Current Issue : July - September Volume : 2012 Issue Number : 3 Articles : 8 Articles
Background: The ability to measure T-cell responses to antigens is proving critical in the field of vaccine\r\ndevelopment and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of\r\ntechnologies exist for this purpose IFNg-ELISpot assays are widely used because of their sensitivity and simplicity.\r\nHowever, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to\r\nyield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay\r\nthat measures antigen-specific T cell responses through changes in monokine gene transcription. The biological\r\namplification of the IFNg signal generated by this assay provides sensitivity comparable to ELISpot, but with the\r\nadvantage that responses can be quantified using small volumes of whole blood.\r\nMethods: Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and\r\nimmunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and\r\ncontrol antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in\r\na TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The\r\ninduction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those\r\nobtained by ELISpot.\r\nResults: Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression\r\nin PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification\r\ngenerated by IFNg-R signaling allows responses to be detected in as little as 25 �µL of whole blood and enables the\r\nassay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.\r\nConclusions: A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation.\r\nAssays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This\r\nassay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when\r\nstorage or transportation is required before processing....
Dexmethylphenidate, otherwise known as d-threo-methylphenidate (D-TMP: (R,R)-(+)-Methyl 2-phenyl-2-(2-piperidyl)acetate), is the dextrorotatory enantiomer of Methylphenidate. In order to assess the safety and efficacy of Dexmethylphenidate, a sensitive analytical method is needed. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay capable of measuring analyte in human plasma was developed and validated to support clinical studies. Venlafaxine Hydrochloride was employed as an Internal Standard. The method of extraction of drug from plasma was done by solid phase extraction (SPE) using DVB-LP polymeric SPE cartridges (30mg, 1ml capacity). Chromatography was performed on X-terra RP C18 (4.6×150mm, 5µm) column using mobile phase constituting Methanol: 5mM ammonium acetate (pH4.00)- 90:10, and detected by mass spectrum (AB Sciex) MRM mode was used for MS/MS detection, Analytical software version is Analyst 1.4.2/1.5.1. The method was linear over a concentration range of 0.201ng/ml to 80.434ng/ml, with a Correlation coefficient (r) was found to be more than 0.999. The mean recovery of dexmethylphenidate is 65.83 % at an overall precision of 7.38 %, with lower limit of quantification set at 0.201ng/ml. This method was simple, fast, specific and exhibited excellent ruggedness....
So many excellent methods are available for the estimation of glucose in blood/blood serum. To carry out this program of estimating glucose in diabetic patient the new method is described here. The chemical method by using o-toluidine which is based upon the spectrophotometric analysis which gives the quantitative determination of glucose from blood/ blood serum....
Background: The aim of this study was to develop an economical ââ?¬Ë?in-houseââ?¬â?¢ single round polymerase chain\r\nreaction (PCR) assay using filter paper-dried blood spots (FP-DBS) for early infant HIV-1 diagnosis and to evaluate its\r\nperformance in an African setting.\r\nMethods: An ââ?¬Ë?in-houseââ?¬â?¢ single round PCR assay that targets conserved regions in the HIV-1 polymerase (pol) gene\r\nwas validated for use with FP-DBS; first we validated this assay using FP-DBS spiked with cell standards of known\r\nHIV-1 copy numbers. Next, we validated the assay by testing the archived FP-DBS (N = 115) from infants of known\r\nHIV-1 infection status. Subsequently this ââ?¬Ë?in-houseââ?¬â?¢ HIV-1 pol PCR FP-DBS assay was then established in Nairobi,\r\nKenya for further evaluation on freshly collected FP-DBS (N = 186) from infants, and compared with findings from\r\na reference laboratory using the Roche AmplicorÃ?® HIV-1 DNA Test, version 1.5 assay.\r\nResults: The HIV-1 pol PCR FP-DBS assay could detect one HIV-1 proviral copy in 38.7% of tests, 2 copies in 46.9%\r\nof tests, 5 copies in 72.5% of tests and 10 copies in 98.1% of tests performed with spiked samples. Using the\r\narchived FP-DBS samples from infants of known infection status, this assay was 92.8% sensitive and 98.3% specific\r\nfor HIV-1 infant diagnosis. Using 186 FP-DBS collected from infants recently defined as HIV-1 positive using the\r\ncommercially available Roche Amplicor v1.5 assay, 178 FP-DBS tested positive by this ââ?¬Ë?in-houseââ?¬â?¢ single-round HIV-1\r\npol PCR FP-DBS PCR assay. Upon subsequent retesting, the 8 infant FP-DBS samples that were discordant were\r\nconfirmed as HIV-1 negative by both assays using a second blood sample.\r\nConclusions: HIV-1 was detected with high sensitivity and specificity using both archived and more recently\r\ncollected samples. This suggests that this ââ?¬Ë?in-houseââ?¬â?¢ HIV-1 pol FP-DBS PCR assay can provide an alternative costeffective,\r\nreliable and rapid method for early detection of HIV-1 infection in infants....
Background: Although G6PD deficiency is the most common genetically determined blood disorder among Iraqis,\r\nits molecular basis has only recently been studied among the Kurds in North Iraq, while studies focusing on Arabs\r\nin other parts of Iraq are still absent.\r\nMethods: A total of 1810 apparently healthy adult male blood donors were randomly recruited from the national\r\nblood transfusion center in Baghdad. They were classified into G6PD deficient and non-deficient individuals based\r\non the results of methemoglobin reduction test (MHRT), with confirmation of deficiency by subsequent enzyme\r\nassays. DNA from deficient individuals was studied using a polymerase chain reaction-Restriction fragment length\r\npolymorphism (PCR-RFLP) for four deficient molecular variants, namely G6PD Mediterranean (563 C�®T), Chatham\r\n(1003 G�®A), A- (202 G�®A) and Aures (143 T�®C). A subset of those with the Mediterranean variant, were further\r\ninvestigated for the 1311 (C�®T) silent mutation.\r\nResults: G6PD deficiency was detected in 109 of the 1810 screened male individuals (6.0%). Among 101 G6PD\r\ndeficient males molecularly studied, the Mediterranean mutation was detected in 75 cases (74.3%), G6PD Chatham\r\nin 5 cases (5.0%), G6PD A- in two cases (2.0%), and G6PD Aures in none. The 1311 silent mutation was detected in\r\n48 out of the 51 G6PD deficient males with the Mediterranean variant studied (94.1%).\r\nConclusions: Three polymorphic variants namely: the Mediterranean, Chatham and A-, constituted more than 80%\r\nof G6PD deficient variants among males in Baghdad. Iraq. This observation is to some extent comparable to other\r\nAsian Arab countries, neighboring Turkey and Iran....
A rapid, simple, and sensitive liquid chromatography- tandem mass spectroscopy method was developed for quantification of Hydroxyzine and its active metabolite Cetirizine in human plasma using Quetiapine as internal standard. The analyte and internal standard was extracted by solid phase extraction using Orochem 30mg, 1ml catridge. Quantification was done on triple quadrapole mass spectrometer employing turbo ion spray mode. The separation of analyte and internal standard was done on Inertsil ODS-3, 100×4.6mm, 5µm column with mobile phase composition of 5mM Ammonium acetate (pH 4.0 adjusted with formic acid): Methanol: Acetonitrile in the ratio of 5:5:90 v/v/v. The total chromatographic runtime was 3min. The linearity range for Hydroxyzine and Cetirizine was 0.250ng/ml-100.093ng/ml, 1.000ng/ml-501.179ng/ml respectively. This method was fully validated for its sensitivity, accuracy, precision, linearity, recovery, matrix effect, dilution integrity, and stability studies....
Motivation: It has been proposed that clustering clinical markers, such as blood test results, can be used to stratify patients.\r\nHowever, the robustness of clusters formed with this approach to data pre-processing and clustering algorithm choices has not\r\nbeen evaluated, nor has clustering reproducibility. Here, wemade use of the NHANES survey to compare clusters generated with\r\nvarious combinations of pre-processing and clustering algorithms, and tested their reproducibility in two separate samples.\r\nMethod: Values of 44 biomarkers and 19 health/life style traits were extracted from the National Health and Nutrition\r\nExamination Survey (NHANES). The 1999ââ?¬â??2002 survey was used for training, while data from the 2003ââ?¬â??2006 survey was\r\ntested as a validation set. Twelve combinations of pre-processing and clustering algorithms were applied to the training set.\r\nThe quality of the resulting clusters was evaluated both by considering their properties and by comparative enrichment\r\nanalysis. Cluster assignments were projected to the validation set (using an artificial neural network) and enrichment in\r\nhealth/life style traits in the resulting clusters was compared to the clusters generated from the original training set.\r\nResults: The clusters obtained with different pre-processing and clustering combinations differed both in terms of cluster\r\nquality measures and in terms of reproducibility of enrichment with health/life style properties. Z-score normalization, for\r\nexample, dramatically improved cluster quality and enrichments, as compared to unprocessed data, regardless of the\r\nclustering algorithm used. Clustering diabetes patients revealed a group of patients enriched with retinopathies. This could\r\nindicate that routine laboratory tests can be used to detect patients suffering from complications of diabetes, although\r\nother explanations for this observation should also be considered.\r\nConclusions: Clustering according to classical clinical biomarkers is a robust process, which may help in patient\r\nstratification. However, optimization of the pre-processing and clustering process may be still required....
The whole blood interferon-gamma assay (QuantiFERON-TB-2G; QFT) has not been fully evaluated as a baseline\r\ntuberculosis screening test in Japanese healthcare students commencing clinical contact. The aim of this study was to compare\r\nthe results from the QFT with those from the tuberculin skin test (TST) in a population deemed to be at a low risk for infection\r\nwith Mycobacterium tuberculosis. Methodology/Principal Findings. Healthcare students recruited at Okayama University\r\nreceived both the TST and the QFT to assess the level of agreement between these two tests. The interleukin-10 levels before\r\nand after exposure to M tuberculosis-specific antigens (early-secreted antigenic target 6-kDa protein [ESAT-6] and culture\r\nfiltrate protein 10 [CFP-10]) were also measured. Of the 536 healthcare students, most of whom had been vaccinated with\r\nbacillus-Calmette-Gue�´ rin (BCG), 207 (56%) were enrolled in this study. The agreement between the QFT and the TST results was\r\npoor, with positive result rates of 1.4% vs. 27.5%, respectively. A multivariate analysis also revealed that the induration\r\ndiameter of the TST was not affected by the interferon-gamma concentration after exposure to either of the antigens but was\r\ninfluenced by the number of BCG needle scars (p = 0.046). The whole blood interleukin-10 assay revealed that after antigen\r\nexposure, the median increases in interleukin-10 concentration was higher in the subgroup with the small increase in\r\ninterferon-gamma concentration than in the subgroup with the large increase in interferon-gamma concentration (0.3 vs. 0 pg/\r\nmL; p = 0.004). Conclusions/Significance. As a baseline screening test for low-risk Japanese healthcare students at their\r\ncourse entry, QFT yielded quite discordant results, compared with the TST, probably because of the low specificity of the TST\r\nresults in the BCG-vaccinated population. We also found, for the first time, that the change in the interleukin-10 level after\r\nexposure to specific antigens was inversely associated with that in the interferon-gamma level in a low-risk population....
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