the first report of human chromosomes was made in 1882 by flemming , in which 2228 chromosomes in the dividing cells of the corneal epithelium were described . forty years later , painter reported that the number of human chromosomes was 48 and that sex was determined according to the presence or absence of the y chromosome . 2n  =  46  was made in 1956 by tjio and levan that the nature of human chromosomes was further established . following this report , the relationships between various congenital disorders and chromosomal abnormalities have been revealed one after another . in 1959 , lejeune et al .   demonstrated that down syndrome is caused by the presence of an extra chromosome 21 , i.e. , trisomy . this finding of chromosome 21-trisomy was a turning point in the subsequent discovery of several other trisomy syndromes , including chromosomes 18 [ 4 , 5 ] , 13 , 8 , and 22 [ 8 , 9 ] . besides the chromosomal number aberrations , abnormalities associated with sex chromosomes , viz . , turner syndrome   and klinefelter syndrome   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   led to the first discovery of chromosomal abnormalities in malignant tumors , the ph chromosome , which is specific to chronic myelogenous leukemia . genetic analysis studies , represented by the human genomic project , led to the application of modern genetics tools in research in 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 have been developed for chromosome testing . in 1971 , q - staining using quinacrine mustard , a fluorescent dye that binds to dna , which was instrumental in the discovery that chromosomes have banding patterns ( stripes ) . later , improved staining techniques , including r- , t- , c- , and g - staining , have appeared . the analysis of the band has further advanced and now it has been developed into high - resolution chromosome banding . furthermore , along with the development of the fluorescent in situ hybridization ( fish ) technique , which involves the mapping of a chromosome using fluorescent labeled probe , it has become possible to identify chromosomal microdeletion and structural abnormalities such as the extra portion of a chromosome and a marker chromosome . fish has also been used in diagnosing abnormalities , caused by chromosomal aneuploidy  which are difficult to detect via conventional test methods . comparative genomic hybridization ( cgh )   and spectral karyotyping ( sky ) , 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 . 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 a technique of drawing images of several hybridization signals with different florescent dyes . combining these two techniques , in 1996 , 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 to create 21 number of colors from n types of fluorescent dyes . therefore , 31 types of colors can be created from five types of fluorescent dyes . in reality , however , because some fluorescence has a wavelength close to that within the infrared 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 macroscopically distinguishable colors ( table  1 ) can be created.table  1combination of fluorescent dyes ( painting probes ) in a chromosome analysis using skychromosomepainting probes1bcd2e3acde4cd5abde6bcde7bc8d9ade10ce11acd12be13ad14b15abc16bd17c18abd19ac20a21de22abcexaeycdea spectrum orange , b texas red , c cy5 , d spectrum green , e cy5.5 combination of fluorescent dyes ( painting probes ) in a chromosome analysis using sky a spectrum orange , b texas red , c cy5 , d spectrum green , e cy5.5 the amount of samples required for the test is 3.0  ml of heparin - 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 . when a chromosome in which the origin can not be identified or the structural abnormality of a partial copy of a chromosome is observed with g - banding , the sky technique makes it possible to recognize each chromosome with different color tones . furthermore , when cells are cultured under conditions in which the image acquisition of cell division is difficult , sky enables bleaching and staining of the specimen after the images of division have been 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 long arm of the y chromosome near the centromere can be detected . the resolution limit of detection is approximately 12  mb , similar to conventional chromosome painting techniques , and minor structural abnormalities of less than 1 band can not 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 high - resolution chromosome banding , instead of using sky alone to macroscopically observe the banding patterns . conventionally , when a chromosome of unknown origin is noticed in a child suffering with some abnormality , a chromosome analysis is performed on both parents in order to make a definitive diagnosis of the chromosome 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 a structural abnormality . conventionally , macroscopic evaluation would be performed based on the banding patterns , in case the informed consent for participation in the karyotype test can not 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 the chromosome of unknown origin without performing karyotyping of the parents , and also can reveal if one of the parents is a carrier of a balanced structural abnormality . refer to table  2 for the indications of evaluation using sky when structural chromosomal abnormalities are actually found via g-banding.table  2usability when analyzing abnormalities that have been found via g - banding by using skyresult of g - bandingkaryotypesupplementary note on skyusability of skyaccessory chromosomeaddidentifies the derivative chromosome of an extra portionderivative chromosomederidentifies the related chromosomesinsertioninsidentifies the derivative chromosome of insertion fragmenta complicated translocationidentifies the related chromosomesgenetic amplificationdimin / hsridentifies the derivative chromosome of homogeneous staining regionduplication??dupidentifies any duplicationreciprocal translocationtsame analysis level of g - bandinginversioninvdifficult to make an analysis that is better than g - bandingdeletiondeldifficult to make an analysis that is better than g - bandingnumerical aberrations+/difficult to make an analysis that is better than g - bandingunknown of derivativemaridentifies a derivative chromosome within the limitations of sensitivitycryptic translocationdetects a derivative chromosome within the limitations of sensitivitythe usability of sky is considered to be highsky can be used for analysis , but it can not be expected to have better analysis results than those of g - bandingsome cases can not be detected using sky usability when analyzing abnormalities that have been found via g - banding by using sky the usability of sky is considered to be high sky can be used for analysis , but it can not be expected to have better analysis results than those of g - banding some cases can not be detected using sky in this section , two cases in which sky can be used are presented , and the benefits and limitations of this technique are discussed . we employed the application of 24-color sky probes [ applied spectral imaging ( asi ) , inc . , asi , carlsbad , ca ] , skypaint hybridization and detection protocol , srl inc . , 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 the trisomy of chromosome 22 . one of the extra chromosomes was distinguished by two colors , and it was deduced to be a derivative chromosome [ der(22 ) ] , likely resulting from the reciprocal translocation in which the long arms of chromosome 11 ( q23.3 ) and chromosome 22 ( q11.2 ) were the breakpoints . therefore , sky is one of the most effective approaches at present for the identification of an extra chromosome of unknown origin . with the use of sky technique , it was possible to make a definitive 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 the parents . this is significant in the clinical practice of genetic counseling for parents to maintain their relationship.fig . 1chromosome analysis on trisomy 11/22 using sky chromosome analysis on trisomy 11/22 using sky the results of the chromosome analysis of 13q - syndrome using sky ( fig . 2 ) indicate structural abnormalities in chromosomes 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 arm of chromosome 4 ( p14 ) can be readily distinguished . this t(2;4 ) , however , may involve [ inv(4)(p15.1p15.32 ) ] , which is a paracentric inversion with breakpoints at p15.1 and p15.32 in the short arm part of chromosome 4 that has been translocated to chromosome 2 , and this necessitates a verification via high - resolution chromosome banding . inversions occurring in the same chromosome are not distinguished with color , so it is impossible to make a definitive diagnosis by using sky . thus in this case , testing of the parents chromosomes via high - resolution chromosome banding is necessary for further detailed diagnosis.fig . 2chromosome analysis of 13q - syndrome using sky showing structural abnormalities in chromosomes 2 , 4 , and 13 chromosome analysis of 13q - syndrome using sky showing structural abnormalities in chromosomes 2 , 4 , and 13 for chromosome 13 , it is possible to diagnose the occurrence of a deletion that is identified as 13q minus syndrome by using sky only , but this does not reveal information about the breakpoints . by simultaneously combining this sky - derived information with the banding patterns to be tested , the karyotype of case 2 is described 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 the conventional method of g - band staining can not be used directly as a stand - alone substitute . thus , more reliable interpretation and diagnosis can be made by combining the information from the two separate techniques , as neither of them can be used for unequivocal diagnosis . when performing tests using sky , it is necessary to understand the characteristics and the limitations of this analysis , and to combine the test results with those of g - band staining or high - resolution chromosome banding for a comprehensive karyotypic diagnosis . 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 the trisomy of chromosome 22 . one of the extra chromosomes was distinguished by two colors , and it was deduced to be a derivative chromosome [ der(22 ) ] , likely resulting from the reciprocal translocation in which the long arms of chromosome 11 ( q23.3 ) and chromosome 22 ( q11.2 ) were the breakpoints . therefore , sky is one of the most effective approaches at present for the identification of an extra chromosome of unknown origin . with the use of sky technique , it was possible to make a definitive 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 the parents . this is significant in the clinical practice of genetic counseling for parents to maintain their relationship.fig .  1chromosome analysis on trisomy 11/22 using sky chromosome analysis on trisomy 11/22 using sky the results of the chromosome analysis of 13q - syndrome using sky ( fig .  2 ) indicate structural abnormalities in chromosomes 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 arm of chromosome 4 ( p14 ) can be readily distinguished . this t(2;4 ) , however , may involve [ inv(4)(p15.1p15.32 ) ] , which is a paracentric inversion with breakpoints at p15.1 and p15.32 in the short arm part of chromosome 4 that has been translocated to chromosome 2 , and this necessitates a verification via high - resolution chromosome banding . inversions occurring in the same chromosome are not distinguished with color , so it is impossible to make a definitive diagnosis by using sky . thus in this case , testing of the parents chromosomes via high - resolution chromosome banding is necessary for further detailed diagnosis.fig . 2chromosome analysis of 13q - syndrome using sky showing structural abnormalities in chromosomes 2 , 4 , and 13 chromosome analysis of 13q - syndrome using sky showing structural abnormalities in chromosomes 2 , 4 , and 13 for chromosome 13 , it is possible to diagnose the occurrence of a deletion that is identified as 13q minus syndrome by using sky only , but this does not reveal information about the breakpoints . by simultaneously combining this sky - derived information with the banding patterns to be tested , it can be diagnosed as a paracentric deletion with breakpoints at q21.2 and q31.2 . the karyotype of case 2 is described 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 the conventional method of g - band staining can not be used directly as a stand - alone substitute . thus , more reliable interpretation and diagnosis can be made by combining the information from the two separate techniques , as neither of them can be used for unequivocal diagnosis . when performing tests using sky , it is necessary to understand the characteristics and the limitations of this analysis , and to combine the test results with those of g - band staining or high - resolution chromosome banding for a comprehensive karyotypic diagnosis . 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 that these 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 expected that other previously unrecognized specific chromosomal translocations are likely to be revealed with the widespread use of this technique . moreover , in recent years , spectral color banding has been developed as a technique that has overcome the limitations of sky   and in this technique g - band differential staining is combined with the sky coloring technology . clinical use of this novel technique is expected to be widespread in the field of clinical genetics in the future and to make significant contributions . considering these points , from the standpoint 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 structural abnormality of a chromosome of unknown origin is detected via g - banding , the combined use of sky makes it possible to identify the origin without performing karyotyping of the parents , which could identify which parent is the carrier of the balanced structural abnormality . therefore , in the clinical practice of genetic counseling , informed consent for a test of 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 of a patient . this advantage could potentially reduce the unnecessary psychological burden on parents with a child who demonstrates a structural abnormality in a chromosome .
spectral karyotyping is a novel technique for chromosome analysis that has been developed based on the approach of the fluorescence in situ hybridization technique . 
 spectral karyotyping makes it feasible to diagnose a variety 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 usage of spectral karyotyping for research in general clinical practice , 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 case reports .