Clinical Chemistry 47: 1622-1626, 2001;
(Clinical Chemistry. 2001;47:1622-1626.)
© 2001 American Association for Clinical Chemistry, Inc.
Diagnosis of Trisomy 21 in Fetal Nucleated Erythrocytes from Maternal Blood by Use of Short Tandem Repeat Sequences
Osamu Samura1,1,
Satoshi Sohda1,
Kirby L. Johnson1,
Barbara Pertl2,
Steven Ralston3,
Laurent C. Delli-Bovi4 and
Diana W. Bianchi1a
1
Division of Genetics, Department of Pediatrics, and
3
Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, New England Medical Center and Tufts University School of Medicine, Boston, MA 02111.
2
Department of Obstetrics and Gynecology, University of Graz, A-8036 Graz, Austria.
4
Womens Health Services, Chestnut Hill, MA 02447.
aAddress correspondence to this author at: Division of Genetics, New England Medical Center, 750 Washington Street, Box 394, Boston, MA 02111. Fax 617-636-1469; e-mail DBianchi{at}Lifespan.org
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Abstract
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Background: The purpose of this study was to determine whether aneuploid fetal nucleated erythrocytes (NRBCs) could be detected in maternal blood through the use of fluorescent PCR amplification with polymorphic short tandem repeat (STR) markers as an alternative or complementary method to analysis by fluorescent in situ hybridization (FISH).
Methods: Peripheral blood samples were obtained from women who had just undergone termination of pregnancy because of fetal trisomy 21 (three cases, 47,XY,+21; four cases, 47,XX,+21). Candidate fetal cells were isolated by flow-sorting by antibodies to the 
chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed by the use of chromosome-specific probes for X, Y, and 21. Fetal NRBCs, as defined by the presence of staining, characteristic morphology, and three chromosome 21 signals, along with maternal leukocytes, defined as negative and two chromosome 21 signals, were micromanipulated separately and subjected to fluorescent PCR amplification of chromosome 21 STR markers (D21S11, D21S1411, and/or D21S1412).
Results: In five of seven cases analyzed, fetal NRBCs were aneuploid, as determined by the presence of triallelic or diallelic peaks of chromosome 21 sequences when compared with sequences from the maternal leukocytes.
Conclusions: Fluorescent PCR amplification of STRs can detect fetal aneuploidy and may be useful in the setting of poor hybridization efficiency with FISH analysis. These results suggest that combined fetal aneuploidy and single-gene diagnoses by the use of DNA microarrays may be feasible in the near future.
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Introduction
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The isolation of intact nucleated fetal cells from maternal blood and subsequent prenatal diagnosis of human aneuploidies have been demonstrated previously (1)(2)(3). The procedure initially involves the enrichment of nucleated erythrocytes (NRBCs),
2
followed by the morphologic identification of candidate fetal cells and the detection of fetal chromosomal abnormalities by fluorescence in situ hybridization (FISH).
The NRBC is one of the target fetal-cell types isolated from maternal peripheral blood for noninvasive genetic diagnosis. The "gold standard" for diagnosis of aneuploidy in fetal cells in maternal blood has been the detection of three chromosome-specific signals in the nucleus by FISH. In some high-risk cases in which sonographic abnormalities suggested fetal trisomy, we have observed many NRBCs that have a characteristic fetal cell morphology (bright -globin staining, a compact mononuclear shape, and symmetrical chromatin condensation), but have been unable to detect FISH signals or detect only a single X-chromosome signal. This may be attributable to the fact that a significant proportion of fetal NRBCs are undergoing apoptosis (4).
The advent of fluorescent PCR technology has enabled a more sensitive determination of product length than nonfluorescent PCR (5). This sensitivity of fluorescent PCR is provided by laser analysis, which allows for sizing at the single base-pair level (6). In addition, fluorescent PCR by the use of polymorphic short tandem repeats (STRs) has recently been applied to the rapid detection of trisomies (7)(8)(9)(10). The sensitivity of this technique approaches the single-cell level (11)(12)(13).
In an attempt to further maximize the potential of genetic analysis from fetal cells after isolation from maternal blood, we applied the techniques of FISH and PCR to the same fetal cell (14). Using this technique, we developed a fetal-cell recycling method to conclusively determine that female -positive NRBCs isolated from maternal blood were fetal in origin using DNA polymorphisms (15). The purpose of the present study was to develop a means of verifying that fetal NRBCs isolated from maternal blood were aneuploid on the basis of polymorphic STR markers by use of fluorescent PCR. This technique, if successful, would allow an alternative or complementary method to FISH in the interphase diagnosis of aneuploidy and may eventually facilitate analysis of fetal cells with DNA microarrays.
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Materials and Methods
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fetal-cell preparation
This work was performed under an institutionally approved protocol. Blood samples (14–16 mL) were collected into EDTA-coated tubes with informed consent from pregnant participants who were undergoing elective termination of pregnancy (17–22 weeks of gestation) because of fetal trisomy 21 (three cases, 47,XY,+21; four cases, 47,XX,+21). Samples were obtained within 2 h after the termination procedure. Separation and identification of NRBCs were performed with a phycoerythrin-conjugated antibody to the chain of fetal hemoglobin and Hoechst 33342 (16)(17). Hoechst 33342-positive and -chain NRBCs were separated by fluorescent-activated cell sorting and deposited onto positively charged microscope slides (Fisher Scientific Worldwide). Each sorted droplet contained both -chain-positive NRBCs and contaminating cells, such as maternal leukocytes (WBCs). Candidate fetal NRBCs were identified on the basis of bright fluorescent staining of the cytoplasm, a large nuclear to cytoplasmic ratio, and morphologic characteristics of the nuclei, including a compact mononuclear shape and symmetrical chromatin condensation. The location of candidate fetal NRBCs on the microscope slide was recorded. The dry slides containing the candidate fetal NRBCs were incubated in freshly prepared methanol–acetic acid (3:1 by volume) for 30 min at room temperature to fix cells to slides. The slides were air dried and kept at room temperature until FISH was performed.
fish procedure
Probes specific for chromosomes X, Y, and 21 (DXZ1, mixture of PHY10, and a mixture of 19.2, 19.2W4, 19.2W5, 18.3W12, respectively; Genzyme Genetics) were directly labeled by nick translation with Cy3-APS (cyanin 3–5-amino-propargyl-2'-deoxyuridine triphosphate; red; Amersham) and fluorescein-12-deoxyuridine triphos phate (green; Boehringer Mannheim). FISH analysis with the X and Y probes was performed as described previously (18)(19). For additional chromosome 21 probe studies, X and Y probes were removed by denaturing the slide with 700 mL/L formamide and 2x standard saline citrate (0.3 mol/L NaCl and 0.03 mol/L sodium citrate) at 72 °C for 2 min. The slide was then rehydrated in an ice-cold ethanol series (70%, 80%, and 95%) for 2 min each. The slide was air-dried, and the chromosome 21 probe was then denatured at 70 °C for 5 min and applied onto the denatured slide. The rehybridization was carried out as described previously (18). Images were visualized under a fluorescence microscope with a triple-band pass filter. Each nucleus was analyzed for the presence of chromosome 21 signals. A cell was identified as fetal if it had three chromosome 21 signals.
recovery of fetal NRBCs and fluorescent pcr amplification
The fetal-cell recycling technique was performed as described previously (14)(15). Between 7 and 20 fetal NRBCs were aspirated into a glass capillary pipette with a micromanipulator (Narishige Co.) under microscopic observation. Similarly, 20 WBCs considered to be maternal in origin were also retrieved from the same slide. All samples were then collected into separate microtubes containing 5 µL of proteinase K solution (400 mg/L proteinase K, 20 mmol/L dithiothreitol, 1.7 µmol/L sodium dodecyl sulfate, 10 mmol/L Tris buffer, 50 mmol/L potassium chloride), covered with 30 µL of mineral oil, and incubated at 37 °C overnight. The proteinase K was then inactivated by heating to 99 °C for 15 min. A multiplex fluorescent PCR assay was applied by the use of STR markers specific for chromosome 21 as described previously (9)(15). In case numbers 3, 4, and 7, two STR markers from chromosome 21 (D21S11 and D21S1411) were analyzed. In all other cases, three STR markers from chromosome 21 (D21S11, D21S1411, and D21S1412) were analyzed. The forward oligonucleotide primers for D21S11 and D21S1412 were 5' end-labeled with 5'-carboxy-fluorescein, whereas the forward primer D21S1411 was labeled with 2',7'-dimethyloxy 4',5'-dichloro 6-carboxy-fluorescein. In brief, PCR amplification was performed in a total volume of 25 µL containing genomic DNA, 200 nM each dNTP, 5–20 pmol of each primer, 10 mM Tris hydrochloride (pH 8.4), 50 mM potassium chloride, 3.0 mM magnesium chloride, and 1.5 U of Platinum Taq polymerase (Life Technologies). After denaturation at 94 °C for 10 min, PCR was carried out for 36 cycles at 94 °C for 48 s, 60 °C for 48 s, and 72 °C for 1 min, with a final extension step at 72 °C for 10 min. The negative controls, consisting of the 5 µL of Tris-EDTA (10 nM Tris-HCl and 1 mM EDTA, pH 8.0) buffer on the slide, underwent the same procedure. An additional negative control of 5 µL of water, in place of DNA, was used to test every PCR mixture. In one case, the negative controls showed the presence of contaminating DNA after PCR; therefore, the results from that experiment were discarded. The PCR products (2 µL) were diluted with 10 µL of distilled, deionized water. One microliter of the 12-µL diluted PCR products was mixed with 2.4 µL of loading buffer and 0.6 µL of Genescan-500 Rox (Applied Biosystems Inc.) containing the reference molecular size standard. Electrophoretic analysis was performed with 5% Long Ranger gels (FMC Bioproducts) and the model 377 DNA sequencer (Applied Biosystems Inc.). The amplification products were analyzed and relative fluorescent intensities calculated with Genescan software (Applied Biosystems Inc.) as described previously (10)(20).
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Results
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The results of the fluorescent PCR analysis are shown in Table 1
. In five of seven cases analyzed, the fetal NRBCs were aneuploid for chromosome 21 by detection of DNA polymorphisms. This determination was made when a pattern of three peaks of fluorescent activities with a ratio of 1:1:1 (trisomic triallelic; Fig. 1
) or a pattern of two peaks with a ratio 2:1 (trisomic dialleic) was detected by at least one set of STR markers for chromosome 21 (Fig. 2
). In addition, when an inherited allele was clearly different from the maternal WBCs, the allele was presumed to be paternally inherited and further proof of the fetal origin of the NRBCs. In cases 1, 3, 4, and 7, a trisomic triallelic pattern with D21S11 markers was detected. All four cases suggest that the trisomy is the result of nondisjunction occurring at maternal meiosis I. In cases 3 and 7, a trisomic triallelic pattern with the D21S1411 marker was detected. In case 1, a trisomic triallelic pattern with the D21S1412 marker was detected. In case 2, a trisomic diallelic pattern with the D21S11 marker was shown. Of note is that the mother in this case had a low-grade mosaicism (3%) for trisomy 21 in her skin fibroblasts. Her peripheral blood karyotype was diploid. Thus, the fetus in case 2 became aneuploid because of a mitotic, not meiotic error. In cases 5 and 6, aneuploidy could not be diagnosed definitively.
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Figure 1. Electrophotograms of amplification products from DNA of fetal NRBCs (A) and maternal WBCs (B) obtained from the same slide in case number 1.
The x axis displays the computed length of PCR products in base pairs, as determined automatically by use of an internal lane standard. The y axis displays fluorescent activity. For the D21S11 and D21S1412 markers, the fetal NRBCs and maternal WBCs both share two alleles and differ at the other allele.
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Figure 2. Electrophotograms of amplification products from DNA of fetal NRBCs (A) and maternal WBCs (B) obtained from the same slide in case number 2.
The x axis displays the computed length of PCR products in base pairs, as determined automatically by use of an internal lane standard. The y axis displays fluorescent activity. For the D21S11 markers, a pattern of two peaks with a ratio 2:1 (trisomic dialleic) was detected.
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Discussion
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To date, the most reliable diagnosis of aneuploidy in intact fetal cells in maternal blood has been accomplished by the observation of three chromosome-specific signals in the nucleus by FISH analysis. If the FISH technique fails to detect chromosome-specific signals in candidate fetal cells in maternal blood, then the PCR technique described here might be an another way of diagnosing fetal aneuploidy. In addition, slide preparations can be suboptimal and hybridization failures can occur when prior immunophenotyping has been performed (21).
Several authors have described the use of the technique of quantitative fluorescent PCR to detect fetal aneuploidy after invasive prenatal diagnosis, such as amniocentesis and chorionic villus sampling (7)(8)(9)(20). We previously developed a fetal-cell recycling method to conclusively determine that female -positive NRBCs isolated from maternal blood were fetal in origin, using DNA polymorphisms (15). In this study, we applied the same techniques on different cases to determine that NRBCs isolated from maternal blood were aneuploid.
As the number of STR markers that are analyzed increases, the determination of aneuploid status becomes more statistically significant. If only one STR marker for chromosome 21 is used, the PCR analysis may be uninformative if the fetal and maternal alleles are identical. However, if four or more STR markers for chromosome 21 are analyzed from a small number of the cells, it is more difficult to obtain optimal conditions for multiplex PCR. Also, with a large number of target sites amplified, the analysis of fluorescence peak position and intensity is difficult to interpret because of peak overlap. Therefore, in this study, we chose to analyze two or three STR markers for chromosome 21 per case.
Candidate fetal NRBCs sharing one or two alleles with the maternal WBCs, but containing another allele that is clearly different, suggests that the cells are indeed fetal in origin. In addition, if candidate fetal NRBCs have three alleles derived from chromosome 21 markers, these cells are conclusively determined to be aneuploid for chromosome 21 and suggest a meiosis I error. In trisomic patients, two different patterns of STR markers were observed: a pattern of two peaks with a ratio of 2:1 (trisomic diallelic) or a pattern of three single peaks (trisomic monoallelic). If enough fetal DNA is present for a quantitative analysis, it is easy to determine the trisomic diallelic pattern. However, if small amounts of target DNA (<10 fetal cells) are present, preferential gene amplification can occur and quantitative analysis might be unreliable.
In five of seven cases analyzed here, retrieved NRBCs were determined to be aneuploid by use of DNA polymorphisms. However, in another two cases we did not get informative results because there were too few NRBCs for quantitative analysis (<10 cells). Whereas single-cell PCR has been used successfully for preimplantation genetic diagnosis, the technique described here requires 10 or more fetal cells in the reaction because of the multiple chemical and physical exposures of the nuclei (i.e., fixation, sorting, the FISH procedures, and manipulation) that may degrade DNA. The requirement of having 10 or more fetal cells in the reaction may limit clinical applications because in most preprocedural blood samples obtained from pregnant women, there are fewer than 10 intact fetal cells present.
In conclusion, fetal-cell recycling, i.e., the isolation of fetal cells from maternal blood followed by FISH and PCR analyses, is informative with respect to fetal aneuploidy determination in some cases. Our data demonstrate that fluorescent PCR amplification of STRs is an alternative way to detect trisomy 21 in candidate fetal NRBCs. Furthermore, we show that genetic analysis of intact fetal cells, once isolated, can be maximized with PCR. This suggests that noninvasive fetal aneuploidy and single gene diagnosis by the use of a combination of FISH, PCR, and DNA microarrays may be feasible in the near future.
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Acknowledgments
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We would like to thank Vincent Falco for the flow-sorting of patient samples and Dr. Michael Berne and Ka Y. Ly for their assistance in analysis of PCR products. This work was supported by NICHD Contract N-HD4–3204 and a research grant from Genzyme Genetics.
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Footnotes
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1 Present address: Department of Obstetrics and Gynecology, Maternity and Perinatal Center, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan. 
2 Nonstandard abbreviations: NRBC, nucleated erythrocyte; FISH, fluorescence in situ hybridization; STR, short tandem repeat; and WBC, leukocyte.
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References
|
|---|
-
Bianchi DW, Mahr A, Zickwolf GK, Houseal TW, Flint AF, Klinger KW. Detection of fetal cells with 47,XY,+ 21 karyotype in maternal peripheral blood. Hum Genet 1992;90:368-370.[Web of Science][Medline]
[Order article via Infotrieve]
-
Gänshirt AD, Börjesson SR, Burschyk M, Dohr A, Garritsen HSP, Helmer E, et al. Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol 1993;30:194-201.
-
Simpson JL, Elias S. Isolating fetal cells from maternal blood. Advances in prenatal diagnosis through molecular technology. JAMA 1993;270:2357-2361.[Abstract/Free Full Text]
-
Sekizawa A, Samura O, Zhen DK, Falco V, Farina A, Bianchi DW. Apoptosis in fetal nucleated erythrocytes circulating in maternal blood. Prenat Diagn 2000;20:886-889.[Web of Science][Medline]
[Order article via Infotrieve]
-
Hattori M, Yoshioka K, Sakaki Y. High-sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations. Electrophoresis 1992;13:560-565.[Web of Science][Medline]
[Order article via Infotrieve]
-
Wells D, Sherlock JK. Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Prenat Diagn 1998;18:1389-1401.[Web of Science][Medline]
[Order article via Infotrieve]
-
Findlay I, Toth T, Matthews P, Marton T, Quirke P, Papp Z. Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis. J Assist Reprod Genet 1998;15:266-275.[Web of Science][Medline]
[Order article via Infotrieve]
-
Pertl B, Yau SC, Sherlock J, Davies AF, Mathew CG, Adinolfi M. Rapid molecular method for prenatal detection of Down’s syndrome. Lancet 1994;343:1197-1198.[Web of Science][Medline]
[Order article via Infotrieve]
-
Pertl B, Kopp S, Kroisel PM, Hausler M, Sherlock J, Winter R, Adinolfi M. Quantitative fluorescence polymerase chain reaction for the rapid prenatal detection of common aneuploidies and fetal sex. Am J Obstet Gynecol 1997;177:899-906.[Web of Science][Medline]
[Order article via Infotrieve]
-
Pertl B, Adinolfi M. Diagnosis of chromosomal aneuploidies using quantitative fluorescent PCR. Lo YMD eds. Clinical applications of PCR 1998:287-299 Humana Press Totowa, NJ. .
-
Findlay I, Taylor A, Quirke P, Frazier R, Urquhart A. DNA fingerprinting from single cells. Nature 1997;389:555-556.[Medline]
[Order article via Infotrieve]
-
Sherlock J, Cirigliano V, Petrou M, Tutschek B, Adinolfi M. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann Hum Genet 1998;62:9-23.[Web of Science][Medline]
[Order article via Infotrieve]
-
Blake D, Tan SL, Ao A. Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol Hum Reprod 1999;5:1166-1175.[Abstract/Free Full Text]
-
Sekizawa A, Samura O, Zhen DK, Falco V, Bianchi DW. Fetal cell recycling: diagnosis of gender and RhD genotype in the same fetal cell retrieved from maternal blood. Am J Obstet Gynecol 1999;181:1237-1242.[Web of Science][Medline]
[Order article via Infotrieve]
-
Samura O, Pertl B, Sohda S, Johnson KL, Sekizawa A, Falco VM, et al. Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin. Hum Genet 2000;107:28-32.[Web of Science][Medline]
[Order article via Infotrieve]
-
DeMaria MA, Zheng YL, Zhen D, Weinschenk NM, Vadnais TJ, Bianchi DW. Improved fetal nucleated erythrocyte sorting purity using intracellular antifetal hemoglobin and Hoechst 33342. Cytometry 1996;25:37-45.[Web of Science][Medline]
[Order article via Infotrieve]
-
Samura O, Sekizawa A, Zhen DK, Falco VM, Bianchi DW. Comparison of fetal cell recovery from maternal blood using high density gradient for initial separation step: 1.090 vs. 1.119 g/ml. Prenat Diagn 2000;20:281-286.[Web of Science][Medline]
[Order article via Infotrieve]
-
Zhen DK, Wang JY, Falco VM, Weber W, Delli-Bovi L, Bianchi DW. Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood. Prenat Diagn 1998;18:1181-1185.[Web of Science][Medline]
[Order article via Infotrieve]
-
Zheng YL, DeMaria M, Zhen D, Vadnais TJ, Bianchi DW. Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: preliminary observations on maternal samples. Prenat Diagn 1995;15:897-905.[Web of Science][Medline]
[Order article via Infotrieve]
-
Pertl B, Kopp S, Kroisel PM, Tului L, Brambati B, Adinolfi M. Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples. J Med Genet 1999;36:300-303.[Abstract/Free Full Text]
-
Oosterwijk JC, Knepfle CFHM, Mesker WE, Vrolijk H, Sloos WCR, Pattenier H, et al. Strategies for rare-event detection: an approach for automated fetal-cell detection in maternal blood. Am J Hum Genet 1998;63:1783-1792.[Web of Science][Medline]
[Order article via Infotrieve]
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Abstract
Introduction
