Classification of fluorescent R-Band metaphase chromosomes using a convolutional neural network is precise and fast in generating karyograms of hematologic neoplastic cells.

Karyotype analysis has a great impact on the diagnosis, treatment and prognosis in hematologic neoplasms. The identification and characterization of chromosomes is a challenging process and needs experienced personal. Artificial intelligence provides novel support tools. However, their safe and reliable application in diagnostics needs to be evaluated. Here, we present a novel laboratory approach to identify chromosomes in cancer cells using a convolutional neural network (CNN). The CNN identified the correct chromosome class for 98.8% of chromosomes, which led to a time saving of 42% for the karyotyping workflow. These results demonstrate that the CNN has potential application value in chromosome classification of hematologic neoplasms. This study contributes to the development of an automatic karyotyping platform.

BACs-on-Beads Assay for the Prenatal Diagnosis of Microdeletion and Microduplication Syndromes.

OBJECTIVE: To evaluate the clinical value of BACs-on-Beads (BoBs) assay in detection of microdeletion and microduplication syndromes. METHODS: A total of 6,814 cases of amniotic fluid cells collected from January 2015 to July 2020 in our hospital were analyzed by chromosomal karyotyping and BoBs assay. Fluorescence in situ hybridization (FISH) or chromosomal microarray analysis (CMA) provided further validation for the cases of microdeletion and microduplication. RESULTS: Thirty microdeletion and microduplication syndromes were identified by BoBs with an incidence of ~1/227, including 22q11.2 microduplication (0.044%, 3/6814), DiGeorge I syndrome (0.044%, 3/6814), 17p11.2 microduplication (0.015%, 1/6814), Smith-Magenis syndrome (0.015%, 1/6814), 17p11.2p11.3 microduplication (0.015%, 1/6814), Williams-Beuren syndrome (0.088%, 6/6814), 7q11.2 microduplication (0.029%, 2/6814), DiGeorge II syndrome (0.015%, 1/6814), 18p11.32p11.21 microduplication (0.015%, 1/6814), Wolf-Hirschhorn syndrome (0.029%, 2/6814), 4p16.3 microduplication (0.015%, 1/6814), Langer-Giedion syndrome (0.015%, 1/6814), Miller-Dieker syndrome (0.015%, 1/6814), Cri du Chat syndrome (0.015%, 1/6814), Xp22.31 microdeletion (0.059%, 4/6814), Prader-Willi syndrome (0.015%, 1/6814). High concordance was obtained between BoBs and FISH or CMA. However, only four cases were detected by chromosomal karyotyping. CONCLUSION: BoBs assay can rapidly detect microdeletion and microduplication syndromes, which compensates the shortcomings of conventional chromosomal karyotyping and greatly improves the efficiency and accuracy of prenatal diagnosis.

Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects.

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.

Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome.

Analysis of the organization of the human genome is vital for understanding genetic diversity, human evolution, and disease pathogenesis. A number of approaches, such as multicolor fluorescence in situ hybridization (FISH) assays, cytogenomic microarray (CMA), and next-generation sequencing (NGS) technologies, are available for simultaneous analysis of the entire human genome. Multicolor FISH-based spectral karyotyping (SKY), multiplex FISH (M-FISH), and Rx-FISH may provide rapid identification of interchromosomal and intrachromosomal rearrangements as well as the origin of unidentified extrachromosomal elements. Recent advances in molecular cytogenetics have made it possible to efficiently examine the entire human genome in a single experiment at much higher resolution and specificity using CMA and NGS technologies. Here, we present an overview of the approaches available for genome-wide analyses. © 2018 by John Wiley & Sons, Inc.

Identification of Small and Non-Small Cell Lung Cancer Markers in Peripheral Blood Using Cytokinesis-Blocked Micronucleus and Spectral Karyotyping Assays.

Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer. There is an urgent need to develop tools to identify individuals at high risk of developing SCLC. We have previously reported that the cytokinesis-blocked micronucleus (CBMN) assay is a strong predictor of non-small cell lung cancer (NSCLC). Here, we investigate the sensitivity of the CBMN endpoints as predictors of SCLC risk. We conducted the CBMN assay on SCLC patients (n = 216), NSCLC patients (n = 173), and healthy controls (n = 204). Per sample, 1,000 binucleated cells (BN) were scored, and 3 endpoints, micronuclei (BN-MN), nucleoplasmic bridges (BN-NPB), and nuclear buds(BN-BUD), were recorded. Spectral karyotyping was also conducted on SCLC patients (n = 116) and NSCLC patients (n = 137) to identify genomic regions unique to each disease. Significantly higher levels of CBMN endpoints were observed in both cancer groups compared to controls. BN-NPBs were significantly higher among SCLC patients compared to NSCLC patients (p < 0.001). Chromosomes 5 and 17 were associated with BN-MN, and chromosomes 5, 18, 20, and 22 were associated with BN-NPBs in SCLC patients. Given the high frequency of chromosome aberrations observed in SCLC, events such as reinsertion of the micronucleus and chromothripsis may be potential mechanisms for the genetic instability in these patients.

Multicolor Spectral Analyses of Mitotic and Meiotic Mouse Chromosomes Involved in Multiple Robertsonian Translocations. II. The NMRI/CD and CD/TA Hybrid Strains.

Multicolor spectral analyses (spectral karyotyping) were performed on mitotic chromosomes of NMRI, CD, and TA mice and on male meiotic chromosomes (diakineses) of NMRI/CD and CD/TA hybrids. All chromosomes, including the various centric (robertsonian) fusions, could be unequivocally identified. Apart from the robertsonian translocations, which were previously detected by conventional banding analyses, no other interchromosomal rearrangements were found in these mice. In both the CD and TA mice, the autosomes 19 and the XY sex chromosomes are not involved in robertsonian translocations. In diakineses of male meiosis of the NMRI/CD hybrid, the 9 expected trivalents were present, whereas in those of the CD/TA hybrids a stable large meiotic multivalent, formed by 15 robertsonian fusion chromosomes and 2 terminally located normal chromosomes, was observed. The specific sequential order of the robertsonian fusion chromosomes found within this meiotic chain was as theoretically predicted. In the majority of diakineses of the NMRI/CD and CD/TA hybrids, the free autosomal bivalent 19 and the XY sex bivalent formed noticeable tight spatial associations.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Multicolor Karyotyping and Fluorescence In Situ Hybridization-Banding (MCB/mBAND).

Multicolor fluorescence in situ hybridization (mFISH) approaches are routine applications in tumor as well as clinical cytogenetics nowadays. The first approach when thinking about mFISH is multicolor karyotyping using human whole chromosome paints as probes; this can be achieved by narrow-band filter-based multiplex-FISH (M-FISH) or interferometer/spectroscopy-based spectral karyotyping (SKY). Besides, various FISH-based banding approaches were reported in the literature, including multicolor banding (MCB/mBAND) the latter being evaluated by narrow-band filters, and using specific software. Here, we describe the combined application of multicolor karyotyping and MCB/mBAND for the characterization of simple and complex acquired chromosomal changes in cancer cytogenetics.

Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt-Hogg-Dubé syndrome.

Hereditary renal cell carcinomas (RCCs) are life-threatening disorders not only for the patients but also for their relatives. Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). The protein product, FLCN, functions as a tumor suppressor, and the affected patients have high risks of developing multiple RCCs. The carcinogenic mechanisms stemming from FLCN dysfunction have been investigated using rodent models and human RCC tissues. However, very limited information has been available about in vitro signaling of human renal cells with genetically mutant FLCN. Herein, we established a new cell line, BHD-F59RSVT, from a BHD patient's chromophobe RCC by transfecting SV40 large T antigen. We investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line. BHD-F59RSVT reflected the patient's FLCN germline mutation, a 3-nt deletion in exon 13 (c.1528_1530delGAG). Neither somatic mutation nor loss of heterozygosity of FLCN was detectable. Chromosome 17p11.2 of the FLCN proximal region demonstrated a trimodal pattern. Genome-wide chromosomal analysis revealed a loss of chromosome 16 and mosaic segmental gains in chromosome 7. BHD-F59RSVT cells were positive when immunostained for cytokeratin 7, supporting their origin from distal convoluted tubules. Western blotting analysis demonstrated severely suppressed FLCN expression at the protein level. The collective findings indicate that the established cell line will be suitable for functional analysis of the typical phenotype of BHD-associated RCC with suppressed FLCN expression.

Hyperspectral backscatter imaging: a label-free approach to cytogenetics.

Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope. Graphical abstract Hyperspectral backscatter imaging for label-free characterization.

Prenatal diagnosis and molecular cytogenetic characterization of de novo pure partial trisomy 6p associated with microcephaly, craniosynostosis and abnormal maternal serum biochemistry.

We present prenatal diagnosis and molecular cytogenetic characterization of de novo pure trisomy 6p22.3 → p25.3 encompassing BMP6 in a fetus associated with microcephaly and craniosynostosis on prenatal ultrasound, abnormal maternal serum biochemistry of a low PAPP-A level in the first-trimester combined test, and a karyotype of 46,XX,der(22)t(6;22)(p22.3;p13)dn. The present case demonstrates the usefulness of rapid prenatal identification of the origin of the extra chromosome material on the short arm of an acrocentric chromosome by spectral karyotyping, fluorescence in situ hybridization and array comparative genomic hybridization. We review the phenotypic abnormality of craniosynostosis in previously reported patients with partial trisomy 6p. We discuss the genotype-phenotype correlation of the involved gene of BMP6 in this case.

Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.

Cytogenetic analysis of a low-grade secondary peripheral chondrosarcoma arising in synovial chondromatosis.

Secondary chondrosarcoma is a malignant chondroid tumor arising in a benign precursor. Synovial chondromatosis is a benign chondroid lesion that rarely transforms to chondrosarcoma. We present the case of a 54-year-old male with the diagnosis of low-grade secondary peripheral chondrosarcoma developed in the context of synovial chondromatosis. Cytogenetics revealed a novel aberration t(1;14)(q23.1~24;q24.1~3). Multicolor banding (mBAND) analysis described the chromosomal regions involved in this translocation with a higher detail. Diagnosis of such borderline lesions is very difficult and cytogenetics is helpful in characterizing these tumors.

[Spectral karyotyping of seven prenatally detected marker chromosomes and complex chromosome aberrations].

OBJECTIVE: To perform spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) and conventional karyotyping on prenatally detected marker chromosomes and complex chromosomal aberrations. METHODS: Five marker chromosomes and 2 complex chromosome aberrations diagnosed by G banding were collected. SKY was performed to verify the composition of marker chromosomes. FISH was used to confirm the diagnosis when necessary. In certain cases, C or N banding technique was employed to verify the composition of chromosomes. Results of ultrasonography and pregnancy outcome were reviewed. RESULTS: Among the 5 marker chromosomes, 2 were large and 3 were medium in size, 4 were de novo and one was inherited from the father. By SKY analysis, 2 marker chromosomes have originated from non-acrocentric chromosomes (4 and 9), whilst the other two have originated from acrocentric chromosomes (21 and 22). The remainder was derived from X chromosome. The SKY results were confirmed by FISH in 3 cases. Four cases have chosen to terminate the pregnancy after genetic counseling. A fetus with inherited paternal marker chromosome was delivered at term, and showed normal development during the first year of life. As for the other 2 cases with complex chromosome aberrations, by SKY examination, one had duplication in chromosome 8 and the other had chromosome rearrangements derived from translocation between chromosomes 2 and 6. In the latter case the fetus was delivered at term but showed developmental retardation at 6 months. CONCLUSION: SKY in combination with FISH can facilitate identification of the origins of marker chromosomes as well as complex chromosomal aberrations. With combined information from ultrasonography, SKY and FISH, effective counseling may be offered to the patients.

Comprehensive cytogenetic study of primary cutaneous gamma-delta T-cell lymphoma by means of spectral karyotyping and genome-wide single nucleotide polymorphism array.

Primary cutaneous gamma-delta T-cell lymphoma (PCGD-TCL), which originates from activated mature gamma-delta T cells with a cytotoxic phenotype is a rare T-cell lymphoproliferative disease. The prognosis of PCGD-TCL has been rather unfavorable due to poor response to conventional chemotherapy, and its molecular features and pathophysiology underlying disease development remain unknown. We report here a case with primarily treatment-resistant PCGD-TCL featuring highly complex cytogenetic and genetic aberrations detected by spectral karyotyping and genome-wide single nucleotide polymorphism (SNP) array. Chromosomal aberrations included several chromosomal translocations involving breakpoints at 9p21, 14q11.2, 14q32.1, or 16q23.1, suggesting the involvement of WWOX, TCL gene cluster, and BCL11B, which are crucial for tumorigenesis in T-cell lymphomas. SNP analysis also identified genome copy number gains and losses in various regions, which can potently deregulate expression of various pro- and anti-oncogenic genes involved in RAS-related protein pathways, PI3K/AKT/MTOR-related pathways, MYC-related signaling, or TP53-related signaling. Thus, this case report may shed some light on the complex molecular abnormalities involved in the development of PCGD-TCL and on information that can aid the search for druggable target molecules in this disease.

Characterization of giant marker and ring chromosomes in a pleomorphic leiomyosarcoma of soft tissue by spectral karyotyping.

Pleomorphic leiomyosarcoma of soft tissue is relatively rare and its cytogenetic and molecular genetic data are scarce. We present a case of pleomorphic leiomyosarcoma arising in the left thigh of a 60-year-old man. Fluorine-18-deoxyglucose positron emission tomography imaging showed a homogenously high uptake within the mass in the proximal left thigh (maximum standardized uptake value, 20.9). Following a core needle biopsy, wide resection of the tumor was performed. Histologically, the tumor was composed of a mixture of spindle cells, polygonal cells and bizarre giant cells forming interlacing bundles and a storiform pattern. Immunohistochemically, the tumor cells were positive for vimentin, smooth muscle actin and desmin. The MIB-1 labeling index was 19.7% in the highest spot. Cytogenetic analysis exhibited a complex karyotype with several numerical and structural alterations, including giant marker and ring chromosomes. Spectral karyotyping demonstrated that giant marker and ring chromosomes were composed of material from the X chromosome. Metaphase-based comparative genomic hybridization analysis showed high-level amplifications of 1q21-q25 and 12q13-q21 and gains of 1p31-p32, 10p11-p13, 17p11 and 19p13. The patient received postoperative adjuvant radiotherapy and doxorubicin-based chemotherapy. No local recurrence or distant metastasis was detected during a follow-up period of 19 months. The clinicopathological, cytogenetic and molecular genetic features of pleomorphic soft tissue leiomyosarcoma are discussed.

Detection of novel genomic aberrations in anaplastic astrocytomas by GTG-banding, SKY, locus-specific FISH, and high density SNP-array.

Astrocytomas represent the largest and most common subgroup of brain tumors. Anaplastic astrocytoma (WHO grade III) may arise from low-grade diffuse astrocytoma (WHO grade II) or as primary tumors without any precursor lesion. Comprehensive analyses of anaplastic astrocytomas combining both cytogenetic and molecular cytogenetic techniques are rare. Therefore, we analyzed genomic alterations of five anaplastic astrocytomas using high-density single nucleotide polymorphism arrays combined with GTG-banding and FISH-techniques. By cytogenetics, we found 169 structural chromosomal aberrations most frequently involving chromosomes 1, 2, 3, 4, 10, and 12, including two not previously described alterations, a nonreciprocal translocation t(3;11)(p12;q13), and one interstitial chromosomal deletion del(2)(q21q31). Additionally, we detected previously not documented loss of heterozygosity (LOH) without copy number changes in 4/5 anaplastic astrocytomas on chromosome regions 5q11.2, 5q22.1, 6q21, 7q21.11, 7q31.33, 8q11.22, 14q21.1, 17q21.31, and 17q22, suggesting segmental uniparental disomy (UPD), applying high-density single nucleotide polymorphism arrays. UPDs are currently considered to play an important role in the initiation and progression of different malignancies. The significance of previously not described genetic alterations in anaplastic astrocytomas presented here needs to be confirmed in a larger series.

Testes genéticos em diagnóstico pré-natal: onde estamos, para onde vamos / Prenatal genetic testing: where we are at, where we are heading to

Este texto tem como objetivo apresentar uma revisão acerca do estado da arte da citogenética convencional e molecular aplicada ao diagnóstico pré-natal, discutindo as aplicações, vantagens e desvantagens dos diferentes métodos, em suas bases teóricas e históricas. Desde 1960, a citogenética convencional, com a análise microscópica dos cromossomos em divisão, vem sendo utilizada como padrão ouro. Entretanto, mesmo adotando essa abordagem, para uma significativa parcela de casos não é possível estabelecer diagnóstico sindrômico definitivo em cerca de metade dos pacientes que apresentam cariótipo normal, na presença de malformações. Para esse grupo, as técnicas moleculares que envolvem estudos em nível genômico poderiam permitir a identificação de novos microarranjos cromossômicos possivelmente responsáveis pelo fenótipo anormal, contribuindo para a caracterização molecular e estabelecimento de um diagnóstico mais preciso, uma abordagem perinatal mais adequada e um aconselhamento genético mais detalhado. Destaca-se o advento das técnicas de FISH, SKY, CGH e array CGH como promissoras aliadas, de forma complementar ao cariótipo convencional

This paper aims at presenting a review of the state of the art of conventional and molecular cytogenetics applied to prenatal diagnosis, the applications, pros and cons of different techniques and their historical and theoretical background. Since 1960, conventional cytogenetics, based on the analysis of chromosomes has been used as a gold standard. However, for a significant proportion of cases it is not possible to establish definitive syndromic diagnosis in about half of the patients with normal karyotype in the presence of malformations. For this group, molecular techniques at the genomic level might allow the identification of new chromosomal areas potentially responsible for the abnormal phenotype, contributing to the molecular characterization and establishment of a more accurate diagnosis and the most appropriate perinatal approach, including a more detailed genetic counseling. The advent of FISH techniques, SKY, CGH and array CGH will be discussed as promising tools to complement cytogenetic diagnosis based on conventional karyotyping

Spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed (1, 2). This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities. SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope (3, 4). SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies (5, 6). SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes (7-10). The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (http://www.spectral-imaging.com/). SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia (11-13). Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models (14). Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).