry, James J.; McDonough, Ann C.; Olson, Betty A.; Drusano, George L.

2004-01-01

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses. PMID:14715540

Gas Control and Thermal Modeling Methods for Pressed Pellet and Fast Rise Thin-Film Thermal Batteries

DTIC Science & Technology

2015-09-01

Thin-Film Thermal Batteries by Frank C Krieger and Michael S Ding Approved for public release; distribution unlimited...Laboratory Gas Control and Thermal Modeling Methods for Pressed Pellet and Fast Rise Thin-Film Thermal Batteries by Frank C Krieger and Michael S...Batteries 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Frank C Krieger and Michael S Ding 5d. PROJECT NUMBER

Multicenter comparison of levels of antibody to the Neisseria meningitidis group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay.

PubMed Central

Carlone, G M; Frasch, C E; Siber, G R; Quataert, S; Gheesling, L L; Turner, S H; Plikaytis, B D; Helsel, L O; DeWitt, W E; Bibb, W F

1992-01-01

There is no standard immunoassay for evaluating immune responses to meningococcal vaccines. We developed an enzyme-linked immunosorbent assay to measure total levels of antibody to Neisseria meningitidis group A capsular polysaccharide. Five laboratories measured the antibody levels in six paired pre- and postvaccination serum samples by using the enzyme-linked immunosorbent assay. Methylated human serum albumin was used to bind native group A polysaccharide to microtiter plate surfaces. The between-laboratory coefficients of variation for pre- and postvaccination sera had ranges of 31 to 91 and 17 to 31, respectively. The mean laboratory coefficients of variation for pre- and postvaccination sera, respectively, were 17 and 11 (Molecular Biology Laboratory, Centers for Disease Control), 12 and 15 (Immunodiagnostic Methods Laboratory, Centers for Disease Control), 22 and 19 (Dana-Farber Cancer Institute), 38 and 38 (Bacterial Polysaccharide Laboratory, U.S. Food and Drug Administration), and 11 and 10 (Praxis Biologics, Inc.). Standardization of this enzyme-linked immunosorbent assay should allow interlaboratory comparison of meningococcal vaccine immunogenicity, thus providing a laboratory-based assessment tool for evaluating meningococcal vaccines. PMID:1734048

Immobilized Lactase in the Biochemistry Laboratory

NASA Astrophysics Data System (ADS)

Allison, Matthew J.; Bering, C. Larry

1998-10-01

Immobilized enzymes have many practical applications. They may be used in clinical, industrial, and biotechnological laboratories and in many clinical diagnostic kits. For educational purposes, use of immobilized enzymes can easily be taught at the undergraduate or even secondary level. We have developed an immobilized enzyme experiment that combines many practical techniques used in the biochemistry laboratory and fits within a three-hour time frame. In this experiment, lactase from over-the-counter tablets for patients with lactose intolerance is immobilized in polyacrylamide, which is then milled into small beads and placed into a chromatography column. A lactose solution is added to the column and the eluant is assayed using the glucose oxidase assay, available as a kit. We have determined the optimal conditions to give the greatest turnover of lactose while allowing the immobilized enzymes to be active for long periods at room temperature.

Thermal Performance Benchmarking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Xuhui; Moreno, Gilbert; Bennion, Kevin

2016-06-07

The goal for this project is to thoroughly characterize the thermal performance of state-of-the-art (SOA) in-production automotive power electronics and electric motor thermal management systems. Information obtained from these studies will be used to: evaluate advantages and disadvantages of different thermal management strategies; establish baseline metrics for the thermal management systems; identify methods of improvement to advance the SOA; increase the publicly available information related to automotive traction-drive thermal management systems; help guide future electric drive technologies (EDT) research and development (R&D) efforts. The thermal performance results combined with component efficiency and heat generation information obtained by Oak Ridge Nationalmore » Laboratory (ORNL) may then be used to determine the operating temperatures for the EDT components under drive-cycle conditions. In FY16, the 2012 Nissan LEAF power electronics and 2014 Honda Accord Hybrid power electronics thermal management system were characterized. Comparison of the two power electronics thermal management systems was also conducted to provide insight into the various cooling strategies to understand the current SOA in thermal management for automotive power electronics and electric motors.« less

Thermal Performance of Biological Substance Systems in Vitro Under Static and Dynamic Conditions at the Cryogenic Test Laboratory, NASA Kennedy Space Center, USA

NASA Technical Reports Server (NTRS)

Augustynowicz, S. D.; Fesmire, James E.; Steinrock, T. (Technical Monitor)

2001-01-01

A unique research program, including a comprehensive study of thermal performance at cryogenic vacuum insulation systems, was performed at the NASA Kennedy Space Center. The main goal was to develop a new soft vacuum system (from 1 torr to 10 torr) that provides an intermediate level of performance (k-value below 4.8 mW/m-K). Liquid nitrogen boil-off methods were used to test conventional materials, novel materials, and certain combinations. The test articles included combinations of aluminum foil, fiberglass paper, polyester fabric, silica aerogel composite blanket, fumed silica, silica aerogel powder, and syntactic foam. A new LCI system was developed at the Cryogenics Test Laboratory. This system performs exceptionally well at soft vacuum levels and nearly as good as an MLI at high vacuum levels. Apparent thermal conductivities for the LCI range from 2 mW/m-K at soft vacuum to 0.1 mW/m-K at high vacuum. Several cryostats were designed, constructed, and calibrated by the Cryogenics Test Laboratory at KSC NASA as part of this research program. The cryostat test apparatus is a liquid nitrogen boil-off calorimeter system for direct measurement of the apparent thermal conductivity at a fixed vacuum level between 5 x 10(exp -5) and 760 torr. The apparatus is also used for transient measurements of temperature profiles. The development of efficient, robust cryogenic insulation systems has been a targeted area of research for a number of years. Improved methods of characterization, testing, and evaluation of complex biological substance systems for cryosurgery and cryobiology are the focus of this paper.

Enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay.

PubMed

Hensley, Tiffany R; Easter, Austin B; Gerdts, Sarah E; De Rosa, Stephen C; Heit, Antje; McElrath, M Juliana; Andersen-Nissen, Erica

2012-09-16

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 μl of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (version 8/2010). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in

Thermal Conductivity within Nanoparticle Powder Beds

NASA Astrophysics Data System (ADS)

Wilson, Mark; Chandross, Michael

Non-equilibrium molecular dynamics is utilized to compute thermal transport properties within nanoparticle powder beds. In the realm of additive manufacturing of metals, the electronic contribution to thermal conduction is critical. To this end, our simulations incorporate the two temperature model, coupling a continuum representation of the electronic thermal contribution and the atomic phonon system. The direct method is used for conductivity determination, wherein thermal gradients between two different temperature heat flux reservoirs are calculated. The approach is demonstrated on several example cases including 304L stainless steel. The results from size distribution variations of mono/poly-disperse systems are extrapolated to predict values at the micron length scale, along with bulk properties at infinite system sizes. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

Validity of urinary monoamine assay sales under the “spot baseline urinary neurotransmitter testing marketing model”

PubMed Central

Hinz, Marty; Stein, Alvin; Uncini, Thomas

2011-01-01

Spot baseline urinary monoamine assays have been used in medicine for over 50 years as a screening test for monoamine-secreting tumors, such as pheochromocytoma and carcinoid syndrome. In these disease states, when the result of a spot baseline monoamine assay is above the specific value set by the laboratory, it is an indication to obtain a 24-hour urine sample to make a definitive diagnosis. There are no defined applications where spot baseline urinary monoamine assays can be used to diagnose disease or other states directly. No peer-reviewed published original research exists which demonstrates that these assays are valid in the treatment of individual patients in the clinical setting. Since 2001, urinary monoamine assay sales have been promoted for numerous applications under the “spot baseline urinary neurotransmitter testing marketing model”. There is no published peer-reviewed original research that defines the scientific foundation upon which the claims for these assays are made. On the contrary, several articles have been published that discredit various aspects of the model. To fill the void, this manuscript is a comprehensive review of the scientific foundation and claims put forth by laboratories selling urinary monoamine assays under the spot baseline urinary neurotransmitter testing marketing model. PMID:21912487

Key Learnings from the Endocrine Disruptor Screening Program (EDSP) Tier 1 Rodent Uterotrophic and Hershberger Assays

PubMed Central

Marty, M Sue; O'Connor, John C

2014-01-01

In 2009, companies began screening compounds using the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). EDSP has two tiers: Tier 1 includes 11 assays to identify compounds with potential endocrine activity. This article describes two laboratories' experiences conducting Tier 1 uterotrophic and Hershberger assays. The uterotrophic assay detects estrogen receptor agonists through increases in uterine weight. The advantages of the uterotrophic rat models (immature vs. adult ovariectomized) and exposure routes are discussed. Across 29 studies, relative differences in uterine weights in the vehicle control group and 17α-ethynylestradiol–positive control group were reasonably reproducible. The Hershberger assay detects androgen receptor (AR) agonists, antagonists, and 5α-reductase inhibitors through changes in accessory sex tissue (AST) weights. Across 23 studies, AST weights were relatively reproducible for the vehicle groups (baseline), testosterone propionate (TP) groups (androgenic response), and flutamide + TP groups (antiandrogenic response). In one laboratory, one and four compounds were positive in the androgenic and antiandrogenic portions of the assay, respectively. Each compound was also positive for AR binding. In the other laboratory, three compounds showed potential antiandrogenic activity, but each compound was negative for AR binding and did not fit the profile for 5α-reductase inhibition. These compounds induced hepatic enzymes that enhanced testosterone metabolism/clearance, resulting in lower testosterone and decreased capacity to maintain AST weights. The Hershberger androgenic and antiandrogenic performance criteria were generally attainable. Overall, the uterotrophic and Hershberger assays were easily adopted and function as described for EDSP screening, although the mode of action for positive results may not be easily determined. PMID:24515841

Measuring antioxidant capacity using the ORAC and TOSC assays.

PubMed

Garrett, Andrew R; Murray, Byron K; Robison, Richard A; O'Neill, Kim L

2010-01-01

Recent epidemiological studies have shown that there may be a link between oxidative stress and the development of several types of chronic diseases. Studies have also shown that diets rich in fruits and vegetables may decrease the incidence of cancer and other chronic diseases. The antioxidant activity of the phytochemicals these foods contain may be partially responsible for the decreased incidence of these diseases in people who regularly consume them. While there are several assays currently used to assess the antioxidant activity of phytochemicals and other antioxidant compounds, two are reviewed here in detail. The first is the oxygen radical absorbance capacity (ORAC) assay, which measures the decrease in fluorescence decay caused by antioxidants, and the second is the total oxyradical scavenging capacity (TOSC) assay, which measures the decrease in ethylene gas production caused by the inhibition of the thermal hydrolysis of ABAP (2,2'-Azobis(2-methyl-(propionamidine) dihydrochloride) by KMBA (alpha-keto-gamma-(methylthio)butyric acid sodium salt) in the presence of antioxidant compounds. These two assays are discussed here, with an in depth review of their methodology and correlation.

Development of a rapid, simple assay of plasma total carotenoids

PubMed Central

2012-01-01

Background Plasma total carotenoids can be used as an indicator of risk of chronic disease. Laboratory analysis of individual carotenoids by high performance liquid chromatography (HPLC) is time consuming, expensive, and not amenable to use beyond a research laboratory. The aim of this research is to establish a rapid, simple, and inexpensive spectrophotometric assay of plasma total carotenoids that has a very strong correlation with HPLC carotenoid profile analysis. Results Plasma total carotenoids from 29 volunteers ranged in concentration from 1.2 to 7.4 μM, as analyzed by HPLC. A linear correlation was found between the absorbance at 448 nm of an alcohol / heptane extract of the plasma and plasma total carotenoids analyzed by HPLC, with a Pearson correlation coefficient of 0.989. The average coefficient of variation for the spectrophotometric assay was 6.5% for the plasma samples. The limit of detection was about 0.3 μM and was linear up to about 34 μM without dilution. Correlations between the integrals of the absorption spectra in the range of carotenoid absorption and total plasma carotenoid concentration gave similar results to the absorbance correlation. Spectrophotometric assay results also agreed with the calculated expected absorbance based on published extinction coefficients for the individual carotenoids, with a Pearson correlation coefficient of 0.988. Conclusion The spectrophotometric assay of total carotenoids strongly correlated with HPLC analysis of carotenoids of the same plasma samples and expected absorbance values based on extinction coefficients. This rapid, simple, inexpensive assay, when coupled with the carotenoid health index, may be useful for nutrition intervention studies, population cohort studies, and public health interventions. PMID:23006902

CLSI-based transference of CALIPER pediatric reference intervals to Beckman Coulter AU biochemical assays.

PubMed

Abou El Hassan, Mohamed; Stoianov, Alexandra; Araújo, Petra A T; Sadeghieh, Tara; Chan, Man Khun; Chen, Yunqi; Randell, Edward; Nieuwesteeg, Michelle; Adeli, Khosrow

2015-11-01

The CALIPER program has established a comprehensive database of pediatric reference intervals using largely the Abbott ARCHITECT biochemical assays. To expand clinical application of CALIPER reference standards, the present study is aimed at transferring CALIPER reference intervals from the Abbott ARCHITECT to Beckman Coulter AU assays. Transference of CALIPER reference intervals was performed based on the CLSI guidelines C28-A3 and EP9-A2. The new reference intervals were directly verified using up to 100 reference samples from the healthy CALIPER cohort. We found a strong correlation between Abbott ARCHITECT and Beckman Coulter AU biochemical assays, allowing the transference of the vast majority (94%; 30 out of 32 assays) of CALIPER reference intervals previously established using Abbott assays. Transferred reference intervals were, in general, similar to previously published CALIPER reference intervals, with some exceptions. Most of the transferred reference intervals were sex-specific and were verified using healthy reference samples from the CALIPER biobank based on CLSI criteria. It is important to note that the comparisons performed between the Abbott and Beckman Coulter assays make no assumptions as to assay accuracy or which system is more correct/accurate. The majority of CALIPER reference intervals were transferrable to Beckman Coulter AU assays, allowing the establishment of a new database of pediatric reference intervals. This further expands the utility of the CALIPER database to clinical laboratories using the AU assays; however, each laboratory should validate these intervals for their analytical platform and local population as recommended by the CLSI. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae.

PubMed

Fitzmaurice, J; Sewell, M; King, C M; McDougall, S; McDonald, W L; O'Keefe, J S

2008-10-01

To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.

Human low density lipoprotein as a substrate for in vitro steroidogenesis assays with fathead minnow ovary explants

EPA Science Inventory

Gonad explant in vitro steroidogenesis assays are used as part of a multifaceted strategy to detect endocrine active chemicals capable of altering steroid hormone synthesis. An in vitro steroidogenesis assay used in our laboratory involves exposing fathead minnow (FHM) gonad exp...

Laboratory Assays in Evaluation of Lynch Syndrome in Patients with Endometrial Carcinoma.

PubMed

Djordjevic, Bojana; Broaddus, Russell R

2016-06-01

This article reviews the main tissue testing modalities for Lynch Syndrome in the pathology laboratory, such as immunohistochemistry and PCR based analyses, and discusses their routine application, interpretation pitfalls, and troubleshooting of common technical performance issues. Discrepancies between laboratory and genetic testing may arise, and are examined in the context of the complexity of molecular abnormalities associated with Lynch Syndrome. The merits of targeted versus universal screening in a changing healthcare climate are addressed. In the absence of comprehensive screening programs, specific tumor topography and histological features that may prompt pathologist-initiated molecular tumor testing are outlined. Copyright © 2016 Elsevier Inc. All rights reserved.

ELECTRONICS UPGRADE TO THE SAVANNAH RIVER NATIONAL LABORATORY COULOMETER FOR PLUTONIUM AND NEPTUNIUM ASSAY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cordaro, J.; Holland, M.; Reeves, G.

The Savannah River Site (SRS) has the analytical measurement capability to perform high-precision plutonium concentration measurements by controlled-potential coulometry. State-of-the-art controlled-potential coulometers were designed and fabricated by the Savannah River National Laboratory and installed in the Analytical Laboratories process control laboratory. The Analytical Laboratories uses coulometry for routine accountability measurements of and for verification of standard preparations used to calibrate other plutonium measurement systems routinely applied to process control, nuclear safety, and other accountability applications. The SRNL Coulometer has a demonstrated measurement reliability of {approx}0.05% for 10 mg samples. The system has also been applied to the characterization of neptuniummore » standard solutions with a comparable reliability. The SRNL coulometer features: a patented current integration system; continuous electrical calibration versus Faraday's Constants and Ohm's Law; the control-potential adjustment technique for enhanced application of the Nernst Equation; a wide operating room temperature range; and a fully automated instrument control and data acquisition capability. Systems have been supplied to the International Atomic Energy Agency (IAEA), Russia, Japanese Atomic Energy Agency (JAEA) and the New Brunswick Laboratory (NBL). The most recent vintage of electronics was based on early 1990's integrated circuits. Many of the components are no longer available. At the request of the IAEA and the Department of State, SRNL has completed an electronics upgrade of their controlled-potential coulometer design. Three systems have built with the new design, one for the IAEA which was installed at SAL in May 2011, one system for Los Alamos National Laboratory, (LANL) and one for the SRS Analytical Laboratory. The LANL and SRS systems are undergoing startup testing with installation scheduled for this summer.« less

Physics Laboratory in UEC

NASA Astrophysics Data System (ADS)

Takada, Tohru; Nakamura, Jin; Suzuki, Masaru

All the first-year students in the University of Electro-Communications (UEC) take "Basic Physics I", "Basic Physics II" and "Physics Laboratory" as required subjects; Basic Physics I and Basic Physics II are calculus-based physics of mechanics, wave and oscillation, thermal physics and electromagnetics. Physics Laboratory is designed mainly aiming at learning the skill of basic experimental technique and technical writing. Although 95% students have taken physics in the senior high school, they poorly understand it by connecting with experience, and it is difficult to learn Physics Laboratory in the university. For this reason, we introduced two ICT (Information and Communication Technology) systems of Physics Laboratory to support students'learning and staff's teaching. By using quantitative data obtained from the ICT systems, we can easily check understanding of physics contents in students, and can improve physics education.

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

PubMed

Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

2008-07-01

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

Engineering of a miniaturized, robotic clinical laboratory

PubMed Central

Nourse, Marilyn B.; Engel, Kate; Anekal, Samartha G.; Bailey, Jocelyn A.; Bhatta, Pradeep; Bhave, Devayani P.; Chandrasekaran, Shekar; Chen, Yutao; Chow, Steven; Das, Ushati; Galil, Erez; Gong, Xinwei; Gessert, Steven F.; Ha, Kevin D.; Hu, Ran; Hyland, Laura; Jammalamadaka, Arvind; Jayasurya, Karthik; Kemp, Timothy M.; Kim, Andrew N.; Lee, Lucie S.; Liu, Yang Lily; Nguyen, Alphonso; O'Leary, Jared; Pangarkar, Chinmay H.; Patel, Paul J.; Quon, Ken; Ramachandran, Pradeep L.; Rappaport, Amy R.; Roy, Joy; Sapida, Jerald F.; Sergeev, Nikolay V.; Shee, Chandan; Shenoy, Renuka; Sivaraman, Sharada; Sosa‐Padilla, Bernardo; Tran, Lorraine; Trent, Amanda; Waggoner, Thomas C.; Wodziak, Dariusz; Yuan, Amy; Zhao, Peter; Holmes, Elizabeth A.

2018-01-01

Abstract The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay‐configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay‐specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti‐herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration‐cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations. PMID:29376134

REVIEW - Thermal Physiology of Laboratory Mice: Defining Thermoneutrality

EPA Science Inventory

In terms of total number of publications, the laboratory mouse (Mus musculus) has emerged as the most popular test subject in biomedical research. Mice are used as models to study obesity, diabetes, eNS diseases and variety of other pathologies. Mice are classified as homeotherms...

Tritium laboratory with multiple purposes at NIPNE Magurele Romania

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matei, L.; Postolache, C.

2008-07-15

The Tritium Laboratory from NIPNE (Romania)) is part of Radioisotope Research and Production Center. The Tritium Laboratory has been in operation since 1960, and carries out R and D activities involving tritium sources in gaseous, liquids and solid state, provides specialized service to CANDU NPP Cernavoda (Romania)), and provides tritium assay services to internal and external customers. The paper presents the activities and perspectives of Tritium Laboratory and its performances in accordance with Quality System Management. (authors)

Thermal and enzymatic recovering of proteins from untanned leather waste.

PubMed

Bajza, Z; Vrucek, V

2001-01-01

The laboratory trials of a process to treat untanned leather waste to isolate valuable protein products are presented. In this comparative study, both thermal and enzymatic treatments of leather waste were performed. The enzymatic method utilizes commercially available alkaline protease at moderate temperatures and for short periods of time. The concentration of the enzyme was 500 units per gram of leather waste which makes the method cost-effective. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis of untanned leather waste is determined. Chemical and physical properties of protein powder products from untanned leather waste were evaluated by spectrophotometric and chromatographic methods and by use of electron microscope. The results of microbiological assays confirm that these products agree to food safety standards. This relatively simple treatment of untanned leather waste may provide a practical and economical solution to the disposal of potentially dangerous waste.

Correlation of predicted and measured thermal stresses on an advanced aircraft structure with similar materials

NASA Technical Reports Server (NTRS)

Jenkins, J. M.

1979-01-01

A laboratory heating test simulating hypersonic heating was conducted on a heat-sink type structure to provide basic thermal stress measurements. Six NASTRAN models utilizing various combinations of bar, shear panel, membrane, and plate elements were used to develop calculated thermal stresses. Thermal stresses were also calculated using a beam model. For a given temperature distribution there was very little variation in NASTRAN calculated thermal stresses when element types were interchanged for a given grid system. Thermal stresses calculated for the beam model compared similarly to the values obtained for the NASTRAN models. Calculated thermal stresses compared generally well to laboratory measured thermal stresses. A discrepancy of signifiance occurred between the measured and predicted thermal stresses in the skin areas. A minor anomaly in the laboratory skin heating uniformity resulted in inadequate temperature input data for the structural models.

Parmitano in U.S. Laboratory

NASA Image and Video Library

2013-07-30

ISS036-E-027387 (29 July 2013) --- European Space Agency astronaut Luca Parmitano, Expedition 36 flight engineer, performs maintenance on the Water Pump Assembly 2 / Thermal Control System (WPA2/TCS) in the Columbus laboratory of the International Space Station.

Parmitano in U.S. Laboratory

NASA Image and Video Library

2013-07-30

ISS036-E-027389 (29 July 2013) --- European Space Agency astronaut Luca Parmitano, Expedition 36 flight engineer, performs maintenance on the Water Pump Assembly 2 / Thermal Control System (WPA2/TCS) in the Columbus laboratory of the International Space Station.

Students' Framing of Laboratory Exercises Using Infrared Cameras

ERIC Educational Resources Information Center

Haglund, Jesper; Jeppsson, Fredrik; Hedberg, David; Schönborn, Konrad J.

2015-01-01

Thermal science is challenging for students due to its largely imperceptible nature. Handheld infrared cameras offer a pedagogical opportunity for students to see otherwise invisible thermal phenomena. In the present study, a class of upper secondary technology students (N = 30) partook in four IR-camera laboratory activities, designed around the…

Carboxyhaemoglobin: a possible reference material for haemoglobin assay.

PubMed Central

Rideout, J M; Louderback, A

1982-01-01

The inter- and intralaboratory quality control of haemoglobin estimation in remote laboratories requires a more rugged control haemolysate than is commercially available. The stabilities of oxyhaemoglobin and carboxyhaemoglobin forms of an ethanediol-containing haemolysate were studied over a three-year period. From the results obtained, carboxyhaemoglobin under nitrogen is proposed as a possible candidate reference material for haemoglobin assay. PMID:7068921

Laboratory measurement of apixaban using anti-factor Xa assays in acute ischemic stroke patients with non-valvular atrial fibrillation.

PubMed

Shin, Hyoshim; Cho, Min-Chul; Kim, Rock Bum; Kim, Chang-Hun; Choi, Nack-Cheon; Kim, Soo-Kyung; Koh, Eun-Ha

2018-02-01

Apixaban is effective and safe for preventing stroke, and its usage has increased exponentially in recent years. However, data concerning the therapeutic range of apixaban is limited. This study determined the trough and peak levels of apixaban-specific anti-factor Xa activity (AFXaA) in acute ischemic stroke patients with non-valvular atrial fibrillation (NVAF) in Korea. The study included 85 patients who received apixaban. Blood samples were taken to measure the trough and peak levels of AFXaA using a chromogenic anti-factor assay, as well as prothrombin time (PT) and activated partial thromboplastin time (aPTT). We also reviewed complications such as major bleeding of patients treated with apixaban. In patients given a 5.0-mg apixaban dose, the median trough and peak levels of AFXaA were 104.5 and 202.0 ng/mL. In patients given a 2.5-mg apixaban dose, the median trough and peak AFXaA levels were 76.0 and 151.0 ng/mL. The PT showed a positive correlation with increased AFXaA activity at both levels (Trough R = 0.486, Peak R = 0.592), but the aPTT had no relationship with AFXaA activity at both levels (Trough R = 0.181, Peak R = 0.129). Two cases with intracranial bleeding belonged to the highest AFXaA quartile (Trough, p = 0.176; Peak, p = 0.053). In conclusion, we determined the trough and peak levels of AFXaA in patients with NVAF while being treated with the apixaban in Korea. Our results could be used as a starting point when setting the reference ranges for laboratories using anti-Xa assay. Large-scale studies are needed to establish the reference range for AFXaA in patients with NVAF.

Validation conform ISO-15189 of assays in the field of autoimmunity: Joint efforts in The Netherlands.

PubMed

Mulder, Leontine; van der Molen, Renate; Koelman, Carin; van Leeuwen, Ester; Roos, Anja; Damoiseaux, Jan

2018-05-01

ISO 15189:2012 requires validation of methods used in the medical laboratory, and lists a series of performance parameters for consideration to include. Although these performance parameters are feasible for clinical chemistry analytes, application in the validation of autoimmunity tests is a challenge. Lack of gold standards or reference methods in combination with the scarcity of well-defined diagnostic samples of patients with rare diseases make validation of new assays difficult. The present manuscript describes the initiative of Dutch medical immunology laboratory specialists to combine efforts and perform multi-center validation studies of new assays in the field of autoimmunity. Validation data and reports are made available to interested Dutch laboratory specialists. Copyright © 2018 Elsevier B.V. All rights reserved.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

PubMed

Sista, Ramakrishna S; Eckhardt, Allen E; Wang, Tong; Graham, Carrie; Rouse, Jeremy L; Norton, Scott M; Srinivasan, Vijay; Pollack, Michael G; Tolun, Adviye A; Bali, Deeksha; Millington, David S; Pamula, Vamsee K

2011-10-01

Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Students' framing of laboratory exercises using infrared cameras

NASA Astrophysics Data System (ADS)

Haglund, Jesper; Jeppsson, Fredrik; Hedberg, David; Schönborn, Konrad J.

2015-12-01

Thermal science is challenging for students due to its largely imperceptible nature. Handheld infrared cameras offer a pedagogical opportunity for students to see otherwise invisible thermal phenomena. In the present study, a class of upper secondary technology students (N =30 ) partook in four IR-camera laboratory activities, designed around the predict-observe-explain approach of White and Gunstone. The activities involved central thermal concepts that focused on heat conduction and dissipative processes such as friction and collisions. Students' interactions within each activity were videotaped and the analysis focuses on how a purposefully selected group of three students engaged with the exercises. As the basis for an interpretative study, a "thick" narrative description of the students' epistemological and conceptual framing of the exercises and how they took advantage of the disciplinary affordance of IR cameras in the thermal domain is provided. Findings include that the students largely shared their conceptual framing of the four activities, but differed among themselves in their epistemological framing, for instance, in how far they found it relevant to digress from the laboratory instructions when inquiring into thermal phenomena. In conclusion, the study unveils the disciplinary affordances of infrared cameras, in the sense of their use in providing access to knowledge about macroscopic thermal science.

Vehicle Thermal Management Models and Tools | Transportation Research |

Science.gov Websites

NREL Models and Tools Vehicle Thermal Management Models and Tools The National Renewable Energy Laboratory's (NREL's) vehicle thermal management modeling tools allow researchers to assess the trade-offs and calculate the potential benefits of thermal design options. image of three models of semi truck cabs. Truck

Design and evaluation of a nondestructive fissile assay device for HTGR fuel samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

McNeany, S. R.; Knoll, R. W.; Jenkins, J. D.

1979-02-01

Nondestructive assay of fissile material plays an important role in nuclear fuel processing facilities. Information for product quality control, plant criticality safety, and nuclear materials accountability can be obtained from assay devices. All of this is necessary for a safe, efficient, and orderly operation of a production plant. Presented here is a design description and an operational evaluation of a device developed to nondestructively assay small samples of High-Temperature Gas-Cooled Reactor (HTGR) fuel. The measurement technique employed consists in thermal-neutron irradiation of a sample followed by pneumatic transfer to a high-efficiency neutron detector where delayed neutrons are counted. In general,more » samples undergo several irradiation and count cycles during a measurement. The total number of delayed-neutron counts accumulated is translated into grams of fissile mass through comparison with the counts accumulated in an identical irradiation and count sequence of calibration standards. Successful operation of the device through many experiments over a one-year period indicates high operational reliability. Tests of assay precision show this to be better than 0.25% for measurements of 10 min. Assay biases may be encountered if calibration standards are not representative of unknown samples, but reasonable care in construction and control of standards should lead to no more than 0.2% bias in the measurements. Nondestructive fissile assay of HTGR fuel samples by thermal-neutron irradiation and delayed-neutron detection has been demonstrated to be a rapid and accurate analysis technique. However, careful attention and control must be given to calibration standards to see that they remain representative of unknown samples.« less

MEASUREMENT OF WIND SPEED FROM COOLING LAKE THERMAL IMAGERY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garrett, A; Robert Kurzeja, R; Eliel Villa-Aleman, E

2009-01-20

The Savannah River National Laboratory (SRNL) collected thermal imagery and ground truth data at two commercial power plant cooling lakes to investigate the applicability of laboratory empirical correlations between surface heat flux and wind speed, and statistics derived from thermal imagery. SRNL demonstrated in a previous paper [1] that a linear relationship exists between the standard deviation of image temperature and surface heat flux. In this paper, SRNL will show that the skewness of the temperature distribution derived from cooling lake thermal images correlates with instantaneous wind speed measured at the same location. SRNL collected thermal imagery, surface meteorology andmore » water temperatures from helicopters and boats at the Comanche Peak and H. B. Robinson nuclear power plant cooling lakes. SRNL found that decreasing skewness correlated with increasing wind speed, as was the case for the laboratory experiments. Simple linear and orthogonal regression models both explained about 50% of the variance in the skewness - wind speed plots. A nonlinear (logistic) regression model produced a better fit to the data, apparently because the thermal convection and resulting skewness are related to wind speed in a highly nonlinear way in nearly calm and in windy conditions.« less

Lessons from Astrobiological Planetary Analogue Exploration in Iceland: Biomarker Assay Performance and Downselection

NASA Technical Reports Server (NTRS)

Gentry, D. M.; Amador, E. S.; Cable, M. L.; Cantrell, T.; Chaudry, N.; Cullen, T.; Duca, Z.; Kirby, J.; Jacobsen, M.; McCaig, H.;

The EXTASE thermal probe: Laboratory investigation and modelling of thermal properties

NASA Astrophysics Data System (ADS)

Kaufmann, E.; Knollenberg, J.; Kargl, G.; Koemle, N. I.

2011-10-01

In recent years space missions including landing devices are getting more important. These missions allow in-situ measurements and lead therefore to information on the structure and behavior of extraterrestrial surface and subsurface layers. Sensors used for this kind of missions have to be adapted to the non-terrestrial environment conditions. The better the properties of the single elements of each sensor are known, the more precise are the results from the data evaluation of in-situ measurements. We present the results of thermal conductivity measurements and simulations done for the fiber compound tube used as structural element for the heating segments of the MUPUS-PEN and EXTASE - a spin-off project of Rosetta/MUPUS.

Laboratory electron exposure of TSS-1 thermal control coating

NASA Technical Reports Server (NTRS)

Vaughn, J. A.; Mccollum, M.; Carruth, M. R., Jr.

1995-01-01

RM400, a conductive thermal control coating, was developed for use on the exterior shell of the tethered satellite. Testing was performed by the Engineering Physics Division to quantify effects of the space environment on this coating and its conductive and optical properties. Included in this testing was exposure of RM400 to electrons with energies ranging from 0.1 to 1 keV, to simulate electrons accelerated from the ambient space plasma when the tethered satellite is fully deployed. During this testing, the coating was found to luminesce, and a prolonged exposure of the coating to high-energy electrons caused the coating to darken. This report describes the tests done to quantify the degradation of the thermal control properties caused by electron exposure and to measure the luminescence as a function of electron energy and current density to the satellite.

Growth hormone: its measurement and the need for assay harmonization.

PubMed

Wood, P

2001-09-01

Serum human growth hormone (hGH) assays show a wide range in bias and in cut-off values for provocative tests, which vary from 13.5 to 35-40 mU/L when they have been established. Studies using novel hGH assays show that methods that are absolutely specific for 22-kDa hGH may not identify bioactive hGH peaks and that 20:22-kDa hGH ratios are increased in acromegaly. Greater harmonization of serum hGH methods can be achieved by: changing from IS 80/505 to IS 98/574, which is calibrated in mass units of recombinant 22-kDa hGH; using monoclonal/polyclonal or polyclonal/polyclonal antibody combinations that measure both 20-kDa and 22-kDa hGH; the development of assays such as the immunofunctional hGH assay which has the convenience of an immunometric assay but gives results that correlate better with bioassays collaboration between manufacturers and laboratories to establish method-related cut-off limits for provocative tests of hGH status.

Key learnings from the Endocrine Disruptor Screening Program (EDSP) Tier 1 rodent uterotrophic and Hershberger assays.

PubMed

Marty, M Sue; O'Connor, John C

2014-02-01

In 2009, companies began screening compounds using the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). EDSP has two tiers: Tier 1 includes 11 assays to identify compounds with potential endocrine activity. This article describes two laboratories' experiences conducting Tier 1 uterotrophic and Hershberger assays. The uterotrophic assay detects estrogen receptor agonists through increases in uterine weight. The advantages of the uterotrophic rat models (immature vs. adult ovariectomized) and exposure routes are discussed. Across 29 studies, relative differences in uterine weights in the vehicle control group and 17α-ethynylestradiol-positive control group were reasonably reproducible. The Hershberger assay detects androgen receptor (AR) agonists, antagonists, and 5α-reductase inhibitors through changes in accessory sex tissue (AST) weights. Across 23 studies, AST weights were relatively reproducible for the vehicle groups (baseline), testosterone propionate (TP) groups (androgenic response), and flutamide + TP groups (antiandrogenic response). In one laboratory, one and four compounds were positive in the androgenic and antiandrogenic portions of the assay, respectively. Each compound was also positive for AR binding. In the other laboratory, three compounds showed potential antiandrogenic activity, but each compound was negative for AR binding and did not fit the profile for 5α-reductase inhibition. These compounds induced hepatic enzymes that enhanced testosterone metabolism/clearance, resulting in lower testosterone and decreased capacity to maintain AST weights. The Hershberger androgenic and antiandrogenic performance criteria were generally attainable. Overall, the uterotrophic and Hershberger assays were easily adopted and function as described for EDSP screening, although the mode of action for positive results may not be easily determined. © 2014 Wiley Periodicals, Inc.

Validation of STA-Liatest D-Di assay for exclusion of pulmonary embolism according to the latest Clinical and Laboratory Standard Institute/Food and Drug Administration guideline. Results of a multicenter management study.

PubMed

Pernod, Gilles; Wu, Haifeng; de Maistre, Emmanuel; Lazarchick, John; Kassis, Jeannine; Aguilar, Carlos; Vera, Pascual M; Palareti, Gualtiero; D'Angelo, Armando

2017-04-01

: Combined clinical pretest probability (PTP) and D-dimer testing have great diagnostic value for pulmonary embolism exclusion. To harmonize performance levels of D-dimer assays available on the market, the Clinical and Laboratory Standard Institute (CLSI) has published a guideline, endorsed by the US Food and Drug Administration (FDA). Such guideline specifies the ideal D-dimer assay characteristic and target population. This study was conducted following the CLSI guideline to upgrade the assay-intended use and obtain FDA clearance of STA-Liatest D-Di assay for pulmonary embolism exclusion in patient with low/moderate PTP. This was an international, multicenter, prospective nonrandomized, noninterventional clinical outcome management study conducted in a standard of care setting. D-dimer assay was performed in consecutive, ambulatory outpatients suspected of pulmonary embolism, with low/moderate PTP, and without medical conditions or in clinical settings known to alter default D-dimer values regardless of the presence of thrombosis using a threshold of 0.5 μg/ml (fibrinogen equivalent units) for venous thromboembolism exclusion. Results were used to determine test performance. Of 1141 patients who underwent D-dimer testing, 1060 had valid results and completed study as planned. STA-Liatest D-Di assay performance has exceeded the CLSI/FDA guidance requirements, with a sensitivity of 97.6% (95% confidence interval: 91.7-99.7%) and a negative predictive value of 99.7% (95% confidence interval: 99.0-100%). STA-Liatest D-Di assay has an excellent performance when used in combination with a PTP score in relevant patients and has the potential to minimize the economic healthcare burden avoiding unnecessary and expensive imaging tests.

Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins ▿

PubMed Central

Carney, Paul J.; Lipatov, Aleksandr S.; Monto, Arnold S.; Donis, Ruben O.; Stevens, James

2010-01-01

During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities. PMID:20660137

Recommended Immunological Assays to Screen for Ricin-Containing Samples.

PubMed

Simon, Stéphanie; Worbs, Sylvia; Avondet, Marc-André; Tracz, Dobryan M; Dano, Julie; Schmidt, Lisa; Volland, Hervé; Dorner, Brigitte G; Corbett, Cindi R

2015-11-26

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

PubMed

Harvey, John J; Chester, Stephanie; Burke, Stephen A; Ansbro, Marisela; Aden, Tricia; Gose, Remedios; Sciulli, Rebecca; Bai, Jing; DesJardin, Lucy; Benfer, Jeffrey L; Hall, Joshua; Smole, Sandra; Doan, Kimberly; Popowich, Michael D; St George, Kirsten; Quinlan, Tammy; Halse, Tanya A; Li, Zhen; Pérez-Osorio, Ailyn C; Glover, William A; Russell, Denny; Reisdorf, Erik; Whyte, Thomas; Whitaker, Brett; Hatcher, Cynthia; Srinivasan, Velusamy; Tatti, Kathleen; Tondella, Maria Lucia; Wang, Xin; Winchell, Jonas M; Mayer, Leonard W; Jernigan, Daniel; Mawle, Alison C

2016-02-01

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a broadly reactive nested reverse transcription-PCR assay to detect murine noroviruses, and investigation of the prevalence of murine noroviruses in laboratory mice in Japan.

PubMed

Kitajima, Masaaki; Oka, Tomoichiro; Tohya, Yukinobu; Katayama, Hiroyuki; Takeda, Naokazu; Katayama, Kazuhiko

2009-09-01

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 approximately 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.

A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-10-01

Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS ® TaqMan ® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT ® HBV Assay (Versant), and 74 specimens tested with Veris and artus ® HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log 10 IU/mL between Veris and Cobas, -0.46 log 10 IU/mL between Veris and RealTime, -0.36 log 10 IU/mL between Veris and Versant, and -0.12 log 10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

Laboratory preparedness in EU/EEA countries for detection of novel avian influenza A(H7N9) virus, May 2013

PubMed Central

Broberg, E; Pereyaslov, D; Struelens, M; Palm, D; Meijer, A; Ellis, J; Zambon, M; McCauley, J; Daniels, R

2015-01-01

Following human infections with novel avian influenza A(H7N9) viruses in China, the European Centre for Disease Prevention and Control, the World Health Organization (WHO) Regional Office for Europe and the European Reference Laboratory Network for Human Influenza (ERLI-Net) rapidly posted relevant information, including real-time RT-PCR protocols. An influenza RNA sequence-based computational assessment of detection capabilities for this virus was conducted in 32 national influenza reference laboratories in 29 countries, mostly WHO National Influenza Centres participating in the WHO Global Influenza Surveillance and Response System (GISRS). Twenty-seven countries considered their generic influenza A virus detection assay to be appropriate for the novel A(H7N9) viruses. Twenty-two countries reported having containment facilities suitable for its isolation and propagation. Laboratories in 27 countries had applied specific H7 real-time RT-PCR assays and 20 countries had N9 assays in place. Positive control virus RNA was provided by the WHO Collaborating Centre in London to 34 laboratories in 22 countries to allow evaluation of their assays. Performance of the generic influenza A virus detection and H7 and N9 subtyping assays was good in 24 laboratories in 19 countries. The survey showed that ERLI-Net laboratories had rapidly developed and verified good capability to detect the novel A(H7N9) influenza viruses. PMID:24507469

Battery Pack Thermal Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pesaran, Ahmad

This presentation describes the thermal design of battery packs at the National Renewable Energy Laboratory. A battery thermal management system essential for xEVs for both normal operation during daily driving (achieving life and performance) and off-normal operation during abuse conditions (achieving safety). The battery thermal management system needs to be optimized with the right tools for the lowest cost. Experimental tools such as NREL's isothermal battery calorimeter, thermal imaging, and heat transfer setups are needed. Thermal models and computer-aided engineering tools are useful for robust designs. During abuse conditions, designs should prevent cell-to-cell propagation in a module/pack (i.e., keep themore » fire small and manageable). NREL's battery ISC device can be used for evaluating the robustness of a module/pack to cell-to-cell propagation.« less

Laboratory investigation of hypercoagulability.

PubMed

Francis, J L

1998-01-01

For many years, the laboratory investigation of patients with thrombophilia has lagged behind that of patients with bleeding diathesis. Improved understanding of the mechanisms that control and regulate coagulation, and the resultant recognition of new defects, have greatly stimulated clinical laboratory interest in this area. Assays to detect resistance to activated protein C; deficiencies of antithrombin, protein C, and protein S; and the presence of antiphospholipid antibodies are widely available and should form part of the investigation of patients that present with idiopathic thrombosis. Such a work-up will likely provide an explanation for thrombosis in 40 to 60% of patients. Abnormalities of fibrinogen and fibrinolysis may explain still more, although such defects are currently considered rare. In addition, presently unrecognized defects almost certainly exist, and the identification of such individuals will undoubtedly improve our understanding of the hemostatic mechanism. Laboratory tests to define the hypercoagulable state are continually being developed. They include whole blood coagulation and platelet function tests and novel activation markers. However, acceptance of these approaches by clinical laboratories has been slow.

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

DOE PAGES

Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

2017-07-19

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

Tritium assay of Li sub 2 O pellets in the LBM/LOTUS experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quanci, J.; Azam, S.; Bertone, P.

1986-01-01

One of the objectives of the Lithium Blanket Module (LBM) program is to test the ability of advanced neutronics codes to model the tritium breeding characteristics of a fusion blanket exposed to a toroidal fusion neutron source. The LBM consists of over 20,000 cylindrical lithium oxide pellets and numerous diagnostic pellets and wafers. The LBM has been irradiated at the Ecole Polytechnique Federale de Lausanne (EPFL) LOTUS facility with a Haefely sealed neutron generator that gives a point deuterium-tritium neutron source up to 5 {times} 10{sup 12} 14-MeV n/s. Both Princeton Plasma Physics Laboratory (PPL) and EPFL assayed the tritiummore » bred at various positions in the LBM. EPFL employed a dissolution technique while PPL recovered the tritium by a thermal extraction method. EPFL uses 0.38-g, 75% TD, lithium oxide diagnostic wafers to evaluate the tritium bred in the LBM. PPPL employs a thermal extraction method to determine the tritium bred in lithium oxide samples. In the initial experiments, diagnostic pellets and wafers were placed at five locations in the LBM central removable test rod at distances of 3, 9, 21, 36, and 48 cm from the front face of the module. The two sets of data for the tritium bred in the LBM along its centerline as a function of distance from the front face of the module were compared with each other, and with the predictions of two-dimensional neutronics codes. 1 ref.« less

Thermal fatigue and thermal shock in bedrock: An attempt to unravel the geomorphic processes and products

NASA Astrophysics Data System (ADS)

Hall, Kevin; Thorn, Colin E.

2014-02-01

Widespread acceptance in science at-large notwithstanding, the ability of thermal stresses to produce thermal fatigue (TF) and/or thermal shock (TS) in bedrock and coarse debris in the field is often doubted. Commonly called insolation weathering in geomorphology, the results of questionable laboratory experiments have led many geomorphologists to consider terrestrial temperatures to be inadequate to generate thermally induced stresses leading to rock failure; the exceptions are the action of fire or lightning. We comprehensively survey the general scientific literature on TF and TS while rigorously scrutinizing that relating to geomorphology. Findings indicate theoretical and experimental information is adequate to establish the feasibility of TF and TS in rock stemming from rock temperatures monitored in the field. While TS may exhibit fracture patterns that are uniquely diagnostic, those of TF lack any such attributes. It would appear unlikely that TF can prepare or weaken rock to increase the likelihood of TS. The question of whether widespread polygonal versus rectilinear cracking is diagnostic of TS is presently an open one as possible explanations invoke process(es) and/or host material(s) and, consequently, to assign palaeoenvironmental significance to such fracture patterns is premature at this time. Further geomorphological laboratory research into TF and TS is merited as sufficient theoretical underpinning already exists. However, laboratory experimentation needs to be much more rigorously defined and executed and is faced with significant hurdles if it is to be effectively linked to field observations.

Assessment of an ELISA Laboratory Exercise

ERIC Educational Resources Information Center

Robinson, David L.; Lau, Joann M.

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) is a powerful immunological technique for quantifying small amounts of compounds and has been used in research and clinical settings for years. Although there are laboratory exercises developed to introduce the ELISA technique to students, their ability to promote student learning has not been…

Germination of Ocotea pulchella (Nees) Mez (Lauraceae) seeds in laboratory and natural restinga environment conditions.

PubMed

Pires, L A; Cardoso, V J M; Joly, C A; Rodrigues, R R

2009-08-01

The germination response of Ocotea pulchella (Nees) Mez seeds to light, temperature, water level and pulp presence is introduced. The laboratory assays were carried out in germination chambers and thermal-gradient apparatus, whereas the field assays were performed in environments with distinct light, temperature and soil moisture conditions within a permanent parcel of Restinga forest of the Parque Estadual da Ilha do Cardoso, Cananéia, São Paulo. The seeds do not exhibit dormancy, they are non photoblastic, and a loss of viability in dry stored seeds can be related to a decrease in water content of the seed. The presence of the pulp and the flooded substratum influenced negatively the germination of O. pulchella seeds tested in the laboratory. Otherwise, light and temperature probably are not limiting factors of the germination of O. pulchella seeds in the natural environment of Restinga. The optimum temperature range for germination of Ocotea pulchella seeds was 20 to 32 degrees C, the minimum or base temperature estimated was 11 degrees C and the maximum ranged between 33 and 42 degrees C. The isotherms exhibited a sigmoidal pattern well described by the Weibull model in the sub-optimal temperature range. The germinability of O. pulchella seeds in the understorey, both in wet and dry soil, was higher than in gaps. Germination was not affected by fluctuations in soil moisture content in the understorey environment, whereas in gaps, germination was higher in wet soils. Thus, the germination of this species involves the interaction of two or more factors and it cannot be explained by a single factor.

The analytical validation of the Oncotype DX Recurrence Score assay

PubMed Central

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score® result (scale: 0–100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time. PMID:27729940

The analytical validation of the Oncotype DX Recurrence Score assay.

PubMed

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX ® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score ® result (scale: 0-100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time.

Annotation of Sequence Variants in Cancer Samples: Processes and Pitfalls for Routine Assays in the Clinical Laboratory.

PubMed

Lee, Lobin A; Arvai, Kevin J; Jones, Dan

2015-07-01

As DNA sequencing of multigene panels becomes routine for cancer samples in the clinical laboratory, an efficient process for classifying variants has become more critical. Determining which germline variants are significant for cancer disposition and which somatic mutations are integral to cancer development or therapy response remains difficult, even for well-studied genes such as BRCA1 and TP53. We compare and contrast the general principles and lines of evidence commonly used to distinguish the significance of cancer-associated germline and somatic genetic variants. The factors important in each step of the analysis pipeline are reviewed, as are some of the publicly available annotation tools. Given the range of indications and uses of cancer sequencing assays, including diagnosis, staging, prognostication, theranostics, and residual disease detection, the need for flexible methods for scoring of variants is discussed. The usefulness of protein prediction tools and multimodal risk-based or Bayesian approaches are highlighted. Using TET2 variants encountered in hematologic neoplasms, several examples of this multifactorial approach to classifying sequence variants of unknown significance are presented. Although there are still significant gaps in the publicly available data for many cancer genes that limit the broad application of explicit algorithms for variant scoring, the elements of a more rigorous model are outlined. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

EPA Science Inventory

The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

GHSI Emergency Radionuclide Bioassay Laboratory Network - Summary of the Second Exercise

PubMed Central

Li, Chunsheng; Bartizel, Christine; Battisti, Paolo; Böttger, Axel; Bouvier, Céline; Capote-Cuellar, Antonio; Carr, Zhanat; Hammond, Derek; Hartmann, Martina; Heikkinen, Tarja; Jones, Robert L.; Kim, Eunjoo; Ko, Raymond; Koga, Roberto; Kukhta, Boris; Mitchell, Lorna; Morhard, Ryan; Paquet, Francois; Quayle, Debora; Rulik, Petr; Sadi, Baki; Sergei, Aleksanin; Sierra, Inmaculada; de Oliveira Sousa, Wanderson; Szabó, Gyula

2017-01-01

The Global Health Security Initiative (GHSI) established a laboratory network within the GHSI community to develop collective surge capacity for radionuclide bioassay in response to a radiological or nuclear emergency as a means of enhancing response capability, health outcomes and community resilience. GHSI partners conducted an exercise in collaboration with the WHO REMPAN (Radiation Emergency Medical Preparedness and Assistance Network) and the IAEA RANET (Response and Assistance Network), to test the participating laboratories (18) for their capabilities in in vitro assay of biological samples, using a urine sample spiked with multiple high-risk radionuclides (90Sr, 106Ru, 137Cs, and 239Pu). Laboratories were required to submit their reports within 72 hours following receipt of the sample, using a pre-formatted template, on the procedures, methods and techniques used to identify and quantify the radionuclides in the sample, as well as the bioassay results with a 95% confidence interval. All of the participating laboratories identified and measured all or some of the radionuclides in the sample. However, gaps were identified in both the procedures used to assay multiple radionuclides in one sample, as well as in the methods or techniques used to assay specific radionuclides in urine. Two third of the participating laboratories had difficulties in determining all the radionuclides in the sample. Results from this exercise indicate that challenges remain with respect to ensuring that results are delivered in a timely, consistent and reliable manner to support medical interventions. Laboratories within the networks are encouraged to work together to develop and maintain collective capabilities and capacity for emergency bioassay, which is an important component of radiation emergency response. PMID:27574317

Rapid mycobacteria drug susceptibility testing using Gel Microdrop (GMD) Growth Assay and flow cytometry.

PubMed

Akselband, Y; Cabral, C; Shapiro, D S; McGrath, P

2005-08-01

Control of multi-drug-resistant tuberculosis has been hampered by the lack of simple, rapid and sensitive methods for assessing bacterial growth and antimicrobial susceptibility. Due to the increasing incidence and high frequency of mutations, it is unlikely that culture methods will disappear in the foreseeable future. Therefore, the need to modernize methods for rapid detection of viable clinical isolates, at a minimum as a gold standard, will persist. Previously, we confirmed the feasibility of using the Gel Microdrop (GMD) Growth Assay for identifying sub-populations of resistant Mycobacteria by testing different laboratory strains. Briefly, this assay format relies on encapsulating single bacterium in agarose microspheres and identifying clonogenic growth using flow cytometry and fluorescent staining. In this study, we modified the GMD Growth Assay to make it suitable for clinical applications. We demonstrated the effectiveness and safety of this novel approach for detecting drug susceptibility in clinically relevant laboratory strains as well as clinical isolates of Mycobacterium tuberculosis. Correlation between results using the GMD Growth Assay format and results using two well characterized methods (Broth Microdilution MIC and BACTEC 460TB) was 87.5% and 90%, respectively. However, due to the inherent sensitivity of flow cytometry and the ability to detect small (<1%) sub-populations of resistant mycobacteria, the GMD Growth Assay identified more cases of drug resistance. Using 4 clinically relevant mycobacterial strains, we assessed susceptibility to primary anti-tuberculosis drugs using both the Broth Microdilution MIC method and the GMD Growth Assay. We performed 24 tests on isoniazid-resistant BCG, Mycobacterium tuberculosis H37Ra and Mycobacterium avium strains. The Broth Microdilution MIC method identified 7 cases (29.1%) of resistance to INH and EMB compared to the GMD Growth Assay which identified resistance in 10 cases (41.6%); in 3 cases (12

Analytical performances of a new enzymatic assay for hemoglobin A1c.

PubMed

Jaisson, Stéphane; Desmons, Aurore; Renard, Benoît; Chevelle, Benjamin; Leroy, Nathalie; Gillery, Philippe

2014-07-01

HbA1c is considered the gold standard for the follow-up of diabetic patients and a new diagnostic tool for diabetes mellitus, which implies the availability of reliable assay methods. We have evaluated a new assay developed by Abbott Laboratories, based on the enzymatic quantification of HbA1c by a fructosyl dipeptide oxidase using Architect analyzers. Precision, linearity, correlation with a HPLC method, accuracy and potential impact interferences on HbA1c measurement have been evaluated. Intra-day and between-day CVs were lower than 1.2% and linearity was excellent from 19 mmol/mol (3.9%) to 163 mmol/mol (17.1%). The results were well correlated with those obtained by the HPLC (Variant II device, kit NU - BioRad): HbA1c [Architect, mmol/mol]=0.986×HbA1c [Variant II, mmol/mol]+0.713 (r=0.998, n=109). This method provided consistent results with IFCC titrated quality control samples. Classical interferences in HbA1c assays (i.e. labile HbA1c, carbamylated hemoglobin, triglycerides or bilirubin) did not have an impact on HbA1c quantification by this method. This new enzymatic assay proved to be a robust and reliable method for HbA1c measurement suitable for routine practice in clinical chemistry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinical Evaluation of Fully Automated Elecsys® Syphilis Assay for the Detection of Antibodies of Treponema pallidum.

PubMed

Li, Dongdong; An, Jingna; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

2016-11-01

The resurgence of syphilis in recent years has become a serious threat to the public health worldwide, and the serological detection of specific antibodies against Treponema pallidum (TP) remains the most reliable method for laboratory diagnosis of syphilis. The performance of the Elecsys ® Syphilis assay, a brand new electrochemiluminescene immunoassay (ECLIA), was assessed by large amounts of samples in this study. In comparison with InTec assay, the Elecsys ® Syphilis assay was evaluated in 146 preselected samples from patients with syphilis, 1803 clinical routine samples, and 175 preselected samples from specific populations with reportedly increased rates of false-positive syphilis test results. Discrepancy samples must be investigated by Mikrogen Syphilis recomline assay. There was an overall agreement of 99.58% between two assays (Kappa = 0.975). The sensitivity and specificity of the Elecsys ® Syphilis assay were 100.0% (95% CI, 96.8-100.0%) and 99.8% (95% CI, 99.5-100.0%), respectively. The Elecsys syphilis assay displays better sensitivity (100%), specificity (99.8%), PPV (98.7%), and NPV (100%) in 2124 samples enrolled, compared with the InTec assay. Considering the excellent ease of use and automation, high throughput, and its superior sensitivity, especially in primary syphilis, the Elecsys ® Syphilis assay could represent an outstanding choice for screening of syphilis in high-volume laboratories. However, more attention was still needed, or the results must be confirmed by other treponemal immunoassays. The new Elecsys ® Syphilis assay is applied to patients with malignant neoplasm or HIV infection. © 2016 Wiley Periodicals, Inc.

Sandia Laboratories in-house activities in support of solar thermal large power applications

NASA Astrophysics Data System (ADS)

Mar, R. W.

1980-03-01

The development of thermal energy storage subsystems for solar thermal large power applications is described. The emphasis is on characterizing the behavior of molten nitrate salts with regard to thermal decomposition, environmental interactions, and corrosion. Electrochemical techniques to determine the ionic species in the melt and for use in real time studies of corrosion are also briefly discussed.

Sandia Laboratories in-house activities in support of solar thermal large power applications

NASA Technical Reports Server (NTRS)

Mar, R. W.

1980-01-01

The development of thermal energy storage subsystems for solar thermal large power applications is described. The emphasis is on characterizing the behavior of molten nitrate salts with regard to thermal decomposition, environmental interactions, and corrosion. Electrochemical techniques to determine the ionic species in the melt and for use in real time studies of corrosion are also briefly discussed.

Characterization of deposits formed on diesel injectors in field test and from thermal oxidative degradation of n-hexadecane in a laboratory reactor

PubMed Central

Venkataraman, Ramya; Eser, Semih

2008-01-01

Solid deposits from commercially available high-pressure diesel injectors (HPDI) were analyzed to study the solid deposition from diesel fuel during engine operation. The structural and chemical properties of injector deposits were compared to those formed from the thermal oxidative stressing of a diesel fuel range model compound, n-hexadecane at 160°C and 450 psi for 2.5 h in a flow reactor. Both deposits consist of polyaromatic compounds (PAH) with oxygen moieties. The similarities in structure and composition of the injector deposits and n-hexadecane deposits suggest that laboratory experiments can simulate thermal oxidative degradation of diesel in commercial injectors. The formation of PAH from n-hexadecane showed that aromatization of straight chain alkanes and polycondensation of aromatic rings was possible at temperatures as low as 160°C in the presence of oxygen. A mechanism for an oxygen-assisted aromatization of cylcoalkanes is proposed. PMID:19091086

Growth and inactivation of Salmonella at low refrigerated storage temperatures and thermal inactivation on raw chicken meat and laboratory media: mixed effect meta-analysis.

PubMed

Smadi, Hanan; Sargeant, Jan M; Shannon, Harry S; Raina, Parminder

2012-12-01

Growth and inactivation regression equations were developed to describe the effects of temperature on Salmonella concentration on chicken meat for refrigerated temperatures (⩽10°C) and for thermal treatment temperatures (55-70°C). The main objectives were: (i) to compare Salmonella growth/inactivation in chicken meat versus laboratory media; (ii) to create regression equations to estimate Salmonella growth in chicken meat that can be used in quantitative risk assessment (QRA) modeling; and (iii) to create regression equations to estimate D-values needed to inactivate Salmonella in chicken meat. A systematic approach was used to identify the articles, critically appraise them, and pool outcomes across studies. Growth represented in density (Log10CFU/g) and D-values (min) as a function of temperature were modeled using hierarchical mixed effects regression models. The current meta-analysis analysis found a significant difference (P⩽0.05) between the two matrices - chicken meat and laboratory media - for both growth at refrigerated temperatures and inactivation by thermal treatment. Growth and inactivation were significantly influenced by temperature after controlling for other variables; however, no consistent pattern in growth was found. Validation of growth and inactivation equations against data not used in their development is needed. Copyright © 2012 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.

Agile Thermal Management STT-RX. Towards High Energy Density, High Conductivity Thermal Energy Storage Composites (PREPRINT)

DTIC Science & Technology

2011-12-01

management system. This paper describes recent development of salt hydrate-based TES composites at the Air Force Research Laboratory. Salt hydrates are...composites. 15. SUBJECT TERMS thermal energy storage, composite, salt hydrate, graphic foam 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...part of a thermal management system. This paper describes recent development of salt hydrate-based TES composites at the Air Force Research

Laboratory Diagnostics of Botulism

PubMed Central

Lindström, Miia; Korkeala, Hannu

2006-01-01

Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined. PMID:16614251

Variability of creatinine measurements in clinical laboratories: results from the CRIC study.

PubMed

Joffe, Marshall; Hsu, Chi-yuan; Feldman, Harold I; Weir, Matthew; Landis, J R; Hamm, L Lee

2010-01-01

Estimating equations using serum creatinine (SCr) are often used to assess glomerular filtration rate (GFR). Such creatinine (Cr)-based formulae may produce biased estimates of GFR when using Cr measurements that have not been calibrated to reference laboratories. In this paper, we sought to examine the degree of this variation in Cr assays in several laboratories associated with academic medical centers affiliated with the Chronic Renal Insufficiency Cohort (CRIC) Study; to consider how best to correct for this variation, and to quantify the impact of such corrections on eligibility for participation in CRIC. Variability of Cr is of particular concern in the conduct of CRIC, a large multicenter study of subjects with chronic renal disease, because eligibility for the study depends on Cr-based assessment of GFR. A library of 5 large volume plasma specimens from apheresis patients was assembled, representing levels of plasma Cr from 0.8 to 2.4 mg/dl. Samples from this library were used for measurement of Cr at each of the 14 CRIC laboratories repetitively over time. We used graphical displays and linear regression methods to examine the variability in Cr, and used linear regression to develop calibration equations. We also examined the impact of the various calibration equations on the proportion of subjects screened as potential participants who were actually eligible for the study. There was substantial variability in Cr assays across laboratories and over time. We developed calibration equations for each laboratory; these equations varied substantially among laboratories and somewhat over time in some laboratories. The laboratory site contributed the most to variability (51% of the variance unexplained by the specimen) and variation with time accounted for another 15%. In some laboratories, calibration equations resulted in differences in eligibility for CRIC of as much as 20%. The substantial variability in SCr assays across laboratories necessitates calibration

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays.

PubMed

Roberts, Karen; Callis, Rowena; Ikeda, Tim; Paunovic, Amalia; Simpson, Carly; Tang, Eric; Turton, Nick; Walker, Graeme

2016-02-01

Since the adoption of Labcyte Echo Acoustic Droplet Ejection (ADE) technology by AstraZeneca in 2005, ADE has become the preferred method for compound dosing into both biochemical and cell-based assays across AstraZeneca research and development globally. The initial implementation of Echos and the direct dosing workflow provided AstraZeneca with a unique set of challenges. In this article, we outline how direct Echo dosing has evolved over the past decade in AstraZeneca. We describe the practical challenges of applying ADE technology to 96-well, 384-well, and 1536-well assays and how AstraZeneca developed and applied software and robotic solutions to generate fully automated and effective cell-based assay workflows. © 2015 Society for Laboratory Automation and Screening.

Proportional and Integral Thermal Control System for Large Scale Heating Tests

NASA Technical Reports Server (NTRS)

Fleischer, Van Tran

2015-01-01

The National Aeronautics and Space Administration Armstrong Flight Research Center (Edwards, California) Flight Loads Laboratory is a unique national laboratory that supports thermal, mechanical, thermal/mechanical, and structural dynamics research and testing. A Proportional Integral thermal control system was designed and implemented to support thermal tests. A thermal control algorithm supporting a quartz lamp heater was developed based on the Proportional Integral control concept and a linearized heating process. The thermal control equations were derived and expressed in terms of power levels, integral gain, proportional gain, and differences between thermal setpoints and skin temperatures. Besides the derived equations, user's predefined thermal test information generated in the form of thermal maps was used to implement the thermal control system capabilities. Graphite heater closed-loop thermal control and graphite heater open-loop power level were added later to fulfill the demand for higher temperature tests. Verification and validation tests were performed to ensure that the thermal control system requirements were achieved. This thermal control system has successfully supported many milestone thermal and thermal/mechanical tests for almost a decade with temperatures ranging from 50 F to 3000 F and temperature rise rates from -10 F/s to 70 F/s for a variety of test articles having unique thermal profiles and test setups.