Development of non-invasive fetal DNA diagnosis from maternal blood

Haruo Takabayashi, Soryu Kuwabara, Toshihiko Ukita, Kazumi Ikawa, Kaoru Yamafuji and Tatsuhiro Igarashi

ADDRESSES:Department of Obstetrics and Gynecology, Kanazawa Medical University, Ishikawa,Japan(H. Takabayashi, MD, S. Kuwabara, MD); Ukita Hospital, Ishikawa, Japan(T. Ukita, MD, K. Yamafuji, MS); Ishikawa Health Service Association, Ishikawa, Japan(K. Ikawa, MS); and Hakuikan Hospital, Toyama, Japan(T. Igarashi, MD).

Correspondence to Dr. Haruo Takabayashi, Department of Obstetrics and Gynecology, Kanazawa Medical University, Daigaku, Uchinada, Kahoku-Gun, Ishikawa 920-02, Japan.

SUMMARY

Several attempts have been made to detect and retrieve fetal nucleated cells including nucleated erythrocytes(NRBCs), leukocytes and trophoblasts in maternal blood. We have recently developed a new method for non-invasive fetal DNA diagnosis from maternal blood. Peripheral blood granulocytes including NRBCs were isolated by a discontinuous density gradient method using Percoll(Pharmasia). NRBCs were found and retrieved at a single cell level using a micromanipulator under a microscope. To determine whether the origin of NRBCs was maternal or fetal, NRBCs were analysed by polymerase chain reaction(PCR) amplification to determine the presence of Y chromosome-specific repeat sequence in mothers carrying male fetuses. We were successful in predicting fetal sex accurately in 10 out of 11 samples taken from maternal blood. This new technique opens up fetal DNA diagnosis from maternal blood during the first trimester of pregnancy to the whole population because there is no risk to the fetus or the mother.

Key words: fetal DNA diagnosis, fetal nucleated erythrocyte, maternal blood, micromanipulation, polymerase chain reaction

INTRODUCTION

Nucleated cells of fetal origin have been reported to be present in maternal blood in the majority of pregnancies. Fetomaternal transfusion is theoretically possible as early as the end of 4 weeks of gestation, when vascularization of the villi and the pumping action of the fetal heart begin. Fetal cells in maternal blood offer an alternative source of specimens to those obtained by invasive techniques such as amniocentesis, chorionic villus sampling, or percutaneous umbilical blood sampling. The non-invasive recovery of fetal cells from maternal blood has great potential to revolutionize fetal medicine. It has been estimated that up to 80 percent of Down syndrome infants are conceived by women under 35 years of age(Holmes,1978). These women are generally not considered candidates for fetal DNA diagnosis, because of the risks of invasive diagnostic procedures. The ability to retrieve fetal DNA information from maternal blood would enable all women to undergo fetal DNA analysis. It is already clear that fetal cells can be isolated from maternal blood by flow sorting or magnetic sorting and can be analysed by PCR or fluorescence in situ hybridization(FISH)(Bianch et al.,1993; Ganshirt-Ahlert et al.,1993; Hamada et al.,1993; Simpson et al.,1993; Zheng et al.,1993). However there is no evidence that fetal cells can be retrieved selectively and individually at a single cell level. NRBCs are a good target cell population for fetal DNA diagnosis because they are unlikely to circulate in the peripheral blood of a normal adult. The purpose of this paper is to report a new technique for the detection, retrieval and analysis of single fetal NRBCs that cross the placental barrier and circulate within the maternal blood.

MATERIALS AND METHODS

Samples(2 ml) of peripheral venous blood were collected with informed consent in EDTA tubes from 60 pregnant women from 4 to 40 weeks of gestation. 20 samples of peripheral venous blood were also obtained from normal adult males or non- pregnant women. The isolation of NRBCs from the peripheral blood was achieved by using a discontinuous density gradient method with Percoll. Percoll is a suspension of polyvinylpyrrolidone coated 21nm diameter silica particles with a density of 1.13g/ml and an osmolality of 20 mosmol/kg. Aliquots of blood collected in EDTA were gently layered onto discontinuous density Percoll gradients. After centrifugation at 400 g for 30 min, the granulocytes with NRBCs at the interface of 1.075 g/ml and 1.085 g/ml Percoll gradients were collected and washed with phosphate-buffered saline. A thin smear was made on a microscope slide. The granulocytes with NRBCs on the slide were stained by the Pappenheim method and examined microscopically for NRBCs. NRBCs were found and retrieved using a micromanipulator under a microscope. PCR(Saiki et al.,1985; Saiki et al.,1988) amplification of the DYZ1 family was used to confirm the fetal origin of nucleated erythrocytes in the maternal blood. We used Taq polymerase and oligonucleotides Y1.1 and Y1.2 encompassing 149 base pairs of a repeated sequence of the Y chromosome for PCR(Kogan et al.,1987; Handyside et al.,1990). 40 cycles of PCR were then carried out: a denaturation step at 94ªÚ for 30 sec; an annealing step at 64ªÚ for 90 sec; and a final polymerase extension step at 72ªÚ for 15 min. 10°æl of the PCR product was subjected to electrophoresis in a 4.5% agarose gel. The gel was visualised under ultraviolet light after it had been stained with ethidium bromide. Controls were set up with a single female fetal NRBC, a single female adult lymphocyte, a single male fetal NRBC, a single male adult lymphocyte and a cell- free medium. Fetal sex as predicted by PCR was compared with the sex of the baby in the 11 cases of delivery in this study.

RESULTS

The presence of NRBCs in the enriched cellular elements from 60 maternal blood samples, as well as from normal adult controls, was analysed. Figure 1 shows the presence of NRBCs in 20 healthy normal males or non-pregnant females and 60 pregnant women at various stages of gestation. NRBCs were found in 33 out of 60 maternal samples. The number of detected NRBCs in each case ranged from 1 to 22 (average 4.1). The data indicated that NRBCs were detected as early as 8 weeks of gestation but not later than 24 weeks of gestation. No NRBCs were found in the 20 adult controls. These fetal NRBCs were successfully retrieved using a micromanipulator under a microscope as shown in Figure 2. Figure 3 shows that the single NRBCs removed from maternal blood were fetal in origin. The Figure confirms that a Y-specific band can be detected in DNA from a single male cell after PCR amplification. PCR analysis, of all 11 samples with a single NRBC, revealed that 5 out of 6 samples from mothers with male fetuses contained Y-specific sequences but none of the 5 samples from mothers with female fetuses contained Y-specific sequences(Table 1). Thus, fetal sex was predicted accurately in 10 out of 11 samples with single NRBCs that were taken from maternal blood.

DISCUSSION

We were successful in retrieving NRBCs from maternal blood using a micromanipulator and also in analysing NRBCs in a single cell level by PCR. Maternal venipuncture is a technically simple, rapid and relatively risk-free method of obtaining fetal cells. Micromanipulation seems to be a rather simple and reliable method of retrieving NRBCs compared to flow sorting or magnetic sorting. Sensitive and specific analytical methods such as PCR and FISH are necessary to study these rare NRBCs. With strict precautions against contamination, this sophisticated new technique would allow the possibility of non-invasive fetal DNA diagnosis from maternal blood and could ultimately be a substitute for invasive techniques. Several biologic questions remain - the way of fetal cell transfer, the biologic meaning of fetal cell transfer and the frequency of fetal cells in maternal blood. At present, we are attempting to analyse NRBCs retrieved from maternal blood using PCR and FISH on interphase cells for fetal DNA diagnosis. Further refinement in methodology is needed in order to apply this new technique to clinical purposes.

ACKNOWLEDGEMENTS

This work was supported by grants from the Ministry of Education, Japan, and Kanazawa Medical University. We wish to thank ª@ª@ª@ª@ª@Ms.Y. Nishida for her expert assistance in the preparation of this manuscript.

REFERENCES

Bianchi, D.W., Zickwolf, G.K., Yih, M.C., et al.(1993).Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood, Prenat. Diagn., 13, 293-300.Ganshirt-Ahlert, D., Borjesson-Stoll, R.,Burschyk, M., et al.(1993). Detection of fetal ª@trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting, Am. J. Reprod. Immunol., 30, 2-3. Hamada, H., Arinami, T., Kubo, T., Hamaguchi, H., Iwasaki, H.(1993). Fetal nucleated cells in maternal peripheral blood: Frequency andrelationship to gestational age, Hum. Genet., 91, 427-432.Handyside, A.H., Kontoglanni, E.H., Hardy, K., Winston, R.M.L.(1990). Pregnancies from biopsied human preimplantation embryos sexed by ª@ Y-specific DNA amplification, Nature, 344, 768-ª@770. Holmes, L.B.(1978). Genetic counselling for the ª@ older pregnant women: New data and questions, ª@N.Engl.J. Med., 298,1419-1421.Kogan, K.C., Doherty, M., Gitschier, J.(1987). ª@ An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences, N. Engl. J. Med., 317,985-990.Saiki, R.K., Scharf, S., Faloona, F., et ª@al.(1985). Enzymatic amplification of °Ë-ª@ª@ª@ª@ globin genomic sequences and restriction site ª@analysis for diagnosis of sickle cell anemia, Science, 230,1350-1354. Saiki, R.K., Gelfand, D., Stoffel, S., et al.(1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science, 239,487-491.Simpson, J.L., Elias, S.(1993). Isolating fetalcells from maternal blood: Adavances inprenatal diagnosis through molecular technology, J. Am. Med. Assoc., 270,2357-2361. Zheng, Y.I., Carter, N.P., Price, C.M., et al.(1993). Prenatal diagnosis from maternalblood: Simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolatedby negative magnetic cell sorting, J. Med. ª@ª@ª@Genet., 30,1051-1056.

CAPTIONS

Table 1: A single nucleated erythrocyte isolation from maternal blood.Figure1: Presence of nucleated erythrocytes in the Percoll-enriched population from peripheral blood.Figure2: Nucleated erythrocyte(NRBC) isolation using a micromanipulator.Arrow indicates a fetal nucleated erythrocyte. (a) a fetal NRBC found on the slide; (b) a fetal NRBC after treatment with a 0.25% trypsin in phosphate-buffered saline;(c) a fetal NRBC detached from slide by holding pipette; (d) a fetal NRBC removed and placed in a biopsy pipette(diameter 10µm). Figure3: Amplification products of the DYZ1 family.A strong 149bp band is present in male cells(arrow).Lane 1, marker; lane 2, no DNA; lane 3, a single nucleated erythrocyte(NRBC) from a pregnant woman with a female fetus at 11 weeks gestation(wg); lane 4, a single NRBC from a pregnant woman with a female fetus at 20 wg; lane 5, a single NRBC from a pregnant woman with a male fetus at 10 wg; lane 6, a single NRBC from a female fetus; lane 8, a single lymphocyte from a female; lane 9, a single NRBC from a male fetus; lane 10, a single lymphocyte from a male; lane 11, 50pg, female DNA; lane 12, 50pg, male DNA; lane 13, marker.