This prenatal testing procedure may provide faster results for perinatologists and more rapid reassurance for high-risk patients. But it should be used as an adjunct to traditional chromosome analysis.
Prenatal diagnosis for Down syndrome and other common chromosomal abnormalities is regarded as one of the cornerstones of prenatal testing. This review will briefly describe the traditional technology of chromosome analysis and introduce fluorescence in situ hybridization (FISH)specifically prenatal interphase FISHas an adjunct to chromosome analysis.
We will discuss the most suitable indications for this additional test and recommend guidelines on how to manage pregnancies when abnormalities are found. And finally, we will review the accuracy and limitations of FISH testing and mention several important caveats.
Prenatal diagnosis for chromosomal abnormalities has been performed since the mid-1960s.1 Traditional chromosome analysis consists of culturing amniotic fluid cells so that they enter metaphasea stage in which the chromosomes are visible as distinct bodies. Results are not available for approximately 7 to 14 days when using the in situ method of culturing and longer if using the flask method. This time reflects the need for amniotic-fluid cells to settle onto coverslips and go through enough divisions to provide a sufficient number of cells for chromosome analysis. The dividing cells are then arrested in metaphase and harvested. The chromosomes are stained, revealing a banding pattern that allows the number of chromosomes and their structure to be analyzed under a light microscope.
The most common chromosomal abnormalities observed in prenatal diagnosis are three copies of chromosomes 13, 18, and 21 (trisomy), and extra or missing sex chromosomes X or Y. Chromosome analysis is considered the standard of care for the diagnosis of these aberrations, which represent approximately two thirds of all prenatally detected chromosomal abnormalities.2
Over the years, the turnaround time for completing chromosome analysis has improved to the point that the time for culturing and analysis now averages 7 to 14 days Although this interval is sufficient for managing most pregnancies, in certain situations a faster turnaround time would greatly help clinicians manage at-risk patients.
The technique of FISH involves hybridization of fluorescently labeled probes to a patient's chromosomal DNA. As shown in Figure 1, FISH can be performed on metaphase chromosomes as well as interphase nuclei (the stage between two successive cell divisions, during which chromosomes are not visible as distinct bodies).
In 1992 Klinger and colleagues designed a prenatal screening test to detect the common chromosomal abnormalities previously described.2 This approach used FISH with probes derived from specific regions of chromosomes 13, 18, 21, X, and Y on uncultured amniotic fluid cells. This technique attempted to ascertain the number of these chromosomes present in the uncultured fetal cells.
Using a fluorescent microscope, interphase FISH slides are analyzed by counting the number of fluorescent signals displayed in each cell. Typically, 50 nuclei are analyzed for each probe. The result is then compared with reportable ranges previously established by the lab, yielding very reliable data about the number of copies of each specific chromosome being studied. This testing allows preliminary detection of trisomy for chromosomes 13, 18, and 21, numerical abnormalities of the sex chromosomes, and triploidy (three sets of all chromosomes, resulting in 69 chromosomes).
Prenatal interphase FISH can be performed on uncultured amniotic fluid, chorionic villi, or fetal blood cells.3 Since tissue culture is not needed, results are usually available 24 to 48 hours from receipt of the sample. Amniotic fluid samples from pregnancies of less than 15 weeks' gestation are usually not recommended for prenatal interphase FISH due to the low number of cells present in the sample.
In the fall of 1997, Vysis, Inc., received FDA clearance for their AneuVysion probe set which consists of two probe mixtures (two probes in one mixture and three probes in the other): (1) a chromosome 13q14 probe labeled in green, mixed with a 21q22 probe labeled in red; and (2) chromosome 18, X, and Y pericentromeric probes labeled in aqua, green, and red, respectively (Figure 1). The FDA recognizes the use of this technology as an adjunct to traditional chromosome analysis.
There are several clinical situations in which patients can benefit from the rapid information provided by prenatal interphase FISH testing.
Advanced maternal age. Age-related risk for numerical chromosomal abnormalities clearly makes advanced maternal age the most common indication for prenatal chromosome analysis. Even though the risk for a numerical chromosomal abnormality increases with advanced maternal age, the number of abnormalities detected in this population is not as high as with these other indications.4 Adjunctive prenatal interphase FISH may not be especially helpful in managing uncomplicated pregnancies in which prenatal diagnosis is performed at gestational ages of less than 20 weeks, however. On the other hand, the rapid results this technology provides can prove invaluable in two other scenarios: pregnancies at increased risk for one of the common chromosomal abnormalities or a prenatal situation with time constraints, which we'll explain further.
Abnormal U/S findings. FISH testing is mostly likely to detect chromosomal abnormalities when ultrasound reveals fetal abnormalities because this is the setting in which chromosome trisomies are so frequently found. Amid anxiety raised by pregnancies complicated by significant U/S findings, the high rate of detecting numerical chromosomal abnormalities that prenatal interphase FISH provides may argue for its use, regardless of gestational age.
Other indications. An abnormal serum screen or known parental carrier of a relevant rearrangement can also indicate a fetus at significant risk for one of the common chromosomal abnormalities.
"Time-sensitive" scenarios. Several time-sensitive prenatal situations can likewise benefit from prenatal interphase FISH: For pregnancies as far along as 20 weeksbecause deadlines may be looming for decisions about pregnancy terminationthe sooner a couple learns whether a fetal chromosomal abnormality is present, the sooner that knowledge can be used to begin managing the pregnancy.
Similarly, because the amniotic fluid from a late-gestation pregnancy contains fewer viable cells that are able to grow in culture, prenatal FISH is particularly useful for pregnancies at 30 or more weeks.5 At the very least, rapid prenatal interphase results can provide limited information that may improve the way the delivery of a fetus with multiple anomalies is managed, even if the chromosome analysis is unsuccessful or unavailable by the time of delivery. Chromosome analysis results should still be pursued after delivery for counseling implications.
Confirmation. Another situation in which rapid turnaround time is advantageous is confirmation of a previously identified chromosomal abnormality. Couples who have received abnormal results from prenatal chromosome analysis may ask if the findings could either be incorrect or have been mistaken for another patient's sample. They may insist upon a confirmatory chromosome analysis before making any decisions about terminating the pregnancy. Provided the previous abnormal chromosome result is detectable using the prenatal interphase FISH test, FISH can be extremely useful in quickly verifying results without having to wait the additional 7 to 14 days for chromosome analysis. In these cases, the chromosome analysis precedes the FISH, so it is not necessary to complete the repeat chromosome analysis. Do initiate cultures, however, in case the FISH results are unsuccessful or do not agree with results of the earlier chromosome analysis.
Although FISH testing can be useful in managing a high-risk pregnancy, a 1993 American College of Medical Genetics Policy Statement maintains that irreversible therapeutic decisions should not be made based on FISH results alone.6 While this statement is currently being revised, at the moment, decisions should be based primarily on U/S findings or metaphase analysis. Therefore, if the clinical indication doesn't include an U/S finding that is prompting an irreversible therapeutic decision to be considered, the final decision should await the results of the chromosome analysis.
Prenatal interphase FISH testing is highly accurate, with reported false-positive and -negative rates usually less than 1%.4,7,8 The main problem, however, is that not all specimens are informative. Uninformative rates will vary among laboratories, but rates of 3% to 10% are considered typical.4 Here are some of the possible causes:
Artifactual misinterpretation. Technical artifact may occur with this technology due to overlapping signals in the nuclei, sub-optimal denaturation or hybridization, and splitting signals (Table 1). Splitting signals are either inherent to the probe or due to separation of sister chromatids. (Chromosomes in nuclei that have undergone DNA synthesis have two sister chromatids that will both hybridize to the probe.)
Potential sources of false FISH results
These artifactual challenges require laboratories to establish reportable ranges based on previous analysis of similar-type samples. The reportable ranges allow the laboratory to recognize the expected range of technical artifact and best interpret the results. A laboratory that performs sufficient quality control and quality assurance procedures as described in the 1999 Standards and Guidelines for Clinical Genetics Laboratories can usually safeguard against misinterpretation due to technical artifact.9
Maternal cell contamination. One of the most significant challenges in interpreting prenatal interphase FISH results is maternal cell contamination (Figure 2). FISH is not specific to fetal cells, as all cells in the sample will potentially hybridize with the probes despite their origin or viability. The presence of maternal cells in a prenatal sampleusually due to contaminating maternal blood cellscan lead to false results, most commonly false-negative ones. The maternal cells can overwhelm a sample to such an extent that the prenatal interphase FISH result really reflects the maternal chromosome complement.
Also, the maternal cells could "dilute" the presence of abnormal fetal cells, leading to a normal result if the percentage of normal maternal cells meets reporting criteria. Usually the risk of maternal cell contamination can be identified by assessing the amount of blood in the pellet from the centrifuged amniotic fluid aliquot. In significantly bloody samples, roughly 50% of the time the fetus will be male and XY nuclei will be observed intermixed with XX nuclei. Analysis of only Y-bearing nuclei will provide results reflecting the fetus. Appropriate analysis of the portion of the slide with only probes to chromosomes 13 and 21 would require rehybridization with a Y probe in a third color (for example, a Y probe labeled in aqua). If the fetus is female or no XY nuclei are seen, then the result is usually considered uninformative because reliable interpretation is not possible, given that maternal and fetal cells cannot be differentiated.10 Keep in mind that chromosome analysis results would be available within 5 to 10 days from the time interphase FISH results are reported.
Although most maternal cell contamination is associated with maternal blood in amniotic fluid, an association between oligohydramnios and overwhelming maternal cell contamination has recently been recognized.11 These samples are not usually bloody, suggesting that the origin of the maternal cells is not maternal blood. It has been reported that approximately 10% of prenatal interphase FISH samples from pregnancies with oligohydramnios can have overwhelming maternal cell contamination.11 In those pregnancies, therefore, cautiously interpret prenatal interphase FISH findings of a "normal female."
The death of a twin. A deceased twin can also complicate the interpretation of interphase FISH results when the amniotic fluid contains cells originating from a dead twin that may be abnormal. For example, a recent case in our laboratory involved a prenatal interphase FISH result consistent with a trisomy 18 female fetus. The chromosome analysis revealed two cell lines: The most prevalent cell line was 47,XX,+13, although each coverslip had at least one cell that was 47,XX,+18. The pregnancy was an IVF twin pregnancy with early demise of one twin. A follow-up abortus sample from the remaining twin showed only 47,XX,+13 cells, which strongly suggests that the deceased twin was 47,XX,+18.With that in mind, for pregnancies involving a demised twin, interpret prenatal interphase FISH results cautiously.
Detection of mosaicism (the presence of more than one cell line in an individual) using prenatal interphase FISH can be extremely difficult, given the above limitations. If a laboratory chooses to release "mosaic" prenatal interphase FISH results, the accuracy of the result will be dependent upon the laboratory's reportable ranges and the risk for maternal cell contamination in that sample. Chromosome analysis results are crucial in interpreting and confirming "mosaic" prenatal interphase FISH results.
Delay of prenatal interphase FISH results and uninformative results can occur. Rehybridization of a sample is sometimes necessary, for example, due to technical problems or when the first of the two slides that most labs typically prepare fails to provide a sufficient number of cells for assessment. The rehybridization would delay results by 1 day; however, results should still be available 48 hours after receipt of the sample.
All prenatal interphase FISH testing should be done in conjunction with traditional metaphase chromosome analysis for several reasons: A normal interphase FISH result does not rule out any structural chromosomal abnormality or numerical abnormalities of chromosomes not detectable by the probes.
An abnormal interphase FISH result does not reveal the mechanism leading to the numerical abnormality. For example, a result consistent with trisomy 21 does not differentiate among a trisomy 21 with three whole copies of the chromosome, trisomy 21 secondary to a Robertsonian translocation, the presence of an unbalanced translocation involving the portion of chromosome 21 that the probe detects, or a supernumerary marker chromosome involving chromosome 21. The risk for recurrence and thus a recommendation for parental studies can vary among the etiologies, as can the severity of the phenotype. Chromosome analysis, on the other hand, not only confirms the interphase FISH results, but also provides the fundamental etiology of the imbalance, allowing for appropriate genetic counseling.
With these caveats in mind, it is safe to say that prenatal interphase FISH is a rapid and reliable method to screen for the common chromosomal abnormalities in prenatal samples. Prenatal interphase FISHas an adjunctis particularly useful in pregnancies that are at significant risk for numerical chromosomal abnormalities involving chromosomes 13, 18, 21, X or Y and prenatal situations that warrant a fast turnaround time. But be aware of the limitations of this testing and of the small risk of false-positive or false-negative results. And finally, be sure to heed the current ACMG Policy Statement and not base irreversible therapeutic decisions upon FISH results alone.
1. Steele MW, Breg WR Jr. Chromosome analysis of human amniotic fluid cells. Lancet. 1966;1:383-385.
2. Klinger K, Landes G, Shook D, et al. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am J Hum Genet. 1992;51:55-65.
3. Bryndorf T, Christensen B, Vad M, et al. Prenatal detection of chromosome aneuploidies in uncultured chorionic villus samples by FISH. Am J Hum Genet. 1996;59:918-926.
4. Estabrooks LL, Sapeta M, Lytle C, et al.. Prenatal interphase FISH using the AneuVysion probe set in over 10,000 samples. Am J Hum Genet. 1999;65(suppl):A162.
5. Hoehn HW. Fluid cell culture. In: Milunsky A, ed. Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment. 4th ed. Baltimore, MD: Johns Hopkins University Press; 1998:128-149.
6. American College of Medical Genetics. Prenatal interphase fluorescence in situ hybridization (FISH) policy statement. Am J Hum Genet. 1993;53:526-527.
7. Ward BE, 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. 1993;52:854-865.
8. Eiben B, Trawicki W, Hammans W, et al. Rapid prenatal diagnosis of aneuploidies in uncultured amniocytes by fluorescence in situ hybridization. Evaluation of >3,000 cases. Fetal Diagn Ther. 1999;14:193-197.
9. American College of Medical Genetics. Clinical Cytogenetics: Interphase/Nuclear Fluorescence In Situ Hybridization (nuc ish). In: Standards and Guidelines for Clinical Genetics Laboratories. 2nd ed, 1999.
10. Christensen B, Bryndorf T, Philip J, et al. Prenatal diagnosis by in situ hybridization on uncultured amniocytes: reduced sensitivity and potential risk of misdiagnosis in blood-stained samples. Prenat Diagn. 1993;13:581-587.
11. Estabrooks LL, Hanna JS, Lamb AN. Overwhelming maternal cell contamination in amniotic fluid samples from patients with oligohydramnios can lead to false prenatal interphase FISH results. Prenat Diagn. 1999;19:178-181.
Laurel Estabrook, Allen LAMB. Prenatal interphase fluorescence in situ hybridization (FISH). Contemporary Ob/Gyn 2000;7:68-87.