Hemoglobin is an oxygen binding protein in erythrocytes. Hemoglobin is composed of four polypeptide chains. During the fetal stage the type of hemoglobin called fetal hemoglobin (HbF) dominates. After birth HbF is replaced by adult hemoglobin (HbA). HbF persists in concentrations less than 1%. Elevated concentration of HbF in adults exists in different conditions, Talassemi for example. When the uterus is damaged and the fetus doesn’t feel well its blood can pass the placenta barrier and enter the blood stream of the mother. A venous blood sample from the mother is analyzed to determine the status of the fetus. Laboratory Medicine Västernorrland already has two methods for analyzing HbF, one routine and one on call. The routine method needed to be replaced and the possibility to use flow cytometry was investigated. In this study, results from flow cytometry using Fetal Cell Count™ kit was compared to the results from the presently used methods, Kleihauer-Betke and HPLC. Cord blood was diluted with venous blood from an adult with the same blood group in various concentrations. A number of tests were performed and showed a fairly good correlation between the different methods. However more tests will be necessary to draw any clear conclusion.

Analytical methods used clinically in Sweden for detection and quantification of fetal hemoglobin (HbF) in maternal blood are either the microscopic method Kleinhauer Betkes test (KBT) or high performance liquid chromatography. A more modern alternative to detect and quantify HbF+ erythrocytes is flow cytometry. The aim of this project was therefore to evaluate the commercial kit "Fetal Cell Count kit" using flow cytometry. The kit used two antibodies; one directed against the specific γ-chain of HbF protein and the other directed against the intracellular enzyme carbanhydrase (CA), which is found in all erythrocytes in adults. The resulting data showed good precision, sensitivity and linearity. A reference interval based on male blood donors was determined to <0.1 % HbF+ erythrocytes and <1.3 %F-cells. The kit is well suited to detect and quantify F-cells. It could be used as a important tool to follow-up patients withβ-thalassemia and sickle cell anemia. However the kit was not as useful for detection and quantification of HbF+ erytrocytes in fetomaternal hemorrhage induced by Rhimmunization.

Orientador: Elenice Deffune / Resumo: A Hemorragia feto-materna (HFM) se dá pela transferência do sangue fetal para o compartimento intravascular materno, devido à ruptura na membrana vásculo-sincicial da placenta. A HFM pode ser responsável pela alosensibilização do sistema imune materno, levando a morbidade e mortalidade de gravidez corrente e/ou de futura, assim como também constitui a base da etiopatogenia de várias afecções, como se verifica na doença hemolítica perinatal que expõe a complicações fetais como, hidropsia, danos cerebrais hipóxicos e morte fetal. Detectar e quantificar hemácias fetais ajuda a prevenir as consequências devida a ocorrência da HFM. Entre os testes diagnósticos utilizados na detecção e quantificação têm - se os mais sensíveis o teste quantitativo de Kleihauer e Betke e o de Citometria de fluxo, sendo o primeiro considerado padrão ouro. Tendo em vista a importância do diagnóstico laboratorial da HFM foi desenvolvida a proposta da construção de imunossensor para detecção de amostras de sangue fetal na corrente sanguínea materna. Foi realizada a produção de anticorpo monoclonal com objetivo de produzir anticorpos contra antígenos de superfícies de hemácias fetais humana e também foram realizadas a construção de unidades sensoriais para detecção de sinal biológico de imunoafinidade entre anticorpos que detectam hemácias fetais e a hemoglobina fetal presente nestas células, a fim de se diagnosticar a ocorrência da HFM. Foram obtidos como resultados a produção de anticorpo monoclonal que... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fetomaternal hemorrhage (FMH) occurs through the transfer of fetal blood into the maternal intravascular compartment, due to rupture in the placenta-syncytial membrane. FMH may be responsible for the alosensitization of the maternal immune system, leading to morbidity and mortality of current and / or future pregnancies, as well as being the basis of the etiopathogenesis of various conditions, as in perinatal haemolytic disease that exposes to fetal complications such as hydrops, hypoxic brain damage, and fetal death. Detecting and quantifying fetal erythrocytes helps to prevent the consequences due to the occurrence of FMH. Among the diagnostic tests used in the detection and quantification are the most sensitive the quantitative test of Kleihauer and Betke and the one of Flow cytometry, being the first considered gold standard. Considering the importance of the laboratory diagnosis of FMH, the proposal of the construction of immunosensor for the detection of fetal blood samples in the maternal blood was developed. The production of a monoclonal antibody was carried out with the objective of producing antibodies against antigens on human fetal red blood cells and also the construction of sensorial units for the detection of biological signal of immunoaffinity between antibodies that detect fetal red blood cells and the fetal hemoglobin present in these cells, in order to diagnose the occurrence of FMH. The results obtained are the production of monoclonal antibody that detec... (Complete abstract click electronic access below) / Mestre