Thesis (M.Tech.: Clinical Technology)- Dept. of Clinical Technology, Durban University of Technology, 2008. xviii, 160 leaves.

Thesis (M.Tech.: Clinical Technology)- Dept. of Clinical Technology, Durban University of Technology, 2008. xviii, 160 leaves. / Anaemia being one of the most severe complications of end stage renal disease is presently being managed with treatment by recombinant erythropoietin (RHuEPO). Recombinant erythropoietin (RHuEPO) produced by recombinant human DNA technology (RHuEPO) is becoming a standard part of therapy in chronic renal failure. In such patients anaemia is a leading cause of morbidity and mortality. Haemodialysis patients have grown dependent on RHuEPO therapy due their anaemia. The assessment of native erythropoietin levels and the detection of antibody levels in blood in the study were carried out to assist in the clear target of the treatment of anaemia. Haemoglobin levels were monitored over a trial period of six months during the RHuEPO therapy. Ferritin, transferrin saturation (T Sats %) and transferrin levels were monitored accordingly. Monitoring of erythropoietin levels in haemodialysis patients has been one of the first done in our population group in South Africa. Forty haemodialysis patients on RHuEPO therapy and ten haemodialysis patients not on RHuEPO therapy and ten healthy individuals from the Haemodialysis unit at Addington Hospital, Durban, South Africa were recruited to participate in the trial. Blood samples were collected then were centrifuged at 5 degrees celsius. Plasma was isolated, stored and subsequently used in enzyme linked immunosorbent assay (ELISA). Two ELISA were set up, one to measure EPO level and another for anti EPO antibodies. The dilutions of 1:50 were selected to detect the presence of antibodies. These have all been done in duplicates. Optical density of each sample was measured using a microplate reader at 450nm. The haemodialysis patients receiving RHuEPO presented with higher EPO levels as compared to the haemodialysis patients not receiving RHuEPO and the healthy individuals. However, in the study the HB levels were not increased over the trial period with higher RHuEPO doses. Higher doses of RHuEPO therapy showed no clear increase in haemoglobin levels. From a total of forty patients, twelve patients (35%) in the months of August and eight (25%) in the month of November were tested positive for antibodies to RHuEPO. Using statistical analysis, no correlation was observed between the antibody levels and the erythropoietin levels. However, we did not test whether the antibodies found were neutralizing or not. Bioassays for EPO may be used for that purpose.

Yes / Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been contro- versial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that sev- eral of these specifically recognize EPOR by Western blot, immunoprecipi- tation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment. / Study was supported by the FP7-Health European commission EpoCan grant (282551).

El objetivo principal de esta tesis fue el de realizar una validación y caracterización de las posibles variables que pueden afectar la metodología empleada para identificar las isoformas presentes en algunas de las especies de EPO (uEPO, rEPO alfa, rEPO beta y el NESP) tras su identificación mediante la técnica de IEF e inmunodetección

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-14T17:12:41Z No. of bitstreams: 1 luciana-carreiras-norte.pdf: 1395438 bytes, checksum: 31a432f41d7dee6ad0313c1c37736102 (MD5) / Made available in DSpace on 2012-11-14T17:12:41Z (GMT). No. of bitstreams: 1 luciana-carreiras-norte.pdf: 1395438 bytes, checksum: 31a432f41d7dee6ad0313c1c37736102 (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Nesta avaliação, realizaram-se ensaios cromatográficos de purificação de eritropoetina humana recombinante, a partir do sobrenadante de cultura de células de mamíferos da linhagem CHO (células de ovário de hamster chinês), produtoras deste biofármaco. Compararam-se as membranas comerciais de troca iônica Sartobind (MA) Q e D, com o objetivo de avaliar a sua aplicabilidade a processos de purificação de EPO. Ambas as membranas apresentaram-se capazes de adsorver EPO nas condições avaliadas. EPO foi detectada nas amostrasde eluído de ambas as membranas, tendo-se verificado que o imunoensaio dotipo dot-blotfoi aquele que forneceu as informações mais conclusivas. Já nas análises de eletroforese, verificou-se um perfil difuso das bandas do eluído das membranas, o que foi creditado à heterogeneidade glicídica da EPO, como anteriormente demonstrado na literatura. / In the present work, the purification of recombinant human erythropoietin (rhEPO) from CHO (Chinese hamster ovary) cell culture supernatant was investigated using anion-exchange chromatography. Commercial membrane adsorbers (Sartobind D and Q) were compared, with the aim of evaluating their aplicability to EPO purification processes. Both membranes were able to adsorb EPO under the conditions evaluated in this work. EPO was detected in the eluate of both membranes. The immunoassay dot-blot was the most interesting analytical tool, giving the most conclusive results. Electrophoretic analyses showeda diffuse band, probably due to glycan heterogeneity in the EPO molecules, as previously reported in the literature.

Made available in DSpace on 2011-05-04T12:36:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Entre os objetivos da Política Nacional de Medicamentos constam o acesso e o financiamento dos denominados medicamentos de dispensação em caráter excepcional (MDCE). Estes, conhecidos por representarem um gasto governamental relativamente alto, se destinam ao tratamento de doenças em sua maioria, crônicas , que atingem uma pequena parcela da população. Para respaldar sua utilização promovendo adequada alocação dos recursos e o uso racional, o Ministério da Saúde publica protocolos clínicos e diretrizes terapêuticas (PCDT), cujo cumprimento é pré-requisito para o acesso dos pacientes a esses medicamentos pelo programa governamental. A adesão dos prescritores aos protocolos é fator imprescindível ao sucesso desta estratégia. O presente estudo busca verificar, mediante critérios objetivos, o grau de cumprimento pelos prescritores dos procedimentos-chave eleitos a partir do PDCT selecionado. Realizado estudo transversal descritivo com prontuário como fonte de dados e sua revisão como estratégia. Tratamento da anemia em pacientes portadores de Insuficiência Renal Crônica (IRC) com o medicamento eritropoetina humana recombinante (EPO) foi eleita como situação traçadora. Escolhidas três unidades de saúde, sendo critérios o número de pacientes acompanhados e a natureza da organização. No banco de dados da Secretaria de Estado de Saúde do Rio de Janeiro, destinado ao acompanhamento da dispensação dos MDCE identificou-se universo de 411 pacientes com CID principal registrado para autorização de dispensação N18.0 e N18.8, que retiraram o medicamento em algum momento entre janeiro de 2004 a dezembro de 2005. Elegidos procedimentos chave do fluxograma de tratamento do PCDT para a condição traçadora como critérios de verificação. Foram revisados prontuários de 202 pacientes renais crônicos que receberam o medicamento por um período de até dois anos de acompanhamento. Quanto ao grau de adesão ao PDCT, 38,1% foram classificados como adequados, 48,5% como regular, 10,4% como insuficiente e 3% como inadequado. Os critérios mais seguidos foram: critério de inclusão valor do hematócrito ou da hemoglobina, realização inicial de hemograma completo e de contagem de plaquetas e exame de monitorização desses últimos. Os menos seguidos foram: dose inicial e suspensão temporária. No geral, há uma boa adesão, porém essa pode ser melhorada. A análise de possíveis barreiras a adesão faz-se mister. / One of the National Medicines Policy goals is to guarantee the access and funding of the group of drugs called medicines of dispensation in exceptional character (MDEC). These expensive drugs are intended for the treatment of diseases - most of them, chronic - that affect a small portion of the population. In this way, they are know to represent high cost to the public service in Brazil. To support the appropriate allocation of resources and rational use, the Health Ministry develops and public the Clinical protocols and standard treatment guidelines (CPSTG) which is essential for the patient access to the governmental program. To perceive this benefits physicians adherence is necessary. The study aims to evaluate, by objective criteria, the degree of physicians’ adherence to CPSTG. A descriptive cross-sectional study was conducted, using medical records review. CPSTG for the treatment of anemia in chronic renal patients with recombinant human erythropoietin (EPO) was chose to evaluation the adherence. Three units of health were chosen to participate due to number of patients and nature of the organization. The population was picked out from the database of the Secretary of State of Health of Rio de Janeiro, which monitor the distribution of MDEC. The universe was 411 patients with principal ICD registered N18.0 and N18.8, which withdrew the drug at some time between January 2004 to December 2005. To verify the adherence to statements from CPSTG was elected key procedures from CPSTG the treatment of anemia in patients with Chronic Renal Failure (CRF) using EPO flowchart. 202 chronic renal patients medical records were reviewed who received the drug for up to two years of monitoring. The characteristics of patients in the study were similar to the patients described in the 2008 Brazilian Census of Dialysis. Among the comorbidities presented by patients, 76.24% had hypertension and 7.42% ischemic heart disease or congestive heart failure. Regarding adherence, 38.1% were classified as adequate, 48.5% regular, 10.4% insufficient and 3% as inadequate. The most followed criterias were: criterion for inclusion of the value of hematocrit or hemoglobin, achieving initial complete blood platelet count monitoring and examination of these latter. The least followed were: initial dose and temporary. It follows that a good adherence was obtained from the study, but this can be improved. It is indispensable the analysis of possible barriers to adherence.