Routine prenatal diagnosis of aneuploidy by FISH studies in high-risk pregnanciesкод для вставкиСкачать
American Journal of Medical Genetics 90:233–238 (2000) Routine Prenatal Diagnosis of Aneuploidy by FISH Studies in High-Risk Pregnancies Baruch Feldman,1 Salah A.D. Ebrahim,2 Sarah L. Hazan,1 Ko Gyi,2 Mark P. Johnson,1,2 Anthony Johnson,1 and Mark I. Evans1,2,3* 1 Division of Reproductive Genetics, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan 2 Department of Pathology, Wayne State University, Detroit, Michigan 3 Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan This study is a prospective clinical trial with fluorescent in situ hybridization (FISH) as a “routine” test for prenatal detection of the most common aneuploidies in high-risk pregnancies. Since April 1996, FISH studies with multicolor, commercially available, specific probes for chromosomes 13, 18, 21, X, and Y have been routinely performed in our cytogenetic laboratory on uncultured chorionic villous samplings (CVS), amniotic fluid samples, or fetal blood obtained by cordocentesis from patients with major or minor fetal anomalies detected by ultrasonography. Among the 4,193 prenatal samples analyzed between April 1996 and June 1998, routine FISH studies were ordered by the referring physicians on 301 (7.2%) cases. Aneuploidies were detected in 32 (10.6%) samples. Fourteen trisomy-21, 10 trisomy-18, 3 trisomy-13, 4 monosomies of X, and 1 case of triploidy were diagnosed by FISH. All 1,505 hybridizations were informative, and all 301 results were available and reported to the referring physicians in 24–48 hr. All relevant FISH results were confirmed by subsequent cytogenetic analysis. In 10 (3.8%) cases with normal FISH results, the final cytogenetic analysis revealed abnormal chromosomal rearrangements that could not be detected by the routine FISH studies. We conclude that rapid FISH analysis of interphase, uncultured fetal cells is an accurate and very sensitive method for routine prenatal diagnosis of the most common aneuploidies in high-risk pregnancies. Am. J. Med. Genet. 90:233–238, 2000. © 2000 Wiley-Liss, Inc. *Correspondence to: Mark I. Evans, M.D., Department of Ob/ Gyn, Hutzel Hospital, 4707 St. Antoine Boulevard, Detroit, MI 48201. E-mail: email@example.com Received 13 July 1999; Accepted 30 September 1999 © 2000 Wiley-Liss, Inc. KEY WORDS: prenatal diagnosis; aneuploidy; FISH INTRODUCTION More than a decade ago, fluorescent in-situ hybridization (FISH) was introduced as a potentially powerful tool in clinical cytogenetics [Cremer et al., 1986; Julien et al., 1986]. The technique has been found to be highly effective for rapidly determining the number of specified chromosomes in interphase cells [Cremer et al., 1988; Lichter et al., 1988]. FISH thus seemed to be especially appealing for the prenatal detection of chromosomal aberrations. Aneuploidies of only 5 chromosomes (13, 18, 21, X, and Y) account for about 65% of all chromosomal abnormalities and 95% of the chromosomal aberrations causing live-born birth defects [Rhoads et al., 1989; Robinson et al., 1990; Whiteman and Klinger, 1991]. Speed is also a very significant advantage in prenatal diagnosis. Traditionally, one of the inherent frustrations of cytogenetic prenatal diagnosis is the length of time it takes to obtain a karyotype from fetal tissue. Although the turnaround time for a traditional cytogenetic study has decreased from 4 weeks to about 10 days, the pressure for rapid results, particularly in the face of fetal anomalies detected by ultrasound, is still tremendous. During the late 1980s and early 1990s, technical issues were the focus of research. Specific probes, determination of cell types suitable for use with FISH, and more effective techniques for cell preparation and signal detection were intensively studied [Philip et al., 1994]. The results of these efforts were technical advances such as commercially available highly specific and reliable probes, direct labeling, and multicolor, computerized signal detection systems [Divane et al., 1994; Jalal et al., 1998]. However, concerns on the sensitivity, specificity and predictive values of the test and lack of uniform laboratory methods produced profound skepticism of the genetics community. In 1993, The American College of Medical Genetics (ACMG) stated that FISH for clinical cytogenetic studies should be considered investigational [American College of Medical Genetics, 1993]. 234 Feldman et al. Several major clinical studies, published in the last 6 years have addressed the accuracy of prenatal detection of the most common aneuploidies by FISH [Bryndorf et al., 1997; Bryndorf et al., 1996; Cacheux et al., 1994; Cooper et al., 1998; Eiben et al., 1998; Klinger et al., 1992; Mercier and Bresson, 1995; Jalal et al., 1998; Verlinsky et al., 1998; Ward et al., 1993]. Most of these studies were limited to uncultured amniocytes. Although most studies were blinded, very few were true prospective clinical trials in which FISH analysis was performed upon the physician’s request, and results were reported to referring physicians and patients as soon as they were available [Eiben et al., 1998; Ward et al., 1993]. A high percentage of samples not suitable for FISH and many uninformative and problematic results were the other major limitations of the previously published studies. The purpose of the current study was to determine the accuracy of FISH in detection of aneuploidies in real clinical practice with very high-risk pregnancies, i.e., those with fetal anomalies detected by ultrasound. We chose this group of patients for three major reasons: (a) these patients can undoubtedly benefit from fast results since they suffer from substantial anxieties; (b) artificial blinding is not indicated. This group of patients allows testing upon the physician’s request and reporting of FISH results in real time without violating the ACMG guidelines, which dictate that “patient–management decisions should not be based on results obtained by FISH alone;” and (c) it allows cytogenetic evaluation of a significant number of normal as well as abnormal cases because the incidence of chromosomal abnormalities was expected to be relatively high in this group. We established a protocol based mainly on the published experience of others combined with our extensive 10 years of experience, which has included pilot studies with a few hundred cases. The study was designed to evaluate the method as a tool in a clinical prenatal diagnosis program. Routine FISH was performed only upon the referring physician’s request and results were officially reported on a real time basis. To avoid the major obstacles of the previous studies, we based our protocol on the use of multicolor commercially available probes, and defined criteria for interpretation to allow very few “problematic” or “uninformative” cases to be reported. MATERIALS AND METHODS Clinical Protocol All routine FISH studies for chromosomes 13, 18, 21, X, and Y that were performed from April 1996 to June 1998 at the Cytogenetic Laboratory at Wayne State University were included in the study. Patients with fetal anomalies detected by ultrasonography having invasive procedures for prenatal karyotyping were counseled about the availability of rapid analysis by FISH compared to standard karyotyping. The limitations of routine prenatal FISH analysis were also explained in detail. Major as well as minor fetal anomalies were included; however, routine FISH analysis was performed only when specifically requested by the referring physician. All fetal cell types including amniocytes, chorionic villous cells, or fetal blood cells obtained by amniocentesis, CVS, or cordocentesis, respectively, were included in the study. The only exclusion criterion was grossly bloody or a heavy stained brownish amniotic fluid sample. There was no minimum total sample size, volume of amniotic fluid, or amount of chorionic villi required to perform both FISH and cytogenetic analyses. FISH results were reported to the referring physician as soon as they were available. The written report included (a) a short description of the technique including the specific probes used; (b) results of the test in numbers of modal signals for each tested chromosome; (c) interpretation of the results including the sex of the fetus based on the sex-chromosomes complement, and the ploidy status of chromosomes 13, 18, and 21; (d) the major limitations of routine prenatal FISH analysis, which are its inability to detect structural chromosomal aberrations, mosaicism, and numerical abnormalities of chromosomes other than on chromosomes 13, 18, 21, X, and Y; (e) the policy statement of the ACMG regarding the investigational nature of the test and its recommendation that no irreversible therapeutic action should be initiated on the basis of FISH results alone. The standard cytogenetic analysis was based on examination of G-banded chromosomes from at least 15 cultured metaphase cells from a minimum of two independent cultures. The results were reported to the referring physician on a separate form, approximately 1 week after the FISH report was issued. Probes The PloidySTAT set (Oncor, Gaithersburg, MD) was used for all our routine FISH studies until July 1997. It was then replaced by the AneuVysion assay kit (Vysis, Downers Grove, IL). Both assays include two sets of multicolor probe mixtures, one for chromosomes 18, X, and Y, and the other for chromosomes 13 and 21. In each mixture the probes are labeled with different fluorophores, which can be visualized on different color spectrums. Technical characteristics, as supplied by the manufacturers of the probes, are listed in Table I. Preparation of Uncultured Cells Amniocytes. For each specimen, 2–4 ml of clear amniotic fluid was centrifuged for 10 min at 1,200 rpm. The pellet was resuspended in 4 ml of trypsin-EDTA, gently vortexed, and incubated at 37°C for 20 min. Following centrifugation at 1,200 rpm for 10 min, the pellet was resuspended by slowly adding 10 ml of prewarmed (37°C) hypotonic solution (0.8% sodium citrate). The tube was then placed in a 37°C incubator for 20 min followed by centrifugation at 1,200 rpm for 10 min. The supernatant was removed and the pellet was resuspended by adding 2 ml of fixative (3:1 mixture of methanol and acetic acid) and gentle mixing. The suspension was kept in refrigerator (2–8°C) for at least 1 hr before preparing slides. Chorionic villous cells. Chorionic villi were thoroughly cleaned under the microscope and 1–2 pieces of Prenatal Diagnosis of Aneuploidy by FISH 235 TABLE I. Probes Used for the Routine FISH Analysis* PloidySTAT™ (Oncor) until 8/97 Chromo Name Probe Mixture 1 18 X Y Probe Mixture 2 13 18 Loci AneuVysion™ (Vysis) since 8/97 Color Name Loci Color Quint-Essential 18 X ␣-Satellite Y ␣-Satellite 18q21.2 X cen Y cen Red Green Yellow CEP 18 CEP X CEP Y 18 cen X cen Y cen Aqua Green Orange Quint-Essential 13 Quint-Essential 21 13q32-q33 21q22.2 Green Red LSI 13 LSI 21 13q14 21q22.13-q22.2 Green Orange *Chromo, Chromosome; ™, Trademark; cen, centromere; CEP, chromosome enumeration probe; LSI, locus specific identifier. cleaned villi were transferred to a 35-mm petri dish for FISH. After pipetting off the medium, 2 ml of prewarmed (37°C) hypotonic solution (1% sodium citrate) were very slowly added and the villi incubated at room temperature (RT) for 15 min. The hypotonic solution was removed and 2 ml of fixative (3:1 mixture of methanol and acetic acid) were added, drop by drop, on the villi. The dish was placed at −4°C for 20 min and the fixative was replaced by 2 ml of fresh solution for 5 min at RT. The fixative was removed and the villi were air-dried for 2 min. The chorionic villi were dissociated by adding 60% acetic acid. Slides can be prepared starting 2 min after the dissociation solution was added. Fetal blood cells. Fetal blood (0.5–1.0 ml) obtained by cordocentesis was mixed with hypotonic solution (0.56% KCl) for 20 min. The sample was centrifuged at 1,000 rpm for 8 min and the pellet was resuspended with the fixative (3:1 mixture of methanol and acetic acid). The suspension was kept in refrigerator (2–8°C) for about 1 hr before preparing slides. ation and interpretation of results were almost always the responsibilities of one very experienced and highly trained laboratory technician. The most-experienced physician in the laboratory evaluated all extended analyses and reported results of all studies performed. At least 50 interphase amniocytes or lymphocytes, or 100 trophoblasts from CVS, were examined for each probe. If available, only nicely rounded cells were selected for analysis. Clusters or overlapping cells were always excluded. Cells with high background or very low signal intensity were also not scored, if possible. The following criteria were chosen for interpretation. Euploidy was diagnosed if at least 85% of the cells were euploids; aneuploidy was diagnosed if at least 85% of the cells had the same aneuploid signals; and if more than 10% of the cells had the same aneuploid signals, the study was extended and 100–200 cells were analyzed. The final diagnosis by FISH was determined by the signals detected in the majority of cells. Mosaicism was suspected in these cases, and the standard cytogenetic analysis was extended as well. Hybridization and Signal Detection Hybridization protocols of Oncor and later Vysis prenatal detection kits were generally followed. Slides were “dropped” according to routine slide-preparation protocol. At least one extra slide was prepared for each sample as a backup. The slide was incubated for 30 min at 37°C in a coplin jar containing prewarmed 40 ml of 2 × SSC (saline sodium citrate). The slide was then dehydrated in 70%, 80%, and 95% ethanol at RT for 2 min each. For denaturation, the slide was immersed in denaturation solution (70% formamide in 2 × SSC) at 70°C for 2 min, and then rehydrated in ethanol as previously described. For hybridization, 10 l of each of the two probe mixtures were applied on marked areas of the slide. The marked areas were covered by coverslips and sealed by rubber cement. The slide was then incubated for 4 to 16 hr at 37°C in a humidified chamber. After incubation the coverslips were removed and the slide was placed in 0.5 × SSC at 72°C for 2–3 min. The slide was then washed in 2 × SSC/0.1%NP-40 at RT for 2 min and air-dried in the dark. DAPI-II counterstain was applied and a coverslip was placed. A filter set specific for different spectrums was used with an Axioscope fluorescence microscope (Zeiss, Oberkochen, Germany) for analysis. Interpretation of Results Although cell preparation and sometimes hybridization were performed by others, the microscopic evalu- RESULTS A total number of 4,193 prenatal samples (3,379 amniocenteses, 802 CVSs, and 12 cordocenteses) were analyzed in our cytogenetic laboratory from April 1996 to June 1998. Routine FISH studies were requested and performed during the time of the study on 301 patients, with 213 minor and 88 major fetal anomalies detected by ultrasonography. The distribution of cases by the invasive procedure performed, and the type of fetal anomalies detected, are summarized in Table II. All studies were done upon the referring physician’s request with no exceptions. However, 2 cases were excluded from routine FISH analysis because of bloody amniotic fluid samples. Utilization of routine FISH analysis almost tripled between 1996 to 1998 (Table TABLE II. Distribution of Cases by Types of Procedures and Fetal Anomalies* Prenatal procedure Amniocentesis CVS Cordocentesis Total Fetal anomalies Minor Major Total 112 100 1 213 (70%) 70 15 3 88 (30%) 182 115 4 301 *CVS, Chorionic villous sampling. 236 Feldman et al. III). All FISH results were available to the referring physicians within 24–48 hr from the time of the procedure. For 52 (17%) cases, the referring physicians requested specifically, and the results were available within 14–18 hr. All 301 cases analyzed by FISH, which included 1,505 hybridizations with five probes per case, were informative. Extended FISH studies were indicated by our interpretation criteria in 7 (2.3%) cases. In 4 cases, 5 to 7 cells of the 50 analyzed had the same aneuploidy signals. In these 4 cases, the aneuploidic cells had three signals for chromosome 21. The extended study revealed only 1–2 more cells with trisomic signals in each of the 4 cases. Thus, based on the protocol, we reported FISH results as euploidy for these cases. The standard cytogenetic analysis was also extended for these cases and the final karyotypes were normal without mosaicism in all 4 cases. The three other studies were extended because the intensity of the signals of chromosome 18 in 2 cases, and X in 1 case, was relatively low. The reported FISH results as well as the final karyotypes were normal in all 3 cases. In the 301 FISH studies, the correct number of signals was detected for all five probes in more than 85% of the analyzed cells. The mean proportions of scored cells that showed the accurate number of signals, in accordance with the final diagnosis by FISH, are summarized in Table IV. Standard cytogenetic analyses revealed 42 (14%) abnormal fetal karyotypes in our study group. We detected 32 aneuploidies involving chromosomes 13, 18, 21, X, and Y, 2 balanced rearrangements, 4 unbalanced rearrangements, and 4 aneuploidies involving chromosomes other than those analyzed by our routine FISH studies (Table V). The incidence of chromosomal abnormalities among patients with major fetal anomalies was twice that of patients with minor anomalies detected by ultrasound (21.6% and 10.8%, respectively, Table V). Of the 32 aneuploidies involving the analyzed chromosomes (14 cases of trisomy-21, 10 of trisomy-18, 3 of trisomy-13, 4 of monosomy-X, and 1 case of triploidy), all were accurately diagnosed by routine FISH studies. All 301 relevant normal and abnormal FISH results were confirmed by subsequent cytogenetic analysis with no false positives or negatives for what the FISH measures. However, from the clinical perspective, routine FISH analysis for the most common aneuploidies missed about 25% of all chromosomal abnormalities. The sensitivity, specificity, and predictive values of the routine FISH as prospective clinical test in our study group are summarized in Table VI. TABLE III. Physicians’ Requests for Routine FISH Analysis During the Study Year 1996 1997 1998 1996–1998 Total no. of procedures No. of FISH studies Percentage of requests 1372 1907 916 4195 58 136 107 301 4.2% 7.1% 11.7% 7.2% TABLE IV. Percentage of Cells With Expected Number of Signals in 50 Representative Normal Cases and All Aneuploidies Chromosome 13 18 21 XX or XY All aneuploidies a Percentage of analyzed cells with expected number of signalsa Range 97.9 ± 1.6% 97.5 ± 1.5% 97.2 ± 3.0% 99.5 ± 1.4% 95.5 ± 3.5% 94.7–100% 94.0–100% 90.0–100% 94.0–100% 86.0–100% Euploid number for euploidies; aneuploid number for aneuploidies. DISCUSSION Traditional cytogenetic analysis detects chromosomal aneuploidies with great accuracy. Its primary advantage is the ability to detect other chromosome rearrangements. Standard karyotyping, however, must be done on metaphase cells following culture time of several days. In certain clinical situations, especially in prenatal diagnosis, waiting for chromosome analysis may place a significant emotional stress on the patient and clinical burden on the referring physician [BerneFromell and Kjessler, 1984; Evans et al., 1988; EversKiebooms et al., 1988]. Apart from being timeconsuming, traditional cytogenetic analysis is also technically demanding, labor intensive, relatively expensive, and requires highly trained analysts. The potential ability of FISH to identify specific chromosomes in uncultured interphase nuclei was determined by early studies. The potential application of FISH technology for rapid routine prenatal diagnosis of the most common aneuploidies has also been established. However, early attempts at aneuploidy detection in uncultured amniocytes suffered from significant limitations caused by probe design, sample preparation, and assay conditions. Several recently published studies demonstrated the applicability of routine FISH analysis as a clinical test for the prenatal detection of aneuploidies. Klinger et al.  and Ward et al.  reported the results of the first major studies comparing aneuploidy detection by FISH with standard cytogenetic analysis. Their studies formed the basis of the clinical protocols for the application of FISH to prenatal diagnosis. However, these two and most of the following studies [Bryndorf et al., 1997; Bryndorf et al., 1996; Cacheux et al., 1994; Cooper et al., 1998; Eiben et al., 1998; Jalal et al., 1998; Mercier and Bresson, 1995; Verlinsky et al., 1998] had several obstacles that delayed wide acceptance of FISH as a highly reliable method for routine prenatal diagnosis. Some authors used probes constructed by their own laboratories [Bryndorf et al., 1997; Bryndorf et al., 1996; Klinger et al., 1992; Ward et al., 1993]. Most cytogenetic laboratories are not qualified to synthesize DNA probes and to perform the necessary quality-control studies. Furthermore, the assay conditions should be modified for each set of probes because the quality and characteristics of the probes are the key factors for successful FISH analysis. All studies were limited to one specific cell type, which is not the case in many clinical prenatal diagnosis programs. Prenatal Diagnosis of Aneuploidy by FISH 237 TABLE V. Chromosomal Abnormalities in the Study Group Fetal anomalies Final cytogenetic diagnosis Aneuploidies of 13, 18, 21, X, Y Other unbalanced rearrangements Balanced rearrangements Total no. of chromosomal abnormalities Normal karyotype Total no. of cases Only two major studies were true prospective clinical trials [Eiben et al., 1998; Ward et al., 1993]. Most studies were artificially blinded at best, and did not evaluate the test in actual clinical settings. Significant numbers of samples were excluded prior to FISH analysis because of blood contamination of amniotic fluid or lack of a sufficient amount of specimen. The final diagnosis in a significant number of FISH studies was “unsuccessful due to failed hybridization” or problematic results due to insufficient number of nuclei for analysis of one or more chromosomes. Of special concern was the fact that, relative to normal karyotypes, FISH missed a higher percentage of aneuploidies due to problematic or unsatisfactory results. For example, 9.8% of samples found to be suitable for FISH in the study of Ward et al.  were reported as uninformative whereas 7.5% of them were potentially detectable aneuploidies. The overall detection rate of the relevant aneuploidies was 73.3% in their study [Ward et al., 1993]. In more recent studies, Bryndorf et al.  could not reproduce these results whereas others [Eiben et al., 1998; Jalal et al., 1998] have demonstrated potential overall detection rates of almost 100%. In most studies, FISH misdiagnosed few false negatives and positives. The present study was generally designed on the basis of previously published major clinical trials and our own long-standing experience [Evans et al., 1994; Evans et al., 1992]. To avoid the obstacles of the recently published studies, we used commercially available multicolor probes. We defined criteria for interpretation of the results to allow very few uninformative cases. All available fetal cell types were included, and the only exclusion criterion was bloody amniotic fluid. Most importantly, however, is the fact that the study was a true prospective clinical trial in which the test was performed upon physician’s request and results were reported as soon as they were available. The present study is the first published clinical trial in which TABLE VI. Characteristics of Routine FISH Analysis as Clinical Test for High-Risk Pregnancies Karyotype FISH Normal Abnormal Total Normal Abnormal Total 259 0 259 10 32 42 269 32 301 Sensitivity, 76.2%; Specificity, 100%; Positive predictive value, 100%; Negative predictive value, 96.3%. Minor Major Total 18 (8.4%) 4 (1.9%) 1 (0.5%) 23 (10.8%) 190 (89.2%) 88 14 (15.9%) 4 (4.6%) 1 (1.1%) 19 (21.6%) 69 (78.4%) 213 32 (10.6%) 8 (2.7%) 2 (0.7%) 42 (14.0%) 259 (86.0%) 301 the modified routine FISH technology, with a potential of 100% accuracy, was integrated into a clinical prenatal diagnosis program. As expected, the incidence of all chromosomal abnormalities in very high risk pregnancies is relatively high (14%), with aneuploidies of 13, 18, 21, X, and Y being the most common finding (76%). As demonstrated by many others, fetal anomaly detected by ultrasound is the most significant risk factor for chromosomal abnormalities. The risk is doubled from about 10 to above 20% in cases with major as compared to minor anomalies. We believe that the major problems of most studies were the result of unsatisfactory criteria for interpretation of results. The different cutoff points for the proportion of cells with identical pattern of signals needed for diagnosis by FISH were between 50–70% in different studies. This cutoff point could be optimized in our study to be as high as 85% due to very reliable probes and optimization of several steps in preparation of cells, hybridization, and detection. The relatively small total number of cells needed for diagnosis, practically 100, gave a much better option to be more selective in cell quality, and was a major factor in the efficiency and accuracy of the test. Our observation that in the vast majority of hybridizations more than 95% of cells showed the accurate number of signals strongly supports our chosen high cutoff point. These modifications were the basis for the zero uninformative FISH results in our group. We cannot comment on the incidence of exclusion due to bloody amniotic fluids because the referring physicians were aware of the exclusion criterion and might preexcluded some specimens. The incidence of bloody fluids referred to our laboratory for standard cytogenetic analysis was less than 1% during the time of the study, thus the significance of that factor is most likely negligible. We are currently studying the routine FISH protocols for bloody specimens, hoping that FISH analysis will be available for all prenatal cases. The sensitivity, specificity, and predictive values for the routine FISH analysis as a method to detect aneuploidies of chromosomes 13, 18, 21, X, and Y in our study group were all 100%. However, the laboratory is requested by the referring health providers to detect all cytogenetically visible chromosomal abnormalities. By that definition, even with 100% accuracy of the test, routine FISH analysis will miss about 25–30% of the detectable cytogenetic abnormalities. Therefore, the future of routine FISH analysis for the prenatal detection of aneuploidies is highly dependent 238 Feldman et al. on protocols with practically zero false positives, and educating physicians and patients about the basis of the unavoidable inherited clinical false negatives. The best proof for the applicability of the test and its usefulness in day-to-day clinical practice is the fact that physicians’ requests for routine FISH analysis almost tripled during the study period. Like any other test, this method is subjected to pitfalls. Even with the most reliable protocols, some rare rearrangements and familial variants, as well as technical errors, could cause false results [Hockstein et al., 1998; Tardy and Toth, 1997; Thangavelu et al., 1998; Verlinsky et al., 1995; Verlinsky et al., 1998]. 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