Hum Genet (2000) 107 : 615–622
DOI 10.1007/s004390000416
O R I G I N A L I N V E S T I G AT I O N
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 94143-0720, USA
e-mail: [email protected],
Tel.: +1-415-4768517, Fax: +1-415-4766145
H-U. G. Weier
Life Sciences Division 74-157, 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
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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 (AneuVysion 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.
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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 BioPrime 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.
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 Ca2+/Mg2+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/MgCl2 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.
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 40–48 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
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
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
9
13
14
15
16
18
21
22
X
Y
Spectrum
Green
FITC
Spectrum
Orange
+
+
+
Cy3
Biotin
(Cy5)
Digoxigenin
(Cy5.5)
+
+
+
+
+
+
+
+
+
+
+
+
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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 80–130 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
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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
620
2500
Metaphase
Intensity [a.u.]
2000
Interphase
1500
Amniocyte
1000
Blastomere
500
0
450
550
650
750
850
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
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
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-
621
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
40–48 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-ES07106-17.
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