IVD Technology Magazine | IVDT Article Index
Originally published January, 1997
Fluorescence in situ hybridization
Steven Seelig and Susan E. Tibedo
Rapidly becoming the standard of care in some cytogenetic evaluations, FISH is an important tool in oncology and genetics.
Fluorescence in situ hybridization (FISH) is an emerging IVD technology ideal for the clinical evaluation and characterization of complex biological specimens--such as blood, amniotic fluid, and solid tumors--for genetic anomalies. Of special value to pathologists and geneticists, FISH facilitates the characterization of a broad range of molecular genetic events, such as aneuploidy, gene amplification, gene deletion, and chromosome translocations, that are difficult to detect with karyotype analysis, PCR, or LCR. FISH-based IVDs have the potential to significantly increase the survival of cancer patients by making possible earlier detection of malignancy and more accurate prognostic assessments following tumor surgery. FISH IVDs also can be applied to prenatal and postnatal genetic analysis. Moreover, the technology is especially useful for simultaneous detection of multiple genetic anomalies in an individual cell, potentially saving assay time and limiting specimen requirements.
Evolution
FISH uses nucleic acid probes--segments of labeled DNA designed to bind, or "hybridize," with the target DNA of a specimen--usually fixed to a glass slide. The probes are labeled with fluorescence molecules to make identification of the probe-target hybrid possible by use of a fluorescence microscope. The hybrid is further analyzed with computer imaging equipment. Since hybridization occurs between two complementary strands of DNA, labeled probes can be used to detect genetic abnormalities, providing valuable information about prenatal disorders, cancer, and other genetic diseases. Unlike other molecular DNA-based tests, which require cell lysis to free nucleic acids for analysis, FISH allows analysis of DNA in situ, that is, in its native, chromosomal form within the cell nucleus. This attribute permits the analysis of chromosomes and genes of individual cells.
A key advantage of FISH is its ability to target only those genetic sequences of interest. In 1986 Joe Gray, PhD, and Dan Pinkel, PhD, while at the Lawrence Livermore Laboratories, found that the inclusion of appropriate blocking DNA sequences in FISH using cloned human genes or unique sequence DNA regions suppressed hybridization of the interspersed repetitive DNA sequences (so-called "junk DNA") scattered throughout the genome. This suppression largely eliminated nonspecific signals. The discovery was patented by the University of California and is licensed exclusively to Vysis, Inc. (Downers Grove, IL).
Development of direct-labeled DNA probes by covalent attachment of the fluorophores to the probe has further simplified the technology: older detection methodologies rely on labeling the probe-target DNA hybrid indirectly by binding a signal-generating moiety (e.g., fluorescein isothiocyanate [FITC]avidin) to a non-signal-generating ligand on the DNA probe (e.g., biotin) (Figure 1). The indirect methods are more complex, require additional steps, and tend to have more nonspecific signals due to the inherent stickiness of the FITC-avidin complex. For example, in a recent study to measure the amplification of HER-2/neu in breast cancer specimens, a comparison of direct-labeled and indirect-labeled probes found a methodological rate of 99.3% with the direct-labeled probe compared with a rate of only 79.7% with the indirect-labeled probe.1 Thus, with the combination of the Pinkel and Gray invention and direct-labeled probes, FISH has the potential to become a routine technology for clinical identification of chromosome anomalies.
Glossary
alpha satellite DNA: Tandem arrays of different copies of an approximately 171-base-pair sequence found at the centromeric region of each human chromosome.
aneuploidy: Deviation from a normal chromosome complement, either fewer (monosomy) or greater (for example, trisomy [three]) than normal.
spot counting: Counting of the distinctive fluorescent signals, or "spots," produced by FISH probes specific to the alpha satellite region of the chromosome when hybridized.
Procedure
FISH, using direct-labeled probes, is a simple procedure consisting of six basic steps (Figure 2). The first step, which involves sample pretreatment, ends with the denaturation of target DNA sequences. Next, the probe is applied to the target area, and the target DNA and probe are hybridized. Following hybridization, the target is washed to remove nonspecifically bound probe. Finally, a counterstain is applied and the results of the hybridization are visualized.
The sample must be prepared in such a way that the probe has access to the target DNA while specimen morphology is preserved. Tissue type, fixation, and sample embedding processes influence the type of pretreatment necessary before denaturation. For lymphocytes, simple denaturation conditions, such as high temperature and low salt or alkali, are adequate. However, formalin-fixed paraffin-embedded tissues require more steps, including deparaffinization and enzymatic and/or chemical treatment to allow the probe access to the target DNA. Hybridization and washing parameters are fairly standard but in some assays must be varied to accommodate the complexity and specificity of some probes.
(A) Color-composite image of an interphase nucleus hybridized with combinations of repetitive-sequence DNA probes labeled with three different fluorophores. The seven chromosomes identified are X (red), Y (aqua), 18 (green), 17 (blue), 12 (violet), 8 (yellow), and 7 (pink).
(B) Metaphase chromosomes stained using three fluorophore labels and combination coding to identify seven pairs of chromosomes.
(C) Normal metaphase hybridized by comparative genomic hybridization.
(D) Sperm cells hybridized with SpectrumOrange CEP 12 and SpectrumGreen CEP 8.
For direct-labeled probes, the results are detected by viewing the samples under a fluorescence microscope with appropriate filters. Indirect detection demands additional labeling steps, which typically require streptavidin or antibody-enzyme conjugates or fluorophore-labeled counterparts, and additional washing steps once the probe is bound to the target. Furthermore, indirect methodologies limit the user's ability to simultaneously score several genetic events in a sample.
A fluorescence microscope that is equipped with a 100-W mercury-arc lamp and oil-immersion fluorescence objectives with numerical apertures > 0.75 is required to view the results of a FISH assay. The probes and counterstain can be visualized separately or simultaneously depending on the filter set used. A single-band-pass filter allows one fluorophore to be viewed; a multi-band-pass filter allows viewing of several different fluorophores.
Many photographic films are available to capture FISH images. Although not impossible, it is usually very difficult to balance the bright probe signals against a dark background to produce acceptable photographic results. A better option is to capture images digitally using a microscope equipped with a digital or video camera. Digital imaging allows not only production of a printed image but also an analysis and enhancement of the image that is not possible with a standard photograph.
Performing FISH assays is not difficult for any technician who is familiar with standard laboratory procedures. However, interpreting FISH assays, especially those used to enumerate chromosomes in interphase nuclei, requires training. The trainee might observe the procedure once, perform it with supervision until competent and comfortable, and finally perform it alone. For interpretation of results, training might include a review of FISH analysis guidelines (developed either in the laboratory or by the assay manufacturer), observation and analysis of results from assays of abnormal and normal specimens (initially under supervision), and finally blinded evaluation of the same slides.
Interpretation becomes more precise with practice. Each laboratory should establish criteria for interpreting the results of a FISH assay based on the types and methods of preparation for the specimens they routinely handle.
By following CLIA and establishing product-specific performance characteristics for FISH probes and reagents, a laboratory can minimize the effects of specimen preparation and assay conditions on the final results. A laboratory can also test its ability to detect a small number of aberrant cells in a specimen that contains mostly normal cells by using appropriate positive and negative controls, creating internal guidelines to standardize the evaluation process, and empirically establishing baselines.
Figure 1. Comparison of direct-labeled and indirect-labeled probe procedures. At the point marked by an asterisk, the fluorophore is covalently linked to a direct-labeled probe; on an indirect-labeled probe, a ligand (e.g., biotin) is linked to the probe and a reagent that carries a detectable label (e.g., FITC-avidin) must be added to the reaction once the probe is bound to detect the probe-target hybrid.
Multievent Capabilities on Individual Cells
Use of multicolored probes allows for simultaneous detection by FISH of multiple genetic events within a single nucleus. A greater number of fluorophores can be used together in direct-labeled probes than in indirect-labeled probes.2 Multievent screening can identify the clonal nature of a specimen as well as the chromosomal rearrangements in metaphase and interphase cells. Additionally, simultaneous target detection can increase assay accuracy by permitting the inclusion of probes that hybridize to control loci (versus test loci). With these probes, it is possible to monitor for hybridization success or failure within each nucleus.
There are several approaches to multievent detection in FISH.2 One is to use a probe labeled with a different spectrally distinct fluorophore for each target detected in the hybridization. At least seven different chromosomes have been detected simultaneously by use of this method.2 Another approach, called color coding, uses different fluorophores singly and in combination to identify more chromosome targets than the number of fluorophores used to label the probes.3 By combining fluorophores, 2N1 targets can be detected (N = number of fluorophore labels).
Recently, all 24 chromosomes have been detected simultaneously in metaphase chromosomes by use of only five fluorophore labels.4,5 An example using three fluorophore labels to determine the aneuploidy of seven different chromosomes in interphase nuclei is shown in Figure A on page 27. When each combination consists of different proportions of each fluorophore label, the number of targets detectable is limited only by the ability to distinguish the different label proportions. For example, eight chromosomes have been reproducibly detected using only two fluorophore labels.2
The scope of FISH probes for detection of genetic defects continues to increase. For example, probes consisting of tandemly repeated human DNA sequences, such as centromeric alpha satellite sequences, are often used to identify or enumerate specific chromosomes. Cloned, unique-sequence, DNA-region probes may be used to detect small regions, or loci, of the genome. Mixtures of such clones may be used to stain, or "paint," large sections of the entire chromosome. Such staining allows analysis of the chromosome number as well as identification of additions and translocations in metaphase cells. The information provided by these probes makes them valuable in assessing the genome for a range of applications, including prenatal/perinatal diagnosis of genetic anomalies, studies of cancer, diagnosis of myeloid disorders, and rare cell analysis.
Prenatal/Perinatal Applications
Chromosomal aneuploidies, such as Down's syndrome, are by far the most common genetic abnormalities associated with birth defects in newborns. Traditional cytogenetics on banded metaphase chromosomes has been the standard prenatal test offered to women at increased risk of having fetuses with chromosomal abnormalities. Cytogenetics can examine all chromosomes for both aneuploidies and structural abnormalities; however, this technique requires a large specimen volume, culture of the fetal cells for several days, isolation of metaphase spreads, and a highly trained technician to analyze the results. Moreover, the method usually takes more than a week to complete. In contrast, FISH performed with a set of chromosome-specific probes covering only the most common aneuploidies (i.e., abnormal number of sex chromosomes or trisomy for chromosomes 13, 18, and 21) can detect these major chromosomal defects with high sensitivity and specificity from uncultured amniocytes in less than 24 hours.6 Thus, in certain clinical situations where rapid detection of the most common aneuploidies is desired, FISH on interphase nuclei provides an initial rapid screen preceding the full cytogenetic evaluation. Results from a FISH assay could be used to prioritize cases in a busy cytogenetics lab by moving those specimens that are positive by FISH to the top of the list for analysis by traditional cytogenetic methods.
Figure 2. The six steps for performing FISH using direct-labeled DNA probes.Standard Cytogenetic Analysis
In the perinatal setting, FISH is used to detect both structural and numerical chromosomal abnormalities. It can play a valuable role in perinatal testing to detect small chromosome deletions often missed by conventional cytogenetics. Examples of such microdeletion syndrome tests include those that detect deletions on chromosome 7 that are associated with Williams syndrome and on chromosome 22 that are associated with DiGeorge and velocardiofacial syndromes.
Cancer Applications
The ability of FISH to rapidly test interphase and metaphase chromosome defects makes it especially useful in the study of cancer. In solid tumors, conventional cytogenetics is rarely used because obtaining metaphases is difficult and those cells that do proceed to mitosis may not be representative of the tumor. Other molecular techniques, such as PCR and Southern, Northern, and Western analysis, require extraction of the tissue. Extraction procedures net both normal and abnormal cells, so sensitivity is lower and quantitation less reliable than with FISH probes.
FISH allows cell-by-cell analysis and thus provides for a more sensitive and reliable assessment of chromosomal aneuploidy, gene amplifications and deletions, and chromosome translocations. A reliable determination of whether a gene is amplified in a specimen is often possible with evaluation of only 20 to 50 cells.
HER-2/neu gene amplification is a significant, independent prognostic indicator for breast cancer recurrence and survival. Amplification and overexpression of HER-2/neu have been correlated with a poor prognosis--a shorter disease-free period following treatment and a shorter overall survival. Studies have also shown that HER-2/neu overexpression is useful as a marker of tumor resistance to chemotherapy and to hormone therapy. Thus, for the management of breast cancers, HER-2/neu amplification has the potential to predict response to treatment and to determine the choice of therapy.
While Southern blot analysis for HER-2/neu gene amplification and immunohistochemistry (IHC) for HER-2/neu protein expression are often used to quantitate gene amplification, each presents technical and interpretative limitations. The results of IHC vary with use of different antibodies and tissue treatment, use of different criteria for positivity, and use of different procedures. Press et al. reported the performance characteristics of several HER-2/neu antibodies to have a sensitivity ranging from 6 to 80%.7 Because of these inherent technical difficulties, the IHC and Southern blot analysis have not been standardized to yield consistent results.
FISH is an alternative technique without the technical and interpretative limitations of Southern blot and IHC. It reliably and accurately indicates gene amplification. For quantitation of HER-2/neu gene amplification, the distinct advantage of FISH is that it assesses the level of amplification directly in the tumor cells while the specimen retains its characteristic morphology.
A group of researchers recently analyzed a cohort of breast cancer specimens by various methods and found FISH to be more sensitive than Southern blot analysis and more accurate than Northern and Western blot analysis and to have a greater applicability and sensitivity in paraffin-embedded tissue than IHC. Against the best of the methodologies (IHC on frozen tissue sections), the sensitivity of FISH on paraffin sections was 97.2%, while its specificity was 100%.1
Myeloid Disorders
Myeloid disorders--including chronic myelogenous leu- kemia (CML), acute myeloid leukemia (AML), myelopro-liferative disorder (MPD), and myelodysplastic syndrome (MDS)--have in common the chromosomal abnormality trisomy 8. Thus, the presence of this trisomy is of prognostic value to the physician. FISH on interphase and metaphase cells analyzed with the Vysis CEP 8 probe (a direct-labeled chromosome-enumerator probe specific to the alpha satellite DNA contained within the centromere region of chromosome 8) was compared to standard cytogenetic analysis in a multicenter, blinded, controlled study. Four laboratories provided a total of 364 archived bone marrow specimens for assay. The results of this study, shown in Table I, exemplify the reliability of FISH technology.
In the management of many hematologic malignancies, bone marrow transplantation (BMT) is a critical therapeutic strategy for successful cure. Following the transplantation, the ability to detect the presence of clonal neoplasms and to assess engraftment by cytogenetics is important. Although in many patients a stable chimeric state evolves between donor and recipient bone marrow, in others an unstable one forms and the malignancy eventually recurs, with an increasing number of host cells appearing in the bone marrow or peripheral circulation.
Approximately one-half of all heterologous BMTs are mismatched with respect to sex. Rapid and accurate identification of the genetic sex of the bone marrow cells offers a simple method for evaluating the donor/recipient status of the bone marrow. While several methods are currently used for this evaluation, enumerating chromosomes X and Y via FISH probes can provide the most rapid and easily quantifiable result.
Using the Vysis direct-label CEP X SpectrumOrange/Y SpectrumGreen DNA FISH probe assay, a pilot study was conducted to evaluate bone marrow specimens in 139 patients who had undergone an opposite-sex BMT.8 The clinical sensitivity for interphase FISH analysis was estimated to be 100%. The estimate for conventional cytogenetics analysis was 77%, and analysis was impeded by hypocellularity in some cases. In 148 patients who had undergone a same-sex BMT, the clinical specificity for interphase FISH analysis was estimated to be 100%. The assay was shown to be highly reproducible, even across laboratories.
Table I. Results of pilot study comparing Vysis CEP 8 (chromosome enumerator probe) FISH interphase analysis with standard cytogenetic analysis for detection of trisomy 8.
Rare Cell Analysis
FISH has been used extensively in attempts to identify fetal cells in the maternal bloodstream for prenatal diagnosis. Cells from amniocentesis or chorionic villus sampling (CVS), traditional sources of fetal material for genetic analysis, are obtained at the expense of a small but significant risk to the fetus. If fetal cells could be obtained from the maternal bloodstream instead, prenatal genetic testing could be noninvasive to the fetus. Such testing, being safer than amniocentesis or CVS, might be used in a greater number of pregnancies.
By screening maternal blood samples using FISH or PCR with probes to the Y chromosome as a marker of male fetal origin, researchers report values ranging from one fetal cell in 105 to one in 109 nucleated maternal cells.9,10 Others have combined IHC detection of fetal hemoglobin with FISH in a technique known as FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms) as a means of identifying female fetal cells.11
Some reports also cite the use of FISH in detection of minimal residual disease in leukemia.12,13 Unlike reverse-transcription PCR (RT-PCR), which requires complex extraction of intact RNA, FISH provides a cell-by-cell analysis of materials simply fixed to a microscope slide. FISH is thus an easy method of quantifying the level of disease present in the specimen.
Practical Considerations
As the diagnostic industry continues to search for creative ways to contain costs, FISH is likely to play an increasing role. FISH offers the ability to obtain, in most cases, definitive results quickly by employing a simple detection strategy that can be interpreted by basic technical staff with proper training.
Automation of FISH analyses will increase assay throughput, reduce technician time, remove operator bias, and simplify the interpretation of complex results. Automated preparation and staining of slides is possible. Image processing methods and instrumentation for the automatic enumeration of chromosomes in interphase nuclei have already been demonstrated. When commercially available, automated "spot counting" will relieve technical personnel of hours of tedious microscope viewing and eliminate errors due to fatigue and misinterpretation or inconsistent application of counting criteria. Karyotyping systems similar to those designed for semiautomated analysis of conventionally stained chromosomes allow the identification of fluorescent-banded chromosomes. These capabilities, once integrated into a complete system, will allow for a turnkey FISH analysis system.
Conclusion
The utility of FISH in clinical research laboratories is growing. In a recent CAP Today article, Arthur Brothman, PhD, director of the Cytogenetics Laboratory at the University of Utah Health Sciences Center, states, "If FISH is not offered in some cytogenetic evaluations, patients are not being given what I believe to be the standard of care."14 Several driving factors are enabling FISH testing to move into mainstream practice in pathology laboratories. These include improvements in methods of cloning unique sequence probes and labeling nucleic acids, simplification of assay procedures to allow for automatic slide staining, high information density through the use of multicolor event detection, sophisticated image analysis tools, and increased knowledge of the clinical importance of genotyping of solid tumors for diagnosis, prognosis, and monitoring.
Acknowledgment
The authors gratefully acknowledge the assistance of Ping H. Hsu, PhD; Walter King, PhD; Dave Lane, PhD; Larry Morrison, PhD; Uwe Muller, PhD; Peter Osella; John Proffitt, PhD; Chris Shasserre; and Doug Taron, PhD, of Vysis, Inc.
References
1. Pauletti G, Godolphin W, Press MF, et al., "Detection and Quantitation of HER-2/neu Gene Amplification in Human Breast Cancer Archival Material Using Fluorescence In Situ Hybridization," Oncogene, 13:6372, 1996.
2. Morrison LE, and Legator MS, "Multi-color Fluorescence In Situ Hybridization Techniques," in An Introduction to Fluorescence In Situ Hybridization, Pinkel D, and Andreev M (eds), New York, John Wiley, in press.
3. Fox JL, Hsu P-H, Legator MS, et al., "Fluorescence In Situ Hybridization: Powerful Molecular Tool for Cancer Prognosis," Clin Chem, 41:15541559, 1995.
4. Speicher MR, Ballard SG, and Ward DC, "Karyotyping Human Chromosomes by Combinatorial Multi-fluor FISH," Nature Genet, 12:368375, 1996.
5. Schröck E, du Manior S, Veldman T, et al., "Multicolor Spectral Karyotyping of Human Chromosomes," Science, 273:494497, 1996.
6. Ward B, Gersen SL, Carelli MP, et al., "Rapid Prenatal Diagnosis of Chromosomal Aneuploidies by Fluorescence In Situ Hybridization: Clinical Experience with 4,500 Specimens," Am J Hum Genet, 52:854865, 1993.
7. Press MF, Hung G, Godolphin W, et al., "Sensitivity of HER-2/neu Antibodies in Archival Tissue Samples: Potential Source of Error in Immunohistochemical Studies of Oncogene Expression," Cancer Res, 54:27712777, 1994.
8. Dewald GW, Schad CR, Christensen ER, et al., "Fluorescence In Situ Hybridization with X and Y Chromosome Probes for Cytogenetic Studies on Bone Marrow Cells after Opposite Sex Transplantation," Bone Marrow Transplantation, 12:149154, 1993.
9. Hamada H, Arinami T, Kubo T, et al., "Fetal Nucleated Cells in Maternal Peripheral Blood: Frequency and Relationship to Gestational Age," Hum Genet, 91:427432, 1993.
10. Reading JP, Huffman JL, Wu JC, et al., "Nucleated Erythrocytes in Maternal Blood: Quantity and Quality of Fetal Cells in Enriched Populations," Hum Reprod, 10:25102515, 1995.
11. Soenen V, Fenaux P, Flactif M, et al., "Combined Immunophenotyping and In Situ Hybridization (FICTION): A Rapid Method to Study Cell Lineage Involvement in Myelodysplastic Syndromes," Brit J Haematol, 90:701706, 1995.
12. White DM, Crolla JA, and Ross FM, "Detection of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukaemia Using Fluorescence In-Situ Hybridization," Brit J Haematol, 91: 10191024, 1995.
13. Zhao L, Chang K-S, Estey EH, et al., "Detection of Residual Leukemia Cells in Patients with Acute Promyelocytic Leukemia by the Fluorescence In Situ Hybridization Method: Potential for Predicting Relapse," Blood, 85:495499, 1995.
14. Check WA, "Cytogenetic Labs Embrace FISH," CAP Today, 10(3):118, 1996.
Steven Seelig, MD, PhD, is chief medical officer and vice president for research and development, and Susan E. Tibedo is a technical writer at Vysis, Inc. (Downers Grove, IL).
