Most DNA fragments used as probes are extracted from bacterial artificial clones (BACs) which contain cloned human genomic DNA sequences in the size of 100–200 Kilobases (Kb). These DNA fragments could be directly labeled by nick translation to incorporate nucleotides coupled with different fluorophores such as coumarins, fluoresceins, rhodamine, and cyanines (Cy3, Cy5, and Cy7) (Morrison et al., 2003). According to the targeted regions and labeling design, FISH probes can be divided into locus-specific probes targeted to specific regions or genes and regional painting probes for specific chromosomal bands, an entire chromosome or whole genome. Commonly used locus-specific probes include alpha repetitive sequences for centromeric regions and single copy sequences for subtelomeric and gene regions. Multi-color locus-specific probes allow simultaneously detection of numerical abnormalities of two to three regions in one FISH assay. For structural rearrangements, locus-specific probes with different fluorophores for two genes or for the 5′ and 3′ regions of a gene have been used to detect “double-fusion” signals resulting from a reciprocal translocation or “break apart” signals from a gene rearrangement, respectively. Painting probes have been used mostly in a research setting to dissect chromosome domains within a nucleus or structural rearrangements in metaphase chromosomes. Figure 1 shows representative FISH applications of locus-specific and chromosome painting probes in the detection of numerical and structural chromosomal abnormalities.

Earlier studies had evaluated signal-to-noise ratios, spatial resolution of the fluorescent signals, and hybridization/detection efficiencies of FISH tests on lymphocytes and aminocytes (Klinger et al., 1992; Ried et al., 1992). These studies led to the commercialization of FISH probes with optimized probe selection and standardized labeling, and the clinical utility of FISH testing in large case series (Ward et al., 1993). To ensure safe and effective diagnostic application, a clinical cytogenetics laboratory needs to establish the analytical and clinical validities for every FISH assay. The analytical validity of a FISH assay is evaluated by its targeted accuracy, sensitivity, specificity, and normal reference ranges following a standardized laboratory procedure (Wolff et al., 2007; Ciolino et al., 2009). FISH testing could be used as an adjunctive assay or a stand-alone diagnostic assay for constitutional and somatic abnormalities. The clinical validity for its intended use should be evaluated by calculating the sensitivity from patients with targeted abnormalities and the specificity from normal controls. Other analytical and clinical considerations include possible false positive or negative results, continuous monitoring of signal variations, periodical evaluation and batch-to-batch comparisons of probe performances (Test and Technology Transfer Committee, 2000).

FISH technology enabled the detection of an increased spectrum of genetic disorders from chromosomal abnormalities to submicroscopic copy number variants (CNVs) and extended the cell-based analysis from metaphases to interphases (Xu and Li, 2013). The analytical resolution of FISH is in the range of 100–200 Kb as determined by the probe size, which is 50-fold higher than the 5–10 megabase (Mb) Giesma banding of a high resolution karyotyping. Locus-specific probes detected submicroscopic CNV and led to the identification of a group of genomic disorders (also termed contiguous gene syndromes or microdeletion syndromes), such as DiGeorge syndrome (OMIM#188400) by a deletion at 22q11.2, Prader-Willi syndrome (OMIM#176270) and Angelman syndrome (OMIM#105830) by a deletion at 15q11.2. FISH can be performed directly on interphase nuclei, which eliminated the time consuming cell culture procedure and extended its diagnostic application toward rapid screening of chromosomal and genomic abnormalities. In the following sections, the diagnostic applications of FISH technology are focused on three main areas: prenatal screening and postnatal diagnosis of constitutional chromosomal abnormalities and submicroscopic pathogenic CNVs, identification and monitoring of acquired chromosomal abnormalities in hematopoietic and solid tumors, and the detection of infectious diseases caused by microbials and parasites.

A Multiplex FISH panel with differentially labeled probes has been developed for prenatal screening of common aneuploidies involving gains or losses of chromosomes X, Y, 13, 18, and 21 (Ried et al., 1992; Ward et al., 1993). Pregnant women with a single indication or combined indications of advanced maternal age, abnormal ultrasound findings, or abnormal maternal serum screening have an increased risk of 4–30% for carrying numerical and structural chromosomal abnormalities; among these abnormalities, 84% were numerical abnormalities mostly detectable by the multiplex FISH panel, and 16% were structural abnormalities required further microarray analysis (Li et al., 2011). For prenatal cases with cardiac anomalies detected by prenatal ultrasound examination, DiGeorge syndrome was detected by FISH. Recently, the application of non-invasive prenatal testing by massive parallel sequencing on maternal cell-free fetal DNA significantly improved the accuracy of aneuploidy screening, which resulted in a 57% decline in invasive prenatal procedures and an increase of diagnostic yield of chromosomal abnormalities (Xu Z. Y. et al., 2013; Meng et al., 2015). Despite these technology advances in prenatal diagnosis, the multiplex FISH panel is still used as an adjunctive assay for rapid detection of common aneuploidies. It should be noted that false positive or negative results as well as maternal cell contamination have been noted in prenatal FISH analysis. Therefore, an irreversible therapeutic action should not be initiated on the basis of FISH results alone. The current guideline recommended that clinical decisions should be made based on two of three pieces of available information: FISH results, conventional cytogenetic analysis and clinical information (Test and Technology Transfer Committee, 2000). Furthermore, aneuploidies and polyploidies have been detected in about 50% of first trimester spontaneous abortions by chromosome analysis and in 35% of products of conception culture failure cases by microarray analysis; it is recognized that an extended FISH panel for chromosomes X/Y/18, 13/21, and 15/16/22 will detect all polyploidies, 84% of aneuploidies, and 69% of multiple aneuploidies causing miscarriages (Zhou et al., 2016).

Developmental delay, intellectual disabilities, and multiple congenital anomalies are present in 1–5% of newborns, and chromosome microarray analysis as the first tier genetic testing has detected a spectrum of cytogenomic abnormalities in 10~20% of these patients (Miller et al., 2010; Li et al., 2015). Analysis of abnormal findings from consecutive pediatric cases observed genomic disorders (microdeletion/microduplication syndromes), subtelomeric rearrangements, interstitial imbalances, chromosomal structural rearrangements, and aneuploidies in about 37, 26, 19, 10, and 8% of these cases, respectively (Xu et al., 2014). Cell-based FISH testing has been a cost-effective adjunctive assay to confirm microarray detected genomic disorders and then to detect carrier statues in a follow-up parental study. Microdeletions can be detected as a loss of one signal in metaphases and interphases, while microduplications can be detected as “twin-spot” like two signals in the interphase nuclei. For subtelomeric rearrangements, a complete set of subtelomeric FISH probes for all human chromosomes was developed (Ning et al., 1996) and have been used routinely as an adjunctive assay in visualizing cryptic and complex subtelomeric rearrangements (Li et al., 2006; Rossi et al., 2009). For many newly defined loci of genomic disorders and interstitial imbalances, there are no commercially available FISH probes. Therefore, “home-brew” targeted BAC clone FISH probes were used for these unique cases (Li et al., 2006; Khattab et al., 2011).

Structural rearrangements like ring chromosomes and small supernumerary marker chromosomes (sSMC) present not only segmental gains or losses but also a mosaic pattern due to their dynamic behavior in mitosis. As shown in Figure 1A, centromeric FISH probes are routinely used to track the changes from dicentric, tricentric, and tetracentric ring chromosomes to loss of the ring through mitosis. Subtelomeric and interstitial FISH probes have been used to define the intactness of the ring chromosome and the level of mosaicism (Zhang et al., 2004; Xu F. et al., 2013). A cytogenomic approach combining chromosome, FISH, and microarray analyses has been recommended for characterizing the genomic structure, mitotic instability, and mechanisms of ring formation for cases with a ring chromosome (Zhang et al., 2012). sSMC are extra centric chromosome fragments usually in the forms of an inverted duplication or a small ring chromosome and present in 0.043% of newborn children. Several sSMC have syndromic phenotypes such as inv dup(22q11.2) for cat-eye syndrome (OMIM*607576) and i(12p) for Pallister Killian syndrome (OMIM#601803), and others like inv dup(15q) and i(18p) can have variable phenotypes (Liehr et al., 2004, 2006). About 30% of sSMC are derived from chromosome 15; the D15S10 or SNRPN probes are routinely used to assess inv dup(15q) (Wang et al., 2015). The euchromatic material in sSMC can be detected by a microarray analysis. A set of pericentric core probes for each arm of human chromosomes has been validated for characterizing unambiguously the chromosomal origin of sSMC and the level of mosaicism (Castronovo et al., 2013).

The discovery of Philadelphia chromosome in chronic myeloid leukemia (CML) followed by the characterization of t(9;22)(q34;q11) with underlying ABL1/BCR gene fusions supported the causative role of chromosomal abnormalities in carcinogenesis and set the foundation for cancer cytogenetics (Mitelman et al., 2007). Cancer is considered a genetic disease at the cellular level resulting from either a progressive process or a one-off catastrophic event (Stephens et al., 2011; Li and Cui, 2016). The two main pathogenetic pathways for hallmarks of cancer development are the inactivation of tumor suppressor genes by deletions, mutations, miRNA upregulation, or epigenetic mechanisms, and the activation or deregulation of oncogenes as a consequence of point mutations, amplification or balanced cytogenetic abnormalities (Vogelstein and Kinzler, 2004; Hanahan and Weinberg, 2011). Recurrent chromosomal abnormalities including translocations, deletions, duplications, and gene amplifications associated with distinct tumor entities have been characterized; specifically designed FISH panels have been widely used in the diagnosis and monitoring of acquired chromosomal abnormalities in hematologic and solid tumors (Hu et al., 2014; Liehr et al., 2015; Mikhail et al., 2016).

Current guidelines recommend an integrated approach for cancer cytogenetic diagnosis (Wolff et al., 2007). In general, both conventional karyotyping and FISH testing are used for initial diagnosis and follow up monitoring of clonal abnormalities. For hematopoietic and lymphoid tumors, the most commonly used FISH probes and disease-specific panels in a clinical cytogenetics laboratory are listed in Table 1. Results from a FISH panel offer a quick evaluation of targeted abnormal patterns and their percentage within the bone marrow cells or leukocytes. Chromosome analysis will then reveal the clonal abnormalities and clonal evolution. For leukemias requiring urgent treatment, such as acute promyelocytic leukemia (APL) caused by the t(15;17)(q24;q21) with underlying PML/RARa fusions, rapid FISH result is mandated for the administration of all-trans retinoic acid (ATRA). Targeted therapy against the ABL1/BCR fusion protein by small molecule tyrosine inhibitors like imatinib mesylate (Gleevec), dasatinib (Sprycel), and nilotinib (Tasigna) has increased the 10-year overall survival from 20 to 80–90% (Li et al., 2013). For many cryptic rearrangements undetectable by routine chromosome analysis, such as t(12;21)(p13;q22) with ETV6/RUNX1 gene fusions, t(4;14)(p16.3;q32) with FGFR3/IGH gene fusions, deletions of 12p13 (ETV6), 13q14 (RB1), and 17p13 (TP53), FISH tests are considered a stand-alone diagnostic assay. Adjunctive use of FISH probes to further define ambiguous or hidden chromosomal abnormalities is required for many cases (Kamath et al., 2008; Massaro et al., 2011). Additionally, FISH is a sensitive and timely method to monitor residual diseases with known clonal abnormality and bone marrow transplantation by sex-mismatch donor at cellular level. Considering some hematologic tumors may be morphologically similar and the abnormalities may not be detected by low-resolution karyotyping and/or in low percentage of leukemic cells, FISH could be important for differential diagnosis between these diseases. For example, cyclin D1 (CCND1) translocation can be detected by FISH as a characteristic abnormality in mantle cell lymphoma, which provides differential diagnosis for morphologically similar chronic lymphoid leukemia (CLL). Furthermore, FISH for nuclear DNA can be combined with immunostaining of cytoplasmic markers for simultaneous identification of chromosomal abnormalities and cell types. For example, IGH translocation is present in multiple myeloma and monoclonal gammopathy of undetermined significance (MM/MGUS) with high frequency, which is usually detected in plasma cells. In a two-step assay with first the hybridization of IGH probe and then immune-staining by fluoresceinisothiocyanate (FITC)-conjugated antibodies against κ- or λ-light chain, the FITC-stained cytoplasm and IGH break apart signals within the nuclei were visualized in plasma cells simultaneously. This modified immuno-FISH was expected to improve the diagnostic accuracy but the low sensitivity limited its application only in follow-up study (Boersma-Vreugdenhil et al., 2003).

FISH tests are widely used in various types of solid tumors. For example, FISH can define gene rearrangements in congenital fibrosarcoma with a novel complex translocation (Marino-Enriquez et al., 2008) and validate subclone markers in heterogeneous melanoma biopsies (Parisi et al., 2011). FISH results can be used to guide cancer treatment. For example, Herceptin-targeted therapy is effectively against HER2 over-expressed breast cancer. For routine clinical specimen, immunohistochemistry, real-time polymerase chain reaction, and FISH were used to assess the HER2 protein level, RNA expression, and DNA copy numbers, respectively. Among these methods, FISH offered a cell-based evaluation for the ratio of HER2 gene copy number to the number of copies of chromosome 17 (HER2/CEP17 ratio). The FISH scoring criteria for HER2/CEP17 ratio and the interpretive guidelines were reported (Hicks et al., 2005). Many targeted therapies for recurrent translocations in various types of solid tumors have been either approved by FDA or are under clinical trials. For example, lapatinib, sorafenib, sunitinib, termsirolimus, and pazopanib have been used for papillary renal cell carcinoma with translocations involving the TFE3 gene at Xp11.2; cixutumumab and mithramycin are in phase II clinical trial for Ewing sarcoma with translocation involving the EWSR1 gene at 22q12 (Li et al., 2013). FISH assays using probes for specific recurrent translocations from different solid tumors could guide effective targeted therapy. FISH tests were also used to evaluate sperm aneuploidy frequencies before and after chemotherapy in patients with testicular cancer and Hodgkin's lymphoma; significantly increased frequencies of aneuploidies for a duration up to 24 months were noted (De Mas et al., 2001; Tempest et al., 2008). It was recommended that genetic counseling about potentially increased reproduction risk from chemotherapy should be offered to cancer patients.

The majority of FISH probes target to specific chromosomal and genomic abnormalities in the human genome. Rapid phylogenetic identification of single microbial cells was achieved using fluorescently labeled oligonucleotides complementary to 16S ribosomal RNA (rRNA) (DeLong et al., 1989). Some segments in the 16S rRNA are invariant in all organisms but phylogenetic group-specific 16S rRNA in different groups of organism can be used as oligonucleotide FISH probes (length 17–34 nucleotides) to identify infectious agents in clinical samples. For example, FISH probes complementary to specific sequence of 16s rRNA can detect malaria infection in blood samples. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probes that detect all five species of Plasmodium known to cause the disease in humans. The sensitivity of this FISH assay is better than the Giemsa staining method. A LED light source may be an available device to read FISH result, which can extend the clinical application of FISH especially in the resource-limited areas. Since rRNA has a short life and is present in a live organism with plenty of copies, FISH should be done in the live pathogens (Shah et al., 2015).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.