RESUMO
BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.
Assuntos
Antígenos de Grupos Sanguíneos , Antígenos de Grupos Sanguíneos/genética , Feminino , Sangue Fetal , Feto , Genótipo , Humanos , Gravidez , Diagnóstico Pré-Natal , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
OBJECTIVE: To examine the feasibility of fetal RHD genotyping at 11-13 weeks' gestation from analysis of circulating cell-free fetal DNA (ccffDNA) in the plasma of RhD negative pregnant women using a high-throughput robotic technique. METHODS: Stored plasma (0.5 ml) from 591 RhD negative women was used for extraction of ccffDNA by a robotic technique. Real-time quantitative polymerase chain reaction (PCR) with probes for exons 5 and 7 of the RHD gene was then used to determine the fetal RHD genotype, which was compared to the neonatal RhD phenotype. RESULTS: In total there were 502 (85.7%) cases with a conclusive result and 84 (14.3%) with an inconclusive result. The prenatal test predicted that the fetus was RhD positive in 332 cases and in all of these the prediction was correct, giving a positive predictive value of 100% (95% CI 96.8-100). The test predicted that the fetus was RhD negative in 170 cases and in 164 of these the prediction was correct, giving a negative predictive value for RhD positive fetuses of 96.5% (95% CI 93.7-99.2). CONCLUSION: The findings demonstrate the feasibility and accuracy of non-invasive fetal RHD genotyping at 11-13 weeks with a high-throughput technique.
Assuntos
DNA/sangue , Sangue Fetal/imunologia , Genótipo , Idade Gestacional , Sistema do Grupo Sanguíneo Rh-Hr/genética , Estudos de Viabilidade , Feminino , Humanos , Fenótipo , Gravidez , Sensibilidade e EspecificidadeRESUMO
Alloimmunization to the blood group antibody anti-RhD (anti-D) is the most common cause of hemolytic disease of the fetus and newborn. Knowledge of fetal D type in women with anti-D makes management of the pregnancy much easier and avoids unnecessary procedures in those women with a D-negative fetus. Fetal D typing can be performed by detection of an RHD gene in cell-free DNA in the plasma of D-negative pregnant women. The technology involves real-time quantitative polymerase chain reactions targeting exons 4, 5, and 10 of RHD, with the exons 4 and 10 tests performed as a multiplex. Testing for SRY in multiplex with the RHD exon 5 test provides an internal control for the presence of fetal DNA when the fetus is male. Fetal D typing has become the standard of care in England in pregnant women with a significant level of anti-D.
Assuntos
DNA/genética , Éxons/genética , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Natal/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/sangue , DNA/imunologia , Inglaterra , Eritroblastose Fetal/sangue , Eritroblastose Fetal/diagnóstico , Eritroblastose Fetal/genética , Éxons/imunologia , Feminino , Humanos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Masculino , Plasma , Gravidez , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Imunoglobulina rho(D)RESUMO
Prenatal fetal sex determination is undertaken in women at high risk of serious genetic disorders affecting a specific sex. Traditionally, this is undertaken by invasive testing, usually chorionic villus sampling, which carries a risk of miscarriage of around 1%. The identification of cell-free fetal DNA in the maternal circulation has allowed the development of 'non-invasive prenatal diagnostic tests', which permit fetal sex determination without risk to the pregnancy.
Assuntos
DNA/sangue , Doenças Genéticas Inatas/diagnóstico , Diagnóstico Pré-Natal/métodos , Análise para Determinação do Sexo , Feminino , Genes sry , Doenças Genéticas Inatas/sangue , Humanos , Masculino , Reação em Cadeia da Polimerase , GravidezRESUMO
Prediction of fetal blood group from DNA is usually performed when the mother has antibodies to RhD, to assess whether the fetus is at risk from hemolytic disease of the fetus and newborn (HDFN). Over the last five years RhD testing on fetal DNA in maternal plasma has been introduced. At the International Blood Group Reference Laboratory (IBGRL) we employ real-time quantitative polymerase chain reaction (RQ-PCR) to detect RHD exons 4, 5, and 10, which also reveals RHDpsi. SRY and, in RhD-negative (RhD-) females, eight biallelic polymorphisms are incorporated in an attempt to provide an internal positive control. Since 2000 we have tested 533 pregnancies for RhD. In 327 pregnancies where the RhD of the infant is known, we had one false-positive and one false-negative result. In 2004 we introduced fetal typing from DNA in maternal plasma for K, Rhc, and RhE, which represent single nucleotide polymorphisms (SNPs) on the KEL and RHCE genes. We have begun trials on an automated method for fetal RhD typing from DNA in maternal plasma. This is designed to test fetal RhD in all pregnant RhD- women, to identify the 40% with an RhD- fetus so that antenatal RhD immunoglobulin (Ig) prophylaxis can be avoided. Similar trials have already been reported by Sanquin Research Laboratories in Amsterdam.
Assuntos
Sangue Fetal/fisiologia , Feto/fisiologia , Genótipo , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/sangue , Feminino , Humanos , Polimorfismo Genético , GravidezRESUMO
BACKGROUND: High-throughput genotyping platforms enable simultaneous analysis of multiple polymorphisms for blood group typing. BLOODchip® ID is a genotyping platform based on Luminex® xMAP technology for simultaneous determination of 37 red blood cell (RBC) antigens (ID CORE XT) and 18 human platelet antigens (HPA) (ID HPA XT) using the BIDS XT software. MATERIALS AND METHODS: In this international multicentre study, the performance of ID CORE XT and ID HPA XT, using the centres' current genotyping methods as the reference for comparison, and the usability and practicality of these systems, were evaluated under working laboratory conditions. DNA was extracted from whole blood in EDTA with Qiagen methodologies. Ninety-six previously phenotyped/genotyped samples were processed per assay: 87 testing samples plus five positive controls and four negative controls. RESULTS: Results were available for 519 samples: 258 with ID CORE XT and 261 with ID HPA XT. There were three "no calls" that were either caused by human error or resolved after repeating the test. Agreement between the tests and reference methods was 99.94% for ID CORE XT (9,540/9,546 antigens determined) and 100% for ID HPA XT (all 4,698 alleles determined). There were six discrepancies in antigen results in five RBC samples, four of which (in VS, N, S and Do(a)) could not be investigated due to lack of sufficient sample to perform additional tests and two of which (in S and C) were resolved in favour of ID CORE XT (100% accuracy). The total hands-on time was 28-41 minutes for a batch of 16 samples. Compared with the reference platforms, ID CORE XT and ID HPA XT were considered simpler to use and had shorter processing times. DISCUSSION: ID CORE XT and ID HPA XT genotyping platforms for RBC and platelet systems were accurate and user-friendly in working laboratory settings.
Assuntos
Antígenos de Plaquetas Humanas/genética , Eritrócitos , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/métodos , Feminino , Humanos , MasculinoRESUMO
Antenatal determination of fetal blood group is important in pregnancies with a significant risk of hemolytic anemia due to maternal alloimmunization. The International Blood Group Reference Laboratory (part of the National Blood Service) in Bristol, UK, provides a fetal blood group genotyping service to obstetricians caring for immunized pregnant women with heterozygous partners. Since 2001, fetal D typing has been offered using free fetal DNA in maternal plasma. Real-time polymerase chain reaction (PCR) assays are performed to detect the RHD gene. To confirm the presence of fetal DNA when RHD is not detected, Y-chromosome sequences are targeted. When a D-negative female fetus is predicted, maternal buffy coat DNA is tested for eight insertion/deletion polymorphisms. Sequences that are absent from the maternal genome are then targeted in maternal plasma and are used to confirm the presence of free fetal DNA in the blood sample. Currently, 283 pregnancies have been tested, of which 50 are awaiting confirmatory results. Fetal D status was correctly predicted in 223 cases, and no result was obtainable in 7 cases. In three cases, serology on cord blood was discrepant with reported results, but all fetuses had received multiple intrauterine transfusions. The new test has significantly reduced the number of invasive procedures carried out in the UK for fetal D grouping. Antenatal anti-D prophylaxis is currently being introduced in the UK to all D-negative women; in the future, detection of fetal RHD sequences in maternal plasma may allow anti-D to be restricted to pregnancies involving a D-positive fetus.
Assuntos
DNA/sangue , Sangue Fetal/imunologia , Diagnóstico Pré-Natal , Imunoglobulina rho(D)/sangue , Antígenos de Plaquetas Humanas/imunologia , Feminino , Previsões , Humanos , Integrina beta3 , Reação em Cadeia da Polimerase , Gravidez , Cuidado Pré-Natal , Púrpura Trombocitopênica Idiopática/prevenção & controle , Isoimunização Rh/prevenção & controle , Sistema do Grupo Sanguíneo Rh-Hr , Imunoglobulina rho(D)/genética , Reino UnidoRESUMO
OBJECTIVES: To assess the accuracy of fetal RHD genotyping using cell-free fetal DNA in maternal plasma at different gestational ages. DESIGN: A prospective multicentre cohort study. SETTING: Seven maternity units in England. PARTICIPANTS: RhD negative pregnant women who booked for antenatal care before 24 weeks' gestation. INTERVENTIONS: Women who gave consent for fetal RHD genotyping had blood taken at the time of booking for antenatal care and, when possible, at other routine visits such as for Down's syndrome screening between 11 and 21 weeks' gestation, at the anomaly scan at 18-21 weeks, and in the third trimester when blood was taken for the routine antibody check. The results of cord blood analysis, done routinely in RhD negative pregnancies, were also obtained to confirm the fetal RHD genotyping. MAIN OUTCOME MEASURES: The accuracy of fetal RHD genotyping compared with RhD status predicted by cord blood serology. RESULTS: Up to four analyses per woman were performed in 2288 women, generating 4913 assessable fetal results. Sensitivity for detection of fetal RHD positivity was 96.85% (94.95% to 98.05%), 99.83% (99.06% to 99.97%), 99.67% (98.17% to 99.94%), 99.82% (98.96% to 99.97%), and 100% (99.59% to 100%) at <11, 11-13, 14-17, 18-23, and >23 completed weeks' gestation, respectively. Before 11 weeks' gestation 16/865 (1.85%) babies tested were falsely predicted as RHD negative. CONCLUSIONS: Mass throughput fetal RHD genotyping is sufficiently accurate for the prediction of RhD type if it is performed from 11 weeks' gestation. Testing before this time could result in a small but significant number of babies being incorrectly classified as RHD negative. These mothers would not receive anti-RhD immunoglobulin, and there would be a risk of haemolytic disease of the newborn in subsequent pregnancies.
Assuntos
DNA/análise , Eritroblastose Fetal/genética , Sangue Fetal/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/sangue , Eritroblastose Fetal/diagnóstico , Eritroblastose Fetal/imunologia , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Genótipo , Idade Gestacional , Humanos , Gravidez , Trimestres da Gravidez , Diagnóstico Pré-Natal , Estudos Prospectivos , Sensibilidade e EspecificidadeAssuntos
Eritroblastose Fetal , Técnicas de Genotipagem/métodos , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/genética , Adulto , Eritroblastose Fetal/sangue , Eritroblastose Fetal/genética , Eritroblastose Fetal/prevenção & controle , Feminino , Humanos , Recém-Nascido , GravidezRESUMO
Identification of the molecular basis of the D polymorphism of the Rh blood group system in the 1990s made it possible to predict D phenotype from DNA. The most valuable application of this has been the determination of fetal D type in pregnant D-negative women with anti-D. Knowledge of fetal D type reveals whether the fetus is at risk of hemolytic disease of the fetus and newborn so that the pregnancy can be managed appropriately. Noninvasive fetal D typing for D-negative pregnant women with anti-D, performed on the small quantity of fetal DNA present in the blood of pregnant women, is now routine practice in several European countries. Noninvasive fetal blood grouping for C, c, E, and K also may be provided as a routine service for alloimmunized pregnant women. In many countries, all D-negative pregnant women are offered anti-D prophylaxis antenatally, yet in a predominantly Caucasian population, about 38% will be carrying a D-negative fetus and will receive the treatment unnecessarily. Large-scale trials to ascertain the accuracy of high-throughput, automated methods suggest that fetal D screening of all D-negative pregnant women is feasible, and it is likely that fetal D screening in D-negative pregnant women will be policy in some European countries within the next few years.
Assuntos
DNA/sangue , Sangue Fetal , Isoanticorpos/genética , Técnicas de Diagnóstico Molecular/métodos , Gravidez/sangue , Diagnóstico Pré-Natal/métodos , Antígenos de Grupos Sanguíneos/genética , Feminino , Genótipo , Humanos , Mães , Imunoglobulina rho(D)RESUMO
Fetuses of women with alloantibodies to RhD (D) are at risk from hemolytic disease of the fetus and newborn, but only if the fetal red cells are D-positive. In such pregnancies, it is beneficial to determine fetal D type, as this will affect the management of the pregnancy. It is possible to predict, with a high level of accuracy, fetal blood group phenotypes from genotyping tests on fetal DNA. The best source is the small quantity of fetal DNA in the blood of pregnant women, as this avoids the requirement for invasive procedures of amniocentesis or chorionic villus sampling (CVS). Many laboratories worldwide now provide noninvasive fetal D genotyping as a routine service for alloimmunized women, and some also test for c, E, C and K.In many countries, anti-D immunoglobulin injections are offered to D-negative pregnant women, to reduce the chances of prenatal immunization, even though up to 40% of these women will have a D-negative fetus. High-throughput, noninvasive fetal D genotyping technologies are being developed so that unnecessary treatment of pregnant women can be avoided. Trials suggest that fetal D typing of all D-negative pregnant women is feasible and should become common practice in the near future.
Assuntos
Isoanticorpos/sangue , Diagnóstico Pré-Natal/métodos , DNA/química , DNA/genética , Feminino , Feto , Genótipo , Humanos , Recém-Nascido , Reação em Cadeia da Polimerase , Gravidez , Imunoglobulina rho(D)RESUMO
BACKGROUND: Alloimmunization against the fetal Kell (KEL1) blood group antigen is gaining importance relative to the Rhesus problem and is the second most important cause of hemolytic disease of the fetus and newborn. Molecular diagnosis for fetal KEL1, which currently involves invasive procedures, is routinely done for accessing whether a fetus is at risk. Here we developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based single allele-based extension reaction (SABER) to examine the fetal KEL1 gene from KEL1-negative pregnant women using cell-free fetal DNA in maternal plasma. METHODS: Thirty-two maternal plasma samples taken at the second and third trimesters of gestation (median: 21.5 weeks) were examined with MALDI-TOF MS-based SABER. The results were confirmed by serological tests on cord blood or polymerase chain reaction (PCR) typing on amniocyte-derived fetal DNA. RESULTS: We were able to detect the fetal KEL1 allele in 11 of the 13 KEL1-positive samples. No false positive results were scored. The paternal KEL1 allele could be correctly determined in 94% of cases (30/32). CONCLUSIONS: Our results indicated that the MALDI-TOF MS-based SABER has been used successfully for the detection of the fetal KEL1 status with the accuracy of 94%. Further, large-scale study, such as multicenter study, can now be explored for clinical application.
Assuntos
DNA/sangue , Sangue Fetal , Sistema do Grupo Sanguíneo de Kell/genética , Espectrometria de Massas , Feminino , Genótipo , Humanos , Gravidez , Segundo Trimestre da Gravidez/sangue , Terceiro Trimestre da Gravidez/sangue , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. DESIGN: Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. SETTING: Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. PARTICIPANTS: Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. MAIN OUTCOME MEASURES: Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. RESULTS: Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. CONCLUSIONS: High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.
Assuntos
DNA/análise , Doenças Fetais/genética , Isoimunização Rh/genética , Imunoglobulina rho(D)/genética , Estudos de Viabilidade , Feminino , Sangue Fetal/química , Técnicas Genéticas/normas , Genótipo , Idade Gestacional , Humanos , Fenótipo , Gravidez , Estudos Prospectivos , Sensibilidade e Especificidade , Procedimentos DesnecessáriosRESUMO
BACKGROUND: When a pregnant woman has an antibody with the potential to cause hemolytic disease of the fetus and newborn, it is beneficial to determine whether her fetus has the corresponding antigen to assess risk. In many countries this is now done routinely for RhD, by testing cell-free fetal DNA in the maternal plasma. Similar tests for K, C, c, and E are reported. STUDY DESIGN AND METHODS: Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL and the C, c, and E alleles of RHCE. These methods were used to test DNA isolated from plasma of pregnant women with antibodies to K, C, c, or E. Accuracy of the tests was determined by comparing results with serologic tests performed on cord red blood cells (RBCs) after delivery or by molecular genotyping on DNA obtained from fetal cells. RESULTS: The K test incorporated an allele-specific primer with two locked nucleic acids and a mismatch. In 70 tests, including 27 K+ fetuses, only one false-negative and no false-positive results were obtained. The C, c, and E tests, performed on 13, 44, and 46 samples, respectively, gave rise to no false results. CONCLUSION: Reliable methods have been developed for predicting fetal K, C, c, and E phenotypes, by testing fetal DNA in the plasma samples of pregnant women whose RBCs lack the corresponding antigens. These methods are now being used routinely in a diagnostic service in the United Kingdom.
Assuntos
DNA/sangue , Sistema do Grupo Sanguíneo de Kell/genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , Adulto , Alelos , Primers do DNA , Erros de Diagnóstico , Feminino , Feto , Genótipo , Humanos , Mães , Reação em Cadeia da Polimerase/normas , Gravidez , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed. METHODS: Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration. RESULTS: The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with > or = 500 microL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3. CONCLUSIONS: This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies.
Assuntos
DNA/sangue , DNA/isolamento & purificação , Feto , Testes Genéticos/métodos , Mães , Automação , Feminino , Feto/metabolismo , Testes Genéticos/normas , Humanos , Relações Materno-Fetais , Reação em Cadeia da Polimerase/métodos , Gravidez , Diagnóstico Pré-Natal/métodos , Diagnóstico Pré-Natal/normasRESUMO
BACKGROUND: The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti-KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti-KEL1 reagents. STUDY DESIGN AND METHODS: Kell genotyping methods, including KEL exon 6 direct sequencing, were applied. KEL cDNA from D1 was sequenced. Flow cytometry was used to assess KEL1 and KEL2 RBC expression. RESULTS: RBCs from the donor, her mother, and an unrelated donor gave weak or negative reactions with some anti-KEL1 reagents. Other Kell-system antigens appeared normal. The three individuals were homozygous for KEL C578 (KEL*2) but heterozygous for a 577A>T transversion, encoding Ser193. They appeared to be KEL*2 homozygotes by routine genotyping methods. Flow cytometry revealed weak KEL1 expression and normal KEL2, similar to that of KEL*2 homozygotes. CONCLUSION: Ser193 in the Kell glycoprotein appears to result in expression of abnormal KEL1, in addition to KEL2. The mutation is not detected by routine Kell genotyping methods and, because of unpredicted KEL1 expression, could lead to a misdiagnosis.
Assuntos
Sequência de Bases/genética , Expressão Gênica , Glicoproteínas/genética , Sistema do Grupo Sanguíneo de Kell/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Adulto , Doadores de Sangue , Análise Mutacional de DNA/métodos , DNA Complementar/genética , Eritroblastose Fetal/genética , Éxons/genética , Feminino , Doenças Fetais/genética , Heterozigoto , Homozigoto , Humanos , Recém-Nascido , MasculinoRESUMO
OBJECTIVE: This study reviews our clinical experience of non-invasive techniques for early sex determination. It assesses the effectiveness of these techniques at reducing invasive prenatal testing for X-linked genetic disease or for ambiguous development of the external genitalia. METHODS: A prospective cohort study of 30 pregnancies was referred to a tertiary unit for prenatal diagnosis. Fetal gender was determined using two non-invasive techniques: analysis of free fetal DNA (ffDNA) in maternal plasma and ultrasound visualisation. The results were compared to fetal gender determined by invasive testing or at birth. RESULTS: Fetal gender was accurately determined by analysis of ffDNA at a mean of 10 + 1 (7 + 6 to 14 + 1) weeks' gestation in all cases. Ultrasound assessment was accurate in 20 of the 23 cases where this was attempted at 12 + 0 (10 + 4 to 14 + 1) weeks' gestation, but could not be determined in the remaining 3 cases. Thirteen of 28 (46%) women chose not to have invasive testing on the basis of these findings. CONCLUSIONS: Both the techniques appear to offer an accurate means of assessing fetal gender, giving parents the option of avoiding invasive testing in the 50% of cases where the fetus would not be affected. The molecular technique is performed at an earlier gestation, but female fetal status is predicted by a negative test result. Ultrasound cannot be applied until 11 weeks' gestation but diagnostic signs are sought in both sexes. Combining these approaches offers a highly sensitive method of non-invasive determination of gender in high-risk pregnancies. Health professionals, clinical geneticists and genetics associates, in particular, who refer women at high risk should be aware of these non-invasive options for prenatal sex determination.
Assuntos
Cromossomos Humanos X , DNA/sangue , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Diagnóstico Pré-Natal/métodos , Análise para Determinação do Sexo/métodos , Estudos de Coortes , Feminino , Idade Gestacional , Humanos , Reação em Cadeia da Polimerase , Gravidez , Estudos Prospectivos , Ultrassonografia Pré-NatalRESUMO
OBJECTIVES: The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis. METHODS: A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th-36th week of gestation. Thirty-eight samples were from ongoing pregnancies of Danish women and 21 samples from 18 pregnant women were stored anonymized samples from the International Blood Group Reference Laboratory, Bristol, United Kingdom. Prediction of fetal RhD type was compared with the serological result obtained after birth. RESULTS: The prediction of the fetal RhD type was in 100% concordance with the serological RhD type from the 16th week of gestation. One sample from the 15th week of gestation was inconclusive. The number of copies of fetal RHD DNA was found to increase with gestational age. Low levels of DNA were found to follow the Poisson distribution (p = 1.0000). CONCLUSION: Our set-up was very reliable for determination of fetal RhD genotype, and thus will be of value in prenatal Rh prophylaxis and in the management of immunized women.