Experimental Study on Separation of Fetal Nucleated Red Blood Cell from the Whole Blood Using Microfluidic Chip / 医用生物力学

Objective To seperate fetal nucleated red blood cells (fNRBCs) from the whole maternal peripheral blood effectively by designing a circular channel microfluidic chip. Methods A microfluidic chip is designed by utilizing the margination in blood flow and the specific adhesion characteristics of immuno-agent anti-CD147. With the whole umbilical cord blood, the effects of different shear forces on the enrichment of fNRBCs was studied by immunofluorescence counting. Results Increasing shear rate in microfluidic chip could improve the number of captured fNRBCs compared with the static adhesion. With the increase of shear rate of blood flow, the number of the captured cells increased at first, and then decreased. Conclusions The use of microfluid chip can effectively seperate fNRBCs from the whole blood. The results provide an experimental reference for the non-invasive prenatal diagnosis research and the exploration on the mechanism of fetal cell migration.

Application of digital polymerase chain reaction technology for noninvasive prenatal test

Recently, noninvasive prenatal test (NIPT) has been adopted as a primary screening tool for fetal chromosomal aneuploidy. The principle of NIPT lies in isolating the fetal fraction of cell-free DNA in maternal plasma and analyzing it with bioinformatic tools to measure the amount of gene from the target chromosome, such as chromosomes 21, 18, and 13. NIPT will contribute to decreasing the need for unnecessary invasive procedures, including amniocentesis and chorionic villi sampling, for confirming fetal aneuploidy because of its higher positive predictive value than that of the conventional prenatal screening method. However, its greater cost than that of the current antenatal screening protocol may be an obstacle to the adoption of this innovative technique in clinical practice. Digital polymerase chain reaction (dPCR) is a novel approach for detecting and quantifying nucleic acid. dPCR provides real-time diagnostic advantages with higher sensitivity, accuracy, and absolute quantification than conventional quantitative PCR. Since the groundbreaking discovery that fetal cell-free nucleic acid exists in maternal plasma was reported, dPCR has been used for the quantification of fetal DNA and for screening for fetal aneuploidy. It has been suggested that dPCR will decrease the cost by targeting specific sequences in the target chromosome, and dPCR-based noninvasive testing will facilitate progress toward the implementation of a noninvasive approach for screening for trisomy 21, 18, and 13. In this review, we highlight the principle of dPCR and discuss its future implications in clinical practice.

Noninvasive prenatal diagnosis ofβ-thalassemia by enriching cell-free fetal DNA in materal plasma / 中国病理生理杂志

AIM:To establish a kind of simple and efficient method for cell-free fetal DNA ( cff-DNA) enrich-ment and to investigate its range of applications and the advantages and disadvantages.METHODS:(1) The single nucleo-tide polymorphisms( SNPs) , which linked to paternalβ-thalassemia mutations, were screened.We analyzed the contact be-tween the SNPs inβ-thalassemia gene ( HBB gene) and haploid type by the Haploview software, and then selected these close SNPs which have higher heterozygosity with the HBB gene.(2) We selected 4 cases of different β-thalassemia muta-tions with their husband, and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma.If the IVS-II-654 mutation was not detected, we detected the SNP which linked to the IVS-II-654 mutation.Similarly, we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma.At the same time, we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma.RESULTS:(1) Nine cases of the SNP ( rs7480526) linked to the mutation at IVS-II-654 in HBB gene, and 11 cases of the SNP ( rs10768683) linked to the muta-tion at CD41-42 in HBB gene were detected.( 2 ) We detected 1 case who inherited the paternal β-thalassemia mutation (IVS-II-654).We did not directly detect patermal IVS-II-654 mutation in maternal plasma, but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100%detection, and 2 cases inherited the paternal β-thalassemia muta-tions (CD41-42) in the maternal plasma by TT-FULL-COLD-PCR and had 100%detection.However, we detected nothing by conventional PCR.CONCLUSION:TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.

Non-invasive prenatal diagnosis of fetal trisomy 21 using cell-free fetal DNA in maternal blood

Since the existence of cell-free fetal DNA (cff-DNA) in maternal circulation was discovered, it has been identified as a promising source of fetal genetic material in the development of reliable methods for non-invasive prenatal diagnosis (NIPD) of fetal trisomy 21 (T21). Currently, a prenatal diagnosis of fetal T21 is achieved through invasive techniques, such as chorionic villus sampling or amniocentesis. However, such invasive diagnostic tests are expensive, require expert technicians, and have a miscarriage risk approximately 1%. Therefore, NIPD using cff-DNA in the detection of fetal T21 is significant in prenatal care. Recently, the application of new techniques using single-molecular counting methods and the development of fetal-specific epigenetic markers has opened up new possibilities in the NIPD of fetal T21 using cff-DNA. These new technologies will facilitate safer, more sensitive and accurate prenatal tests in the near future. In this review, we investigate the recent methods for the NIPD of fetal T21 and discuss their implications in future clinical practice.

Noninvasive Prenatal Diagnosis Using Cell-free Fetal Nucleic Acids in the Maternal Circulation / 대한주산의학회잡지

Cell-free fetal nucleic acids in the maternal circulation can be broadly divided into fetal DNA and RNA that originate from apoptotic placenta cells. Cell-free fetal nucleic acids can be detected from 4-5 weeks gestation and are undetectable in the maternal circulation after delivery. Therefore, cell-free fetal nucleic acids have been proposed as a potential material for non-invasive prenatal diagnosis (NIPD), which poses no risk to mother and child. The clinical applications of this technology fall into three categories: first, early sex determination in cases at high risk of X-linked disorders or congenital adrenal hyperplasia requiring follow-up testing or antenatal treatment; second, detection of specific paternally inherited monogenic diseases in families with high genetic risk; and third, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis of aneuploidy, particularly Down syndrome. Fetal sex determination is already performed in routine clinical care for high-risk individuals in some countries. Many researchers have explored the possibility of cell-free fetal nucleic acids on NIPD of monogenic diseases and aneuploidy. Promising results have been reported from studies using the combination of markers and the application of various experimental methods. Although these technologies can raise ethical, social, and legal concerns, a reliable noninvasive test using cell-free fetal nucleic acids may in future form a part of national antenatal programs for detection of Down syndrome and other common genetic disorders.

Noninvasive Prenatal Diagnosis using Cell-Free Fetal DNA in Maternal Plasma: Clinical Applications

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

Diagnóstico pré-natal utilizando sangue materno / Prenatal diagnosis using maternal blood

As técnicas de diagnóstico pré-natal têm evoluído de forma acelerada, em especial aquelas que implicam menor invasão fetal. Nesse cenário, grande importância tem sido dispensada às técnicas de detecção de DNA fetal livre (DNA-fl) no sangue materno, as quais apresentam melhor relação custo-benefício quando comparadas às técnicas de enriquecimento e isolamento de células fetais. O DNA-fl pode ser avaliado de forma qualitativa ou quantitativa. Na primeira forma, detectam-se seqüências gênicas fetais de herança exclusivamente paterna com o intuito de selecionar fetos portadores de determinada doença, como a mutação para a fibrose cística ou a identificação de características fetais diferentes das maternas, como a incompatibilidade sanguínea RhD. Assim, os recursos propedêuticos invasivos ficariam reservados apenas para uma pequena parcela dessa população. Na segunda forma, quantifica-se a concentração de DNA-fl para se definir gestantes sob risco para eventos desfavoráveis relacionados a alterações da interface materno-fetal, como abortamento, trabalho de parto pré-termo e pré-eclâmpsia. Cabe às instituições de atenção pré-natal terciária a pesquisa e a aplicação desses recursos, objetivando a redução dos custos e sua maior disponibilização para a sociedade, conseqüentemente oferecendo diagnósticos mais precoces e menores taxas de morbimortalidade materna e fetal.

Prenatal diagnostic techniques are evolving greatly, especially noninvasive procedures. Great importance have been given to the free fetal DNA (ff-DNA) detection in maternal blood presents better cost/benefits relation when compared to enrichment and isolation of fetal cells. The ff-DNA can be analyzed qualitatively or quantitatively forms. In the first form, the fetal paternally derived genetic sequences are detected in order to select which fetuses are affected by determined disease, as the mutation for cystic fibrosis, of to identify fetal characterístics different from his mother's, as in RHD incompatibility. Then, invasive procedures could only be used in part of this population aiming more therapeutics aspects than diagnosis. In the second form, the ff-DNA quantilication can define pregnancies under risk for undesirable outcomes related to anomalies in the maternal-fetal interface, such as abortion, preterm labor and pre-eclampsia. It's expected that tertiary prenatal care centers research can work with those procedures in order to offer early diagnosis and lowest rates of fetomaternal morbimortality.