Alloimmunisation becomes a problem when serological discrepancies occur in matching antigens between donors and patients for blood transfusion. The rate of alloimmunisation has been increased especially in multiply transfused patients. Blood group genotyping (BGG) is a DNA-based assay that aids in reducing this situation. Currently, many platforms of BGG have become available, in which every technique has its own advantages and disadvantages. All these platforms lack the ability to identify novel alleles that might have an unknown clinical significance. The advent of next-generation sequencing (NGS) offers identification of the unprecedented alleles due to its basis of sequence-based typing. Moreover, it provides an extreme high-throughput which may be able to screen many donors and patients in a single run. In this project, two approaches have been developed in generating sequencing libraries followed by sequencing on the Ion Torrent Personal Genome MachineTM platform (Ion PGMTM). The first approach was amplicon-based target selection using Ion AmpliseqTM Custom Panel, designated as Human Erythrocyte Antigens and Human Platelet Antigens Panel (HEA and HPA Panel). This panel assay screens 11 blood group systems, as well as 16 human platelet antigens. The outcome was extraordinary, in particular four novel alleles had been identified out of 28 samples, one in the RHCE gene 208C > T (Arg70Trp) in exon 2 and three in the KEL gene. The first SNP was 331G > A (Ala111Thr) in exon 4. The second SNP was 1907C > T (Ala636Val) in exon 17 and the third SNP was 2165T > C (Leu722Pro) in exon 19. However, some issues occurred regarding co-amplification of homologous genes. The second approach was a long-range polymerase chain reaction (LR-PCR) based approach. This method provided a high resolution assay by amplification of entire genes, including the non-coding area, of the Kell and Rh blood group systems. The Kell blood group was initially utilised as a model in order to apply the same approach on the Rh system. Most alleles encoding the antigens of the Kell blood group, especially the high prevalence ones, were identified. The Rh LR-PCR approach was carried out by amplification of the RHD and RHCE genes with seven amplicons. For five RhD-positive samples no mutations were observed within the coding areas. On the other hand, five serotyped weak D samples were genotyped as; two weak D type 1, two weak D type 2 and one DAR3.1 weak partial D 4.0 (RHD*DAR3.01). Regarding the RHCE, the following antigens (C, c, E, c) were predicted properly from the sequencing data. Moreover, the RHCE*ceVS.02 was identified. 64 and 39 intronic SNPs were identified in RHD and RHCE genes, respectively. The intronic SNPs assisted the genotyping process by identifying the haplotype of interest. Interestingly, the novel allele identified in the RHCE gene by the HEA and HPA Panel was confirmed to belong to the RHCE gene by the LR-PCR approach, indicating the panel misaligned it to the RHD gene. In conclusion, NGS paves the way to be an alternative substitution to the previous molecular techniques. It would supplant the conventional serology for typing blood for transfusion.

Les travaux présentés dans cette thèse s’intéressent à l’immobilisation de biomolécules pour le développement d’outils d’analyse multiparamétrique pour la caractérisation d’échantillons biologiques et le diagnostic, sur un support de type biopuce couplé à une détection colorimétrique.Un premier axe de recherche concerne le développement de tests d’hybridation d’acides nucléiques et d’immunotests à haut-débit automatisés sur plaque de filtration. Cette méthode a permis la mise au point d’un test de génotypage automatisé pour le dépistage transfusionnel haut-débit (génotypage érythrocytaire étendu) en collaboration avec l’Établissement Français du Sang Rhône-Alpes (EFS-RA). Il permet d’analyser 96 échantillons en quatre heures, et de caractériser six génotypes par échantillon. Cet outil a fait l’objet d’une validation sur un panel de 293 donneurs.La seconde partie des travaux présentés s’intéresse au développement d’un procédé d’immobilisation d’oligonucléotides sur un polymère particulier (PolyshrinkTM) pour l’élaboration d’un système d’analyse miniaturisé. Plusieurs stratégies d’activation ont été envisagées et ont abouti à la mise au point d’une technique d’immobilisation d’oligonucleotides in situ dans des plots d’hydrogel. La méthode de fabrication permet d’obtenir une matrice de plots d’hydrogel de 60 µm de diamètre et d’une hauteur de 6 µm en moyenne. En outre, il a été démontré que les oligonucléotides immobilisés dans les plots pouvaient détecter de façon quantitative et sélective les cibles complémentaires présentes dans l’échantillon analysé en utilisant une détection par colorimétrie ou par chimiluminescence. / The work reported in this thesis focuses on biomolecules immobilization for the development of multiparametric analysis tools on a biochip coupled with a colorimetric detection, applied to the characterization of biological samples and to diagnosis.The first concern was the development of high-throughput automated hybridization tests and immunotests on a filtration plate. This method led to the elaboration of an automated platform for extended blood group genotyping in collaboration with the Etablissement Français du Sang Rhône-Alpes (EFS-RA). It enables to analyze 96 samples in four hours and to characterize six genotypes per sample. Its analytical performances were validated on a panel of 293 blood donors.The second part of this work aimed to elaborate a new strategy for oligonucleotide immobilization on an innovative polymer (PolyshrinkTM) for the development of miniaturized analysis systems. Several approaches were evaluated and led to an in-situ immobilization of oligonucleotides in hydrogel dots technique. This method leads to 6 µm hydrogel dots with a diameter of 60 µm. Moreover it was demonstrated that such immobilized oligonucleotides were able to detect targets specifically and quantitatively using either a chemiluminescent or a colorimetric detection.

Les travaux présentés dans cette thèse font suite à celle de Melle LE GOFF. Ils se concentrent sur la technologie HIFI brevetée et développée pendant ses travaux. Une première partie du travail présenté dans ce manuscrit concerne le test HIFI Blood 96™ et plus particulièrement les améliorations et les évolutions apportées au test afin d'en faire un véritable outil de génotypage, multiparamétrique et haut-débit pouvant être installé dans les banques de sang dans le but de constituer des inventaires de sang génotypé de façon étendue, participant ainsi à améliorer la sécurité transfusionnelle. Il permet de caractériser 96 échantillons sur 15 polymorphismes (divisés en deux panels) associés aux groupes sanguins en approximativement 4h30. Cette plateforme a fait l'objet d'une étude de validation à moyenne échelle sur 583 donneurs pour le panel 1 et 190 donneurs pour le panel 2. La deuxième partie des travaux décrit l'adaptation de la technologie HIFI appliquée au diagnostic des pathologies respiratoires, avec le développement d'une autre plateforme, ReSynPlex, en partenariat avec 3 équipes de recherche de Grenoble / The work reported in this thesis follows the one undertaken by Ms LE GOFF. It is focused on HIFI technology, which is patented and developed during her thesis. The first part of this work concerns the HIFI Blood 96™ test, and particularly the improvements and developments adduced to the test to make it a real diagnostic tool, multiparametric and high-throughput which can be implemented in blood banks in order to constitute negative antigen inventories, thus contributing to improve blood safety. It allows to characterize 96 samples on 15 polymorphisms (divided in two panels) associated to blood group systems in approximately 4.5 hours. A mesoscale validation study has been conducted on 583 samples for panel 1 and 190 samples for panel 2. The second part of this work describes the adaptation of HIFI technology applied to diagnosis of respiratory tract infections, with the development of another platform, ReSynPlex, in partnership with 3 research teams in Grenoble