published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Sickle cell disease is an inherited blood cell disorder that affects about 100,000 people in the US and results in high cost of medical care exceeding $1.1 billion annually. Sickle cell patients suffer from unpredictable, painful vaso-occlusive crises. Portable, costeffective approaches for diagnosis and monitoring sickle blood activities are important for a better management of the disease and reducing the medical cost. In this research, a mobile application controlled, impedance-based flow cytometer is developed for the diagnosis of sickle cell disease. Calibration of the portable device is performed using a component of known impedance value. The preliminary test results are then compared to those obtained by a commercial benchtop impedance analyzer for further validation. With the developed portable flow cytometer, experiments are performed on two sickle cell samples and a healthy cell sample. The acquired results are subsequently analyzed with MATLAB scripts to extract single-cell level impedance information as well as statistics of different cell conditions. Significant differences in cell impedance signals are observed between sickle cells and normal cells, as well as between sickle cells under hypoxia and normoxia conditions. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Over the last 50 years, flow cytometry has evolved from a modest cell counter into an invaluable analytical tool that measures an ever-expanding variety of phenotypes. Flow cytometers interrogate passing samples with laser light and measure the elastically scattered photons to ascertain information about sample size, granularity, and basic morphology. Obtaining molecular information, however, requires the addition of exogenous fluorescent labels. These labels, although a power tool, have numerous challenges and limitations such as large emission spectra and cellular toxicity. To move beyond fluorescent labels in microscopy, a variety of techniques that probe the intrinsic Raman vibrations within a sample have been developed, such as coherent anti-Stokes Raman scattering (CARS) and Raman microspectroscopy. In this dissertation, I present the first development of a label-free flow cytometer that measures the elastically scattered photons and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, a coherent Raman technique that probes a large region of the Raman spectrum simultaneously, provides rich molecularly-sensitive information. Furthermore, I present its application to sorting polymer microparticles and its use in two example biological applications: monitoring lipid bodies within cultures of Saccharomyces cerevisiae, a model yeast with numerous human homologs, and monitoring the affect of nitrogen starvation on Phaeodactylum tricornutum, a diatom, which is being genetically engineered to efficiently produce biofuels.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care. / AFRIKAANSE OPSOMMING: Inleiding: Vloeisitometrie tegnologie het verskeie tradisionele laboratorium toetse vervang as gevolg van beter akuraadheid, sensitiwiteit en vinniger beskikbaarheid van resultate in ‘n kliniese omgewing, veral kliniese proewe. Vloeisitometrie is ‘n kragtige tegniek om chemiese (fluorokroom byvoeging) en fisiese (sel grote en kompleksiteit) karakter eienskappe van individuele selle te meet. Met die toename in gebruik en gewildheid van hiedie instrumente, neem die behoefde toe vir gevorderde kwaliteit kontroles, gestandardiseerde prosedures, met profesionele toets programme tesame met metode ontwikkeling. Klein verstellings aan instrument parameters beinvloed die betroubaarheid, herhaalbaarheid en sensitiwiteit van ‘n sitometer en moet gemonitor (en dokumenteer) word om identiese kondisies van leesings op ‘n daaglikse basis te verseker. Dit kan bereik word deur in te skakel met ‘n interne kwaliteits versekerings program [IQA: “Internal Quality Control”] en/of ‘n eksterne kwaliteits versekerings program [EQA: “External Quality Control”] te volg. Op die oomblik is daar geen sulke kwaliteits versekerings programme in Suid Afrika en/of in die verarmende Afrika lande beskikbaar nie. MIV is ‘n wêreldwye bekommernis en laboratoriums en klinieke in hierdie gedeeltes van die land verlang ‘n dringende behoefdte vir sulke “IQA” programme om kwaliteit van instrumentasie en akkurate pasiënt resultate te verseker wat tot beter behandeling van pasiënte lei. Kwaliteit versekerings programme soos “CD Chex®” en “UK Nequas” is beskikbaar, maar baie probleme met verwysing na monster integriteit as gevolg van tydsame vervoer en aflewering kondisies word hiermee geassosieër. Die behoefte het ontstaan vir ‘n laboratorium in Suid Afrika wat direk die omliggende laboratoriums, hospitale en klinieke kan voorsien met gestabiliseerde blood monsters wat gebruik kan word as “IQA”. Die vervoer en aflewerings kondisies van hierdie monsters sal aansienlik verbeter as gevolg van die korter aflewerings afstand wat direk die beperkte temperatuur wisseling beinvloed. Doel van studie: Die doelwit van hoofstuk een is om vir die leser ‘n inleiding te gee tot terminologie en konsepte van immunologie en die immune sisteem. Hoofstuk twee beskyf die impak wat gestabiliseerde heelbloed het op die kliniese immunologie met betrekking tot kwaliteit beheer en kwaliteit versekering. Die doelwit van hierdie studie is om heelbloed te stabiliseer sodat die rakleeftyd meer as 30 dae is en sodoende as verwysings-materiaal kontroles vir Suid Afrikaanse immunofenotipering kan dien. Dit is ‘n verdere doelwit om hierdie tuis-gestabiliseerde kontrole monsters te gebruik as “IQA” verwysings materiaal in verarmende Afrika lande. Die doelwit van hoofstuk vier is om limfosiete te stimuleer om verskeie aktiverings merkers uit te druk op hul selmembrane en dan te stabiliseer en dié te gebruik as Kwaliteits Kontroles vir die meer gespesialiseerde immunologiese toetse. Studie ontwerp: Hoofstuk drie beskryf ‘n aangepaste en verbeterde metode van heel bloed stabiliseering. Stabiliteit word ondersoek in ‘n verskyndenheid konsentrasies van ‘n primêre stabiliseerings agent (chromium chloried heksahidraat) en inkubasie periodes met paraformaldehied as tweede stabiliseerings agent word deeglik gedokumenteer. Bloedmonsters van gesonde indiwidië (n=10) was gestabiliseer en gemonitor vir roetine MIV membraanoppervlak antigene oor ‘n periode van 40 dae. Hierdie monsters (n=10) was gelees en geanaliseer op ‘n BD FACSCalibur™ en vergelyk met ‘n BD FACSCount™ vloeisitometer instrument. Drie gestabiliseerde heelbloed monsters (n=3) was gemonitor vir ‘n periode vir so lank moontlik die fenotipiese selmembraan molekules identifiseerbaar was en die kwantiteit bepaalbaar was. Hierdie drie monsters was gemeet op beide instrumente. As ‘n addisionele doelwit, was hierdie drie gestabiliseerde monsters ondersoek om as moontlike kalibrasie materiaal (verteenwoordig ‘n normale bloedmonster) te dien vir die BD FACSCount™ instrument in die oggende voor pasiënt monsters gelees kan word. In hoofstuk vier was limfosiete geϊsoleer en geaktiveer met ‘n verskyndenheid stimulante om optimale aktiveerings-antigene uit te druk op T helper selmembrane (byvoorbeeld CD25, CD69, HLA-DR en CD40 Ligand). Hierdie geaktiveerde monsters was geanaliseer op die BD FACSCalibur™ en daarna gestabiliseer. Na stabilisasie van die geaktiveerde limfosiet monsters was dit gemonitor oor ‘n tydperk so lank moontlik data plotte leesbaar en selpopulasies identifiseerbaar was. Hierdie monsters kan dien as ‘n moontlike “IQA” toets stel vir ‘n meer gespesialiseerde immunologiese aktiveerings kontrole doeleindes. Resultate: In hoofstuk drie; tien individiële gestabiliseerde heelbloed monsters het gedui op geen-beduidende P waardes (P > 0.05) vir CD3, CD4 en CD8 persentasies en absolute waardes; gemeet vanaf DAG 3 vergelykbaar tot-en-met DAG 40. Met korrelasie statistiek en vergelyking van die BD FACSCalibur™ met die FACSCount™ instrumente, is die volgende opgemerk; R2 = 0.9848 vir die CD4 absolute waardes en ‘n R2 = 0.9636 vir die CD8 absolute waardes. Drie gestabiliseerde monsters (n=3) was gemonitor vir MIV roetine fenotipeering tot en met DAG 84. Die selpopulasies was duidelik identifiseerbaar en die kwantitatief meetbaar op albei instrumente (BD FACSCalibur™ en BD FACSCount™). Hoofstuk vier: geaktiveerde T helper lymphosiete het 25 – 35% membraan CD40 Ligand uitgedruk op hul selmembrane. Die stimulant van keuse was ionomysien teen ‘n optimale konsentrasie van 4μM. Die optimale inkubasie tydperk was vier ure by 37°C in 5% CO2 kondisie. Ses uur inkubasie in 4μM ionomysien by 37°C in ‘n 5% CO2 omgewing was optimal vir die CD69 selmembraan uitdrukking en het 84.21% opgelewer. Vir CD25 selmembraan uitdrukking was die selle vir ses ure met phietoheamagglutinin (PHA) gestimuleer by 37°C in 5% CO2 kondisie en het 43% CD25 selmembraan uitdrukking opgelewer. HLA-DR selmembraan uitdrukking: selle was vir ses ure saam met PHA by 37°C in 5% CO2 kondisie inkubeer en het 43.32% opgelewer. CD40 Ligand aktivering/gestabiliseerde limfosiete het tot en met dag 23 stabiliteit getoon. Die ligand was duidelik identifiseerbaar en kwantifiseerbaar. Geaktiveerde lymphosiete wat CD69, CD25 en HLA-DR selmembraan merkers uitdruk het na die stabiliseerings proses stabiliteit getoon tot-en-met dag 16. Gevolgtrekking: Die doel van hierdie studie was om verwysingskontroles voor te berei sodat dit vars heelbloed naboots met uitkenbare eienskappe vir kliniese situasies. ‘n Toets kontrolestel met verwysings materiaal vir drie vlakke (byvoorbeeld ‘n lae, medium en hoë kontrole) absolute selwaardes en persentasies kan voorberei word vir roetine immunologiese fenotiperings merkers (CD3/CD4/CD8/CD45). Meer gespesialiseerde kontrolestelle vir meer spesifieke doeleindes kan opgemaak word wat ‘n verskydenheid van limfosiet aktiveringsmerkers bevat met byvoorbeeld ‘n “nul”, lae en hoë verwysings kontrole daarin. Hierdie heelbloed kan dien as “aktiveerde interne kwaliteits verwysings materiaal” en kan gebruik word om nuut aangestelde laboratorium werkers en nuut gekwalifiseerde studente op te lei. Hierdie verwysings materiaal / kontroles kan aangewend word vir bevoegdheids doeleindes (byvoorbeeld vir SANAS akkreditasie doeleindes), vir metode ontwikkeling, vir sagteware toetsing, vir paneel opstelling en instrument verstellings doeleindes. Die kontroles moet ‘n verskydenheid eienskappe bevat om effektief te wees. Byvoorbeeld, stabiliteit tydens storing, gewenslik meer as ‘n paar weke, herhaalbaar en maklik handteerbaar. Hierdie kontroles sal inligting voorsien op ‘n daaglikse basis tydens wisseling van tegnieke of instrumentasie wat akuraatheid beinvloed en op die ou-end direk pasiënt versorging bevoordeel.

Indiana University-Purdue University Indianapolis (IUPUI) / The non-hematopoietic, stationary stromal cells located inside and surrounding skin-draining lymph nodes play a key role in regulating immune responses. We studied distinct populations of lymph node stromal cells from both human subjects and animal models in order to describe their phenotype and function. In the mouse model, we studied two distinct populations: an endothelial cell population expressing Ly51 and MHC-II, and an epithelial cell population expressing the epithelial adhesion molecule EpCAM. Analysis of intra-nodal and extra-nodal lymph node (CD45-) stromal cells through flow cytometry and qPCR provides a general phenotypic profile of the distinct populations. My research focused on the EpCAM+ epithelial cell population located in the fat pad surrounding the skin draining lymph nodes. The EpCAM+ population has been characterized by surface marker phenotype, anatomic location, and gene expression profile. This population demonstrates the ability to inhibit the activation and proliferation of both CD4 and CD8 T cells. This population may play a role in suppressing overactive inflammation and auto-reactive T cells that escaped thymic deletion. The other major arm of my project consisted of identifying a novel endothelial cell population in human lymph nodes. Freshly resected lymph nodes were processed into single cell suspensions and selected for non-hematopoietic CD45- stromal cells. The unique endothelial population expressing CD34 HLA-DR was then characterized and analyzed for anatomic position, surface marker expression, and gene profiles. Overall, these studies emphasize the importance of stationary lymph node stromal cells to our functioning immune systems, and may have clinical relevance to autoimmune diseases, inflammation, and bone marrow transplantation.