<p>During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population.</p><p>In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (<i>E. coli</i>) but eukaryotic organisms (<i>P. pastoris</i>) were included.</p><p>The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof both<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible for<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures of<i>E. coli.</i></p><p>Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population.</p><p>In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses.</p><p><b>Key words</b>: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,<i>E. coli</i>,<i>P. pastoris</i>, inclusion body, food preservatives,viability, membrane potential</p>

The main goal in biopharmaceutical production is achieving high volumetric productivity while maintaining product quality (i.e. glycosylation). The objectives of this project were to explore the use of dielectric analysis in the early detection of cell demise and to analyze the impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and glycosylation. Measurements of changes in the polarizability of individual cells can be performed in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked according to their polarizability and recorded as a force index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer. Discrete cell sub-populations were identified as cells passed through the various stages of apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in the mitochondrial metabolism linked with early apoptosis for both inducers. In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination with 1 mM uridine or 1 mM uridine + 8 μM MnCl2 was added to the basal and feed medium for two CHO cell lines to determine their impact on the biopharmaceutical production and the glycosylation process. The results showed that the addition of all three precursors combined increased UDP-Gal, which increased and maintained the galactosylation index during the bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a further increase in the UDP-Gal concentration did not increase the galactosylation. A negative impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which was linked to the CHO-EG2 cell line being DHFR-/-. This work presents a dielectric detection method to monitor early changes in the cell metabolism and information for shifting and maintaining galactosylation during biopharmaceutical production. / February 2016

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2011 / BACKGROUND: Human Immunodeficiency Virus (HIV) is a global epidemic with growing numbers of people on highly active anti‐retroviral therapy (HAART) programmes. Effectiveness of treatment needs to be monitored to ensure the uncompromised well being of patients. This is currently done using both Viral Load (VL) and CD4 cell counts for HAART initiation and follow‐up. Although VL is the best predictor of disease progression it is often too expensive for monitoring patients in resource‐limited settings. There is thus a need for a cheaper, more accessible alternative to monitor long term patient response to therapy. METHODS: This study evaluated the use of a recently described flow cytometric assay of CD38 expression (previously developed at the Johannesburg Flow Cytometry Reference Laboratory) in a cohort of HIV+ patients failing 1st line therapy, who were subsequently enrolled onto 2nd line HAART. CD38 and CD8 were “piggy ‐backed” onto the PLG/CD4 protocol and mean fluorescence intensity (MFI) of the CD8/38 expression was monitored longitudinally. Patterns of CD38 expression were compared to 1st line treatment observations to establish equivalence in the predictive power of CD38 expression of fluctuation in viral load on 2nd line treatment patients. In addition, the effect of sample age on assay accuracy was tested before implementation of the CD38 assay at a secondary testing site. RESULTS: The patterns observed in the cohort of 2nd line therapy patients mirrored patterns previously seen in 1st line therapy with 55% of patients showing a continuous decline in CD38 MFI that mimicked changes in VL. The remaining 33% of patients had non‐specific increases in CD38 MFI without concurrent increases in VL and one patient showed irregular VL and CD38 MFI (non‐responder). The CD38 assay showed acceptable accuracy and reproducibility up to 48 hours after venesection (%CV<5%). Implementation at the secondary testing site was successful with 98% similarity (%CV<5%) compared to the reference laboratory. CONCLUSION: CD38 monitoring of 2nd line therapy patients showed comparable patterns to observations in 1st line therapy patients. The assay proved stable over time and easy to implement at another PLG/CD4 testing facility. As such, the CD38 assay offers a cost‐effective, reliable real time supplementary test to long‐term VL monitoring of HIV infected patients on the national ART programme.

Rheumatoid arthritis (RA) is a chronic, heterogeneous, autoimmune disease that causes inflammation of synovial joints leading to pain, stiffness and swelling. If left untreated, RA results in irreversible joint destruction and long term disability. Initial treatment with glucocorticoids and other immunosuppressive agents suppresses inflammation. However, many of these drugs are not well-tolerated due to extensive side effects or are simply ineffective. The discovery of tumour necrosis factor-α (TNF) as a key mediator of inflammation in RA led to the development of monoclonal anti-TNF antibody therapy. Since then, other biologic drugs targeting immune pathways have been developed for RA, including interleukin-6 (IL-6) blockade, B cell depletion, and T cell co-stimulation blockade. Not all patients will respond to their first biologic drug and currently there is no way to predict which patient will respond to each different class of drug. Generally, 3 – 6 months are required to determine clinical efficacy, during which time joint inflammation proceeds. Therefore, discovering biomarkers to predict treatment response is a research priority. Biologic drugs target immune pathways. As single cell technology advances and has increasing capacity to identify subtle changes in many cell subsets, I hypothesise that studying the blood immune cell landscape will define cellular biomarker profiles relevant to each individual patient’s disease.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 13, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-161).

Platelets play a pivotal role in hemostasis and thrombosis. It induces vascular retraction and clot formation through platelets activation and signal transmission, which promote ligands expressing on the surface of platelets, such as glycoproteins and P-selectin. Some surface glycoproteins gatherd to form complexes after activation. It bound to extracellular receptors such as collagen and thrombin to induce aggregation, which could also be induced by releasing agonists, such as arachidonic acid, adenosine diphosphate, epinephrine, serotonin and fibrinogen, to active the nearby platelets. The standard process of plateletapheresis in the Blood Center was to hold the platelets in still for one hour before stored on a vibrator. The process of holding platelets still for one hour before storage was omitted in some hospitals. It was not clear whether to omit the process has any effect on the quality of platelets. The expressions of P-selectin and vWF receptor, CD42b (Gp Ib£\) on platelets were analyzed by flow cytometry in this study. No significant differences (p= 0.77 and p= 0.62, respectively) were found. Similar results were obtained when functions of platelets were evaluated by agonists. It was concluded that leaving the platelets in room temperature for one hour to recover before keeping it on a vibrator would not enhance the functions of platelets aggregation significantly.

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 192-206).