Complex systems are those which exhibit one or more of the following inter-related behaviours: 1. Nonlinear behaviour: the component parts do not act in linear ways, that is the superposition of the actions of the parts is not the output of the system. 2. Emergent behaviour: the output of the system may be inexpressible in terms of the rules or equations of the component parts. 3. Self-organisation: order appears from the chaotic interactions of individuals and the rules they obey. 4. Layers of description: in which a rule may apply at some higher levels of description but not at lower layers. 5. Adaptation: in which the environment becomes encoded in the rules governing the structure and/or behaviour of the parts (in this case strictly agents) that undergo selection in which those that are by some measure better become more numerous than those that are not as “fit”. A single cell is a complex system: we cannot explain all of its behaviour as simply the sum of its parts. Similarly, DNA structures, social networks, cancers, the brain, and living beings are intricate complex systems. This thesis tackles all of these topics from a complex systems approach. I have skirted some of the philosophical issues of complex systems and mainly focussed on appropriate tools to analyse these systems, addressing important questions such as: • What is the best way to extract information from DNA? • How can we model and analyse mutations in DNA? • Can we determine the likely spread of both viruses and ideas in social networks? • How can we model the growth of cancer? • How can we model and analyse interactions between genes in such living systems as the fruit fly, cancers, and humans? • Can complex systems techniques give us some insight into the human brain? / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290759 / Thesis (Ph.D.)-- School of Electrical and Electronic Engineering, 2007

Complex systems are those which exhibit one or more of the following inter-related behaviours: 1. Nonlinear behaviour: the component parts do not act in linear ways, that is the superposition of the actions of the parts is not the output of the system. 2. Emergent behaviour: the output of the system may be inexpressible in terms of the rules or equations of the component parts. 3. Self-organisation: order appears from the chaotic interactions of individuals and the rules they obey. 4. Layers of description: in which a rule may apply at some higher levels of description but not at lower layers. 5. Adaptation: in which the environment becomes encoded in the rules governing the structure and/or behaviour of the parts (in this case strictly agents) that undergo selection in which those that are by some measure better become more numerous than those that are not as “fit”. A single cell is a complex system: we cannot explain all of its behaviour as simply the sum of its parts. Similarly, DNA structures, social networks, cancers, the brain, and living beings are intricate complex systems. This thesis tackles all of these topics from a complex systems approach. I have skirted some of the philosophical issues of complex systems and mainly focussed on appropriate tools to analyse these systems, addressing important questions such as: • What is the best way to extract information from DNA? • How can we model and analyse mutations in DNA? • Can we determine the likely spread of both viruses and ideas in social networks? • How can we model the growth of cancer? • How can we model and analyse interactions between genes in such living systems as the fruit fly, cancers, and humans? • Can complex systems techniques give us some insight into the human brain? / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290759 / Thesis (Ph.D.)-- School of Electrical and Electronic Engineering, 2007

Complex systems are those which exhibit one or more of the following inter-related behaviours: 1. Nonlinear behaviour: the component parts do not act in linear ways, that is the superposition of the actions of the parts is not the output of the system. 2. Emergent behaviour: the output of the system may be inexpressible in terms of the rules or equations of the component parts. 3. Self-organisation: order appears from the chaotic interactions of individuals and the rules they obey. 4. Layers of description: in which a rule may apply at some higher levels of description but not at lower layers. 5. Adaptation: in which the environment becomes encoded in the rules governing the structure and/or behaviour of the parts (in this case strictly agents) that undergo selection in which those that are by some measure better become more numerous than those that are not as “fit”. A single cell is a complex system: we cannot explain all of its behaviour as simply the sum of its parts. Similarly, DNA structures, social networks, cancers, the brain, and living beings are intricate complex systems. This thesis tackles all of these topics from a complex systems approach. I have skirted some of the philosophical issues of complex systems and mainly focussed on appropriate tools to analyse these systems, addressing important questions such as: • What is the best way to extract information from DNA? • How can we model and analyse mutations in DNA? • Can we determine the likely spread of both viruses and ideas in social networks? • How can we model the growth of cancer? • How can we model and analyse interactions between genes in such living systems as the fruit fly, cancers, and humans? • Can complex systems techniques give us some insight into the human brain? / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290759 / Thesis (Ph.D.)-- School of Electrical and Electronic Engineering, 2007

Complex systems are those which exhibit one or more of the following inter-related behaviours: 1. Nonlinear behaviour: the component parts do not act in linear ways, that is the superposition of the actions of the parts is not the output of the system. 2. Emergent behaviour: the output of the system may be inexpressible in terms of the rules or equations of the component parts. 3. Self-organisation: order appears from the chaotic interactions of individuals and the rules they obey. 4. Layers of description: in which a rule may apply at some higher levels of description but not at lower layers. 5. Adaptation: in which the environment becomes encoded in the rules governing the structure and/or behaviour of the parts (in this case strictly agents) that undergo selection in which those that are by some measure better become more numerous than those that are not as “fit”. A single cell is a complex system: we cannot explain all of its behaviour as simply the sum of its parts. Similarly, DNA structures, social networks, cancers, the brain, and living beings are intricate complex systems. This thesis tackles all of these topics from a complex systems approach. I have skirted some of the philosophical issues of complex systems and mainly focussed on appropriate tools to analyse these systems, addressing important questions such as: • What is the best way to extract information from DNA? • How can we model and analyse mutations in DNA? • Can we determine the likely spread of both viruses and ideas in social networks? • How can we model the growth of cancer? • How can we model and analyse interactions between genes in such living systems as the fruit fly, cancers, and humans? • Can complex systems techniques give us some insight into the human brain? / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290759 / Thesis (Ph.D.)-- School of Electrical and Electronic Engineering, 2007

Complex systems are those which exhibit one or more of the following inter-related behaviours: 1. Nonlinear behaviour: the component parts do not act in linear ways, that is the superposition of the actions of the parts is not the output of the system. 2. Emergent behaviour: the output of the system may be inexpressible in terms of the rules or equations of the component parts. 3. Self-organisation: order appears from the chaotic interactions of individuals and the rules they obey. 4. Layers of description: in which a rule may apply at some higher levels of description but not at lower layers. 5. Adaptation: in which the environment becomes encoded in the rules governing the structure and/or behaviour of the parts (in this case strictly agents) that undergo selection in which those that are by some measure better become more numerous than those that are not as “fit”. A single cell is a complex system: we cannot explain all of its behaviour as simply the sum of its parts. Similarly, DNA structures, social networks, cancers, the brain, and living beings are intricate complex systems. This thesis tackles all of these topics from a complex systems approach. I have skirted some of the philosophical issues of complex systems and mainly focussed on appropriate tools to analyse these systems, addressing important questions such as: • What is the best way to extract information from DNA? • How can we model and analyse mutations in DNA? • Can we determine the likely spread of both viruses and ideas in social networks? • How can we model the growth of cancer? • How can we model and analyse interactions between genes in such living systems as the fruit fly, cancers, and humans? • Can complex systems techniques give us some insight into the human brain? / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1290759 / Thesis (Ph.D.)-- School of Electrical and Electronic Engineering, 2007

Pesquisa que analisa questões técnicas, éticas e jurídicas relacionadas com o uso informatizado de dados genéticos na persecução criminal que suscitam a elaboração de regulamentações técnicas legais para o desejável equilíbrio entre garantias e direitos individuais e os de interesse coletivo relacionados com segurança pública. A automatização de dados de caráter pessoal tem trazido preocupações aos governantes de diversos países, levando-os a adotar medidas legais sobre o tema. Os avanços da genética e da informática possibilitaram a criação de bancos de dados de DNA voltados à identificação criminal que, por serem eficazes no combate à criminalidade, tornaram-se aspiração para muitos Estados, como é o caso brasileiro. Sem que se olvidem ou que se exaltem as potenciais benesses sociais, na criação de bancos de dados de DNA devem ser valorados outros aspectos que também permeiam a questão e que podem ferir suscetibilidades, direitos e garantias individuais. Dentre estes se destacam os de vieses técnicos e éticos concernentes à possibilidade de uso indevido de informações genômicas contidas na molécula de DNA, além dos aspectos jurídicos relacionados com garantias e direitos individuais e coletivos. A presente investigação estuda elementos técnicos relacionados com a análise de polimorfismos do DNA que autorizam seu uso como método de identificação humana, amplamente empregado na atualidade pela Medicina Legal e pela Criminalística para determinação de parentesco biológico e elucidação de crimes. São analisadas características estruturais e funcionais de bancos de dados genéticos e as principais questões técnicas, éticas e legais relacionadas com a coleta de materiais biológicos, com os cuidados de preservação e garantia de autenticidade, com a qualidade dos serviços laboratoriais usados para obtenção de perfis genéticos e com o valor probante da prova pericial formada. É avaliada a importância dos bancos de dados criminais de DNA para a investigação policial e para a persecução judicial, sopesando-se os interesses da segurança pública e os de preservação da privacidade dos sujeitos afetados. São também comparativamente examinados os principais dos bancos de dados de identificação genética criminal já em funcionamento no mundo e suas características atinentes aos sujeitos e tipos de delitos que neles são incluídos; o tempo de permanência dos dados; seu gerenciamento e o armazenamento de vestígios e de amostras-referência. São ainda apontados os parâmetros técnicos e legais mínimos a serem considerados para a criação e o estabelecimento de um banco de dados desse gênero. É estudada pormenorizadamente a proposta feita pela SENASP/MJ para a implantação de um banco nacional de perfis de DNA criminal no Brasil, aos moldes do consagrado CODIS norte-americano. Os resultados desta pesquisa sugerem que, ao se considerar que os direitos e garantias individuais não têm caráter absoluto frente a interesses públicos legítimos, a criação de um banco de dados criminais de DNA no Brasil é viável através da edição de uma lei estabelecedora dos limites das medidas restritivas das prerrogativas individuais e que regule minuciosamente seu funcionamento. / Research that analyses juridical, ethical and technical questions related to the digital use of genetic data at criminal prosecutions that engender the elaboration of legal and technical regulation to the desirable balance among individual rights and guarantees and those of collective interests related to public security. Personal data automation has brought concerns to several countries governments, leading them to adopt legal measures about the theme. Enhancements at genetics and information technology areas had made possible the creation of DNA databases related to criminal identification that, due to their efficacy at criminal combat, have become an aspiration to many States, such as Brazil. Without neither forgetting nor magnifying its potential social benefits, at DNAs database creation other aspects, that are also involved and that could hurt individual susceptibilities, rights and guarantees, should be valued. Among these, it should be emphasized those of technical and ethical concerns related to the improper use of DNAs genomic information, besides juridical aspects related to individual and collective rights and guarantees. The present investigation studies technical elements related to DNA polymorphisms analysis that allow its use as an Human Identification Method, largely employed nowadays at Criminalistics and Forensic Medicine to determine biological kinships and crime scene elucidations. We analyze genetic databases functional and structural characteristics, and the main legal, ethical and technical questions related to biological samples collection, to their preservation and authenticity guarantee, to the involved laboratories quality, and to the probative value of the formed forensic proof. Its also evaluated DNA criminal databases importance to police investigation and judicial prosecution, considering both the public security interest and the privacy preservation of the affected individuals. The main genetic identification databases already working around the world are also comparatively analyzed, as well as their characteristics, such as: what kinds of individuals and faults are included at database; for how long this data stays at the bank; how it is managed and how the storage of evidences and reference samples is done. We also point the minimum legal and technical parameters that should be considered to the creation and establishment of such a database. Its studied in details the SENASP/MJ proposal to implement a national bank of criminal DNA profiles in Brazil, similar to the American CODIS. The results of our study suggest that, considering that individual rights and guarantees dont have absolute character front legitimate public interests, the creation of a criminal DNA database in Brazil is practicable through the edition of some law that would establish the limits to individual prerogatives and also minutely regulate its operation.

Criminology is a science dealing with the protection of citizens and state from infringement. Criminology uses mostly biological or genetic methods for crime detection. Forensic traces which are collected by forensic experts on the scene are the key items of those methods. Forensic genetics is among the most important forensic subdisciplines. Forensic genetics uses DNA analysis for identification. The main aims of this study are description and importance of biological, anthropological and genetic methods in criminology, different ways of forensic identification, division and collection of forensic traces, characterization and course of forensic DNA analysis and DNA profiling. Key words Criminology, forensic methods, forensic identification, forensic trace, forensic biology, anthropology and genetics, information systems, forensic DNA analysis, DNA profile.

Neste estudo, foi avaliada a relação entre polimorfismos dos genes da leptina (LEP) e receptores da leptina (LEPR) e parâmetros antropométricos, leptinemia glicemia e lipídeos séricos, em indivíduos da população brasileira. Foram incluídos 238 indivíduos com idade entre 30 e 80 anos. Foram medidos o índice de massa corporal (IMC), a cintura abdominal (CA) e a razão cintura quadril (RCQ). Amostras de sangue periférico foram obtidas para análise do perfil bioquímico e extração de DNA. Os polimorfismos de nuleotideo único (SNPs) LEP G-2548A e LEPR Lys109Arg, Gln223Arg e Lys656Asn foram detectados por PCR-RFLP. Os SNPs LEPR Lys109Arg e Gln223Arg foram associados com obesidade e com IMC e CA aumentados (p<0.05). Estes polimorfismos também foram associados com leptina e glicose elevada (p<0,05). O perfil lipídico sérico foi influenciado pelo polimorfismo LEPR Lys109Arg (p<0.05). A relação entre os SNPs LEPR Lys109Arg e Gln223Arg e o perfil lipídico foi modificada pelo gênero. Os haplótipos LEP G-2548/ LEPR Lys109Arg foram relacionados com diferenças no IMC de obesos. Os haplotipos LEPR Lys109Arg/Gln223Arg foram associados com diferenças na CA e glicemia e lipídeos séricos. Em conclusão, os polimorfismos LEPR Lys109Arg e Gln223Arg estão associados com obesidade e alterações de leptina, glicose e lipídeos circulantes de forma dependente do gênero. / We have assessed the relationship between polymorphisms of the leptin (LEP) and the leptin receptor (LEPR) genes and anthropometric parameters, plasma leptin and glucose and serum lipids in individuals of the Brazilian population. We included 238 individuais with 30 to 80 years. Body mass index (BMI), abdominal circumference (AC) and waist-to-hip ratio (WHR) were measured. Peripheral blood samples were collected for analysis of the biochemical profile and DNA extraction. The single nucleotide polymorphisms (SNP) LEP G-2548A and LEPR Lys109Arg, Gln223Arg and Lys656Asn were detected by PCR-RFLP. The SNPs LEPR Lys109Arg and Gln223Arg were associated with obesity and with increased BMI and AC (p <0.05). These polymorphisms were also associated with increase leptin and glucose (p<0,05). The serum lipid profile was influenced by the LEPR Lys 1 09Arg (p<0.05). The relationship between the SNPs LEPR Lys 1 09Arg and Gln223Arg and the lipid profile was modified by gender. The haplotypes LEP G-2548A1 LEPR Lys109Arg were related with differences on BMI in obese group. The haplotypes LEPR Lys109Arg/Gln223Arg were associated with differences on AC, glucose and serum lipids. In conclusion, the LEPR Lys109Arg and Gln223Arg polymorphisms are associated with obesity and alterations in blood leptin, glucose and lipids in a gender-dependent manner.

A síndrome de Noonan (SN) é uma doença de herança autossômica, relativamente frequente na população e que apresenta heterogeneidade genética. Caracteriza-se por dismorfismos faciais, baixa estatura, pescoço curto/alado, alterações cardíacas, deformidades esternais e criptorquia. A SN apresenta sobreposição dos achados clínicos com outras síndromes mais raras, denominadas síndromes Noonan-like (SNL): síndrome cardio-facio-cutânea (CFC), síndrome de Costello (SC), neurofibromatose-síndrome de Noonan (NFSN), síndrome de Noonan com manchas lentiginosas/síndrome de LEOPARD (SL), síndrome de Noonan-like com perda de cabelos anágenos (SNL-PCA) e síndrome de Noonan-like com leucemia mielomonocítica juvenil (SNL-LMMJ). As SN e SNL decorrem de mutações em genes pertencentes à via de sinalização RAS/MAPK alguns dos quais são protooncogenes, o que tem despertado o interesse na caracterização do risco de desenvolvimento de neoplasias nessas síndromes. Os objetivos deste estudo visam o sequencimento conjunto dos genes PTPN11, SOS1, RAF1, KRAS, SHOC2, BRAF e HRAS em pacientes com diagnóstico clínico das SN e SNL a fim de: determinar a frequência de mutação; estabelecer uma correlação genótipo-fenótipo; estabelecer um fluxograma para o estudo molecular a partir dos hotspots; e avaliar se a variabilidade fenotípica apresentada nos pacientes com SN pode ser explicada pela presença de mutações em mais de um gene da via RAS/MAPK. Foram avaliados 194 probandos - 152 com SN e 42 com SNL (19 CFC, 15 NFNS, 4 CS e 4 LS). Mutações foram identificadas em 99 pacientes 80 com SN (53%); 19 com SNL. Apenas um paciente com SN apresentou mutação em dois genes da via RAS/MAPK (PTPN11 e SOS1). O estudo molecular na SN mostrou, assim como na literatura, um maior envolvimento do gene PTPN11 (34%), seguido dos genes SOS1 (12%) e RAF1 (7%). A comparação dos achados clínicos, levando em consideração as alterações gênicas, também confirma as correlações já descritas na literatura; entre elas observamos que pacientes com SN e mutação no gene: PTPN11, apresentaram maior frequência de estenose pulmonar valvar (EPV) e baixa estatura; SOS1, apresentaram maior frequência de EPV; RAF1, apresentaram maior frequência de miocardiopatia hipertrófica, e menor frequência de EPV e déficit intelectual; SHOC2, apresentaram anormalidades de cabelo. Na nossa casuística também foram observados alguns achados raros: craniosinostose, tumor expansivo em fossa posterior e a primeira descrição de um tumor sólido (schwanomatose) em um paciente com mutação no gene KRAS. A partir deste estudo foi possível estabelecer um fluxograma para investigação molecular devido à correlação genótipo-fenótipo, que embora não seja totalmente precisa, explica parte da grande variabilidade clínica observada em especial quanto ao tipo de cardiopatia. A presença de mutações em diferentes genes da via RAS/MAPK não é frequente, além de não agravar o fenótipo e não explicar a variabilidade fenotípica observada. Embora um grande avanço no conhecimento sobre SN e SNL tenha sido alcançado, vários aspectos precisam ser melhor elucidados, como: caracterização precisa da propensão ao desenvolvimento de neoplasias, estudos que permitam avaliar as consequências biológicas das mutações, identificação de outros genes responsáveis, assim como outros fatores genéticos e/ou ambientais que possam explicar a variabilidade clínica inter e intrafamilial / Noonan syndrome (NS) is a relatively common, autosomal dominant disease that presents a marked genetic heterogeneity. It is characterized by facial dysmorphisms, short stature, webbed/short neck, cardiac abnormalities, esternal anomalies and cryptorchidism. NS shows clinical overlap of some of its findings with other rarer syndromes, known as Noonan-like syndromes (NLS): cardio-facio-cutaneous syndrome (CFC), Costello syndrome (CS), neurofibromatosis-Noonan syndrome (NFNS), Noonan syndrome with lentiginous stains/LEOPARD syndrome (LS), Noonan-like syndrome with loose anagen hair (NLS-LAH) and Noonan-like syndrome with juvenile myelomonocytic leukemia (NLS-JMML). NS and NLS are related to mutations in genes belonging of RAS-MAPK signaling pathway. Some of these genes are classified as proto-oncogenes. This fact also arouses the interest in the characterization of the risk for cancer development in this group of patients. The objectives of this study are to sequence the genes associated with NS and NLS (PTPN11, SOS1, RAF1, KRAS, SHOC2, HRAS and BRAF) in patients that fulfilled clinical diagnostic criteria for NS or NLS to: determine the frequency of the mutations; establish a genotype-phenotype correlation; estabilish a flowchart for molecular study from the hotspots; and evaluate when the phenotypic variability presented in NS patients can be explained by the presence of mutations in more than one gene of the RAS/MAPK pathway. This study evaluated 194 probands 152 with NS e 42 with NLS (19 CFC, 15 NFSN, 4 SC e 4 SL). Mutations were identified in 99 patients 80 with NS (53%), 19 with NLS. Only one patient presented mutation in two different genes of the RAS/MAPK pathway (PTPN11 and SOS1). The molecular analysis showed a predominance of mutations in the PTPN11 gene (34%), followed by the SOS1 (12%) and RAF1 (7%) genes in patients with NS, in accordance with the literature. Patients with NS and mutation in the: PTPN11 gene, presented a higher frequency of valvular pulmonary stenosis (VPS) and short stature; SOS1 gene, showed a higher frequency of VPS; RAF1 gene, showed a higher frequency of hypertrophic cardiomyopathy and lower frequency of VPS and intellectual deficit; and SHOC2 gene, showed more hair abnormalities. This genotype-phenotype correlations is also concordant with the literature. In our study, we also observed some rare finds in some patients: craniosynostosis, expansive tumor in the posterior fossa and the first description of a solid tumor (schwanomatose) in a patient with NS and KRAS gene mutation. Based on this genotype-phenotype correlation, we have proposed a flowchart for molecular investigation. The presence of mutation in more them one gene of the RAS/MAPK pathway was not frequent; additionally, it did not aggravate the phenotype and could not explain the phenotypic variability observed. Although a great advance in the knowledge about SN and SNL has been achieved, several aspects remain to be clarified, as the exact risk for cancer development, functional studies to assess the biological consequences of the mutations and the identification of other genes, as well as other genetic and/or environmental factors that influence the interand intrafamilial clinical variability

INTRODUÇÃO: A determinação do genótipo RHD fetal no plasma materno é um teste de diagnóstico pré-natal não invasivo oferecido a gestantes RhD negativo que apresentam potencial de sensibilização e/ou Doença Hemolítica Perinatal. Atualmente, este exame é realizado de rotina em diversos países, mas não no Brasil. A Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) oferece atendimento terciário a gestantes RhD negativo, com monitorização dos títulos de anticorpos irregulares, administração da imunoglobulina anti-D e/ou terapêutica fetal, quando necessários. OBJETIVO: Avaliar a acurácia do teste semiautomatizado para determinação do genótipo RHD fetal no plasma materno. METODOLOGIA: Foram coletadas prospectivamente amostras de sangue de 220 gestantes RhD negativo, com idade gestacional entre 8-28 semanas. O plasma foi obtido em no máximo 2 horas após a coleta, e uma alíquota de 1 mL foi submetida à extração de ácidos nucléicos no equipamento automatizado MagNA Pure Compact (Roche), empregando o kit Large Volume. O DNA extraído foi submetido a PCR em tempo real (Step One Plus - Applied Biosystems), usando o protocolo do grupo SAFE, que tem como alvos os éxons 5 e 7 do gene RHD. RESULTADOS: Ocorreu exclusão de 35 amostras devido a problemas pré-analíticos, aborto ou desconhecimento do fenótipo do recém-nascido. Entre as 185 amostras analisadas, 130 (70,2%) foram genotipadas como RHD+ e 55 (29,8%) RHD-. Os resultados obtidos foram comparados com a fenotipagem do cordão umbilical, e houve concordância completa (100%). Sete amostras exibiram amplificação exclusiva para o éxon 7. Essas amostras foram submetidas aos protocolos em PCR convencional, e PCR em tempo real específico para o pseudogene RHD. Ambos os ensaios apresentaram os mesmos resultados: cinco positivos e dois negativos. Nesses mesmos 7 casos, após extração da camada de leucócitos materna, os protocolos foram repetidos, e o resultado confirmou que cinco mães eram RHD. As duas amostras com resultado negativo foram submetidas ao protocolo Multiplex, envolvendo os éxons 3-9 do gene RHD, com resultados negativos, confirmando que as mães são verdadeiramente RHD- portanto o sinal do éxon 7 é provindo dos fetos que são D variantes. CONCLUSÃO: O método para a determinação do RHD fetal no plasma materno descrito demonstrou ser rápido, de fácil execução, alta precisão e reprodutível, além de indicar possíveis variantes RHD em nossa população / BACKGROUND: Fetal RHD genotype determination in maternal plasma is a noninvasive prenatal diagnostic test performed in RhD negative pregnant women at risk of alloimmunization and/or Hemolytic Disease of Fetus and Newborn. Currently, this test is routinely performed in many countries but not in Brazil. The Department of Obstetrics at Hospital das Clínicas, São Paulo University Medical School provides tertiary antenatal care for RhD negative pregnant women including anti-D immunoglobulin administration, antibody levels monitoring and intrauterine treatment if necessary. AIMS: To validate the accuracy of a semi-automated test for fetal RHD genotype determination in maternal plasma. METHODS: Two-hundred and twenty blood samples were prospectively collected between 8 and 28 weeks of gestational age. Plasma processing was performed within 2 hours after blood collection, and nucleic acids were extracted from 1mL aliquots with an automated extraction platform (MagNA Pure Compact Roche) and the Large Volume kit. RHD gene exons 5 and 7 were amplified with real-time PCR (Step One Plus - Applied Biosystems) using the SAFE group protocol. RESULTS: Thirty-five samples were excluded due to pre-analytical problems, miscarriage and missing follow-up. In the remaining 185 samples, 130 (70.2%) were genotyped as RhD+ and 55 (29.8%) RhD-. Comparison with umbilical cord blood group phenotype showed 100% concordance. Seven samples showed amplification for exon 7 only. These were further investigated with conventional and real-time PCR with an specific protocol for RHD? pseudogene: 5 were positive and 2, negative. In these 7 cases, maternal buffy-coat DNA analysis also confirmed that 5 women were RHD?. In the remaining 2 cases, a multiplex protocol directed at RHD gene exons 3-9 confirmed that both mothers were truly RhD negative so exon 7 signal comes from the fetuses, further found to harbor D variants. CONCLUSION: The present study demonstrates that fetal RHD determination in maternal plasma is a fast, easy-to-perform and reproducible technique with high accuracy in our population. Moreover, it helps in the identification of possible RHD variants in our population