Improvement of platelet ionized calcium measurements by implementation and standardization of a ratiometric technique using blue laser flow cytometry / by Darren H.M. Hickerson.
| Author/creator | Hickerson, Darren H. M. author. |
| Other author | Kalmus, Gerhard W., degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 1999. |
| Description | xiiii, 219 leaves : illustrations ; 28 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | Abnormal platelet basal intracellular ionized calcium concentration ([Ca²⁺]i) and altered Ca²⁺ translocation upon agonist stimulation have been associated with a variety of disease states. Current techniques to measure [Ca²⁺]i), have flaws which hinder the potential for accurate, routine clinical analysis. This project sought to improve and simplify [Ca²⁺]i measurements in platelets by adapting the Fluo-3 and Fura Red technique of Novak and Rabinovitch (1994), thus bringing the advantages ofratiometric analysis to the now ubiquitous blue laser flow cytometers. Improvements included optimization of handling protocols, development and introduction of a novel direct agonist injection system, use of logarithmically amplified fluorescence detection, and software-based ratiometry. Application of the technique revealed new information that three heterogeneously Ca²⁺-dye staining subpopulations (SPs) of platelets may coexist in samples from normal donors. These SPs correlated with characteristics of normal, prematurely activated, and aged or damaged platelets. Simultaneous, multiparametric activation kinetics analysis demonstrated a tentative time course for in vitro Ca²⁺ flux, microparticle generation, CD62 neo-expression, degranulation, and shape change (estimated from the "size" parameter) from a single activation reaction. In the first known platelet or ratiometric application of Fluo-4 with Fura Red, Fluo-4 was compared to Fluo-3. Fluo-4 was shown to be brighter, but not more sensitive than Fluo-3. |
| General note | Submitted to the faculty of the Department of Biology. |
| General note | Advisor: Gerhard W. Kalmus |
| Dissertation note | M.S. East Carolina University 1999 |
| Bibliography note | Includes bibliographical references (leaves 207-219). |
| Genre/form | Academic theses. |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |
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|---|---|---|---|---|
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