- Email: [email protected]
Prenatal diagnosis of severe combined immunodeficiency
Volume 101 Number 6
aspirates of infants with respiratory distress will provide a more sensitive and specific site of colonization of infants at risk for developing early-onset disease, although in a n infant with respiratory distress the correlation between isolation of GBS from a tracheal aspirate and invasive GBS disease is unknown. We thank Stephen George, Ph.D., for the statistical analysis and John Gilbert for editorial assistance, and we gratefully acknowledge the study nurses from our obstetric department and the nursing staff of the newborn center-whose cooperation allowed us to complete this study.
Clinical and laboratory observations
6.
7.
8.
9.
REFERENCES 1. Merestein GB, Todd WA, Brown G, Yost CC, and Luzier T: Group B hemolytic streptoccus: Randomized controlled treatment study at term, Obstet Gynecol 55:315, 1980. 2. Yow MD, Mason EO, Leeds LJ, Thompson PK, Clark D J, and Gardner AE: Ampicillin prevents intrapartum transmission of group B streptococcus, JAMA 41:1245, 1979. 3. Baker CF, Clark D J, and Barrett FF: Selective broth medium for isolation of group B streptococci, Appl Microbiol 26:884, 1973. 4. Mason EO, Wong P, and Barrett FF: Evaluation of four methods for detection of group B streptococcal colonization, J Clin Microbiol 4:429, 1976. 5. Gray BM, Pass MA, and Dillion HC Jr: Laboratory and field
10.
I I.
12.
13.
995
evaluation of selective media for isolation of group B strepto cocci, J Clin Microbiol 9:466, 1979. Romero R, and Wilkinson HW: Identification of group B streptococci by immunofluorescence staining, Appl Microbiol 28:199, 1974. Lancefield RC: Serologic differentiation of specific types of bovine hemolytic streptococci (group B), J Exp Med 59:44I, 1934. Herbert GA, Pitman B, McKinney RM, and Cherry WB: The preparation and physiochemical characterization of fluorescent antibody reagents, Atlanta, GA, 1972, United States Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control. Hall RT, Barnes W, Krishnan L, Harris D J, Rhodes PG, Fayez J, and Miller GL: Antibiotic treatment of parturient women colonized with group B streptococci, Am J Obstet Gynecol 124:630, 1976. Levin EB, and Amstey MS: Natural history of group B streptococcus colonization and its therapy during pregnancy, Am J Obstet Gynecol 139:512, 198l. Anthony BF, Okada BM, and Hobel C J: Epidemiology of group B streptococcus: Maternal and nosocomial sources for infant acquisition, J PEDIATR95:431, 1979. Ancona R J, Ferrieri P, and Williams PP: Maternal factors that enhance the acquisition of group B streptococci by newborn infants, J Med Microbiol 13:273, 1980. Pass MA, Gray BM, Khare S, et al: Prospective studies of group B streptococcal infections in infants, J PEDIATR95:437, 1979.
Prenatal diagnosis of severe combined immunodeficiency A. Durandy, Y. Dumez, D. Guy-Grand, C. Oury, R. Henrion, and C. Griscelli, Paris, France
ANTENATAL DIAGNOSIS of severe combined immunodeficiency ( S C I D ) associated with a defect of adenosine desaminase was achieved seven years ago by assay of enzymatic activity in cultured amniotic cells.~ The development of fetoscopy has led to the availability of pure fetal blood, allowing the antenatal diagnosis of hemoglobinopathies, 2 hemophiliafl and chronic granulomatous disease. 4 We report here the prenatal diagnosis of S C I D in a fetus at risk, based on T- and B-lymphocyte marker analysis. The immunologic abnormality has been confirmed by studies performed after termination of pregnancy. From the Groupe de Recherche d'lmmunologie et de Rhumatologie Pkdiatrique INSERM 2, Hbpital des Enfants Malades, and the Clinique Universitaire de Port-Royal, Groupe Hospitalier Cochin. Reprint address: A. Durandy, 1NSERM U 132, Hbpital des Enfants Malades, 149 rue de Sbvres, 75720 Paris Cbdex 15, France.
0022-3476/82/120995+03500.30/0 9 1982 The C. V. Mosby Co.
METHODS Blood from 37 nonimmunodeficient fetuses and from the propositi was obtained at fetoscopy according to a method described by Rodeck and Campbell. 5 Punctures were performed between 18 and 22 weeks' gestation in the vessels of the umbilical cord insertion, permitting aspiration of 100 to 700 #1 of fetal blood into preservative-free heparin. We investigated the immunologic status of 35 presumably nonimmunodeficient fetuses by taking advantage of fetal blood sampling justified for diagnosis of hemoglobinopathies. Because in those conditions only an aliquot (100 to 150 ~zl) of fetal blood was available for immunologic tests, only limited investigations were performed for each fetus. Fetoscopy was also performed, at the same term of gestation, for two fetuses at risk for severe combined immunodeficiency. 6 Purity of fetal blood was estimated by red blood cell size analysis with a Coulter channel analyzer and was later
996
Clinical and laboratory observations
The Journal of Pediatrics December 1982
Table. Immunologic studies of 18 to 22-week control fetuses and of a fetus affected with S C I D
Fetuses at risk for SCID
Adults Tests Lymphocytes/~l Lymphocyte markers % T (OKT3) Helper-inducer (OKT4) Cytotoxie-suppressor (OKT8) Immature T (OKT6) Anti-F(ab) IgG Anti-~ chain Anti-6 chain Anti-common DR Anti-~2-microglobulin PHA-induced proliferation (cpm X 10-3) Nonstimulated PHA
(n =
20)
Control Fetuses*
2,200 _+ 0,700
4,800 + 1,200
(15)
77.5 _+ 9.9 49.3 _+ 3.2 31.4 _+ 4.7
53.7 _+ 8.4 36.9 • 5.1 24.5 _+ 8.2
(12) (8) (8)
0.1 10.2 9.1 7.5 14.7 95.8
NT 13.7 _+ 2.5 16.6 _+ 5.0 14.2 • 2.9 18.2 • 10.1 94.7 + 5.6
1.1 + 0.9 57.5 • 31.0
• 0.5 _+ 3.7 _+ 4.7 _+ 3.8 • 5.1 • 2.7
0.5 +_ 0.4 52.1 _+ 25.8
1
I
3,000
Propositus fetus affected with SCID Fetal blood [ After termination
3,600
200
300
65.0 NT NT
NT NT NT
< 1 NT NT
0 0 < 1
(15) (11) ( 11 ) (11) (15)
NT NT 11.5 NT NT NT
NT NT 8.0 NT 16.7 87.6
NT NT 0 NT NT NT
< 1 0 0 0 1 NT
(7) (7)
0.8 35.7
NT NT
NT NT
1.2 1.5
*Number of fetuses tested shown in parentheses. corroborated by the Kleihauer test. Mononuclear cells were isolated on a Fieoll-Hypaque gradient, washed, and analyzed for the markers of T- and B-lymphocytes. Monoclonal antibodies specific for T-lymphocytes or T-cell subsets (OKT3, OKT4, and OKT8 antibodies, Ortho Pharmaceutical Corp., Raritan, N.J.) were used in an indirect fluorescence test. B-lymphocytes were revealed by rhodamine-labeled F ( a b ) ' 2 fragments of anti-~z heavy chains or of anti-F(ab) IgG antisera or rhodamine-labeled antiserum anti-6 heavy chains (Nordic Laboratory, Tilburg, The Netherlands). Monoclonal antibodies specific for ~2-microglobulin (Sera Lab Crawley Down, Sussex, England) borne by all leukocytes or for common H L A - D R antigen (OKIa, Ortho Pharmaceutical Corp.), expressed by B-lymphocytes and most monocytes, were also used in an indirect immunofluorescence test. Each fluorescence study was performed on cell suspension in a micromethod using 4 to 30 x 103 leukocytes, and at least 250 cells were examined. Proliferative response of 40 x 103 leukocytes from fetuses or adults to phytohemagglutinin (10 ~ g / m l ) (Difco Laboratories, Detroit, Mich.) was evaluated by incorporation of tritiated thymidine during the last 18 hours of a three-day culture. 6 Histologic examination of fetal tissues was performed after fixation in Carnoy, Bouin, or Zenker solutions. Slides were stained with methyl green pironin, May Grunwald Giemsa, or alcian blue. CASE REPORT Prenatal diagnosis was attempted in a patient who had previously given birth to a boy affected with a severe combined immune deficiency. This child died at 4 months of age with interstitial
pneumonia, protracted diarrhea, and a maternofetal graft-vs-host disease. Immunologic investigations, performed immediately before death, showed agammaglobulinemia (IgG 0.2 gm/L; IgA and IgM not detectable), a subnormal number of lymphocytes (l,300/ul), but an absence of PHA-induced proliferation. Adenosine desaminase and nucleoside phosphorylase activities were found to be within normal ranges in child and parents. Postmortem examination revealed a severe lymphoid depletion of spleen, lymph nodes, and thymus, which did not contain Hassall corpuscles. In the absence of other documented cases in the family, the mode of inheritance could not be defined. RESULTS In the 37 fetuses investigated for diagnosis of hemoglobinopathies or diagnosis of S C I D , contamination with maternal blood as judged by red blood cell size and the Kleihauer test was nil or below 3% in 31 samples and below 10% in six others. In the 35 presumably nonimmunodeficient fetuses, T- and B-lymphocyte membrane markers, including common H L A - D R and /32-microglobulin, were easily detectable by immunofluorescence (Table). These percentages were comparabie to those found in adults, except for a moderate increase of B-cell number and a slight decrease of T-cell number. Respective proportions of O K T 4 (helper subset) and O K T 8 (suppressive-cytotoxic subset) among T-cell populations (OKT3 positive cells) were also similar in fetuses and adults./32-Microglobulin and H L A - D R antigens were normally found on fetal leukocyte membranes. PHA-induced proliferative responses were identical in fetuses and control adults. In the two fetuses at risk for S C I D , immunologic studies showed no detectable immunologic defect (Table), which has been completely confirmed after birth.
Volume 1O1 Number 6
Pure fetal blood (500 #1) was obtained at the twentysecond week of gestation from the propositus (fetus) at risk for SCID. We observed a profound lymphopenia (200/tzl) characterized by a complete absence of mature T- and B-lymphocytes (Table). Termination of pregnancy was performed by local injection of prostaglandin E2. The fetus (a male) measured 18 cm from crown to rump and weighed 550 gm, corresponding to the presumed term. The alymphocytosis was confirmed in blood (300 ~1). Not T- and B-lymphocyte markers were detected and, as expected, PHA-induced proliferation was nil (Table). The macroscopic study showed no anomaly except for the weight of th e thymus (0.107 gin). At the histologic examination, the lobulation of the thymus was normal, but it contained no lymphocytes and only a few Hassall corpuscles. T h e spleen was deprived of white pulp; the lymph nodes, normally located, were devoid of lymphocytes. No lymphocytes were observed in the gut mucosa and appendix. DISCUSSION The diagnosis of SCID associated with enzyme abnormality has been accomplished during intrauterine life by amniotic cell enzymatic study, l In SCID without detectable enzyme deficiency, diagnosis remains possible in the prenatal period by analysis of T- and B-lymphocyte markers of pure fetal blood obtained at fetoscopy. We herein report the first prenatal diagnosis of SCID characterized by a profound lymphopenia and an absence of Tand B-lymphocyte surface markers. We believe that these results, compared to normal values observed in nonimmunodeficient fetuses, including two fetuses at risk for SCID studied previously,6justified the termination of pregnancy. The diagnosis was confirmed by histologic examination, which showed a complete lymphocyte depletion of lymphoid organs. The lymphoid organs of 22-week fetuses contain easily detectable lymphocytes already organized in the spleen (white pulp) and lymph nodes.7 In the propositus, thymus weight was one-tenth the norm for the age of gestation? The detection of lymphocyte membrane markers may permit antenatal diagnosis of several types of SCID, characterized by either absence of both T and B cells (alymphocytosis with agammagiobulinemia) or a defect of precursor T-lymphocytes.9 Furthermore, it may allow the prenatal diagnosis of X-linked agammaglobulinemia characterized by a complete absence of surface immunoglobulin-bearing cells. We used the same method to detect other membrane-associated antigens, Such as histocompatilility antigens A-B and DR and 3z-microglobulin, known to be poorly expressed on leukocytes of some Patients with SCID. 1~One fetus at risk for this disease was examined for prenatal diagnosis; normal expression of HLA and 32-
Clinical and laboratory observations
997
microglobulin was observed and was confirmed after birth. 6 We must stress that prenatal diagnosis of immune deficiency can be performed only when a previously affected child in the family has been investigated. The precise abnormality of lymphocyte markers must be known to allow a prenatal diagnosis in a further pregnancy. The mode of inheritance of an immune deficiency must also be taken into consideration because, in X-linked inherited disease, fetoscopy would be proposed only in male fetuses of pregnant women at risk. Other immune deficiencies are not yet detectable in the prenata ! period using detection of surface antigen markers of T- and B-lymphocytes or proliferative response of T-lymphocytes induced by mitogens. For example, prenatal diagnosis of ataxia telangiectasia and Wiskott-Aldrich syndrome cannot be performed because they are not characterized by an absence of a defined lymphocyte population or by a constant immunologic abnormality. REFERENCES 1. Hirschhorn R, Beratis N, and Rosen FS: Adenosine desaminase deficiency in a child diagnosed prenatally, Lancet 1:73, 1975. 2. Dubart A, Gooseens M, Beuzard Y, Monplaisir N, Testa U, Basset P, and Rosa J: Prenatal diagnosis of hemoglobinopathies: Comparison of the results obtained by isoelectric focusing of hemoglobinsand by chromatography of radioactive globinchains, Blood 56:1092, 1980. 3. Firshein S, Hoyer L, Lazarchick J, Forget B, Hobbins J, Clyne L, Pitlick F, Muir A, Merkatz I, and Mahoney M: Prenatal diagnosis of classic hemophilia, N Engl J Med 300:937, 1979. 4. Newburger PE, Cohen HJ, Rothschild SB, Hobbins C, Malawista SE, and Mahoney M: Prenatal diagnosis of chronic granulomatous disease, N Engl J Med 300:178, 1979. 5. Rodeck CH, and Campbell CH: Umbilical cord insertion as source of pure fetal blood for prenatal diagnosis, Lancet 1:1244, 1979. 6. Durandy A, Dumez Y, Oury C, and Griscelli C: Prenatal testing for inherited immune deficiencies by fetal blood sampling, Prenatal Diagnosis (in press). 7. Stites P, Caldwell J, Carr M, and Fudenberg H: Ontogeny of immunity in humans, Clin Immunol Immunopathol 4:519, 1975. 8. Bellanti J: General immunobiology:Concepts of immunobiology, in: immunology. Philadelphia, 1971, W. B. Saunders Co.,p 13, 9. Griscelli C, Durandy A, Virelizier JL, Ballet J J, and Daguillard F: Selective defect of precursor T cells in SCID with B lymphocytes. J PEDIATR93:404, 1978. 10. Griscelti C, Durandy A, Virelizier JL, Hors J, Lepage V, and Colombani J: Impaired cell to cell interaction in partial combined immunodeficiencywith variable expressionof HLA antigens, in Seligmann M, and Hitzig WH, editors: Primary immunodeficiencies,INSERM Symposium No. 16, Amsterdam 1980, Elsevier/North Holland Biomedical Press, p 499.
aspirates of infants with respiratory distress will provide a more sensitive and specific site of colonization of infants at risk for developing early-onset disease, although in a n infant with respiratory distress the correlation between isolation of GBS from a tracheal aspirate and invasive GBS disease is unknown. We thank Stephen George, Ph.D., for the statistical analysis and John Gilbert for editorial assistance, and we gratefully acknowledge the study nurses from our obstetric department and the nursing staff of the newborn center-whose cooperation allowed us to complete this study.
Clinical and laboratory observations
6.
7.
8.
9.
REFERENCES 1. Merestein GB, Todd WA, Brown G, Yost CC, and Luzier T: Group B hemolytic streptoccus: Randomized controlled treatment study at term, Obstet Gynecol 55:315, 1980. 2. Yow MD, Mason EO, Leeds LJ, Thompson PK, Clark D J, and Gardner AE: Ampicillin prevents intrapartum transmission of group B streptococcus, JAMA 41:1245, 1979. 3. Baker CF, Clark D J, and Barrett FF: Selective broth medium for isolation of group B streptococci, Appl Microbiol 26:884, 1973. 4. Mason EO, Wong P, and Barrett FF: Evaluation of four methods for detection of group B streptococcal colonization, J Clin Microbiol 4:429, 1976. 5. Gray BM, Pass MA, and Dillion HC Jr: Laboratory and field
10.
I I.
12.
13.
995
evaluation of selective media for isolation of group B strepto cocci, J Clin Microbiol 9:466, 1979. Romero R, and Wilkinson HW: Identification of group B streptococci by immunofluorescence staining, Appl Microbiol 28:199, 1974. Lancefield RC: Serologic differentiation of specific types of bovine hemolytic streptococci (group B), J Exp Med 59:44I, 1934. Herbert GA, Pitman B, McKinney RM, and Cherry WB: The preparation and physiochemical characterization of fluorescent antibody reagents, Atlanta, GA, 1972, United States Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control. Hall RT, Barnes W, Krishnan L, Harris D J, Rhodes PG, Fayez J, and Miller GL: Antibiotic treatment of parturient women colonized with group B streptococci, Am J Obstet Gynecol 124:630, 1976. Levin EB, and Amstey MS: Natural history of group B streptococcus colonization and its therapy during pregnancy, Am J Obstet Gynecol 139:512, 198l. Anthony BF, Okada BM, and Hobel C J: Epidemiology of group B streptococcus: Maternal and nosocomial sources for infant acquisition, J PEDIATR95:431, 1979. Ancona R J, Ferrieri P, and Williams PP: Maternal factors that enhance the acquisition of group B streptococci by newborn infants, J Med Microbiol 13:273, 1980. Pass MA, Gray BM, Khare S, et al: Prospective studies of group B streptococcal infections in infants, J PEDIATR95:437, 1979.
Prenatal diagnosis of severe combined immunodeficiency A. Durandy, Y. Dumez, D. Guy-Grand, C. Oury, R. Henrion, and C. Griscelli, Paris, France
ANTENATAL DIAGNOSIS of severe combined immunodeficiency ( S C I D ) associated with a defect of adenosine desaminase was achieved seven years ago by assay of enzymatic activity in cultured amniotic cells.~ The development of fetoscopy has led to the availability of pure fetal blood, allowing the antenatal diagnosis of hemoglobinopathies, 2 hemophiliafl and chronic granulomatous disease. 4 We report here the prenatal diagnosis of S C I D in a fetus at risk, based on T- and B-lymphocyte marker analysis. The immunologic abnormality has been confirmed by studies performed after termination of pregnancy. From the Groupe de Recherche d'lmmunologie et de Rhumatologie Pkdiatrique INSERM 2, Hbpital des Enfants Malades, and the Clinique Universitaire de Port-Royal, Groupe Hospitalier Cochin. Reprint address: A. Durandy, 1NSERM U 132, Hbpital des Enfants Malades, 149 rue de Sbvres, 75720 Paris Cbdex 15, France.
0022-3476/82/120995+03500.30/0 9 1982 The C. V. Mosby Co.
METHODS Blood from 37 nonimmunodeficient fetuses and from the propositi was obtained at fetoscopy according to a method described by Rodeck and Campbell. 5 Punctures were performed between 18 and 22 weeks' gestation in the vessels of the umbilical cord insertion, permitting aspiration of 100 to 700 #1 of fetal blood into preservative-free heparin. We investigated the immunologic status of 35 presumably nonimmunodeficient fetuses by taking advantage of fetal blood sampling justified for diagnosis of hemoglobinopathies. Because in those conditions only an aliquot (100 to 150 ~zl) of fetal blood was available for immunologic tests, only limited investigations were performed for each fetus. Fetoscopy was also performed, at the same term of gestation, for two fetuses at risk for severe combined immunodeficiency. 6 Purity of fetal blood was estimated by red blood cell size analysis with a Coulter channel analyzer and was later
996
Clinical and laboratory observations
The Journal of Pediatrics December 1982
Table. Immunologic studies of 18 to 22-week control fetuses and of a fetus affected with S C I D
Fetuses at risk for SCID
Adults Tests Lymphocytes/~l Lymphocyte markers % T (OKT3) Helper-inducer (OKT4) Cytotoxie-suppressor (OKT8) Immature T (OKT6) Anti-F(ab) IgG Anti-~ chain Anti-6 chain Anti-common DR Anti-~2-microglobulin PHA-induced proliferation (cpm X 10-3) Nonstimulated PHA
(n =
20)
Control Fetuses*
2,200 _+ 0,700
4,800 + 1,200
(15)
77.5 _+ 9.9 49.3 _+ 3.2 31.4 _+ 4.7
53.7 _+ 8.4 36.9 • 5.1 24.5 _+ 8.2
(12) (8) (8)
0.1 10.2 9.1 7.5 14.7 95.8
NT 13.7 _+ 2.5 16.6 _+ 5.0 14.2 • 2.9 18.2 • 10.1 94.7 + 5.6
1.1 + 0.9 57.5 • 31.0
• 0.5 _+ 3.7 _+ 4.7 _+ 3.8 • 5.1 • 2.7
0.5 +_ 0.4 52.1 _+ 25.8
1
I
3,000
Propositus fetus affected with SCID Fetal blood [ After termination
3,600
200
300
65.0 NT NT
NT NT NT
< 1 NT NT
0 0 < 1
(15) (11) ( 11 ) (11) (15)
NT NT 11.5 NT NT NT
NT NT 8.0 NT 16.7 87.6
NT NT 0 NT NT NT
< 1 0 0 0 1 NT
(7) (7)
0.8 35.7
NT NT
NT NT
1.2 1.5
*Number of fetuses tested shown in parentheses. corroborated by the Kleihauer test. Mononuclear cells were isolated on a Fieoll-Hypaque gradient, washed, and analyzed for the markers of T- and B-lymphocytes. Monoclonal antibodies specific for T-lymphocytes or T-cell subsets (OKT3, OKT4, and OKT8 antibodies, Ortho Pharmaceutical Corp., Raritan, N.J.) were used in an indirect fluorescence test. B-lymphocytes were revealed by rhodamine-labeled F ( a b ) ' 2 fragments of anti-~z heavy chains or of anti-F(ab) IgG antisera or rhodamine-labeled antiserum anti-6 heavy chains (Nordic Laboratory, Tilburg, The Netherlands). Monoclonal antibodies specific for ~2-microglobulin (Sera Lab Crawley Down, Sussex, England) borne by all leukocytes or for common H L A - D R antigen (OKIa, Ortho Pharmaceutical Corp.), expressed by B-lymphocytes and most monocytes, were also used in an indirect immunofluorescence test. Each fluorescence study was performed on cell suspension in a micromethod using 4 to 30 x 103 leukocytes, and at least 250 cells were examined. Proliferative response of 40 x 103 leukocytes from fetuses or adults to phytohemagglutinin (10 ~ g / m l ) (Difco Laboratories, Detroit, Mich.) was evaluated by incorporation of tritiated thymidine during the last 18 hours of a three-day culture. 6 Histologic examination of fetal tissues was performed after fixation in Carnoy, Bouin, or Zenker solutions. Slides were stained with methyl green pironin, May Grunwald Giemsa, or alcian blue. CASE REPORT Prenatal diagnosis was attempted in a patient who had previously given birth to a boy affected with a severe combined immune deficiency. This child died at 4 months of age with interstitial
pneumonia, protracted diarrhea, and a maternofetal graft-vs-host disease. Immunologic investigations, performed immediately before death, showed agammaglobulinemia (IgG 0.2 gm/L; IgA and IgM not detectable), a subnormal number of lymphocytes (l,300/ul), but an absence of PHA-induced proliferation. Adenosine desaminase and nucleoside phosphorylase activities were found to be within normal ranges in child and parents. Postmortem examination revealed a severe lymphoid depletion of spleen, lymph nodes, and thymus, which did not contain Hassall corpuscles. In the absence of other documented cases in the family, the mode of inheritance could not be defined. RESULTS In the 37 fetuses investigated for diagnosis of hemoglobinopathies or diagnosis of S C I D , contamination with maternal blood as judged by red blood cell size and the Kleihauer test was nil or below 3% in 31 samples and below 10% in six others. In the 35 presumably nonimmunodeficient fetuses, T- and B-lymphocyte membrane markers, including common H L A - D R and /32-microglobulin, were easily detectable by immunofluorescence (Table). These percentages were comparabie to those found in adults, except for a moderate increase of B-cell number and a slight decrease of T-cell number. Respective proportions of O K T 4 (helper subset) and O K T 8 (suppressive-cytotoxic subset) among T-cell populations (OKT3 positive cells) were also similar in fetuses and adults./32-Microglobulin and H L A - D R antigens were normally found on fetal leukocyte membranes. PHA-induced proliferative responses were identical in fetuses and control adults. In the two fetuses at risk for S C I D , immunologic studies showed no detectable immunologic defect (Table), which has been completely confirmed after birth.
Volume 1O1 Number 6
Pure fetal blood (500 #1) was obtained at the twentysecond week of gestation from the propositus (fetus) at risk for SCID. We observed a profound lymphopenia (200/tzl) characterized by a complete absence of mature T- and B-lymphocytes (Table). Termination of pregnancy was performed by local injection of prostaglandin E2. The fetus (a male) measured 18 cm from crown to rump and weighed 550 gm, corresponding to the presumed term. The alymphocytosis was confirmed in blood (300 ~1). Not T- and B-lymphocyte markers were detected and, as expected, PHA-induced proliferation was nil (Table). The macroscopic study showed no anomaly except for the weight of th e thymus (0.107 gin). At the histologic examination, the lobulation of the thymus was normal, but it contained no lymphocytes and only a few Hassall corpuscles. T h e spleen was deprived of white pulp; the lymph nodes, normally located, were devoid of lymphocytes. No lymphocytes were observed in the gut mucosa and appendix. DISCUSSION The diagnosis of SCID associated with enzyme abnormality has been accomplished during intrauterine life by amniotic cell enzymatic study, l In SCID without detectable enzyme deficiency, diagnosis remains possible in the prenatal period by analysis of T- and B-lymphocyte markers of pure fetal blood obtained at fetoscopy. We herein report the first prenatal diagnosis of SCID characterized by a profound lymphopenia and an absence of Tand B-lymphocyte surface markers. We believe that these results, compared to normal values observed in nonimmunodeficient fetuses, including two fetuses at risk for SCID studied previously,6justified the termination of pregnancy. The diagnosis was confirmed by histologic examination, which showed a complete lymphocyte depletion of lymphoid organs. The lymphoid organs of 22-week fetuses contain easily detectable lymphocytes already organized in the spleen (white pulp) and lymph nodes.7 In the propositus, thymus weight was one-tenth the norm for the age of gestation? The detection of lymphocyte membrane markers may permit antenatal diagnosis of several types of SCID, characterized by either absence of both T and B cells (alymphocytosis with agammagiobulinemia) or a defect of precursor T-lymphocytes.9 Furthermore, it may allow the prenatal diagnosis of X-linked agammaglobulinemia characterized by a complete absence of surface immunoglobulin-bearing cells. We used the same method to detect other membrane-associated antigens, Such as histocompatilility antigens A-B and DR and 3z-microglobulin, known to be poorly expressed on leukocytes of some Patients with SCID. 1~One fetus at risk for this disease was examined for prenatal diagnosis; normal expression of HLA and 32-
Clinical and laboratory observations
997
microglobulin was observed and was confirmed after birth. 6 We must stress that prenatal diagnosis of immune deficiency can be performed only when a previously affected child in the family has been investigated. The precise abnormality of lymphocyte markers must be known to allow a prenatal diagnosis in a further pregnancy. The mode of inheritance of an immune deficiency must also be taken into consideration because, in X-linked inherited disease, fetoscopy would be proposed only in male fetuses of pregnant women at risk. Other immune deficiencies are not yet detectable in the prenata ! period using detection of surface antigen markers of T- and B-lymphocytes or proliferative response of T-lymphocytes induced by mitogens. For example, prenatal diagnosis of ataxia telangiectasia and Wiskott-Aldrich syndrome cannot be performed because they are not characterized by an absence of a defined lymphocyte population or by a constant immunologic abnormality. REFERENCES 1. Hirschhorn R, Beratis N, and Rosen FS: Adenosine desaminase deficiency in a child diagnosed prenatally, Lancet 1:73, 1975. 2. Dubart A, Gooseens M, Beuzard Y, Monplaisir N, Testa U, Basset P, and Rosa J: Prenatal diagnosis of hemoglobinopathies: Comparison of the results obtained by isoelectric focusing of hemoglobinsand by chromatography of radioactive globinchains, Blood 56:1092, 1980. 3. Firshein S, Hoyer L, Lazarchick J, Forget B, Hobbins J, Clyne L, Pitlick F, Muir A, Merkatz I, and Mahoney M: Prenatal diagnosis of classic hemophilia, N Engl J Med 300:937, 1979. 4. Newburger PE, Cohen HJ, Rothschild SB, Hobbins C, Malawista SE, and Mahoney M: Prenatal diagnosis of chronic granulomatous disease, N Engl J Med 300:178, 1979. 5. Rodeck CH, and Campbell CH: Umbilical cord insertion as source of pure fetal blood for prenatal diagnosis, Lancet 1:1244, 1979. 6. Durandy A, Dumez Y, Oury C, and Griscelli C: Prenatal testing for inherited immune deficiencies by fetal blood sampling, Prenatal Diagnosis (in press). 7. Stites P, Caldwell J, Carr M, and Fudenberg H: Ontogeny of immunity in humans, Clin Immunol Immunopathol 4:519, 1975. 8. Bellanti J: General immunobiology:Concepts of immunobiology, in: immunology. Philadelphia, 1971, W. B. Saunders Co.,p 13, 9. Griscelli C, Durandy A, Virelizier JL, Ballet J J, and Daguillard F: Selective defect of precursor T cells in SCID with B lymphocytes. J PEDIATR93:404, 1978. 10. Griscelti C, Durandy A, Virelizier JL, Hors J, Lepage V, and Colombani J: Impaired cell to cell interaction in partial combined immunodeficiencywith variable expressionof HLA antigens, in Seligmann M, and Hitzig WH, editors: Primary immunodeficiencies,INSERM Symposium No. 16, Amsterdam 1980, Elsevier/North Holland Biomedical Press, p 499.