Multilocus genetic analysis of single interphase cells by spectral imaging

Transcription

1 Hum Genet (2000) 107 : DOI /s ORIGINAL INVESTIGATION J. Fung H.-U. G. Weier J. D. Goldberg R. A. Pedersen Multilocus genetic analysis of single interphase cells by spectral imaging Received: 21 June 2000 / Accepted: 26 September 2000 / Published online: 15 November 2000 Springer-Verlag 2000 Abstract Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types. J. Fung ( ) J. D. Goldberg R. A. Pedersen Reproductive Genetics Unit, Department of Obstetrics, Gynecology and Reproductive Sciences, Box 0720, 533 Parnassus Avenue, University of California, San Francisco, CA , USA jlfung@itsa.ucsf.edu, Tel.: , Fax: H-U. G. Weier Life Sciences Division , University of California, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Introduction Chromosome abnormalities, whether numerical or structural, are common causes of congenital malformations and spontaneous abortions. Cytogenetic analysis of 1,000 spontaneous abortions showed about half of these abortuses (463/1,000) carrying recognized chromosome abnormalities (Hassold et al. 1980). Numerical aberrations involving partial or entire chromosomes have detrimental effects on mammalian embryonic, fetal, and perinatal development. The most common chromosomal abnormalities found in human abortuses are trisomy 16, triploidy, or 45, XO (Turner syndrome) (Sadler 1995). Women over age 35 have a significantly higher risk of chromosomally abnormal conceptuses, since the incidence of aneuploid germ cells increases with age. Abnormal chromosome numbers may originate from nondisjunction of chromosomes at first or second meiotic division. Monosomy for chromosomes other than the sex chromosomes does not lead to live birth, since it causes death at a very early stage of gestation. Trisomy occurs in at least 4% of all clinically recognized pregnancies, although few such fetuses survive to term (Hassold and Jacobs 1984). The rate of chromosomal abnormality among in vitro fertilized embryos is higher than in spontaneous abortions (Dailey et al. 1996; Munné et al. 1995), suggesting that many chromosomally abnormal embryos are eliminated before the pregnancies are recognized. Couples who are at risk of having children with genetic disorders and undergo in vitro fertilization may benefit from new methods for diagnosing genetic disease during the earliest stages of development, i.e., before implantation into the uterus. This procedure has been termed preimplantation genetic diagnosis (PGD) (Handyside et al. 1990, 1998; Grifo 1992). PGD includes micromanipulation of oocytes or embryos, removing polar bodies from mature oocytes (Verlinsky et al. 1994; Verlinsky and Evsikov 1999), or biopsying one or more blastomeres from the developing embryo (Hardy et al. 1990; Munné et al. 1998a, b). Accurate determination of chromosome

2 616 number and ploidy status of oocytes prior to fertilization, of embryos prior to transfer, and of fetuses during the first trimester is expected to increase the efficiency of assisted reproductive techniques and the proportion of normal fullterm births. Genetic analysis of single cells is then performed either by PCR (Wells and Sherlock 1998) or fluorescence in situ hybridization (FISH) (Conn et al. 1998; Munné et al. 1998b). However, few of the cells available from these sources are found in metaphase, and the chromosomes cannot be analyzed by conventional karyotyping, i.e., chromosome banding. Therefore, the procedure must be able to unambiguously determine the chromosomal composition of interphase nuclei. For diagnosis of numerical chromosome abnormalities, FISH has provided a rapid method with unambiguous results and fewer contamination problems than PCR (Tkachuk et al. 1991). FISH also has been applied very successfully to the rapid identification of aneuploidies in uncultured cells from amniotic fluid for prenatal diagnosis (Eiben et al. 2000; Feldman et al. 2000; Gersen et al. 1995; Pergament et al. 2000). Implicated in hyperdiploid embryos that survive to term, chromosomes 13, 18, 21, X, or Y have been proposed as targets for the rapid prenatal detection of aneuploid cells (Ward et al. 1993). However, these five chromosomes represent only about 38% of cases of spontaneous abortions caused by the presence of an extra chromosome (Hassold and Jacobs 1984). Ideally, one would like to detect aneuploidy involving any of the 24 human chromosomes. Thus, an analytical method to enumerate as many chromosomes as possible in only a few interphase cells is desirable. Presently, commercially available hybridization kits allow enumeration of five chromosomes in interphase cells using filter-based fluorescence microscope systems [X, Y, 13, 18, 21 (Aneu- Vysion assay kit, Vysis) or 13, 16, 18, 21, 22 (MultiVysion PB multicolor probe panel, Vysis)]. Repeated hybridization on single interphase cells must be applied in order to score additional chromosomes (Munné et al. 1998b). Furthermore, several groups recently reported the less invasive isolation of fetal cells from maternal peripheral blood samples between weeks 13 and 17 of gestation (Cheung et al. 1996; Zheng et al. 1993). Unfortunately, fetal cells are rare in the maternal circulation, and cells obtained by these procedures are extremely difficult to culture. Therefore, our study using spectral imaging was prompted by a need to recognize the majority of numerical chromosome aberrations in the few available fetal or embryonic interphase cells. Spectral imaging is a powerful technique in which standard emission filters in a fluorescence microscope are replaced with an interferometer to record high-resolution spectra from fluorescently stained specimens. This bioimaging system combines the techniques of fluorescence optical microscopy, charged coupled device imaging, Fourier spectroscopy, and software for digital image analysis. The power of this technology has been demonstrated by specific staining of all 24 human chromosomes in metaphase spreads, termed spectral karyotyping (SKY) (Garini et al. 1996; Schröck et al. 1996). SKY has been applied in cancer studies (Schröck et al. 1996; Zitzelsberger et al. 1999), prenatal diagnosis (Ning et al. 1999), human oocytes and polar bodies (Márquez et al. 1998), and artificially condensed chromatin of blastomeres (Willadsen et al. 1999). For the analysis of interphase cells, however, whole-chromosome painting probes are inappropriate because the chromosomal domains overlap in the nucleus. We previously reported hybridization of a 7-chromosomes probe set (chromosomes 10, 14, 16, 18, 22, X, and Y) and an 11-chromosomes probe set (chromosomes 3, 4, 7, 13, 14, 16, 18, 21, 22, X, and Y) to lymphocyte interphase nuclei and blastomeres using the spectral imaging system and filter-based quantitative imaging processing system (Fung et al. 1998a). Our results demonstrated that the spectral imaging system provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei. However, we were able to detect only seven chromosomes in lymphocyte interphase nuclei and failed to analyze the blastomeres. Therefore, we focused our most recent efforts on developing a 10-chromosomes probe set for detection of DNA targets most frequently associated with aneuploidy and spontaneous abortions (chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y). We investigated the effects of cell preparations (blastomeres from abnormal human preimplantation embryos, and uncultured amniocytes from amniocentesis) and directed effort towards software development (building the reference spectra library file). Using this approach, we are now able to detect and count copies of ten chromosomes in single interphase cells, thereby demonstrating that spectral imaging can be an important technique for genetic analysis in non-dividing cells. Materials and methods Slides preparation and pretreatment Metaphase spreads from white blood cells Metaphase spreads were made from phytohemagglutinin-stimulated short-term cultures of normal male lymphocytes according to the procedure described by Harper and Saunders (1981). Fixed cells were dropped on ethanol-cleaned slides in a CDS-5 Cytogenetic Drying Chamber (Thermatron Industries) at 25 C and 45 50% relative humidity. Metaphase spreads and interphase nuclei were checked using a phase microscope. If cytoplasmic residue was visible around the spreads, the remaining cells were washed several more times with fixative before being dropped. Ideally, the chromosome metaphase spread appeared dark black by phase-contrast microscopy. If metaphase spreads appeared glassy, it suggested that the cells had dried too slowly. We then decreased the chamber s humidity by 2 5%. If metaphase spreads appeared gray, it suggested the cells had dried too fast and we increased the chamber s humidity by 2 5%. Slides were stored in boxes for at least 2 weeks at room temperature, then in sealed plastic bags under nitrogen gas at 20 C until used. Blastomeres All procedures followed protocols approved by the UCSF Committee on Human Research regarding use of embryos for research.

3 617 Prior written consent was obtained from all donors. All embryos used for this study had arrested development or were morphologically abnormal. The zona pellucida of embryos was removed in 0.5% pronase after which the embryo was placed in Ca 2+ /Mg 2+ - free PBS until the blastomeres came apart during repeated pipetting of the embryo. Individual blastomeres were incubated in a hypotonic solution of 1% Na-citrate, 6 mg/ml bovine serum albumin in water for 5 min before being placed on microscope slides and fixed with a solution of methanol/acetic acid (3/1 or 1/1, v/v) (Tarkowski 1966). Throughout the procedure, the nucleus was observed in a phase-contrast microscope and its location on the slide was marked using a carbide- or diamond-tip pen following fixation. The slide was then dehydrated in three consecutive baths of 70%, 80% and 100% ethanol for 2 min each before it was used for FISH or stored at 20 C. Human uncultured amniocytes Amniocytes were donated for research under an approved protocol in accordance with guidelines set by the UCSF Committee on Human Research. Uncultured amniocytes were obtained from 1 2 ml of amniotic fluid and fixed on slides within a few hours of amniocentesis. The cells were pelleted and then resuspended in a hypotonic solution consisting of 0.3% KCl for 30 min at 37 C. Several drops of ice-cold fixative (methanol/acetic acid, 3/1, v/v) were added and gently mixed, after which the cells were spun down. The cells were resuspended with ice-cold fixative and pelleted several times. Then, the cells were dropped on fixative-cleaned slides above a boiling water bath. The slides were air dried and aged in 2 SSC at 37 C for 1 h. Cells were pretreated with pepsin (50 µg/ml pepsin in 0.01 N HCl) at 37 C for 13 min, 1 PBS at room temperature for 5 min, and then postfixed in 1% formaldehyde in 1 PBS/MgCl 2 at room temperature for 5 min and washed in PBS. Finally, the slides were dehydrated in 70%, 80%, and 100% ethanol for 2 min each. The slides were used for FISH or stored at 20 C. Probe preparation Probes specific for repeated DNA on chromosome 15 (CEP15, satellite III, D15Z1, Vysis), chromosome X (CEPX, alpha satellite, DXZ1, Vysis), and chromosome Y (CEPY, satellite III, DYZ1, Vysis) were labeled with either a green or red fluorochrome (spectrum green or spectrum orange, respectively). The probe specific for satellite II DNA of chromosome 16 was prepared from clone phur195 (Moyzis et al. 1987). The probe specific for repeated DNA of chromosome 9 was prepared from flow-sorted chromosome 9 by in vitro amplification, similar to the scheme described earlier (Weier et al. 1991), and primers used were specific for satellite III DNA (H.-U.G. Weier, unpublished data). Locus-specific DNA probes for chromosome 13 (YAC 900g6), chromosome 14 (YAC 886a3), chromosome 18 (YAC 945b6), chromosome 21 (YAC 141g6), and chromosome 22 (YAC 849e9) were obtained from yeast artificial chromosome (YAC) clones. YAC clones were obtained from the Genethon/CEPH library (Weissenbach et al. 1992) and purchased from Research Genetics. The DNA from YACs was isolated using pulsed-field gel electrophoresis (PFGE) and amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) (Cassel et al. 1997; Fung et al. 1998b; Weier et al. 1994). All PCR products were purified by chloroform extraction, precipitated in 2-propanol and resuspended in 1 TE buffer, ph 7.2. The DNAs from clones specific for chromosomes 9, 13, 14, 16, 18, 21, and 22 were labeled by random priming (BioPrime Kit, GIBCO/LTI, Gaithersburg, Md.) incorporating biotin-14-dctp (part of the Bio- Prime Kit), digoxigenin-11-dutp (Roche Molecular Biochemicals, Indianapolis, Ind.), fluorescein-12-dutp (Roche Molecular Biochemicals) (Weier et al. 1994), or Cy3-dUTP (Amersham, Arlington Heights, Ind.). Table 1 lists the components of our 10-chromosomes probe set and the respective labeling and detection scheme. Briefly, bound biotinylated probes were detected with avidin-cy5, and bound digoxigenin-labeled probes were detected with Cy5.5-conjugated antibodies against digoxin (Sigma, St. Louis, Mo.). Between 0.5 and 3 µl of each probe along with of 1 µl human COT1DNA (1 mg/ml, GIBCO/LTI) and 1 µl salmon sperm DNA (20 mg/ml, 3-5, Boulder, Co.) were precipitated with 1 µl glycogen (Roche Molecular Biochemicals, 1 mg/ml) and 1/10 volume of 3 M sodium acetate in two volumes of 2-propanol, air dried and resuspended in 3 µl water, before 7 µl of hybridization master mix [78.6% formamide (FA, GIBCO/LTI), 14.3% dextran sulfate in 2.9 SSC, ph 7.0 (1 SSC is 150 mm NaCl, 15 mm Na citrate)], were added. This gave a total hybridization mixture of 10 µl. In situ hybridization The hybridization mixture was denatured at 76 C for 7 min, then allowed to pre-anneal at 37 C for 60 min. The slides were denatured at 76 C for 5 min in 70% FA/2 SSC, ph 7.0, dehydrated in 70%, 80%, and 100% ethanol for 2 min each step, and allowed to air dry. The hybridization mixture was applied to the slides, covered with a glass coverslip, and sealed with rubber cement. The hybridization proceeded at 37 C for h in a moisture chamber. Following hybridization, the slides were washed at 43 C three times in 50% FA/2 SSC for 10 min each followed by two washes in 2 SSC for 10 min each, one wash in 0.4 SSC for 5 min, and a final wash in 0.1% Tween 20 /4 SSC at room temperature for 2 min. Next, 80 µl of blocking reagent (vial 2, SKY kit, Applied Spectral Imaging, Migdal Haemek, Israel) were applied to each slide, and slides were covered with a plastic coverslip, and incubated at 37 C for 45 min. After removal of the coverslip, 80 µl of buffer I (vial 3, SKY kit, containing avidin-cy5 and mouse-antidigoxin) were added to each slide. The slides were then incubated at 37 C for 45 min and washed three times in 0.1% Tween 20/4 SSC at room temperature for 5 min each on a shaking platform. Next, 80 µl of buffer II (vial 4, SKY kit, containing goatanti-mouse antibody conjugated to Cy5.5) were applied to each Table 1 Fluorochrome labeling scheme for chromosomespecific DNA probes. Probes labeled with biotin or digoxigenin were detected with avidin-cy5 and Cy5.5-conjugated antibodies against digoxin, respectively Chromosome Spectrum FITC Spectrum Cy3 Biotin Digoxigenin Green Orange (Cy5) (Cy5.5) X + + Y +

4 618 slide, and slides were incubated at 37 C for 45 min in the dark. Slides were washed three times in 0.1% Tween 20/4 SSC at room temperature for 5 min each on a shaker, and finally, the slides were mounted in 10 µl of 4,6-diamidino-2-phenylindole (DAPI, vial 5, SKY kit). Spectral imaging detection Spectral images were acquired using an SD200 SpectraCube spectral imaging system (Applied Spectral Imaging) (Fung et al. 1998a; Garini et al. 1996; Schröck et al. 1996). The SD200 imaging system attached to a Nikon E600 microscope consisted of an optical head (Sagnac interferometer) coupled to a multi-line CCD camera (Hamamatsu, Bridgewater, N.J.) to capture images at discrete interferometric steps. The images were stored as a stack in a Pentium 586/300 MHz computer. The multiple-band-pass filter set used for fluorochrome excitation was custom-designed (SKY-1, Chroma Technology, Brattleboro, Vt.) to provide broad emission bands (giving a fractional spectral reading from ~450 nm to ~850 nm). Using a xenon light source, the spectral image was generated by acquiring interferometric frames per object. Next, each interferogram was Fourier-transformed, producing the fluorescence spectrum for each pixel of the image. DAPI images were recorded using a DAPI-specific optical filter set. Sample emission spectra were measured in the visible and near-infrared spectral range simultaneously at all points in the microscopic image. The spectral information was displayed by assigning red, green or blue colors (RGB color image) to three areas of interest in the spectrum. Based on the measured spectrum for each signal domain, a spectral classification algorithm comparing the measured spectra with reference spectra allowed the assignment of a pseudocolor to all points in the image that had the same spectrum. This algorithm forms the basis for chromosome identification by spectral karyotyping. Thus, a classification color image and a karyotype table were obtained. Building the reference spectra library file The probe set shown in Table 1 was hybridized on metaphase spreads to build the reference spectra library. This library was essential for karyotyping metaphase spreads and interphase nuclei. It is built by analyzing a metaphase plate with known chromosome identities. The spectra of the pure dyes, i.e., the signals on chromosome 9, 15, 21, 22, and Y, were stored as reference spectra. Then, we built a combinatorial table using the probe combinations listed in Table 1 and assigned pseudocolors to each chromosome type. Results Metaphase spread analysis In the present study, six fluorochromes (spectrum green, FITC, spectrum orange, Cy3, Cy5, and Cy5.5) were used to label DNA probes. The emission maxima of spectrum green, FITC, spectrum orange (or Cy3), Cy5, and Cy5.5 are 530 nm, 525 nm, 592 nm, 678 nm, and 702 nm, respectively. A previous publication suggested a wavelength difference of about 20 nm between the emission maxima of spectrum orange and Cy3 fluorochrome (Carter 1996). However, the emission spectra of Cy3 probes prepared inhouse and the commercially available spectrum-orangelabeled DNA probes were found to be identical. Therefore, these two fluorochromes were indistinguishable. One inverted DAPI image of a metaphase spread prepared from short-term lymphocyte cultures from a healthy male individual is shown in Fig. 1A, and Fig. 1B is the corresponding RGB color image acquired by the spectral imaging system. The signals in the RGB color image were selected by manually drawing red contour lines around each signal domain. To make the signals in Fig. 1B, C clear, the red contour lines are not shown in both of them, but only in Fig. 1A. A total of 18 signals were counted. After the spectrum of each signal was compared to the reference spectra library, the classification color image (Fig. 1C) and karyotype table (Fig. 1D) were constructed. The size and shape of the signals in the classification color image (Fig. 1C) and the karyotype table (Fig. 1D) were the same as the size and shape of each contour (red, Fig. 1A). In the karyotype table (Fig. 1D), chromosomal signals were grouped such that signals from the RGB color image (Fig. 1B) were aligned with corresponding images from the classification color image (Fig. 1C). This normal karyotype (Fig. 1D) showed two copies each of chromosome 9, chromosome 13, chromosome 14, chromosome 15, chromosome 16, chromosome 18, chromosome 21, chromosome 22, and one copy each of the X and Y chromosomes, as expected for a normal male metaphase spread. Interphase cell analysis Once a combinatorial table was built from a normal male metaphase spread, we hybridized the probe set on three different human interphase cell types. Most interphase cells, especially the uncultured amniocytes, showed elevated levels of background fluorescence compared to lymphocyte metaphases. Figure 2 shows the background fluorescence after hybridization from different cells. The signal amplitude for uncultured amniocytes is shown in Fig. 2 at one half that of the other signals, and the strength of background level was: uncultured amniocytes>>blastomeres>interphase cells from lymphocytes>metaphase cells from lymphocytes. Since the spectrum of background fluorescence differed from the reference spectra, it could easily be distinguished and eliminated using the spectral imaging system. Figure 1E shows the RGB image of a normal male lymphocyte nucleus with its classification color image shown in Fig. 1F. A nonspecific signal (at the 8 o clock position) was not scored, because its spectrum differed greatly from all reference spectra. The karyotype table (data not shown) showed a normal karyotype with a total of 18 signals representing the eight autosomal targets and one copy each of chromosomes X and Y. The interphase nuclei from lymphocytes hybridized with our probes demonstrated good hybridization efficiency and were karyotyped as being normal. The 10-chromosomes probe set was also hybridized to human uncultured amniocytes. Figure 1G shows the RGB color image of a hybridized amniocyte; its classification image is shown in Fig. 1H. This normal male cell showed a total of 18 signals representing eight autosomal targets

5 619 Fig. 1A J Fluorescence in situ hybridization (FISH) results using different human cell types. A The inverted DAPI image of a lymphocyte metaphase spread with contour lines indicating the position of the chromosome-specific signals. B The red, green or blue (RGB) color image of the metaphase spread. C The classification color image of the metaphase spread. D The karyotype table: autosomal signals are grouped in two pairs with the left member of each pair from B, and the right member from C. E The RGB color image of a lymphocyte interphase nucleus. F The classification color image corresponding to E. G The RGB color image of an amniocyte interphase nucleus. H The classification color image corresponding to G. I The RGB color image of a binucleated blastomere. J The classification color image corresponding to I and one copy each of chromosomes X and Y (karyotype not shown). It should be noted that the nuclei of uncultured amniocytes were much smaller than those of interphase lymphocytes and blastomeres after fixation. The fixation of uncultured amniocytes on slides turned out to be somewhat difficult. Most nuclei were not flattened out well enough, presenting a problem due to the limited focal depth. Figure 1G showed signals in close proximity to one another. Overlapping signal domains were a problem in uncultured amniocytes, in which only about 20% of all cells showed interpretable spreads. One of our main objectives was to demonstrate the spectral imaging-based enumeration of ten different chromosome types in single blastomere cells from human preimplantation embryos. Figure 1I, J (RGB color image and classification image, respectively) shows the results

6 620 Intensity [a.u.] of our spectral imaging analysis of one such blastomere. This cell, a binucleated blastomere, was found to be an abnormal male cell displaying a total of 29 signals. The classification image indicated a large nucleus (top, Fig. 1J) with signals corresponding to two chromosomes 9, four chromosomes 13, four chromosomes 14, two chromosomes 15, two chromosomes 16, five chromosomes 18, three chromosomes 21, two chromosomes 22, and one X chromosome. A smaller nucleus (bottom, Fig. 1J) contained hybridization targets equivalent to one chromosome 15, one chromosome 18, one X chromosome, and one Y chromosome. Based on the spectral imaging results, this blastomere was considered a hyperdiploid male cell [9(2), 13(4), 14(4), 15(3), 16(2), 18(6), 21(3), 22(2), X(2), Y(1)]. All blastomeres fixed for this study (N=25) spread very well. Fourteen nuclei (56%) showed clearly interpretable hybridization results, and most of them were karyotyped as abnormal, since all those cells were from one-pronuclei (PN) and three-pn human embryos and had arrested development or were morphologically abnormal. The signals from 11 nuclei (44%) were faint. This may be related to the quality of the embryos, since all of them were developing abnormally. Alternatively, we could not exclude the possibility that either the method of fixation was suboptimal or the efficiency of FISH on blastomeres is lower than that on either lymphocytes or amniocytes. Because the number of blastomeres with karyotyping resulting from each embryo was very limited, chromosomal mosaicism was not addressed in this study. Discussion Metaphase Interphase Amniocyte Blastomere Wavelength [nm] Fig. 2 The emission spectra of background fluorescence on slides from four different cell types after hybridization (metaphase cells from lymphocytes, interphase cells from lymphocytes, uncultured amniocytes, and blastomeres). The signal for uncultured amniocyte is shown at one half the scale of the other signals In our previous publication (Fung et al. 1998a), we only detected seven chromosomes in interphase nuclei from lymphocytes using the SpectraCube software. In this study, we tested our 10-chromosomes probe set on three cell types, lymphocytes, amniocytes, and human blastomeres. We also used the SkyView software instead of SpectraCube software. The subtraction of background is done automatically using the SkyView software. The signals then were selected by drawing contour lines manually around the recognized signals. In some cases, we selected a dot that was not the signal. For example, there is a dot between the position of 8 and 9 o clock in Fig.1E at an intensity similar to other signals. However, it would not be recognized as a signal since it exhibited a different spectrum from the reference spectra library. The results demonstrated that spectral imaging system can identify and count ten chromosome-specific targets in interphase cells even in the presence of relatively high levels of autofluorescence or non-specified signals, especially uncultured amniocytes. On the basis of these findings, we anticipate that the spectral imaging system can provide information for patient decision-making that will augment current modalities for preimplantation and prenatal diagnosis. Specifically, the ability to assess multiple chromosome types in interphase cells can enhance the value of early screening of conceptuses. This is particularly true for preimplantation diagnosis of aneuploidies. Owing to the high frequency of numerical chromosome anomalies in early embryos (Munné and Cohen 1998), many of the embryos ordinarily transferred after assisted reproductive technology procedures are genetically abnormal (Munné et al. 1998c). Screening such embryos by PGD could both enhance implantation rates per embryo transferred (by selecting out monosomic conceptuses) and decrease spontaneous abortion rates (by selecting out trisomic conceptuses) (Munné et al. 1999). The detection and enumeration of chromosome-specific signal domains in interphase cells is often complicated by reduced penetration of probe molecules into the interphase nuclei, overlapping or overly spread signals, and high levels of nuclear autofluorescence. Some of these problems appear to be caused by inappropriate cell fixation and choice of probes. In order to obtain high-efficiency of FISH, meticulous slide preparation was essential. In this study, we applied three different methods to fix each of the three different types of cells. All fixations and cell spreads of lymphocytes were performed under controlled environmental conditions inside a Cytogenetic Drying Chamber at 25 C and 45 50% relative humidity. This highly controlled environment contributes greatly to the reproducibility of this technique. While developing our probe set for spectral imaging analysis of interphase cells, we had to optimize several hybridization parameters such as target DNA, probe labeling, fluorochrome selection, and cell preparation. The ideal probe set should consist of bright, single-copy locus-specific probes rather than DNA repeat probes to avoid crosshybridization and domain clustering. If a probe target, such as the satellite III chromosome Y-specific target, were too big (Wyrobek et al. 1994), it might easily spread over too great an area in the interphase nucleus and impair the spectral imaging analysis. On the other hand, when a probe such as the chromosome 22-specific probe (YAC 849e9) caused cross-hybridization (Fung et al. 1999), we attempted to adjust the hybridization stringency and block-

7 ing protocol before evaluating another chromosome-specific probe. To suppress cross-hybridization signals generated by YAC 849e9, for example, it was sufficient to add an increased amount of human COT-1 DNA to the probe mixture. We also found that not all ratio-labeling schemes worked with the same efficiency. Often, one fluorochrome yielded stronger signals than the other fluorochrome when both were bound on the same DNA. For example, the intensity of probes detected with Cy5.5 was usually much stronger than the intensity of Cy3-labeled probes. This effect was probably a combination of different quantum efficiencies, probe labeling index, energy transfer and detection sensitivity. In filter-based microscope systems, signals from weaker probes are typically enhanced through longer exposure times. The SD-200 spectral imaging system, however, uses the same exposure time for each interferogram in an exposure series. To adjust the ratio of Cy3 fluorescence to Cy5 or Cy5.5 fluorescence, the Cy3 probes were thus used at a higher concentration than their Cy5 counterparts. Another complicating factor was that the positions of emission peaks in the spectrum were found to be slightly different from cell type to cell type. For example, the difference between emission maxima of Cy5 and Cy5.5 on interphase nuclei measured less than the difference on metaphase spreads. While this observation suggested some influence of hybridization target environment and/or autofluorescence levels on fluorescence emission spectra, it also emphasized that selection of DNA labels, i.e., fluorochromes, remains an important issue for spectral imaging analysis. While the high spectral resolution of spectral imaging now offers a reliable way to record fluorescence profiles for the various probes and thus to discriminate specific signals from background noise, exposure times to acquire this large amount of information are still in the order of minutes. Often, fluorescence had bleached significantly after acquisition of two images. A substantial increase in the quality and number of DNA probes that can be hybridized and enumerated simultaneously seems feasible once additional reporter molecules become commercially available, such as nanocrystals or quantum beads (Bruchez et al. 1998; Chan and Nie 1998) or dyes from other suppliers. Recently, Munné et al. (1998b) described FISH techniques in which one can analyze nine probe targets in two consecutive hybridizations on day-3 human preimplantation embryos, allowing embryo transfer on the same day as the analysis. In our study, the hybridization time was h, but the time could be shortened if this new technique is applied to PGD. The protocol is otherwise useful for blastocyst transfer. On the other hand, for routine clinical use for in vitro fertilization, there are many parameters that still need to be optimized. When only one or two blastomeres can be analyzed, potential mosaicism in embryos becomes a significant problem which should be addressed by subsequent amniocentesis or chorionic villus sampling. Therefore, this new technique will be more useful and reliable in prenatal diagnosis rather than preimplantation genetic diagnosis. In conclusion, while further improvements in fixation, probe selection and fluorochrome technology remain to be accomplished, the results shown here demonstrate the utility of spectral imaging for evaluating numerical chromosomal anomalies in interphase cells. In addition to expanding the number of chromosomes that can be analyzed, this approach could facilitate the accuracy of FISH analysis in interphase cells by allowing multiple probes to be used for a subset of chromosomes in the complement. Acknowledgements Supported by grants from the U.C. Energy Institute, the U.C. BioSTAR Program and Geron Corporation (to R.A.P.). We gratefully acknowledge the technical support from Applied Spectral Imaging, Inc., Carlsbad, California. J.F. was supported in part by a NIEHS training grant 5-T32-ES References 621 Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281: Carter N P (1996) Fluorescence in situ hybridization-state of the art. Bioimaging 4:41 51 Cassel M, Munné S, Fung J, Weier H-UG (1997) Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells. 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