- Email: [email protected]
Molecular cytogenetics: An essential component of modern prenatal diagnosis
Molecular cytogenetics: An essential component of modern prenatal diagnosis W. Allen Hogge, MD, .... a Urvashi Surti, Phi), b' c, d SallyJ. Kochmar, MS, c Patricia Mowery-Rushton, Phl), c and Kathleen Cumbie, BS c Pittsburgh, Pennsylvania
Traditional cytogenetic studies with high-resolution banding techniques have been the mainstay of prenatal diagnosis for >20 years. However, this approach is limited by the resolution of light microscopy, and it requires cultured cells, necessitating a significant delay in obtaining chromosome studies. The advent of molecular cytogenetics, or fluorescence in situ hybridization, has added an adjunctive tool to overcome both these limitations. During a 16-month period 35 prenatal diagnosis cases had molecular cytogenetic studies performed; 71% of the evaluations were informative. We present five of these cases to illustrate the benefits of this technique for clinical prenatal diagnosis. (Am J Obstet Gynecol 1996;175:352-7.)
Key words: Prenatal diagnosis, fluorescence in situ hybridization, molecular cytogenetics During the past 20 years, since the introduction of chromosome banding, chromosome analysis has become the mainstay of prenatal diagnosis. Routine and highresolution cytogenetic studies permit detection of both numeric and structural chromosomal abnormalities. However, changes involving <5 million bp are difficult or impossible to identify with standard or traditional cytogenetic methods. Likewise, chromosomal alterations with indistinct banding patterns, such as marker chromosomes or de novo or cryptic translocations, are often difficult to interpret)' 2 Many of these difficulties have been circumvented by the availability of chromosome-specific deoxyribonucleic acid probes and the technique known as fluorescence in situ hybridization. This technique allows the detection of submicroscopic, or cryptic, translocations, the characterization of marker chromosomes, and the diagnosis of microdeletion syndromes?' 4We present the results of our initial experience with the introduction of fluorescence in situ hybridization into a prenatal diagnosis program to emphasize the benefits of the technique in providing precise and accurate diagnostic information. At the same time our preliminary data point out the limitations of fluorescence in situ hybridization that must be considered when applying it as a clinical tool.
From the Departments of Obstetrics, Gynecology, and Reproductive Sciences~ and Pathology, ~ University of Pittsburgh, the Department of Geneticsf Magee-Womens Hospital, and Magee-Womens Research Institute, a Presented as Associate Fellow at the Fifty-eighth Annual Meeting of The South Atlantic Association of Obstetricians and Gynecologists, Lake Buena Vista, Florida, January 27-30, 1996. Reprint requests: W. Allen Hogge, MD, Department of Genetics, MageeWomens Hospital, 300 Halket St., Pittsburgh, PA 15213. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 ~5.00 + 0 6/6/74753
352
Methods Fluorescence in situ hybridization. Cells obtained were processed and slides were prepared with a protocol modified from Pinkle and Gray.5 Chromosome-specific probes obtained from Oncor (Gaithersburg, Md.) were used for all experiments, and protocols provided by the manufacturer were used. Slides were viewed under a Zeiss (Zeiss, Germany) Axiophot microscope equipped with an HBO (Osram, Allentown, Pa.) 100/2W mercury lamp. Cell identification and signal counting were performed at a magnification of xl000 with oil with both triple and single bandpass filters. The decision to use fluorescence in situ hybridization as a diagnostic toot in a prenatal case was made by a clinical geneticist after a complete review of the pertinent clinical history. The major criteria used in this decision were (1) equivocal cytogenetic results likely to be clarified be molecular studies (for example, marker chromosomes or high suspicion of a cryptic translocation on light microscopy), (2) ultrasonographic malformations highly suggestive of a microdeletion syndrome (for example, conotruncal heart defect as a component of DiGeorge sequence), and (3) ultrasonographic malformations highly suggestive of a specific chromosome defect and for which a rapid diagnostic result would provide a benefit in obstetric management (for example, suspected trisomy 18 and evidence of fetal compromise). To maximize the diagnostic accuracy of fluorescence in situ hybridization in these clinical settings, very strict scoring criteriawere utilized (Table I). These criteria are established to prevent false-positive results but do so at a loss of sensitivity because of a significant "noninformative" rate. All cases having fluorescence in situ hybridization evaluation from its introduction into our clinical program (June 1, 1994) until Oct. 31, 1995, were analyzed.
Volume 175, Number 2 AmJ ObstetGynecol
Hogge et al. 353
Table I. Scoring criteria, fluorescent.in situ hybridization Metaphase cell analysis Minimum of 20 cells examined. Each cell analyzed must clearly display internal control signal (if applicable). Acceptable error rate: 5% (1/20). If >2 cells per 20 are not consistent, then 30 additional cells are analyzed. An external control (normal individual) is run in parallel with each patient sample. Interphase cell analysis Minimum of 50 cells examined. External control of same tissue type, if possible, or peripheral blood preparation is run in parallel vdth each patient sample. Normal result: 80% of cells with normal signal number. Abnormal result: 70% of cells with a consistent abnormal number of signals (for example, 70% with 3 signals for chromosome 21 = trisomy 21). Noninformative: Any other distribution of signals or control sample does not meet scoring criteria.
Results During the period of this study (June 1, 1994, to Oct. 31, 1995), 35 cases had molecular cytogenetic evaluations. In 25 instances a precise diagnosis could be made. Of the remaining 10 cases, 7 were uninformative because of insufficient cells and 3 did not meet guidelines (Table I). Of note, only one of the noninformative cases was a third-trimester sample. There were 3 abnormal results in these noninformative cases (triploidy, trisomy 21, trisomy 18). The triploidy sample did have three signals for chromosome 18 but was noninformative for chromosome 13. Following are five cases that illustrate the benefits and limitations of introducing fluorescence in situ hybridization into a prenatal diagnosis program.
Case reports Case 1. A 40-year-old woman, gravida 2, para 1, was seen at 12 weeks' gestation for chorionic villus sampling (CVS). Ultrasonographic age at the time of the procedure was 11.0 weeks. Traditional cytogenetics revealed 10% mosaicism for a marker chromosome. To rule out placental mosaicism, amniocentesis was performed at 15.9 weeks' gestation, and the initial 50 cells analyzed revealed no evidence of the marker chromosome. However, molecular studies (fluorescence in situ hybridization) on the original CVS slides revealed the marker to be an isochromosome for the p arm of chromosome 12 [i(12p)] (Fig. 1). This chromosomal abnormality is present in the Pallister-Killian syndrome, a disorder characterized by dysmorphic facial features, mental retardation, and in a small percentage of cases, diaphragmatic hernia and congenital heart disease. Because of the diagnosis of i (12p) by fluorescence in situ hybridization, an additional 13 clones were examined from the amniocentesis. One cell was found to contain the i (12p) marker. Because the marker chromosome is known to be lost in the process of cell culture, a repeat amniocentesis was performed at 19.9 weeks' gestation, and analysis of uncultured amniocytes by fluorescence in situ hybridization revealed 31% mosaicism for i(12p). The patient subsequently spontaneously aborted the pregnancy at 21.1 weeks' gestation. At autopsy clinical examination confirmed the diagnosis of Pallister-Killian syndrome, and molecular studies indicated 16% to 35% mosaicism in the various fetal tissues analyzed. Case 2. A history of an unbalanced familial translocation in a nephew was the initial reason for referral of this
21-year-old primigravid woman. 6 At referral the patient was 21.6 weeks' gestation by ultrasonographic evaluation, which also revealed complex congenital heart disease in the fetus. Echocardiography confirmed the diagnosis of tetralogy of Fallot. Chromosome analysis was performed by cordocentesis because of the late gestation, and traditional cytogenetics indicated an apparently normal 46,XY karyotype. Because the nephew was known to have cryptic 9q;17p translocation involving the telomeric portions of these two chromosomes, fluorescence in situ hybridization analysis was performed with a probe for the telometric (17pl 3.3) region of the short arm of chromosome 17(D17S34). Fig. 2 depicts the balanced translocation found in our patient and seen only on fluorescence in situ hybridization analysis. Only one signal was seen in the metaphases examined from the fetus, confirming an unbalanced cryptic translocation with partial monosomy for 17p material and partial trisomy for 9q material. The patient subsequently terminated the pregnancy. At autopsy the only additional finding was hypospadias. Case 3. This amniotic fluid sample was sent to our laboratory for chromosome analysis because of advanced maternal age and fetal gastroschisis detected by ultrasonography. Traditional cytogenetics indicated a normal 46,XX karyotype. The patient spontaneously aborted a male fetus at 17 weeks' gestation, and autopsy confirmed both external and internal genitalia to be male. Paraffin blocks were obtained from file autopsy, and molecular cytogenetic analysis was perfbrmed with deoxyribonucleic acid probes specific for the X c~-satellite (DXZ1) and a Yclassic satellite (DYZ1). Two copies of the X chromosome were found in all cells analyzed, and no copies of the Y chromosome were detected. Molecular studies are underway to search for the presence of the gene for the testis-determining factor (SR?0. Case 4. A 22-year-old woman, gravida 2, para 1, had an ultrasonographic examination at 16 weeks at her initial prenatal visit when uncertain of menstrual dating. Fetal anatomy appeared normal. An admission for pyelonephritis at 30.7 weeks' gestation prompted a repeat ultrasonographic study, which revealed measurements consistent with 28.5 weeks (15th percentile), polyhydramnios (amniotic fluid index 32.7 cm), and a two-vessel umbilical cord. Nine days later a genetics Consultation was obtained, and an amniocentesis was performed for chromosome evaluation and lung maturity. A biophysical profile on the day of admission indicated absent fetal breathing
354
Hogge et al.
August 1996 AmJ Obstet Gynecol
chl~o~ 12 a l p h a - s a 4 c e l l i t e D12Z3
/
Fig. 1. Fluorescence in situ hybridization study ofinterphase cells in case 1 demonstrating three signals with use of 12c~ satellite probe.
Fig. 2. Metaphase cell analysis from patient with balanced cryptic translocation in case 2 with fluorescent probe for 17p13.3. One signal is seen on E group chromosome corresponding to p arm of chromosome 17. Second signal was present on telomeric region of q arm of C group chromosome determined to be No. 9, indicating translocation of this chromosome material from other chromosome 17. a n d p o o r tone. Loss o f variability a n d s p o n t a n e o u s fetal h e a r t rate d e c e l e r a t i o n s p r o m p t e d an e m e r g e n c y cesare a n section. A 1216 g m infant with clinical features o f trisomy 18 was delivered. F l u o r e s c e n t in situ hybridiza-
tion o n the c u l t u r e d a m n i o t i c fluid cells with a c h r o m o s o m e 18 cz-satellite p r o b e (D18Z1) i n d i c a t e d t h r e e copies o f c h r o m o s o m e 18. Case 5. A history o f early-onset p r e e c l a m p s i a (23
Volume 175, Number 2 AmJ Obstet Gynecol
weeks' gestation) and ultrasonographic findings of a thickened placenta prompted referral of this 16-year-old gravida 2, para 1 woman. Amniocentesis was performed and fluorescent in situ hybridization with a chromosome 13qi4-specific probe (RB1) and a chromosome 21q22specific probe (D21S65) indicated two copies of both chromosome 13 and chromosome 21, excluding the diagnosis of triploidy. The patient was subsequently delivered of a 624 gm infant because of worsening preeclampsia at 24.5 weeks.
Comment
The successful culture and cytogenetic analysis of amniotic fluid cells in the early 1970s opened a new era in fetal assessment. The field was further expanded a decade later with the extension of prenatal diagnosis into the first trimester with CVS. In spite of the high degree of accuracy of cytogenetic diagnoses with amniocytes or chorionic villi, certain problems of interpretation remain. These include de novo marker chromosomes, "placental" mosaicism, and discrepancies between prenatal cytogenetic results and those obtained from the newborn. These five cases illustrate the significant benefit of the new technique of molecular cytogenetics in clarifying confusing or discrepant results obtained with the traditional banding method of chromosome analysis. Fluorescence in situ hybridization takes advantage of molecular probes that are specific for a defined region of cytogenetic interest. It is based on microscopic visualization of copy n u m b e r and therefore is sensitive well beyond what can be seen on traditionally banded chromosomes with a light microscope. 4 In addition to its increased sensitivity, fluorescence in situ hybridization allows a more rapid diagnosis because it can be performed on uncultured cells. Case 1 illustrates the benefits of fluorescence in situ hybridization in the evaluation of marker chromosomes suspected to be isochromosome 12p by traditional banding methods. A precise diagnosis of the chromosome material present allowed a specific clinical diagnosis to be made. The case also points out the unique difficulties associated with the prenatal diagnosis of the Pallister-Killian syndrome. The isochromosome is often lost during the process of cell culture. 7 Prenatal diagnosis is possible by fluorescence in situ hybridization analysis of uncultured cells obtained by CVS or amniocentesisP' 9 The finding of mosaicism in CVS material that is not present in the fetus has been termed "confined placental mosaicism. ''1° Initially thought to represent a source of inaccurate prenatal diagnostic results and a limitation of CVS, it is now recognized as a biologic p h e n o m e n o n that in many cases has clinical significance.11It is essential that these abnormalities be fully evaluated to detect those circumstances with clinical significance (true mosaicism and uniparental disomy), a2' as Case 2 is indicative of the power of the technique in the diagnosis of previously unexplained dysmorphic genetic
Hogge et al. 355
disorders. By traditional cytogenetic methods both the affected relative and our patient's fetus appeared to have normal chromosome n u m b e r and structure. However, molecular analysis revealed a familial cryptic translocafion involving the telomeres of 9q and 17p. Both affected individuals were found to have an unbalanced translocation resulting in partial monosomy for 17p and partial trisomy for 9q. The presence of an apparent laboratory error raises specific concerns regarding procedure and laboratory techniques, particularly in the case of sex discrepancies that can result from maternal cell contamination or mislabeling of samples. Case 3 illustrates that in some circumstances the cytogenetic results are not in error and represent abnormalities that are beyond the resolution of traditional methods of analysis. In this case of a 46,XX male only the gene (s) responsible for maleness may be present, most likely translocated onto a non-Ychromosome. Cases 4 and 5 represent the benefit that fluorescence in situ hybridization can have with respect to rapid diagnosis. Obstetric management decisions can be made on the basis of precise diagnostic information. In many circumstances, such as case 4, it will obviate the need to subject the patient to multiple diagnostic and therapeutic interventions. Had a genetics consultation been requested in a timely fashion, more than a week of intensive evaluation and a subsequent surgical procedure could have been avoided. The introduction of molecular cytogenefics has, and will continue to, markedly expand the diagnostic capabilities in prenatal diagnosis. Axly program providing prenatal diagnosis must have the capabilities to perform these sensitive techniques if accurate and precise diagnostic information is to be provided to patients undergoing CVS, amniocentesis, or other prenatal diagnostic testing. At the same time it must be recognized that fluorescence in situ hybridization has significant limitations, including a high noninformative rate and a significant risk of misdiagnosis. Fluorescence in situ hybridization should only be used in cases with a high suspicion for a specific chromosome abnormality and in consultation with a clinical geneticist knowledgable in the indications for and limitations of molecular cytogenefics. REFERENCES
1. Benn PA, Hsu LYE Incidence and significance of supernumerary marker chromosomes in prenatal diagnosis. Am J Hum Genet 1984;36:1092-102. 2. Warburton D. De novo balanced chromosome rearrangement and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints. AmJ Hum Genet 1991;49:995-1013. 3. Ledbetter DH. Cryptic translocations of telomere integrity. AmJ Hum Genet 1992;51:451-6. 4. Callan DF, Eyre H, Yip MY, FreemantleJ, Haan EA. Molecular cytogenetics and clinical studies of 42 patients with marker chromosomes. Am J Hum Genet 1992;43:709-15. 5. Pinkel D, GrayJW. Cytogenetic analysis by in situ hybridization with fluorescently labelled nucleic acid probes: Cold Springs Harbor Symposium. Quant Biol 1986;51:151-7.
356
Hogge et al.
6. Estop AM, Mowery-Rushton PA, Cieply KM, Kochmar sJ, Sherer CR, Clemens M, et al. Identification of an unbalanced cryptic tranlocation t(9;17)(q34.3;p13.3) in a child with dysmorphic featnres.J Med Genet 1995;32:819-22. 7. Blancato JK, Hunt M, George J, Katz J, Meck JM. Prenatal diagnosis of tetrasomy 12p by in situ hybridization: varying levels of mosaicism in different fetal tissues. Prenat Diagn 1992;12:979-83. 8. Los FJ, Van Opstal D, Schol MP, Gaillard jLj, Brandenburg H, Van Den Ouweland AMW, et al. Prenatal diagnosis of mosaic tetrasomy 12p/trisomy 12p by fluorescent in situ hybridization in amniotic fluid cells: a case report of Pallister-Kiliian syndrome. Prenat Diagn 1995;15:1155-9. 9. Bernert J, Barrels I, Gatz G, Hansmann I, Heyat M, Niedmann PD, et al. Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS. AmJ Med Genet 1992; 42:74%50. 10. Kalousek DK, Dill FJ. Chromosomal mosaicism confined to the placenta in human conception. Science 1983;221:665-7. 11. Kalousek DK. Confined placental mosaicism and intrauterine development. Pediatr Pathol 1990;10:69-77. 12. Cassidy SB, Lai L, Erickson RP, Magnuson L, Thomas E, Gendron R, et al. Trisomy 15 with 10ss of the paternal 15 as a cause of Prader Willi syndrome due to maternal disomy. AmJ Hum Genet 1992;51:701-8. 13. Teshima I, Kalousek DK, Vekemans MJ], Markovic V, Cox DM, Dallaire L, et al. Chromosome mosaicism in CVS and amniocentesis samples (Canadian multicentre trial). Prenat Diagn 1992;12:443-59.
Discussion I)1~ LEO PLOU~E, JR., Augusta, Georgia. Fluorescence in situ hybridization is truly one of the most exciting recent developments in the field of reproductive medicine a n d cytogenetics. It combines the long-established techniques developed for cytogenetic studies with the recent breakthroughs accomplished in molecular medicine. Seld o m has a wedding of such techniques yielded clinically applicable results in so little time. There are several variations on the technique. O n e aspect concerns the material studied. Fluorescence in situ hybridization can be p e r f o r m e d o n metaphase cells, which has b e e n the standard approach to cytogenetic studies. A good example of this was shown by Dr. Hogge in the second case, involving a familial translocation. This approach requires full culture of cells, as has traditionally b e e n required for the production of a karyotype. Additionally, fluorescence in situ hybridization can be d o n e o n interphase cells, where the chromatin is still c l u m p e d in the nucleus. This was demonstrated in the first case, involving the diagnosis of the Pallister-Killian syndrome. This technique can be applied directly to a cell preparation a n d can provide a rapid diagnosis, in some cases within a few hours. This technique has recently b e e n applied to single cells for preimplantation diagnosis. The second aspect involves the selection of labeling material. This has r a n g e d from radioisotopes to the use of fluorescent labels. The latter has quickly become the preferred technique. It avoids the use of radioactive materials in the laboratory and, because fluorochromes of different colors can b e u s e d , it allows the c o n c u r r e n t use of different molecular probes. A third a n d most critical e l e m e n t is the appropriate selection of probes. This has to be based on specific criteria. It involves an in-depth knowledge of the disorder
August 1996 AmJ Obstet Gynecol
suspected a n d appropriate considerations of conditions which may lead to false-positive or false-negative results. With all of its promises, fluorescence in situ hybridization is still the object of some controversy. Some have advocated its use o n a routine basis for all cases where a karyotype is ordered. Others have highlighted the shortcomings of t h e technique a n d the potential for misinterpretation. They have emphasized the n e e d for concurrent traditional karyotype studies. There is clearly a n e e d for specific criteria as to when the technique should be used. Today Dr. Hogge a n d colleagues have shared with us their experience over a 16-month interval with the use of fluorescence in situ hybridization. We are told this involved 35 cases, with 25 cases being considered as informative. Dr. Hogge further presented us with five specific cases where fluorescence in situ hybridization was felt to be of great assistance. These are exciting cases that highlight the benefits of this diagnostic approach. I will briefly c o m m e n t o n the cases presented a n d then outline general questions about the technique. Dr. Hogge's team must be c o m m e n d e d for the thoroughness of their investigations in elucidating a case of the Pallister-Killian syndrome. We could only find two other such examples in the literature. The second case clearly highlights the increased sensitivity gained in studying a karyotype by fluorescence in situ hybridization. The third case highlights the additional information that can be gained through fluorescence in situ hybridization in cases of intersex disorders. Further, fluorescence in situ hybridization in this case would be indicated with specific probes for SRY, the gene currently proposed as the testicular-determining factor. I c a n n o t refrain at this point from interjecting a concern our group has had for some time regarding the use of fluorescence in situ hybridization in the workup of intersex disorders, particularly when there is an X chromosome a n d a fragment of a n o t h e r yet-to-be-identified sex chromosome. Some laboratories have routinely b e e n using fluorescence in situ hybridization probes targeting the SRY sequence. This sequence is located quite far u p the short arm of the Y chromosome. These laboratories have reported these cases as being negative for Ymaterial. This clearly constitutes a misuse of the technique. The search for the presence of Y material is linked to the increased likelihood of gonadal malignancies in these individuals. T h e p o r t i o n of the Y chromosome linked to the oncogenic potential is currently located very close to the centromere, or central portion, of the Y chromosome. Absence of SRY does n o t m e a n that there is no Y material present a n d these patients may still be at risk for dysgerminomas a n d gonadoblastomas. It is therefore essential for the clinician to d e m a n d that centromeric probes be used in these cases. The last two cases present good examples of the rapid diagnostic potential offered through fluorescence in situ hybridization. We suspect both these cases involved the study of interphase cells. This is of great assistance to the clinician, particularly in d e t e r m i n i n g m a n a g e m e n t or m o d e of delivery.
Volume 175, Number 2 AmJ Obstet Gynecol
What p e r c e n t a g e o f the total v o l u m e do these 35 cases represent? What were the criteria used to p r o c e e d with fluorescence in situ hybridization in the c u r r e n t patient populatio~a and what general criteria would the authors propose f r o m now on? I would also like Dr. H o g g e to c o m m e n t on his opinion about the c o n c u r r e n t n e e d for karyotypic studies, particularly if fluorescence in situ hybridization is applied to interphase cells. I would also ask h i m to share his concerns a b o u t the potential for falsepositive and false-negative results. As stated before, a major advantage of fluorescence in situ hybridization is the ability to study interphase cells and obtain rapid results. I would also ask Dr. H o g g e to share with us the least time interval n e e d e d for his group to make a diagnosis t h r o u g h fluorescence in situ hybridization. In conclusion, fluorescence in situ hybridization has greatly e n l a r g e d the diagnostic a r m a m e n t a r i u m available to the clinician. Dr. H o g g e and his group have provided us with excellent examples o f this practical application of this technique. This is a rapidly evolving area that will require close m o n i t o r i n g by the clinician for its appropriate application. DR. Ron~_, V. W~E, Charlotte, N o r t h Carolina. Please c o m m e n t on the limitations and potentials of fluorescence in situ hybridization with fetal cells in maternal blood. DR. HOGGE (Closing). I would like to c o m m e n t on the case (case 3) I left out in the interest of time. This was a case we did mostly for o u r own benefit because this apparently was a false laboratory result. O u r cytogenetics indicated a 46,XX result. T h e fetus had o t h e r anomalies and that p r e g n a n c y was terminated. At pregnancy termination the fetus was f o u n d to be male both externally and internally. Rather than accept the possibilities o f maternal cell c o n t a m i n a t i o n or laboratory m i x u p of samples, we looked m o r e deeply into causes and f o u n d no evid e n c e for Y c h r o m o s o m e material with a Y c e n t r o m e r e probe. We are now interested in looking to see whether SRY is present somewhere else in the c h r o m o s o m e s of this fetus. W h a t does this r e p r e s e n t of o u r tOtal volume? I was trying to calculate that as you were asking the question. We do a b o u t 2000 prenatal cases a year, so this would probably r e p r e s e n t about 1% of our cases. We do n o t do t h e m for routine cytogenetics on patients having amniocentesis for advanced m a t e r n a l age to get a rapid answer; we would n o t do t h e m in cases of a b n o r m a l multiplemarker screen for rapid answers unless we are dealing with a circumstance where we are close to the 24-week cutoff p o i n t for t e r m i n a t i o n of pregnancy in Pennsylvania. We would use fluorescence in situ hybridization un-
Hooge et al.
357
der that circumstance, but that would be an extremely rare indication in our institution. W h a t are the criteria? We have fairly strict criteria at Magee-Womens Hospital and the University of Pittsburgh for the use of fluorescence in situ hybridization; that is, it's n o t o r d e r e d unless it's a p p r o v e d by the director of the laboratory or me. We d o n ' t think this is a t e c h n i q u e that is ready for " p r i m e time." One, you n e e d to know the particular syndromes you can look for with fluorescence in situ hybridization, and I would use the example of the heart defect earlier. We h a p p e n e d to know that particular patient r e p r e s e n t e d a case with a translocation. In o t h e r similar circflmstances there are u n i q u e deletions that are involved with tetralogy of Fallot, and we would know to look for those particular ones. D o i n g routine fluorescence in situ hybridization ,with 13, 18, or 21 probes would miss all those cases. We use it in the case of fetal anomalies in which there are specific c h r o m o s o m e abnormalities we know to look for, knowing full well we will miss some abnormalities. We use it specifically in late pregnancy, that is, the third trimester. In circumstances in which there is a high suspicion of trisomy 18, we use it to assist in p l a n n i n g for delivery to avoid caesarean section in cases of severe intrauterine growth restriction. We use it in those circumstances in which we have a family or clinical history to p o i n t us in a direction, as in case 2, which had the translocation. It is n o t a t e c h n i q u e that should be used routinely. It has a n u m b e r of limitations. You d o n ' t want 30% uninformative cases for routine cytogenetics, so it should n o t be d o n e for that. It should never be d o n e and used in the absence of a c o n c u r r e n t karyotype, as Dr. Plouffe stated before. We will use it only in that circumstance of trisomy 18 to avoid caesarean delivery, given that we have o t h e r features to support the diagnosis of trisomy 18. False-positive and false-negative results are b o t h significant problems, and I p o i n t e d out how often that can occur. In our laboratory the least time tO a result is about 48 hours. It can be d o n e in a m o r e rapid fashion, but our laboratory does a better j o b given a m i n i m u m of 48 hours to do it. In terms of how it is going to be used for fetal cells in maternal blood, I think it has the high potential to be useful. T h e difficulty is that we n e e d a large n u m b e r of cells for analysis, given that a significant n u m b e r of normal cells will have too many signals and a significant n u m b e r of a b n o r m a l cells will have too few signals. W h e n dealing with the small n u m b e r of fetal cells we are able to isolate f r o m a m a t e r n a l samples at this point, there is a , m u c h h i g h e r likelihood of making a wrong diagnosis than a correct diagnosis.
Traditional cytogenetic studies with high-resolution banding techniques have been the mainstay of prenatal diagnosis for >20 years. However, this approach is limited by the resolution of light microscopy, and it requires cultured cells, necessitating a significant delay in obtaining chromosome studies. The advent of molecular cytogenetics, or fluorescence in situ hybridization, has added an adjunctive tool to overcome both these limitations. During a 16-month period 35 prenatal diagnosis cases had molecular cytogenetic studies performed; 71% of the evaluations were informative. We present five of these cases to illustrate the benefits of this technique for clinical prenatal diagnosis. (Am J Obstet Gynecol 1996;175:352-7.)
Key words: Prenatal diagnosis, fluorescence in situ hybridization, molecular cytogenetics During the past 20 years, since the introduction of chromosome banding, chromosome analysis has become the mainstay of prenatal diagnosis. Routine and highresolution cytogenetic studies permit detection of both numeric and structural chromosomal abnormalities. However, changes involving <5 million bp are difficult or impossible to identify with standard or traditional cytogenetic methods. Likewise, chromosomal alterations with indistinct banding patterns, such as marker chromosomes or de novo or cryptic translocations, are often difficult to interpret)' 2 Many of these difficulties have been circumvented by the availability of chromosome-specific deoxyribonucleic acid probes and the technique known as fluorescence in situ hybridization. This technique allows the detection of submicroscopic, or cryptic, translocations, the characterization of marker chromosomes, and the diagnosis of microdeletion syndromes?' 4We present the results of our initial experience with the introduction of fluorescence in situ hybridization into a prenatal diagnosis program to emphasize the benefits of the technique in providing precise and accurate diagnostic information. At the same time our preliminary data point out the limitations of fluorescence in situ hybridization that must be considered when applying it as a clinical tool.
From the Departments of Obstetrics, Gynecology, and Reproductive Sciences~ and Pathology, ~ University of Pittsburgh, the Department of Geneticsf Magee-Womens Hospital, and Magee-Womens Research Institute, a Presented as Associate Fellow at the Fifty-eighth Annual Meeting of The South Atlantic Association of Obstetricians and Gynecologists, Lake Buena Vista, Florida, January 27-30, 1996. Reprint requests: W. Allen Hogge, MD, Department of Genetics, MageeWomens Hospital, 300 Halket St., Pittsburgh, PA 15213. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 ~5.00 + 0 6/6/74753
352
Methods Fluorescence in situ hybridization. Cells obtained were processed and slides were prepared with a protocol modified from Pinkle and Gray.5 Chromosome-specific probes obtained from Oncor (Gaithersburg, Md.) were used for all experiments, and protocols provided by the manufacturer were used. Slides were viewed under a Zeiss (Zeiss, Germany) Axiophot microscope equipped with an HBO (Osram, Allentown, Pa.) 100/2W mercury lamp. Cell identification and signal counting were performed at a magnification of xl000 with oil with both triple and single bandpass filters. The decision to use fluorescence in situ hybridization as a diagnostic toot in a prenatal case was made by a clinical geneticist after a complete review of the pertinent clinical history. The major criteria used in this decision were (1) equivocal cytogenetic results likely to be clarified be molecular studies (for example, marker chromosomes or high suspicion of a cryptic translocation on light microscopy), (2) ultrasonographic malformations highly suggestive of a microdeletion syndrome (for example, conotruncal heart defect as a component of DiGeorge sequence), and (3) ultrasonographic malformations highly suggestive of a specific chromosome defect and for which a rapid diagnostic result would provide a benefit in obstetric management (for example, suspected trisomy 18 and evidence of fetal compromise). To maximize the diagnostic accuracy of fluorescence in situ hybridization in these clinical settings, very strict scoring criteriawere utilized (Table I). These criteria are established to prevent false-positive results but do so at a loss of sensitivity because of a significant "noninformative" rate. All cases having fluorescence in situ hybridization evaluation from its introduction into our clinical program (June 1, 1994) until Oct. 31, 1995, were analyzed.
Volume 175, Number 2 AmJ ObstetGynecol
Hogge et al. 353
Table I. Scoring criteria, fluorescent.in situ hybridization Metaphase cell analysis Minimum of 20 cells examined. Each cell analyzed must clearly display internal control signal (if applicable). Acceptable error rate: 5% (1/20). If >2 cells per 20 are not consistent, then 30 additional cells are analyzed. An external control (normal individual) is run in parallel with each patient sample. Interphase cell analysis Minimum of 50 cells examined. External control of same tissue type, if possible, or peripheral blood preparation is run in parallel vdth each patient sample. Normal result: 80% of cells with normal signal number. Abnormal result: 70% of cells with a consistent abnormal number of signals (for example, 70% with 3 signals for chromosome 21 = trisomy 21). Noninformative: Any other distribution of signals or control sample does not meet scoring criteria.
Results During the period of this study (June 1, 1994, to Oct. 31, 1995), 35 cases had molecular cytogenetic evaluations. In 25 instances a precise diagnosis could be made. Of the remaining 10 cases, 7 were uninformative because of insufficient cells and 3 did not meet guidelines (Table I). Of note, only one of the noninformative cases was a third-trimester sample. There were 3 abnormal results in these noninformative cases (triploidy, trisomy 21, trisomy 18). The triploidy sample did have three signals for chromosome 18 but was noninformative for chromosome 13. Following are five cases that illustrate the benefits and limitations of introducing fluorescence in situ hybridization into a prenatal diagnosis program.
Case reports Case 1. A 40-year-old woman, gravida 2, para 1, was seen at 12 weeks' gestation for chorionic villus sampling (CVS). Ultrasonographic age at the time of the procedure was 11.0 weeks. Traditional cytogenetics revealed 10% mosaicism for a marker chromosome. To rule out placental mosaicism, amniocentesis was performed at 15.9 weeks' gestation, and the initial 50 cells analyzed revealed no evidence of the marker chromosome. However, molecular studies (fluorescence in situ hybridization) on the original CVS slides revealed the marker to be an isochromosome for the p arm of chromosome 12 [i(12p)] (Fig. 1). This chromosomal abnormality is present in the Pallister-Killian syndrome, a disorder characterized by dysmorphic facial features, mental retardation, and in a small percentage of cases, diaphragmatic hernia and congenital heart disease. Because of the diagnosis of i (12p) by fluorescence in situ hybridization, an additional 13 clones were examined from the amniocentesis. One cell was found to contain the i (12p) marker. Because the marker chromosome is known to be lost in the process of cell culture, a repeat amniocentesis was performed at 19.9 weeks' gestation, and analysis of uncultured amniocytes by fluorescence in situ hybridization revealed 31% mosaicism for i(12p). The patient subsequently spontaneously aborted the pregnancy at 21.1 weeks' gestation. At autopsy clinical examination confirmed the diagnosis of Pallister-Killian syndrome, and molecular studies indicated 16% to 35% mosaicism in the various fetal tissues analyzed. Case 2. A history of an unbalanced familial translocation in a nephew was the initial reason for referral of this
21-year-old primigravid woman. 6 At referral the patient was 21.6 weeks' gestation by ultrasonographic evaluation, which also revealed complex congenital heart disease in the fetus. Echocardiography confirmed the diagnosis of tetralogy of Fallot. Chromosome analysis was performed by cordocentesis because of the late gestation, and traditional cytogenetics indicated an apparently normal 46,XY karyotype. Because the nephew was known to have cryptic 9q;17p translocation involving the telomeric portions of these two chromosomes, fluorescence in situ hybridization analysis was performed with a probe for the telometric (17pl 3.3) region of the short arm of chromosome 17(D17S34). Fig. 2 depicts the balanced translocation found in our patient and seen only on fluorescence in situ hybridization analysis. Only one signal was seen in the metaphases examined from the fetus, confirming an unbalanced cryptic translocation with partial monosomy for 17p material and partial trisomy for 9q material. The patient subsequently terminated the pregnancy. At autopsy the only additional finding was hypospadias. Case 3. This amniotic fluid sample was sent to our laboratory for chromosome analysis because of advanced maternal age and fetal gastroschisis detected by ultrasonography. Traditional cytogenetics indicated a normal 46,XX karyotype. The patient spontaneously aborted a male fetus at 17 weeks' gestation, and autopsy confirmed both external and internal genitalia to be male. Paraffin blocks were obtained from file autopsy, and molecular cytogenetic analysis was perfbrmed with deoxyribonucleic acid probes specific for the X c~-satellite (DXZ1) and a Yclassic satellite (DYZ1). Two copies of the X chromosome were found in all cells analyzed, and no copies of the Y chromosome were detected. Molecular studies are underway to search for the presence of the gene for the testis-determining factor (SR?0. Case 4. A 22-year-old woman, gravida 2, para 1, had an ultrasonographic examination at 16 weeks at her initial prenatal visit when uncertain of menstrual dating. Fetal anatomy appeared normal. An admission for pyelonephritis at 30.7 weeks' gestation prompted a repeat ultrasonographic study, which revealed measurements consistent with 28.5 weeks (15th percentile), polyhydramnios (amniotic fluid index 32.7 cm), and a two-vessel umbilical cord. Nine days later a genetics Consultation was obtained, and an amniocentesis was performed for chromosome evaluation and lung maturity. A biophysical profile on the day of admission indicated absent fetal breathing
354
Hogge et al.
August 1996 AmJ Obstet Gynecol
chl~o~ 12 a l p h a - s a 4 c e l l i t e D12Z3
/
Fig. 1. Fluorescence in situ hybridization study ofinterphase cells in case 1 demonstrating three signals with use of 12c~ satellite probe.
Fig. 2. Metaphase cell analysis from patient with balanced cryptic translocation in case 2 with fluorescent probe for 17p13.3. One signal is seen on E group chromosome corresponding to p arm of chromosome 17. Second signal was present on telomeric region of q arm of C group chromosome determined to be No. 9, indicating translocation of this chromosome material from other chromosome 17. a n d p o o r tone. Loss o f variability a n d s p o n t a n e o u s fetal h e a r t rate d e c e l e r a t i o n s p r o m p t e d an e m e r g e n c y cesare a n section. A 1216 g m infant with clinical features o f trisomy 18 was delivered. F l u o r e s c e n t in situ hybridiza-
tion o n the c u l t u r e d a m n i o t i c fluid cells with a c h r o m o s o m e 18 cz-satellite p r o b e (D18Z1) i n d i c a t e d t h r e e copies o f c h r o m o s o m e 18. Case 5. A history o f early-onset p r e e c l a m p s i a (23
Volume 175, Number 2 AmJ Obstet Gynecol
weeks' gestation) and ultrasonographic findings of a thickened placenta prompted referral of this 16-year-old gravida 2, para 1 woman. Amniocentesis was performed and fluorescent in situ hybridization with a chromosome 13qi4-specific probe (RB1) and a chromosome 21q22specific probe (D21S65) indicated two copies of both chromosome 13 and chromosome 21, excluding the diagnosis of triploidy. The patient was subsequently delivered of a 624 gm infant because of worsening preeclampsia at 24.5 weeks.
Comment
The successful culture and cytogenetic analysis of amniotic fluid cells in the early 1970s opened a new era in fetal assessment. The field was further expanded a decade later with the extension of prenatal diagnosis into the first trimester with CVS. In spite of the high degree of accuracy of cytogenetic diagnoses with amniocytes or chorionic villi, certain problems of interpretation remain. These include de novo marker chromosomes, "placental" mosaicism, and discrepancies between prenatal cytogenetic results and those obtained from the newborn. These five cases illustrate the significant benefit of the new technique of molecular cytogenetics in clarifying confusing or discrepant results obtained with the traditional banding method of chromosome analysis. Fluorescence in situ hybridization takes advantage of molecular probes that are specific for a defined region of cytogenetic interest. It is based on microscopic visualization of copy n u m b e r and therefore is sensitive well beyond what can be seen on traditionally banded chromosomes with a light microscope. 4 In addition to its increased sensitivity, fluorescence in situ hybridization allows a more rapid diagnosis because it can be performed on uncultured cells. Case 1 illustrates the benefits of fluorescence in situ hybridization in the evaluation of marker chromosomes suspected to be isochromosome 12p by traditional banding methods. A precise diagnosis of the chromosome material present allowed a specific clinical diagnosis to be made. The case also points out the unique difficulties associated with the prenatal diagnosis of the Pallister-Killian syndrome. The isochromosome is often lost during the process of cell culture. 7 Prenatal diagnosis is possible by fluorescence in situ hybridization analysis of uncultured cells obtained by CVS or amniocentesisP' 9 The finding of mosaicism in CVS material that is not present in the fetus has been termed "confined placental mosaicism. ''1° Initially thought to represent a source of inaccurate prenatal diagnostic results and a limitation of CVS, it is now recognized as a biologic p h e n o m e n o n that in many cases has clinical significance.11It is essential that these abnormalities be fully evaluated to detect those circumstances with clinical significance (true mosaicism and uniparental disomy), a2' as Case 2 is indicative of the power of the technique in the diagnosis of previously unexplained dysmorphic genetic
Hogge et al. 355
disorders. By traditional cytogenetic methods both the affected relative and our patient's fetus appeared to have normal chromosome n u m b e r and structure. However, molecular analysis revealed a familial cryptic translocafion involving the telomeres of 9q and 17p. Both affected individuals were found to have an unbalanced translocation resulting in partial monosomy for 17p and partial trisomy for 9q. The presence of an apparent laboratory error raises specific concerns regarding procedure and laboratory techniques, particularly in the case of sex discrepancies that can result from maternal cell contamination or mislabeling of samples. Case 3 illustrates that in some circumstances the cytogenetic results are not in error and represent abnormalities that are beyond the resolution of traditional methods of analysis. In this case of a 46,XX male only the gene (s) responsible for maleness may be present, most likely translocated onto a non-Ychromosome. Cases 4 and 5 represent the benefit that fluorescence in situ hybridization can have with respect to rapid diagnosis. Obstetric management decisions can be made on the basis of precise diagnostic information. In many circumstances, such as case 4, it will obviate the need to subject the patient to multiple diagnostic and therapeutic interventions. Had a genetics consultation been requested in a timely fashion, more than a week of intensive evaluation and a subsequent surgical procedure could have been avoided. The introduction of molecular cytogenefics has, and will continue to, markedly expand the diagnostic capabilities in prenatal diagnosis. Axly program providing prenatal diagnosis must have the capabilities to perform these sensitive techniques if accurate and precise diagnostic information is to be provided to patients undergoing CVS, amniocentesis, or other prenatal diagnostic testing. At the same time it must be recognized that fluorescence in situ hybridization has significant limitations, including a high noninformative rate and a significant risk of misdiagnosis. Fluorescence in situ hybridization should only be used in cases with a high suspicion for a specific chromosome abnormality and in consultation with a clinical geneticist knowledgable in the indications for and limitations of molecular cytogenefics. REFERENCES
1. Benn PA, Hsu LYE Incidence and significance of supernumerary marker chromosomes in prenatal diagnosis. Am J Hum Genet 1984;36:1092-102. 2. Warburton D. De novo balanced chromosome rearrangement and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints. AmJ Hum Genet 1991;49:995-1013. 3. Ledbetter DH. Cryptic translocations of telomere integrity. AmJ Hum Genet 1992;51:451-6. 4. Callan DF, Eyre H, Yip MY, FreemantleJ, Haan EA. Molecular cytogenetics and clinical studies of 42 patients with marker chromosomes. Am J Hum Genet 1992;43:709-15. 5. Pinkel D, GrayJW. Cytogenetic analysis by in situ hybridization with fluorescently labelled nucleic acid probes: Cold Springs Harbor Symposium. Quant Biol 1986;51:151-7.
356
Hogge et al.
6. Estop AM, Mowery-Rushton PA, Cieply KM, Kochmar sJ, Sherer CR, Clemens M, et al. Identification of an unbalanced cryptic tranlocation t(9;17)(q34.3;p13.3) in a child with dysmorphic featnres.J Med Genet 1995;32:819-22. 7. Blancato JK, Hunt M, George J, Katz J, Meck JM. Prenatal diagnosis of tetrasomy 12p by in situ hybridization: varying levels of mosaicism in different fetal tissues. Prenat Diagn 1992;12:979-83. 8. Los FJ, Van Opstal D, Schol MP, Gaillard jLj, Brandenburg H, Van Den Ouweland AMW, et al. Prenatal diagnosis of mosaic tetrasomy 12p/trisomy 12p by fluorescent in situ hybridization in amniotic fluid cells: a case report of Pallister-Kiliian syndrome. Prenat Diagn 1995;15:1155-9. 9. Bernert J, Barrels I, Gatz G, Hansmann I, Heyat M, Niedmann PD, et al. Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS. AmJ Med Genet 1992; 42:74%50. 10. Kalousek DK, Dill FJ. Chromosomal mosaicism confined to the placenta in human conception. Science 1983;221:665-7. 11. Kalousek DK. Confined placental mosaicism and intrauterine development. Pediatr Pathol 1990;10:69-77. 12. Cassidy SB, Lai L, Erickson RP, Magnuson L, Thomas E, Gendron R, et al. Trisomy 15 with 10ss of the paternal 15 as a cause of Prader Willi syndrome due to maternal disomy. AmJ Hum Genet 1992;51:701-8. 13. Teshima I, Kalousek DK, Vekemans MJ], Markovic V, Cox DM, Dallaire L, et al. Chromosome mosaicism in CVS and amniocentesis samples (Canadian multicentre trial). Prenat Diagn 1992;12:443-59.
Discussion I)1~ LEO PLOU~E, JR., Augusta, Georgia. Fluorescence in situ hybridization is truly one of the most exciting recent developments in the field of reproductive medicine a n d cytogenetics. It combines the long-established techniques developed for cytogenetic studies with the recent breakthroughs accomplished in molecular medicine. Seld o m has a wedding of such techniques yielded clinically applicable results in so little time. There are several variations on the technique. O n e aspect concerns the material studied. Fluorescence in situ hybridization can be p e r f o r m e d o n metaphase cells, which has b e e n the standard approach to cytogenetic studies. A good example of this was shown by Dr. Hogge in the second case, involving a familial translocation. This approach requires full culture of cells, as has traditionally b e e n required for the production of a karyotype. Additionally, fluorescence in situ hybridization can be d o n e o n interphase cells, where the chromatin is still c l u m p e d in the nucleus. This was demonstrated in the first case, involving the diagnosis of the Pallister-Killian syndrome. This technique can be applied directly to a cell preparation a n d can provide a rapid diagnosis, in some cases within a few hours. This technique has recently b e e n applied to single cells for preimplantation diagnosis. The second aspect involves the selection of labeling material. This has r a n g e d from radioisotopes to the use of fluorescent labels. The latter has quickly become the preferred technique. It avoids the use of radioactive materials in the laboratory and, because fluorochromes of different colors can b e u s e d , it allows the c o n c u r r e n t use of different molecular probes. A third a n d most critical e l e m e n t is the appropriate selection of probes. This has to be based on specific criteria. It involves an in-depth knowledge of the disorder
August 1996 AmJ Obstet Gynecol
suspected a n d appropriate considerations of conditions which may lead to false-positive or false-negative results. With all of its promises, fluorescence in situ hybridization is still the object of some controversy. Some have advocated its use o n a routine basis for all cases where a karyotype is ordered. Others have highlighted the shortcomings of t h e technique a n d the potential for misinterpretation. They have emphasized the n e e d for concurrent traditional karyotype studies. There is clearly a n e e d for specific criteria as to when the technique should be used. Today Dr. Hogge a n d colleagues have shared with us their experience over a 16-month interval with the use of fluorescence in situ hybridization. We are told this involved 35 cases, with 25 cases being considered as informative. Dr. Hogge further presented us with five specific cases where fluorescence in situ hybridization was felt to be of great assistance. These are exciting cases that highlight the benefits of this diagnostic approach. I will briefly c o m m e n t o n the cases presented a n d then outline general questions about the technique. Dr. Hogge's team must be c o m m e n d e d for the thoroughness of their investigations in elucidating a case of the Pallister-Killian syndrome. We could only find two other such examples in the literature. The second case clearly highlights the increased sensitivity gained in studying a karyotype by fluorescence in situ hybridization. The third case highlights the additional information that can be gained through fluorescence in situ hybridization in cases of intersex disorders. Further, fluorescence in situ hybridization in this case would be indicated with specific probes for SRY, the gene currently proposed as the testicular-determining factor. I c a n n o t refrain at this point from interjecting a concern our group has had for some time regarding the use of fluorescence in situ hybridization in the workup of intersex disorders, particularly when there is an X chromosome a n d a fragment of a n o t h e r yet-to-be-identified sex chromosome. Some laboratories have routinely b e e n using fluorescence in situ hybridization probes targeting the SRY sequence. This sequence is located quite far u p the short arm of the Y chromosome. These laboratories have reported these cases as being negative for Ymaterial. This clearly constitutes a misuse of the technique. The search for the presence of Y material is linked to the increased likelihood of gonadal malignancies in these individuals. T h e p o r t i o n of the Y chromosome linked to the oncogenic potential is currently located very close to the centromere, or central portion, of the Y chromosome. Absence of SRY does n o t m e a n that there is no Y material present a n d these patients may still be at risk for dysgerminomas a n d gonadoblastomas. It is therefore essential for the clinician to d e m a n d that centromeric probes be used in these cases. The last two cases present good examples of the rapid diagnostic potential offered through fluorescence in situ hybridization. We suspect both these cases involved the study of interphase cells. This is of great assistance to the clinician, particularly in d e t e r m i n i n g m a n a g e m e n t or m o d e of delivery.
Volume 175, Number 2 AmJ Obstet Gynecol
What p e r c e n t a g e o f the total v o l u m e do these 35 cases represent? What were the criteria used to p r o c e e d with fluorescence in situ hybridization in the c u r r e n t patient populatio~a and what general criteria would the authors propose f r o m now on? I would also like Dr. H o g g e to c o m m e n t on his opinion about the c o n c u r r e n t n e e d for karyotypic studies, particularly if fluorescence in situ hybridization is applied to interphase cells. I would also ask h i m to share his concerns a b o u t the potential for falsepositive and false-negative results. As stated before, a major advantage of fluorescence in situ hybridization is the ability to study interphase cells and obtain rapid results. I would also ask Dr. H o g g e to share with us the least time interval n e e d e d for his group to make a diagnosis t h r o u g h fluorescence in situ hybridization. In conclusion, fluorescence in situ hybridization has greatly e n l a r g e d the diagnostic a r m a m e n t a r i u m available to the clinician. Dr. H o g g e and his group have provided us with excellent examples o f this practical application of this technique. This is a rapidly evolving area that will require close m o n i t o r i n g by the clinician for its appropriate application. DR. Ron~_, V. W~E, Charlotte, N o r t h Carolina. Please c o m m e n t on the limitations and potentials of fluorescence in situ hybridization with fetal cells in maternal blood. DR. HOGGE (Closing). I would like to c o m m e n t on the case (case 3) I left out in the interest of time. This was a case we did mostly for o u r own benefit because this apparently was a false laboratory result. O u r cytogenetics indicated a 46,XX result. T h e fetus had o t h e r anomalies and that p r e g n a n c y was terminated. At pregnancy termination the fetus was f o u n d to be male both externally and internally. Rather than accept the possibilities o f maternal cell c o n t a m i n a t i o n or laboratory m i x u p of samples, we looked m o r e deeply into causes and f o u n d no evid e n c e for Y c h r o m o s o m e material with a Y c e n t r o m e r e probe. We are now interested in looking to see whether SRY is present somewhere else in the c h r o m o s o m e s of this fetus. W h a t does this r e p r e s e n t of o u r tOtal volume? I was trying to calculate that as you were asking the question. We do a b o u t 2000 prenatal cases a year, so this would probably r e p r e s e n t about 1% of our cases. We do n o t do t h e m for routine cytogenetics on patients having amniocentesis for advanced m a t e r n a l age to get a rapid answer; we would n o t do t h e m in cases of a b n o r m a l multiplemarker screen for rapid answers unless we are dealing with a circumstance where we are close to the 24-week cutoff p o i n t for t e r m i n a t i o n of pregnancy in Pennsylvania. We would use fluorescence in situ hybridization un-
Hooge et al.
357
der that circumstance, but that would be an extremely rare indication in our institution. W h a t are the criteria? We have fairly strict criteria at Magee-Womens Hospital and the University of Pittsburgh for the use of fluorescence in situ hybridization; that is, it's n o t o r d e r e d unless it's a p p r o v e d by the director of the laboratory or me. We d o n ' t think this is a t e c h n i q u e that is ready for " p r i m e time." One, you n e e d to know the particular syndromes you can look for with fluorescence in situ hybridization, and I would use the example of the heart defect earlier. We h a p p e n e d to know that particular patient r e p r e s e n t e d a case with a translocation. In o t h e r similar circflmstances there are u n i q u e deletions that are involved with tetralogy of Fallot, and we would know to look for those particular ones. D o i n g routine fluorescence in situ hybridization ,with 13, 18, or 21 probes would miss all those cases. We use it in the case of fetal anomalies in which there are specific c h r o m o s o m e abnormalities we know to look for, knowing full well we will miss some abnormalities. We use it specifically in late pregnancy, that is, the third trimester. In circumstances in which there is a high suspicion of trisomy 18, we use it to assist in p l a n n i n g for delivery to avoid caesarean section in cases of severe intrauterine growth restriction. We use it in those circumstances in which we have a family or clinical history to p o i n t us in a direction, as in case 2, which had the translocation. It is n o t a t e c h n i q u e that should be used routinely. It has a n u m b e r of limitations. You d o n ' t want 30% uninformative cases for routine cytogenetics, so it should n o t be d o n e for that. It should never be d o n e and used in the absence of a c o n c u r r e n t karyotype, as Dr. Plouffe stated before. We will use it only in that circumstance of trisomy 18 to avoid caesarean delivery, given that we have o t h e r features to support the diagnosis of trisomy 18. False-positive and false-negative results are b o t h significant problems, and I p o i n t e d out how often that can occur. In our laboratory the least time tO a result is about 48 hours. It can be d o n e in a m o r e rapid fashion, but our laboratory does a better j o b given a m i n i m u m of 48 hours to do it. In terms of how it is going to be used for fetal cells in maternal blood, I think it has the high potential to be useful. T h e difficulty is that we n e e d a large n u m b e r of cells for analysis, given that a significant n u m b e r of normal cells will have too many signals and a significant n u m b e r of a b n o r m a l cells will have too few signals. W h e n dealing with the small n u m b e r of fetal cells we are able to isolate f r o m a m a t e r n a l samples at this point, there is a , m u c h h i g h e r likelihood of making a wrong diagnosis than a correct diagnosis.