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Blood sampling
Published in R. C. Richard Davison, Paul M. Smith, James Hopker, Michael J. Price, Florentina Hettinga, Garry Tew, Lindsay Bottoms, Sport and Exercise Physiology Testing Guidelines: Volume I – Sport Testing, 2022
Ronald J. Maughan, Susan M. Shirreffs
There is some debate as to which, if any, concentration measurements should be corrected for changes in blood or plasma volume. The modern Coulter counter incorporates an autosampler and spectrophotometer, which permits automated measurement of haemoglobin concentration. While this automation has considerable attractions, including a high level of accuracy in the measures of red cell count and haemoglobin concentration, care must be taken in the interpretation of the measures of cell volume. The diluent commonly used in the preparation of samples for analysis is not isotonic with normal human blood plasma: Isoton II has an osmolality of about 340 mOsmol·kg−1, compared with an osmolality of human plasma of about 285–290 mOsmol·kg−1. Because the red cell membrane is freely permeable to water, a rapid equilibration will take place on mixing of blood with the diluent, leading to a change (in the case of Isoton II there will be a decrease) in the red cell volume. The measured volume is therefore different, by an amount proportional to the difference in osmolality between the plasma and the diluent, from the volume of the cells while in the circulation. In situations where the plasma osmolality changes substantially, as during intense or prolonged exercise, this will invalidate measures made using automated cell counting procedures (Watson and Maughan, 2014).
Alternative Methodologies to Animal Testing
Published in Nicola Loprieno, Alternative Methodologies for the Safety Evaluation of Chemicals in the Cosmetic Industry, 2019
Two assays for testing in vitro teratogens have been used by the National Toxicology Program (NTP) in the U.S.: The MOT assay measures the attachment of ascitic mouse ovarian tumor cells (MOT cells) to a lectin (concanavalin A-) coated surface. Cellular adhesiveness is a generic property of embryonic cells; therefore, it is assumed that inhibition of adhesiveness by a chemical indicates teratogenic potential. The test chemical is added to a suspension of MOT cells which have been exposed to radiolabeled thymidine, and a plastic sheet coated with concanavalin A is placed in the culture vessel. Over a period of hours, the MOT cells normally adhere to the lectin-coated surface. Inhibition of this phenomenon by teratogens can be assessed by counting the radioactivity associated with the plastic sheets and by comparison with control values.The HEMP assay measures the growth and proliferation of Human Embryonic Palatal Mesenchyme (HEMP) cells in the presence of test agents. Growth and division of cells is a fundamental process in developing tissues, and inhibition of this would be indicative of developmental toxicity. The HEMP cells are plated at a low density in tissue culture dishes. After 24 h, the test agent is added, and the cultures are maintained for an additional 72 h without a media change. At the end of this period, the number of cells present is counted using a Coulter counter.
Flow Cytometric Analysis of Fluorescent Estrogen Binding in Cancer Cell Suspensions
Published in Louis P. Pertschuk, Sin Hang Lee, Localization of Putative Steroid Receptors, 2019
Chris Benz, Israel Wiznitzer, Sin Hang Lee
Flow cytometry (FCM) began over 25 years ago as a tool to automatically count and size individual cells from a flowing cell suspension. By the mid 1950s, this early tool was commercially available as the Coulter Counter®. Ten years later, with the aid of more sophisticated spectrophotometry detection and computer coupling, multiparameter analysis of DNA or protein staining cellular constituents could be performed on cells flowing at the rate of 500/sec. These developments resulted in the later availability of the Ortho Cytofluorograph®. Fluorescent dye emission rather than absorption was utilized to increase the signal-to-noise ratio, and the demonstration of stoichiometric binding between fluorochromes and DNA expanded the application of FCM to producing DNA histograms and analyzing the growth of tumor cell suspensions for percent G1, S, and G2/M phase cells. Most recently, the Becton-Dickinson Fluorescence Activated Cell Sorter (FACS)® introduced electrostatic sorting of individual cells based on the analysis of light scatter (cell size) or fluorescence intensity. The application of this new and expanding field to medical and biologic problems has been extensively reviewed by Melamed et al.14 It is now clear that FCM can be successfully used to study the binding of a wide variety of fluorescently labeled ligands to their membrane, cytoplasmic, or nuclear receptors in intact cells.15
Prevalence, risk factors and outcome of Mycoplasma pneumoniae infection among children in Uganda: a prospective study
Published in Paediatrics and International Child Health, 2021
Rebecca Nantanda, Freddie Bwanga, Irene Najjingo, Grace Ndeezi, James K Tumwine
The blood for IgM serological tests was immediately transferred into a cold box at 4°C. The IgM used was Demeditec Mycoplasma pneumoniae IgM ELISA DEMYCM0350 (Demeditec Diagnostics GmbH, Germany). The sample for CBC was kept at room temperature until transferral to the laboratory. All samples reached the laboratory within 8 hours of collection. The Coulter counter method was used to measure the total and differential white cell count. The sputum quality was assessed both macroscopically to determine whether it was saliva and by the Gram stain method. Gram stain quantified the number of organisms in the sputum, and all samples with >10 squamous epithelial cells per low power field were considered to be of poor quality. The PCR results from such samples were not analysed for M. pneumoniae. In these participants, only the results of the IgM for M. pneumoniae were considered.
Thymus gland size during recovery from complicated severe acute malnutrition: a prospective study of the role of probiotics
Published in Paediatrics and International Child Health, 2019
Nicolette Nabukeera-Barungi, Benedikte Grenov, Henrik Friis, Betty Lanyero, Hanifa Namusoke, Ezekiel Mupere, Kim F. Michaelsen, Christian Mølgaard, Vibeke B. Christensen, Maren Johanne Rytter
Complete blood counts and HIV tests were undertaken on admission, and plasma C-reactive protein (CRP) was determined on admission, at discharge and at the 8-week follow-up visit. Venepunctures were used to draw 4 ml of blood which was placed in heparinised vacutainer tubes (Becton Dickinson, Franklin lakes, NJ, USA). Samples for complete blood count were analysed using a Coulter counter and expressed as cells/mm3. Rapid tests were used for HIV serology using the Determine HIV-1/2 kit (Abbott Laboratories, USA), and positive samples were confirmed with the HIV 1/2 STAT-PAK Dipstick Assay kit (Chembio Diagnostic systems Inc., USA). In order to confirm their HIV status, an HIV DNA/PCR test was undertaken in all children under 18 months with a positive serology test. Plasma was obtained by centrifuging at 1300–2200 G for 10 min, then stored at −80°C until shipped on dry ice to the Department of Nutrition Exercise and Sports, University of Copenhagen, Denmark. Plasma CRP was measured by a highly sensitive kit on an ABX Pentra 400 (Horiba, France).
Measuring the Systemic Inflammatory Response to On- and Off-Pump Coronary Artery Bypass Graft (CABG) Surgeries Using the Tryptophan/Kynurenine Pathway
Published in Journal of Investigative Surgery, 2022
Ahmed Farouk, Rasha A. Hamed, Saeid Elsawy, Nashwa F. Abd El Hafez, Farag M. Moftah, Muammar A. Y. Nassar, Fify Alfy Gabra, Tahia H. Saleem
From each patient, 5 mL of venous blood was withdrawn according to the schedule: 24 h preoperative, immediately after complete revascularization, 10 min before the end of operation and 72 h postoperative. About 3 mL of each sample was collected on heparin tube for Trp and Kyn estimation while 2 mL was collected in plain collection tubes for IL-6 determination. WBCs were analyzed by coulter counter as a routine investigation. Serum and plasma were separated by centrifugation at 3500 rpm for 15 min at 4 °C, and then stored in −20 °C freezer. Plasma acidic protein was precipitated by sulfosalicylic acid and centrifugation, for determination of Trp. The free amino acids remaining in the supernatant were used for analysis by HPLC using ion exchange column.