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1
Poon, Terence CW, Tony SK Mok, Anthony TC Chan та ін. "Quantification and Utility of Monosialylated α-Fetoprotein in the Diagnosis of Hepatocellular Carcinoma with Nondiagnostic Serum Total α-Fetoprotein". Clinical Chemistry 48, № 7 (2002): 1021–27. http://dx.doi.org/10.1093/clinchem/48.7.1021.
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Abstract Background: At concentrations <500 μg/L, serum α-fetoprotein (AFP) has low specificity in the diagnosis of hepatocellular carcinoma (HCC), but monosialylated AFP (msAFP) is more specific for HCC. We describe two strategies for quantitative analysis of msAFP and explore their diagnostic accuracy in cases of HCC with nondiagnostic serum total AFP concentrations. Methods: We first used isoelectric focusing, Western blot, and densitometry (IEF-Western blot assay). We then developed a second assay, a novel glycosylation immunosorbent assay (GISA), based on the specificity of sialyltransferase and immunosorbent technology. Both assays were used to measure msAFP and msAFP percentage relative to total AFP in sera with nondiagnostic AFP concentrations from 36 patients with newly diagnosed HCC and from 18 patients with liver cirrhosis. Results: The msAFP percentages and concentrations were significantly higher in the HCC patient group regardless of the quantification methods. The msAFP concentrations and msAFP percentages obtained by the two assays were highly correlated (r = 0.70 and 0.49, respectively). For discrimination of HCC with nondiagnostic serum total AFP from liver cirrhosis, the areas under the ROC curves were 0.81 (95% confidence interval, 0.70–0.92) for msAFP by IEF-Western blot assay, 0.73 (0.58–0.87) for msAFP by GISA, 0.89 (0.80–0.97) for msAFP percentage by IEF-Western blot assay, and 0.74 (0.59–0.89) for msAFP percentage by GISA. Conclusions: Both the serum concentration and percentage of msAFP are potential diagnostic markers for HCC with nondiagnostic AFP. GISA can quantify a specific glycoform of a serologic marker.
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Rybak, R., L. Faldikova, M. Faldyna, M. Machatkova, and J. Rubes. "Bull and boar sperm DNA integrity evaluated by sperm chromatin structure assay in the Czech Republic." Veterinární Medicína 49, No. 1 (2012): 1–8. http://dx.doi.org/10.17221/5668-vetmed.
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Analysis of sperm parameters is very important for predicting the outcome of assisted reproductive techniques and is necessary for determination of fertility potential of males tested for artificial insemination. In our study we have determined the level of bull and boar sperm DNA damage by Sperm Chromatin Structure Assay (SCSA). This test is based on increased susceptibility of altered DNA (strand breaks) in sperm nuclear chromatinto in situ denaturation measured by flow cytometry after staining with acridine orange (AO). Sperm chromatin damage was quantified by percentages of spermatozoa with detectable DNA Fragmentation Index – DFI divided into moderate (m-DFI) and high (h-DFI) DFI. Percentage of immature cells (HDS; cells with High DNA Stainability) was also evaluated. We measured sperm SCSA parameters in a total of 37 bulls in two groups from different localities and 68 boar samples from one locality. Significantly higher percentage of spermatozoa with detectable DFI was detected in six bulls (16.2%) and a significantly higher percentage of immature cell forms (HDS) was found in other six bulls (16.2%) among all tested bulls. The mean percentages of spermatozoa with h-DFI and HDS of bulls from the second group were statistically higher than those from the first group (P < 0.01). Five boars (7.4%) of all tested boars had significantly higher percentage of spermatozoa with DFI and 18 boars (26.5%) had significantly higher percentage of sperm with HDS compared to the other boars. Both percentages of spermatozoa with DFI and HDS were significantly higher in one boar compared to the others. Boars had significantly higher percentages of spermatozoa with h-DFI and HDS (P < 0.0001) in comparison to bulls. For individual bulls, the highest percentages of spermatozoa with DFI and HDS were 20.8% and 3.5%, respectively while for boars these were 17.6% and 10.2%, respectively. No significant correlations were found between percentages of spermatozoa with DFI and HDS. This sensitive procedure seems to be convenient as additional method for semen quality detection in farm animals before their exploitation in breeding.
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Nathan, David M., Amanda Griffin, Francesca M. Perez, Erica Basque, Lily Do, and Barbara Steiner. "Accuracy of a Point-of-Care Hemoglobin A1c Assay." Journal of Diabetes Science and Technology 13, no. 6 (2019): 1149–53. http://dx.doi.org/10.1177/1932296819836101.
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Point-of-care test (POCT) HbA1c assays provide rapidly available results for clinical decision-making. Accuracy and precision must be established. Venous blood samples from 300 patients were assayed for HbA1c by a laboratory technician (“laboratory assessment”) with the POCT Alere Afinion™ assay and a laboratory (Premier AffinityTM) assay. POCT results from 402 patients’ fingerstick samples assayed by nine nontechnician staff (“clinical assessment”) were compared with the laboratory assay. The laboratory assessment showed tight correlation ( r2= .977, P < .001) between the assays. Mean absolute and relative differences were 0.01 percentage points and 2.1%, respectively. CVs for the POCT and laboratory assays were <2% and <1%, respectively. The clinical assessment also showed a tight correlation between the assays ( r2= .978, P < .001), with mean absolute and relative differences of 0.2 percentage points and 3.41%, respectively. CV for the POCT assay was <2%. The POCT performed acceptably compared to the laboratory assay under realistic clinical conditions.
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Zavala H., Andrea, Emilio Hormazabal U., Gloria Montenegro R., et al. "Effects of extracts from Maytenus on Aegorhinus superciliosus (Coleoptera: Curculionidae) and Hippodamia convergens (Coleoptera: Coccinellidae)." Revista Colombiana de Entomología 43, no. 2 (2017): 233. http://dx.doi.org/10.25100/socolen.v43i2.5948.
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The insecticidal effects of five ethanolic extracts produced from three species of the genus Maytenus: M. boaria leaf (MBL), M. boaria bark (MBB), M. boaria seed (MBS), M. disticha leaf (MDL) and M. magellanica leaf (MML) were evaluated on the lady beetle Hippodamia convergens (Coleoptera: Coccinellidae) and on the pest of berry Aegorhinus superciliosus (Coleoptera: Curculionidae). The anti-feeding effects of the extracts on the latter were also evaluated. Residual application was used, with five concentrations for each species of insect and ten replications of each assay. To evaluate anti-feeding action in adult A. superciliosus, choice (C) and no-choice (NC) experiments were established to determine the percentage of leaf area consumed. The assays lasted for 120 hours, with the mortality and anti-feeding effect monitored every 24 hours. The highest percentage of mortality in the two insect species was recorded with the MBS ethanolic extract: H. convergens presented 82 %, with LC50: 32 mg/ml; while A. superciliosus presented 85 % in the choice assay and 86 % in the no-choice assay, with LC50: 23 mg/ml. In both assays, the mortality increased with exposure time, reaching its highest at 120 hours. The lowest mortality was obtained with MBB extract in choice (C) and with MML extract in no-choice (NC) assays. In the choice assay, the highest percentage of leaf area consumed was recorded with MBB extract, while in the no-choice assay the highest percentage of consumed was with MML extract, which presented higher values even than the control with no application. The lowest leaf percentage consumed, in both assays, was recorded with MBS extract at the highest concentrations (20 and 30 % w/v).
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Park, Joonhong, Taewon Bae, Yonggon Cho, Dalsik Kim, and Jaehyeon Lee. "Analytical Performance of the Sysmex HISCL HBsAg Assay and Comparison with the Roche Elecsys HBsAg II Quant Assay in the Quantification of Hepatitis B Surface Antigen." Medicina 57, no. 12 (2021): 1307. http://dx.doi.org/10.3390/medicina57121307.
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Background and Objectives: This study aims to estimate the analytical performance of the Sysmex HISCL HBsAg assay and to assess the analytical correlation with the Roche Elecsys HBsAg II quant assay with clinical samples and the WHO International Standard (IS). Materials and Methods: The intra-assay precision, linearity, assay limitation, accuracy, and comparative evaluation of the HISCL HBsAg assay were estimated. Results: Extrapolating from the plot of the average total allowable error versus the reference value, an accuracy goal of 20% would be achieved around a limit of quantification (LoQ) of 0.014867 IU/mL. The percentage of biases for each level of the WHO IS measured by the two assays were less than 15%, except for the WHO 3rd IS, for which the HISCL HBsAg assay achieved a percentage of bias of 33%. In the comparative evaluation, Passing–Bablok regression analysis did not reveal any significant deviation from linearity between the two assays (y = −48.6998 + 1.9206x; p = 0.79 by the CUSUM test for linearity). The mean difference of the quantitative HBsAg level between the two assays was 1762.5 IU/mL in the Bland–Altman plot. Conclusions: The HISCL HBsAg assay, with a highly sensitive LoQ of 0.03 IU/mL, showed similar analytical performance in HBsAg quantification to the Elecsys HBsAg II quant assay and may be helpful in obtaining better diagnoses and therapeutic strategies for treating HBV infections.
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Blincko, Stuart, and Raymond Edwards. "Non-separation assay for glycohemoglobin." Clinical Chemistry 44, no. 6 (1998): 1302–8. http://dx.doi.org/10.1093/clinchem/44.6.1302.
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Abstract The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD ±0.03)%HbA1 +0.6(SD ±0.3), Sy‖x = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD ±0.04)%HbA1c + 1.8(SD ±0.3), Sy‖x = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was &lt;2% (intraassay, range 5–22% GHb) and &lt;4.2% (interassay, range 4–16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 μmol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results.
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Himabindu K and Vijayalakshmi A. "In-vitro Acetylcholinesterase and antioxidant activity of Ficus dalhousie and Melissa parviflora Benth." International Journal of Research in Pharmaceutical Sciences 11, no. 4 (2021): 8065–70. http://dx.doi.org/10.26452/ijrps.v11i4.4817.
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Ficus dalhousie and Melissa parviflora Benth both plants have been used as Tranquiliser, Relaxants, Nervine tonic and Calming aids throughout the world. The present study was aimed to identify the antioxidant potential of the Ethyl acetate and Hydro alcoholic extract of these plants by in vitro methods. Anti-Alzheimer activity of the plant extract were screened by Acetylcholinesterase (AChE) inhibition and antioxidant by DPPH and Hydrogen oxide. The results of the assays were calculated by the percentage inhibition of these free radicals. In Acetylcholinesterase (AChE) assay inhibitory potentials of Ficus dalhousie exhibited 73.34 ± 1.12%, whereas in Melissa arviflora it was 86.88± 2.12%. In DPPH radical scavenging assay the percentage inhibition was 77.87 ± 2.02% in Ficus dalhousie and 76.92± 1.32% in Melissa arviflora. Hydrogen peroxide scavenging assay the percentage inhibition was 86.56 ± 1.05% in Ficus dalhousie and 80.75± 1.92% was in Melissa arviflora. In all the research assays, the extract showed a concentration dependent increase in free radical scavenging activity. The results revealed that the antioxidant effects of the plant extract might be due to the presence of phenol and flavonoid compounds.
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Nachreiner, R. F., S. J. Oschmann, L. E. Edqvist, and J. I. Richards. "Factors affecting skim milk progesterone assay results." American Journal of Veterinary Research 53, no. 7 (1992): 1085–89. http://dx.doi.org/10.2460/ajvr.1992.53.7.1085.
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Summary Five studies were performed to determine factors affecting progesterone concentration in skim milk. Results of the first study indicated that progesterone concentration was higher in skim milk of samples kept 16 hours in an ice bath (0 C) than of those left at room temperature (21 C). In the second study, this temperature effect was found to be reversible, with skim milk progesterone concentration increasing when whole milk samples were cooled prior to centrifugation. In the third study, [3H]-labeled progesterone was used to determine the relationship between fat content of foremilk (the first milk obtained from the teats), midmilk (milk obtained midway through milking), and strippings (milk obtained immediately after milking machines have been removed) samples and temperature (4 C and 21 C) on the percentage of progesterone in the skim milk fraction. The relationship between percentage of butterfat and percentage of progesterone in skim milk was linear when the log of these variables was used for calculations. In the fourth study, assayable progesterone in the skim milk fraction of foremilk, midmilk, and strippings was affected by temperature. In the fifth study, a multiple-regression procedure was used to determine the amount of variation in percentage of radioactive progesterone in the skim milk - fraction. Independent variables (whole milk butterfat and temperature of incubation [1, 3, 13, 22, 37, and 50 C]) and the natural log of each variable, were entered into a step-wise multiple-regression analysis. The log of the temperature and percentage of butterfat of whole milk at the time of centrifugation accounted for 89.2% (r2 = 0.892) of the variation in the log of the progesterone concentration in the skim milk fractions. The equation describing this relationship was: log percentage of progesterone in the skim milk fraction = 4.046 — 0.144 × (log of temperature of whole milk sample) × 0.688 × (log percentage of butterfat in whole milk sample). The loss of progesterone from skim milk fractions of warm whole milk samples is possibly a physical phenomenon dependent on the temperature of the sample and its percentage of butterfat. A nomograph was created to allow others to use these variables in making adjustments in progesterone concentrations.
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Suhandynata, Raymond T., Melissa A. Hoffman, Michael J. Kelner, Ronald W. McLawhon, Sharon L. Reed, and Robert L. Fitzgerald. "Multi-Platform Comparison of SARS-CoV-2 Serology Assays for the Detection of COVID-19." Journal of Applied Laboratory Medicine 5, no. 6 (2020): 1324–36. http://dx.doi.org/10.1093/jalm/jfaa139.
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Abstract Background COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that is responsible for the 2019 coronavirus pandemic. Acute infections should be diagnosed by polymerase chain reaction (PCR) based tests, but serology tests can demonstrate previous exposure to the virus. Methods We compared the performance of the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays using 179 negative participants to determine negative percentage agreement (NPA) and in 60 SARS-CoV-2 PCR-confirmed positive patients to determine positive percentage agreement (PPA) at 3 different time frames following a positive SARS-CoV-2 PCR result. Results At ≥15 days, the PPA (95% CI) was 100 (86.3–100)% for the Diazyme IgM/IgG panel, 96.0 (79.7–99.9)% for the Roche total Ig assay, and 100 (86.3–100)% for the Abbott IgG assay. The NPA (95% CI) was 98.3 (95.2–99.7)% for the Diazyme IgM/IgG panel, 99.4 (96.9–100)% for the Roche total Ig assay, and 98.9 (96.0–99.9)% for the Abbott IgG assay. When the Roche total Ig assay was combined with either the Diazyme IgM/IgG panel or the Abbott IgG assay, the positive predictive value was 100% while the negative predictive value remained greater than 99%. Conclusions Our data demonstrates that the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays have similar clinical performances. We demonstrated a low false-positive rate across all 3 platforms and observed that false positives observed on the Roche platform are unique compared to those observed on the Diazyme or Abbott assays. Using multiple platforms in tandem increases the PPVs, which is important when screening populations with low disease prevalence.
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Bergami, Federica, Francesca Arena, Eleonora Francesca Pattonieri, et al. "Performance of Whole Blood Stimulation Assays for the Quantification of SARS-CoV-2 Specific T-Cell Response: A Cross-Sectional Study." Diagnostics 12, no. 6 (2022): 1509. http://dx.doi.org/10.3390/diagnostics12061509.
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Since the identification of the new severe acute respiratory syndrome virus 2 (SARS-CoV-2), a huge effort in terms of diagnostic strategies has been deployed. To date, serological assays represent a valuable tool for the identification of recovered COVID-19 patients and for the monitoring of immune response elicited by vaccination. However, the role of T-cell response should be better clarified and simple and easy to perform assays should be routinely introduced. The main aim of this study was to compare a home-made assay for whole blood stimulation with a standardized ELISpot assay design in our laboratory for the assessment of spike-specific T-cell response in vaccinated subjects. Even if a good correlation between the assays was reported, a higher percentage of responder subjects was reported for immunocompromised subjects with ELISpot assay (56%) than home-made whole blood stimulation assay (33%). Additionally, three commercial assays were compared with our home-made assay, reporting a good agreement in terms of both positive and negative results.
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Aubry, Aurélien, Baptiste Demey, Catherine François, et al. "Longitudinal Analysis and Comparison of Six Serological Assays up to Eight Months Post-COVID-19 Diagnosis." Journal of Clinical Medicine 10, no. 9 (2021): 1815. http://dx.doi.org/10.3390/jcm10091815.
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Background: There is much data available concerning the initiation of the immune response after SARS-CoV-2 infection, but long-term data are scarce. Methods: We thus longitudinally evaluated and compared the total and neutralizing immune response of 61 patients to SARS-CoV-2 infection up to eight months after diagnosis by RT–PCR using several commercial assays. Results: Among the 208 samples tested, the percentage of seropositivity was comparable between assays up to four months after diagnosis and then tended to be more heterogeneous between assays (p < 0.05). The percentage of patients with a neutralizing titer decreased from 82% before two months postdiagnosis to 57% after six months. This decrease appeared to be more marked for patients under 65 years old and those not requiring hospitalization. The percentage of serology reversion at 6 months was from 11% with the WANTAI total assay to over 39% with the ABBOTT IgG assay. The neutralizing antibody titers decreased in parallel with the decrease of total antibody titers, with important heterogeneity between assays. Conclusions: In conclusion, serological tests show equivalent sensitivity in the first months after the diagnosis of SARS-CoV-2 infection, but their performance later, postinfection, must be considered when interpreting the results.
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Bhavya, sri Khagga, Ghanukota Srija, and Mogili Sumakanth. "Novel titrimetric method for the determination of rosuvastatin in pure form." World Journal of Biology Pharmacy and Health Sciences 13, no. 2 (2023): 001–4. https://doi.org/10.5281/zenodo.7948477.
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In this experiment assay of Rosuvastatin was performed by titrimetric method. The titration performed is a complexometric titration. The complexometric titration used is an easy, inexpensive volumetric titration method. This method is based on rection of calcium with a solution of disodium ethylenediaminotetraacetate (EDTA). Complex of calcium and EDTA is formed. Erichrome black Tis used as an indicator in this complexometric titration. At pH 12-13, Erichrome black T changes the color from pink to blue. The appearance blue color is determined to be the endpoint of this experiment. The titrations were carried out in triplicates and the average reading was taken to calculate the percentage assay. The percentage purity was found to be 103.05%
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Cao, Guohua, and Ronald L. Prior. "Comparison of different analytical methods for assessing total antioxidant capacity of human serum." Clinical Chemistry 44, no. 6 (1998): 1309–15. http://dx.doi.org/10.1093/clinchem/44.6.1309.
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Abstract Three assays were compared for the determination of total antioxidant capacity in human serum: the oxygen radical absorbance capacity (ORAC) assay, the Randox Trolox-equivalent antioxidant capacity (Randox-TEAC) assay, and the ferric reducing ability (FRAP) assay. There was a weak but significant linear correlation between serum ORAC and serum FRAP. There was no correlation either between serum ORAC and serum TEAC or between serum FRAP and serum TEAC. The effect of dilution on the serum TEAC value and the use of inhibition percentage at a fixed time, without considering the length of inhibition time in the quantitation of results, adversely affected the Randox-TEAC assay. The FRAP assay is simple and inexpensive but does not measure the SH-group-containing antioxidants. The ORAC assay has high specificity and responds to numerous antioxidants. By utilizing different extraction techniques in the ORAC assay, one can remove serum proteins and also make some gross differentiation between aqueous and lipid-soluble antioxidants. However, the ORAC assay requires ∼60 min more than the FRAP or Randox-TEAC assay to quantitate results.
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Swadźba, Jakub, Tomasz Anyszek, Andrzej Panek, Agnieszka Chojęta, Kinga Wyrzykowska, and Emilia Martin. "Head-to-Head Comparison of 5 Anti-SARS-CoV-2 Assays Performance in One Hundred COVID-19 Vaccinees, over an 8-Month Course." Diagnostics 12, no. 6 (2022): 1426. http://dx.doi.org/10.3390/diagnostics12061426.
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The immunoassays used to measure anti-spike SARS-CoV-2 antibodies are widely available on the market. However, their performance in COVID-19 vaccinees is not yet adequately assessed. Our study provides a head-to-head comparison of five methods: Abbott’s S1-RBD IgG, Roche’s S1-RBD total antibody, Euroimmun’s S1 IgG, and DiaSorin’s TrimericS IgG and S1/S2 IgG assays. Testing was performed in one hundred vaccinated subjects, at eight timepoints over eight months after vaccination. The results differed substantially between methods; however, they correlated strongly and demonstrated the individuals’ responses to both doses of vaccination and the waning of humoral immunity after eight months. Importantly, we encountered a high percentage of results above the assay-specific upper quantitation limit (UQL) for undiluted samples. This was the most pronounced for the Roche’s and Euroimmun’s assays. The Abbott’s assay showed the lowest percentage of results above the UQL. We also attempted to find a common way to establish antibody concentrations that might be classified as high. However, this resulted in between 10% and 100% of such results for different methods on day 240′. This highlights the need for an assay-specific approach for adjusting the cut-offs that may indicate COVID-19 immunity.
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Nhu, Quynh Bach Thi, Linh Le Thi Thuy, Hong Thi Nguyen, et al. "HCV RNA Quantification by a Domestic Commercial Assay: A Case Study among People Who Inject Drugs in Vietnam." Diagnostics 13, no. 22 (2023): 3456. http://dx.doi.org/10.3390/diagnostics13223456.
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The desired performance of nucleic acid testing (NAT) may vary if used for disease diagnosis or for the evaluation of the therapeutic efficacy of a treatment, although in most cases, the same assay is used. However, these tests may not be affordable in many situations including in low/middle income countries that in response have developed domestic assays. Given the example of HCV NAT among people who inject drugs in Vietnam, we aimed at evaluating a domestic assay versus an FDA- and CE-approved assay. This cross-evaluation revealed that (i) the domestic assay had a poorer sensitivity with a threshold of detection above 104 IU/mL, and (ii) the FDA-approved assay had a percentage of false negative results close to 1%. Together, in the present study, the domestic assay had a performance compatible with diagnosis purposes (given that this population was 70% HCV seropositive) but not compatible with HCV treatment monitoring (given that treatment failures are rare and the observed viremia frequently below the threshold of detection). This study highlights the need for a proper evaluation of HCV RNA domestic assays in order to efficiently contribute to the WHO HCV elimination target by 2030.
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Layton, Alice, Larry McKay, Dan Williams, Victoria Garrett, Randall Gentry, and Gary Sayler. "Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water." Applied and Environmental Microbiology 72, no. 6 (2006): 4214–24. http://dx.doi.org/10.1128/aem.01036-05.
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ABSTRACT Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r 2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.
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Aleskerova, L. E., S. G. Vasilieva, A. S. Morozov, A. D. Ismailov, and E. S. Lobakova. "Bioluminescence Toxicity Assay of Polyethylenimine-Based Sorbents." Biotekhnologiya 36, no. 3 (2020): 73–81. http://dx.doi.org/10.21519/0234-2758-2020-36-3-73-81.
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The toxicity of polyethylenimine-based sorbents and their extracts was evaluated and their effect on the bioluminescence of Photobacterium phosphoreum photobacteria was studied. These test bacteria are commonly used as objects to determine the toxicity of various materials. The analyzed materials were synthesized by cross-linking PEI with diethylene glycol diglycidyl ether (DGDE) at mass contents of the latter of 1.9-120.0% with subsequent freezing. It was found that the degree of luminescence inhibition in the P. phosphoreum cells depended on the PEI/DGDE ratio in the sorbent. The sorbents with high DGDE content (60-120%) did not affect the cell luminescence activity, while those with a lower percentage of the cross-linker (0.9-30%) exerted a pronounced inhibitory effect on luminescence of photobacteria according to the data obtained via the standard biotesting method. It was also established that the inhibitory effect of sorbents with a lower DGDE percentage (<30%) in a phosphate buffer was significantly lower than in salt solutions. Water and ethanol extracts of sorbents with the DGDE mass percentage of more than 15% did not significantly inhibit the luminescence of P. phosphoreum during 1 h of incubation. Immobilization of P. phosphoreum cells on the surface and internal parts of the studied sorbents was observed by the method of scanning electron microscopy. bioluminescence, biotest, toxicity analysis, photobacteria, polymer sorbents, polyethylenimine This study was funded by the Russian Science Foundation (Grant no. 16-14-00112).
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Qian, Guanhua, and Tinghe Yu. "Nanosecond Electric Pulses Induce Early and Late Phases of DNA Damage and Cell Death in Cisplatin-Resistant Human Ovarian Cancer Cells." BioMed Research International 2018 (August 8, 2018): 1–7. http://dx.doi.org/10.1155/2018/4504895.
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Chemoresistance is a challenge for management of ovarian cancer, and therefore the response of resistant cells to nanosecond electric pulses (nsEP) was explored. Human ovarian cancer cell line COC1 and the cisplatin-resistant subline COC1/DDP were subjected to nsEP (32 ns, 10 kV/cm, 10 Hz pulse repletion frequency, and 10 min exposure duration), and then the cellular responses were followed. The percentages of dead cells and of comet-formed cells in the alkaline assay displayed two peak levels (i.e., 2 and 8 h after nsEP exposure), with the highest value noted at 8 h; the percentage of comet-formed cells in the neutral assay was increased at 8 h; the apoptotic percentage was increased at 8 h, with collapse of the mitochondrial membrane potential and the activation of caspase-3 and caspase-9. The comet assay demonstrated DNA single-strand break at 2 h and double-strand break at 8 h. nsEP resulted in lower cytotoxicity in COC1/DDP cells compared with COC1 cells. These findings indicated that nsEP induced early and late phases of DNA damage and cell death, and these two types of cell death may have distinct applications to treatments of chemoresistant ovarian cancers.
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Machado, Hanna Karla Andrade Guapyassú, Carolina Steller Wagner Martins, Vanda Jorgetti, Rosilene Motta Elias, and Rosa Maria Affonso Moysés. "Chronic kidney disease is a main confounding factor for 25-vitamin D measurement." Brazilian Journal of Nephrology 42, no. 1 (2020): 94–98. http://dx.doi.org/10.1590/2175-8239-jbn-2019-0053.
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Abstract Background: Current guidelines recommend assessment of 25-vitamin D status in patients with chronic kidney disease (CKD). Although significant differences among assays have been described, the impact of CKD on this variability has never been tested. Methods: We tested the variability between two 25-vitamin D assays in patients with CKD (eGFR < 60 mL/min/1.73m2) who had consecutive 25-vitamin D measurements in 2015 (Assay 1 - Diasorin LIASON 25 TOTAL - D assay®) and 2016 (Assay 2 - Beckman Coulter Unicel Xl 800®). The cohort consisted of 791 adult patients (122 with normal renal function and 669 with CKD - 33, 30, and 37% in stages 3, 4, and 5 on dialysis, respectively). Results: Levels of 25-vitamin D were lower and the prevalence of hypovitaminosis D using assay 1 was higher than using assay 2 in patients with CKD, regardless of similar levels of calcium, phosphate, and parathyroid hormone. As kidney function decreased, the percentage of disagreement between the assays increased. Conclusion: There is a noteworthy variability between assays in patients with CKD such that the diagnosis of hypovitaminosis D is modified. The mechanism behind this result is still unclear and might be due to a possible interference in the analytical process. However, the clinical significance is unquestionable, as the supplementation of vitamin D can be erroneously prescribed to these patients.
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Squire, Christine R., and William D. Fraser. "Thyroid Stimulating Hormone Measurement Using a Third Generation Immunometric Assay." Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 32, no. 3 (1995): 307–13. http://dx.doi.org/10.1177/000456329503200308.
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After an initial evaluation of the standard procedure for performance of a third generation TSH (thyroid stimulating hormone) assay (Amerlite TSH-30) modifications were made to standardize the timing of measurement of light emission following signal reagent addition. By adopting this optimized procedure, a significant improvement in assay sensitivity was achieved when compared to a second generation TSH assay (DAKO). Using the optimized assay the sensitivity was 0·003 mU/L (20 replicates of zero) or 0·009 mU/L [22% CV (coefficient of variation) from the precision profile]. Recovery of added TSH and parallelism of the assay were good. A significant negative bias was detected for the Amerlite TSH-30 assay when compared to the DAKO assay (log y = 0·92 log x − 0·33, n = 210). Excellent discrimination was achieved between euthyroid, hypothyroid and thyrotoxic subjects. A high percentage of thyrotoxic patients had undetectable TSH and the spread of values between thyrotoxic and euthyroid was greater with the third generation assay. In patients receiving thyroxine therapy a higher percentage had detectable TSH values. The optimized Amerlite TSH 30 assay offers improved assay performance when compared to a second generation assay.
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Falcone, F., M. Marinelli, L. Minguzzi, et al. "Tumor Markers and Lung Cancer: Guidelines in a Cost-limited Medical Organization." International Journal of Biological Markers 11, no. 2 (1996): 61–66. http://dx.doi.org/10.1177/172460089601100201.
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The aim of our study was to evaluate the cost of the tumor marker assays most widely used in pneumological practive and the effectiveness of the percentage of DRG-based reimbursements absorbed by these assays. For this purpose we assessed the cost of lung tumor marker assays in Emilia Romagna compared to the DRG-based reimbursement of inpatients affected by lung diseases in whom the use of tumor markers is indicated. As an example, we evaluated the cost/effectiveness of the CEA assay in the differential diagnosis of 68 pleural effusions from 46 patients (20 benign diseases, 26 malignant). Because the CEA assay was not a substitute for cytology when this was not diagnostic, 41.3% of the resources were not efficiently spent. If the marker assay had been performed only in cases with negative cytology, we could have spared 14 of 46 tests. Moreover, since the expense lies predominantly in the cost of reagents (81.23%), we suggest as a routine procedure to collect and store samples for tumor marker assay in all cases; the test should be performed in a selected population of patients with negative cytology and “suspect” clinical outcome.
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Masters, P. W., R. G. Jones, D. A. Purves, E. H. Cooper, and J. M. Cooney. "Commercial assays for serum osteocalcin give clinically discordant results." Clinical Chemistry 40, no. 3 (1994): 358–63. http://dx.doi.org/10.1093/clinchem/40.3.358.
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Abstract Serum samples from 9 healthy controls and from subjects with primary hyperparathyroidism (n = 5), Paget disease (n = 3), pregnancy (n = 5), glucocorticoid therapy (n = 5), postmenopausal osteoporosis (n = 10), and renal failure (n = 10) were used to assess the clinical agreement among eight commercially available assay kits for osteocalcin (OC). These kits differ in their assay configurations (six radioimmunoassays, two immunoradiometric assays), standards (five bovine, three human), and antibodies (six polyclonal, two monoclonal). Individual results were divided by the mean OC of the control subjects for each assay and expressed as percentage deviations. The expected wide variation in absolute OC concentrations between kits was only partially reduced by this transformation. Agreement was equally poor when absolute OC concentrations were compared with the reference ranges quoted by the manufacturers. The discordance was particularly marked in renal failure, presumably because of immunoreactive fragments, and in osteoporosis. Systematic differences could not be attributed to assay format, species source of standard, or antibody specificity. We conclude that results cannot be compared between assays even when normalized against healthy subjects, and that standardization is needed.
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Stieber, F., J. Howard, D. Manissero, et al. "Evaluation of a lateral-flow nanoparticle fluorescence assay for TB infection diagnosis." International Journal of Tuberculosis and Lung Disease 25, no. 11 (2021): 917–22. http://dx.doi.org/10.5588/ijtld.21.0391.
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BACKGROUND: Programmatic management of TB infection is a critical component of the WHO End TB Strategy. Interferon-gamma release assays (IGRAs) overcome some limitations of the tuberculin skin test, but implementation of IGRA testing in low-resource settings is challenging.METHODS: In this feasibility study, we evaluated performance of a novel digital lateral-flow assay, the QIAreach® QuantiFERON® TB (QIAreach-QFT) test, against the QuantiFERON®-TB Gold Plus (QFT-Plus) assay. A population with a mix of risk factors for TB infection (111 donors) were sampled over multiple days. A total of 207 individual blood samples were tested according to the manufacturer´s instructions.RESULTS: The overall percentage agreement was 95.6% (two-sided 95% CI 91.8–98), with a positive percentage agreement (i.e., sensitivity) of 100% (95% CI 94.7–100) and a negative percentage agreement (i.e., specificity) of 95.6% (95% CI 90.6–98.4). All QFT-Plus positive specimens with TB1-Nil and TB2-Nil values less than 1 IU/ml tested positive on QIAreach-QFT.CONCLUSIONS: QIAreach QFT is a deployable, accurate testing solution for decentralised testing. It has the potential to overcome key hurdles for TB infection screening in high-burden settings thus helping to achieve the WHO End TB programme goals.
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MILLER, DAVID J., JILL M. DEMERS, ANDREA G. BRAUNDMEEER, and MELISSA L. BEHRENS. "The Use of Two Fluorescent Dyes to Identify Sperm in a Competitive Binding Assay to Oocytes." Journal of Andrology 19, no. 6 (1998): 650–56. http://dx.doi.org/10.1002/j.1939-4640.1998.tb02074.x.
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ABSTRACT: The relationship of most sperm laboratory assays to male fertility is inconsistent. Assays that measure traits required to fertilize oocytes are expected to have the most predictive value. A new assay that measures the competitive ability of two sperm samples to bind to oocytes was developed. Two populations of sperm were labeled using a pair of lipophilic dyes. A concentration of 75 μM of the two dyes, DiQ (4‐[4‐(dihexadecylamino)styryl]‐N‐methylquinolinium iodide; an orangered dye) and DiOC16 (3,3′‐dihexadecyloxacar‐bocyanine perchlorate; a yellow‐green dye), intensely stained 66 and 73% of sperm, respectively, without affecting sperm motility or oocyte‐binding ability. Because sperm could be stained with fluorescent dyes, sperm from two semen samples were mixed together in a droplet, and oocytes were added to allow sperm to bind oocytes competitively. Oocytebound sperm from each sample were counted. Binding was specific; nonspecific sperm binding was estimated by sperm bound to two‐cell mouse embryos and averaged one to three sperm per embryo. Staining with DiQ or DiOC16 did not affect oocyte‐binding ability since more than 80% of the sperm bound were stained with either dye. Furthermore, if different ratios of DiQ‐ or DiOC16‐stained sperm from the same ejaculate were prepared in droplets and oocytes were added, the percentage of sperm bound to the oocytes reflected the percentage of sperm in the droplet; there was no differential effect of either dye. This assay used fixed oocytes because sperm bound equally to fixed and fresh bovine oocytes. This competitive oocyte‐binding assay allows one to make a series of pairwise comparisons between a group of males or to include an internal control sample in spermoocyte binding assays. This assay may allow more accurate prediction of the oocyte‐binding ability of sperm.
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Nandita, R., M. Jeevitha, Kaarthikeyan Gurumoorthy, Rajeshkumar Shanmugham, Sudarsan Ravichandran, and Kethiswar Raj. "Anti-Inflammatory Activity and Free Radical Scavenging Activity of Tridax Procumbens Leaves-Based Chitosan Gel." Journal of Pharmacy and Bioallied Sciences 16, Suppl 4 (2024): S4081—S4084. https://doi.org/10.4103/jpbs.jpbs_1439_24.
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ABSTRACT Introduction: Tridax procumbens (TP) has been used as an anticoagulant and wound healer. Known by other names as Tridax daisy or coatbuttons, TP belongs to flowering plant species of Asteraceae family. The current study is conducted to analyze the anti-inflammatory and antioxidant activity of TP leaves extract-based chitosan (TPC) gel. Materials and Methods: Anti-inflammatory activity of TPC gel was assessed by bovine serum albumin (BSA) denaturation assay and egg albumin (EA) denaturation assay. Free radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay and hydroxyl radical scavenging (H2O2) assay. Results: Both BSA and EA assay showed that the TPC gel (test gel) demonstrated maximum inhibition at 50 μL concentration and lowest inhibition at 10 μL similar to commercially available gel. The antioxidant activity assays revealed that both standard and the test gel exhibited high inhibitory percentage at all tested concentrations. Conclusion: The anti-inflammatory and antioxidant assay results demonstrate that the investigated compounds, notably TP leaves extract-based chitosan gel, have the potential to be effective agents in moderating oxidative stress and lowering inflammation.
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Welsh, Allison W., Christopher B. Moeder, Sudha Kumar, et al. "Standardization of Estrogen Receptor Measurement in Breast Cancer Suggests False-Negative Results Are a Function of Threshold Intensity Rather Than Percentage of Positive Cells." Journal of Clinical Oncology 29, no. 22 (2011): 2978–84. http://dx.doi.org/10.1200/jco.2010.32.9706.
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Purpose Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. Methods An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. Results Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/μg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. Conclusion Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.
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Giritharan, G., N. Ramakrishnappa, A. Balendran, K. M. Cheng, and R. Rajamahendran. "Development of in vitro tests to predict fertility of bulls." Canadian Journal of Animal Science 85, no. 1 (2005): 47–52. http://dx.doi.org/10.4141/a03-114.
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The overall objective was to develop an in vitro test to predict fertility of bulls in the field. We investigated the bull effect on in vitro embryo production, zona binding and acrosome reaction, and the correlation of this effect to field fertility meas ured by 60–90 d non-return rate. Frozen semen from three separate ejaculates of eight unrelated young bulls, obtained from an artificial insemination (AI) center, was used. On thawing, ejaculates from each bull were pooled, motile sperm were selected and (a) subjected to immunofluorescent assay at 0 and 4 h of incubation in capacitation medium to assess acrosome status, (b) used in an in vitro fertilization assay system to assess cleavage and blastocyst production rates, and (c) sperm-zona binding assay was carried out to determine the number of sperm bound to the zona pellucida of mature oocytes. Percentage of pre-freeze motile sperm (PrFM) and non-return rate data were obtained from the AI center. PrFM, percentage of acrosome reacted sperm at 0 h (AR1), increase in percentage of acrosome reacted sperm after 4 h (InAR) and sperm-zona binding rates (ZB) differed (P < 0.05) among sperm samples obtained from different young bulls. Significant correlations (P < 0.05) were observed between PrFM and AR1 (r = -0.31), InAR (r = 0.36), and ZB (r = 0.32). AR1 was negatively correlated to ZB (r = -0.27) and cleavage rate (r = -0.20), InAR was positively correlated with ZB (r = 0.31) and cleavage rate (r = 0.26). None of the in vitro tests was correlated with non-return rate. These findings indicate that along with pre-freeze motility, a combination of in vitro tests including the percentage of spontaneously acrosome reacted sperm at thawing, might be useful in predicting bull field fertility. Such a combination of assays, however, has yet to be determined. Key words: Field fertility, acrosome reaction, zona binding, IVF, fertility assay
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Lau, R. C. H. "Antibody level of New Zealand children immunized with the triple vaccine DTP (diphtheria-tetanus-pertussis)." Epidemiology and Infection 101, no. 2 (1988): 405–10. http://dx.doi.org/10.1017/s0950268800054352.
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SUMMARYEnzyme-linked immunosorbent assay (ELISA) tests were used to measure IgG antibody levels in 2638 New Zealand children who had been immunized with the triple vaccine DTP. The percentage of children immune to diphtheria decreased with age. The percentage of children immune to tetanus varied from 67.1 to 55.0%. The percentage of children with measurable antibody to pertussis increased with age. The mean percentages of children with measurable antibody or immunity to one or more DTP components were 34.2% (with 3 components), 34.4% (2 components), and 78.1% (1 component). It appears the immunization strategy for diphtheria and tetanus is satisfactory for herd immunity in New Zealand children. However, the current pertussis strategy may not be providing adequate immunity to 5-year-olds in this country.
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Geiter, Sabine, Markus Graf, and Helga Vetr. "Correlation between Two ADAMTS-13 Activity Assays Based on Different Principles." Blood 110, no. 11 (2007): 3159. http://dx.doi.org/10.1182/blood.v110.11.3159.3159.
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Abstract Defects in ADAMTS-13, the von Willebrand Factor (vWF) cleaving protease, are thought to be the main cause for the microvascular thrombotic disorder TTP (thrombotic thrombocytopenic purpura) that is in more than 90% of cases fatal if not treated early and appropriately. Usually this disease is clinically diagnosed, but in recent years the need for rapid and reliable diagnostic tests for ADAMTS-13 levels has increased. We present here the comparison of two commercially available assays for quantification of ADAMTS-13 activity both suitable for routine analysis but based on different principles. The two assays differ in their test principle and the readout system as follows: Assay 1 is a fluorogenic assay using a FRETS-vWF73 substrate and a kinetic measurement (TECHNOZYM®ADAMTS-13 ELISA); with this assay ADAMTS13 Antigen can also be determined in a second step. Assay 2 is a chromogenic assay and detects the cleaved vWF73 substrate by a specific monoclonal antibody (TECHNOZYM®ADAMTS-13 Activity ELISA). Citrated plasma of normal donors (n=7), of pooled normal plasma (n=15) and of TTP patients (n=14) were tested in both assays. Results are reported in both assays as percentage of normal activity. The standard for both assays is prepared from a pool of 100 normal donors and defined as 100%. The samples comprised a range from 0.2% up to 107% activity. The overall correlation coefficient between the two different activity assays was 0.96. 5 samples were found to have less than 5% activity in both assays. These results show that data obtained by the new TECHNOZYM®ADAMTS-13 Activity ELISA correlate very well with the fluorogenic assay (TECHNOZYM®ADAMTS-13 ELISA) in spite of the fact that these assays are based on very different principles.
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Nazarian, Aaron A., Ivonne L. Archibeque, Yen H. Nguyen, Paul Wang, Angus M. Sinclair, and David A. Powers. "Characterization of Bispecific T-cell Engager (BiTE®) Antibodies with a High-Capacity T-cell Dependent Cellular Cytotoxicity (TDCC) Assay." Journal of Biomolecular Screening 20, no. 4 (2014): 519–27. http://dx.doi.org/10.1177/1087057114561405.
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The Bispecific T-cell Engager (BiTE®) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors.
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Miyamoto, S., M. Irahara, K. Ushigoe, A. Kuwahara, H. Sugino, and T. Aono. "Effects of activin on hormone secretion by single female rat pituitary cells: analysis by cell immunoblot assay." Journal of Endocrinology 161, no. 3 (1999): 375–82. http://dx.doi.org/10.1677/joe.0.1610375.
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We investigated the effect of activin A on secretion of LH, FSH, and prolactin (PRL) by female cultured rat pituitary cells at the single-cell level by means of the cell immunoblot assay. Anterior pituitary cells from 8-week-old female rats were preincubated with or without activin A for 24 h, after which they were monodispersed and immediately used for cell immunoblot assay. The percentages of LH-, FSH- and PRL-immunoreactive cell blots detected were 5.5, 5.3 and 43.1%, respectively, of all pituitary cells applied to the transfer membrane. The percentage of LH-secreting cells and mean LH secretion per cell did not change after treatment with activin. In contrast, activin significantly increased the percentage of FSH-secreting cells and mean FSH secretion per cell to 136.0 and 114. 5% respectively. In addition, activin significantly decreased the percentage of PRL-secreting cells and mean PRL secretion per cell to 52.2 and 72.0% respectively. These results suggest that (1) activin A has effects on female rat pituitary cells that increase not only the amount of FSH secretion per cell but also the number of FSH-secreting cells, and (2) activin A decreases both the amount of PRL secretion per cell and the number of PRL-secreting cells.
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Calvaresi, Emilia C., and Jonathan R. Genzen. "Evaluating Percentage-Based Reporting of Glucose-6-Phosphate Dehydrogenase (G6PD) Enzymatic Activity." American Journal of Clinical Pathology 154, no. 2 (2020): 248–54. http://dx.doi.org/10.1093/ajcp/aqaa040.
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Abstract Objectives The World Health Organization recommends measurement of glucose-6-phosphate dehydrogenase (G6PD) activity before initiation of 8-aminoquinoline therapy. A new drug for malaria prophylaxis and treatment (tafenoquine) is contraindicated in patients with G6PD deficiency or unknown G6PD status given its prolonged half-life. Assessments of percentage of normal G6PD activity using laboratory-specific result distributions are not widely available, making tafenoquine-eligibility decisions potentially challenging. Methods Using an institutional review board–exempt protocol, a data set of quantitative G6PD results was retrieved from a national reference laboratory. G6PD testing was previously performed at 37 °C using an automated enzymatic assay configured on a Roche cobas c501 chemistry analyzer. Results Overall, 52,216 results from patients 18 years and older and 6,397 results from patients younger than 18 years were obtained. A modified adjusted male median of 12.7 U/g Hb was derived for adult males in this assay configuration. Result distributions showed higher G6PD activity in neonates. Conclusions Retrospective data analysis can be used to determine laboratory-specific normal G6PD activity values in clinical populations and thus can assist in clinical-eligibility considerations for 8-aminoquinoline treatment.
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Zakaria, Nurhisyam, Mohamad Mahdzir, Mahfuzah Yusoff, Norhafiza Mohd Arshad, Khalijah Awang, and Noor Nagoor. "Cytotoxic Effects of Pinnatane A Extracted from Walsura pinnata (Meliaceae) on Human Liver Cancer Cells." Molecules 23, no. 11 (2018): 2733. http://dx.doi.org/10.3390/molecules23112733.
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Background: Pinnatane A from the bark of Walsura pinnata was investigated for its anti-cancer properties by analyzing the cytotoxic activities and cell cycle arrest mechanism induced in two different liver cancer cell lines. Methods: A 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to analyze the pinnatane A selectivity in inducing cell death in cancer and normal cells. Various biological assays were carried out to analyze the anti-cancer properties of pinnatane A, such as a live/dead assay for cell death microscopic visualization, cell cycle analysis using propidium iodide (PI) to identify the cell cycle arrest phase, annexin V-fluorescein isothiocyanate (annexin V-FITC)/PI flow cytometry assay to measure percentage of cell populations at different stages of apoptosis and necrosis, and DNA fragmentation assay to verify the late stage of apoptosis. Results: The MTT assay identified pinnatane A prominent dose- and time-dependent cytotoxicity effects in Hep3B and HepG2 cells, with minimal effect on normal cells. The live/dead assay showed significant cell death, while cell cycle analysis showed arrest at the G0/G1 phase in both cell lines. Annexin V-FITC/PI flow cytometry and DNA fragmentation assays identified apoptotic cell death in Hep3B and necrotic cell death in HepG2 cell lines. Conclusions: Pinnatane A has the potential for further development as a chemotherapeutic agent prominently against human liver cells.
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Grunberg, Steven M., Anna Marie Daniels, Helmut Muensch, et al. "Correlation of meningioma hormone receptor status with hormone sensitivity in a tumor stem-cell assay." Journal of Neurosurgery 66, no. 3 (1987): 405–8. http://dx.doi.org/10.3171/jns.1987.66.3.0405.
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✓ Several investigators have detected progesterone receptors in a high percentage of meningioma specimens and have noted progesterone receptors to be more common than estrogen receptors in these specimens. However, a functional significance of such hormone receptor positivity in control of meningioma growth has not been described. This paper describes a paired test of the estrogen and progesterone receptor assay as the biochemical assay and of the human tumor stem-cell clonogenic assay (HTSCCA) as the functional assay in 17 meningioma specimens. Only one (6%) of the 17 specimens was estrogen receptor-positive, while 11 (69%) of 16 specimens were progesterone receptor-positive. The HTSCCA revealed that only two (15%) of 13 specimens were sensitive to estradiol while five (31%) of 16 specimens were sensitive to progesterone. Comparison of progesterone results for the 15 specimens on which both hormone receptor assay and HTSCCA were performed revealed correlation in a majority of cases; four specimens were positive for both assays and five specimens were negative for both assays. No specimen that was negative for progesterone receptors was sensitive to progesterone by HTSCCA. These results suggest that the hormone receptor and sensitivity pattern of meningiomas may differ from that of breast cancer, and that progesterone addition or ablation may be a reasonable therapeutic approach for meningiomas.
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Giovannini, Catia, Fabrizia Suzzi, Francesco Tovoli, et al. "Low-Baseline PD1+ Granulocytes Predict Responses to Atezolizumab–Bevacizumab in Hepatocellular Carcinoma." Cancers 15, no. 6 (2023): 1661. http://dx.doi.org/10.3390/cancers15061661.
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Introduction: Immune check point inhibitors have recently entered the armamentarium of advanced hepatocellular carcinoma (HCC) treatment. Among them, the combination of atezolizumab plus bevacizumab has pushed it a step forward; however, a number of patients still present primary non-responses without any biomarker to predict responses to different options. Here, we aimed to identify a putative baseline biomarker to predict the response to atezolizumab–bevacizumab, by investigating whether baseline PD1+ and PD-L1+ peripheral granulocyte percentages might offer a non-invasive, cheap, and easily feasible assay. Methods: A prospective Italian cohort of 34 patients treated by atezolizumab–bevacizumab was tested to assay the baseline percentage of peripheral granulocytes and their PD1 and PD-L1 expression. The neutrophil to lymphocyte ratio (NLR) was also considered, and all data were compared with the clinical course of patients. Results: A low-baseline PD1+ peripheral granulocyte percentage turned out to predict responder patients (mean ±SD of PD1+ granulocyte percentage in responders versus non-responders: 9.9 ± 9.1 vs. 29.2 ± 17.6; student’s t-test, p < 0.01). In line, patients identified by a low PD1+ granulocyte percentage displayed a longer TTP (log-rank test, p < 0.0001). A lower granulocyte percentage on total white blood cells, irrespective of PD1 or PD-L1 expression, is also associated with responses to atezolizumab–bevacizumab (log-rank test, p < 0.05). No predictive value was observed for either the PD-L1+ granulocyte percentage or NLR. Conclusions: A low-baseline PD1+ peripheral granulocyte percentage is associated with responses to atezolizumab–bevacizumab treatment in advanced HCC. These findings encourage evaluating this minimally invasive, cheap, and easy test in further independent cohorts and outlining the relevance of innate immunity in the response to immune-checkpoint inhibitors.
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Vimalavathini, R., Arthi M, Arunkumar M, and D. Gnanavelou. "In vitro anti-urolithic activity of Chandraprabavathi – A herbo-mineral formulation." World Journal of Advanced Research and Reviews 3, no. 3 (2019): 038–40. https://doi.org/10.5281/zenodo.4309970.
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Chandraprbhavati (CPV) is an Ayurvedic formulation clinically used in the management of urinary calculi. The present study was undertaken to investigate the <em>in-vitro</em> antiurolithic activity of CPV by artificial urine and nucleation assay. Cystone demonstrated better percentage inhibition of calcium oxalate crystals formation than CPV in nucleation assay, however CPV exhibited more inhibition of super saturation of calcium oxalate crystals in artificial urine assay than cystone. The percentage inhibition of calcium oxalate crystals formation increased in dose dependent manner for both the drugs. Thus our study demonstrates primary evidence for CPV possessing antiurolithiatic property <em>in vitro</em>.
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Osei-Safo, Dorcas, Ibrahim Chikowe, Jerry Joe Ebow Kingsley Harrison, Daniel Konadu Yeboah, and Ivan Addae-Mensah. "Further Validation of a Rapid Screening Semiquantitative Thin-Layer Chromatographic Method for Marketed Antimalarial Medicines for Adoption in Malawi." Journal of Analytical Methods in Chemistry 2018 (2018): 1–7. http://dx.doi.org/10.1155/2018/2130390.
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A recently developed semiquantitative thin-layer chromatographic (SQ-TLC) assay has been employed in postmarketing surveillance of antimalarial medicines used in Malawi prior to HPLC assay. Both methods gave analogous results in a significant majority of the samples, with a good correlation (r = 0.9012) for the active pharmaceutical ingredients of the dosage forms assayed. Artemether-containing medicines had the highest percentage (92.67%) of samples with comparable results for both assays. The lowest percentage (66.67%) was observed in artesunate-containing medicines. The SQ-TLC method was validated for specificity, accuracy, precision, linearity, and stability according to the International Conference on Harmonisation guidelines, with the results falling within acceptable limits. For specificity, retention factor values of the test samples and reference standards were comparable, while accuracy and precision of 91.1 ± 5.7% were obtained for all samples. The method was linear in the range 1.0–2.0 µg/spot with a correlation coefficient ofr = 0.9783. Stability tests also fell within acceptable limits. In this study, we present the validation of the SQ-TLC method and propose its adoption as a rapid screening tool for field estimation of the quality of antimalarial and other essential medicines in Malawi and other parts of the developing world prior to a more accurate HPLC assay.
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Ward, Matthew D., Kristin E. Mullins, Elizabeth Pickett, et al. "Performance of 4 Automated SARS-CoV-2 Serology Assay Platforms in a Large Cohort Including Susceptible COVID-19–Negative and COVID-19–Positive Patients." Journal of Applied Laboratory Medicine 6, no. 4 (2021): 942–52. http://dx.doi.org/10.1093/jalm/jfab014.
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Abstract Background Anti–SARS-CoV-2 serological responses may have a vital role in controlling the spread of the disease. However, the comparative performance of automated serological assays has not been determined in susceptible patients with significant comorbidities. Methods In this study, we used large numbers of samples from patients who were negative (n = 2030) or positive (n = 112) for COVID-19 to compare the performance of 4 serological assay platforms: Siemens Healthineers Atellica IM Analyzer, Siemens Healthineers Dimension EXL Systems, Abbott ARCHITECT, and Roche cobas. Results All 4 serology assay platforms exhibited comparable negative percentage of agreement with negative COVID-19 status ranging from 99.2% to 99.7% and positive percentage of agreement from 84.8% to 87.5% with positive real-time reverse transcriptase PCR results. Of the 2142 total samples, only 38 samples (1.8%) yielded discordant results on one or more platforms. However, only 1.1% (23/2030) of results from the COVID-19–negative cohort were discordant. whereas discordance was 10-fold higher for the COVID-19–positive cohort, at 11.3% (15/112). Of the total 38 discordant results, 34 were discordant on only one platform. Conclusions Serology assay performance was comparable across the 4 platforms assessed in a large population of patients who were COVID-19 negative with relevant comorbidities. The pattern of discordance shows that samples were discordant on a single assay platform, and the discordance rate was 10-fold higher in the population that was COVID-19 positive.
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Sagala, Evayanti Meiliana, and Jansen Silalahi. "Wound Healing Activities of Hydrolyzed Virgin Coconut Oil (HVCO) and Fucoidan Combination: An In Vitro Assay." Asian Journal of Pharmaceutical Research and Development 7, no. 3 (2019): 40–45. http://dx.doi.org/10.22270/ajprd.v7i3.532.
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combination, in the NIH 3T3 cell line using in vitro assay, and compared with single HVCO and single fucoidan. Methods: NIH 3T3 Cell viability and proliferation were assessed using the MTT method, migration activity was assessed using scratch wound healing assays and expression of COX-2 and VEGF protein were determined using immunocytochemistry (ICC). Results: The results from the proliferative activity assay show that the effective concentrations for all samples were 31.25 μg /ml. NIH 3T3 cells migration activity assay showed that the best combination of the HVCO and fucoidan was 50:50. From COX 2 and VEGF protein expression test results, the combination of HVCO and fucoidan has a higher percentage of expression than single HVCO or single fucoidan Conclusion: The results reveal that the combination of HVCO and fucoidan has better wound healing activity than single HVCO or single fucoidan
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Feng, Zheng, Michael Schlichting, Christoph Helwig, et al. "Comparative study of two PD-L1 expression assays in patients with non-small cell lung cancer (NSCLC)." Journal of Clinical Oncology 35, no. 15_suppl (2017): e20581-e20581. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20581.
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e20581 Background: Response to anti–PD-L1/PD-1 therapy in patients with NSCLC has been associated with tumor PD-L1 expression. Avelumab is a fully human anti‒PD-L1 IgG1 antibody that has shown antitumor activity in advanced NSCLC, with a trend for greater efficacy in patients with PD-L1+ vs PD-L1– NSCLC tumors, as assessed using a proprietary PD-L1 assay in development (Dako, Carpinteria, CA; based on antibody clone 73-10, Merck KGaA, Darmstadt, Germany). This study compares the analytical performance of the 73-10 assay with the FDA-approved PD-L1 IHC 22C3 pharmDx diagnostic assay (Dako) in NSCLC tumors. Methods: Formalin-fixed paraffin-embedded NSCLC tumor samples were obtained from a commercial source and from a study of first-line (1L) avelumab in patients with advanced NSCLC (NCT01772004). Tumor membrane PD-L1 expression was assessed with the 73-10 and 22C3 assays by immunohistochemistry. Correlation between assays was determined at different PD-L1 cut-off levels. Results: In initial staining of commercial NSCLC samples, the 73-10 assay showed a broad dynamic range. Of 148 commercial samples, the 73-10 assay with ≥1%, ≥50% and ≥80% cut-offs classified 64.2%, 36.5% and 23.6% of samples as PD-L1+, respectively, whereas 20.3% were PD-L1+ with the 22C3 assay at a pre-specified ≥50% cut-off. The overall percentage agreement between assays was 83.8% using the ≥50% cut-off for both assays, and 93.9% using the ≥80% cut-off for 73-10 and ≥50% for 22C3. In follow-up studies, 83 study samples from the 1L NSCLC cohort were evaluable with both assays. With the 73-10 assay at ≥1%, ≥50%, and ≥80% cut-offs, 79.5%, 45.8%, and 31.3% of study tumors, respectively, were PD-L1+; with the 22C3 assay at ≥1% and ≥50% cut-offs, 59.0% and 21.7% were PD-L1+. Conclusions: Using a high tumor cell PD-L1 staining cut-off, the 73-10 and 22C3 assays showed highly concordant staining of NSCLC samples, with similar sensitivity observed with an ≥80% cut-off for 73-10 and ≥50% for 22C3. Using a low frequency of tumor PD-L1 expression, data suggested that the 73-10 assay had greater sensitivity than the 22C3 assay. These results provide a rationale for additional studies using the 73-10 assay at various cut-offs within the avelumab trial program. Clinical trial information: NCT01772004.
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Jovanović, Miloš, Zorica Drinić, Dubravka Bigović, Ana Alimpić-Aradski, Sonja Duletić-Laušević, and Katarina Šavikin. "In vitro antineurodegenerative activity and in silico predictions of blood-brain barrier penetration of Helichrysum plicatum flower extract." Lekovite sirovine, no. 40 (2020): 45–51. http://dx.doi.org/10.5937/leksir2040045j.
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This study aimed to assess the antineurodegenerative and antioxidant activity of Helichrysum plicatum flower extract, as well as to identify extract ingredients with acceptable pharmacokinetic parameters such as gastrointestinal absorption, blood-brain barrier permeation, and P-glycoprotein-mediated effusion for optimal therapeutic brain exposure. Antioxidant activity was evaluated by ABTS, FRAP, and b-carotene bleaching assays, while antineurodegenerative activity was tested using acetylcholinesterase (AChE) and tyrosinase (TYR) inhibitory activity assays. In the ABTS test, the dry extract at the highest applied concentration (500 µg/mL) showed better or similar antioxidant activity compared to the standards. In the b-carotene assay, all applied concentrations of the extract showed significantly higher activity than vitamin C. No concentration-dependent activity was observed in the AChE assay, while in the TYR assay the lowest extract concentration (100 µg/mL) showed the highest percentage of inhibition (27.92 %). Pharmacokinetic parameters of compounds were predicted by in silico SwissADME online tool in accordance by the rules of drug-likeness. According to the pharmacokinetic properties, we concluded that pentoxymethoxylated flavones may represent CNS drug candidates for further studies.
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Thakkar, Yax, Kaushal Joshi, Christina Hickey, et al. "The BlueScreen HC assay to predict the genotoxic potential of fragrance materials." Mutagenesis 37, no. 1 (2022): 13–23. http://dx.doi.org/10.1093/mutage/geac004.
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Abstract BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.
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Trambas, Christina, John W. Pickering, Martin Than, et al. "Impact of High-Sensitivity Troponin I Testing with Sex-Specific Cutoffs on the Diagnosis of Acute Myocardial Infarction." Clinical Chemistry 62, no. 6 (2016): 831–38. http://dx.doi.org/10.1373/clinchem.2015.252569.
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Abstract BACKGROUND High-sensitivity cardiac troponin I (hs-cTnI) assays show sex-dependent differences in the 99th percentile of healthy populations, with concentrations in women approximately 50% lower. The adoption of sex-specific cutoffs seems appropriate, although it is not yet clear what effect these will have on acute myocardial infarction (AMI) diagnosis and management. METHODS We conducted a retrospective pre- and postchangeover analysis of troponin I testing in the 6 months before and after moving from the contemporary Abbott Architect TnI assay (cTnI) to hs-cTnI at 2 tertiary centers in Australia and New Zealand. The cTnI cutoff was 30 ng/L for both sexes, whereas a female-specific cutoff of 16 ng/L was adopted upon changeover to hsTnI. RESULTS Changeover from the cTnI assay to the hs-cTnI assay increased the number of female patients with increased troponin I concentrations at both sites (from 29.7% to 34.9% and from 22.4% to 30.8%; P &lt; 0.001). There was no statistically significant change in the number of men with increased concentrations in the same time period (P = 0.09). The increased percentage of women with increased troponin I was not associated with an increase in the number of women with AMI diagnoses at either center. Angiographic data available from 1 center showed no change in the percentage of angiograms performed in women. CONCLUSIONS Although increasing the proportion of women with increased troponin I, adopting sex-specific cutoffs with the hs-cTnI assay did not lead to an increase in AMI diagnoses in females, or in the number of women undergoing angiography.
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Ismail, Hammad, Ammara Rasheed, Ihsan-ul Haq, et al. "Five Indigenous Plants of Pakistan with Antinociceptive, Anti-Inflammatory, Antidepressant, and Anticoagulant Properties in Sprague Dawley Rats." Evidence-Based Complementary and Alternative Medicine 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/7849501.
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Five medicinal plants of Pakistan were investigated for their antinociceptive, anti-inflammatory, antidepressant, and anticoagulant potential. Antinociceptive activity was estimated by hot plate and writhing assay. In hot plate assay, Quercus dilatata (52.2%) and Hedera nepalensis (59.1%) showed moderate while Withania coagulans (65.3%) displayed a significant reduction in pain. On the other hand, in writhing assay, Quercus dilatata (49.6%), Hedera nepalensis (52.7%), and Withania coagulans (62.0%) showed comparative less activity. In anti-inflammatory assays crude extracts showed significant edema inhibition in a dose dependent manner. In carrageenan assay, the highest activity was observed for Withania coagulans (70.0%) followed by Quercus dilatata (66.7%) and Hedera nepalensis (63.3%). Similar behavior was observed in histamine assay with percentage inhibitions of 74.3%, 60.4%, and 63.5%, respectively. Antidepressant activity was estimated by forced swim test and the most potent activity was revealed by Withania coagulans with immobility time 2.2s (95.9%) followed by Hedera nepalensis with immobility time 25.3s (53.4%). Moreover, the crude extracts of Fagonia cretica (74.6%), Hedera nepalensis (73.8%), and Phytolacca latbenia (67.3%) showed good anticoagulant activity with coagulation times 86.9s, 84.3s, and 67.5s, respectively. Collectively, the results demonstrate that these five plants have rich medicinal constituents which can be further explored.
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van Beers, Eduard J., Leigh Samsel, Laurel G. Mendelsohn, et al. "Imaging Flow Cytometry for Fully Automated Quantification of Percentage of Sickled Cells in Sickle Cell Anemia." Blood 120, no. 21 (2012): 2105. http://dx.doi.org/10.1182/blood.v120.21.2105.2105.
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Abstract Abstract 2105 Introduction In preclinical and early phase pharmacologic trials in sickle cell anemia (SCA) the percentage of sickled cells after deoxygenation, in a so called sickling assay, has been used as an outcome variable. Although this sickling assay is a highly useful test, this method has the disadvantage of being subjective, operator-dependent and with low sensitivity and high variability due to the lack of automated method to quantitate the percentage of sickled cells from a large sample. Imaging flow cytometry is an emerging technology with potential to improve this assay. Therefore, we have explored the capability of this new technique to discriminate sickled cells from unsickledcells in this assay. Methods This study had regional ethics committee approval, and all patients gave written informed consent. To perform the sickling assay peripheral blood was drawn, diluted 1:100 with HemOx buffer (TCS Scientific Corp., Southampton, PA, USA) and aliquoted onto a 96 well plate and placed in a glovebox in hypoxic conditions (2% oxygen) for 2 hours (or as otherwise specified). After incubation, samples were fixed with glutaraldehyde, washed and placed on ice before analyzing the samples with imaging flow cytometry. Cells were analyzed on the ImageStreamx imaging flow cytometer (Amnis Corporation, Seattle, Washington, USA). Data were acquired using the INSPIRE acquisition software and using the 60X objective. Data from a minimum of 5000 cells were collected for each sample and analyzed using IDEAS 5.0 software. Single in-focus cells were identified using data from the brightfield images using various masks. As a learning population for the IDEAS software we hand tagged populations of sickled and unsickledcells. Results Using various combinations of pre-defined and self-defined masks we found that shape-ratio (the ratio between the shortest width of the mask divided by the longest part of the mask) identified our hand-tagged populations the best. We customized the algorithm with tight masks and a spot count feature to eliminate doublets and other artifacts. We were able to identify sickled or unsickled cells and a continuum between the two morphological extremes (figure 1a,1b). We selected three different shape ratio cut-off values for further analysis (table 1). To test the classifying algorithm, we spiked normal control blood with different amounts of SCA blood before incubation under variable hypoxia. At 3% oxygen the relation between percentage of sickled cells and percentage of SCA blood in the sample was strong (a Spearman rho for all cut-off values higher than 0.925) and significant (P≤0.001). At 4% oxygen the relation was less strong (Spearman rho higher than 0.725 for all cut-off values) but still significant (P≤0.05). As an additional validation, we found (as expected) lower percentages of hypoxia-induced sickling in blood from patients with SCA according to the level of fetal hemoglobin (HbF) expression (figure 1c). At all shape ratio cut-off values HbF percentage seems to suppress the amount of cells identified as sickled. While additional experiments are underway in an attempt to validate this finding, we preliminarily observe that fetal hemoglobin has a large effect on this flow cytometry sickling assay. Experiments assessing how sensitive this new technique is in detecting the effect of the anti-sickling agent 5-hydroxymethyl-2-furfural (Aes-103) are ongoing. Conclusion This study shows that imaging flow cytometryhas potential as a fully automated, operator independent method to quantify sickled cells in a sickling assay. While additional experiments are ongoing, our early data suggest that the presented technique seems discriminative enough to identify patient dependent and independent differences in sickling capacity of SCA red cells. Disclosures: No relevant conflicts of interest to declare.
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Martinez, Emily M., Samuel D. Klebanoff, Stephanie Secrest, et al. "High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 7 (2018): 603–12. http://dx.doi.org/10.1177/2472555218768745.
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High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell–dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell–mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.
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Mallick, Manas K., Bhanwar S. Choudhary, and Gnananandh Budi. "Geological Reserve Estimation of Limestone Deposit: A Comparative Study Between ISDW and OK." Modelling, Measurement and Control C 81, no. 1-4 (2020): 72–77. http://dx.doi.org/10.18280/mmc_c.811-413.
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Geostatistics plays an important role for reserve estimation in mining industry. Geostatistical tools became popular because of its high degree of accuracy and time saving process for estimation. The uncertainty of geological deposit can be populated by geo-statistical tools. The limestone ore deposit was studied in this paper. The assay value of individual constituents of limestone ore i.e CaO, SiO2, Al2O3 and Fe2O3 were determined for a block by using Inverse Square Distance Weighting (ISDW) method. The average assay value of those individual constituents were 45.85, 15.94, 1.56 and 0.82 percentage respectively. The assay value of CaO was also estimated by two linear method of estimation i.e ISDW and Ordinary Kriging (OK). The assay value of CaO were determined 45.85 and 44.67 percentage respectively. The assay values were properly validated and concluded accordingly. The application of ISDW and OK were implemented to build the resource model together in order to assess the uncertainty of the deposit. Grade estimation by using different geo-statistical techniques are done by SURPAC mine planning software.
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Akao, Nobuaki, Yoshihisa Goto, Kaoru Kondo, and Yoshisuke Tsuda. "Changing chemosusceptibility in the second-stage larvae of Toxocara canis by long-term incubation." Journal of Helminthology 67, no. 2 (1993): 145–50. http://dx.doi.org/10.1017/s0022149x00013031.
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AbstractSecond-stage larvae of Toxocara canis were maintained in vitro for one year. Susceptibility of the larvae to drugs was evaluated by means of minimal larvicidal concentration (MLC) and larval bursting percentage. MLCs of citral and decanoic acid were almost constant throughout all stages of incubation. However, bursting percentage markedly varied within the first 20 weeks of incubation. Therefore, while larvae are available for use in the MLC assay at any stage of incubation, those beyond the first 20 weeks after incubation should be used for the bursting assay to obtain reproducible results.
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Amalia, Latifa, Retno Murwanti, Triana Hertiani, and Kurnia Rahayu Purnomo. "Evaluation of the Potential In Vitro effects of Plantago major L. on Wound Healing in Human Umbilical Vein Endothelial Cells (HUVEC)." Indonesian Journal of Cancer Chemoprevention 15, no. 2 (2025): 87. https://doi.org/10.14499/indonesianjcanchemoprev15iss2pp87-95.
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The treatment of skin wounds remains a major concern in the field of medicine, particularly in the case of chronic wounds resulting from various disorders such as diabetes. The utilization of herbs or herbal preparations for the purpose of healing skin wounds presents a therapeutic challenge within the realm of traditional medicine. Plantago major L. is known to have bioactive compounds that have wound healing activity such as aucubin. This study aimed to determine the in vitro wound healing potential of Plantago major L. extract (PLE). The study involved several assays, including phytochemical examination of PLE using TLC, cell viability testing using MTT assay, and wound healing testing using scratch assay on human umbilical vein endothelial cells (HUVEC). The results confirmed the presence of aucubin as one of the compounds in PLE. It was observed that PLE with 125 μg/mL exhibited the highest wound closure percentage at 90.66%. This study shows that PLE possesses wound healing capabilities.Keywords: Plantago major L., PLE, cytotoxic assay, wound scratch assay, HUVEC.
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Arteaga Figueroa, Lluvia, Leticia Barbosa Navarro, Martin Patiño Vera, and Vera L. Petricevich. "Preliminary Studies of the Immunomodulator Effect of theBougainvillea xbuttianaExtract in a Mouse Model." Evidence-Based Complementary and Alternative Medicine 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/479412.
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Bougainvillea xbuttianais used as an analgesic in folk medicine in Mexico. The purpose of the present study was to determine the effects of the ethanolic extract fromB. xbuttianaon macrophages activities. The phytochemical screening was performed for determine the presence of alkaloids, flavonoids, triterpenes, and saponins. The effects ofB. xbuttianawere analyzed using the macrophages activities as determined by the H2O2release, spreading and phagocytic index, vacuoles formation percentage, and mediators production. The viability percentage was determined in live cells after fixing and staining with crystal violet. The presence of H2O2in macrophages was performed by using the peroxidase-phenol red solution. The cytokine production was determined by two assays, ELISA for detection of IL-6, IL-10, and IFN-γand biological assay for TNF detection. The results showed that theBxbextract dose-dependent manner produces (a) an increase in levels of H2O2and spreading and vacuoles formation percentages, (b) a decrease in phagocytic index and in the amounts of TNF, IL-6, and IFN-γ, and (c) an increase significant in IL-10 and NO production. This study indicates that the ethanolic extract fromBougainvillea xbuttianawas able to activate macrophages. The combination of these results suggests that this extract has an immunomodulator effect.