1076
Letters
to
the Editor
HLA TYPING OF AMNIOTIC-FLUID CELLS APPLIED TO PRENATAL DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA
SIR,-We have studied HLA-A and HLA-B antigens on fibroblasts from embryonic tissues in triploid abortuses to determine the origin of the extra chromosome set.1 Microcytotoxicity2 and micro-complement-fixation3 tests were used for HLA typing. We had some difficulty when typing amnioticfluid cells which are mainly epithelial. We abandoned the micro-complement-fixation test and improved the microcytotoxicity technique mainly by careful selection of specific antisera having no non-specific reactions against this cell type and by choosing rabbit complement which was non-toxic to epithelial cells. HLA typing is done on trypsinised amniotic-fluid cells in two stages. The microcytotoxicity test requires only 2x 105 cells for complete typing of HLA A and B antigens; then the results of microcytotoxicity are confirmed by microsbsorption techniques,4 the presence of each A and B antigen bei,’g tested separately with a suspension of 106 cells for each test. Microabsorption is highly specific and especially sensitive for the B locus.
second pregnancy of a woman who had had an affected boB with salt-wasting crises. First of all the affected child, the par ents, and three living grandparents were HLA typed (Dr Fauchet, Rennes). The results were informative enough to suggest that prenatal diagnosis should be attempted. A fibroblast cell line was initiated from the affected child and used for the selection of antisera. Amniocentesis was done at 17 weeks of pregnancy and amniotic-fluid cells were cultured.’ HLA typing of trypsimscd amniotic-fluid cells was done on day 15 after amniocentesis bB microcytotoxicity and gave clear results for the two HLA A loci and for one B locus and a weak reaction for the second B (B8) locus. The complete results of HLA typing were obtained, on trypsinised amniotic-fluid cells, 20 davs after amniocentesis by microabsorption against two HLA A antisera (Al and A3) and four HLA B antisera (B8, B12, B14, B40 Lymphocytes from the parents which had been stored in liquid nitrogen were used in these tests. A clear-cut response "Ias obtained (figure) and showed, on the basis of HLA B genotvping, that the fetus was heterozygote for the gene of 21-hvdroxylase deficiency and therefore expected to be healthy. z
We thank Prof. J. Dausset and Prof. M. Fellous for helpful suggestions, Prof. J. Hors for providing the specific antisera, and Piof B. Le Marec and Dr A. Guillou for referring the de Recherches de Biologie Prénatale (INSERM U. 73), Château de Longchamp, 75016 Paris, France
Groupe
C.A.H. case.
PHILIPPE COUILLIN HENRIETTE NICOLAS JOELLE BOUÉ ANDRÉ BOUÉ
ONE OR TWO STAGE ASSAY FOR FACTOR VIII? factor-VIII preparations are now available for of patients with hxmophilia. Some are made by non-profit organisations, some by commercial firms. The purification procedures vary, although most of them start from cryoprecipitate followed by aluminium-hydroxide adsorption, ethanol fractionation, and aminoacid or polyethylene glycol
SIR,-Many
the
treatment
precipitation.
C. A. H.
family tree. Triangle=prenatal diagnosis.
Fetal HLA typing on amniotic-fluid cells has been done in twin pregnancies, for paternal ascertainment in cases of artificial insemination by donor for autosomal recessive disease, and for prenatal diagnosis of congenital adrenal hyperplasia (C.A.H.). Dupont and others5.6 found a close genetic linkage between HLA and C.A.H. due to 21-hydroxylase deficiency, an autosomal recessive disease. In twenty families with at least two children, tested in our laboratory, no genetic recombination between HLA B and the 21-hydroxylase deficiency gene was observed. The close genetic linkage between HLA B and the enzyme deficiency should permit prenatal diagnosis of C.A.H. by HLA typing of amniotic-fluid cells. Prenatal diagnosis of C.A.H. was attempted during the 1. 2.
Couillin, P., Hors, J., Boué, J., Boué, A. Hum. Genet. 1978, 41, 35. Mittal, K. K., Mickey, H. R., Sougal, D. P., Terasaki, P. Transplantation,
3.
Dao, V. L, Hors, J., Sasportes, M., Netter, R., Colombani, J. J. Biol. Stan-
1968, 6, 913. dard
1974, 2, 121.
Fellous, M., Kamoun, M., Wiels, J., Dausset, J., Clements, G., Zenthen, J., Klein, G. Immunogenetics, 1977, 5, 423 5. Dupont, B., Oberfield, S. E., Smithwick, E. M., Lee, T. D., Levine, L. S. Lancet, 1977,11, 1309. 6. Levine, L. S., Zachmann, M., New, M I., Prader, A., Pollack, M., O’Neill, G. J., Yang, S. Y, Oberfield, S. E., Dupont, B. New Engl. J. Med. 1978,
Kirkwood et al.’ have reported a discrepancy between one and two stage factor-VIII assays for measuring F VIII:C in concentrates. The two-stage assay gave a 20‘c higher value for F vii! :c content when tested against a reference plasma. BB’e have made a study both in vitro and in vivo of six commeroat preparations of factor-VIII concentrate. F vm:c was measured by one2 and two stage3 assays by two independent techmcians. with, as reference standard, a normal pool from the plasma of fifty male blood-donors. This reference plasma contained a factor-vm activity of 1.01 i.u. when assayed against the second international F VIII standard (National Institute for Biological Standards and Control, London). In-vivo recovery was measured in severe hxmophilic children who required a single injection of F VIII for ear)}’ hxmorrhages either in joints or in muscles. Citrated plasma samples drawn before treatment and 15, 30, and 60 min after infusion were tested for F VIII activity by one-stage assav.’ The highest factor-VIII activity was always found in the 60 m’n sample and this value was used in the calculation of recoven. Recovery was defined by expressing actual F vm:c as a ’( of theoreticalF vm:c. Theoretical F vm:c was calculated bl dividing the units of F vrn:c injected by the plasma B olu:i1e (calculated on the basis of body-weight and hxmatocni The table shows the results of F VIII:C measurement in concentrate by one and two stage assays. Except for product 6, an av er-
4.
299, 911.
7. Boué, A. in Prenatal Diagnosis (edited by A. Boué I N S E R M 1976 1 Kirkwood, T. B. L., Barrowcliffe, T. W. Br J. Hœmat 1978, 40, 333 2. Soulier, J. P , Larrieu, M J. Sang, 1953, 24, 205 3. Biggs, R., Eveling, J., Richards, G. Br.JHœmat. 1955, 1, 20 4. Allain, J. P., Frommel, D. Blood, 1976, 47, 973.
Letters
to
the Editor
HLA TYPING OF AMNIOTIC-FLUID CELLS APPLIED TO PRENATAL DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA
SIR,-We have studied HLA-A and HLA-B antigens on fibroblasts from embryonic tissues in triploid abortuses to determine the origin of the extra chromosome set.1 Microcytotoxicity2 and micro-complement-fixation3 tests were used for HLA typing. We had some difficulty when typing amnioticfluid cells which are mainly epithelial. We abandoned the micro-complement-fixation test and improved the microcytotoxicity technique mainly by careful selection of specific antisera having no non-specific reactions against this cell type and by choosing rabbit complement which was non-toxic to epithelial cells. HLA typing is done on trypsinised amniotic-fluid cells in two stages. The microcytotoxicity test requires only 2x 105 cells for complete typing of HLA A and B antigens; then the results of microcytotoxicity are confirmed by microsbsorption techniques,4 the presence of each A and B antigen bei,’g tested separately with a suspension of 106 cells for each test. Microabsorption is highly specific and especially sensitive for the B locus.
second pregnancy of a woman who had had an affected boB with salt-wasting crises. First of all the affected child, the par ents, and three living grandparents were HLA typed (Dr Fauchet, Rennes). The results were informative enough to suggest that prenatal diagnosis should be attempted. A fibroblast cell line was initiated from the affected child and used for the selection of antisera. Amniocentesis was done at 17 weeks of pregnancy and amniotic-fluid cells were cultured.’ HLA typing of trypsimscd amniotic-fluid cells was done on day 15 after amniocentesis bB microcytotoxicity and gave clear results for the two HLA A loci and for one B locus and a weak reaction for the second B (B8) locus. The complete results of HLA typing were obtained, on trypsinised amniotic-fluid cells, 20 davs after amniocentesis by microabsorption against two HLA A antisera (Al and A3) and four HLA B antisera (B8, B12, B14, B40 Lymphocytes from the parents which had been stored in liquid nitrogen were used in these tests. A clear-cut response "Ias obtained (figure) and showed, on the basis of HLA B genotvping, that the fetus was heterozygote for the gene of 21-hvdroxylase deficiency and therefore expected to be healthy. z
We thank Prof. J. Dausset and Prof. M. Fellous for helpful suggestions, Prof. J. Hors for providing the specific antisera, and Piof B. Le Marec and Dr A. Guillou for referring the de Recherches de Biologie Prénatale (INSERM U. 73), Château de Longchamp, 75016 Paris, France
Groupe
C.A.H. case.
PHILIPPE COUILLIN HENRIETTE NICOLAS JOELLE BOUÉ ANDRÉ BOUÉ
ONE OR TWO STAGE ASSAY FOR FACTOR VIII? factor-VIII preparations are now available for of patients with hxmophilia. Some are made by non-profit organisations, some by commercial firms. The purification procedures vary, although most of them start from cryoprecipitate followed by aluminium-hydroxide adsorption, ethanol fractionation, and aminoacid or polyethylene glycol
SIR,-Many
the
treatment
precipitation.
C. A. H.
family tree. Triangle=prenatal diagnosis.
Fetal HLA typing on amniotic-fluid cells has been done in twin pregnancies, for paternal ascertainment in cases of artificial insemination by donor for autosomal recessive disease, and for prenatal diagnosis of congenital adrenal hyperplasia (C.A.H.). Dupont and others5.6 found a close genetic linkage between HLA and C.A.H. due to 21-hydroxylase deficiency, an autosomal recessive disease. In twenty families with at least two children, tested in our laboratory, no genetic recombination between HLA B and the 21-hydroxylase deficiency gene was observed. The close genetic linkage between HLA B and the enzyme deficiency should permit prenatal diagnosis of C.A.H. by HLA typing of amniotic-fluid cells. Prenatal diagnosis of C.A.H. was attempted during the 1. 2.
Couillin, P., Hors, J., Boué, J., Boué, A. Hum. Genet. 1978, 41, 35. Mittal, K. K., Mickey, H. R., Sougal, D. P., Terasaki, P. Transplantation,
3.
Dao, V. L, Hors, J., Sasportes, M., Netter, R., Colombani, J. J. Biol. Stan-
1968, 6, 913. dard
1974, 2, 121.
Fellous, M., Kamoun, M., Wiels, J., Dausset, J., Clements, G., Zenthen, J., Klein, G. Immunogenetics, 1977, 5, 423 5. Dupont, B., Oberfield, S. E., Smithwick, E. M., Lee, T. D., Levine, L. S. Lancet, 1977,11, 1309. 6. Levine, L. S., Zachmann, M., New, M I., Prader, A., Pollack, M., O’Neill, G. J., Yang, S. Y, Oberfield, S. E., Dupont, B. New Engl. J. Med. 1978,
Kirkwood et al.’ have reported a discrepancy between one and two stage factor-VIII assays for measuring F VIII:C in concentrates. The two-stage assay gave a 20‘c higher value for F vii! :c content when tested against a reference plasma. BB’e have made a study both in vitro and in vivo of six commeroat preparations of factor-VIII concentrate. F vm:c was measured by one2 and two stage3 assays by two independent techmcians. with, as reference standard, a normal pool from the plasma of fifty male blood-donors. This reference plasma contained a factor-vm activity of 1.01 i.u. when assayed against the second international F VIII standard (National Institute for Biological Standards and Control, London). In-vivo recovery was measured in severe hxmophilic children who required a single injection of F VIII for ear)}’ hxmorrhages either in joints or in muscles. Citrated plasma samples drawn before treatment and 15, 30, and 60 min after infusion were tested for F VIII activity by one-stage assav.’ The highest factor-VIII activity was always found in the 60 m’n sample and this value was used in the calculation of recoven. Recovery was defined by expressing actual F vm:c as a ’( of theoreticalF vm:c. Theoretical F vm:c was calculated bl dividing the units of F vrn:c injected by the plasma B olu:i1e (calculated on the basis of body-weight and hxmatocni The table shows the results of F VIII:C measurement in concentrate by one and two stage assays. Except for product 6, an av er-
4.
299, 911.
7. Boué, A. in Prenatal Diagnosis (edited by A. Boué I N S E R M 1976 1 Kirkwood, T. B. L., Barrowcliffe, T. W. Br J. Hœmat 1978, 40, 333 2. Soulier, J. P , Larrieu, M J. Sang, 1953, 24, 205 3. Biggs, R., Eveling, J., Richards, G. Br.JHœmat. 1955, 1, 20 4. Allain, J. P., Frommel, D. Blood, 1976, 47, 973.
