Cell Biochem Biophys
DOI 10.1007/s12013-011-9285-2

REVIEW PAPER

Chromosome Analysis Using Spectral Karyotyping (SKY)George Imataka • Osamu ArisakaÓ The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract Spectral karyotyping is a novel technique forchromosome analysis that has been developed based on theapproach of the fluorescence in situ hybridization technique. Spectral karyotyping makes it feasible to diagnose avariety of diseases, because of its technology in painting
each of the 24 human chromosomes with different colors.
In recent years, it has become possible to adopt the usageof spectral karyotyping for research in general clinicalpractice, and its usability has attracted particular attention
in the diagnosis of different diseases. In this review, we
will explain the principle of the spectral karyotyping, as
well as its specificity and limitation in detecting the genetic
defects within clinical application by presenting two casereports.
Keywords Spectral karyotyping  Fluorescent in situ
hybridization  Chromosome SKY  Multicolor FISH

Introduction
The first report of human chromosomes was made in 1882
by Flemming, in which 22–28 chromosomes in the dividing cells of the corneal epithelium were described [1].
Forty years later, Painter reported that the number ofhuman chromosomes was 48 and that sex was determinedaccording to the presence or absence of the Y chromosome[1]. It was not until the report of ‘‘2n = 46’’ was made in1956 by Tjio and Levan that the nature of human chromosomes was further established [1]. Following this report,
G. Imataka (&)  O. Arisaka
Department of Pediatrics, Dokkyo Medical University,
880 Mibu, Shimotsuga, Tochigi 321-0293, Japane-mail: geo@dokkyomed.ac.jp

the relationships between various congenital disorders andchromosomal abnormalities have been revealed one after
another. In 1959, Lejeune et al. [2] demonstrated that
Down syndrome is caused by the presence of an extra
chromosome 21, i.e., trisomy [3]. This finding of chromosome 21-trisomy was a turning point in the subsequentdiscovery of several other trisomy syndromes, including
chromosomes 18 [4, 5], 13 [6], 8 [7], and 22 [8, 9]. Besides
the chromosomal number aberrations, abnormalities associated with sex chromosomes, viz., Turner syndrome [10]and Klinefelter syndrome [11] and other chromosomes[5, 12] have been reported.
An increased understanding of malignant diseases in the
1960s and the advent of better cell culture methods
including a method of culturing peripheral blood [13] led to
the first discovery of chromosomal abnormalities inmalignant tumors, the Ph1 chromosome, which is specificto chronic myelogenous leukemia [14]. Genetic analysis
studies, represented by the Human Genomic Project [15],led to the application of modern genetics tools in researchin biological phenomena such as the development, evolution, and aging, as well as abnormal cellular differentiation
including cancers.
Therefore, in the course of studying human chromosomes for more than a century, many new techniques havebeen developed for chromosome testing. In 1971,
Caspersson, et al. [16] developed Q-staining using quinacrine mustard, a fluorescent dye that binds to DNA, which
was instrumental in the discovery that chromosomes havebanding patterns (stripes). Later, improved staining techniques, including R-, T-, C-, and G-staining, haveappeared. The analysis of the band has further advanced
and now it has been developed into high-resolution chromosome banding [17]. Furthermore, along with thedevelopment of the fluorescent in situ hybridization (FISH)

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technique [18], which involves the mapping of a chromosome using fluorescent labeled probe, it has become possible to identify chromosomal microdeletion and structuralabnormalities such as the extra portion of a chromosome
and a marker chromosome. FISH has also been used indiagnosing abnormalities, caused by chromosomal aneuploidy—which are difficult to detect via conventional test
methods.
Comparative genomic hybridization (CGH) [19] andspectral karyotyping (SKY) [20], which are based on the
FISH technique, are relatively recent and improved techniques of chromosomal analysis. SKY as described here
refers to the multicolor-FISH technique, which makes it
possible to identify 24 human chromosomes via single
hybridization and then to stain them with different colors.
In this article, the analysis of chromosomal abnormalities
using the SKY technique and its limitation for detection
will be discussed.

Table 1 Combination of fluorescent dyes (painting probes) ina chromosome analysis usingSKY

Principle of SKY
Two techniques are used as the basic principles of FISH:
chromosome painting and multicolor fluorescence. The
former is a technique of drawing an entire image of certain
chromosome by using fluorescent signals. The latter is atechnique of drawing images of several hybridization signals with different florescent dyes. Combining these twotechniques, in 1996, the SKY technique was developed bySchrock et al. [20].
In SKY, the color emission of chromosomes is determined by the combination of painting probes and (fluorescent dyes). In this technique, new colors can be
developed by extracting a pair of different fluorescent dyes
from among the five types of fluorescent dyes, spectrum
orange, Texas red, Cy5, spectrum green, Cy5.5, and mixing
each pair together. Thus, it is theoretically possible tocreate 2N-1 number of colors from N types of fluorescentdyes. Therefore, 31 types of colors can be created from five
types of fluorescent dyes. In reality, however, becausesome fluorescence has a wavelength close to that within theinfrared spectrum, a two-dimensional imaging spectroscopy system (e.g., the Spectral Bio-Imaging System
SD-200, Applied Spectral Imaging Ltd. Israel) needs to be
used to process spectral images so that 24 macroscopicallydistinguishable colors (Table 1) can be created.
The amount of samples required for the test is 3.0 ml ofheparin-treated blood in the case of chromosome testing for
regular congenital abnormalities, and 1.0 ml of bone marrow fluid and 5.0 ml of blood in the case of blood disorders. Currently, no standardized method is available
for describing chromosome karyotypes based on SKY
analysis.

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A spectrum orange, B Texas red,
C Cy5, D spectrum green,
E Cy5.5

Chromosome

Painting probes

1

BCD

2

E

3

ACDE

4

CD

5

ABDE

6

BCDE

7

BC

8

D

9

ADE

10

CE

11

ACD

12

BE

13

AD

14

B

15
16

ABC
BD

17

C

18

ABD

19

AC

20

A

21

DE

22

ABCE

X

AE

Y

CDE

Characteristics of SKY in Clinical Application
When a chromosome in which the origin cannot be identified or the structural abnormality of a partial copy of achromosome is observed with G-banding, the SKY technique makes it possible to recognize each chromosome
with different color tones. Furthermore, when cells arecultured under conditions in which the image acquisition ofcell division is difficult, SKY enables bleaching andstaining of the specimen after the images of division havebeen verified.
SKY provides the advantage of easy visual interpretation when analyzing results, but it is not possible to evaluate structural abnormalities, such as inversion, deletion,
insertion, and duplication in the same chromosome,because these are shown with the same color. Also, neither
the Q-positive segment nor the satellite region of the longarm of the Y chromosome near the centromere can bedetected. The resolution limit of detection is approximately
1–2 Mb, similar to conventional chromosome painting
techniques, and minor structural abnormalities of less than
1 band cannot be detected. Therefore, in order to verify
the site of chromosomal breakage, it is usually necessary to
use SKY beforehand in combination with G-banding orCell Biochem Biophys

high-resolution chromosome banding, instead of using SKY
alone to macroscopically observe the banding patterns.
Furthermore, SKY provides the following notableadvantages. Conventionally, when a chromosome ofunknown origin is noticed in a child suffering with someabnormality, a chromosome analysis is performed on bothparents in order to make a definitive diagnosis of thechromosome in question. If a balanced structural abnormality is detected in the chromosome of either of the parents, then it is definitively diagnosed as the origin of such astructural abnormality. Conventionally, macroscopic evaluation would be performed based on the banding patterns,
in case the informed consent for participation in the
karyotype test cannot be obtained from the parents, and in
the case of de novo translocation. In such cases, SKY has
the advantage of being capable of readily clarifying thechromosome of unknown origin without performing karyotyping of the parents, and also can reveal if one of theparents is a carrier of a balanced structural abnormality.
This advantage of SKY is very useful in the clinical practiceof genetic counseling [9]. Refer to Table 2 for the indications of evaluation using SKY when structural chromosomal abnormalities are actually found via G-banding.

Case 1: Trisomy 11/22
The results of chromosome analysis on Trisomy 11/22
using SKY are shown in Fig. 1 [8, 9]. SKY was performed
after an extra marker chromosome was detected in group G
chromosomes via G-banding and the results showed thetrisomy of chromosome 22. One of the extra chromosomes
was distinguished by two colors, and it was deduced to be aderivative chromosome [der(22)], likely resulting from thereciprocal translocation in which the long arms of chromosome 11 (q23.3) and chromosome 22 (q11.2) were thebreakpoints. The karyotype of this case is described as?der(22)t(11:22)(q23.3;q11.2). Therefore, SKY is one of
the most effective approaches at present for the identification of an extra chromosome of unknown origin. Withthe use of SKY technique, it was possible to make adefinitive diagnosis of trisomy 11/22 in the child without
performing any chromosome testing on the parents, i.e.,
without examining the possibility of structural abnormalities that would have been inherited from either one of theparents. This is significant in the clinical practice of genetic
counseling for parents to maintain their relationship.
Case 2: 13q-Syndrome

Case and Review
In this section, two cases in which SKY can be used arepresented, and the benefits and limitations of this techniqueare discussed. We employed the application of 24-color
SKY probes [Applied Spectral Imaging (ASI), Inc., ASI,
TM
Carlsbad, CA], skypaint hybridization and detection
protocol, SRL Inc., Tokyo, Japan.

The results of the chromosome analysis of 13q-syndrome
using SKY (Fig. 2) indicate structural abnormalities inchromosomes 2, 4, and 13 [12, 21]. For chromosomes 2
and 4, the reciprocal translocation t(2;4) with breakpoints
on the long arm of chromosome 2 (q24.2) and the short armof chromosome 4 (p14) can be readily distinguished. Thist(2;4), however, may involve [inv(4)(p15.1p15.32)], which
is a paracentric inversion with breakpoints at p15.1 andTable 2 Usability when analyzing abnormalities that have been found via G-banding by using SKY
Result of G-banding

Karyotype

Supplementary note on SKY

Usability of SKY

Accessory chromosome

add

Identifies the derivative chromosome of an extra portiona

Derivative chromosome

derIdentifies the related chromosomesaInsertion

ins

Identifies the derivative chromosome of insertion fragmentaIdentifies the related chromosomesaA complicated translocation
Genetic amplification
Duplication?

dimin/hsr?dup

Identifies the derivative chromosome of homogeneous staining region
Identifies any duplicationa

Reciprocal translocationtSame analysis level of G-bandingb

Inversion

inv

Difficult to make an analysis that is better than G-bandingcDeletiondelDifficult to make an analysis that is better than G-bandingc

Numerical aberrations?/-

Difficult to make an analysis that is better than G-bandingc

Unknown of derivative

mar

Identifies a derivative chromosome within the limitations of sensitivitycDetects a derivative chromosome within the limitations of sensitivityc

Cryptic translocationaa

The usability of SKY is considered to be highb

SKY can be used for analysis, but it cannot be expected to have better analysis results than those of G-bandingcSome cases cannot be detected using SKY

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Cell Biochem Biophys

G-band staining or high-resolution chromosome banding
for a comprehensive karyotypic diagnosis.

Conclusion

Fig. 1 Chromosome analysis on Trisomy 11/22 using SKY

p15.32 in the short arm part of chromosome 4 that has beentranslocated to chromosome 2, and this necessitates averification via high-resolution chromosome banding.
Inversions occurring in the same chromosome are notdistinguished with color, so it is impossible to make adefinitive diagnosis by using SKY. Thus in this case,testing of the parents’ chromosomes via high-resolution
chromosome banding is necessary for further detailed
diagnosis.
For chromosome 13, it is possible to diagnose the occurrence of a deletion that is identified as 13q minus syndromeby using SKY only, but this does not reveal information
about the breakpoints. By simultaneously combining thisSKY-derived information with the banding patterns to betested, it can be diagnosed as a paracentric deletion withbreakpoints at q21.2 and q31.2. The karyotype of Case 2 isdescribed as 46,XX, der(2)t(2:4)(q24.2;p14)inv(4) (p15.
1p15.32), der(4)t(2;4), del(13)(q21.2q31.2) or 46,XX,t(2;4)(q24.2;p14), del(13)(q21.2q31.2). As stated above, it is difficult to make a definitive diagnosis from the results of a SKY
analysis alone, and also one needs to be careful, because theconventional method of G-band staining cannot be useddirectly as a stand-alone substitute. Thus, more reliableinterpretation and diagnosis can be made by combining theinformation from the two separate techniques, as neither ofthem can be used for unequivocal diagnosis.
When performing tests using SKY, it is necessary tounderstand the characteristics and the limitations of thisanalysis, and to combine the test results with those of

Fig. 2 Chromosome analysis of 13q-syndrome using SKY showing
structural abnormalities in chromosomes 2, 4, and 13

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With the introduction of SKY, a new technology of chromosome analysis, the accuracy of testing is improved and it
now becomes possible to devise new clinical studies that
previously had not been feasible with conventional testing
methods. T-Band regions such as 1p, 11p15, 16p, 19p, 19q,
and 22q are known as sites that are difficult to observe as
bands of contrasting colors, even though it is believed thatthese regions contain many genes, according to conventional chromosome analysis. Furthermore, the macroscopic
analysis of that band with the human eye involves a limitation in recognizing minor reciprocal translocations, and it
is also possible that they may go unnoticed. Identification
via SKY coloring has overcome the weakness of the conventional band pattern diagnosis method, and it is expectedthat other previously unrecognized specific chromosomaltranslocations are likely to be revealed with the widespreaduse of this technique.
Moreover, in recent years, spectral color banding has beendeveloped as a technique that has overcome the limitations of
SKY [22] and in this technique G-band differential staining
is combined with the SKY coloring technology. Clinical useof this novel technique is expected to be widespread in thefield of clinical genetics in the future and to make significant
contributions. However, the cost of this technique still
remains prohibitory. Considering these points, from thestandpoint of genetic counseling, incorporation of SKY into
G-band chromosome banding or high-resolution chromosome banding as needed would be the most practical technique of chromosome testing. In fact, when the structuralabnormality of a chromosome of unknown origin is detected
via G-banding, the combined use of SKY makes it possible toidentify the origin without performing karyotyping of theparents, which could identify which parent is the carrier ofthe balanced structural abnormality. Therefore, in the clinical practice of genetic counseling, informed consent for a testof congenital chromosomal abnormalities can be readily
obtained from the parents. Furthermore, an evaluation of the
SKY results is made based on the colors that are displayed, so
it is easy for a physician to explain the results to the parents ofa patient. This advantage could potentially reduce theunnecessary psychological burden on parents with a child
who demonstrates a structural abnormality in a chromosome.
Acknowledgments The authors would like to thank SRL Inc.,Tokyo, Japan for executing cytogenic analysis of SKY and FISH
technique. This study was supported by Dokkyo Medical University
Investigator-Investigated Research Grant (No. 2010-01-6).

Cell Biochem Biophys
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