Methods of plasma catecholamine measurement including radioimmunoassay

Fluorometric and radioenzymatic assays are presently the most widely used techniques for the estimation of plasma, urine, and tissue catecholamines. The fluorometric assay lacks specificity and sensitivity. The radioenzymatic assay is significantly more sensitive and specific but is technically very complex, time consuming, and expensive. A newer methodology has been developed by modification of a 125I radioimmunoassay for metanephrine. The assay utilizes an antibody that specifically binds metanephrine. Plasma and urinary epinephrine and norepinephrine are detected by conversion to metanephrine with the enzymes catechol-O-methyl-transferase (COMT) and phenylethanolamine-N-methyltransferase (PNMT). The major advantages of the radioimmunoassay are the savings in cost and time. The radioenzymatic assay utilizes an expensive tritium-labeled compound, S-adenosylmethionine, and requires multiple organic solvent-extraction steps, thin-layer chromatography, and liquid scintillation counting. The radioimmunoassay requires only one extraction with alumina to aid in specificity and to concentrate the catecholamines. Sample detection is by gamma counting. The radioenzymatic assay is presently the reference method for catecholamines and is best suited for small numbers of samples where sample volume is limited and exquisite sensitivity is required. The radioimmunoassay is rapid, has sufficient sensitivity, specificity, and precision for most applications and is best applied to the analysis of large numbers of samples.

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Comparison of Four Commercially Available Point-of-Care Tests to Detect Antibodies against Canine Parvovirus in Dogs

Viruses ◽

10.3390/v13010018 ◽

2020 ◽

Vol 13 (1) ◽

pp. 18

Author(s):

Michèle Bergmann ◽

Mike Holzheu ◽

Yury Zablotski ◽

Stephanie Speck ◽

Uwe Truyen ◽

...

Keyword(s):

Test Performance ◽

Point Of Care ◽

Reference Method ◽

Canine Parvovirus ◽

P Value ◽

Important Indicator ◽

Specificity And Sensitivity ◽

Specific Pathogen ◽

The One ◽

Point Of Care Tests

Measuring antibodies to evaluate dogs´ immunity against canine parvovirus (CPV) is useful to avoid unnecessary re-vaccinations. The study aimed to evaluate the quality and practicability of four point-of-care (POC) tests for detection of anti-CPV antibodies. The sera of 198 client-owned and 43 specific pathogen-free (SPF) dogs were included; virus neutralization was the reference method. Specificity, sensitivity, positive and negative predictive value (PPV and NPV), and overall accuracy (OA) were calculated. Specificity was considered to be the most important indicator for POC test performance. Differences between specificity and sensitivity of POC tests in the sera of all dogs were determined by McNemar, agreement by Cohen´s kappa. Prevalence of anti-CPV antibodies in all dogs was 80% (192/241); in the subgroup of client-owned dogs, it was 97% (192/198); and in the subgroup of SPF dogs, it was 0% (0/43). FASTest® and CanTiCheck® were easiest to perform. Specificity was highest in the CanTiCheck® (overall dogs, 98%; client-owned dogs, 83%; SPF dogs, 100%) and the TiterCHEK® (overall dogs, 96%; client-owned dogs, 67%; SPF dogs, 100%); no significant differences in specificity were observed between the ImmunoComb®, the TiterCHEK®, and the CanTiCheck®. Sensitivity was highest in the FASTest® (overall dogs, 95%; client-owned dogs, 95%) and the CanTiCheck® (overall dogs, 80%; client-owned dogs, 80%); sensitivity of the FASTest® was significantly higher compared to the one of the other three tests (McNemars p-value in each comparison: <0.001). CanTiCheck® would be the POC test of choice when considering specificity and practicability. However, differences in the number of false positive results between CanTiCheck®, TiterCHEK®, and ImmunoComb® were minimal.

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Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay

Toxins ◽

10.3390/toxins13010010 ◽

2020 ◽

Vol 13 (1) ◽

pp. 10

Author(s):

Ilenia Drigo ◽

Elena Tonon ◽

Simone Pascoletti ◽

Fabrizio Anniballi ◽

Suzanne R. Kalb ◽

...

Keyword(s):

Limit Of Detection ◽

Animal Health ◽

Lethal Dose ◽

Reference Method ◽

Mouse Bioassay ◽

Peptide Fragments ◽

Mass Spectrometers ◽

Detection And Identification ◽

Maldi Biotyper ◽

Sensitivity Specificity

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66–100%), 96.08% (95% CI: 86.54–99.52%), and 93.33% (95% CI: 78.25–98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15–99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

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SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility

10.1101/2020.07.03.20145383 ◽

2020 ◽

Author(s):

Pia Jokela ◽

Anu E Jääskeläinen ◽

Hanna Jarva ◽

Tanja Holma ◽

Maarit J Ahava ◽

...

Keyword(s):

Nucleic Acid ◽

Emergency Departments ◽

Large Scale ◽

Tertiary Care ◽

Reference Method ◽

Preliminary Evaluation ◽

Specificity And Sensitivity ◽

Department Evaluation ◽

Test Result ◽

Respiratory Specimens

AbstractRapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.

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A Quantitative Method for Determining Soil Populations of Streptomyces and Differentiating Potential Potato Scab-Inducing Strains

Plant Disease ◽

10.1094/pdis.1998.82.6.631 ◽

1998 ◽

Vol 82 (6) ◽

pp. 631-638 ◽

Cited By ~ 26

Author(s):

Kenneth L. Conn ◽

Edlira Leci ◽

Giora Kritzman ◽

George Lazarovits

Keyword(s):

Potato Scab ◽

Colony Morphology ◽

Malt Extract ◽

Facilitated Diffusion ◽

Thaxtomin A ◽

Streptomyces Spp ◽

Plastic Bags ◽

Large Numbers ◽

History Of ◽

Layer Chromatography

A procedure is described for estimating Streptomyces populations in soil. Soils are air-dried, 10g quantities are shaken in plastic bags containing 0.1% water agar and homogenized with a Stomacher homogenizer, serial dilutions are plated on a semi-selective culture (STR) medium and incubated for 2 weeks at 22°C, and the Streptomyces colonies are enumerated. Use of STR medium reduced the bacterial and fungal colonies recovered from soil to levels below that of the Streptomyces spp. while not affecting the number of Streptomyces colonies compared with those enumerated on yeast malt extract medium. A procedure for screening large numbers of Streptomyces strains for thaxtomin production, a phytotoxin recognized as a virulence marker in S. scabies, is also described. Strains are grown on oatmeal medium, and the thaxtomin is extracted from the medium by facilitated diffusion and detected by miniature thin layer chromatography. S. scabies and S. acidiscabies strains (approximately 130 from Ontario and 70 from other locations in North America) that produced thaxtomin did not form aerial mycelia or sporulate on STR medium within 2 weeks at 22°C. Ontario S. scabies strains that produced thaxtomin A also produced melanin on STR medium. All S. scabies strains from scab lesions that produced thaxtomin A had this colony morphology, whereas only 4 to 9% of strains from soil with this colony morphology produced thaxtomin A. Using these procedures, we determined that the population of thaxtomin-producing S. scabies in soil from a potato field in Ontario with a history of potato scab was about 20,000 CFU/g soil.

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Urea-Fibrinogen Slide Coagulase Test – A Simple Alternative Method for the Rapid Identification of Staphylococcus aureus

Journal of Gandaki Medical College-Nepal ◽

10.3126/jgmcn.v10i1.17903 ◽

2017 ◽

Vol 10 (1) ◽

pp. 5-10

Author(s):

Binita Koirala Sharma ◽

S Gokhale ◽

K Sharma

Keyword(s):

Staphylococcus Aureus ◽

Sensitivity And Specificity ◽

Reference Method ◽

Cost Effective ◽

Rapid Identification ◽

Accurate Identification ◽

Predictive Values ◽

Negative Results ◽

Animal Pathogens ◽

Sensitivity Specificity

Introduction: The accurate identification of Staphylococcus aureus clinical isolates requires a series of tests. Morphological features and slide coagulase test are two criteria on which S. aureus are identified. Resort to tube coagulase test is sought when results of slide coagulase test are equivocal or doubtful. Both coagulase tests detect the enzymes that convert fibrinogen into fibrin. Human, rabbit or sheep pooled plasma is used as substrate for both tests. Slide coagulase test is simpler and faster as compared to tube coagulase test. The plasma could be carrier of many human and animal pathogens like HIV, HBV, HCV etc. Storage of plasma for longer duration is fraught with chances of contamination. Improperly stored plasma can lead to false positive or negative results. Citrated plasma may be unsuitable for this test if contaminated with citrate utilizing bacteria. Considering the role of S. aureus as a common etiological agent in nosocomial and community infections, there is a need of implementing rapid, easy and cost-effective phenotypic test.Objectives: Considering the disadvantages and risks associated with fresh plasma, this study aims to launch for safer, more reliable substitute with longer shelf life that may provide reliable results for prompt identification of S. aureus by slide coagulase test.Methods: The present work evaluates slide coagulase test (SCT), and urea fibrinogen slide coagulase test (UF-SCT) for S. aureus detection considering Tube coagulase test (TCT) as the reference method. Sensitivity, specificity, positive predictive value and negative predictive values of SCT and UF-SCT were calculated using TCT as gold standard. Results: A total of 150 staphylococcal isolates from different clinical specimens ere selected for the evaluation of coagulase tests. All the specimens were subjected to SCT, UF-SCT and TCT. The UF-SCT showed better sensitivity (95.04%), specificity (100%), PPV (100%), and NPV (82.85%) with reference to TCT. UF-SCT showed similar sensitivity and specificity to SCT. None of the isolates were negative in UF-SCT and positive in SCT. Since UF-SCT does not incorporate plasma directly and at the same time has a very good sensitivity and specificity, it is recommended that UF-SCT could replace SCT for identification of S. aureus.Conclusion: The findings of present study shall encourage laboratories to use the urea-fibrinogen slide coagulase test routinely for the rapid identification of S aureus.Journal of Gandaki Medical College Vol. 10, No. 1, 2017, Page: 5-10

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The Validation of the Sample6 DETECTTM HT/L for AOAC Research Institute

Journal of AOAC International ◽

10.5740/jaoacint.17-0484 ◽

2018 ◽

Vol 101 (5) ◽

pp. 1584-1592

Author(s):

Kakolie Banerjee ◽

Brittney Pierson ◽

Chuxuan Hu ◽

Elijah Carrier ◽

Lauren Malsick ◽

...

Keyword(s):

Pathogen Detection ◽

Incubation Temperature ◽

Detection System ◽

Reference Method ◽

Culture Method ◽

The Elderly ◽

Sample Volume ◽

Test Portion ◽

Environmental Surfaces ◽

The Matrix

Abstract Background: Listeria spp. are an important foodborne human pathogen because of their ability to cause disease and high mortality in individuals, particularly pregnant women, neonates, the elderly, immunocompromised individuals, and children. The Sample6 DETECTTM HT/L Kit is a semi-automated qualitative pathogen detection system designed to detect Listeria spp. (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. marthii) in environmental samples using the Sample6 BioIlluminationTM technology. Objective: The study was done to evaluate the Sample6 DETECT HT/L Kit. The assay was evaluated for inclusivity, exclusivity, robustness, product consistency, and stability, and a matrix study of one environmental surface. Methods: The performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration reference culture method for Listeria using an unpaired study design. Results: The Sample6 DETECT HT/L assay correctly identified all 50 inclusivity isolates and correctly excluded all 30 nontarget strains evaluated. The assay was not affected by minor variations in incubation temperature and time, or sample volume. Results across three production lots spanning the shelf life of the assay were consistent. In the matrix study, the Sample6 DETECT HT/L for Listeria correctly identified each test portion for the presence or absence of Listeria, and there were no statistically significant differences between candidate and reference method results. Conclusions: The data collected in this study demonstrate that the Sample6 DETECT HT/L assay is a reliable method for the detection of Listeria spp. on stainless-steel environmental surfaces after 22 h of enrichment.

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Bolus Residue Scale: An Easy-to-Use and Reliable Videofluoroscopic Analysis Tool to Score Bolus Residue in Patients with Dysphagia

International Journal of Otolaryngology ◽

10.1155/2015/780197 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

Nathalie Rommel ◽

Charlotte Borgers ◽

Dirk Van Beckevoort ◽

Ann Goeleven ◽

Eddy Dejaeger ◽

...

Keyword(s):

Rating Scale ◽

Intraclass Correlation ◽

Correlation Coefficients ◽

Posterior Pharyngeal Wall ◽

High Specificity ◽

Analysis Tool ◽

Pharyngeal Wall ◽

Specificity And Sensitivity ◽

Low Sensitivity ◽

Sensitivity Specificity

Background. We aimed to validate an easy-to-use videofluoroscopic analysis tool, the bolus residue scale (BRS), for detection and classification of pharyngeal retention in the valleculae, piriform sinuses, and/or the posterior pharyngeal wall.Methods. 50 randomly selected videofluoroscopic images of 10 mL swallows (recorded in 18 dysphagia patients and 8 controls) were analyzed by 4 experts and 6 nonexpert observers. A score from 1 to 6 was assigned according to the number of structures affected by residue. Inter- and intrarater reliabilities were assessed by calculation of intraclass correlation coefficients (ICCs) for expert and nonexpert observers. Sensitivity, specificity, and interrater agreement were analyzed for different BRS levels.Results. Intrarater reproducibility was almost perfect for experts (mean ICC 0.972) and ranged from substantial to almost perfect for nonexperts (mean ICC 0.835). Interjudge agreement of the experts ranged from substantial to almost perfect (mean ICC 0.780), but interrater reliability of nonexperts ranged from substantial to good (mean 0.719). BRS shows for experts a high specificity and sensitivity and for nonexperts a low sensitivity and high specificity.Conclusions. The BRS is a simple, easy-to-carry-out, and accessible rating scale to locate pharyngeal retention on videofluoroscopic images with a good specificity and reproducibility for observers of different expertise levels.

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Challenges in vitamin D analysis / Izazovi u analizi vitamina D

Journal of Medical Biochemistry ◽

10.2478/v10011-012-0016-z ◽

2012 ◽

Vol 31 (4) ◽

pp. 326-332 ◽

Cited By ~ 1

Author(s):

Mustafa Serteser ◽

Abdurrahman Coskun ◽

Tamer C Inal ◽

Ibrahim Unsal

Keyword(s):

Vitamin D ◽

Clinical Laboratory ◽

Direct Detection ◽

Reference Method ◽

Detection Methods ◽

Binding Assays ◽

External Quality Assessment Scheme ◽

Specificity And Sensitivity ◽

Competitive Protein Binding ◽

Phosphorus Levels

Summary Vitamin D is an important determinant for the regulation of calcium and phosphorus levels and mineralization of the bone. The most reliable indicator of vitamin D status is the measurement of plasma or serum 25OH-D concentration. Several studies reported discrepancies between the results of assays. These high variabilities in 25OH-D measurements are due to used assay technologies and lack of standardization against the reference materials. Different assays have been employed for the measurement of 25OHD levels: Competitive Protein Binding Assays, immunoassays, direct detection methods. Choosing an assay platform is important both for clinical laboratory professionals and researchers, and several factors affect this process. Recently, liquid chromatography and tandem mass spectrometry is an alternative method to traditional assays and provides higher specificity and sensitivity than many assays; therefore, it has been suggested as a candidate reference method for circulating 25OH-D3. Standardization of methods for the quantification of 25OH-D by using the human-based samples would reduce the inter-method variability. The best way for laboratories to demonstrate the accuracy of their results is by participating in the external quality assessment scheme. Standardization of the assays is also required to provide clinicians with the accurate tools to diagnose hypovitaminosis. In addition, assay-specific decision limits are needed to define appropriate thresholds of treatment.

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Fluorometric Determination of Plasma 11-Hydroxycorticosteroids. I. Rapid Procedure for Clinical Screening

Clinical Chemistry ◽

10.1093/clinchem/19.7.710 ◽

1973 ◽

Vol 19 (7) ◽

pp. 710-717 ◽

Cited By ~ 15

Author(s):

Luis E Mejer ◽

Roberta C Blanchard

Keyword(s):

Small Groups ◽

Excitation Wavelength ◽

Plasma Samples ◽

Fluorometric Determination ◽

Clinical Screening ◽

Rapid Procedure ◽

Specificity And Sensitivity ◽

Accuracy And Precision ◽

Sensitivity Specificity

Abstract A method proposed by Kitabchi and Kitchell [Anal. Biochem. 34, 529 (1970)] for the fluorometric determination of plasma 11-hydroxycorticosteroids has been modified. It is simplified by eliminating centrifugations, by processing all samples consecutively (rather than in small groups), by using disposable test tubes, and by prealkalinizing standards and blanks as well as plasma samples. Specificity and sensitivity are increased by measuring fluorescence at 520 nm, with an excitation wavelength of 470 nm. Effects of prealkalinization and time of fluorescence development on final cortisol values were studied. Large fluorescence increases are possible after 60 min of fluorescence development. Cortisol recoveries were not changed by the use of phase-separating filter paper nor were cortisol values altered by partial aging of the fluorescence reagent. Sensitivity, specificity, accuracy, and precision of the proposed method are reported.

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The Validation of the Dai Nippon Medi·Ca SA Method for AOAC Research Institute Performance Tested MethodsSM Certification

Journal of AOAC International ◽

10.5740/jaoacint.17-0487 ◽

2018 ◽

Vol 101 (5) ◽

pp. 1522-1533

Author(s):

Mai Shimizu ◽

Kentaro Takenaka ◽

Takeo Suzuki ◽

Aya Miyasaka ◽

Taiki Matsuda ◽

...

Keyword(s):

Reference Method ◽

Sample Volume ◽

Contamination Level ◽

Stability Studies ◽

Cooked Ham ◽

Aoac Official ◽

The Mean ◽

Food Matrices ◽

Mean Differences ◽

Medium Method

Abstract A ready-made dry medium method for Staphylococcus aureus count, the Medi·Ca SA method incubated at 35 or 37°C, was compared with the Baird-Parker method (AOAC Official MethodSM975.55) for 11 food matrices: raw beef, raw ground beef, raw lamb, cooked ham, raw salmon, frozen prawn, fresh chilled pasta, pasteurized milk, natural cheese, cream puff, and potato salad. The mean difference between the two methods at each contamination level for each matrix was <0.5 log10, and the 95% confidence intervals on the mean differences fell within the range of −0.50 to 0.50. Standard deviation of repeatability and RSDr values of the Medi·Ca SA method were generally the same level as those of the Baird-Parker method, and r2 ranged from 0.98 to 1.00. Product consistency and stability studies showed little variability between productions lots and a shelf-life of 16 months. Incubation time within the range of 22–26 h and variations to the sample volume did not adversely affect the results. These results showed that the Medi·Ca SA method is a reasonable alternative to the reference method for selected food matrices and makes it possible to simultaneously detect and enumerate S. aureus in only 24�h.

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