Caracterización genética de plaquetas: aporte a la práctica clínica y a la Medicina Transfusional en Córdoba

Abstract

La transfusión de plaquetas (PQT) es necesaria para prevenir y tratar las hemorragias en pacientes trombocitopénicos o con disfunción plaquetaria grave. En Argentina, la mayor parte del suministro de sangre depende de los donantes de reemplazo familiares, ya que las donaciones altruistas y habituales son excepcionales. Este es el principal motivo por el que los donantes de PQT son escasos. En este contexto, la implementación de un programa de captación de donantes de PQT es fundamental a fin de contar con una fuente permanente de PQT para satisfacer las necesidades de la práctica clínica. Por otro lado, aunque la trombocitopenia fetal y neonatal aloinmune (FNAIT), provocada por la aloinmunización materna frente a los antígenos PQT fetales heredados del padre que sean diferentes de los maternos, ha sido reconocida como la principal causa de morbilidad y mortalidad hemorrágica primaria en fetos y recién nacidos, los programas de cribado para detectar embarazos de riesgo aún no se han implementado en ningún país. El objetivo principal de este trabajo fue implementar y validar técnicas moleculares para la genotipificación de antígenos plaquetarios humanos (HPA), a los fines de contar con herramientas moleculares a nivel regional, y constituir el primer registro de donantes de PQT en Argentina. Para ello, se reclutó a donantes de sangre voluntarios y altruistas que asistieron a la Fundación Banco Central de Sangre desde julio de 2016 a julio de 2017, y se caracterizó su genotipo HPA. Además, con el objetivo de resaltar la importancia de diagnosticar la FNAIT en los entornos de atención de la salud fetal y perinatal y demostrar la utilidad de las herramientas moleculares en el diagnóstico precoz de esta entidad clínica, se realizó un estudio de frecuencia de genotipos HPA en 200 mujeres embarazadas de nuestro medio que se encontraban cursando el primer semestre de embarazo y se evaluó la incidencia de casos de FNAIT. Los resultados de la implementación y verificación de las técnicas moleculares se correlacionaron un 100 % con los resultados de referencia esperados. Se reclutó y caracterizó una cohorte de 500 donantes y se creó la primera base de datos con sus genotipos HPA. Asimismo, se identificó a 8/500 donantes (1,6%) HPA-1a negativos y se describen, por primera vez, las variantes alélicas de los sistemas HPA-4b, -6b y -9b en nuestra población. En el estudio de gestantes se detectaron los alelos más frecuentemente asociados a FNAIT 14 en población caucásica. Se identificaron 3/200 (1,5%) mujeres embarazadas HPA-1a- negativas, detectándose incompatibilidad entre el neonato y su madre en dos casos. Solo uno de los neonatos requirió transfusión de PQTS. Además de estos casos, se detectaron otros 3 casos de FNAIT: uno debido a incompatibilidad en el sistema HPA-3, otro en el sistema HPA-15 y un tercer caso en el sistema HPA-1. Las técnicas moleculares implementadas en esta tesis para la genotipificación HPA han demostrado ser herramientas confiables y robustas para aplicarse tanto en la realización de estudios de genotipificación a gran escala, como así también, para estudiar situaciones clínicas concretas, como la FNAIT. Los esfuerzos realizados en este trabajo de tesis para el reclutamiento, caracterización y registro de donantes voluntarios de PQT, culminaron en la obtención de la primera fuente sostenible de PQT para transfusiones compatibles del país. Sorprendentemente, identificamos un mayor porcentaje de donantes HPA-1a-negativos en comparación con los datos previamente obtenidos en la población de Argentina. Este trabajo constituye el primer reporte de genotipos HPA de baja frecuencia en población de mujeres embarazada y de casos de FNAIT confirmados por evidencia molecular en Argentina, indicando la relevancia del diagnóstico precoz de esta entidad clínica. Dado que el retraso en el diagnóstico de FNAIT podría tener consecuencias graves en el feto y en los recién nacidos, las estrategias para abordar la salud materna, fetal y perinatal, así como las políticas de prevención dirigidas a reducir la morbilidad y mortalidad fetal y neonatal, deben enfocarse en implementar programas para identificar embarazos de riesgo y así reducir las complicaciones relacionadas con la trombocitopenia en fetos y recién nacidos.

Platelet (PLT) transfusion is necessary to prevent and treat haemorrhages in thrombocytopenic patients or those with severe platelet dysfunction. In Argentina, blood supplies from voluntary non‐remunerated blood donors remain dependent on family replacement donors since altruistic repetitive donations are exceptional. This is the main reason why PLT donors are very scarce. In this context, the implementation of a PLT donor recruitment program is essential to have a permanent source of PLTs to fulfil clinical needs. On the other hand, even though Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) caused by maternal alloimmunization against fetal PLT antigens inherited from the father but absent in the mother constitutes the main cause of primary haemorrhagic morbidity and mortality in fetuses and newborns, screening programs to detect pregnancies at risk have not been implemented in any country so far. The main objective of this study was to implement and validate molecular techniques for genotyping of human platelet antigens (HPA) in order to have local molecular tools and establish the first PQT-donor registry in Argentina. For this purpose, voluntary and altruistic blood donors who attended at the Fundación Banco Central de Sangre from July 2016 to July 2017 were recruited and their HPA genotype was characterized. In addition, in order to highlight the importance of diagnosing FNAIT in fetal and perinatal health care settings and demonstrate the usefulness of molecular tools in early diagnosis of this clinical entity, we analysed the frequency of HPA genotypes in 200 pregnant women going through the first semester of pregnancy and the incidence of FNAIT. The results of the implementation and verification of the molecular techniques correlated 100% with the expected reference results. A cohort of 500 donors was recruited and characterized, and the first registry of HPA-genotypes was elaborated. Likewise, 8/500 HPA- 1a negative donors (1.6%) were identified and allelic variants of the HPA-4b, -6b and -9b systems were described for the first time in Argentina. In the study of pregnant women, the alleles most frequently associated with FNAIT were detected in the Caucasian population. Three out of 200 (1.5%) HPA-1a-negative pregnant women were identified, detecting incompatibility between neonate and mother in two cases. Only one of the neonates required 16 PLT transfusion. In addition, other 3 cases of FNAIT were detected; one was due to incompatibility in the HPA-3 system, another in HPA-15 system, and the third case in the HPA-1 system. Molecular techniques implemented in this thesis for HPA genotyping have proven to be reliable and robust tools to be applied in large-scale genotyping studies and to study specific clinical situations, such as FNAIT. The efforts made in this thesis work for recruitment, characterization and registration of voluntary PLT donors culminated in the creation of the first sustainable source of PQTS for compatible transfusions in Argentina. Surprisingly, we identified a higher percentage of HPA-1a-negative donors compared to previous data of the Argentine population. This work constitutes the first report of low frequency HPA genotypes in a population of pregnant women and FNAIT cases confirmed by molecular evidence in Argentina, indicating the relevance of its early diagnosis. Given that the delay in detection of FNAIT could have serious consequences on fetuses and newborns, strategies to improve maternal, fetal and perinatal health, as well as prevention policies to reduce fetal and neonatal morbidity and mortality should focus on implementing programs to identify pregnancies at risk and thus, reduce complications related to thrombocytopenia in fetuses and newborns.

Platelet (PLT) transfusion is necessary to prevent and treat haemorrhages in thrombocytopenic patients or those with severe platelet dysfunction. In Argentina, blood supplies from voluntary non‐remunerated blood donors remain dependent on family replacement donors since altruistic repetitive donations are exceptional. This is the main reason why PLT donors are very scarce. In this context, the implementation of a PLT donor recruitment program is essential to have a permanent source of PLTs to fulfil clinical needs. On the other hand, even though Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) caused by maternal alloimmunization against fetal PLT antigens inherited from the father but absent in the mother constitutes the main cause of primary haemorrhagic morbidity and mortality in fetuses and newborns, screening programs to detect pregnancies at risk have not been implemented in any country so far. The main objective of this study was to implement and validate molecular techniques for genotyping of human platelet antigens (HPA) in order to have local molecular tools and establish the first PQT-donor registry in Argentina. For this purpose, voluntary and altruistic blood donors who attended at the Fundación Banco Central de Sangre from July 2016 to July 2017 were recruited and their HPA genotype was characterized. In addition, in order to highlight the importance of diagnosing FNAIT in fetal and perinatal health care settings and demonstrate the usefulness of molecular tools in early diagnosis of this clinical entity, we analysed the frequency of HPA genotypes in 200 pregnant women going through the first semester of pregnancy and the incidence of FNAIT. The results of the implementation and verification of the molecular techniques correlated 100% with the expected reference results. A cohort of 500 donors was recruited and characterized, and the first registry of HPA-genotypes was elaborated. Likewise, 8/500 HPA- 1a negative donors (1.6%) were identified and allelic variants of the HPA-4b, -6b and -9b systems were described for the first time in Argentina. In the study of pregnant women, the alleles most frequently associated with FNAIT were detected in the Caucasian population. Three out of 200 (1.5%) HPA-1a-negative pregnant women were identified, detecting incompatibility between neonate and mother in two cases. Only one of the neonates required 16 PLT transfusion. In addition, other 3 cases of FNAIT were detected; one was due to incompatibility in the HPA-3 system, another in HPA-15 system, and the third case in the HPA-1 system. Molecular techniques implemented in this thesis for HPA genotyping have proven to be reliable and robust tools to be applied in large-scale genotyping studies and to study specific clinical situations, such as FNAIT. The efforts made in this thesis work for recruitment, characterization and registration of voluntary PLT donors culminated in the creation of the first sustainable source of PQTS for compatible transfusions in Argentina. Surprisingly, we identified a higher percentage of HPA-1a-negative donors compared to previous data of the Argentine population. This work constitutes the first report of low frequency HPA genotypes in a population of pregnant women and FNAIT cases confirmed by molecular evidence in Argentina, indicating the relevance of its early diagnosis. Given that the delay in detection of FNAIT could have serious consequences on fetuses and newborns, strategies to improve maternal, fetal and perinatal health, as well as prevention policies to reduce fetal and neonatal morbidity and mortality should focus on implementing programs to identify pregnancies at risk and thus, reduce complications related to thrombocytopenia in fetuses and newborns.