The Kanazawa Medical University Methodology and Experience to Date in Isolating and Analyzing Fetal Cells in Maternal Blood

Haruo Takabayashi, Soryu Kuwabara, Toshihiko Ukita, Kazumi Ikawa, Kaoru Yamafuji and Saori Habuta

Department of Obstetrics and Gynecology, Kanazawa Medical University,Uchinada,Kahoku,Ishikawa 920-02,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 SRL Ltd., Tokyo, Japan(S. Habuta, MS).

Key words: fetal DNA diagnosis, fetal nucleated erythrocyte, maternal blood, micro manipulation, PCR, FISH, PEP, CGH, FDD-MB, single cell analysis

8th International Conference on Early Prenatal Diagnosis Goa, India, June 4-8, 1996

Mr.Chairman, ladies & gentlemen.

I am very pleased to have this opportunity of talking to you today and presenting an overview of our research called The Kanazawa Medical University methodology and experience to date in isolating and analyzing fetal cells in maternal blood. Our lab is at Kanazawa Medical University, located between the sea of Japan and Kahoku Lake. Kanazawa is on the main island of Honshu in Japan.

Nucleated cells of fetal origin have been reported to be present in maternal blood in the majority of pregnancies. Several attempts have been made to detect and retrieve fetal nucleated cells including nucleated erythrocytes, leukocytes and trophoblasts in maternal blood. Because the number of fetal nucleated cells in maternal blood is extremely limited, there have been some trials using flow sorting or magnetic sorting as the collection method. Nucleated erythrocytes are a good target cell population for fetal DNA diagnosis because they are unlikely to circulate in the peripheral blood of a normal adult. We have recently developed a new method for non-invasive fetal DNA diagnosis from maternal blood. We were successful in retrieving nucleated erythrocytes from maternal blood using a micromanipulator and also in analyzing nucleated erythrocytes on a single cell level by PCR,FISH and PEP-PCR. The purpose of this presentation is to report a new technique for the detection, retrieval and analysis of single fetal nucleated erythrocytes that cross the placental barrier and circulate within the maternal blood.

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 ability to retrieve fetal DNA information from maternal blood would enable all women to undergo fetal DNA analysis. The non-invasive recovery of fetal cells from maternal blood has great potential to revolutionize fetal medicine.

The Herzenberg study demonstrated convincing evidence that fetal lymphocytes circulated in maternal blood as early as the 15th weeks of gestation. Additional studies have described the persistence of fetal lymphocytes in maternal blood for as long as 5 years post-partum. The relative longevity of these cells creates difficulties in establishing with certainty that the isolated cells derive from the current pregnancy. For this reason, many investigators are not actively pursuing isolation of fetal lymphocytes. As early as 1893, Schmorl described the frequent presence of trophoblasts in the lungs of women who had died of eclampsia. Douglas et al. demonstrated syncytiotrophoblasts in the circulating blood of pregnant women from 18 weeks of gestation until term and concluded that this migration of trophoblasts was a normal process in pregnancy. Goodfellow and Taylor documented circulating trophoblast cells in peripheral venous blood samples from six out of ten pregnant women. Trophoblasts were extracted by differential centrifugation and identified by indirect immunofluorescence with antitrophoblast microvillus antibody. An additional monoclonal antibody, H315, was initially described as specific for the recognition of trophoblast cell surface antigens. Although a preliminary study described a high percentage of H315 positive cells in the blood of pregnant women, these cells were subsequently shown to be maternal lymphocytes that had adsorbed the H315 antigen.

Nucleated erythrocytes are a good target cell population for fetal cell sorting experiments because they are unlikely to circulate in the peripheral blood of a normal adult. They are, however, present in significant numbers in the blood of early fetuses. Nucleated erythrocytes, by definition, contain a nucleus, and have a full complement of nuclear genes. Since nucleated erythrocytes are nearly completely differentiated , isolated nucleated erythrocytes are likely to derive from the pregnancy being studied. Finally, there are a thousand fold more red cells than white cells available for analysis after feto-maternal transfusion.

S-6 You can find nucleated erythrocytes very easily in fetal blood as shown in this slide.

S-7 Fetal erythrocytes usually contain HbF, so we can distinguish fetal erythrocytes from maternal erythrocytes by using HbF staining.

S-8 This slide shows the incidence of nucleated erythrocytes in the cord blood. It seems difficult to find nucleated erythrocytes in late gestational period even in the fetal blood.

S-9 This slide shows the strategy of our research. Samples of peripheral venous blood were collected with informed consent in EDTA tubes from pregnant women . The isolation of nucleated erythrocytes from the peripheral blood was achieved by using a discontinuous density gradient method with Percoll. A thin smear was made on a microscope slide. The granulocytes with nucleated erythrocytes on the slide were stained by the May-Giemsa method and examined microscopically for nucleated erythrocytes. Nucleated erythrocytes were found and retrieved using a micromanipulator under a microscope.

S-10 This slide shows how to isolate nucleated erythrocytes using Percoll. Aliquots of blood collected in EDTA were gently layered onto discontinuous density Percoll gradients. After centrifugation , the granulocytes with nucleated erythrocytes at the interface of Percoll gradients were collected and washed.

S-11 This slide shows the actual way to isolate nucleated erythrocytes using Percoll.

S-12 A thin smear is made on a microscope slide as shown in this slide.

S-13 Several nucleated erythrocytes are shown in fetal blood or in maternal blood or in Percoll-enriched population from maternal blood. A nucleated erythrocyte is indicated by the arrow in this slide. This nucleated erythrocyte is an orthochromatic erythroblast. The orthochromatic erythroblast is the most clearly differentiated nucleated erythrocyte, and is almost the same size as a mature erythrocyte. The nucleus/cytoplasm ratio is low, and the nucleus is extremely concentrated and non-structural.

S-14 You can find nucleated erythrocytes easily after the enrichment procedure in several stages of pregnancy as shown in this slide.

S-15 These are other nucleated erythrocytes after the enrichment procedure.

This is a micromanipulator that we are now using in our lab and Miss Yamafuji. She is a specialist for retrieving nucleated erythrocytes using a micromanipulator. Many scientists from all over the world have visited our lab to learn how to retrieve nucleated erythrocytes.

S-17 These fetal nucleated erythrocytes were successfully retrieved using a micromanipulator under a microscope as shown in this slide. The arrow indicates fetal nucleated erythrocytes.

S-18 The presence of nucleated erythrocytes in the enriched cellular elements from 91 maternal blood samples, as well as from normal adult controls was analyzed. This slide shows the presence of nucleated erythrocytes in 20 healthy normal males or non-pregnant females and 91 pregnant women at various stages of gestation and 19 peruperas. Nucleated erythrocytes were found in 82 out of 91 maternal samples. The earliest stage of gestation at which nucleated erythrocytes were detected was 5 weeks. And then nucleated erythrocytes were detected in all cases later than 8 weeks of gestation. Nucleated erythrocytes were found in 13 out of 19 perupera blood samples . No nucleated erythrocytes were found in the 20 adult controls.

S-19 The number of detected nucleated erythrocytes from 7 ml of maternal blood is shown in this slide. The average number increases according gestational weeks and then decreases after delivery.

S-20 PCR amplification of the DYZ1 family was used to confirm the fetal origin of nucleated erythrocytes in the maternal blood. 40 cycles of PCR were then carried out.

S-21 This slide shows that the single nucleated erythrocyte removed from maternal blood was fetal in origin. Fetal sex as predicted by PCR was compared with the sex of the baby at delivery. The figure confirms that a Y-specific band can be detected in DNA from a single male cell after PCR amplification.

S-22 PCR analysis, on all 11 samples with a single nucleated erythrocyte, revealed that 5 out of 6 samples from mothers carrying male fetuses contained Y-specific sequences, but none of the five samples from mothers carrying female fetuses contained Y-specific sequences. Thus, fetal sex was predicted accurately in 10 of 11 samples with a single nucleated erythrocyte that was taken from maternal blood.

S-23 TSD family was also successfully amplified by PCR .

S-24 PEP employs random 15-base oligonucleotides as primers, Taq polymerase and modified PCR reaction conditions. The probability of amplifying any sequence within the genome by this method is estimated to be at least 0.78, although significant locus-dependent variation affects amplification efficiency. We utilized this whole genome amplification system for prenatal single-cell analysis of Tay-Sacks disease and Duchenne or Becker muscular dystrophy and ZFX/ZFY sequences, and have assessed the potential application of this system to non-invasive prenatal DNA diagnosis using fetal nucleated erythrocytes obtained from maternal blood.

S-25 A single nucleated erythrocyte was collected into a microtube containing 5 µl of an alkaline lysis solution according to the method described by Li et al. DNA was extracted from the cell and amplified by the method of primer extension pre-amplification described by Zhang et al. 50 primer-extension cycles were performed in a thermocycler. Each sample was then divided into aliquots, upon which multiple DNA analyses were performed using the PCR.

S-26 We were also successful in applying the PEP-PCR technique to the May-Giemsa-stained nucleated erythrocytes of fetuses. DYZ1 and TSD exon in fetal nucleated erythrocytes are shown in this slide. DYZ1 bands could be detected in the DNA from single male cells after PCR and PEP-PCR. None of the DNA from single female cells contained DYZ1 bands. TSD bands could be detected in the DNA from single cells after PCR and PEP-PCR.

S-27 This slide is from Dr.Sekizawa, a Japanese medical doctor. He learned our technique 2 years ago and then applied it to the analysis of Duchenne or Becker muscular dystrophy exon and RhD exon. Sex and each DMD exon in fetal nucleated erythrocytes are shown in this slide. In ZFX/ZFY loci analyses, the nucleated erythrocyte has four fragments, and is confirmed to be male and of fetal origin. The maternal lymphocyte has two fragments, and is confirmed to be female and of maternal origin. This fetus showed no exon deletion.

S-28 A lymphocyte from a patient who had DMD with a known deletion of exon 50 was analyzed using the same procedure. 7 dystrophin exons and the ZFX/ZFY loci were simultaneously examined by PEP reactions. The deletion of exon 50 was correctly detected both in analysis from PEP reactions and in DNA analysis. Examination of the ZFX/ZFY loci of the lymphocyte showed that the patient was genotypically male.

S-29 Sex and RhD exon 7 in fetal nucleated erythrocytes are shown in this slide. The nucleated erythrocytes were confirmed to be of fetal origin and RhD positive.

The development of fluorescence in situ hybridization has opened the door to tests specifically designed to rapidly facilitate prenatal identification of the major chromosomal aneuploidies. Chromosome-specific DNA probe sets now exist for most human chromosomes. When used in conjunction with a fluorescent dye, hybridization of these probes can be used to identify specific chromosomes of interest. This technique can be applied to metaphase chromosome spreads. More importantly, in the context of analysis of fetal cells recovered from maternal circulation, numerical aberrations in interphase nuclei become very apparent. Although highly extended compared to metaphase chromosomes, each chromosome in the interphase nucleus occupies a discrete focal domain. After hybridization to fluorescently labeled chromosome-specific DNA probes, each chromosome is detected as a colored dot using engineered probes that are specific for that chromosome. The number of dots indicates the number of copies present in that specific chromosome. Thus, three dots after hybridization to a chromosome 21 probe set indicates trisomy 21. Different colored dots are produced by labeling each probe with a different fluorescent dye, so that multiple chromosomes can be analyzed simultaneously.

S-31 This slide shows a result from fluorescence in situ hybridization. Nucleated erythrocytes have been hybridized with a Y chromosome centromeric probe.

S-32 This slide also shows a result from fluorescence in situ hybridization. Nucleated erythrocytes have been hybridized with a 21 chromosome centromeric probe.

At present, we are attempting to analyze nucleated erythrocytes retrieved from maternal blood using PEP-CGH for fetal DNA diagnosis. It is also thought that the micromanipulation method will become more necessary if PEP-CGH analysis on the single cell level becomes possible in the near future. How do we envision CGH on single fetal cells? If you have an abnormal single cell that you've isolated from a pregnant woman, say with trisomy 18, you can take that cell and do PEP and whole genome PCR and label your amplification product with a red dye or rhodamine. At the same time, you can take a normal single cell from a male and do whole cell genome PCR with PEP and label it with a green dye. You mix those two together, and you use them as a probe essentially to a normal metaphase spread. Any areas that are different between these two and the normal metaphase will show up as red. So in this technique the entire chromosome 18 should look red because it is imbalanced. There is more material from chromosome 18 in one cell than in the other cell and in the normal metaphase. Therefore, this will be very useful, not only to detect aneuploidy, but to detect more subtle rearrangements that would require a karyotype and a lot of dividing cells to analyze it, and that's not possible with a single cell isolated from maternal blood.

S-34 This slide shows a model of fetal medicine in the near future. Finally, fetal cells in maternal blood could result in widespread screening for aneuploidy and single cell gene disorders.

S-35 Meanwhile, several biological questions remain: the mechanism of fetal cell transfer, the biologic meaning of fetal cell transfer and the frequency of fetal cells in maternal blood. What specific cells cross the placenta? When during the pregnancy do they enter the maternal circulation? What is their life span in maternal blood? More research is needed to determine the biology of fetomaternal transfer.

S-36 We were successful in retrieving nucleated erythrocytes from maternal blood using a micromanipulator and also in analyzing nucleated erythrocytes on a single cell level by PCR, FISH and PEP-PCR. 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. In closing, I would just like to acknowledge the members of my laboratory group. This is obviously not work that I could do by myself; We have a large number of hard working and dedicated people on this project.

Before closing this lecture, I would like to inform you about my own web page on the Net. You can find our research resources in the web. I look forward to seeing you again on the web. Thank you for your attention.