Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado
The prenatal diagnosis of chromosomal abnormalities had a great evolution over time. It began with the first chromosomal cell culture in amniotic fluid in 1966, and was initially used to identify numerical chromosomal abnormalities, particularly Trisomy 21. With the advent of cytogenetic banding tec...
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2023
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Universidad de Buenos Aires |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
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Español |
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spa |
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QF-PCR Diagnóstico prenatal rápido Aneploudía Ciencias de la vida |
| spellingShingle |
QF-PCR Diagnóstico prenatal rápido Aneploudía Ciencias de la vida Canonero, Ivana Beatriz Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| topic_facet |
QF-PCR Diagnóstico prenatal rápido Aneploudía Ciencias de la vida |
| description |
The prenatal diagnosis of chromosomal abnormalities had a great evolution over time.
It began with the first chromosomal cell culture in amniotic fluid in 1966, and was initially used to identify numerical chromosomal abnormalities, particularly Trisomy 21. With the advent of cytogenetic banding techniques, fundamental chromosomal abnormalities began to be detected.
In this way, the practice of the karyotype in the amniotic cells between 15 and 17 weeks of gestation, became the primary cytogenetic technique in prenatal diagnosis for two decades, but the cytogenetic diagnosis was confined to the second trimester of gestation. In the early 1980s, prenatal diagnosis during the first trimester was introduced, incorporating chorionic biopsy starting at 12 weeks of gestation, and this led to early identification of fetal chromosomal abnormalities in the first trimester, with earlier diagnosis.
First, the invasive test was indicated in all those with advanced maternal age, due to the connection between aneuploidies a consecuence of a meiotic nondisjunction event and maternal age, but this strategy only identified 30% of trisomies, with a very high number of invasive tests with normal results and a large subdiagnosis of aneuploidies.
With the improvement of screening techniques for patients at risk, ultrasound screening strategies with highly sensitive sonographic markers were determined, which increased the aneuploidy detection rate by up to 90%. Adding biochemical markers in maternal blood (incorporation of free chorionic gonadotropin subunit dosage and protein A of pregnancy), increased the aneuploidy detection rate to 95%.
In recent years, advanced non-invasive screening for the detection of aneuploidies in cell free fetal DNA in maternal circulation has been incorporated, raising the detection rate to 99%, with a false positive rate of 0.01%.
Thus, by implementing these screening methods, the number of invasive procedures was significantly reduced and it was possible to identify patients at high risk of aneuploidy.
Once the prenatal material has been obtained through an invasive procedure, the samples need to be cultured for several days to obtain a cell division that allows stopping the process in metaphase, to then make sample of the chromosomes with different cytogenetic techniques that allow their analysis under an optical microscope. This prenatal karyotyping process takes approximately 15-21 days for amniotic fluid and 7-10 days for chorionic biopsy culture (short or direct culture and long culture). This delay in the result generates a lot of anguish and uncertainty in the parents, and in the health team, when waiting for a result to define a prognosis, behavior for fetal treatment, or define the need for other fetal prenatal studies.
For this reason, to shorten the waiting time for the result, the rapid diagnosis technique for aneuploidies was incorporated, called fluorescent quantitative polymerase chain reaction or QF-PCR. This molecular technique evaluates STRs that allows evaluating aneuploidies of chromosomes 21, 18, 13, X and Y within 24 hours of extracting the prenatal sample.
If the QF-PCR is normal, the karyotype allows evaluation of the other chromosomes, and if it is altered, the karyotype allows confirming whether it is a free trisomy or the product of a structural rearrangement.
When the pregnancy has a high risk of aneuploidies, or fetal malformations, and the QF-PCR and karyotype studies were normal, there is a comparative genomic hybridization microarray study that allows the investigation of DNA microdeletions and duplications with a resolution 100 times higher than that of karyotype.
The main objective was to establish the most effective genetic diagnostic strategies for fetuses with altered screening and/or with congenital malformations, which are diagnosed prenatally, combining screening methods, cytogenetic and molecular diagnostic techniques.
The secondary objective was to evaluate and compare the diagnostic performance of the QF-PCR in comparison with the conventional cytogenetic technique to identify advantages, limitations, challenges of counseling after its implementation.
The present study included 596 prenatal samples that underwent prenatal karyotype and QF-PCR. The diagnostic performance for the detection of aneuploidies 21, 18, 13, X and Y was comparable, without significant differences, with the advantage of rapid diagnosis within 24 hours of QF-PCR and the advantage of evaluating the chromosome structure with the karyotype.
The possibility of replacing direct chorionic biopsy culture with QF-PCR + long chorionic culture was raised.
The molecular techniques of QF-PCR, prenatal karyotype and prenatal microarray are complementary.
It is concluded that in all pregnant patients with an altered combined screening of the first trimester or the presence of fetal malformations, or a positive Fetal DNA Test (NIPT) an invasive diagnostic test (chorionic biopsy or amniocentesis) is indicated.
In every prenatal sample, QF-PCR and prenatal karyotype should be performed. If the QF-PCR identifies an aneuploidy, this result can be reported after 24 hours, while the karyotype is awaited to define whether it is a structural abnormality or a free trisomy.
If the QF-PCR results normal, and an aneuploidy is suspected in another chromosome different from those evaluated by this technique, such anomaly will be identified in the karyotype.
If both studies were normal, or a microdeletion/microduplication syndrome was suspected, the prenatal microarray study would proceed.
It is concluded that this proposal for a combination of screening methods and molecular techniques allows a greater performance of prenatal diagnosis, to define prognosis, counseling and therapeutic conduct in an early and efficient manner. |
| author2 |
Cuestas, Eduardo |
| author_facet |
Cuestas, Eduardo Canonero, Ivana Beatriz |
| format |
Tesis de maestría Tesis de maestría acceptedVersion |
| author |
Canonero, Ivana Beatriz |
| author_sort |
Canonero, Ivana Beatriz |
| title |
Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| title_short |
Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| title_full |
Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| title_fullStr |
Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| title_full_unstemmed |
Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| title_sort |
estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado |
| publisher |
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica |
| publishDate |
2023 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_8030 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_8030.dir/8030.PDF |
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| spelling |
I28-R145-HWA_80302025-12-15 The prenatal diagnosis of chromosomal abnormalities had a great evolution over time. It began with the first chromosomal cell culture in amniotic fluid in 1966, and was initially used to identify numerical chromosomal abnormalities, particularly Trisomy 21. With the advent of cytogenetic banding techniques, fundamental chromosomal abnormalities began to be detected. In this way, the practice of the karyotype in the amniotic cells between 15 and 17 weeks of gestation, became the primary cytogenetic technique in prenatal diagnosis for two decades, but the cytogenetic diagnosis was confined to the second trimester of gestation. In the early 1980s, prenatal diagnosis during the first trimester was introduced, incorporating chorionic biopsy starting at 12 weeks of gestation, and this led to early identification of fetal chromosomal abnormalities in the first trimester, with earlier diagnosis. First, the invasive test was indicated in all those with advanced maternal age, due to the connection between aneuploidies a consecuence of a meiotic nondisjunction event and maternal age, but this strategy only identified 30% of trisomies, with a very high number of invasive tests with normal results and a large subdiagnosis of aneuploidies. With the improvement of screening techniques for patients at risk, ultrasound screening strategies with highly sensitive sonographic markers were determined, which increased the aneuploidy detection rate by up to 90%. Adding biochemical markers in maternal blood (incorporation of free chorionic gonadotropin subunit dosage and protein A of pregnancy), increased the aneuploidy detection rate to 95%. In recent years, advanced non-invasive screening for the detection of aneuploidies in cell free fetal DNA in maternal circulation has been incorporated, raising the detection rate to 99%, with a false positive rate of 0.01%. Thus, by implementing these screening methods, the number of invasive procedures was significantly reduced and it was possible to identify patients at high risk of aneuploidy. Once the prenatal material has been obtained through an invasive procedure, the samples need to be cultured for several days to obtain a cell division that allows stopping the process in metaphase, to then make sample of the chromosomes with different cytogenetic techniques that allow their analysis under an optical microscope. This prenatal karyotyping process takes approximately 15-21 days for amniotic fluid and 7-10 days for chorionic biopsy culture (short or direct culture and long culture). This delay in the result generates a lot of anguish and uncertainty in the parents, and in the health team, when waiting for a result to define a prognosis, behavior for fetal treatment, or define the need for other fetal prenatal studies. For this reason, to shorten the waiting time for the result, the rapid diagnosis technique for aneuploidies was incorporated, called fluorescent quantitative polymerase chain reaction or QF-PCR. This molecular technique evaluates STRs that allows evaluating aneuploidies of chromosomes 21, 18, 13, X and Y within 24 hours of extracting the prenatal sample. If the QF-PCR is normal, the karyotype allows evaluation of the other chromosomes, and if it is altered, the karyotype allows confirming whether it is a free trisomy or the product of a structural rearrangement. When the pregnancy has a high risk of aneuploidies, or fetal malformations, and the QF-PCR and karyotype studies were normal, there is a comparative genomic hybridization microarray study that allows the investigation of DNA microdeletions and duplications with a resolution 100 times higher than that of karyotype. The main objective was to establish the most effective genetic diagnostic strategies for fetuses with altered screening and/or with congenital malformations, which are diagnosed prenatally, combining screening methods, cytogenetic and molecular diagnostic techniques. The secondary objective was to evaluate and compare the diagnostic performance of the QF-PCR in comparison with the conventional cytogenetic technique to identify advantages, limitations, challenges of counseling after its implementation. The present study included 596 prenatal samples that underwent prenatal karyotype and QF-PCR. The diagnostic performance for the detection of aneuploidies 21, 18, 13, X and Y was comparable, without significant differences, with the advantage of rapid diagnosis within 24 hours of QF-PCR and the advantage of evaluating the chromosome structure with the karyotype. The possibility of replacing direct chorionic biopsy culture with QF-PCR + long chorionic culture was raised. The molecular techniques of QF-PCR, prenatal karyotype and prenatal microarray are complementary. It is concluded that in all pregnant patients with an altered combined screening of the first trimester or the presence of fetal malformations, or a positive Fetal DNA Test (NIPT) an invasive diagnostic test (chorionic biopsy or amniocentesis) is indicated. In every prenatal sample, QF-PCR and prenatal karyotype should be performed. If the QF-PCR identifies an aneuploidy, this result can be reported after 24 hours, while the karyotype is awaited to define whether it is a structural abnormality or a free trisomy. If the QF-PCR results normal, and an aneuploidy is suspected in another chromosome different from those evaluated by this technique, such anomaly will be identified in the karyotype. If both studies were normal, or a microdeletion/microduplication syndrome was suspected, the prenatal microarray study would proceed. It is concluded that this proposal for a combination of screening methods and molecular techniques allows a greater performance of prenatal diagnosis, to define prognosis, counseling and therapeutic conduct in an early and efficient manner. Fil: Canonero, Ivana Beatriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Cuestas, Eduardo Canonero, Ivana Beatriz 2023-10-23 El diagnóstico prenatal de anomalías cromosómicas, tuvo una gran evolución en el tiempo. Comenzó con el primer cultivo celular cromosómico en líquido amniótico en el año 1966, siendo usado para identificar anomalías cromosómicas numéricas, particularmente Trisomía 21. Con el advenimiento de las técnicas de bandeo citogenético, comenzaron a detectarse anomalías cromosómicas estructurales. De este modo, y la realización del cariotipo en las células amnióticas se volvió la técnica citogenética primaria en diagnóstico prenatal durante dos décadas, pero el diagnóstico citogenético estaba confinado al segundo trimestre de la gestación. A comienzos de 1980, se introdujo el diagnóstico prenatal durante el primer trimestre del embarazo, incorporándose la biopsia corial a partir de las 12 semanas de gestación, y esto llevó a la identificación temprana de anomalías cromosómicas fetales al primer trimestre, con un diagnóstico más precoz. Primeramente la prueba invasiva estaba indicada en toda paciente con edad materna avanzada, debido a la correlación entre aneuploidías por un evento de no disyunción meiótica y edad materna, pero esta estrategia solamente identificaba el 30% de las trisomías, con un número muy elevado de pruebas invasivas con resultados normales y un gran subdiagnóstico de aneuploidías. Con el perfeccionamiento de las técnicas de cribado, se determinaron estrategias de screening ecográfico con marcadores sonográficos muy sensibles, que elevaron la tasa de detección de aneuploidías a un 90%. Agregando marcadores bioquímicos en sangre materna(incorporación del dosaje de la subunidad libre de gonadotrofina coriónica y proteína A del embarazo), aumentó la tasa de detección de aneuploidías a un 95%. En los últimos años se incorporó el screening avanzado no invasivo de detección de aneuploidías en ADN libre fetal en circulación materna, elevando la tasa de detección a un 99%. De este modo implementando estos métodos de screening, se redujo en forma significativa el número de procedimientos invasivos y fue posible identificar a las pacientes de alto riesgo de aneuploidías. Una vez obtenido el material prenatal mediante un procedimiento invasivo, las muestras necesitan ser cultivadas durante varios días para obtener una división celular que permita detener el proceso en metafase, para luego hacer extendidos de los cromosomas con diferentes técnicas citogenéticas que permiten su análisis al microscopio óptico. Este proceso del cariotipo prenatal tiene una demora de aproximadamente 15-21 días para el líquido amniótico y de 7-10 días para el cultivo de biopsia corial (cultivo corto o directo y cultivo largo). Esta demora del resultado genera mucha angustia e incertidumbre en los padres, y en el equipo de salud, al momento de esperar un resultado para definir un pronóstico, conducta o tratamiento fetal, o definir la necesidad de otros estudios prenatales fetales. Por este motivo para acortar el tiempo de espera del resultado, se incorporó la técnica de diagnóstico rápido de aneuploidías, llamada reacción en cadena de la polimerasa cuantitativa fluorescente o QF-PCR. Esta técnica molecular evalúa STRs que permite detectar aneuploidías de los cromosomas 21, 18, 13, X e Y, dentro de las 24 hs de extraída la muestra prenatal. Si la QF-PCR resulta normal, el cariotipo permite evaluar los otros cromosomas, y si resulta alterada, el cariotipo permite confirmar si se trata de una trisomía libre, o producto de un rearreglo estructural. Cuando el embarazo tiene un alto riesgo de aneuploidías, o malformaciones fetales, y los estudios de QF-PCR y cariotipo resultaron normales, existe el estudio de microarray de hibridación genómica comparada que permite investigar microdeleciones y duplicaciones del ADN con una resolución 100 veces superior al cariotipo. El objetivo principal fue establecer las estrategias de diagnóstico genético más efectivas para los fetos con cribado alterado y/o con malformaciones congénitas, que se diagnostican prenatalmente, combinando métodos de screening, técnicas de diagnóstico citogenético y molecular. El objetivo secundario fue evaluar y comparar el rendimiento diagnóstico de la técnica de QF-PCR en comparación con la técnica convencional citogenética para identificar ventajas, limitaciones y desafíos del asesoramiento tras su implementación. En el presente estudio se incluyeron 596 muestras prenatales a las que se les realizó cariotipo prenatal y QF-PCR. El rendimiento diagnóstico para la detección de las aneuploidías 21, 18, 13, X e. Y fue comparable, sin diferencias significativas, con la ventaja del diagnóstico rápido dentro de las 24 hs de la QF-PCR y la ventaja de evaluar la estructura cromosómica con el cariotipo. Se planteó la posibilidad de reemplazar el cultivo de biopsia corial directo por la QF-PCR + el cultivo largo coriónico. Se concluye que en toda paciente embarazada ante un screening combinado alterado del primer trimestre o presencia de malformaciones fetales, o un Test de ADN fetal (NIPT) positivo está indicada la prueba invasiva diagnóstica(biopsia coriónica o amniocentesis). Sobre dicha muestra prenatal se debiera realizar QF-PCR y cariotipo prenatal. Si la QF-PCR identificara una aneuploidía, ya a las 24 hs se puede reportar dicho resultado mientras se espera el cariotipo para definir si se trata de una anomalía estructural o una trisomía libre. Si la QF-PCR resultara normal, y se sospechara una aneuploidía en otro cromosoma diferente a los que evalúa dicha técnica, dicha anomalía será identificada en el cariotipo. Si ambos estudios resultaran normales, o se sospechara un síndrome de microdeleción/duplicación se avanzaría con el estudio de microarray prenatal. Se concluye que esta propuesta de combinación de métodos de screening y técnicas moleculares permite un mayor rendimiento diagnóstico prenatal para la detección de anomalías cromosómicas, para definir pronóstico, asesoramiento y conducta terapéutica en forma precoz y eficiente. application/pdf Vazquez, Martín Cerretini, Roxana Fernández Gianotti, Tomás QF-PCR Diagnóstico prenatal rápido Aneploudía spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Magíster de la Universidad de Buenos Aires en Biología Molecular Estudio de efectividad diagnóstica de la reacción en cadena de la polimerasa cuantitativa fluorescente en fetos con cribado prenatal alterado info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_8030 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_8030.dir/8030.PDF |