RESUMEN
BACKGROUND: Transfusion-induced alloimmunization has severe clinical consequences including haemolytic transfusion reactions, impaired transfused RBCs longevity and greater difficulty in finding compatible blood. Molecular analysis of genomic DNA now permits prediction of blood group phenotypes based on identification of single nucleotide polymorphisms. Implementation of molecular technologies in donor centres would be helpful in finding RBC units for special patient populations, but DNA extraction remains an obstacle to donor genotyping. MATERIALS AND METHODS: We propose a simple method compatible with high throughput that allows blood group genotyping using a multiplex commercial kit without the need for DNA extraction. The principle relies on pre-PCR treatment of whole blood using heating/cooling procedure in association with a recombinant hotstart polymerase. RESULTS: In a prospective analysis, we yielded 5628 alleles identification and designated 63 donors with rare blood, that is either negative for a high-frequency antigen or with a rare combination of common antigens. CONCLUSION: The procedure was optimized for simplicity of use in genotyping platform and would allow not only to supply antigen-matched products to recipients but also to find rare phenotypes. This methodology could also be useful for establishing a donor repository for human platelet antigens (HPA)-matched platelets since the same issues are involved for patients with neonatal alloimmune thrombocytopenia or post-transfusion purpura.
Asunto(s)
Donantes de Sangre , Técnicas de Genotipaje/métodos , Polimorfismo de Nucleótido Simple , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodosRESUMEN
TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors).
Asunto(s)
Antígenos de Neoplasias/inmunología , Autoanticuerpos/análisis , Inmunoensayo/métodos , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Antígenos de Neoplasias/sangre , Autoanticuerpos/inmunología , Humanos , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodosRESUMEN
N-Acetyl oligonucleotides and their prodrugs were synthesized on photolabile solid support. Tm studies showed a decrease of hydridization for N-acetyl A and G and an increase for N-acetyl C. In cells extract, acetyl groups were hydrolysed.
Asunto(s)
Oligonucleótidos/síntesis química , Profármacos/síntesis química , Acetilación , Secuencia de Bases , Hidrólisis , Indicadores y Reactivos , Conformación de Ácido Nucleico , Nucleósidos/química , Oligonucleótidos/química , Profármacos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.
Asunto(s)
Oligonucleótidos/farmacocinética , Profármacos/farmacocinética , Nucleótidos de Pirimidina/farmacocinética , Timidina/análogos & derivados , Biotransformación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
MALDI-TOF mass spectrometry was used to analyze oligonucleotides still anchored to long-chain alkylamine controlled-pore glass (LCAA-CPG) through a photolabile linker. This technique is useful to follow supported chemical reactions in real time and monitor by-products formation.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Oligonucleótidos Antisentido/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Vidrio/química , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , FotoquímicaRESUMEN
DNA microarrays are a powerful experimental tool for the detection of specific genomic sequences and are invaluable to a broad array of applications: clinical diagnosis, personalized medicine, drug research and development, gene therapy, food control technologies, and environmental sciences. Alloimmunization to human platelet antigens (HPAs) is commonly responsible for neonatal alloimmune thrombocytopenia, post-transfusional purpura and platelet transfusion refractoriness. Using DNA microarrays, we developed a diagnosis to type the biallelic HPA-1 platelet group. The region for the human genomic DNA sequence that contains the polymorphism responsible for HPA-1 alleles was amplified by polymerase chain reaction (PCR). The expected DNA fragments were hybridized on DNA microarrays, and the data were analyzed using specially developed software. Our initial results show that the two HPA-1 antigens polymorphisms containing a single base difference were detected using DNA microarrays.
Asunto(s)
Antígenos de Plaqueta Humana/sangre , Antígenos de Plaqueta Humana/genética , Análisis Mutacional de ADN/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Reacción en Cadena de la Polimerasa/instrumentación , Polimorfismo de Nucleótido Simple/genética , Análisis Mutacional de ADN/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Estudios de Factibilidad , Genotipo , Humanos , Hibridación Fluorescente in Situ/métodos , Integrina beta3 , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. We found a good fit between the calculated kinetics curves and the experimental points. The oligonucleotide prodrug was fully metabolized to yield the dodecathymidine phosphorothioate likely through a carboxyesterase mediated mechanism.