American Journal of Medical Genetics M394-397 (1992)
UNCULTURED BLOOD SMEARS HYBRIDIZED WITH ALPHA SATELLITE PROBES TO DIAGNOSE 45,XIN SPONTANEOUSLY ABORTED FETUSES C. von Kap-hen, Thaddeus E. Kelly and Wendy L. Golden Department of Pediatrics, Division of Medical Genetics, University of Virginia School of Medicine, Charlottesville'
Traditional cytogenetic analysis of material from spontaneous abortions often fails due to non-viability of the tissues in cell culture. The 45,X constitution is common in early pregnancy loss. Fluorescent in situ hybridization (FISH) was used to diagnose 45,X in uncultured blood smears obtained from 2 cases of spontaneous pregnancy loss with fetal hydrops. The ability of chromosome specific alpha satellite probes to detect aneuploidy in uncultured cells should greatly increase the successful detection of chromosomal abnormalities in early abortuses.
Fetal blood smears contain a large number of nucleated cells. Small amounts of peripheral blood can easily be obtained from the placenta or the umbilical vein. Aneuploidies can be detected in both mitotic cells and interphase nuclei using fluorescent in situ hybridization (FISH) (Anastasi et al., 1990; Burns et al., 1985). This study explored whether fetal blood smears could be used as a source of non-mitotic cells for determination of sex chromosome aneuploidy by FISH. The results obtained demonstrate the usefulness of blood smears in the rapid detection of fetal aneuploidy.
KEY W0RDS:Alpha satellite probes, FISH, peripheral blood smears, Ullrich-Turner syndrome, hydrops fetalis.
MATERIALS AND METHODS Slide Making and In Situ Hybridization
INTRODUCTION Chromosome abnormalities are detected in approximately one half of all clinical miscarriages (Boue and Boue, 1973; Warburton et al., 1979). Standard cytogenetic techniques require mitotic cells for chromosome analysis; however, cells capable of growth in culture are not always available due to bacterial contamination and non-viability of aborted tissue. Received for publication May 11,1992; revision received August
3, 1992.
'Address reprint requests to C. von Kap-hem, Division of Medical Genetics, University Hospital, Box 386, Charlottesville VA 22908. 0 1992 Wiley-Liss, Inc.
Blood smears were made on Fisher Biotech ProbeOn Plus slides which were air dried and stained for 4 min. in Wright's stain (BDH). The slides were destained in 3 changes of fix (3:l Methanol: glacial acetic acid) prior to hybridization. Fibroblasts derived from diaphragm (Case 1) were cultured on coverslips and harvested in situ by the usual techniques. Except for Wright's staining, fibroblasts were treated to the same conditions as smears. The slides were pretreated with RNase, digested with pepsin A (Sigma,
45,X Detected With Hybridized Probes
20 p g / m l in 0.01 N HC1) for 10 minutes and dehydrated in an ethanol series (70, 85, 100 vol. %). FISH was performed according to the protocol of Pinkel et al. (1986). Slides were denatured in 70% formamide in 2XSSC (pH7.0) at 70°C for 2 minutes and dehydrated in a cold ethanol series. The X or Y biotinylated alpha satellite probes ( 1 . 5 , ~1; Oncor, Gaithersburg, MD) were added to 30 u , 1s of a hybridization mixture composed of 55 % formamide, 10% dextran sulfate and l,ug(,ul herring tests DNA in 2XSSC. The mix was denatured at 70°C for 5 minutes and applied to each slide. The slides were incubated overnight at 37°C. Post hybridization washing was carried out according to the Oncor chromosome in situ kit instructions. Cytochemical Detection and Scoring Fluorescein-labeledavidin (VectorLaboratories, Burlingame, CA) was bound to the biotin-labeled hybridized probe. The signal was enhanced by treatment with biotinylated goat anti-avidin (Vector), followed by a second layer of fluoresceinated avidin. The slides were coverslipped with antifade (Johnson et.al., 1981) containing l,ug/ml of DAPI and l/ug/ml of propidium iodide. Signals were scored by exciting fluorescein (Olympus B-cube). Photography was done using Kodak Ektachrome P800/1600 color slide film, developed to push 2 for high contrast. Intact nuclei were located under UV fluorescence. The number of discrete signals was then determined by exciting fluorescein on a background of propidium iodide. Randomly selected blood smears from both males and females (controls) were coded and included with the experimental slides to ensure against scoring bias. Chromosome analysis was carried out by a separate observer. CLINICAL REPORTS Case 1: A 27-year-old G3P2 woman presented at 20 weeks gestation with a complaint of loss of fetal movement. Ultrasound disclosed fetal death and massive hydrops fetalis. At delivery the female fetus was noted to be massively edematous, but without specific external malformations. There was a moderately severe degree of maceration of fetal tis-
395
sues. Several drops of blood were obtained from a placental vein; at autopsy a piece of diaphragm was collected for fibroblast culture. The autopsy findings were consistent with the diagnosis of Turner syndrome. Case 2: A 25-year-old G2P1 woman presented at 19 weeks gestation by post-LMP-dating because of loss of previously noted fetal movement. An ultrasound study documented massive fetal hydrops with cystic hygroma and utero fetal death. Following delivery, the female fetus was noted to be macerated, edematous, but without external malformations. Several drops of blood were collected from an umbilical vein. An autopsy was not authorized. RESULTS The alpha satellite probes specific for the X and Y chromosomes were selected for in situ hybridization on the basis of the clinical findings suggestive of Ullrich-Turner syndrome. These were used on uncultured blood smears, as well as cultured fibroblasts. Both probes demonstrated a high efficiency of hybridization (approximately 98 % of interphase cells contained one or more signals). Case 1: The blood smear gave a single signal for the X chromosome. Cells were not available for a second smear so absence of the Y was demonstrated in an outgrowth of fibroblasts from a diaphragmatic specimen (Table I, Fig. lc,d). The fibroblast culture established from Case 1 was shown by chromosome analysis to have a 45,Xkaryotype with no evidence of mosaicism. Case 2: The X probe yielded a single signal, but no signal was found with the Y probe (Table I, Fig le, f). A 45,X chromosome constitution was confirmed by chromosome analysis of a blood culture. Smears from a control female and control male gave the expected number of signals (Table I, Fig. la,b). DISCUSSION Aneuploidies can be clearly detected in fetal blood smears by FISH. This technique reduced the
396
von Kap hem et al.
risk of failure to obtain a diagnosis in 2 miscarriages by successfully employing non-mitotic cells from potentially non-viable fetal tissue. Hence, an explanation was provided the coupIes for their pregnancy loss. In Case 1, however, cultured fibroblasts were necessary to make this study fully informative since only the X probe was applied to a
Blood smears are simple to prepare and require only a small volume of blood, making this technique potentially useful as a screening test for sex and autosomd aneuploidies. This spscific protocol produces discrete, brilliant signals with a minimum of background, thereby lending itself to automation far more readily than conventional chromosome analysis.
smear.
Figure 1. In situ hybridization using biotinylated alpha satellite probes to the X chromosome (a, c, e) and to the Y chromosome (b, d, f). a: blood smear from control female; note the 2 hybridization signals. b: blood smear from control male; note only 1 hybridization signal. c;d: samples obtained from Case 1. Note hybridization in the blood smear with the X probe (c) and the absence of signal in the fibroblast with the Y probe (d). e;f: blood smears from Case 2. Note hybridization with the X probe (e) and the absence of signal with the Y probe (0.
45,X Detected With Hybridized Probes
Currently, this technique is most useful when a clinical diagnosis suggests the chromosome specific probes to be used. Whenever possible a complete karyotypic analysis should also be attempted to rule out deletions and rearrangements which the centromeric probes cannot detect.
397
ACKNOWLEDGMENTS We would like to thank Dr. Mark Love11 for his suggestions and Dr. E. J. Ampleford for Critical reading of the manuscript. We thank Linda Davis for typing the manuscript.
TABLE I: Cell8 Oblcrvcd with Fluorcrcnce after Hybridiution with Biotinylatcd Alpha Satellite Probes to h e X and Y Chromosomes.
% of Nuclei with Spot Number
TISSUE TYPE
DNA PROBE
NO. OF CELLS
0
1
2
3
1.7
0.40
0
0
CASE 1 Blood Smear
X
711
1.3
96.6
Fibroblasts
Y
100
100
0
Con~rold Fibroblasts
Y
100
0
100
0
0
Blood Smear
X
300
1 .o
95.I
3.3
0
Blood Smear
Y
100
100
0
0
0
Control d Blood Smear
Y
205
2.0
95.0
3 .O
0
X
310
0.5
5.5
94.0
0
CASE 2
Control P Blood Smear
REFERENCES Anastasi J, Le Beau MM, Vardiman JW,Westbrook CA (1990): Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe. Am J Pathol 136: 131-139. Boue J, Boue A (1973): Chromosome analysis of two consecutive abortuses in each of 43 women. Humangenetik 19:275-280. Bums J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee J O'D (1985): Sensitive system for visualizing biotinylated DNA probes hybridized in situ: Rapid sex determination of intact cells. J Clin Pathol 38: 1085-1092.
Johnson GD, de C Nogueira Araujo GM (1981): A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43: 349-350. W (1986): Cytogenetic Pinkel D, Straume T, Gray J analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934-2938. Warburton D, Susser M, Stein Z, mine J (1979): Genetic and epidemiologic investigation of spontaneous abortion: relevance to clinical practice. In Golbus MS, Hall BD (eds.) "Diagnostic Approaches to the Malformed Fetus, Abortus, Stillborn and Deceased Newborn". BD:OAS (5A): 127-136.
UNCULTURED BLOOD SMEARS HYBRIDIZED WITH ALPHA SATELLITE PROBES TO DIAGNOSE 45,XIN SPONTANEOUSLY ABORTED FETUSES C. von Kap-hen, Thaddeus E. Kelly and Wendy L. Golden Department of Pediatrics, Division of Medical Genetics, University of Virginia School of Medicine, Charlottesville'
Traditional cytogenetic analysis of material from spontaneous abortions often fails due to non-viability of the tissues in cell culture. The 45,X constitution is common in early pregnancy loss. Fluorescent in situ hybridization (FISH) was used to diagnose 45,X in uncultured blood smears obtained from 2 cases of spontaneous pregnancy loss with fetal hydrops. The ability of chromosome specific alpha satellite probes to detect aneuploidy in uncultured cells should greatly increase the successful detection of chromosomal abnormalities in early abortuses.
Fetal blood smears contain a large number of nucleated cells. Small amounts of peripheral blood can easily be obtained from the placenta or the umbilical vein. Aneuploidies can be detected in both mitotic cells and interphase nuclei using fluorescent in situ hybridization (FISH) (Anastasi et al., 1990; Burns et al., 1985). This study explored whether fetal blood smears could be used as a source of non-mitotic cells for determination of sex chromosome aneuploidy by FISH. The results obtained demonstrate the usefulness of blood smears in the rapid detection of fetal aneuploidy.
KEY W0RDS:Alpha satellite probes, FISH, peripheral blood smears, Ullrich-Turner syndrome, hydrops fetalis.
MATERIALS AND METHODS Slide Making and In Situ Hybridization
INTRODUCTION Chromosome abnormalities are detected in approximately one half of all clinical miscarriages (Boue and Boue, 1973; Warburton et al., 1979). Standard cytogenetic techniques require mitotic cells for chromosome analysis; however, cells capable of growth in culture are not always available due to bacterial contamination and non-viability of aborted tissue. Received for publication May 11,1992; revision received August
3, 1992.
'Address reprint requests to C. von Kap-hem, Division of Medical Genetics, University Hospital, Box 386, Charlottesville VA 22908. 0 1992 Wiley-Liss, Inc.
Blood smears were made on Fisher Biotech ProbeOn Plus slides which were air dried and stained for 4 min. in Wright's stain (BDH). The slides were destained in 3 changes of fix (3:l Methanol: glacial acetic acid) prior to hybridization. Fibroblasts derived from diaphragm (Case 1) were cultured on coverslips and harvested in situ by the usual techniques. Except for Wright's staining, fibroblasts were treated to the same conditions as smears. The slides were pretreated with RNase, digested with pepsin A (Sigma,
45,X Detected With Hybridized Probes
20 p g / m l in 0.01 N HC1) for 10 minutes and dehydrated in an ethanol series (70, 85, 100 vol. %). FISH was performed according to the protocol of Pinkel et al. (1986). Slides were denatured in 70% formamide in 2XSSC (pH7.0) at 70°C for 2 minutes and dehydrated in a cold ethanol series. The X or Y biotinylated alpha satellite probes ( 1 . 5 , ~1; Oncor, Gaithersburg, MD) were added to 30 u , 1s of a hybridization mixture composed of 55 % formamide, 10% dextran sulfate and l,ug(,ul herring tests DNA in 2XSSC. The mix was denatured at 70°C for 5 minutes and applied to each slide. The slides were incubated overnight at 37°C. Post hybridization washing was carried out according to the Oncor chromosome in situ kit instructions. Cytochemical Detection and Scoring Fluorescein-labeledavidin (VectorLaboratories, Burlingame, CA) was bound to the biotin-labeled hybridized probe. The signal was enhanced by treatment with biotinylated goat anti-avidin (Vector), followed by a second layer of fluoresceinated avidin. The slides were coverslipped with antifade (Johnson et.al., 1981) containing l,ug/ml of DAPI and l/ug/ml of propidium iodide. Signals were scored by exciting fluorescein (Olympus B-cube). Photography was done using Kodak Ektachrome P800/1600 color slide film, developed to push 2 for high contrast. Intact nuclei were located under UV fluorescence. The number of discrete signals was then determined by exciting fluorescein on a background of propidium iodide. Randomly selected blood smears from both males and females (controls) were coded and included with the experimental slides to ensure against scoring bias. Chromosome analysis was carried out by a separate observer. CLINICAL REPORTS Case 1: A 27-year-old G3P2 woman presented at 20 weeks gestation with a complaint of loss of fetal movement. Ultrasound disclosed fetal death and massive hydrops fetalis. At delivery the female fetus was noted to be massively edematous, but without specific external malformations. There was a moderately severe degree of maceration of fetal tis-
395
sues. Several drops of blood were obtained from a placental vein; at autopsy a piece of diaphragm was collected for fibroblast culture. The autopsy findings were consistent with the diagnosis of Turner syndrome. Case 2: A 25-year-old G2P1 woman presented at 19 weeks gestation by post-LMP-dating because of loss of previously noted fetal movement. An ultrasound study documented massive fetal hydrops with cystic hygroma and utero fetal death. Following delivery, the female fetus was noted to be macerated, edematous, but without external malformations. Several drops of blood were collected from an umbilical vein. An autopsy was not authorized. RESULTS The alpha satellite probes specific for the X and Y chromosomes were selected for in situ hybridization on the basis of the clinical findings suggestive of Ullrich-Turner syndrome. These were used on uncultured blood smears, as well as cultured fibroblasts. Both probes demonstrated a high efficiency of hybridization (approximately 98 % of interphase cells contained one or more signals). Case 1: The blood smear gave a single signal for the X chromosome. Cells were not available for a second smear so absence of the Y was demonstrated in an outgrowth of fibroblasts from a diaphragmatic specimen (Table I, Fig. lc,d). The fibroblast culture established from Case 1 was shown by chromosome analysis to have a 45,Xkaryotype with no evidence of mosaicism. Case 2: The X probe yielded a single signal, but no signal was found with the Y probe (Table I, Fig le, f). A 45,X chromosome constitution was confirmed by chromosome analysis of a blood culture. Smears from a control female and control male gave the expected number of signals (Table I, Fig. la,b). DISCUSSION Aneuploidies can be clearly detected in fetal blood smears by FISH. This technique reduced the
396
von Kap hem et al.
risk of failure to obtain a diagnosis in 2 miscarriages by successfully employing non-mitotic cells from potentially non-viable fetal tissue. Hence, an explanation was provided the coupIes for their pregnancy loss. In Case 1, however, cultured fibroblasts were necessary to make this study fully informative since only the X probe was applied to a
Blood smears are simple to prepare and require only a small volume of blood, making this technique potentially useful as a screening test for sex and autosomd aneuploidies. This spscific protocol produces discrete, brilliant signals with a minimum of background, thereby lending itself to automation far more readily than conventional chromosome analysis.
smear.
Figure 1. In situ hybridization using biotinylated alpha satellite probes to the X chromosome (a, c, e) and to the Y chromosome (b, d, f). a: blood smear from control female; note the 2 hybridization signals. b: blood smear from control male; note only 1 hybridization signal. c;d: samples obtained from Case 1. Note hybridization in the blood smear with the X probe (c) and the absence of signal in the fibroblast with the Y probe (d). e;f: blood smears from Case 2. Note hybridization with the X probe (e) and the absence of signal with the Y probe (0.
45,X Detected With Hybridized Probes
Currently, this technique is most useful when a clinical diagnosis suggests the chromosome specific probes to be used. Whenever possible a complete karyotypic analysis should also be attempted to rule out deletions and rearrangements which the centromeric probes cannot detect.
397
ACKNOWLEDGMENTS We would like to thank Dr. Mark Love11 for his suggestions and Dr. E. J. Ampleford for Critical reading of the manuscript. We thank Linda Davis for typing the manuscript.
TABLE I: Cell8 Oblcrvcd with Fluorcrcnce after Hybridiution with Biotinylatcd Alpha Satellite Probes to h e X and Y Chromosomes.
% of Nuclei with Spot Number
TISSUE TYPE
DNA PROBE
NO. OF CELLS
0
1
2
3
1.7
0.40
0
0
CASE 1 Blood Smear
X
711
1.3
96.6
Fibroblasts
Y
100
100
0
Con~rold Fibroblasts
Y
100
0
100
0
0
Blood Smear
X
300
1 .o
95.I
3.3
0
Blood Smear
Y
100
100
0
0
0
Control d Blood Smear
Y
205
2.0
95.0
3 .O
0
X
310
0.5
5.5
94.0
0
CASE 2
Control P Blood Smear
REFERENCES Anastasi J, Le Beau MM, Vardiman JW,Westbrook CA (1990): Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe. Am J Pathol 136: 131-139. Boue J, Boue A (1973): Chromosome analysis of two consecutive abortuses in each of 43 women. Humangenetik 19:275-280. Bums J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee J O'D (1985): Sensitive system for visualizing biotinylated DNA probes hybridized in situ: Rapid sex determination of intact cells. J Clin Pathol 38: 1085-1092.
Johnson GD, de C Nogueira Araujo GM (1981): A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43: 349-350. W (1986): Cytogenetic Pinkel D, Straume T, Gray J analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934-2938. Warburton D, Susser M, Stein Z, mine J (1979): Genetic and epidemiologic investigation of spontaneous abortion: relevance to clinical practice. In Golbus MS, Hall BD (eds.) "Diagnostic Approaches to the Malformed Fetus, Abortus, Stillborn and Deceased Newborn". BD:OAS (5A): 127-136.
