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PRENATAL DIAGNOSIS USING FETAL CELLS AND FREE FETAL DNA IN MATERNAL BLOOD
METABOLIC AND GENETIC SCREENING
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PRENATAL DIAGNOSIS USING FETAL CELLS AND FREE FETAL DNA IN MATERNAL BLOOD Wolfgang Holzgreve, MD, MS, Drhc, and Sinuhe Hahn, PhD
Research in prenatal diagnosis over the past decade has been characterized by a focus on the development of safe and accurate alternatives to current invasive procedures. This is largely due to the high falsepositive rates, which is of the order of 5%, of current noninvasive methods such as serum analyte screening or ultrasonography for the detection of fetal chromosomal aneuploidies. A additional factor that has to be considered is the demographic change that has taken place in the developed world, whereby women are generally of a more advanced age when considering childbearing. The latter is, of course, associated with an increased risk for a fetal aneuploidy of maternal origin. As these couples generally only have one or two children, many are reluctant to expose it to the procedure-related risk of an invasive prenatal diagnostic test. A further issue is that no current noninvasive method is able to detect Mendelian genetic disorders affecting the fetus. There is no doubt that the availability of the human genome sequence will revolutionize medical genetics and thereby also greatly extend the scope of prenatal diagnosis. Hence, these various factors have helped to fuel the current quest for practical efficacious noninvasive alternatives that are capable of detecting a wide spectrum of fetal genetic issues. Two methods that have emerged as being capable of addressing these issues are the isolation of fetal cells from the blood of pregnant women as well as the recent discovery of cell-free fetal DNA in the maternal circulation. From the Department of Obstetrics and Gynecology, University of Basel, Basel, Switzerland
CLINICS IN PERINATOLOGY VOLUME 28 * NUMBER 2 *JUNE 2001
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SUCCESSFUL ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD Even though there is considerable historic evidence for the presence of fetal cells in the maternal circulation of trophoblasts in the lungs of women who had died from such as the discovery by the German pathologist Schmorl at the turn of the 19th century, their reliable isolation from maternal blood has proved to be a difficult technical ~hallenge.~,~~ This is due, on the one hand, to the scarcity of fetal cells in maternal blood, which is of the order of 1 in lo6 to 1 in lo7 maternal nucleated cells.32Furthermore, a suitable fetal target cell had to be chosen. Trophoblasts have been determined to be less than ideal candidates, as the lack of specific antibodies makes them difficult to enrich for. Furthermore, since many are multinucleate, they are less suited for fetal 27 Fetal lymphocytes, on the other hand, have chromosomal analy~is.~, the ability to persist for years postpartum, and hence, there is a real risk of obtaining cells from a previous ~ r e g n a n c y . ~ , ~ ~ Consequently, the cell type that has emerged as the most suitable is the fetal erythroblasts, also termed nucleated red blood cell (NRBC).31,33 This cell type is abundant in the early fetal circulation and rare in the periphery of normal adults. Furthermore, in contrast to fetal lymphocytes, it is short-lived. Fetal NRBCs can also be potentially identified by the expression of proteins, which are more abundant in fetal NRBCs than adult ones, such as the fetal and embryonic hemoglobins. Their high level expression of other antigens, such as the transferring receptor (CD71) and glycophorin A (GPA) also greatly facilitates their easy en27 ri~hment.~, Because of their scarcity, adequate enrichment strategies had to developed to enable their retrieval from maternal blood samples. The two most widely used technologies are fluorescent activated cell sorting (FACS),pioneered by the group of Bianchi? and the magnetic equivalent, MACS or magnetic cell sorting, which was introduced for this task by our Both technologies rely on the interaction of specific monoclonal antibodies with the target cell. In the instance of FACS, these antibodies are labeled with a fluorescent dye. Generally for the isolation of fetal NRBCs, anti-gamma globin antibodies are used.'O These cells are also doubled labeled with a fluorescent dye that binds to the nucleus. The sorting on the basis of these two parameters ensures that nucleated red cells are retrieved and not denucleated fetal erythrocytes. Suitably dual labeled fluorescing cells are retrieved from the mixture by the aid of a computer-assisted laser technology. Cell sorting by MACS employs antibodies that are conjugated with micromagnetic particles. Here antibodies that bind to the cell surface are used, such as anti-CD71 or anti-GPA, and not intra-cellularly such as for the fetal her no glob in^.^^, 33, 53 The retention of suitably antibody bound cells is facilitated by a strong magnetic field during which unbound cells are flushed out by several washing steps.
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Several other methods that have been explored include differential density gradients, selective lysis of maternal cells, and charge flow ~eparation.~, 27 Unlike MACS and FACS, neither of these others methods has been tested in any large-scale trials. Another technical obstacle that had to be overcome was how to analyze the chromosomal content of the few fetal cells retrieved from the maternal circulation. Because these fetal NRBCs were not actively dividing and because of their small number, it would be impossible to use standard cytogenetic methods. This issue was addressed by the then newly developed method of fluorescent in-situ hybridization, whereby single interphase chromosomes could be rendered visible by the use of chromosome specific fluorescently labeled DNA probes and a suitably equipped fluorescent micros~ope.~~ By the combination of these two separate enrichment and identification strategies, we and others in the early 1990s were able to identify fetal aneuploidies using fetal cells enriched from maternal blood prior to an invasive procedure.6,24, 46 THE NIH FUNDED NIFTY STUDY
These encouraging findings and further reports presented at a seminal meeting held in 1994 no doubt encouraged the National Institutes of Health (NIH) to explore the feasibility of using this technology for the identification of fetal aneuploidies. For this purpose, they initiated the so-called NIFTY study (National Institutes of Child Health and Development Fetal Cell Isolation Study), the aim of which was to determine the relative efficacy of these emerging technologies for the identification of fetal aneupl~idies.'~ At the close of the first phase of this study, close to 3000 blood samples obtained from pregnant women who had an elevated risk for l 8 Two bearing a fetus with a chromosomal aberration were e~amined.~, of the participating laboratories used MACS forms of enrichment, whereas two other groups used different FACS protocols. The preliminary analysis of these data indicated that fetal aneuploidies could be detected with a higher specificity than current noninvasive methods. There is also some suggestion that MACS enrichment procedures may be more efficient than comparable FACS ones. These issues will be resolved in the second phase of this study. This study has also highlighted the possibility of examining multiple fetal chromosomes by multi-color FISH" or by repeatedly examining the same cell in multiple rounds of FISH.59,6o In our own studies, in which we have focused mainly on the determination of fetal sex, we have obtained sensitivities close to 60% when using one XY positive cell as being indicative that the women was pregnant with a son.26The specificity, however, was quite low, being of the order of 70%. This could be increased to more than 95% if three or more XY positive cells were observed, which was, however, accompa-
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nied by a concomitant drop in sensitivity to below 20%. This result implies that more emphasis will have to be placed on improving the efficiency of fetal cell recovery, for which purpose we have investigated the use of various density gradients as well as new more specific antibodies.", 53 A further important issue addressed by this study is the question of whether pregnant women would feel coerced to undergo an involuntary prenatal diagnosis by the introduction of such new techn~logies.~~ This study, and our own extension, has indicated that no such reservation exists. On the contrary, many couples, especially those involved in an assisted reproductive technology program would welcome the introduction of such a noninvasive prenatal diagnostic methodology. In the extension of this study, several new developments will be addressed, including the examination of fetal Mendelian genetic disorders as well as the possibility of culturing fetal hemopoietic progenitor cells isolated from the maternal circulation. THE ANALYSIS OF FETAL MENDELIAN GENETIC DISORDERS USING PCR
It is probably fitting that the first report that clearly indicated the feasibility of using fetal NRBCs isolated from maternal blood for the analysis of mendelian genetic disorders was made by Kan and Dozy:7 as this group had originally pioneered the prenatal analysis of fetal 52 genetic disorders, specifically hemogl~binopathies.~~, In their landmark article, Cheung and colleagues used a very similar MACS enrichment procedure as to that established by our group, in that fetal NRBCs were enriched for using anti-CD71 antib~dies.'~ Putative fetal NRBCs were identified by immunohistochemistry for zeta globin and isolated by micromanipulation under a microscope. Pooled fetal NRBCs were then examined for the particular paternal and maternal hemoglobin mutations in question. The correct fetal genotype was made in all of the cases examined. This study, hence, strongly indicated the scope of future examinations. Indeed, in a recent collaboration with DiNaro and colleagues, we were able to confirm the validity of this approach, by correctly identifying the fetal genoytpe in all six of the cases examined.20Similar observation for other fetal loci have also been made by other researchers including those made by the groups of Takabayashi, Sekizawa, and von EggelingZ7 Our own investigations have focused on several different fetal loci. On the one hand, it has been our conviction that fetal NRBCs cannot be reliably identified solely on the basis of a purported fetal antigen, such a zeta globin, but that the irrevocable identification can only be made by a genetic analysis. For this purpose, we have investigated the use of microsatellite patterns, also termed genetic finger printing. By the use of such loci, we have shown that fetal cells can be reliably distinguished from maternal cells.22
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These studies also indicated that the PCR analysis of single NRBCs is particularly prone to allele drop out, a phenomenon, whereby only one allele of a pair is represented because of inadequate amplification of the other allele. We were, however, able to show that the analvsis of at least five single fetal NRBCS would yield a diagnostic accuraci of over 990 22, 25 In addition, as a model system we have explored the feasibility of determining the fetal rhesus D genotype in pregnancies at risk for fetal hemolytic disease because of a rhesus D incompatibility. Because the rhesus D gene is absent in rhesus D individuals, the PCR assay is relatively simple, being akin to the demonstration of the fetal Y chromosome in a female genetic ba~kground.~ For this purpose, we developed a multiplex PCR assay in which both the presence of the fetal rhesus D status and sex, by an examination for the Y chromosome, could be detected in single isolated cells.54Our examination of 19 samples obtained from rhesus D pregnant women indicated that NRBCs could be identified in 14 of them. This result implies that using current enrichment strategies that NRBCs may not always be detectable. In every one of these 14 instances, however, we were able to determine correctly the fetal genotype for both loci examined. Our study furthermore showed that approximately half of the NRBCs in maternal blood were of fetal origin, thereby settling an old and contentious issue.27 A current drawback of these investigations has been the actual micromanipulation of the putative fetal NRBCs from the microscope slide. To date all investigations have used finely drawn microcapillaries with which the identified is lifted off the slide; a tedious and difficult 29 For this purpose, we have examined the use of new laserprocedure.27, mediated micromanipulation In the one system developed by Liotta and colleagues at the NIH,21 the target cell is fixed covalently to a thin plastic film by the pulse of an infrared laser. This membrane is conveniently located at the base of a cap, which fits to standard PCR reaction vessels, thereby permitting the rapid analysis of the transferred cells. In our hands, it has been difficult reliably to isolate individual cells by this method. Indeed, this system seems to be more suited for the capture of multiple individually identified cells from tissue sections. The other system, as described by Schutze and c011eagues,4~relies on a laser pulse that propels the target cell directly into a PCR reaction vessel located above the microscope slide. Although somewhat more tricky to use than the other laser capture system, this technology does seem to permit the reliable isolation of single cells from the enriched preparation on the microscope slide. /I.
CULTURE OF FETAL CELLS
The feasibility of culturing fetal erythroid progenitor cells from maternal blood was first alluded to in a short report made by Lo et al.45
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This tantalizing possibility would permit the elimination of several serious limitations associated with the analysis of isolated fetal cells. First, the problem of allele drop out no longer is relevant when dealing with a large clonal expansion of cells. Furthermore, the ability of analyse cells undergoing active cell division raises the possibility of obtaining a full karyotype, still the gold standard for many cytogeneticists, by a noninvasive manner. As attractive as this goal may be, it has proved to be quite elusive with few successful reports being substantiated by competing labora55 Another problem with most approaches that have been pubtorie~.'~, lished to date is that the maternal blood sample used for the culture experiment was obtained after an invasive procedure, or even after the termination of pregnancy, which is known to lead to a massive influx of fetal cells into the maternal periphery.56Those attempts to use samples, which have not been pre-enriched for fetal progenitor cells by such artificial means, have generally been less s u c c e ~ s f u l36. ~ ~ ~ This failure is commonly attributed to the excessive expansion of maternal cells, which out compete the few fetal cells in the culture preparation. In an attempt to address this issue, several groups have been investigating whether different cytokine combinations would permit the preferential expansion of fetal progenitor cells over the maternal cells present in the enriched cell fraction. Our own studies, using fetal blood samples have indicated that early fetal hemopoietic progenitor cells have a greater expansion potential than adult progenitor cells and that this can be boosted by culturing the cells with a combination of the cytokines flt-3 ligand and thrombop ~ e i t i nSimilar . ~ ~ observations regarding the culture of fetal blood samples has been made by the group of Fisk in London.14 Conversely, Bohmer and colleagues have determined that the application of interleukin-3 (IL-3) into the culture medium may promote the outgrowth of fetal erythroid progenitor^.'^ In another experimental setting, this group has determined that transforming growth factor-b (TGF-b) can influence globin expression of adult erythroid progenitor cells.12 All these experiments have been performed on fetal blood samples or artificial mixtures, and most attempts to generate fetal cell cultures from maternal blood samples have failed. This is most evident in recent reports by Huber et a1,36who scanned more than 750,000 cultured cells by FISH for the X and Y chromosomes without detecting any cells from pregnancies bearing male fetuses. Similarly, the group of Ferguson Smith in Oxford has not been able to detect in fetal cells in maternal bloodderived culture^.'^ A further issue that should be addressed is whether this test will be cost- and time-effective, as the it is doubtful whether the difficult laborintensive and expensive 2-week culture period and subsequent genetic analysis will be within the framework of modern health care schemes. Hence, it is foreseeable that considerable technical and social hurdles will have to be overcome before the use of this option becomes widespread.
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THE PRESENCE OF FREE FETAL DNA IN MATERNAL BLOOD
It is doubtful that when the Swiss plant physiologists Anker and Stroun first reported their observation that cell-free DNA could be detected in the fluids of various organisms that they could have imagined the diverse Dennis Lo and colleagues extended upon the observation that free tumor-derived DNA could be detected in the plasma of cancer patients,I6 by reasoning that since the placenta shared numerous traits with invasive malignant tissue, that free fetal DNA may also be present in the circulation of pregnant women.44This turned out to indeed be the instance as male fetal DNA was in fact readily detectable in maternal serum and plasma samples.44 A caveat of this approach is that since free maternal DNA is also present in the maternal circulation, only those fetal genetic loci can be examined which are not present in the maternal genome (e.g., the Y chromosome and the rhesus D gene in rhesus d pregnant women).28,41 Despite these limitations, this technology has proved to be very useful for determining the fetal sex in pregnancies at risk for an X-linked disorder and fetal rhesus D status in pregnancies at risk for hemolytic disease of the newborn. By using the sensitive multiplex PCR we have established for the analysis of single fetal cells, we have been able to simultaneously examine for these two fetal loci in maternal plasma samples.62Our studies, which confirm observations made by Faas, Lo, Bischoff and colleagues, demonstrate that this method can be reliably used for the determination of fetal loci on second trimester plasma samples.41 A further extension of this work was made by Amicucci and coworkers, who showed that this approach can be used to detect the presence or absence of paternal mutations in heterozygous Mendelian disorders.2 In this manner, if the paternal mutation cannot be detected, it can safely be assumed that the fetus will be an unaffected heterozygote for the disorder in question. Of course, this method can only be used where the disorder being investigated is the result of different paternal and maternal mutations. Because of the ease with which this test can be performed, it is foreseeable that it will form the basis for the first series of clinically applicable fetal genetic tests on a noninvasive basis. FETAL CELL TRAFFIC AND FREE FETAL DNA AND FETAL ANEUPLOIDY
Previous studies using FISH on enriched fetal cells performed by our group and others had shown that the numbers of cells with three signals indicative of a fetal aneuploidy were significantly elevated in pregnancies with aneuploid fetuses when compared with normal
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matched controls.', 24,46 This result implied that there may be more fetal cells present in the maternal circulation in pregnancies with certain fetal chromosomal aberrations. To address this issue more closely, Bianch and colleagues developed a quantitative PCR procedure that relied on the incorporation of radiolabeled nucleotides into the PCR product.* The assessment of the amount radio-label would then provide a measure of the quantity of target input material, in this instance a Y chromosome specific sequence. In their study, in which they examined the total amount of nucleated fetal cells in unsorted maternal blood samples, they observed approximately 2- to 5-fold more fetal nucleated cells in fetuses affected by trisomy 21 and trisomy 13 when compared with normal male fetuses. No similar elevation was observed for trisomy 18.*These data, hence, strongly suggested that certain fetal aneuploidies may lead to the presence of more fetal cells in the maternal periphery, thereby loading the system in favor of their detection. In an analogous manner, but by using a more exact real-time PCR assay, the so-called Taqman system,3O whereby each cycle of the PCR amplification is actively monitored, Lo and colleagues have shown that the levels of free fetal DNA are very low at the beginning of pregnancy and increase dramatically towards term.40By the use of this technology, it was natural that Lo and Bianchi would also examine the levels of free fetal DNA in fetuses affected by trisomy 21.42Their study showed that these levels were elevated approximately 2-fold in fetuses affected by Downs' syndrome when compared with matched controls. As we had established a similar assay in our laboratory, we performed a similar examination,2*but extended it to examine other fetal aneuploidies in collaboration with the group of Laird Jackson in Philadelphia.61Remarkably, we observed a very similar pattern to that observed by Bianchi an colleagues for fetal cell numbers: in that we determined significant elevations for fetuses with trisomy 21 and trisomy 13 but not for trisomy These results could be attributed in part to the placental structure peculiar to these aneuploidies, in that those of fetuses with trisomy 21 and trisomy 13 are usually of a normal size but are characterized by several structural lesions, whereas those of trisomy 18 are invariably of a smaller size. Taken together, these results do suggest that the quantitative analysis of fetal cells and free fetal DNA in maternal blood may be helpful in screening for certain fetal aneuploidies. FETAL CELL TRAFFIC AND FREE FETAL DNA IN PREECLAMPSIA
A novel observation we have made in our laboratory is that significantly elevated numbers of erythroblasts were observed in the peripheral blood of pregnant women with preeclamp~ia.~~ Because at that time we were not certain as to whether these erythroblasts were of fetal or
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maternal origin, we performed a case control study in which only pregnancies with singleton male fetuses were examined.34This study again showed that significantly elevated numbers of NRBCs were present in the periphery of pregnant women with preeclampsia when compared with the control cohort. Since we were, however, exclusively studying pregnancies with male fetuses, we could next examine these samples with FISH for the X and Y chromosomes to determine the proportion of male fetal cells. This analyses irrevocably showed that indeed a large proportion of the erythroblasts were of fetal origin and that correspondingly there was a significant influx of fetal cells into the maternal periphery in pree~lampsia.~~ In a separate analysis, the group of Lo examined the levels of free fetal DNA in serum samples obtained from preeclamptic patients.43This study obtained remarkably similar results to our study regarding fetal cells, in that significantly elevated amounts of free fetal DNA were 43 present in the circulation of pregnant women with pree~lampsia.~~, By using the same real-time PCR, we had established for the quantiwe next tative analysis of free fetal DNA from aneuploid fetuses:' examined free fetal levels in a large cohort of serum samples collected from the institutions of Sibai in Memphis and Ylikorkola in Helsinki.63 This study, which was performed in a blinded manner, showed that indeed free fetal levels were significantly elevated in preeclamptic pregnancies when compared with the control cohort. As we were, however, also measuring the total amount of free DNA in these samples, which is to a large extent (over 95%) derived from the mother, we were able to make a rather fortuitous observation, in that we found that these DNA levels were also similarly elevated. Furthermore, we were able to show that these increments corresponded to disease severity. In addition, we were able to show that the increments in free fetal and maternal DNA corresponded to each other in pregnancies affected by preeclampsia but not in normal ~ r e g n a n c i e s . ~ ~ This finding addresses some fundamental issues regarding the cause of preeclampsia, in that we show both the fetal and maternal compartments are affected by the disorder and that the degree of this correlates with disease severity. The fact that the levels of free fetal and maternal DNA correlate to each other in preeclampsia can best be reconciled to a model whereby localized placental damage leads to a more systemic damage of maternal tissues. Such a mechanism has been proposed by Redman and colleagues whereby the release of placental micro-particles, .~~ also termed SMPs, leads to damage of the maternal e n d ~ t h e l i u mAs such, the release of free fetal DNA could be speculated to be associated with placental breakdown and release of SMPs, which would then lead to endothelial damage and the subsequent release of free maternal DNA. Further experiments will be necessary to test this new hypothesis. FUTURE DIRECTIONS
It is clear from the above discourse that fetal cells in the maternal periphery do exist and that proof of concept studies have shown that
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they can be used for the analysis of fetal aneuploidies and inherited Mendelian genetic disorders. It is, however, also quite clear that in order for their analysis to provide an effective noninvasive method for prenatal diagnosis, several quantum improvements have to be made. These include optimized enrichment protocols that are not as labor intensive as current ones. The provision of automated MACS devices is a step in the right direction. A further serious current drawback is the inability to efficiently recognize putative fetal NRBCs. Here it is envisaged to use automated imaging devices combined with some form of immunohistochemistry. The rapid PCR analysis of single fetal cells could be improved by the adaptation of laser micromanipulation protocols and robotic PCR cyclers. The improvement of whole genome amplification procedures will no doubt also greatly facilitate this type of analysis. There is no doubt that the discovery of cell-free fetal DNA will greatly aid in the rapid determination of fetal loci such as rhesus D. The quantitation of this material by real-time PCR may also provide an additional screening aid for fetal aneuploidies and perhaps other pregnancy related disorders such as preeclampsia. References 1. Al-Mufti R, Hambley H, Farzaneh F, et al: Investigation of maternal blood enriched for fetal cells: Role in screening and diagnosis of fetal trisomies. Am J Med Genet 85:6675, 1999 2. Amicucci P, Gennarelli M, Novelli G, et al: Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 46:301-302, 2000 3. Aubin JT, Le Van Kim C, Mouro I, et al: Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification, Br J Haematol 98:35&364, 1997 4. Bianchi DW Current knowledge about fetal blood cells in the maternal circulation. J Perinatol Med 26:175-185, 1998 5. Bianchi DW, Flint AF, Pizzimneti MF, et al: Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci USA 873279-3283, 1990 6. Bianchi DW, Mahr A, Zickwolf GK, et al: Detection of fetal cells with 47,XY,+21 karyotype in maternal peripheral blood. Hum Genet 90:36%370,1998 7. Bianchi DW, Simpson JL, Jackson LG, et al: Fetal cells in maternal blood: NIFTY clinical trial interim analysis. Prenat Diagn 19:994-995, 1999 8. Bianchi DW, Williams JM, Sullivan LM, et al: PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 615322-829, 1997 9. Bianchi DW, Zickwolf GK, Weil GJ, et al: Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 93:705-708, 1996 10. Bianchi DW, Zickwolf GK, Yih MC, et a1 Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood. Prenat Diag 13:293-300, 1993 11. Bischoff FZ, Lewis DE, Nguyen DD, et al: Prenatal diagnosis with use of fetal cells isolated from maternal blood: Five-color fluorescent in situ hybridization analysis on flow-sorted cells for chromosomes X, Y, 13, 18, and 21. Am J Obstet Gynecol 179:203-209, 1998 12. Bohmer RM, Campbell TA, Bianchi DW: Selectively increased growth of fetal hemoglobin-expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta. Blood 95:2967-2974, 2000 13. Bohmer RM, Johnson KL, Bianchi D W Differential effects of interleukin-3 on fetal and adult erythroid cells in culture: Implications for the isolation of fetal cells from maternal blood. Prenat Diagn 20:640447, 2000
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14. Campagnoli C, Fisk N, Overton T, et al: Circulating hematopoietic progenitor cells in first trimester fetal blood. Blood 95:1967-1992, 2000 15. Chen H, Griffin DK, Jestice K, et a1 Evaluating the culture of fetal erythroblasts from maternal blood for non-invasive prenatal diagnosis. Prenat Diagn 18:883-892, 1998 16. Chen X, Bonnefoi H, Diebold-Berger S, et al: Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer. Clin Cancer Res 59297-2303, 1999 17. Cheung MC, Goldberg JD, Kan YW: Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood. Nat Genet 14:264-268, 1996 18. de la Cruz F, Shifrin H, Elias S, et al: Low false-positive rate of aneuploidy detection using fetal cells isolated from maternal blood. Fetal Diagn Ther 13:380, 1998 19. de la Cruz F, Shifrin H, Elias S, et a1 Prenatal diagnosis by use of fetal cells isolated from maternal blood. Am J Obstet Gynecol 173:1354-1355, 1995 20. DiNaro E, Ghezzi F, Vitucci A, et al: Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 6:571-574, 2000 21. Emmert-Buck MR, Bonner W, Smith PD, et a1 Laser capture microdissection. Science 274998-1001,1996 22. Garvin AM, Holzgreve W, Hahn S Highly accurate analysis of heterozygous loci by single cell PCR. Nucleic Acids Res 26:3468-3472, 1998 23. Ganshirt D, Smeets FW, Dohr A, et al: Enrichment of fetal nucleated red blood cells fi-om the maternal circulation for prenatal diagnosis: Experiences with triple density gradient and MACS based on more than 600 cases. Fetal Diagn Ther 13: 276-286, 1998 24. Ganshirt-Ahlert D, Borejesson-Stoll R, Burschyk M, et al: Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol 303194-201, 1993 25. Hahn S, Garvin A, DiNaro E, et al: Allele drop out can occur in alleles differing by a single nucleotide and is not alleviated by preamplification nor minor template increments. Genetic Testing 2351-355, 1998 26. Hahn S, Kiefer V, Brombacher V, et a1 Fetal cells in maternal blood An update from Basel. Eur J Obstet Gynecol Reprod Biol 85:lOl-104, 1999 27. Hahn S, Sant R, Holzgreve W Fetal cells in maternal blood: Current and future perspectives. Mol Hum Reprod 4:515-521, 1998 28. Hahn S, Zhong XU, Biirk MR, et al: Multiplex and realtime quantitative PCR on fetal DNA in maternal plasma: A comparison with fetal cells isolated from maternal blood. In Circulating DNA in Plasma. Ann NY Acad Sci 906148-152,2000 29. Hahn S, Zhong XU, Troeger C, et al: Current applications of single cell PCR. Cell Mol Life Sci 579G105, 2000 30. Heid C, Stevens J, Livak K, et al: Real time quantitative PCR. Genome Res 6:8&994, 1996 31. Herzenberg LA, Bianchi DW, Schroder J, et al: Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Natl Acad Sci USA 76:1453-1455, 1979 32. Holzgreve W, DiNaro E, Garvin AM, et al: Prenatal diagnosis using fetal cells enriched from maternal blood Croat Med J 39:115-120, 1998 33. Holzgreve W, Garritsen HS, Ganshirt Ahlert D: Fetal cells in the maternal circulation. J Reprod Med 37410418, 1998 34. Holzgreve W, Ghezzi F, DiNaro E, et a1 Disturbed fetomaternal cell traffic in preeclampsia. Obstet Gynecol 91: 669-672, 1998 35. Holzgreve W, Hahn S Novel molecular biological approaches for the diagnosis of preeclampsia. Clin Chem 45:451-452, 1999 36. Huber K, Bittner J, Worofka B, et a1 Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells. Prenat Diagn 20479-486,2000 37. Kan YW, Dozy Ah4: Antenatal diagnosis of sickle-cell anaemia by DNA analysis of amniotic-fluid cells. Lancet 2:910-912, 1978 38. Klinger K, Landes G, Shook D, et al: Rapid detection of chromosome aneuploidies in
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uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am J Hum Genet 51:5565, 1992 39. Knight M, Redman CW, Linton EA, et al: Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. Br J Obstet Gynaecol 105:632440, 1998 40. Lo DYM, Tein MSC, Lau TK, et a1 Quantitative analysis of fetal DNA in maternal plasma and serum: Implications for noninvasive prenatal diagnosis. Am J Hum Genet 62768-775. 1998 41. Lo YM: Fetal DNA in maternal plasma. Ann NY Acad Sci 906:141-147, 2000 42. Lo YM, Lau TK, Zhang J, et al: Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 45:1747-1751, 1999 43. Lo YM, Leung TN, Tein MS, et al: Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 453184-188, 1999 44. Lo YMD, Corbetta N, Chamberlain PF, et a1 Presence of fetal DNA in maternal plasma and serum. Lancet 350:485487, 1999 45. Lo YMD, Morey AL, Wainscoat JS, et a1 Culture of fetal erythroid cells from maternal peripheral blood. Lancet 344: 264-265, 1999 46. Price TO, Elias S, Wachtel SS, et a1 Prenatal diagnosis with fetal cells isolated from matemal blood by multiparameter flow cytometry. Am J Obstet Gynecol 165:17311737, 1991 47. Sch&orl G: Pathologisch - anatomische Untersuchungen ueber Publereklampsie. Vogel, Leipzig, 1893 48. Schroder 1. Tilikainen A, De La Chauelle A Fetal leucocvtes in maternal circulation after de1i;ery. Transplantation 17:34c360, 1974 49. Schiitze K, Clement-Sengenwald A: Catch and move: Cut or fuse. Nature, 368:667669, 1994 50. Smits G, Holzgreve W, Hahn S: An examination of different Percoll density gradients in combination with MACS for the enrichment of fetal erythroblasts. Arch Gynecol Obstet 2633160-163, 2000 51. Stroun M, Maurice P, Vasioukhin V, et al: The origin and mechanism of circulating DNA. Ann NY Acad Sci 906:161-168,2000 52. Taylor JM, Dozy A, Kan YW, et a1 Genetic lesion in homozygous alpha thalassaemia (hydrops fetalis). Nature 251:392-393, 1974 53. Troeger C, Holzgreve W, Hahn S A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS. Prenatal Diag 19:521-526, 2000 54. Troeger C, Zhong XY, Burgemeister R, et al: Approximately half the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod 5:1162-1165, 1999 55. Valerio D, Aiello R, Altieri V, et al: Culture of fetal erythroid progenitor cells from maternal blood for non-invasive prenatal genetic diagnosis Prenat Diagn 16:10731082, 1996 56. Valerio D, Altieri V, Cavallo D, et al: Detection of fetal trisomy 18 by short-term culture of maternal peripheral blood. Am J Obstet Gynecol 183:222-225, 2000 57. Wyrsch A, dalle Carbonare V, Jansen W, et al: Umbilical cord blood from preterm human fetuses is rich in committed and primitive hematopoietic progenitors with high proliferative and self-renewal capacity. Exp Hematol271338-1345, 1999 58. Zamerowski S, Lumley M, Arreola RA, et al: The psychosocial impact on highrisk pregnant women of a noninvasive prenatal diagnostic test. Fetal Diagn Ther 14:125-126, 1999 59. Zhen DK, Wang JY, Falco VM, et al: Poly-FISH A technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood Prenat Diagn 18:1181-1185, 1998 60. Zheng YL, Carter NP, Price CM, et al: Prenatal diagnosis from maternal blood: Simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting. J Med Genet 30:1051-1056, 1993 61. Zhong XY, Biirk MR, Troeger C, et al: Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 20:795-798, 2000
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62. Zhong XY, Holzgreve W, Hahn S: Detection of fetal rhesus D and sex from fetal DNA in maternal plasma by multiplex PCR. Br J Obstet Gynaecol 107:766-769, 2000 63. Zhong XU, Laivouri H, Livingston JC, et al: Elevation of both maternal and fetal extracellular circulatory DNA concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 184414-419, 2001
Address reprint requests to Wolfgang Holzgreve, MD, MS, Drhc Chairman and Director Department of Obstetrics and Gynecology University of Basel Schanzenstrasse 46, CH 4031 Basel Switzerland e-mail: wholzgreve8uhbs.ch
0095-5108/01 $15.00
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PRENATAL DIAGNOSIS USING FETAL CELLS AND FREE FETAL DNA IN MATERNAL BLOOD Wolfgang Holzgreve, MD, MS, Drhc, and Sinuhe Hahn, PhD
Research in prenatal diagnosis over the past decade has been characterized by a focus on the development of safe and accurate alternatives to current invasive procedures. This is largely due to the high falsepositive rates, which is of the order of 5%, of current noninvasive methods such as serum analyte screening or ultrasonography for the detection of fetal chromosomal aneuploidies. A additional factor that has to be considered is the demographic change that has taken place in the developed world, whereby women are generally of a more advanced age when considering childbearing. The latter is, of course, associated with an increased risk for a fetal aneuploidy of maternal origin. As these couples generally only have one or two children, many are reluctant to expose it to the procedure-related risk of an invasive prenatal diagnostic test. A further issue is that no current noninvasive method is able to detect Mendelian genetic disorders affecting the fetus. There is no doubt that the availability of the human genome sequence will revolutionize medical genetics and thereby also greatly extend the scope of prenatal diagnosis. Hence, these various factors have helped to fuel the current quest for practical efficacious noninvasive alternatives that are capable of detecting a wide spectrum of fetal genetic issues. Two methods that have emerged as being capable of addressing these issues are the isolation of fetal cells from the blood of pregnant women as well as the recent discovery of cell-free fetal DNA in the maternal circulation. From the Department of Obstetrics and Gynecology, University of Basel, Basel, Switzerland
CLINICS IN PERINATOLOGY VOLUME 28 * NUMBER 2 *JUNE 2001
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SUCCESSFUL ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD Even though there is considerable historic evidence for the presence of fetal cells in the maternal circulation of trophoblasts in the lungs of women who had died from such as the discovery by the German pathologist Schmorl at the turn of the 19th century, their reliable isolation from maternal blood has proved to be a difficult technical ~hallenge.~,~~ This is due, on the one hand, to the scarcity of fetal cells in maternal blood, which is of the order of 1 in lo6 to 1 in lo7 maternal nucleated cells.32Furthermore, a suitable fetal target cell had to be chosen. Trophoblasts have been determined to be less than ideal candidates, as the lack of specific antibodies makes them difficult to enrich for. Furthermore, since many are multinucleate, they are less suited for fetal 27 Fetal lymphocytes, on the other hand, have chromosomal analy~is.~, the ability to persist for years postpartum, and hence, there is a real risk of obtaining cells from a previous ~ r e g n a n c y . ~ , ~ ~ Consequently, the cell type that has emerged as the most suitable is the fetal erythroblasts, also termed nucleated red blood cell (NRBC).31,33 This cell type is abundant in the early fetal circulation and rare in the periphery of normal adults. Furthermore, in contrast to fetal lymphocytes, it is short-lived. Fetal NRBCs can also be potentially identified by the expression of proteins, which are more abundant in fetal NRBCs than adult ones, such as the fetal and embryonic hemoglobins. Their high level expression of other antigens, such as the transferring receptor (CD71) and glycophorin A (GPA) also greatly facilitates their easy en27 ri~hment.~, Because of their scarcity, adequate enrichment strategies had to developed to enable their retrieval from maternal blood samples. The two most widely used technologies are fluorescent activated cell sorting (FACS),pioneered by the group of Bianchi? and the magnetic equivalent, MACS or magnetic cell sorting, which was introduced for this task by our Both technologies rely on the interaction of specific monoclonal antibodies with the target cell. In the instance of FACS, these antibodies are labeled with a fluorescent dye. Generally for the isolation of fetal NRBCs, anti-gamma globin antibodies are used.'O These cells are also doubled labeled with a fluorescent dye that binds to the nucleus. The sorting on the basis of these two parameters ensures that nucleated red cells are retrieved and not denucleated fetal erythrocytes. Suitably dual labeled fluorescing cells are retrieved from the mixture by the aid of a computer-assisted laser technology. Cell sorting by MACS employs antibodies that are conjugated with micromagnetic particles. Here antibodies that bind to the cell surface are used, such as anti-CD71 or anti-GPA, and not intra-cellularly such as for the fetal her no glob in^.^^, 33, 53 The retention of suitably antibody bound cells is facilitated by a strong magnetic field during which unbound cells are flushed out by several washing steps.
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Several other methods that have been explored include differential density gradients, selective lysis of maternal cells, and charge flow ~eparation.~, 27 Unlike MACS and FACS, neither of these others methods has been tested in any large-scale trials. Another technical obstacle that had to be overcome was how to analyze the chromosomal content of the few fetal cells retrieved from the maternal circulation. Because these fetal NRBCs were not actively dividing and because of their small number, it would be impossible to use standard cytogenetic methods. This issue was addressed by the then newly developed method of fluorescent in-situ hybridization, whereby single interphase chromosomes could be rendered visible by the use of chromosome specific fluorescently labeled DNA probes and a suitably equipped fluorescent micros~ope.~~ By the combination of these two separate enrichment and identification strategies, we and others in the early 1990s were able to identify fetal aneuploidies using fetal cells enriched from maternal blood prior to an invasive procedure.6,24, 46 THE NIH FUNDED NIFTY STUDY
These encouraging findings and further reports presented at a seminal meeting held in 1994 no doubt encouraged the National Institutes of Health (NIH) to explore the feasibility of using this technology for the identification of fetal aneuploidies. For this purpose, they initiated the so-called NIFTY study (National Institutes of Child Health and Development Fetal Cell Isolation Study), the aim of which was to determine the relative efficacy of these emerging technologies for the identification of fetal aneupl~idies.'~ At the close of the first phase of this study, close to 3000 blood samples obtained from pregnant women who had an elevated risk for l 8 Two bearing a fetus with a chromosomal aberration were e~amined.~, of the participating laboratories used MACS forms of enrichment, whereas two other groups used different FACS protocols. The preliminary analysis of these data indicated that fetal aneuploidies could be detected with a higher specificity than current noninvasive methods. There is also some suggestion that MACS enrichment procedures may be more efficient than comparable FACS ones. These issues will be resolved in the second phase of this study. This study has also highlighted the possibility of examining multiple fetal chromosomes by multi-color FISH" or by repeatedly examining the same cell in multiple rounds of FISH.59,6o In our own studies, in which we have focused mainly on the determination of fetal sex, we have obtained sensitivities close to 60% when using one XY positive cell as being indicative that the women was pregnant with a son.26The specificity, however, was quite low, being of the order of 70%. This could be increased to more than 95% if three or more XY positive cells were observed, which was, however, accompa-
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nied by a concomitant drop in sensitivity to below 20%. This result implies that more emphasis will have to be placed on improving the efficiency of fetal cell recovery, for which purpose we have investigated the use of various density gradients as well as new more specific antibodies.", 53 A further important issue addressed by this study is the question of whether pregnant women would feel coerced to undergo an involuntary prenatal diagnosis by the introduction of such new techn~logies.~~ This study, and our own extension, has indicated that no such reservation exists. On the contrary, many couples, especially those involved in an assisted reproductive technology program would welcome the introduction of such a noninvasive prenatal diagnostic methodology. In the extension of this study, several new developments will be addressed, including the examination of fetal Mendelian genetic disorders as well as the possibility of culturing fetal hemopoietic progenitor cells isolated from the maternal circulation. THE ANALYSIS OF FETAL MENDELIAN GENETIC DISORDERS USING PCR
It is probably fitting that the first report that clearly indicated the feasibility of using fetal NRBCs isolated from maternal blood for the analysis of mendelian genetic disorders was made by Kan and Dozy:7 as this group had originally pioneered the prenatal analysis of fetal 52 genetic disorders, specifically hemogl~binopathies.~~, In their landmark article, Cheung and colleagues used a very similar MACS enrichment procedure as to that established by our group, in that fetal NRBCs were enriched for using anti-CD71 antib~dies.'~ Putative fetal NRBCs were identified by immunohistochemistry for zeta globin and isolated by micromanipulation under a microscope. Pooled fetal NRBCs were then examined for the particular paternal and maternal hemoglobin mutations in question. The correct fetal genotype was made in all of the cases examined. This study, hence, strongly indicated the scope of future examinations. Indeed, in a recent collaboration with DiNaro and colleagues, we were able to confirm the validity of this approach, by correctly identifying the fetal genoytpe in all six of the cases examined.20Similar observation for other fetal loci have also been made by other researchers including those made by the groups of Takabayashi, Sekizawa, and von EggelingZ7 Our own investigations have focused on several different fetal loci. On the one hand, it has been our conviction that fetal NRBCs cannot be reliably identified solely on the basis of a purported fetal antigen, such a zeta globin, but that the irrevocable identification can only be made by a genetic analysis. For this purpose, we have investigated the use of microsatellite patterns, also termed genetic finger printing. By the use of such loci, we have shown that fetal cells can be reliably distinguished from maternal cells.22
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These studies also indicated that the PCR analysis of single NRBCs is particularly prone to allele drop out, a phenomenon, whereby only one allele of a pair is represented because of inadequate amplification of the other allele. We were, however, able to show that the analvsis of at least five single fetal NRBCS would yield a diagnostic accuraci of over 990 22, 25 In addition, as a model system we have explored the feasibility of determining the fetal rhesus D genotype in pregnancies at risk for fetal hemolytic disease because of a rhesus D incompatibility. Because the rhesus D gene is absent in rhesus D individuals, the PCR assay is relatively simple, being akin to the demonstration of the fetal Y chromosome in a female genetic ba~kground.~ For this purpose, we developed a multiplex PCR assay in which both the presence of the fetal rhesus D status and sex, by an examination for the Y chromosome, could be detected in single isolated cells.54Our examination of 19 samples obtained from rhesus D pregnant women indicated that NRBCs could be identified in 14 of them. This result implies that using current enrichment strategies that NRBCs may not always be detectable. In every one of these 14 instances, however, we were able to determine correctly the fetal genotype for both loci examined. Our study furthermore showed that approximately half of the NRBCs in maternal blood were of fetal origin, thereby settling an old and contentious issue.27 A current drawback of these investigations has been the actual micromanipulation of the putative fetal NRBCs from the microscope slide. To date all investigations have used finely drawn microcapillaries with which the identified is lifted off the slide; a tedious and difficult 29 For this purpose, we have examined the use of new laserprocedure.27, mediated micromanipulation In the one system developed by Liotta and colleagues at the NIH,21 the target cell is fixed covalently to a thin plastic film by the pulse of an infrared laser. This membrane is conveniently located at the base of a cap, which fits to standard PCR reaction vessels, thereby permitting the rapid analysis of the transferred cells. In our hands, it has been difficult reliably to isolate individual cells by this method. Indeed, this system seems to be more suited for the capture of multiple individually identified cells from tissue sections. The other system, as described by Schutze and c011eagues,4~relies on a laser pulse that propels the target cell directly into a PCR reaction vessel located above the microscope slide. Although somewhat more tricky to use than the other laser capture system, this technology does seem to permit the reliable isolation of single cells from the enriched preparation on the microscope slide. /I.
CULTURE OF FETAL CELLS
The feasibility of culturing fetal erythroid progenitor cells from maternal blood was first alluded to in a short report made by Lo et al.45
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This tantalizing possibility would permit the elimination of several serious limitations associated with the analysis of isolated fetal cells. First, the problem of allele drop out no longer is relevant when dealing with a large clonal expansion of cells. Furthermore, the ability of analyse cells undergoing active cell division raises the possibility of obtaining a full karyotype, still the gold standard for many cytogeneticists, by a noninvasive manner. As attractive as this goal may be, it has proved to be quite elusive with few successful reports being substantiated by competing labora55 Another problem with most approaches that have been pubtorie~.'~, lished to date is that the maternal blood sample used for the culture experiment was obtained after an invasive procedure, or even after the termination of pregnancy, which is known to lead to a massive influx of fetal cells into the maternal periphery.56Those attempts to use samples, which have not been pre-enriched for fetal progenitor cells by such artificial means, have generally been less s u c c e ~ s f u l36. ~ ~ ~ This failure is commonly attributed to the excessive expansion of maternal cells, which out compete the few fetal cells in the culture preparation. In an attempt to address this issue, several groups have been investigating whether different cytokine combinations would permit the preferential expansion of fetal progenitor cells over the maternal cells present in the enriched cell fraction. Our own studies, using fetal blood samples have indicated that early fetal hemopoietic progenitor cells have a greater expansion potential than adult progenitor cells and that this can be boosted by culturing the cells with a combination of the cytokines flt-3 ligand and thrombop ~ e i t i nSimilar . ~ ~ observations regarding the culture of fetal blood samples has been made by the group of Fisk in London.14 Conversely, Bohmer and colleagues have determined that the application of interleukin-3 (IL-3) into the culture medium may promote the outgrowth of fetal erythroid progenitor^.'^ In another experimental setting, this group has determined that transforming growth factor-b (TGF-b) can influence globin expression of adult erythroid progenitor cells.12 All these experiments have been performed on fetal blood samples or artificial mixtures, and most attempts to generate fetal cell cultures from maternal blood samples have failed. This is most evident in recent reports by Huber et a1,36who scanned more than 750,000 cultured cells by FISH for the X and Y chromosomes without detecting any cells from pregnancies bearing male fetuses. Similarly, the group of Ferguson Smith in Oxford has not been able to detect in fetal cells in maternal bloodderived culture^.'^ A further issue that should be addressed is whether this test will be cost- and time-effective, as the it is doubtful whether the difficult laborintensive and expensive 2-week culture period and subsequent genetic analysis will be within the framework of modern health care schemes. Hence, it is foreseeable that considerable technical and social hurdles will have to be overcome before the use of this option becomes widespread.
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THE PRESENCE OF FREE FETAL DNA IN MATERNAL BLOOD
It is doubtful that when the Swiss plant physiologists Anker and Stroun first reported their observation that cell-free DNA could be detected in the fluids of various organisms that they could have imagined the diverse Dennis Lo and colleagues extended upon the observation that free tumor-derived DNA could be detected in the plasma of cancer patients,I6 by reasoning that since the placenta shared numerous traits with invasive malignant tissue, that free fetal DNA may also be present in the circulation of pregnant women.44This turned out to indeed be the instance as male fetal DNA was in fact readily detectable in maternal serum and plasma samples.44 A caveat of this approach is that since free maternal DNA is also present in the maternal circulation, only those fetal genetic loci can be examined which are not present in the maternal genome (e.g., the Y chromosome and the rhesus D gene in rhesus d pregnant women).28,41 Despite these limitations, this technology has proved to be very useful for determining the fetal sex in pregnancies at risk for an X-linked disorder and fetal rhesus D status in pregnancies at risk for hemolytic disease of the newborn. By using the sensitive multiplex PCR we have established for the analysis of single fetal cells, we have been able to simultaneously examine for these two fetal loci in maternal plasma samples.62Our studies, which confirm observations made by Faas, Lo, Bischoff and colleagues, demonstrate that this method can be reliably used for the determination of fetal loci on second trimester plasma samples.41 A further extension of this work was made by Amicucci and coworkers, who showed that this approach can be used to detect the presence or absence of paternal mutations in heterozygous Mendelian disorders.2 In this manner, if the paternal mutation cannot be detected, it can safely be assumed that the fetus will be an unaffected heterozygote for the disorder in question. Of course, this method can only be used where the disorder being investigated is the result of different paternal and maternal mutations. Because of the ease with which this test can be performed, it is foreseeable that it will form the basis for the first series of clinically applicable fetal genetic tests on a noninvasive basis. FETAL CELL TRAFFIC AND FREE FETAL DNA AND FETAL ANEUPLOIDY
Previous studies using FISH on enriched fetal cells performed by our group and others had shown that the numbers of cells with three signals indicative of a fetal aneuploidy were significantly elevated in pregnancies with aneuploid fetuses when compared with normal
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matched controls.', 24,46 This result implied that there may be more fetal cells present in the maternal circulation in pregnancies with certain fetal chromosomal aberrations. To address this issue more closely, Bianch and colleagues developed a quantitative PCR procedure that relied on the incorporation of radiolabeled nucleotides into the PCR product.* The assessment of the amount radio-label would then provide a measure of the quantity of target input material, in this instance a Y chromosome specific sequence. In their study, in which they examined the total amount of nucleated fetal cells in unsorted maternal blood samples, they observed approximately 2- to 5-fold more fetal nucleated cells in fetuses affected by trisomy 21 and trisomy 13 when compared with normal male fetuses. No similar elevation was observed for trisomy 18.*These data, hence, strongly suggested that certain fetal aneuploidies may lead to the presence of more fetal cells in the maternal periphery, thereby loading the system in favor of their detection. In an analogous manner, but by using a more exact real-time PCR assay, the so-called Taqman system,3O whereby each cycle of the PCR amplification is actively monitored, Lo and colleagues have shown that the levels of free fetal DNA are very low at the beginning of pregnancy and increase dramatically towards term.40By the use of this technology, it was natural that Lo and Bianchi would also examine the levels of free fetal DNA in fetuses affected by trisomy 21.42Their study showed that these levels were elevated approximately 2-fold in fetuses affected by Downs' syndrome when compared with matched controls. As we had established a similar assay in our laboratory, we performed a similar examination,2*but extended it to examine other fetal aneuploidies in collaboration with the group of Laird Jackson in Philadelphia.61Remarkably, we observed a very similar pattern to that observed by Bianchi an colleagues for fetal cell numbers: in that we determined significant elevations for fetuses with trisomy 21 and trisomy 13 but not for trisomy These results could be attributed in part to the placental structure peculiar to these aneuploidies, in that those of fetuses with trisomy 21 and trisomy 13 are usually of a normal size but are characterized by several structural lesions, whereas those of trisomy 18 are invariably of a smaller size. Taken together, these results do suggest that the quantitative analysis of fetal cells and free fetal DNA in maternal blood may be helpful in screening for certain fetal aneuploidies. FETAL CELL TRAFFIC AND FREE FETAL DNA IN PREECLAMPSIA
A novel observation we have made in our laboratory is that significantly elevated numbers of erythroblasts were observed in the peripheral blood of pregnant women with preeclamp~ia.~~ Because at that time we were not certain as to whether these erythroblasts were of fetal or
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maternal origin, we performed a case control study in which only pregnancies with singleton male fetuses were examined.34This study again showed that significantly elevated numbers of NRBCs were present in the periphery of pregnant women with preeclampsia when compared with the control cohort. Since we were, however, exclusively studying pregnancies with male fetuses, we could next examine these samples with FISH for the X and Y chromosomes to determine the proportion of male fetal cells. This analyses irrevocably showed that indeed a large proportion of the erythroblasts were of fetal origin and that correspondingly there was a significant influx of fetal cells into the maternal periphery in pree~lampsia.~~ In a separate analysis, the group of Lo examined the levels of free fetal DNA in serum samples obtained from preeclamptic patients.43This study obtained remarkably similar results to our study regarding fetal cells, in that significantly elevated amounts of free fetal DNA were 43 present in the circulation of pregnant women with pree~lampsia.~~, By using the same real-time PCR, we had established for the quantiwe next tative analysis of free fetal DNA from aneuploid fetuses:' examined free fetal levels in a large cohort of serum samples collected from the institutions of Sibai in Memphis and Ylikorkola in Helsinki.63 This study, which was performed in a blinded manner, showed that indeed free fetal levels were significantly elevated in preeclamptic pregnancies when compared with the control cohort. As we were, however, also measuring the total amount of free DNA in these samples, which is to a large extent (over 95%) derived from the mother, we were able to make a rather fortuitous observation, in that we found that these DNA levels were also similarly elevated. Furthermore, we were able to show that these increments corresponded to disease severity. In addition, we were able to show that the increments in free fetal and maternal DNA corresponded to each other in pregnancies affected by preeclampsia but not in normal ~ r e g n a n c i e s . ~ ~ This finding addresses some fundamental issues regarding the cause of preeclampsia, in that we show both the fetal and maternal compartments are affected by the disorder and that the degree of this correlates with disease severity. The fact that the levels of free fetal and maternal DNA correlate to each other in preeclampsia can best be reconciled to a model whereby localized placental damage leads to a more systemic damage of maternal tissues. Such a mechanism has been proposed by Redman and colleagues whereby the release of placental micro-particles, .~~ also termed SMPs, leads to damage of the maternal e n d ~ t h e l i u mAs such, the release of free fetal DNA could be speculated to be associated with placental breakdown and release of SMPs, which would then lead to endothelial damage and the subsequent release of free maternal DNA. Further experiments will be necessary to test this new hypothesis. FUTURE DIRECTIONS
It is clear from the above discourse that fetal cells in the maternal periphery do exist and that proof of concept studies have shown that
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they can be used for the analysis of fetal aneuploidies and inherited Mendelian genetic disorders. It is, however, also quite clear that in order for their analysis to provide an effective noninvasive method for prenatal diagnosis, several quantum improvements have to be made. These include optimized enrichment protocols that are not as labor intensive as current ones. The provision of automated MACS devices is a step in the right direction. A further serious current drawback is the inability to efficiently recognize putative fetal NRBCs. Here it is envisaged to use automated imaging devices combined with some form of immunohistochemistry. The rapid PCR analysis of single fetal cells could be improved by the adaptation of laser micromanipulation protocols and robotic PCR cyclers. The improvement of whole genome amplification procedures will no doubt also greatly facilitate this type of analysis. There is no doubt that the discovery of cell-free fetal DNA will greatly aid in the rapid determination of fetal loci such as rhesus D. The quantitation of this material by real-time PCR may also provide an additional screening aid for fetal aneuploidies and perhaps other pregnancy related disorders such as preeclampsia. References 1. Al-Mufti R, Hambley H, Farzaneh F, et al: Investigation of maternal blood enriched for fetal cells: Role in screening and diagnosis of fetal trisomies. Am J Med Genet 85:6675, 1999 2. Amicucci P, Gennarelli M, Novelli G, et al: Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 46:301-302, 2000 3. Aubin JT, Le Van Kim C, Mouro I, et al: Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification, Br J Haematol 98:35&364, 1997 4. Bianchi DW Current knowledge about fetal blood cells in the maternal circulation. J Perinatol Med 26:175-185, 1998 5. Bianchi DW, Flint AF, Pizzimneti MF, et al: Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci USA 873279-3283, 1990 6. Bianchi DW, Mahr A, Zickwolf GK, et al: Detection of fetal cells with 47,XY,+21 karyotype in maternal peripheral blood. Hum Genet 90:36%370,1998 7. Bianchi DW, Simpson JL, Jackson LG, et al: Fetal cells in maternal blood: NIFTY clinical trial interim analysis. Prenat Diagn 19:994-995, 1999 8. Bianchi DW, Williams JM, Sullivan LM, et al: PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 615322-829, 1997 9. Bianchi DW, Zickwolf GK, Weil GJ, et al: Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 93:705-708, 1996 10. Bianchi DW, Zickwolf GK, Yih MC, et a1 Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood. Prenat Diag 13:293-300, 1993 11. Bischoff FZ, Lewis DE, Nguyen DD, et al: Prenatal diagnosis with use of fetal cells isolated from maternal blood: Five-color fluorescent in situ hybridization analysis on flow-sorted cells for chromosomes X, Y, 13, 18, and 21. Am J Obstet Gynecol 179:203-209, 1998 12. Bohmer RM, Campbell TA, Bianchi DW: Selectively increased growth of fetal hemoglobin-expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta. Blood 95:2967-2974, 2000 13. Bohmer RM, Johnson KL, Bianchi D W Differential effects of interleukin-3 on fetal and adult erythroid cells in culture: Implications for the isolation of fetal cells from maternal blood. Prenat Diagn 20:640447, 2000
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Address reprint requests to Wolfgang Holzgreve, MD, MS, Drhc Chairman and Director Department of Obstetrics and Gynecology University of Basel Schanzenstrasse 46, CH 4031 Basel Switzerland e-mail: wholzgreve8uhbs.ch