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In Vitro Alternative Methods for the Assessment of Dermal Irritation and Inflammation
Published in David W. Hobson, Dermal and Ocular Toxicology, 2020
David W. Hobson, James A. Blank
Problems with serum protein precipitation have been reported using the acidic isopropanol solution as described above.39 This problem can be circumvented by changing the formazan product solubilization solution to one that includes sodium dodecyl sulfate (SDS).39,44 Modifications to the method as described by Hansen and co-workers are reported to decrease assay variability and to increase assay sensitivity.44 These modifications include the following: Add 25 μl of the 5 mg/ml MTT stock per 100 μl of medium instead of 10 μl.Replace the acidic isopropanol with an SDS-N,N-dimethyl formamide (DMF) mixture. The mixture is prepared by dissolving a 20% w/v solution of SDS in a 50% DMF-deionized water solution. The pH of the resulting solution is adjusted to 4.7 by the addition of 2.5% of 80% acetic acid and 2.5%of 1 N hydrochloric acid.After a 2-h 37°C incubation with MTT, 100 μl of the acidified SDS-DMF solution is added. The plates are then incubated overnight at 37°C and the optical densities at 570 nm determined. This procedure does not require mixing before measuring OD.
Cytokine Measurements in Disease
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
David Heney, Rosamonde E. Banks, John T. Whicher, Stuart W. Evans
Bioassays are generally more sensitive than immunoassays for cytokines. The characteristics of calibration curves vary between different assays, and limits of sensitivity must be set so that only the portion of the curve producing acceptable precision is used. Sensitivity is determined not only by the assay sensitivity, but also by the dilution of the biological sample which may be needed to overcome inhibitory or cytotoxic effects of the sample. Similarly, in immunoassays, the dilution necessary to decrease nonspecific background to acceptable levels may result in a higher minimum detectable level.
Cervical Neoplasia, Human Papilloma Virus and Psychoneuroimmunology
Published in Herman Friedman, Thomas W. Klein, Andrea L. Friedman, Psychoneuroimmunology, Stress, and Infection, 2020
Michael H. Antoni, Karl Goodkin
Much remains to be demonstrated regarding the biological factors responsible for the initiation and progression of CIN. Prospective studies of the rates of progression among various HPV subtypes with repeated measures on larger samples are warranted. Use of emerging laboratory techniques like the polymerase chain reaction may improve subtype assay sensitivity and specificity. Also, documentation of other potential infectious co-factors including cytomegalovirus, chlamydia, gonorrhea, syphilis and even retro-viruses must be done to isolate the impact of HPV infection. The stepwise, well-defined progression of CIN renders it amenable to research investigating the concomitant factors (i.e., immunologic status) associated with the promotion of HIV-1 infection. Lymphomas and Kaposi’s sarcoma represent traditional opportunistic neoplasias in AIDS. Cervical neoplasias are part of the list of such malignancies which identify those HIV-1 infected individuals as having AIDS and as candidates for imminent progression to other AIDS-related conditions due to the documented immunosuppression and HPV infections (as potential co-factors of HIV progression) which are probable criteria for the manifestation of CIN.
Testing strategies used in the diagnosis of rare inherited bleeding disorders
Published in Expert Review of Hematology, 2023
[17–20]Screening tests like the prothrombin time (PT) and activated partial thromboplastin time (APTT) are the most commonly ordered initial tests in evaluation of suspected bleeding disorders; the assays assess the different coagulation pathways (Figure 1). However, the PT and APTT have limitations in their sensitivity to mild coagulation factor deficiencies, which vary based on reagent/instrument combinations and may result in a missed diagnosis [21–26]. Laboratory professionals are generally aware of these limitations; however, the majority of clinicians are not aware of limitations of assay sensitivity. Examples of sensitivity of PT and APTT to commonly ordered coagulation factor assays are shown in Figure 2a–h. The most commonly performed coagulation factor assay is the one-stage factor assay [27], where the assay end point is the detection of a fibrin clot. Chromogenic assays, where the end point is the detection of the release of a chromogenic substrate, are more expensive, have limited availability, and are rarely performed except for the diagnosis of HA and HB [28] and to monitor selected extended half-life coagulation factor concentrates.
The urocortin peptides: biological relevance and laboratory aspects of UCN3 and its receptor
Published in Critical Reviews in Clinical Laboratory Sciences, 2022
Norah J. Alghamdi, Christopher T. Burns, Roland Valdes
Measurement of mature UCN3 in urine also shows variability when normalized to the urinary creatinine. A study published by Gozal et al. used a proteomic and mass spectrometry technique to detect and quantify urinary biomarkers for OSA from a 2 D-gel electrophoresis. Enzyme-linked immunosorbent assay (ELISA) and western blot methods were also used to confirm the findings. The study reported cutoffs, dynamic range, and precision for the electrophoresis, mass spectrometry and western blot assays. The ELISA assay was developed using antibodies raised against the C-terminal epitope of the UCN3 sequence; however, details of the peptide structure used to develop the antibodies and the specificity with other urocortins were not reported. The assay sensitivity, dynamic range, and precision study were reported for ELISA. The results indicated that urinary UCN3 levels significantly increased in OSA (6.9 ± 0.4 pg/mL/mg creatinine) compared with the control group (3.8 ± 0.2 pg/mL/mg creatinine) [63].
Gender roles are related to cortisol habituation to repeated social evaluative stressors in adults: secondary analyses from a randomized controlled trial
Published in Stress, 2021
Andrew W. Manigault, Ryan C. Shorey, Haley Appelmann, Katrina R. Hamilton, Matt C. Scanlin, Robert-Paul Juster, Peggy M. Zoccola
Salivary cortisol was collected four times per lab visit, including samples taken immediately before the TSST and 25, 35, and 60 min after the TSST. Participants were given Salivettes (Sarstedt, Inc., Newton, N.C.) and instructed to saturate the swab with saliva for up to 3 min. Instructions were given to ensure that swabs were not touched by hand. Samples were immediately stored at −20 °C after collection and then transferred to a −80 °C freezer until processed. All samples were centrifuged and assayed in duplicate (values averaged) at Ohio University facilities with standard enzyme-linked immunoassay procedures (Salimetrics, LLC, State College, PA). The assay sensitivity was less than 0.007 ug/dL, inter-assay coefficients of variation were less than 11.0%, and intra-assay coefficients of variation were less than 7.0%.