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Mosaic and hypermodal cells in amniotic fluid cell cultures
Mosaic and hypermodal cells in amniotic fluid cell cultures CHARLES ELLEN
R. KING, MAGENIS,
GERALD SUSAN Kansas
M.D.
PRESCOTT.
CATHERINE
M.D. D.M.D.
OLSON, BENNETT,
City,
Kansas,
B.S. B.S.
and Portland,
Oregon
Karyotypic results from a series of 975 consecutive amniotic fluid specimens studied for prenatal diagnosis are reported. In only 32% of specimens were all cells analyzed eyploid. Two tliirds (63%) of the specimens studied were hypomodal in at least one cell; 25% of the studies were hypomodal in three or more cells. mcontrast, only 3.5% of the studies were hypermodal in one or more Celli. Most frequently an additional chromosome 2 was present -in these hypermodai cells. Cells with other than a diploid chromosome constitution may be mistakenly identified as evidence of mosaic& This is especially likely when’ the involved chromosome isone associated with a clinically defined syndrome. (AM. J. OBSTET. GYNECOL. 136:383, 1980.)
analysis of amniotic fluid cells for antenatal diagnosis is a well-established procedure. The techniques used are complex and require careful attention to detail to avoid diagnostic errors. A review of our experience provides information which may aid when mosaicism is questioned with amniotic fluid cell cultures. This ‘is especially important since both falsepositive and false-negative examples of mosaicism have been reported in amniotic fluid cell cultures.” ’
CYTOGENETIC
Material and methods The patients in this report represent a consecutive series of 975 patients who have undergone antenatal genetic diagnosis at the University of Oregon Health Sciences Center. The essential aspects of this process have been reviewed previously,3, 4 but details important to the present discussion will be provided. Following sonograhy at 14 to 15 weeks’ gestation, amniocentesis is performed with sterile technique. Approximately 15 to 20 cc of amniotic fluid are removed. The specimen is From the Department of Obstetrics and Gynecology, University of Kansas Medical Center, and the Division of Medical Genetics, University of Oregon Health Sciences center. Receizjed for publication Accepted April
Ortober
I I, 1978.
23, 1979.
R+nint requests: Charles R. King, M.D., Department of Ob$etrics and Gynecology, U+versity of Kansas Medical Center, 39th U Rainbow, Kansas City, Kansas 66103. OOOZ-9378/80/030383+03$00.30/0
@ 1980 The C. V. Mosby
Co.
centrifuged at 600 rpm for 10 minutes. Cells are resuspended in one sixth of the original amniotic fluid volume and placed in F-10 medium supplemented with 15% fetal calf serum, glutamine, and gentamycin. Specimens are grown in Falcon T-30 plastic Basks in a water-jacketed COz incubator. Cells are subcultured and harvested independently from each of the flasks with trypsin: Following hypotonic treatment with 0.075M KC1 in a dilute aqueous solution of fetal calf serum, the cells are fixed with acetic acid/methanol and slides are prepared. Cells are banded with either quinacrine mustard, trypsin-Wright’s, or both. Studies are generally completed within 20 days following the amniocentesis. A minimum of 20 cells are counted and at least three karyotypes are made from each specimen. Studies are extended if questionable results are obtained.
Result+ and comment The indications for study in these patients are quite similar to those in other published series.5, 6 Advanced maternal age was the most frequent indication for study. Other indications were history of a previous chromosome error, neural tube defect, or biochemical genetic disease. Likewise, the observed frequency of chromosome aberrations (2.2%, Table I) is in agreement with previously published studies. However, it appears that fewer sex chromosome aberrations may have been identified. This may simply be a reflection of 383
384
King et al.
February
Am. J. Obstet.
Table I. <:ht-otnosorne abnormalities in 975 consecutive amniotic Huid specimens
Table II. Distribttrion hypermodal c-ells
Table III. Amniotic hypermodal cells
of‘ hypotnodal
Huiti cultures
Cdl I + 1” +1 + I4 +X +21
+c: + I9
and
with two or more
Crll 2
~Xl 3
+12 +4 +D +c:
+ Marker* + Market + Marker
:it,2’% 01 the sludirs detnonstrateci at lcast otte Ii!WII. Seven amniotic: specimens were studied with two otmore hypertnocial cells (Table III). Specimen I had two c-ells each \cith an aclditional chromosome I?. On tollrnv-up this infant is normal. Pregnancies 2. 3 and 5 arc’ rtill in pt-ogress. Pregnancies 4 and 6 resulted in ttormal oftbprittg. Fluid f-t-om the seventh study had 1 cell Lvith an ,ttlditional chromosome 10. One of the rnarkct. (.hromosontes was ciefiniteiy art F ,group rhrotnosomc, but banding did ttot identify whrrhrr ;I t 9 or ‘20. I‘hc third cell contained an F size chrnmosomr hut tto Iurrticr clclitieation WiLS possible. In short. none ot rhcsc infants has been abnormal. but thrcxt prrgnancirs cotirinuc. I2dditional chromosomes in hypct-modal tells rqxc’scnr a nonrandom distribution (Titblc IV). StatisriA atiatvsis” of this tlistrihurion contirms that a tiotirat1dom change is prcst’nr. (:hromosonie L’ ~vas prvsctnt ci~nific~anrl~ more frequentI\ than cxpccrcci h\- chanct (p ( 0.05). .A similar finding was reported h) Peakman and associates.!’ \Vhy is chromosome 2 so t’rcquentl~ the additional ctirottiosotiie in rhese atieupioid crtls? Furthet- $tncl\ is ttccesaar) to ciefinitively answer this cluesCon. but ir ih possible that specific- grnrtic material which cntiantes tong-term in vitro gro\vlh is totinrl on this chromostmtc. If this is correct. then identification and prcferctttial activation of. this tnatcriai shottld promote in vitro growth of amniotic fluid cells. ‘Ilie tnajor concern with interpretation of’ culrurail~ produced atteupioid~~ is possible confusion with mosaicistn. Obviously, conseling and the decisions ot parents may he quite different. dependent on whether a mosaic fetus ot- cultural at~ruploici~ is present. WC have not &set-Ted an abnormal infant or fetus v-hen onlv one cell ~vas ancuploid, but this could occut.. since abnormal cmhr)onic- cells have been observed to grow slowly in vitro.” \Vhrn tnosaicism is identified. the abnormal cell line niay represent a minimal estimate of its ;ic-total in \ iv0 f rcquetic 1 Hoehn and colleaguesg” h-ave suggested ttiar tht technique ot amniotic lluid culture affects the mtmhet of anueploici cells. l‘hq suggested that cells cultured with the in situ coverslip technique demonstrated less anueploidv and fewer structural rearrangements. We have not made this cotnparison. since in situ culturing its not perfortned with these specimens. However, if the gain ot an additional chromosome is nonrandom (‘l‘able I\‘). it then becomes more difticult to explain this phenomenon as entirety the result 01 a chance occ cttyrencc’ due to ;I specific culture technique. I‘he final interpretation of prenatal cytogenetic studies must depend on accuracy and attention to detail. pertnodal
.vo.
+ Market
*Marker chromosomes were not specifically identifiable as To chromosome number, but may havr been identified bv group as nntrtl in the test. sample 5i/c. itt- it ma! indic-ate 3 subtle ciiffercnce bct\Veeti this population and others prc\ iottsly reported. Aneuploid\ as an artifact ot’culturc has been hrieH? cotnment~d r~I)on in prcviou4 reporr\,‘. ‘. ’ which get)craI1~ ham Ix~ti onI> single case reports. The absrract b\ Peakman and associates” has reporttxl the results ot’ an initial stud\ of this phenomenon. 11s cat1 be scett from .I‘able I I. Iwo thirds of all cultut-cs contain at least ottc attcuploitl cell and one ot ever)’ four cultures has three or mot-e aneuploid c-ells. Hypomoclal cells account for- tl~c. majority of these changes. Nowever.
1, 1980
Gyecol.
Volume 136
Mosaic
Number
3
Table
IV. Extra
1
2
3
chromosome
4
5
6
7
in hypermodal
8
9
IO
amniotic
11
cells
in amniotic
fluid
385
fluid cells
X .Y
x x
x
12
I3
14
When aneuploid cells are identified their frequency must be defined. Generally more cells must be studied. Ifaneuploidy is present in more than one in situ colony or more than one culture Bask. the case for mosaicism is much stronger. In some cases a repeat amniocentesis and study may be indicated. This will not completely “explain away” the observed aneuploidy but it reduces the likelihood of’ mosaicism. When the involved chromosome is associated with an observed chromosomal aneuploidy syndrome the possibility of mosaicism is more critical, although survival has been observed in an otherwise nonviable trisomy when mosaicism was present. Parents need to be advised of the possibility of
and hypermodal
15
16
17
x
x
18
19
20
x
x
x s x
21
22
X
x x x X X X X X x .Y
Y
Marker
mosaicism when apparent cultural aneuploidy is observed. Frequently mosaicism will not be present, but only when fully informed of this possibility will some parents elect to terminate a possibly affected pregnancy. Generally a professional judgment regarding the presence or absence of mosaicism must be made and discussed with the family. The counselor will frequently need to provide additional psychologic and professional support to these families.
Addendum Pregnancies 2,3, and 5, reported as in progress, have all ended with the birth of normal infants. Thus all reported pregnancies have produced normal infants.
REFERENCES
1. A&. P. Von Koskull, H.. and Ryznanen, M.: A case of 46,Xx/47,Xx +G mosaicism in amniotic fluid not confirmed in the fetus, Clin Genet. 13: 103, 1978. 2. Ladda, R., Hildebrandt, R., and Dobello, Y.: Sex chromosomal mosaicism undetected by prenatal study, J. Pediatr. 90:841. 1977. 3. Prescott, G. H., Pernoll, M. L., Hecht, F., and Nicholas, A.: A prenatal diagnosis clinic: an initial report, AM. J. OBSTET.
GYNECOL.
116:942,
1973.
4. Pernoll, M. L., Prescott, G. H.. Hecht, F., and Olson, C. L.: Prenatal diagnosis: Practice, pitfalls, and progress, Obstet. Gynecol. 44~773, 1974. 5. Epsetin, C. J., and Golbus, M. S.: Prenatal diagnosis of genetic diseases, Am. Sci. 65:703, 1977. 6. Galjaard, H.: European experience with prenatal diagnosis of congenital disease: A survey of 6121 cases, Cytogenet. Cell Genet. 16:453, 1976. 7. COX, B. M., Niewczas-Late, V., Riffell, M. I., and Hamer-
ton, J. L.: Chromosomal mosaicism in diagnostic amniotic fluid cell cultures. Pediatr. Res. 8:679, 1974. 8. Kohn, G., Mennuti, M. T., Kaback. M., Schwartz, R. M.. Chemke, J., Goldman, B., and Mellman. W. J.: Chromosomal mosaicism in amniotic cell culture. Isr. J. Med. Sci. 11:476,
1975.
9. Peakman, D. C., Moreton, M. D., Corn, B. J., and Robinson. A.: Chromosomal mosaicism in amniotic fluid cell cultures. Pediatr. Res. 12:455. 1978. 10. Slatter, M. J.: Comparative validity of the chi square and two modified chi square goodness of lit tests for small but equal expected frequencies, Biometrika 53:619, 1966. 11. Cure, S., Boue, J., and Boue, A.: Growth characteristics of human embryonic cell lines with chromosome anomalies, Biomedicine 21:233, 1974. 12. Hoehn, H., Bryant, E. M., Karp, L. E., and Martin, G. M.: Cultivated cells from diagnostic amniocentesis in second trimester pregnancies, Clin. Genet. 7:29, 1975.
Tenth Annual Meeting of the Wisconsin Association for Perinatal Care The Wisconsin Association for Perinatal Care announces the Tenth Annual Wisconsin Association for Perinatal Care Meeting, March 16, 17, and 18, 1980, at the Holidome in Stevens Point, Wisconsin. For information write: Wisconsin Association for Perinatal Care Statewide Office, 1010 Mound St., Madison, Wisconsin 53715.
R. KING, MAGENIS,
GERALD SUSAN Kansas
M.D.
PRESCOTT.
CATHERINE
M.D. D.M.D.
OLSON, BENNETT,
City,
Kansas,
B.S. B.S.
and Portland,
Oregon
Karyotypic results from a series of 975 consecutive amniotic fluid specimens studied for prenatal diagnosis are reported. In only 32% of specimens were all cells analyzed eyploid. Two tliirds (63%) of the specimens studied were hypomodal in at least one cell; 25% of the studies were hypomodal in three or more cells. mcontrast, only 3.5% of the studies were hypermodal in one or more Celli. Most frequently an additional chromosome 2 was present -in these hypermodai cells. Cells with other than a diploid chromosome constitution may be mistakenly identified as evidence of mosaic& This is especially likely when’ the involved chromosome isone associated with a clinically defined syndrome. (AM. J. OBSTET. GYNECOL. 136:383, 1980.)
analysis of amniotic fluid cells for antenatal diagnosis is a well-established procedure. The techniques used are complex and require careful attention to detail to avoid diagnostic errors. A review of our experience provides information which may aid when mosaicism is questioned with amniotic fluid cell cultures. This ‘is especially important since both falsepositive and false-negative examples of mosaicism have been reported in amniotic fluid cell cultures.” ’
CYTOGENETIC
Material and methods The patients in this report represent a consecutive series of 975 patients who have undergone antenatal genetic diagnosis at the University of Oregon Health Sciences Center. The essential aspects of this process have been reviewed previously,3, 4 but details important to the present discussion will be provided. Following sonograhy at 14 to 15 weeks’ gestation, amniocentesis is performed with sterile technique. Approximately 15 to 20 cc of amniotic fluid are removed. The specimen is From the Department of Obstetrics and Gynecology, University of Kansas Medical Center, and the Division of Medical Genetics, University of Oregon Health Sciences center. Receizjed for publication Accepted April
Ortober
I I, 1978.
23, 1979.
R+nint requests: Charles R. King, M.D., Department of Ob$etrics and Gynecology, U+versity of Kansas Medical Center, 39th U Rainbow, Kansas City, Kansas 66103. OOOZ-9378/80/030383+03$00.30/0
@ 1980 The C. V. Mosby
Co.
centrifuged at 600 rpm for 10 minutes. Cells are resuspended in one sixth of the original amniotic fluid volume and placed in F-10 medium supplemented with 15% fetal calf serum, glutamine, and gentamycin. Specimens are grown in Falcon T-30 plastic Basks in a water-jacketed COz incubator. Cells are subcultured and harvested independently from each of the flasks with trypsin: Following hypotonic treatment with 0.075M KC1 in a dilute aqueous solution of fetal calf serum, the cells are fixed with acetic acid/methanol and slides are prepared. Cells are banded with either quinacrine mustard, trypsin-Wright’s, or both. Studies are generally completed within 20 days following the amniocentesis. A minimum of 20 cells are counted and at least three karyotypes are made from each specimen. Studies are extended if questionable results are obtained.
Result+ and comment The indications for study in these patients are quite similar to those in other published series.5, 6 Advanced maternal age was the most frequent indication for study. Other indications were history of a previous chromosome error, neural tube defect, or biochemical genetic disease. Likewise, the observed frequency of chromosome aberrations (2.2%, Table I) is in agreement with previously published studies. However, it appears that fewer sex chromosome aberrations may have been identified. This may simply be a reflection of 383
384
King et al.
February
Am. J. Obstet.
Table I. <:ht-otnosorne abnormalities in 975 consecutive amniotic Huid specimens
Table II. Distribttrion hypermodal c-ells
Table III. Amniotic hypermodal cells
of‘ hypotnodal
Huiti cultures
Cdl I + 1” +1 + I4 +X +21
+c: + I9
and
with two or more
Crll 2
~Xl 3
+12 +4 +D +c:
+ Marker* + Market + Marker
:it,2’% 01 the sludirs detnonstrateci at lcast otte Ii!WII. Seven amniotic: specimens were studied with two otmore hypertnocial cells (Table III). Specimen I had two c-ells each \cith an aclditional chromosome I?. On tollrnv-up this infant is normal. Pregnancies 2. 3 and 5 arc’ rtill in pt-ogress. Pregnancies 4 and 6 resulted in ttormal oftbprittg. Fluid f-t-om the seventh study had 1 cell Lvith an ,ttlditional chromosome 10. One of the rnarkct. (.hromosontes was ciefiniteiy art F ,group rhrotnosomc, but banding did ttot identify whrrhrr ;I t 9 or ‘20. I‘hc third cell contained an F size chrnmosomr hut tto Iurrticr clclitieation WiLS possible. In short. none ot rhcsc infants has been abnormal. but thrcxt prrgnancirs cotirinuc. I2dditional chromosomes in hypct-modal tells rqxc’scnr a nonrandom distribution (Titblc IV). StatisriA atiatvsis” of this tlistrihurion contirms that a tiotirat1dom change is prcst’nr. (:hromosonie L’ ~vas prvsctnt ci~nific~anrl~ more frequentI\ than cxpccrcci h\- chanct (p ( 0.05). .A similar finding was reported h) Peakman and associates.!’ \Vhy is chromosome 2 so t’rcquentl~ the additional ctirottiosotiie in rhese atieupioid crtls? Furthet- $tncl\ is ttccesaar) to ciefinitively answer this cluesCon. but ir ih possible that specific- grnrtic material which cntiantes tong-term in vitro gro\vlh is totinrl on this chromostmtc. If this is correct. then identification and prcferctttial activation of. this tnatcriai shottld promote in vitro growth of amniotic fluid cells. ‘Ilie tnajor concern with interpretation of’ culrurail~ produced atteupioid~~ is possible confusion with mosaicistn. Obviously, conseling and the decisions ot parents may he quite different. dependent on whether a mosaic fetus ot- cultural at~ruploici~ is present. WC have not &set-Ted an abnormal infant or fetus v-hen onlv one cell ~vas ancuploid, but this could occut.. since abnormal cmhr)onic- cells have been observed to grow slowly in vitro.” \Vhrn tnosaicism is identified. the abnormal cell line niay represent a minimal estimate of its ;ic-total in \ iv0 f rcquetic 1 Hoehn and colleaguesg” h-ave suggested ttiar tht technique ot amniotic lluid culture affects the mtmhet of anueploici cells. l‘hq suggested that cells cultured with the in situ coverslip technique demonstrated less anueploidv and fewer structural rearrangements. We have not made this cotnparison. since in situ culturing its not perfortned with these specimens. However, if the gain ot an additional chromosome is nonrandom (‘l‘able I\‘). it then becomes more difticult to explain this phenomenon as entirety the result 01 a chance occ cttyrencc’ due to ;I specific culture technique. I‘he final interpretation of prenatal cytogenetic studies must depend on accuracy and attention to detail. pertnodal
.vo.
+ Market
*Marker chromosomes were not specifically identifiable as To chromosome number, but may havr been identified bv group as nntrtl in the test. sample 5i/c. itt- it ma! indic-ate 3 subtle ciiffercnce bct\Veeti this population and others prc\ iottsly reported. Aneuploid\ as an artifact ot’culturc has been hrieH? cotnment~d r~I)on in prcviou4 reporr\,‘. ‘. ’ which get)craI1~ ham Ix~ti onI> single case reports. The absrract b\ Peakman and associates” has reporttxl the results ot’ an initial stud\ of this phenomenon. 11s cat1 be scett from .I‘able I I. Iwo thirds of all cultut-cs contain at least ottc attcuploitl cell and one ot ever)’ four cultures has three or mot-e aneuploid c-ells. Hypomoclal cells account for- tl~c. majority of these changes. Nowever.
1, 1980
Gyecol.
Volume 136
Mosaic
Number
3
Table
IV. Extra
1
2
3
chromosome
4
5
6
7
in hypermodal
8
9
IO
amniotic
11
cells
in amniotic
fluid
385
fluid cells
X .Y
x x
x
12
I3
14
When aneuploid cells are identified their frequency must be defined. Generally more cells must be studied. Ifaneuploidy is present in more than one in situ colony or more than one culture Bask. the case for mosaicism is much stronger. In some cases a repeat amniocentesis and study may be indicated. This will not completely “explain away” the observed aneuploidy but it reduces the likelihood of’ mosaicism. When the involved chromosome is associated with an observed chromosomal aneuploidy syndrome the possibility of mosaicism is more critical, although survival has been observed in an otherwise nonviable trisomy when mosaicism was present. Parents need to be advised of the possibility of
and hypermodal
15
16
17
x
x
18
19
20
x
x
x s x
21
22
X
x x x X X X X X x .Y
Y
Marker
mosaicism when apparent cultural aneuploidy is observed. Frequently mosaicism will not be present, but only when fully informed of this possibility will some parents elect to terminate a possibly affected pregnancy. Generally a professional judgment regarding the presence or absence of mosaicism must be made and discussed with the family. The counselor will frequently need to provide additional psychologic and professional support to these families.
Addendum Pregnancies 2,3, and 5, reported as in progress, have all ended with the birth of normal infants. Thus all reported pregnancies have produced normal infants.
REFERENCES
1. A&. P. Von Koskull, H.. and Ryznanen, M.: A case of 46,Xx/47,Xx +G mosaicism in amniotic fluid not confirmed in the fetus, Clin Genet. 13: 103, 1978. 2. Ladda, R., Hildebrandt, R., and Dobello, Y.: Sex chromosomal mosaicism undetected by prenatal study, J. Pediatr. 90:841. 1977. 3. Prescott, G. H., Pernoll, M. L., Hecht, F., and Nicholas, A.: A prenatal diagnosis clinic: an initial report, AM. J. OBSTET.
GYNECOL.
116:942,
1973.
4. Pernoll, M. L., Prescott, G. H.. Hecht, F., and Olson, C. L.: Prenatal diagnosis: Practice, pitfalls, and progress, Obstet. Gynecol. 44~773, 1974. 5. Epsetin, C. J., and Golbus, M. S.: Prenatal diagnosis of genetic diseases, Am. Sci. 65:703, 1977. 6. Galjaard, H.: European experience with prenatal diagnosis of congenital disease: A survey of 6121 cases, Cytogenet. Cell Genet. 16:453, 1976. 7. COX, B. M., Niewczas-Late, V., Riffell, M. I., and Hamer-
ton, J. L.: Chromosomal mosaicism in diagnostic amniotic fluid cell cultures. Pediatr. Res. 8:679, 1974. 8. Kohn, G., Mennuti, M. T., Kaback. M., Schwartz, R. M.. Chemke, J., Goldman, B., and Mellman. W. J.: Chromosomal mosaicism in amniotic cell culture. Isr. J. Med. Sci. 11:476,
1975.
9. Peakman, D. C., Moreton, M. D., Corn, B. J., and Robinson. A.: Chromosomal mosaicism in amniotic fluid cell cultures. Pediatr. Res. 12:455. 1978. 10. Slatter, M. J.: Comparative validity of the chi square and two modified chi square goodness of lit tests for small but equal expected frequencies, Biometrika 53:619, 1966. 11. Cure, S., Boue, J., and Boue, A.: Growth characteristics of human embryonic cell lines with chromosome anomalies, Biomedicine 21:233, 1974. 12. Hoehn, H., Bryant, E. M., Karp, L. E., and Martin, G. M.: Cultivated cells from diagnostic amniocentesis in second trimester pregnancies, Clin. Genet. 7:29, 1975.
Tenth Annual Meeting of the Wisconsin Association for Perinatal Care The Wisconsin Association for Perinatal Care announces the Tenth Annual Wisconsin Association for Perinatal Care Meeting, March 16, 17, and 18, 1980, at the Holidome in Stevens Point, Wisconsin. For information write: Wisconsin Association for Perinatal Care Statewide Office, 1010 Mound St., Madison, Wisconsin 53715.