- Email: [email protected]
MONOSPECIFIC ANTISERA, HYBRIDOMA ANTIBODIES, AND IMMUNOASSAYS FOR CYSTIC FIBROSIS PROTEIN
313 TABLE II-LABETALOL PATIENTS POSITIVE FOR AMA
MONOSPECIFIC ANTISERA, HYBRIDOMA ANTIBODIES, AND IMMUNOASSAYS FOR CYSTIC FIBROSIS PROTEIN
I
I
* The two figures indicate the duration of labetalol therapy in months for two successive tests. During the interval between the two tests the patient became AMA positive.
contrast, of the 6 AMA
positive patients on treatment regimens
which excluded labetalol, 4 already displayed serum AMA when first tested so that the influence of their therapy on the development of these autoantibodies could not be determined. Of the remaining 2 patients in this group, 1 acquired AMA (1:8) after treatment with oxprenolol, prazosin, and cyclopenthiazide, while the other 1 was AMA positive (1:16) only once in nine tests carried out over a 43-month period of continuous treatment with propranolol, methyldopa, and cyclopenthiazide. AMA developing in labetalol-treated patients were of very low titre in all but one instance and did not appear to be rising in titre on subsequent testing. In one patient (table n, no 4) titres were negative before initiation of labetalol therapy and again after 9 months of treatment, and then were found to be positive after 23 months treatment, with titres rising rapidly over the next 3 months to 1:128. The AMA positive labetalol-treated patients were fully investigated (ECG, chest X-ray, liver function, and screening for autoimmune diseases) but no abnormalities attributable to their drug therapy were found. In particular there was no liver dysfunction, myocarditis, pericarditis, arthritis, or skin reactions. AMA are rare in healthy subjects, occurring in only 0-5% of our 2600 controls,3 and their development as a result of drug therapy is worrying. For example, AMA are associated with primary biliary cirrhosis, where over 90% of patients are positive,4 in other liver conditions (transiently), and in up to 8% of patients with systemic lupus erythematosus and other autoimmune diseases. None of our labetalol-treated AMApositive patients had evidence of these diseases. The drug ’Venocuran’ used in the treatment of varicose veins and containing phenopyrazone, cardiac glycosides, and horsechestnut extract, has been implicated in the induction of AMA6 in up to 90% of long-term users of this drug some of whom had a
syndrome designated "pseudolupus". Our AMA positive patients had been on labetalol for many months before these antibodies developed and they were usually taking a high dose of the drug. The development of AMA has not yet posed a clinical problem in patients on labetalol ; nevertheless screening of such patients for AMA antibody and the search for possible clinical complications seems warranted. This study was supported by the National Heart Foundation of New Zealand and by the Medical Research Council of New Zealand.
Department of Medicine,
University of Auckland School of Medicine; and Hypertension Clinic, Auckland Hospital,
J. DOUGLAS WILSON ROGER J. BOOTH JOCELYN Y. BULLOCK
Auckland, New Zealand
D. GRAEME CAMPBELL
4. Domach D. Autoimmunity in liver disease. In: Schwartz RS ed. Progress in clinical immunology: Vol I. New York; Grune & Stratton, 1972: 45-70. 5. Doniach D. Delhanty J, Lindquist HJ, Catterall RD. Mitochondrial and other tissue autoantibodies in patients with biological false positive reactions for syphilis. Clin Exp Immunol 1970; 6: 871-84. 6. Grob PJ, Muller-Schoop JW, Hacks MA, Joller-Jemelka HI. Drug-induced pseudolupus. Lancet 1975; ii: 144-48.
SIR,-We have found the cystic fibrosis protein (CFP), demonstrated by isoelectric focusing of human serum, to be a specific marker for the identification of CF homozygotes and obligate heterozygote carriers of the defective gene responsible for this disorder.’ The existence of the CFP and its ability to distinguish obligate heterozygotes and homozygotes from normal unaffected controls have been independently confirmed by three groups,2-4 including Manson and Brockwho described a quantitative immunoassay for CF based on guineapig antiserum monospecific for CFP. We have also succeeded in raising monospecific antisera to CFP and wish to report on our development of both xenogeneic polyvalent monospecific mouse antisera and hybridoma antibodies to CFP. Mice were studied because success in raising mouse antisera would indicate the feasibility of producing large quantities of hybridoma antibody ; also the immunisation schedule, route of injection, and proper chemical state of the immunogen for the production of high quality antisera could be worked out cheaply using this
species. BALB--c mice were immunised intraperitoneally weekly for 3-6 weeks with CFP. The CFP was first partly purified by isolating IgG-CFP complexes from serum with Staphylococcus aureus protein A covalently linked to ’Sepharose CL-4B’. Protein A specifically removes IgG from human serum,5 and CFP (which is found non-covalently linked to IgG) is removed with it.The use of protein A allowed us to eliminate trace serum components such as kallikrein and plasmin whose presence might make the production of monospecific antisera more difficult, since they have isoelectric points (pI) close to that of the CFP. !, CFP was further purified by electrofocusing of the CFP-IgG complexes.6 The CFP region was then excised and emulsified in isotonic acetate buffer, pH 4.7(rather than saline4) to ensure that the CFP remained dissociated from IgG when injected. The resulting hyperimmune mouse sera routinely produced two lines of precipitation against CF homozygote or heterozygote sera and one line against normal control sera (figure, top) when analysed by counterimmunoelectrophoresis (CIE) or immunoelectrophoresis.8 The antisera were rendered monospecific by absorption with CFP-negative human serum or IgG to remove unwanted antibodies (figure, bottom). Complete concordance was obtained between results for CFP positivity by isoelectric focusing and CIE when twenty control, ten homozygote, and eight obligate heterozygote sera were analysed. The precipitation line against heterozygote sera was usually closer to the antigen well and fainter than the line obtained against CF homozygote sera, indicating that the heterozygous sera contained less CFP. Preliminary results using rocket immunoelectrophoresis confirmed the results of CIE, as well as those of Manson and Brock,4by establishing a quantitative difference in the amount of CFP in the homozygote and heterozygote sera. CIE, however, is 1. Wilson
GB, Fudenberg HH, Jahn TL. Studies on cystic fibrosis using isoelec-
I: an assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum. Pediat Res 1975; 9: 635-40. 2. Scholey J, Applegarth DA, Davidson AGF, Wong LTK. Detection of cystic fibrosis protein by electrofocusing. Pediat Res 1978; 12: 800-01. 3. Tulley GW, Nevin BG, Young IR, Nevin NC. Detection of cystic fibrosis protein by isoelectric focusing of serum. Pediat Res 1979; 13: 1078. 4. Manson JC, Brock DJH. Development of a quantitative immunoassay for the cystic fibrosis gene. Lancet 1980; i: 330-31. 5. Hjelm H, Hjelm K, Sjöquist, J. Protein A from Staphylococcus aureus: Its isolation by affinity chromatography and its use as an immunosorbent for the isolation of immunoglobulins. FEBS Lett 1972; 28: 73-75. 6. Wilson GB. Cystic fibrosis protein, a confirmed diagnostic marker for detecting heterozygote carriers: Significance in relation to future screening and to a proposed defect in alpha2-macroglobulin. Pediat Res 1979; 13:
tric
focusing
1079-81. 7.
Austen KF. The use of isoelectric focusing to study components of the human plasma kinin-forming system. Ann NY Acad Sci
Spagg J, Kaplan AP, 1973; 209: 372-83.
8. Oudin
J, Williams CA. Precipitation analysis by diffusion in gels. In: Williams CA, Chase MW, eds. Methods in immunology and immunochemistry: vol III. New York: Academic Press, 1973: 103-374.
314 INDOMETHACIN FOR PERSISTENT DUCTUS ARTERIOSUS
SIR,-Dr Firth and Dr Pickering (July 19,
p.
144) correctly
that the response of the neonatal ductus to indomethacin is greatest when the drug is administered in the first 2 weeks after birth.1 My updated experience with indomethacin treatment for persistent ductus arteriosus (PDA) in the very-lowbirthweight infant (mean gestation 28 weeks, mean weight 976 g) is shown in the table. If medical ductus closure is attempted after the first 2 weeks of life the results are disappointing and surgical ligation is often necessary. state
RESPONSE TO INDOMETHACIN BY AGE
Results obtained when either cystic fibrosis homozygote (CF), heterozygote carrier (H), or normal unaffected control (N) sera were analysed for reactivity against hyperimmune sera obtained by immunising mice with purified CFP by CIE. Top set of wells contained mouse antiserum and bottom set contained human serum. The anode was at the bottom. Top.-Results obtained using two different antisera before absorption to remove non-specific antibodies. Bottom.-Results obtained using antisera no. 2 rendered monospecific after absorption with CFP-negative normal control serum. Note that the precipitation line obtained for the heterozygote carrier (indicated by the arrow) is closer to the antigen well and fainter than the line obtained for the CF homozygote sample, indicating less CFP in the former sample. (The haloes around the antigen wells are artifacts.)
quicker than rocket immunoelectrophoresis (90 min to run) requires less antiserum. CIE could therefore serve as a rapid qualitative immunoassay for detecting carriers. Hybridoma clones were prepared by fusing spleen cells from and
mice with non-immunoglobulin-secreting murine myeloma cells (line S194/S-XXO BU.1), employing standard techniques.9,lo Hybridoma antibody to CFP could be readily demonstrated in concentrated culture supernatants by CIE. We are continuing to use CIE as the method of choice for antibody detection in attempts to isolate single hybridoma clones for the establishment of monoclonal antibody-producing cell lines. Our results to date indicate that mice, as well as guineapigs, can be used to produce antibodies to CFP and confirm that CFP levels in serum and possibly other biological fluids can be used to detect and distinguish CF homozygote and CF obligate heterozygote subjects.
immune
by
USPHS grants HD-09938 and
Charleston, South Carolina 29403, U.S.A.
GREGORY B. WILSON
Research CA-25746.
supported
in part
Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, 9. Köhler
G, Milstein C. Continuous cultures of fused cells secreting antibody
of predefined specificity. Nature 1975; 256: 495-96. 10. Vaughan VL, Hansen D, Stadler J. Parameters of polyethylene glycolinduced cell fusion and hybridization in lymphoid cell lines. Somatic Cell Genet 1976; 2: 537-44.
Despite the apparently good results of early treatment indomethacin should not be given lightly to the newborn. Prostaglandin inhibition is likely to alter blood flow to many body organs. Transient renal and electrolyte disturbancesl·2 and necrotising enterocolitis3 have been reported as complications. Recently despite ductus closure two infants treated in Belfast developed necrotising enterocolitis and one of these died. Retrolental fibroplasia is at least a theoretical possibility,4 and bleeding problems3·5 and bilirubin displacement from albumin6 may also occur. For these reasons I feel that certain criteria should be fulfilled before indomethacin is used to treat newborn babies with PDA.7 First, the ductus shunt should be large and have failed to respond to conservative measures, so that ligation becomes the only alternative.l Full evaluation of the infant with echocardiography or cardiac catheterisation will be necessary to determine the size of the left-to-right shunt. I accept a leftatrial/aorta ratio of 1.5/1 or greater as evidence of a large ductus shunt.7 Secondly, biochemical monitoring of coagulation, renal function, and bilirubin is advisable.1,6 I do not administer indomethacin unless the platelet count is >100 000/1, prothombin time <20 s, partial thromboplastin time <70 s, urine output >2 ml/kg/h, serum creatinine <130 mol/1 (1-5 mg/dl), and bilirubin <150 fLmol/l (8 mgldl).t.7 Thirdly, I always obtain the verbal consent of the mother before giving indomethacin. It is my firm opinion that this potent drug should be administered only in neonatal intensive-care units where the appropriate investigations are readily available and where treated babies can be closely followed up. Neonatal paediatricians must guard against a repetition of therapeutic misadventures of the past-tragedies with chloramphenicol, sulphonamides, and oxygen are not too distant memories. Royal Maternity Hospital, Belfast BT12 6BB
1.
H. L. HALLIDAY
Hirata T, Brady JP. Indomethacin therapy for large patent ductus arteriosus in the very low birth weight infant: results and complications. Pediatrics 1979; 64: 154-59. 2. Heymann MA, Rudolph AM, Silverman NH. Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis. N Engl J Med 1976; 295: 530-33. 3. Harinck E, van Ertbruggen I, Senders RC, Moulaert AJ. Problems with indomethacin for ductus closure. Lancet 1977; ii: 245. 4. Rudolph AM, Heymann MA. Medical treatment of the ductus arteriosus.
Halliday HL,
Hosp Pract 1977; 12: 57-65. 5. Friedman Z, Whitman V, Maisels MJ, Berman W Jr, Marks KH, Vessel ES. Indomethacin disposition and indomethacin-induced platelet dysfunction in premature infants. J Clin Phasmacol 1978; 18: 272-79. 6. Kitterman JA. Patent ductus arteriosus: current clinical status. Arch Dis Child 1980; 55: 106-09. 7. Halliday HL. Assessment of left-to-right shunt in neonatal ductus arteriosus. M.D. thesis, Queen’s University Belfast, 1980.
MONOSPECIFIC ANTISERA, HYBRIDOMA ANTIBODIES, AND IMMUNOASSAYS FOR CYSTIC FIBROSIS PROTEIN
I
I
* The two figures indicate the duration of labetalol therapy in months for two successive tests. During the interval between the two tests the patient became AMA positive.
contrast, of the 6 AMA
positive patients on treatment regimens
which excluded labetalol, 4 already displayed serum AMA when first tested so that the influence of their therapy on the development of these autoantibodies could not be determined. Of the remaining 2 patients in this group, 1 acquired AMA (1:8) after treatment with oxprenolol, prazosin, and cyclopenthiazide, while the other 1 was AMA positive (1:16) only once in nine tests carried out over a 43-month period of continuous treatment with propranolol, methyldopa, and cyclopenthiazide. AMA developing in labetalol-treated patients were of very low titre in all but one instance and did not appear to be rising in titre on subsequent testing. In one patient (table n, no 4) titres were negative before initiation of labetalol therapy and again after 9 months of treatment, and then were found to be positive after 23 months treatment, with titres rising rapidly over the next 3 months to 1:128. The AMA positive labetalol-treated patients were fully investigated (ECG, chest X-ray, liver function, and screening for autoimmune diseases) but no abnormalities attributable to their drug therapy were found. In particular there was no liver dysfunction, myocarditis, pericarditis, arthritis, or skin reactions. AMA are rare in healthy subjects, occurring in only 0-5% of our 2600 controls,3 and their development as a result of drug therapy is worrying. For example, AMA are associated with primary biliary cirrhosis, where over 90% of patients are positive,4 in other liver conditions (transiently), and in up to 8% of patients with systemic lupus erythematosus and other autoimmune diseases. None of our labetalol-treated AMApositive patients had evidence of these diseases. The drug ’Venocuran’ used in the treatment of varicose veins and containing phenopyrazone, cardiac glycosides, and horsechestnut extract, has been implicated in the induction of AMA6 in up to 90% of long-term users of this drug some of whom had a
syndrome designated "pseudolupus". Our AMA positive patients had been on labetalol for many months before these antibodies developed and they were usually taking a high dose of the drug. The development of AMA has not yet posed a clinical problem in patients on labetalol ; nevertheless screening of such patients for AMA antibody and the search for possible clinical complications seems warranted. This study was supported by the National Heart Foundation of New Zealand and by the Medical Research Council of New Zealand.
Department of Medicine,
University of Auckland School of Medicine; and Hypertension Clinic, Auckland Hospital,
J. DOUGLAS WILSON ROGER J. BOOTH JOCELYN Y. BULLOCK
Auckland, New Zealand
D. GRAEME CAMPBELL
4. Domach D. Autoimmunity in liver disease. In: Schwartz RS ed. Progress in clinical immunology: Vol I. New York; Grune & Stratton, 1972: 45-70. 5. Doniach D. Delhanty J, Lindquist HJ, Catterall RD. Mitochondrial and other tissue autoantibodies in patients with biological false positive reactions for syphilis. Clin Exp Immunol 1970; 6: 871-84. 6. Grob PJ, Muller-Schoop JW, Hacks MA, Joller-Jemelka HI. Drug-induced pseudolupus. Lancet 1975; ii: 144-48.
SIR,-We have found the cystic fibrosis protein (CFP), demonstrated by isoelectric focusing of human serum, to be a specific marker for the identification of CF homozygotes and obligate heterozygote carriers of the defective gene responsible for this disorder.’ The existence of the CFP and its ability to distinguish obligate heterozygotes and homozygotes from normal unaffected controls have been independently confirmed by three groups,2-4 including Manson and Brockwho described a quantitative immunoassay for CF based on guineapig antiserum monospecific for CFP. We have also succeeded in raising monospecific antisera to CFP and wish to report on our development of both xenogeneic polyvalent monospecific mouse antisera and hybridoma antibodies to CFP. Mice were studied because success in raising mouse antisera would indicate the feasibility of producing large quantities of hybridoma antibody ; also the immunisation schedule, route of injection, and proper chemical state of the immunogen for the production of high quality antisera could be worked out cheaply using this
species. BALB--c mice were immunised intraperitoneally weekly for 3-6 weeks with CFP. The CFP was first partly purified by isolating IgG-CFP complexes from serum with Staphylococcus aureus protein A covalently linked to ’Sepharose CL-4B’. Protein A specifically removes IgG from human serum,5 and CFP (which is found non-covalently linked to IgG) is removed with it.The use of protein A allowed us to eliminate trace serum components such as kallikrein and plasmin whose presence might make the production of monospecific antisera more difficult, since they have isoelectric points (pI) close to that of the CFP. !, CFP was further purified by electrofocusing of the CFP-IgG complexes.6 The CFP region was then excised and emulsified in isotonic acetate buffer, pH 4.7(rather than saline4) to ensure that the CFP remained dissociated from IgG when injected. The resulting hyperimmune mouse sera routinely produced two lines of precipitation against CF homozygote or heterozygote sera and one line against normal control sera (figure, top) when analysed by counterimmunoelectrophoresis (CIE) or immunoelectrophoresis.8 The antisera were rendered monospecific by absorption with CFP-negative human serum or IgG to remove unwanted antibodies (figure, bottom). Complete concordance was obtained between results for CFP positivity by isoelectric focusing and CIE when twenty control, ten homozygote, and eight obligate heterozygote sera were analysed. The precipitation line against heterozygote sera was usually closer to the antigen well and fainter than the line obtained against CF homozygote sera, indicating that the heterozygous sera contained less CFP. Preliminary results using rocket immunoelectrophoresis confirmed the results of CIE, as well as those of Manson and Brock,4by establishing a quantitative difference in the amount of CFP in the homozygote and heterozygote sera. CIE, however, is 1. Wilson
GB, Fudenberg HH, Jahn TL. Studies on cystic fibrosis using isoelec-
I: an assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum. Pediat Res 1975; 9: 635-40. 2. Scholey J, Applegarth DA, Davidson AGF, Wong LTK. Detection of cystic fibrosis protein by electrofocusing. Pediat Res 1978; 12: 800-01. 3. Tulley GW, Nevin BG, Young IR, Nevin NC. Detection of cystic fibrosis protein by isoelectric focusing of serum. Pediat Res 1979; 13: 1078. 4. Manson JC, Brock DJH. Development of a quantitative immunoassay for the cystic fibrosis gene. Lancet 1980; i: 330-31. 5. Hjelm H, Hjelm K, Sjöquist, J. Protein A from Staphylococcus aureus: Its isolation by affinity chromatography and its use as an immunosorbent for the isolation of immunoglobulins. FEBS Lett 1972; 28: 73-75. 6. Wilson GB. Cystic fibrosis protein, a confirmed diagnostic marker for detecting heterozygote carriers: Significance in relation to future screening and to a proposed defect in alpha2-macroglobulin. Pediat Res 1979; 13:
tric
focusing
1079-81. 7.
Austen KF. The use of isoelectric focusing to study components of the human plasma kinin-forming system. Ann NY Acad Sci
Spagg J, Kaplan AP, 1973; 209: 372-83.
8. Oudin
J, Williams CA. Precipitation analysis by diffusion in gels. In: Williams CA, Chase MW, eds. Methods in immunology and immunochemistry: vol III. New York: Academic Press, 1973: 103-374.
314 INDOMETHACIN FOR PERSISTENT DUCTUS ARTERIOSUS
SIR,-Dr Firth and Dr Pickering (July 19,
p.
144) correctly
that the response of the neonatal ductus to indomethacin is greatest when the drug is administered in the first 2 weeks after birth.1 My updated experience with indomethacin treatment for persistent ductus arteriosus (PDA) in the very-lowbirthweight infant (mean gestation 28 weeks, mean weight 976 g) is shown in the table. If medical ductus closure is attempted after the first 2 weeks of life the results are disappointing and surgical ligation is often necessary. state
RESPONSE TO INDOMETHACIN BY AGE
Results obtained when either cystic fibrosis homozygote (CF), heterozygote carrier (H), or normal unaffected control (N) sera were analysed for reactivity against hyperimmune sera obtained by immunising mice with purified CFP by CIE. Top set of wells contained mouse antiserum and bottom set contained human serum. The anode was at the bottom. Top.-Results obtained using two different antisera before absorption to remove non-specific antibodies. Bottom.-Results obtained using antisera no. 2 rendered monospecific after absorption with CFP-negative normal control serum. Note that the precipitation line obtained for the heterozygote carrier (indicated by the arrow) is closer to the antigen well and fainter than the line obtained for the CF homozygote sample, indicating less CFP in the former sample. (The haloes around the antigen wells are artifacts.)
quicker than rocket immunoelectrophoresis (90 min to run) requires less antiserum. CIE could therefore serve as a rapid qualitative immunoassay for detecting carriers. Hybridoma clones were prepared by fusing spleen cells from and
mice with non-immunoglobulin-secreting murine myeloma cells (line S194/S-XXO BU.1), employing standard techniques.9,lo Hybridoma antibody to CFP could be readily demonstrated in concentrated culture supernatants by CIE. We are continuing to use CIE as the method of choice for antibody detection in attempts to isolate single hybridoma clones for the establishment of monoclonal antibody-producing cell lines. Our results to date indicate that mice, as well as guineapigs, can be used to produce antibodies to CFP and confirm that CFP levels in serum and possibly other biological fluids can be used to detect and distinguish CF homozygote and CF obligate heterozygote subjects.
immune
by
USPHS grants HD-09938 and
Charleston, South Carolina 29403, U.S.A.
GREGORY B. WILSON
Research CA-25746.
supported
in part
Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, 9. Köhler
G, Milstein C. Continuous cultures of fused cells secreting antibody
of predefined specificity. Nature 1975; 256: 495-96. 10. Vaughan VL, Hansen D, Stadler J. Parameters of polyethylene glycolinduced cell fusion and hybridization in lymphoid cell lines. Somatic Cell Genet 1976; 2: 537-44.
Despite the apparently good results of early treatment indomethacin should not be given lightly to the newborn. Prostaglandin inhibition is likely to alter blood flow to many body organs. Transient renal and electrolyte disturbancesl·2 and necrotising enterocolitis3 have been reported as complications. Recently despite ductus closure two infants treated in Belfast developed necrotising enterocolitis and one of these died. Retrolental fibroplasia is at least a theoretical possibility,4 and bleeding problems3·5 and bilirubin displacement from albumin6 may also occur. For these reasons I feel that certain criteria should be fulfilled before indomethacin is used to treat newborn babies with PDA.7 First, the ductus shunt should be large and have failed to respond to conservative measures, so that ligation becomes the only alternative.l Full evaluation of the infant with echocardiography or cardiac catheterisation will be necessary to determine the size of the left-to-right shunt. I accept a leftatrial/aorta ratio of 1.5/1 or greater as evidence of a large ductus shunt.7 Secondly, biochemical monitoring of coagulation, renal function, and bilirubin is advisable.1,6 I do not administer indomethacin unless the platelet count is >100 000/1, prothombin time <20 s, partial thromboplastin time <70 s, urine output >2 ml/kg/h, serum creatinine <130 mol/1 (1-5 mg/dl), and bilirubin <150 fLmol/l (8 mgldl).t.7 Thirdly, I always obtain the verbal consent of the mother before giving indomethacin. It is my firm opinion that this potent drug should be administered only in neonatal intensive-care units where the appropriate investigations are readily available and where treated babies can be closely followed up. Neonatal paediatricians must guard against a repetition of therapeutic misadventures of the past-tragedies with chloramphenicol, sulphonamides, and oxygen are not too distant memories. Royal Maternity Hospital, Belfast BT12 6BB
1.
H. L. HALLIDAY
Hirata T, Brady JP. Indomethacin therapy for large patent ductus arteriosus in the very low birth weight infant: results and complications. Pediatrics 1979; 64: 154-59. 2. Heymann MA, Rudolph AM, Silverman NH. Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis. N Engl J Med 1976; 295: 530-33. 3. Harinck E, van Ertbruggen I, Senders RC, Moulaert AJ. Problems with indomethacin for ductus closure. Lancet 1977; ii: 245. 4. Rudolph AM, Heymann MA. Medical treatment of the ductus arteriosus.
Halliday HL,
Hosp Pract 1977; 12: 57-65. 5. Friedman Z, Whitman V, Maisels MJ, Berman W Jr, Marks KH, Vessel ES. Indomethacin disposition and indomethacin-induced platelet dysfunction in premature infants. J Clin Phasmacol 1978; 18: 272-79. 6. Kitterman JA. Patent ductus arteriosus: current clinical status. Arch Dis Child 1980; 55: 106-09. 7. Halliday HL. Assessment of left-to-right shunt in neonatal ductus arteriosus. M.D. thesis, Queen’s University Belfast, 1980.