Prenat. Diagn. 19: 620–627 (1999)
Cytogenetic Aspects of the Canadian Early and Mid-trimester Amniotic Fluid Trial (CEMAT) Elizabeth J. T. Winsor1*, Darrell J. Tomkins2, Dagmar Kalousek3, Sandra Farrell4, Philip Wyatt5, Yao-Shan Fan6, Ronald Carter7, Hungshu Wang8, Louis Dallaire9, Patrice Eydoux10, J. Philip Welch11, Angelika Dawson12, Jim C. C. Lin13, Joel Singer3, JoAnn Johnson14 and R. Douglas Wilson15 1 Department of Laboratory Medicine and Pathobiology, The Toronto Hospital, Eaton 3-301, 200 Elizabeth Street, Toronto, Ontario, Canada, M5G 2C4 2 Cytogenetics Laboratory, 5B4.49 WC Mackenzie HSC, University of Alberta Hospital, 8440 112th Street, Edmonton, Alberta, Canada, T6G 2B7 3 BC’s Children & Women Hospital, Rm L225-4480 Oak Street, Vancouver, British Columbia, Canada, V6H 3V4 4 Department of Laboratory Medicine, The Credit Valley Hospital, 2200 Eglinton Ave W, Mississauga, Ontario, Canada, L5M 2N1 5 Department of Genetics, North York General Hospital, 4001 Leslie Street, Toronto, Ontario, Canada, M2K 1E1 6 Cytogenetics Laboratory, LHSC Victoria Campus, 375 South Street, London, Ontario, Canada, N6A 4G5 7 HSC 3N15 McMaster Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5 8 Department of Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1 9 Hopital Ste Justine, 3175 Cote Ste-Catherine, Montreal, Quebec, Canada, H3T 1C5 10 Laboratoire Biologie du Development, Hospital Robert-Debre, 8 Blvd Serrurier, Paris 75019, France 11 Atlantic Research Centre, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9 12 Section of Genetics & Metabolism, Children’s Hospital HSC, 685 William Avenue, Winnipeg, Manitoba, Canada, R3E 0Z2 13 Cytogenetics Laboratory, University of Alberta Hospital, 8440 112 Street, Edmonton, Alberta, Canada, T6G 2B7 14 Department of Obstetrics and Gynecology, The Toronto Hospital, Eaton 6-216, 200 Elizabeth Street, Toronto, Ontario, Canada, M5G 2C4 15 Department of Medical Genetics, BCWH 2H30, 4500 Oak Street, Vancouver, British Columbia, Canada, V6H 3N1
Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0–12+6 weeks (EA) and 1999 fluids at 15+0–16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks’ gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus. Copyright 1999 John Wiley & Sons, Ltd. : early amniocentesis; chromosome culture; maternal cell contamination
INTRODUCTION The CEMAT trial protocol, pregnancy outcome and summary of the cytogenetic findings have previously been published (The Canadian Early and Midtrimester Amniocentesis Trial Group, 1998). A significant increase in talipes equinovarus was found in the early amniocentesis group compared with midtrimester amniocentesis (1.3 per cent versus 0.1 per cent, p=0.0001). There was also a significant difference *Correspondence to: E. J. T. Winsor, Clinical Cytogenetics Laboratory, The Toronto Hospital, Eaton 3-301, 200 Elizabeth Street, Toronto M5G 2C4, Canada. E-mail: [email protected]. on.ca Contract/grant sponsor: Medical Research Council of Canada Clinical Trials Committee.
CCC 0197–3851/99/070620–08$17.50 Copyright 1999 John Wiley & Sons, Ltd.
in total fetal losses for early amniocentesis compared with mid-trimester amniocentesis (7.6 per cent versus 5.9 per cent; difference 1.7 per cent, one-sided CI 2.98 per cent, p=0.012). This paper provides a detailed comparison between amniotic fluids obtained at 11+0– 12+6 weeks with those obtained at 15+0–16+6 with regard to cytogenetic results, culture success, rate of cell growth, maternal cell contamination, mosaicism and accuracy.
MATERIALS AND METHODS Patients were randomized to early (11+0–12+6 weeks) (EA) or mid-trimester amniocentesis (15+0–16+6) (MA) and the procedure was scheduled for the earliest Received 23 November 1998 Revised 1 February 1999 Accepted 2 February 1999
available appointment within the assigned gestation. The protocol specified that 11 ml of amniotic fluid should be obtained from EA patients and 20 ml from MA patients. If less than 8 ml of fluid was obtained, this was defined as a procedure failure, but chromosome analysis was attempted on all fluids received in the laboratory, irrespective of fluid volume. Chromosome results from patients with fetal demise prior to the amniocentesis or patients having testing outside the study are not included in this report. Initial analysis for the cytogenetic data was based on study allocation to EA or MA regardless of actual calculated gestation. Some subsequent analyses included only fluids obtained within the gestational windows (11+0–12+6 and 15+0–16+6 weeks). Gestation at the time of amniocentesis was calculated from the crown–rump length at randomization. Culture and analysis of amniotic fluids were performed according to the routine methods of each of the 12 participating cytogenetics laboratories. The procedure with regard to discard or inclusion in the chromosome culture of the first aliquot of fluid varied between centres. With the exception of one centre (7 per cent of total patients), there was no difference in the discard procedure within centres for EA and MA fluids. Cells were either harvested in situ or were subcultured. A standard analysis was defined as a minimum of 10 cells from at least two primary vessels. Cultures were assigned to one of the following three categories: (1) failure, if no result was reported to the physician; (2) inadequate, if a result was reported but there was a notation that the cells available for analysis did not meet reporting standards; or (3)
Cytogenetic Aspects of the Canadian Early and Mid-trimester Amniotic Fluid Trial (CEMAT) Elizabeth J. T. Winsor1*, Darrell J. Tomkins2, Dagmar Kalousek3, Sandra Farrell4, Philip Wyatt5, Yao-Shan Fan6, Ronald Carter7, Hungshu Wang8, Louis Dallaire9, Patrice Eydoux10, J. Philip Welch11, Angelika Dawson12, Jim C. C. Lin13, Joel Singer3, JoAnn Johnson14 and R. Douglas Wilson15 1 Department of Laboratory Medicine and Pathobiology, The Toronto Hospital, Eaton 3-301, 200 Elizabeth Street, Toronto, Ontario, Canada, M5G 2C4 2 Cytogenetics Laboratory, 5B4.49 WC Mackenzie HSC, University of Alberta Hospital, 8440 112th Street, Edmonton, Alberta, Canada, T6G 2B7 3 BC’s Children & Women Hospital, Rm L225-4480 Oak Street, Vancouver, British Columbia, Canada, V6H 3V4 4 Department of Laboratory Medicine, The Credit Valley Hospital, 2200 Eglinton Ave W, Mississauga, Ontario, Canada, L5M 2N1 5 Department of Genetics, North York General Hospital, 4001 Leslie Street, Toronto, Ontario, Canada, M2K 1E1 6 Cytogenetics Laboratory, LHSC Victoria Campus, 375 South Street, London, Ontario, Canada, N6A 4G5 7 HSC 3N15 McMaster Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5 8 Department of Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1 9 Hopital Ste Justine, 3175 Cote Ste-Catherine, Montreal, Quebec, Canada, H3T 1C5 10 Laboratoire Biologie du Development, Hospital Robert-Debre, 8 Blvd Serrurier, Paris 75019, France 11 Atlantic Research Centre, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9 12 Section of Genetics & Metabolism, Children’s Hospital HSC, 685 William Avenue, Winnipeg, Manitoba, Canada, R3E 0Z2 13 Cytogenetics Laboratory, University of Alberta Hospital, 8440 112 Street, Edmonton, Alberta, Canada, T6G 2B7 14 Department of Obstetrics and Gynecology, The Toronto Hospital, Eaton 6-216, 200 Elizabeth Street, Toronto, Ontario, Canada, M5G 2C4 15 Department of Medical Genetics, BCWH 2H30, 4500 Oak Street, Vancouver, British Columbia, Canada, V6H 3N1
Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0–12+6 weeks (EA) and 1999 fluids at 15+0–16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks’ gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus. Copyright 1999 John Wiley & Sons, Ltd. : early amniocentesis; chromosome culture; maternal cell contamination
INTRODUCTION The CEMAT trial protocol, pregnancy outcome and summary of the cytogenetic findings have previously been published (The Canadian Early and Midtrimester Amniocentesis Trial Group, 1998). A significant increase in talipes equinovarus was found in the early amniocentesis group compared with midtrimester amniocentesis (1.3 per cent versus 0.1 per cent, p=0.0001). There was also a significant difference *Correspondence to: E. J. T. Winsor, Clinical Cytogenetics Laboratory, The Toronto Hospital, Eaton 3-301, 200 Elizabeth Street, Toronto M5G 2C4, Canada. E-mail: [email protected]. on.ca Contract/grant sponsor: Medical Research Council of Canada Clinical Trials Committee.
CCC 0197–3851/99/070620–08$17.50 Copyright 1999 John Wiley & Sons, Ltd.
in total fetal losses for early amniocentesis compared with mid-trimester amniocentesis (7.6 per cent versus 5.9 per cent; difference 1.7 per cent, one-sided CI 2.98 per cent, p=0.012). This paper provides a detailed comparison between amniotic fluids obtained at 11+0– 12+6 weeks with those obtained at 15+0–16+6 with regard to cytogenetic results, culture success, rate of cell growth, maternal cell contamination, mosaicism and accuracy.
MATERIALS AND METHODS Patients were randomized to early (11+0–12+6 weeks) (EA) or mid-trimester amniocentesis (15+0–16+6) (MA) and the procedure was scheduled for the earliest Received 23 November 1998 Revised 1 February 1999 Accepted 2 February 1999
available appointment within the assigned gestation. The protocol specified that 11 ml of amniotic fluid should be obtained from EA patients and 20 ml from MA patients. If less than 8 ml of fluid was obtained, this was defined as a procedure failure, but chromosome analysis was attempted on all fluids received in the laboratory, irrespective of fluid volume. Chromosome results from patients with fetal demise prior to the amniocentesis or patients having testing outside the study are not included in this report. Initial analysis for the cytogenetic data was based on study allocation to EA or MA regardless of actual calculated gestation. Some subsequent analyses included only fluids obtained within the gestational windows (11+0–12+6 and 15+0–16+6 weeks). Gestation at the time of amniocentesis was calculated from the crown–rump length at randomization. Culture and analysis of amniotic fluids were performed according to the routine methods of each of the 12 participating cytogenetics laboratories. The procedure with regard to discard or inclusion in the chromosome culture of the first aliquot of fluid varied between centres. With the exception of one centre (7 per cent of total patients), there was no difference in the discard procedure within centres for EA and MA fluids. Cells were either harvested in situ or were subcultured. A standard analysis was defined as a minimum of 10 cells from at least two primary vessels. Cultures were assigned to one of the following three categories: (1) failure, if no result was reported to the physician; (2) inadequate, if a result was reported but there was a notation that the cells available for analysis did not meet reporting standards; or (3)
