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Primed in situ labeling for rapid prenatal diagnosis
Primed in situ labeling for rapid prenatal diagnosis GopalRao V.N. Velagelati, PhD,a Lee P. Shulman, MD,b Owen P. Phillips, MD,b Sugandhi A. Tharapel, PhD,a and Avirachan T. Tharapel, PhDa,b Memphis, Tennessee OBJECTIVE: Our purpose was to assess the feasibility of primed in situ labeling for analysis of prenatal diagnostic specimens. STUDY DESIGN: Prenatal diagnostic specimens were chosen at random for analysis without knowledge of clinical indication. Primed in situ labeling with primers for chromosomes 18, 21, X, and Y was performed separate from conventional cytogenetic analyses. All clinical management considerations were based solely on conventional cytogenetic analyses. RESULTS: Forty-one samples were analyzed by primed in situ labeling: 35 direct preparations of chorionic villi and 6 uncultured amniotic fluid samples. In all cases analysis confirmed the particular chromosome number determined by conventional cytogenetic analysis. CONCLUSIONS: Although conventional metaphase studies remain the standard for prenatal cytogenetic analyses, the preliminary feasibility study finds primed in situ labeling to be a rapid and reliable adjunctive diagnostic technique applicable for prenatal diagnosis in certain clinical situations. Further study is needed to assess the efficacy of primed in situ labeling in comparison to fluorescent in situ hybridization and conventional cytogenetic analyses for prenatal diagnoses. (Am J Obstet Gynecol 1998;178:1313-20.)
Key words: Prenatal diagnosis, cytogenetics, in situ hybridization
Advances in prenatal diagnosis over the past several decades have witnessed the progression to earlier diagnostic procedures and more rapid availability of results after the procedure. The development of chorionic villus sampling (CVS) in the 1980s allowed for safe and effective first-trimester prenatal diagnosis, with direct analysis of cytotrophoblasts permitting a preliminary cytogenetic result being available within 18 to 36 hours after the procedure.1 Indeed, recent studies have sought to assess the safety and efficacy of so-called “early” amniocentesis performed in the late first and early second trimesters.2 However, cytogenetic analysis of early and conventional amniocentesis specimens requires culture methods; this results in several days passing between procedure and initial results, a delay that could have significant clinical implications when fetal aneuploidy is strongly suggested and results in patient anxiety. In an effort to expedite initial or preliminary results, fluorescent in situ hybridization has been applied to the detection of specific aneuploidies in uncultured amniotic fluid specimens.3, 4 This technique combines cytogenetic From the Departments of Pediatricsa and Obstetrics and Gynecology,b University of Tennessee, Memphis. Presented at the Sixty-fifth Annual Meeting of The Central Association of Obstetricians and Gynecologists, Scottsdale, Arizona, October 29–November 1, 1997. Reprint requests: Lee P. Shulman, MD, Department of Obstetrics and Gynecology, University of Tennessee, Memphis, 853 Jefferson Ave., Room E-102, Memphis, TN 38103-2896. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/6/90345
and molecular techniques and permits the detection of specific aneuploidies or specific nucleotide sequences in interphase and metaphase nuclei. Because it cannot, as yet, provide complete karyotypic information, it has been used primarily as an adjunctive test to conventional cytogenetic analysis. However, certain technical features of fluorescent in situ hybridization, such as the need for α-satellite repeat probes, and the lack of specific α-satellite probes for chromosomes 13 and 21 have somewhat limited its use for prenatal diagnosis. Accordingly, another procedure, known as primed in situ labeling, was developed that also combines molecular and cytogenetic techniques and provides an even more rapid and accurate assessment of specific nucleotide sequences and chromosome complements.5 Primed in situ labeling has been applied for the detection of aneuploidies in somatic cells and spermatozoa and for the characterization of marker chromosomes.6-8 We sought to determine whether primed in situ labeling and “dual” primed in situ labeling could be used for the detection of specific chromosome numbers in cells obtained from first- and second-trimester prenatal diagnostic procedures. Material and methods Cytogenetic specimens for primed in situ labeling analysis were randomly chosen from among secondtrimester amniotic fluid and first-trimester CVS specimens submitted to the cytogenetic laboratory of the University of Tennessee, Memphis, by reproductive ge1313
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Table I. Primer sequences, reannealing temperatures, and optimal concentrations from four chromosomes studied by primed in situ labeling Primer X Y 18 21
Sequence 5´-GTTCAGCTCTGTGAGTGAAA-3´ 5´-TCCATTCGATTCCATTTTTTTCGAGAA-3´ 5´-ATGTGTGTCCTCAAAG-3´ 5´-TGATGTGTGTACCCAGCC-3´
neticists (O.P.P., L.P.S.) of the Division of Reproductive Genetics, Department of Obstetrics and Gynecology, University of Tennessee, Memphis. Specimens were chosen for analysis without knowledge of the indication for prenatal diagnosis. In cases of amniotic fluid analyses, 2 to 3 ml were removed for analyses before cultures were set up for conventional cytogenetic analyses. For CVS specimens cell suspensions from the direct harvest were used. For each cell type slides were prepared for analyses without the need for slide coating or pretreatment. At no time did primed in situ labeling analyses diminish the capability to determine fetal karyotype by conventional cytogenetic methods. Primed in situ labeling reactions were carried on all specimens on the same day as slide preparation with use of primers targeting the pericentromeric α-satellite sequences of chromosomes 18, 21, X, and Y (Research Genetics, Huntsville, Ala.). The primer sequences, reannealing temperatures, and optimal concentrations of each primer are found in Table I. Primed in situ labeling and dual primed in situ labeling reactions were carried out according to published protocols9 with slight modifications. Briefly, for each slide the reaction mixture was prepared to a final volume of 50 µl containing 100 to 300 pmol of primer, 0.2 mmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate, 0.02 mmol/L each of deoxythymidine triphosphate and digoxigenin-11-deoxyuridine triphosphate (Boehringer-Mannheim, Indianapolis), 50 mmol/L potassium chloride, 10 mmol/L Tris–hydrochloric acid (pH 8.3), 1.5 mmol/L magnesium chloride, 0.01% bovine serum albumin, and 2 to 2.5 µl of Taq deoxyribonucleic acid (DNA) polymerase. The reaction was carried out on a programmable heating plate, MISHA (Shandon-Lipshaw, Pittsburgh). The reaction cycle consisted of an initial 15-minute reannealing period at a temperature specific for a particular primer and a final extension of 30 minutes at 72° C. The reaction was halted by immersing the slides in a 72° C bath containing 500 mmol/L sodium chloride and 50 mmol/L ethylenediaminetetraacetic acid (pH 8) for 3 minutes. Slides were then transferred to 4× saline–sodium cit-
Reannealing temperature (° C)
Optimum concentration (pmol/L)
68 56 65 61
150 150 100 100
rate buffer/0.2% Tween-20 at room temperature for 3 minutes and exposed to fluorescein-labeled antidigoxigenin antibody (20 µg/ml) for 10 minutes at 37° C. The slides were twice washed in 4× saline–sodium citrate buffer/0.2% Tween-20 for 3 minutes each, counterstained with propidium iodide (0.02 µg/ml), and examined under an Olympus Vanox microscope equipped with a CytoVision Probe Image Analysis System (Applied Imaging, Pittsburgh). For dual primed in situ labeling analyses, slides were transferred to 1× NT buffer (50 mmol/L Tris–hydrochloric acid (pH 7.2), 10 mmol/L magnesium sulfate, 100 µmol/L dithiothreitol, and 150 µg/ml bovine serum albumin) and washed twice before being treated with a dideoxynucleosides mixture (10 µmol/L each of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate; 4 µl of 10× NT buffer; and 2 U of Klenow enzyme) for 10 minutes at 37° C to block the free 3´ ends of the elongation fragments generated by the first primed in situ labeling reaction. Slides were washed in a “stop” solution and twice in 1× NT buffer at room temperature. After excess fluid was drained, slides were placed on MISHA at a specific reannealing temperature dependent on the particular primer. The second reaction mixture (identical to the first except for different primer and biotin-11-dUTP) was applied to the slide and the reaction was carried out similar to the first reaction. Detection was carried out with use of a 1:1 mixture of fluorescein-labeled antidigoxigenin antibody and rhodamine-labeled antiavidin; the slides were then counterstained with 4´,6´-diamidino-2phenylindole. Hybridization scoring criteria. A minimum of 50 nuclei were scored by two independent and experienced cytogeneticists (G.V.N.V., S.A.T., A.T.T.). All nuclei were evaluated with either a 60× or 100× oil objective. Clumped and overlapping nuclei, nuclei with no clear boundaries or with attached cytoplasm and cell membrane, and nuclei with irregular shapes were not included in the scoring process. A nucleus is considered monosomic if one signal is observed, disomic when two signals are viewed, and tri-
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Fig. 1. Scored cell from uncultured amniotic fluid cell specimen demonstrating two X signals from primed in situ labeling reaction. Fetal X complement was confirmed by conventional cytogenetic analysis.
Fig. 2. Scored cell from a directly prepared cytotrophoblastic cell specimen demonstrating two chromosome 18 signals. Disomy 18 confirmed by conventional cytogenetic analysis.
somic when three signals are detected; cells with four or more signals are also recorded. All slides were coded in a blinded fashion with only a study number provided to the scorers. By use of criteria reported by Divane et al.,4 a particular chromosome number (complement) was assigned when ≥70% of hybridized nuclei displayed the same number of signals. All primed in situ labeling results were recorded well before results of conventional cytogenetic analyses were available.
core cells and fetal fibroblasts. Directly prepared cytotrophoblastic cells from this specimen were evaluated by primed in situ labeling for chromosomes 18, 21, X, and Y, which was performed by cytogeneticists who were not involved in the cytogenetic evaluation of chorionic villi or slide preparation and who were unaware of the trisomic fetal complement. In all 41 cases primed in situ labeling and dual primed in situ labeling correctly predicted fetal gender (Fig. 1) and chromosome 18 (Fig. 2) and 21 complements as determined by conventional cytogenetics. Mean percentage of signals from pregnancies characterized by euploid complements and from the case of fetal trisomy 21 are found in Tables II through VI.
Results Forty-one samples were included in this study, including 6 amniotic fluid specimens and 35 direct preparations of chorionic villi. Among the amniotic fluid cases, four samples were obtained from women at increased risk for fetal chromosome abnormalities or neural tube defects as determined by maternal serum screening and two samples from women undergoing amniocentesis because of advanced maternal age (≥35 years old at estimated date of delivery). Of the 35 chorionic villi specimens, 32 were obtained because of advanced maternal age, two from women who were previously delivered of infants with aneuploidy and one from a woman at increased risk (1:4) for cystic fibrosis. Thirty-four of the samples were studied with primers for chromosomes 18, X, and Y and five samples (two amniotic fluid and three chorionic villi) were studied with use of the modified dual primed in situ labeling method. Of the five samples studied by the dual technique, only primers for chromosomes X and Y were used. These 40 samples were from women carrying fetuses with normal chromosome complements. One sample (direct preparation of chorionic villi) was obtained from a woman who underwent CVS for advanced maternal age (age 38 years) and was found to be carrying a female fetus with trisomy 21, as detected by direct analysis of chorionic villi and later confirmed by analysis of cultured mesenchymal
Comment Analysis of metaphase spreads remains the standard diagnostic method for almost all prenatal diagnostic cases in spite of the development and relatively widespread use of rapid adjunctive cytogenetic analyses such as fluorescent in situ hybridization. The limitations of these adjunctive methods, including the inability of fluorescent in situ hybridization and primed in situ labeling to provide complete karyotypic information and concerns about the sensitivity and specificity of these techniques, require that clinical management for almost all prenatal diagnostic cases be based on results from conventional cytogenetic analyses of metaphase spreads. Indeed, the development of CVS and the ability to use cytotrophoblastic cells for rapid, yet complete, fetal karyotypic analyses has allowed for more early and rapid cytogenetic analyses for women at increased risk for fetal aneuploidy. However, amniocentesis remains the most popular method for prenatal diagnosis, and analysis of amniotic fluid cells requires culture methods that now result in a 7- to 14-day interval (for most laboratories) from sample procurement to karyotypic results.
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Table II. Mean distribution of signals in samples characterized by 46,XX complements Percentage of cells demonstrating signal number No. of signals
Chromosome X
0 1 2 3 ≥4
5 5 85 4 1
Chromosome Y 90 6 2 1 1
Table V. Mean distribution of signals in samples characterized by 46,XX complements evaluated by dual primed in situ labeling Percentage of cells demonstrating signal number
Chromosome 18 3 4 88 4 1
Table III. Mean distribution of signals in samples characterized by 46,XY complements Percentage of cells demonstrating signal number
No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
3 5 89 2 1
90 4 3 1 1
Table VI. Mean distribution of signals in sample obtained from woman carrying fetus with 47,XX,+21 complement Percentage of cells demonstrating signal number
No. of signals
Chromosome X
0 1 2 3 ≥4
Chromosome Y
3 88 7 1 1
8 87 3 1 1
Chromosome 18 3 5 89 2 1`
Table IV. Mean distribution of signals in samples characterized by 46,XY complements evaluated by dual primed in situ labeling Percentage of cells demonstrating signal number No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
3 91 5 1 0
2 91 3 2 1
In recent years fluorescent in situ hybridization has been used to obtain rapid cytogenetic results from prenatal diagnostic specimens. Klinger et al.3 analyzed 526 amniotic fluid samples for chromosomes 13, 18, 21, X, and Y by fluorescent in situ hybridization; numeric abnormalities involving these chromosomes account for the majority of fetal chromosome abnormalities detected by CVS and amniocentesis.10 In this study fluorescent in situ hybridization correctly predicted specific chromosome complements in all samples. More recently, Divane et al.4 demonstrated successful application of a simultaneous five-color (probe) fluorescent in situ hybridization technique to assess chromosomes 13, 18, 21, X, and Y in 30 amniotic fluid samples. However, fluorescent in situ hybridization is limited by
No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
6 9 85 0 0
91 9 0 0 0
Chromosome 21 8 4 3 85 0
several technical factors. First, and most important, analyses do not provide complete karyotypic information but rather assess specific loci on specific chromosomes. Although the majority of fetal chromosome abnormalities involve nondisjunction of chromosomes 21, 18, 13, X, and Y,10 fluorescent in situ hybridization does not permit the detection of most structural and some numerical chromosome abnormalities. Second, the lack of specificity of some repeat probes (e.g., chromosomes 13 and 21) and uncoupling between the hybridization signal and chromosome count lead to difficulties in interpreting signal counts,9 although the use of different telomeric probes specific for chromosomes 13 and 21 can bypass the diagnostic problems associated with lack of centromeric probe specificity. Third, although fluorescent in situ hybridization does significantly decrease the time between sample procurement and result, the process requires considerable laboratory and interpretative resources, especially in cases where specific α-satellite probes are not applicable for the needed diagnosis.6 Primed in situ labeling is based on the reannealing of specific oligonucleotide primers to the target sequences in situ and subsequent extension of the primer sequence by Taq DNA polymerase in the presence of labeled nucleotides. The high efficiency of this technique for discriminating α-satellite DNA sequences has been well documented and has been applied for the analysis of somatic tissues and sperm.6-8 The advantages of primed in situ la-
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beling over conventional fluorescent in situ hybridization analysis include increased sensitivity and specificity, especially in the detection of aneuploidies involving chromosomes 13 and 21.6, 9, 11, 12 Primed in situ labeling uses the high specificity between primers and their genomic targets; it is thus possible to design specific primers for individual centromere detection of chromosomes 13 and 21 without cross-hybridization.13 Second, primed in situ labeling requires a far lesser time from sample procurement to diagnostic result. With semiautomated protocols, reactions can be completed in less than an hour after slide preparation, with even less time required when probes of moderate to high copy sequences, such as α-satellite DNA, are used. Another benefit of the shorter reaction time of primed in situ labeling over fluorescent in situ hybridization is that the shortened reaction time reduces the background hybridization, thereby making the signal less ambiguous and improving sensitivity and specificity of the analysis.9 This shortened reaction time represents potential diagnostic advantages for certain critically ill newborns, the evaluation of selected pregnancies characterized by structural or growth abnormalities, and allaying some patient anxiety by providing rapid and preliminary, albeit incomplete, information concerning common fetal aneuploidies. Our current study, to the best of our knowledge, is the first series limited to the evaluation of prenatal diagnostic specimens by primed in situ labeling. Our results from this blinded preliminary feasibility study find primed in situ labeling to correctly predict selected chromosome complements in all specimens. This technique, like fluorescent in situ hybridization, is limited to the detection of specific aneuploidies and is likewise limited in detecting structural and less common numeric chromosome abnormalities. In addition, the, sensitivity and specificity of primed in situ labeling for chorionic villi and amniotic fluid analyses remain to be determined. However, we believe that this technique will be found to be a rapid and effective adjunctive diagnostic method for specific clinical situations. Indeed, having both primed in situ labeling and fluorescent in situ hybridization available should improve our ability to detect numeric and structural chromosome abnormalities in a far shorter period of time than is currently possible,14 although these techniques should not now be used in lieu of conventional cytogenetics for prenatal diagnostic cases. Accordingly, we believe that primed in situ labeling deserves consideration as an adjunctive cytogenetic analysis applicable for obtaining rapid, but limited, preliminary results from selected prenatal diagnostic specimens. REFERENCES
1. Phillips OP, Tharapel AT, Lerner JL, Park VM, Wachtel SS, Shulman LP. Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling. Am J Obstet Gynecol 1996;174:850-5.
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2. Shulman LP, Elias S, Phillips OP, Grevengood C, Dungan JS, Simpson JL. Amniocentesis performed at 14 weeks gestation or earlier: comparison with first-trimester transabdominal chorionic villus sampling. Obstet Gynecol 1994;83:543-8. 3. Klinger K, Landes G, Shook D, Harvey R, Lopez L, Locke P, 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. 4. Divane A, Carter NP, Spathas DH, Ferguson-Smith MA. Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization. Prenat Diagn 1994;14:1061-9. 5. Koch J, Kolvraa S, Petersen K, Gregersen N, Bolund L. Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ. Chromosome 1989;98:259-65. 6. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. Use of the primed in situ labeling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum Genet 1995;95:12-7. 7. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. PRINS as a method for rapid chromosomal labeling on human spermatozoa. Mol Reprod Dev 1995;40:333-7. 8. Velagaleti GVN, Tharapel SA, Martens PR, Tharapel AT. Rapid identification marker chromosomes using primed in situ hybridization (PRINS). Am J Med Genet 1997;71:130-3. 9. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. Selection of chromosome-specific primers and their use in simple and double PRINS techniques for rapid in situ identification of human chromosomes. Cytogenet Cell Genet 1995;70:138-42. 10. Hook EB. The impact of aneuploidy upon public health: mortality and morbidity associated with human chromosome abnormalities. In: Dellarco VL, Voytek PE, Hollaender A, editors. Aneuploidy etiology and mechanisms. New York: Plenum Press; 1985. p. 7-33. 11. Gosden J, Lawson D. Rapid chromosome identification by oligonucleotide-primed in situ DNA synthesis (PRINS). Hum Mol Genet 1994;3:931-6. 12. Gosden J, Lawson D. Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ. Cytogenet Cell Genet 1995;68:57-60. 13. Charlieu JP, Murgue B, Laurent A-M, Marçais B, Bellis M, Roizes G. Discrimination between alpha-satellite DNA sequences from chromosomes 13 and 21 by using polymerase chain reaction. Genomics 1992;14:515-6. 14. Hindkjaer J, Brandt CA, Koch J, Lund TB, Kolvraa S. Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting). Chromosome Res 1995;3:41-4.
Discussion DR. MARK JOHNSON, Detroit, Michigan. Although primed in situ labeling is a well-described and well-used technique in molecular cytogenetics, it has received little attention in the field of prenatal diagnosis. With pressures to report results more quickly in prenatal testing, use of molecular-based technologies such as fluorescent in situ hybridization for aneuploid screening has, if anything, been overused by individuals who are not familiar with the limitations of such techniques. In this study the authors run the risk of perpetuating such misconceptions to the less well informed when they say that “fluorescent in situ hbridization has recently been used to obtain rapid cytogenetic results from prenatal specimens.” This reflects the common misconception among lay practitioners that fluorescent in situ hybridization screening alone is sufficient for prenatal diagnosis,
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where in fact it really only represents a preliminary screen for the presence or absence of specific probed-for molecular sequences. Indeed, in our experience, we have found that many cytogenetically abnormal prenatal cases would have been missed with use of molecular fluorescent in situ hybridization techniques alone without confirmation by standard cultured karyotype. In a recent multicenter international collaborative report using data from 142,367 prenatal diagnostic specimens from eight centers in four countries, we found 3807 (2.67%) karyotypic abnormalities, of which 64.4% would have been detected with use of current fluorescent in situ hybridization probes for chromosomes 13, 18, 21, X, and Y, whereas 36.4% would have been missed without traditional karyotyping. Although screening for the major aneuploid syndromes was successful in our collaborative study, supporting the power of molecular techniques, more subtle structural aberrations and unbalanced rearrangements not involving the probed-for sequences are routinely missed. Unfortunately, more than half these missed rearrangements would have had a significant clinical impact for the fetus. It is therefore important to remember that molecular-based techniques may prove to be a useful adjunct in our diagnostic armamentarium but do not obviate the necessity of a full, banded karyotype. The authors emphasize the speed and increased sensitivity of primed in situ labeling compared with standard fluorescent in situ hybridization techniques. No one questions the capacity of primed in situ labeling to provide rapid and reliable results; however, the authors fall a bit short in providing a compelling argument for better sensitivity and specificity on the basis of the data presented in their study. One intriguing issue is that they are highly critical of fluorescent in situ hybridization because of the use of probes against highly repetitive sequences of chromosome-specific centromeric α-satellite DNA and the reported problems in the past of cross-hybridization, particularly between chromosomes 13 and 21. However, this valid concern of several years ago has been rectified, and for the last 2 years laboratories have routinely used probes for chromosome-specific telomeric sequences for chromosomes in which cross-hybridization was a problem. Interestingly, in their Methods, the investigators describe the use of primed in situ labeling primers directed against the same pericentric α-satellite regions they had just described to be problematic in fluorescent in situ hybridization. I would therefore ask the investigators for additional information based on their data to support their conclusion that primed in situ labeling offers better sensitivity and specificity over fluorescent in situ hybridization on the basis of their data. The investigators are part of a large, well-established and well-known, reputable prenatal diagnosis program. In the current study they present their apparent early experiences in establishing the primed in situ labeling technique in prenatal specimens. The small sample size of 35 CVSs and 6 amniocenteses represents more a pilot study
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than a conclusive report validating translation of a technique to a new clinical application. I was therefore hoping that investigators could update us and provide us with more substantial numbers to support the use of this technique on the basis of their continuing accumulation of prenatal cases in their laboratory, in particular with use of multiprimed in situ labeling. At Wayne State we are also looking at this exciting new technique but are not quite ready to replace our current use of direct and standard fluorescent in situ hybridization for aneuploid screening. One obvious technical concern in molecular techniques is successful probe hybridization and signal recognition. In the current study the investigators were successful in achieving hybridization and polymerization resulting in clear signal recognition on analysis for each of their primers. I would ask the authors if in their developing experience with this technique have their results continued to improve to the level or beyond that achieved with fluorescent in situ hybridization, supporting their position that the primed in situ labeling technique is comparable or better than fluorescent in situ hybridization approaches? In conclusion, I would repeat my concerns that practitioners understand the limitations of such techniques and that rapid molecular approaches such as primed in situ labeling and fluorescent in situ hybridization do not obviate the need for a complete, banded karyotype after CVS or amniocentesis. DR. PAUL TOMICH, Maywood, Illinois. The authors are part of a large, well-respected prenatal diagnosis program and have made important contributions in the field of prenatal genetics. Hence this presentation should be appreciated as a potentially important feasibility study concerning the role of primed in situ labeling in prenatal diagnosis. Primed in situ DNA synthesis since its introduction in 1989 by Koch has proved to be valuable for mapping and investigating repeated sequences in chromosomes and is thought to have some advantages over fluorescent in situ hybridization. It has been used in the detection of aneuploidies in somatic cells and in spermatozoa. Its role as a prenatal diagnostic procedure has not yet been established. The authors have reported their initial experience in using primed in situ labeling in 41 prenatal diagnosis specimens, 35 from CVS and 6 from amniotic fluid specimens. The indications for the CVS were (1) 32 women with advanced maternal age, (2) 2 women with previous children with chromosomal abnormalities, and (3) 1 woman at increased risk for cystic fibrosis. The amniotic fluid specimens were obtained because of abnormal screening testing results in 4 cases and advanced maternal age in 2 cases. Single primed in situ labeling was performed on 35 specimens and dual primed in situ labeling was performed on 2 amniotic fluid specimens and 3 CVS specimens. The conclusions are as follows. Primed in situ labeling can be rapidly done; same day service is possible. The au-
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thors claim that in their study there was an 86% to 90% success rate. The technique is as good as or better than fluorescent in situ hybridization and offers the advantage of a more rapid test, with results available more quickly. The authors claim that it offers superior sensitivity and specificity compared with fluorescent in situ hybridization. What are the problems with this study? The first has to do with the numbers. Thirty-five CVS specimens and 6 amniotic fluid specimens are an insufficient number on which to support any meaningful conclusions. I would think that at least 50 to 100 specimens from both CVS and amniocenteses would be necessary before reporting on the “reliability” of a technique. Next, only one case was probed for chromosome 21, which was already known to be from a specimen for an aneuploidy with trisomy 21. Again, the authors need to use primed in situ labeling for all major aneuploidies (chromosomes 21, 18, 13, X, and Y) in larger numbers before supporting their thesis that this technique is as good as or better than fluorescent in situ hybridization. The authors report a success rate of 86% to 90% with primed in situ labeling; this does not surpass the 90% to 95% rate commonly achieved by fluorescent in situ hybridization. One other issue not mentioned in the study is the issue of cost: perhaps Dr. Shulman could provide data on the cost of fluorescent in situ hybridization versus primed in situ labeling at the University of Tennessee or from national data that he may be aware of. I am not a geneticist. However, I am a perinatologist with an interest in prenatal diagnosis and I treat and counsel patients when they have either been found to have an anomaly on ultrasonography or have previously had a chromosomally abnormal child, are older than 35 years, or have abnormal triple testing. With regard to use of primed in situ labeling in CVS specimens, I do not see that the current practice of doing full studies should be redesigned to do this technique as well: rapid turnaround time is not really an issue. For those situations where amniotic fluid specimens are to be used, what is the clinician to do, make clinical decisions on the results returned in 1 day or await full studies before counseling or treating the patient? Whether we are talking about fluorescent in situ hybridization or primed in situ labeling in the prenatal diagnosis arena, we need to remember that such screening does not obviate the need for traditional karyotyping in such specimens. At the present time, faced with the dilemma of whether there may be a chromosomal abnormality and facing the need to counsel parents concerning the results of such studies, I will await final karyotype studies (at our cytogenetic laboratory fluorescent in situ hybridization through an outside laboratory is back in 3 to 4 days and full chromosomal studies in 7 days), fully appreciating that the “wait” is nerve wracking and anxiety producing for parents, but 100% reliable information is absolutely imperative in such circumstances. This study should be looked on as no more than a preliminary feasibility study. The authors are encouraged to
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pursue additional studies on this topic to identify what role primed in situ labeling may have in the field of prenatal diagnosis. It is one thing to present data as an initial experience and quite another for anyone to make the leap to conclude after this presentation that the role of this technique in prenatal diagnosis has now been demonstrated or to suggest that either fluorescent in situ hybridization or primed in situ labeling should replace traditional chromosomal analysis. I hope that no one leaving here goes to their hospital or cytogenetics laboratory and says, “I want a primed in situ labeling, get me a fluorescent in situ hybridization” instead of “How quickly can you get the chromeme studies done and back to me?” DR. NORMAN GINSBURG, Chicago, Illinois. The fluorescent in situ hybridization and primed in situ labeling techniques do have a role in confirming or helping to speed along the diagnosis. There are often times when amniocentesis takes a long time. We get a few cells that are confirmatory of an abnormality, and this is a technique that can analyze many more cells at a more rapid rate. The same goes for CVS as well, particularly when people are coming to a time where availability of a firsttrimester termination is rapidly coming to an end. DR. SHULMAN (Closing). Let me add that in no way should anyone leave this room today asking for anything but a complete cytogenetic analysis. Not only is it prudent perinatal medicine, we’re also dealing with subjects of standards of care; in fact, I don’t think any of us in this room would base any clinical management on anything other than a complete karyotypic analysis of the fetus. Let me also say that I also did not come to bury or to ruin fluorescent in situ hybridization but rather to praise it and improve it; in fact, this is indeed an initial feasibility study. If you did ask your laboratory to provide you with a primed in situ labeling analysis, they would probably say, “A what?,” primarily because the machinery that is required for this is not currently available in the United States; it’s been our work with the Pelastor group in Belgium that has allowed us to have the machine, the reagents, not so much the primers, that has allowed us to do this initial feasibility study. As far as the telomeric sequences, Dr. Johnson is absolutely right. That is being used more and more to differentiate chromosomes 13 and 21. There were two cases in which we’ve used nontelomeric, noncentromeric specific sequences to get very nice preparations looking at either chromosome 13, chromosome 21, and chromosome X; in fact, the real power of the primed in situ labeling reaction is that we can take very small, unique sequences that are not found anywhere else within the genome and have a very powerful in situ hybridization technique that we can use truly as an injunctive diagnostic method. Dr. Johnson is correct. Our numbers for this particular preliminary study are small and actually represent sensitivities that are at least no better than those of fluorescent in situ hybridization.
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We have now done well over 150 cases. We are now finding, in fact, sensitivity well in the range of between 93% and 97%; in fact, we now have gone one step further. Instead of relying on 70% of cells to call a particular complement, we are now using 80% showing a particular complement before we will call it as that complement. We have stopped doing further studies on prenatal tissue for the time being; we’re stopping at about 150, primarily because our laboratory is now using primed in situ labeling for gene sequencing. Hopefully next year we’ll have some interesting data to present with that. I don’t have a clue what the cost of primed in situ labeling is. I can tell you that the machines that are re-
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quired cost somewhere in the range of $75,000 to $90,000 and in a sense it’s a very fancy but modified PCR machine that allows us to anneal, reanneal, and add the tack (phonetic) polymerase. I would not alter my management on the basis of a primed in situ labeling. I would provide information on a full karyotypic analysis; I believe within the next year or so when these machines are available may, in fact, represent an improvement in our ability to provide in situ hybridization preliminary information for those particular patients. Hopefully, as we learn and are able to apply this technique more, in fact, we will use it for more complete structural and numeric abnormality assessments for fetal and even for adult karyotypes.
Key words: Prenatal diagnosis, cytogenetics, in situ hybridization
Advances in prenatal diagnosis over the past several decades have witnessed the progression to earlier diagnostic procedures and more rapid availability of results after the procedure. The development of chorionic villus sampling (CVS) in the 1980s allowed for safe and effective first-trimester prenatal diagnosis, with direct analysis of cytotrophoblasts permitting a preliminary cytogenetic result being available within 18 to 36 hours after the procedure.1 Indeed, recent studies have sought to assess the safety and efficacy of so-called “early” amniocentesis performed in the late first and early second trimesters.2 However, cytogenetic analysis of early and conventional amniocentesis specimens requires culture methods; this results in several days passing between procedure and initial results, a delay that could have significant clinical implications when fetal aneuploidy is strongly suggested and results in patient anxiety. In an effort to expedite initial or preliminary results, fluorescent in situ hybridization has been applied to the detection of specific aneuploidies in uncultured amniotic fluid specimens.3, 4 This technique combines cytogenetic From the Departments of Pediatricsa and Obstetrics and Gynecology,b University of Tennessee, Memphis. Presented at the Sixty-fifth Annual Meeting of The Central Association of Obstetricians and Gynecologists, Scottsdale, Arizona, October 29–November 1, 1997. Reprint requests: Lee P. Shulman, MD, Department of Obstetrics and Gynecology, University of Tennessee, Memphis, 853 Jefferson Ave., Room E-102, Memphis, TN 38103-2896. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/6/90345
and molecular techniques and permits the detection of specific aneuploidies or specific nucleotide sequences in interphase and metaphase nuclei. Because it cannot, as yet, provide complete karyotypic information, it has been used primarily as an adjunctive test to conventional cytogenetic analysis. However, certain technical features of fluorescent in situ hybridization, such as the need for α-satellite repeat probes, and the lack of specific α-satellite probes for chromosomes 13 and 21 have somewhat limited its use for prenatal diagnosis. Accordingly, another procedure, known as primed in situ labeling, was developed that also combines molecular and cytogenetic techniques and provides an even more rapid and accurate assessment of specific nucleotide sequences and chromosome complements.5 Primed in situ labeling has been applied for the detection of aneuploidies in somatic cells and spermatozoa and for the characterization of marker chromosomes.6-8 We sought to determine whether primed in situ labeling and “dual” primed in situ labeling could be used for the detection of specific chromosome numbers in cells obtained from first- and second-trimester prenatal diagnostic procedures. Material and methods Cytogenetic specimens for primed in situ labeling analysis were randomly chosen from among secondtrimester amniotic fluid and first-trimester CVS specimens submitted to the cytogenetic laboratory of the University of Tennessee, Memphis, by reproductive ge1313
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Table I. Primer sequences, reannealing temperatures, and optimal concentrations from four chromosomes studied by primed in situ labeling Primer X Y 18 21
Sequence 5´-GTTCAGCTCTGTGAGTGAAA-3´ 5´-TCCATTCGATTCCATTTTTTTCGAGAA-3´ 5´-ATGTGTGTCCTCAAAG-3´ 5´-TGATGTGTGTACCCAGCC-3´
neticists (O.P.P., L.P.S.) of the Division of Reproductive Genetics, Department of Obstetrics and Gynecology, University of Tennessee, Memphis. Specimens were chosen for analysis without knowledge of the indication for prenatal diagnosis. In cases of amniotic fluid analyses, 2 to 3 ml were removed for analyses before cultures were set up for conventional cytogenetic analyses. For CVS specimens cell suspensions from the direct harvest were used. For each cell type slides were prepared for analyses without the need for slide coating or pretreatment. At no time did primed in situ labeling analyses diminish the capability to determine fetal karyotype by conventional cytogenetic methods. Primed in situ labeling reactions were carried on all specimens on the same day as slide preparation with use of primers targeting the pericentromeric α-satellite sequences of chromosomes 18, 21, X, and Y (Research Genetics, Huntsville, Ala.). The primer sequences, reannealing temperatures, and optimal concentrations of each primer are found in Table I. Primed in situ labeling and dual primed in situ labeling reactions were carried out according to published protocols9 with slight modifications. Briefly, for each slide the reaction mixture was prepared to a final volume of 50 µl containing 100 to 300 pmol of primer, 0.2 mmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate, 0.02 mmol/L each of deoxythymidine triphosphate and digoxigenin-11-deoxyuridine triphosphate (Boehringer-Mannheim, Indianapolis), 50 mmol/L potassium chloride, 10 mmol/L Tris–hydrochloric acid (pH 8.3), 1.5 mmol/L magnesium chloride, 0.01% bovine serum albumin, and 2 to 2.5 µl of Taq deoxyribonucleic acid (DNA) polymerase. The reaction was carried out on a programmable heating plate, MISHA (Shandon-Lipshaw, Pittsburgh). The reaction cycle consisted of an initial 15-minute reannealing period at a temperature specific for a particular primer and a final extension of 30 minutes at 72° C. The reaction was halted by immersing the slides in a 72° C bath containing 500 mmol/L sodium chloride and 50 mmol/L ethylenediaminetetraacetic acid (pH 8) for 3 minutes. Slides were then transferred to 4× saline–sodium cit-
Reannealing temperature (° C)
Optimum concentration (pmol/L)
68 56 65 61
150 150 100 100
rate buffer/0.2% Tween-20 at room temperature for 3 minutes and exposed to fluorescein-labeled antidigoxigenin antibody (20 µg/ml) for 10 minutes at 37° C. The slides were twice washed in 4× saline–sodium citrate buffer/0.2% Tween-20 for 3 minutes each, counterstained with propidium iodide (0.02 µg/ml), and examined under an Olympus Vanox microscope equipped with a CytoVision Probe Image Analysis System (Applied Imaging, Pittsburgh). For dual primed in situ labeling analyses, slides were transferred to 1× NT buffer (50 mmol/L Tris–hydrochloric acid (pH 7.2), 10 mmol/L magnesium sulfate, 100 µmol/L dithiothreitol, and 150 µg/ml bovine serum albumin) and washed twice before being treated with a dideoxynucleosides mixture (10 µmol/L each of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate; 4 µl of 10× NT buffer; and 2 U of Klenow enzyme) for 10 minutes at 37° C to block the free 3´ ends of the elongation fragments generated by the first primed in situ labeling reaction. Slides were washed in a “stop” solution and twice in 1× NT buffer at room temperature. After excess fluid was drained, slides were placed on MISHA at a specific reannealing temperature dependent on the particular primer. The second reaction mixture (identical to the first except for different primer and biotin-11-dUTP) was applied to the slide and the reaction was carried out similar to the first reaction. Detection was carried out with use of a 1:1 mixture of fluorescein-labeled antidigoxigenin antibody and rhodamine-labeled antiavidin; the slides were then counterstained with 4´,6´-diamidino-2phenylindole. Hybridization scoring criteria. A minimum of 50 nuclei were scored by two independent and experienced cytogeneticists (G.V.N.V., S.A.T., A.T.T.). All nuclei were evaluated with either a 60× or 100× oil objective. Clumped and overlapping nuclei, nuclei with no clear boundaries or with attached cytoplasm and cell membrane, and nuclei with irregular shapes were not included in the scoring process. A nucleus is considered monosomic if one signal is observed, disomic when two signals are viewed, and tri-
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Fig. 1. Scored cell from uncultured amniotic fluid cell specimen demonstrating two X signals from primed in situ labeling reaction. Fetal X complement was confirmed by conventional cytogenetic analysis.
Fig. 2. Scored cell from a directly prepared cytotrophoblastic cell specimen demonstrating two chromosome 18 signals. Disomy 18 confirmed by conventional cytogenetic analysis.
somic when three signals are detected; cells with four or more signals are also recorded. All slides were coded in a blinded fashion with only a study number provided to the scorers. By use of criteria reported by Divane et al.,4 a particular chromosome number (complement) was assigned when ≥70% of hybridized nuclei displayed the same number of signals. All primed in situ labeling results were recorded well before results of conventional cytogenetic analyses were available.
core cells and fetal fibroblasts. Directly prepared cytotrophoblastic cells from this specimen were evaluated by primed in situ labeling for chromosomes 18, 21, X, and Y, which was performed by cytogeneticists who were not involved in the cytogenetic evaluation of chorionic villi or slide preparation and who were unaware of the trisomic fetal complement. In all 41 cases primed in situ labeling and dual primed in situ labeling correctly predicted fetal gender (Fig. 1) and chromosome 18 (Fig. 2) and 21 complements as determined by conventional cytogenetics. Mean percentage of signals from pregnancies characterized by euploid complements and from the case of fetal trisomy 21 are found in Tables II through VI.
Results Forty-one samples were included in this study, including 6 amniotic fluid specimens and 35 direct preparations of chorionic villi. Among the amniotic fluid cases, four samples were obtained from women at increased risk for fetal chromosome abnormalities or neural tube defects as determined by maternal serum screening and two samples from women undergoing amniocentesis because of advanced maternal age (≥35 years old at estimated date of delivery). Of the 35 chorionic villi specimens, 32 were obtained because of advanced maternal age, two from women who were previously delivered of infants with aneuploidy and one from a woman at increased risk (1:4) for cystic fibrosis. Thirty-four of the samples were studied with primers for chromosomes 18, X, and Y and five samples (two amniotic fluid and three chorionic villi) were studied with use of the modified dual primed in situ labeling method. Of the five samples studied by the dual technique, only primers for chromosomes X and Y were used. These 40 samples were from women carrying fetuses with normal chromosome complements. One sample (direct preparation of chorionic villi) was obtained from a woman who underwent CVS for advanced maternal age (age 38 years) and was found to be carrying a female fetus with trisomy 21, as detected by direct analysis of chorionic villi and later confirmed by analysis of cultured mesenchymal
Comment Analysis of metaphase spreads remains the standard diagnostic method for almost all prenatal diagnostic cases in spite of the development and relatively widespread use of rapid adjunctive cytogenetic analyses such as fluorescent in situ hybridization. The limitations of these adjunctive methods, including the inability of fluorescent in situ hybridization and primed in situ labeling to provide complete karyotypic information and concerns about the sensitivity and specificity of these techniques, require that clinical management for almost all prenatal diagnostic cases be based on results from conventional cytogenetic analyses of metaphase spreads. Indeed, the development of CVS and the ability to use cytotrophoblastic cells for rapid, yet complete, fetal karyotypic analyses has allowed for more early and rapid cytogenetic analyses for women at increased risk for fetal aneuploidy. However, amniocentesis remains the most popular method for prenatal diagnosis, and analysis of amniotic fluid cells requires culture methods that now result in a 7- to 14-day interval (for most laboratories) from sample procurement to karyotypic results.
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Table II. Mean distribution of signals in samples characterized by 46,XX complements Percentage of cells demonstrating signal number No. of signals
Chromosome X
0 1 2 3 ≥4
5 5 85 4 1
Chromosome Y 90 6 2 1 1
Table V. Mean distribution of signals in samples characterized by 46,XX complements evaluated by dual primed in situ labeling Percentage of cells demonstrating signal number
Chromosome 18 3 4 88 4 1
Table III. Mean distribution of signals in samples characterized by 46,XY complements Percentage of cells demonstrating signal number
No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
3 5 89 2 1
90 4 3 1 1
Table VI. Mean distribution of signals in sample obtained from woman carrying fetus with 47,XX,+21 complement Percentage of cells demonstrating signal number
No. of signals
Chromosome X
0 1 2 3 ≥4
Chromosome Y
3 88 7 1 1
8 87 3 1 1
Chromosome 18 3 5 89 2 1`
Table IV. Mean distribution of signals in samples characterized by 46,XY complements evaluated by dual primed in situ labeling Percentage of cells demonstrating signal number No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
3 91 5 1 0
2 91 3 2 1
In recent years fluorescent in situ hybridization has been used to obtain rapid cytogenetic results from prenatal diagnostic specimens. Klinger et al.3 analyzed 526 amniotic fluid samples for chromosomes 13, 18, 21, X, and Y by fluorescent in situ hybridization; numeric abnormalities involving these chromosomes account for the majority of fetal chromosome abnormalities detected by CVS and amniocentesis.10 In this study fluorescent in situ hybridization correctly predicted specific chromosome complements in all samples. More recently, Divane et al.4 demonstrated successful application of a simultaneous five-color (probe) fluorescent in situ hybridization technique to assess chromosomes 13, 18, 21, X, and Y in 30 amniotic fluid samples. However, fluorescent in situ hybridization is limited by
No. of signals
Chromosome X
Chromosome Y
0 1 2 3 ≥4
6 9 85 0 0
91 9 0 0 0
Chromosome 21 8 4 3 85 0
several technical factors. First, and most important, analyses do not provide complete karyotypic information but rather assess specific loci on specific chromosomes. Although the majority of fetal chromosome abnormalities involve nondisjunction of chromosomes 21, 18, 13, X, and Y,10 fluorescent in situ hybridization does not permit the detection of most structural and some numerical chromosome abnormalities. Second, the lack of specificity of some repeat probes (e.g., chromosomes 13 and 21) and uncoupling between the hybridization signal and chromosome count lead to difficulties in interpreting signal counts,9 although the use of different telomeric probes specific for chromosomes 13 and 21 can bypass the diagnostic problems associated with lack of centromeric probe specificity. Third, although fluorescent in situ hybridization does significantly decrease the time between sample procurement and result, the process requires considerable laboratory and interpretative resources, especially in cases where specific α-satellite probes are not applicable for the needed diagnosis.6 Primed in situ labeling is based on the reannealing of specific oligonucleotide primers to the target sequences in situ and subsequent extension of the primer sequence by Taq DNA polymerase in the presence of labeled nucleotides. The high efficiency of this technique for discriminating α-satellite DNA sequences has been well documented and has been applied for the analysis of somatic tissues and sperm.6-8 The advantages of primed in situ la-
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beling over conventional fluorescent in situ hybridization analysis include increased sensitivity and specificity, especially in the detection of aneuploidies involving chromosomes 13 and 21.6, 9, 11, 12 Primed in situ labeling uses the high specificity between primers and their genomic targets; it is thus possible to design specific primers for individual centromere detection of chromosomes 13 and 21 without cross-hybridization.13 Second, primed in situ labeling requires a far lesser time from sample procurement to diagnostic result. With semiautomated protocols, reactions can be completed in less than an hour after slide preparation, with even less time required when probes of moderate to high copy sequences, such as α-satellite DNA, are used. Another benefit of the shorter reaction time of primed in situ labeling over fluorescent in situ hybridization is that the shortened reaction time reduces the background hybridization, thereby making the signal less ambiguous and improving sensitivity and specificity of the analysis.9 This shortened reaction time represents potential diagnostic advantages for certain critically ill newborns, the evaluation of selected pregnancies characterized by structural or growth abnormalities, and allaying some patient anxiety by providing rapid and preliminary, albeit incomplete, information concerning common fetal aneuploidies. Our current study, to the best of our knowledge, is the first series limited to the evaluation of prenatal diagnostic specimens by primed in situ labeling. Our results from this blinded preliminary feasibility study find primed in situ labeling to correctly predict selected chromosome complements in all specimens. This technique, like fluorescent in situ hybridization, is limited to the detection of specific aneuploidies and is likewise limited in detecting structural and less common numeric chromosome abnormalities. In addition, the, sensitivity and specificity of primed in situ labeling for chorionic villi and amniotic fluid analyses remain to be determined. However, we believe that this technique will be found to be a rapid and effective adjunctive diagnostic method for specific clinical situations. Indeed, having both primed in situ labeling and fluorescent in situ hybridization available should improve our ability to detect numeric and structural chromosome abnormalities in a far shorter period of time than is currently possible,14 although these techniques should not now be used in lieu of conventional cytogenetics for prenatal diagnostic cases. Accordingly, we believe that primed in situ labeling deserves consideration as an adjunctive cytogenetic analysis applicable for obtaining rapid, but limited, preliminary results from selected prenatal diagnostic specimens. REFERENCES
1. Phillips OP, Tharapel AT, Lerner JL, Park VM, Wachtel SS, Shulman LP. Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling. Am J Obstet Gynecol 1996;174:850-5.
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2. Shulman LP, Elias S, Phillips OP, Grevengood C, Dungan JS, Simpson JL. Amniocentesis performed at 14 weeks gestation or earlier: comparison with first-trimester transabdominal chorionic villus sampling. Obstet Gynecol 1994;83:543-8. 3. Klinger K, Landes G, Shook D, Harvey R, Lopez L, Locke P, 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. 4. Divane A, Carter NP, Spathas DH, Ferguson-Smith MA. Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization. Prenat Diagn 1994;14:1061-9. 5. Koch J, Kolvraa S, Petersen K, Gregersen N, Bolund L. Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ. Chromosome 1989;98:259-65. 6. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. Use of the primed in situ labeling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum Genet 1995;95:12-7. 7. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. PRINS as a method for rapid chromosomal labeling on human spermatozoa. Mol Reprod Dev 1995;40:333-7. 8. Velagaleti GVN, Tharapel SA, Martens PR, Tharapel AT. Rapid identification marker chromosomes using primed in situ hybridization (PRINS). Am J Med Genet 1997;71:130-3. 9. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. Selection of chromosome-specific primers and their use in simple and double PRINS techniques for rapid in situ identification of human chromosomes. Cytogenet Cell Genet 1995;70:138-42. 10. Hook EB. The impact of aneuploidy upon public health: mortality and morbidity associated with human chromosome abnormalities. In: Dellarco VL, Voytek PE, Hollaender A, editors. Aneuploidy etiology and mechanisms. New York: Plenum Press; 1985. p. 7-33. 11. Gosden J, Lawson D. Rapid chromosome identification by oligonucleotide-primed in situ DNA synthesis (PRINS). Hum Mol Genet 1994;3:931-6. 12. Gosden J, Lawson D. Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ. Cytogenet Cell Genet 1995;68:57-60. 13. Charlieu JP, Murgue B, Laurent A-M, Marçais B, Bellis M, Roizes G. Discrimination between alpha-satellite DNA sequences from chromosomes 13 and 21 by using polymerase chain reaction. Genomics 1992;14:515-6. 14. Hindkjaer J, Brandt CA, Koch J, Lund TB, Kolvraa S. Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting). Chromosome Res 1995;3:41-4.
Discussion DR. MARK JOHNSON, Detroit, Michigan. Although primed in situ labeling is a well-described and well-used technique in molecular cytogenetics, it has received little attention in the field of prenatal diagnosis. With pressures to report results more quickly in prenatal testing, use of molecular-based technologies such as fluorescent in situ hybridization for aneuploid screening has, if anything, been overused by individuals who are not familiar with the limitations of such techniques. In this study the authors run the risk of perpetuating such misconceptions to the less well informed when they say that “fluorescent in situ hbridization has recently been used to obtain rapid cytogenetic results from prenatal specimens.” This reflects the common misconception among lay practitioners that fluorescent in situ hybridization screening alone is sufficient for prenatal diagnosis,
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where in fact it really only represents a preliminary screen for the presence or absence of specific probed-for molecular sequences. Indeed, in our experience, we have found that many cytogenetically abnormal prenatal cases would have been missed with use of molecular fluorescent in situ hybridization techniques alone without confirmation by standard cultured karyotype. In a recent multicenter international collaborative report using data from 142,367 prenatal diagnostic specimens from eight centers in four countries, we found 3807 (2.67%) karyotypic abnormalities, of which 64.4% would have been detected with use of current fluorescent in situ hybridization probes for chromosomes 13, 18, 21, X, and Y, whereas 36.4% would have been missed without traditional karyotyping. Although screening for the major aneuploid syndromes was successful in our collaborative study, supporting the power of molecular techniques, more subtle structural aberrations and unbalanced rearrangements not involving the probed-for sequences are routinely missed. Unfortunately, more than half these missed rearrangements would have had a significant clinical impact for the fetus. It is therefore important to remember that molecular-based techniques may prove to be a useful adjunct in our diagnostic armamentarium but do not obviate the necessity of a full, banded karyotype. The authors emphasize the speed and increased sensitivity of primed in situ labeling compared with standard fluorescent in situ hybridization techniques. No one questions the capacity of primed in situ labeling to provide rapid and reliable results; however, the authors fall a bit short in providing a compelling argument for better sensitivity and specificity on the basis of the data presented in their study. One intriguing issue is that they are highly critical of fluorescent in situ hybridization because of the use of probes against highly repetitive sequences of chromosome-specific centromeric α-satellite DNA and the reported problems in the past of cross-hybridization, particularly between chromosomes 13 and 21. However, this valid concern of several years ago has been rectified, and for the last 2 years laboratories have routinely used probes for chromosome-specific telomeric sequences for chromosomes in which cross-hybridization was a problem. Interestingly, in their Methods, the investigators describe the use of primed in situ labeling primers directed against the same pericentric α-satellite regions they had just described to be problematic in fluorescent in situ hybridization. I would therefore ask the investigators for additional information based on their data to support their conclusion that primed in situ labeling offers better sensitivity and specificity over fluorescent in situ hybridization on the basis of their data. The investigators are part of a large, well-established and well-known, reputable prenatal diagnosis program. In the current study they present their apparent early experiences in establishing the primed in situ labeling technique in prenatal specimens. The small sample size of 35 CVSs and 6 amniocenteses represents more a pilot study
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than a conclusive report validating translation of a technique to a new clinical application. I was therefore hoping that investigators could update us and provide us with more substantial numbers to support the use of this technique on the basis of their continuing accumulation of prenatal cases in their laboratory, in particular with use of multiprimed in situ labeling. At Wayne State we are also looking at this exciting new technique but are not quite ready to replace our current use of direct and standard fluorescent in situ hybridization for aneuploid screening. One obvious technical concern in molecular techniques is successful probe hybridization and signal recognition. In the current study the investigators were successful in achieving hybridization and polymerization resulting in clear signal recognition on analysis for each of their primers. I would ask the authors if in their developing experience with this technique have their results continued to improve to the level or beyond that achieved with fluorescent in situ hybridization, supporting their position that the primed in situ labeling technique is comparable or better than fluorescent in situ hybridization approaches? In conclusion, I would repeat my concerns that practitioners understand the limitations of such techniques and that rapid molecular approaches such as primed in situ labeling and fluorescent in situ hybridization do not obviate the need for a complete, banded karyotype after CVS or amniocentesis. DR. PAUL TOMICH, Maywood, Illinois. The authors are part of a large, well-respected prenatal diagnosis program and have made important contributions in the field of prenatal genetics. Hence this presentation should be appreciated as a potentially important feasibility study concerning the role of primed in situ labeling in prenatal diagnosis. Primed in situ DNA synthesis since its introduction in 1989 by Koch has proved to be valuable for mapping and investigating repeated sequences in chromosomes and is thought to have some advantages over fluorescent in situ hybridization. It has been used in the detection of aneuploidies in somatic cells and in spermatozoa. Its role as a prenatal diagnostic procedure has not yet been established. The authors have reported their initial experience in using primed in situ labeling in 41 prenatal diagnosis specimens, 35 from CVS and 6 from amniotic fluid specimens. The indications for the CVS were (1) 32 women with advanced maternal age, (2) 2 women with previous children with chromosomal abnormalities, and (3) 1 woman at increased risk for cystic fibrosis. The amniotic fluid specimens were obtained because of abnormal screening testing results in 4 cases and advanced maternal age in 2 cases. Single primed in situ labeling was performed on 35 specimens and dual primed in situ labeling was performed on 2 amniotic fluid specimens and 3 CVS specimens. The conclusions are as follows. Primed in situ labeling can be rapidly done; same day service is possible. The au-
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thors claim that in their study there was an 86% to 90% success rate. The technique is as good as or better than fluorescent in situ hybridization and offers the advantage of a more rapid test, with results available more quickly. The authors claim that it offers superior sensitivity and specificity compared with fluorescent in situ hybridization. What are the problems with this study? The first has to do with the numbers. Thirty-five CVS specimens and 6 amniotic fluid specimens are an insufficient number on which to support any meaningful conclusions. I would think that at least 50 to 100 specimens from both CVS and amniocenteses would be necessary before reporting on the “reliability” of a technique. Next, only one case was probed for chromosome 21, which was already known to be from a specimen for an aneuploidy with trisomy 21. Again, the authors need to use primed in situ labeling for all major aneuploidies (chromosomes 21, 18, 13, X, and Y) in larger numbers before supporting their thesis that this technique is as good as or better than fluorescent in situ hybridization. The authors report a success rate of 86% to 90% with primed in situ labeling; this does not surpass the 90% to 95% rate commonly achieved by fluorescent in situ hybridization. One other issue not mentioned in the study is the issue of cost: perhaps Dr. Shulman could provide data on the cost of fluorescent in situ hybridization versus primed in situ labeling at the University of Tennessee or from national data that he may be aware of. I am not a geneticist. However, I am a perinatologist with an interest in prenatal diagnosis and I treat and counsel patients when they have either been found to have an anomaly on ultrasonography or have previously had a chromosomally abnormal child, are older than 35 years, or have abnormal triple testing. With regard to use of primed in situ labeling in CVS specimens, I do not see that the current practice of doing full studies should be redesigned to do this technique as well: rapid turnaround time is not really an issue. For those situations where amniotic fluid specimens are to be used, what is the clinician to do, make clinical decisions on the results returned in 1 day or await full studies before counseling or treating the patient? Whether we are talking about fluorescent in situ hybridization or primed in situ labeling in the prenatal diagnosis arena, we need to remember that such screening does not obviate the need for traditional karyotyping in such specimens. At the present time, faced with the dilemma of whether there may be a chromosomal abnormality and facing the need to counsel parents concerning the results of such studies, I will await final karyotype studies (at our cytogenetic laboratory fluorescent in situ hybridization through an outside laboratory is back in 3 to 4 days and full chromosomal studies in 7 days), fully appreciating that the “wait” is nerve wracking and anxiety producing for parents, but 100% reliable information is absolutely imperative in such circumstances. This study should be looked on as no more than a preliminary feasibility study. The authors are encouraged to
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pursue additional studies on this topic to identify what role primed in situ labeling may have in the field of prenatal diagnosis. It is one thing to present data as an initial experience and quite another for anyone to make the leap to conclude after this presentation that the role of this technique in prenatal diagnosis has now been demonstrated or to suggest that either fluorescent in situ hybridization or primed in situ labeling should replace traditional chromosomal analysis. I hope that no one leaving here goes to their hospital or cytogenetics laboratory and says, “I want a primed in situ labeling, get me a fluorescent in situ hybridization” instead of “How quickly can you get the chromeme studies done and back to me?” DR. NORMAN GINSBURG, Chicago, Illinois. The fluorescent in situ hybridization and primed in situ labeling techniques do have a role in confirming or helping to speed along the diagnosis. There are often times when amniocentesis takes a long time. We get a few cells that are confirmatory of an abnormality, and this is a technique that can analyze many more cells at a more rapid rate. The same goes for CVS as well, particularly when people are coming to a time where availability of a firsttrimester termination is rapidly coming to an end. DR. SHULMAN (Closing). Let me add that in no way should anyone leave this room today asking for anything but a complete cytogenetic analysis. Not only is it prudent perinatal medicine, we’re also dealing with subjects of standards of care; in fact, I don’t think any of us in this room would base any clinical management on anything other than a complete karyotypic analysis of the fetus. Let me also say that I also did not come to bury or to ruin fluorescent in situ hybridization but rather to praise it and improve it; in fact, this is indeed an initial feasibility study. If you did ask your laboratory to provide you with a primed in situ labeling analysis, they would probably say, “A what?,” primarily because the machinery that is required for this is not currently available in the United States; it’s been our work with the Pelastor group in Belgium that has allowed us to have the machine, the reagents, not so much the primers, that has allowed us to do this initial feasibility study. As far as the telomeric sequences, Dr. Johnson is absolutely right. That is being used more and more to differentiate chromosomes 13 and 21. There were two cases in which we’ve used nontelomeric, noncentromeric specific sequences to get very nice preparations looking at either chromosome 13, chromosome 21, and chromosome X; in fact, the real power of the primed in situ labeling reaction is that we can take very small, unique sequences that are not found anywhere else within the genome and have a very powerful in situ hybridization technique that we can use truly as an injunctive diagnostic method. Dr. Johnson is correct. Our numbers for this particular preliminary study are small and actually represent sensitivities that are at least no better than those of fluorescent in situ hybridization.
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We have now done well over 150 cases. We are now finding, in fact, sensitivity well in the range of between 93% and 97%; in fact, we now have gone one step further. Instead of relying on 70% of cells to call a particular complement, we are now using 80% showing a particular complement before we will call it as that complement. We have stopped doing further studies on prenatal tissue for the time being; we’re stopping at about 150, primarily because our laboratory is now using primed in situ labeling for gene sequencing. Hopefully next year we’ll have some interesting data to present with that. I don’t have a clue what the cost of primed in situ labeling is. I can tell you that the machines that are re-
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quired cost somewhere in the range of $75,000 to $90,000 and in a sense it’s a very fancy but modified PCR machine that allows us to anneal, reanneal, and add the tack (phonetic) polymerase. I would not alter my management on the basis of a primed in situ labeling. I would provide information on a full karyotypic analysis; I believe within the next year or so when these machines are available may, in fact, represent an improvement in our ability to provide in situ hybridization preliminary information for those particular patients. Hopefully, as we learn and are able to apply this technique more, in fact, we will use it for more complete structural and numeric abnormality assessments for fetal and even for adult karyotypes.