1293 TRANSMISSION OF HEPATITIS B BY IMMUNE SERUM GLOBULIN
SIR,-Accounts of the transmission of hepatitis B by occasional lots of immune serum globulin (ISG)’-5 and of the detection of HBsAg in occasional lots of ISG6,7 have tended not to address the method of preparation of the ISG or its physicochemical purity or whether the plasma source for the globulin had been tested for HBsAg by sensitive third-generation tests. Answers to these questions are necessary because of the,lack of association between ISG prepared by Cohn cold ethanol fractionation and the transmission of hepatitis B in the past,8 the evidence that HBsAg-negative ISG prepared from HBsAgpositive plasma did not transmit hepatitis B to volunteers,8 and the absence of HBsAg in ISG prepared from HBsAg-negative plasma9 and from HBsAg-positive plasma in some cases.’o," Additional factors which should be considered include the possibility of false-positive tests for HBsAg (the assays used in most of these studies were standardised for testing serum, rather than the more viscous ISG) and the fact that the hepatitis B virus and HBsAg can coexist in varying ratios in both plasma and plasma derivatives from infected persons. 12 To clarify these issues, we obtained from a U.S. manufacturer a sample of ISG which had been implicated in an outbreak of hepatitis B. This ISG had been prepared from plasma collected and pooled before U.S. regulations required that individual plasma units be tested for HBsAg by sensitive test methods. At the time of collection, all plasma units were reported to be negative by the relatively insensitive counterelectrophoresis test. This ISG was manufactured from pooled plasma by Cohn-Oncley cold ethanol fractionation. A sample of the final ISG preparation in powder form was reconstituted with distilled water as a 10% suspension (w/v). Testing for HBsAg by radioimmunoassay (RIA) and reversed passive hw-magglutination on this preparation revealed positive results (endpoint dilution titre 1:10 and titre 1:32, respectively). Neutralisation with anti-HBs confirmed these positive results. Anti-HBcAg was present in this reconstituted preparation with an end-point dilution titre by RIA of 1:1000. Anti-HBs was detected by RIA, but at too low a titre to be confirmed by neutralisation with HBsAg. No intact viral particles were seen when this ISG was examined by immune electronmicroscopy. Analysis by ultracentrifugation (kindly done by Mr A. M.
1.
Young,
Bureau of
of ISG. 10 ml of
a
Biologics)
revealed
a
sedimentation profile
12% suspension of this ISG
was given intraan amount similar chimpanzee, colony-born patients in the outbreak in which this lot had
U.S.
venously to that given to been implicated. Weekly to a
serum samples from this chimpanzee revealed the transmission of hepatitis B to the chimpanzee, with HBsAg (RIA) detectable from weeks 9 to 13 after inoculation. Anti-HBc (RIA) was first detected at week 12, and anti-HBs (RIA) at week 13. Serum aspartate and alanine aminotransferase levels were increased from weeks 12 to 16. Histopathological changes were detected at one of the monthly liver biopsies (week 13) and were characterised by lymphocytic infiltration, parenchymal cell angulation, and eosinophilic degeneration with acidophilic bodies. No other chimpanzees housed in this facility were inoculated or infected with hepatitis B virus during this time. This study documents the transmission of hepatitis B by ISG made from plasma collected before the U.S. requirement to test donor plasma for HBsAg by sensitive tests. Although the intravenous route may have enhanced the transmission of hepatitis B, this study confirms the presence of HBV in an ISG preparation which had been associated with a hepatitis B outbreak among humans receiving it by the intramuscular route. ISG fractionated by the Cohn-Oncley cold ethanol procedure is often thought to be unable to transmit hepatitis B because of the combination of Cohn-Oncley fractionation and neutral-’ isation of HBV by anti-HBs present in the ISG; however, the anti-HBs may not neutralise the HBV if high concentrations of HBV, low concentrations of anti-HBs, or both are present in the original plasma pool. Such ISG might become more prevalent as donors with high titre anti-HBs are excluded from the manufacture of hepatitis B immune globulin and their plasma is no longer used in preparing ISG; however, this does not seem to be happening in the U.S. probably because of the elimination of HBsAg-positive plasma from the preparation of ISG by sensitive test methods (unpublished).
RISK OF HEPATITIS B TRANSMISSION BY ISG
Morgado AF, da Fonte JG. An outbreak of hepatitis attributable to inoculation with contaminated gamma globulin. Bull Pan Am Health Org 1979;
13: 177-86. FL, Crovari P, de Flora S. Hepatitis B in subjects treated with a drug containing immunoglobulins. J Infect Dis 1977; 135: 252-58. 3. Nakamura S, Sato T. Acute hepatitis B after administration of gammaglobu2. Petrilli
lin. Lancet 1976; i: 478. 4. John JT, Ninan GT, Rajagopalan MS, et al. Epidemic hepatitis B caused by commercial human immunoglobulin. Lancet 1979; 1: 1074. 5. de Silva LC, Sette H, Antonacio F, Lopes JD. Commercial gammaglobulin (CGG) as a possible vehicle of transmission of HBsAg in familial clustering. Rev Inst Med Trop Sao Paulo 1977; 19: 352-54. 6. Meneghetti F, Pornaro E, Bertaggia A, Ongaro G. HBsAg investigation in commercial preparations containing gamma globulins. G Mal Infett 1977; 29: 1027-30. 7. Dioguardi N, de Franchis R. Hepatitis-B surface antigen in commercial gamma-globulin. Lancet 1975; ii: 816. 8. Murray R, Diefenbach WCL, Geller H, Leone NC, Ratner F. The problem of reducing the danger of serum hepatitis from blood and blood products. NY State J Med 1955; 55: 1145-50. 9. Hoofnagle JH, Gerety RJ, Barker LF. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-13. 10. Holland PV, Alter HJ, Purcell RH, Lander JT, Sgouris JT, Schmidt PJ. Hepatitis B antigen (HBAg) and antibody (anti-HBAg) in cold ethanol fractions of human plasma. Transfusion 1972; 12: 363-70. 11. Trepo C, Hantz O, Jacquier MF, Nemoz G, Cappel R, Trepo D. Different fates of hepatitis B virus markers dunng plasma fractionation. Vox Sang
HBsAg-positive lots of ISG are rare. In a study of 1278 lots of ISG prepared by nineteen U.S. manufacturers between 1962 and 1974, only 10 lots (0-8%) contained HBsAg, and all of these were produced by two manufacturers. 13 No lot of ISG submitted to the Bureau of Biologics for release since 1973 has been HBsAg-positive9 (and unpublished); all have had detectable anti-HBs. The chances of an HBsAg-positive lot of ISG occurring are even less likely now, since the 1975 requirement that all donor plasma be tested for HBsAg by third generation methods. Thus, U.S. regulations requiring testing of donor plasma for HBsAg by sensitive methods appear to have prevented the manufacture of "high risk" ISG such as that described here (table). Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20205, U.S.A.
EDWARD TABOR ROBERT J. GERETY
1978; 35: 143-48. 12.
Barker LF. The prevalence of hepatitis B surface antigen in commercially prepared plasma products. J Lab Clin Med 1976; 88: 102-13.
Hoofnagle JH, Gerety RJ, Thiel J,
13.
Barker LF. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-13.
Hoofnagle JH, Gerety RJ,
’
SIR,-Accounts of the transmission of hepatitis B by occasional lots of immune serum globulin (ISG)’-5 and of the detection of HBsAg in occasional lots of ISG6,7 have tended not to address the method of preparation of the ISG or its physicochemical purity or whether the plasma source for the globulin had been tested for HBsAg by sensitive third-generation tests. Answers to these questions are necessary because of the,lack of association between ISG prepared by Cohn cold ethanol fractionation and the transmission of hepatitis B in the past,8 the evidence that HBsAg-negative ISG prepared from HBsAgpositive plasma did not transmit hepatitis B to volunteers,8 and the absence of HBsAg in ISG prepared from HBsAg-negative plasma9 and from HBsAg-positive plasma in some cases.’o," Additional factors which should be considered include the possibility of false-positive tests for HBsAg (the assays used in most of these studies were standardised for testing serum, rather than the more viscous ISG) and the fact that the hepatitis B virus and HBsAg can coexist in varying ratios in both plasma and plasma derivatives from infected persons. 12 To clarify these issues, we obtained from a U.S. manufacturer a sample of ISG which had been implicated in an outbreak of hepatitis B. This ISG had been prepared from plasma collected and pooled before U.S. regulations required that individual plasma units be tested for HBsAg by sensitive test methods. At the time of collection, all plasma units were reported to be negative by the relatively insensitive counterelectrophoresis test. This ISG was manufactured from pooled plasma by Cohn-Oncley cold ethanol fractionation. A sample of the final ISG preparation in powder form was reconstituted with distilled water as a 10% suspension (w/v). Testing for HBsAg by radioimmunoassay (RIA) and reversed passive hw-magglutination on this preparation revealed positive results (endpoint dilution titre 1:10 and titre 1:32, respectively). Neutralisation with anti-HBs confirmed these positive results. Anti-HBcAg was present in this reconstituted preparation with an end-point dilution titre by RIA of 1:1000. Anti-HBs was detected by RIA, but at too low a titre to be confirmed by neutralisation with HBsAg. No intact viral particles were seen when this ISG was examined by immune electronmicroscopy. Analysis by ultracentrifugation (kindly done by Mr A. M.
1.
Young,
Bureau of
of ISG. 10 ml of
a
Biologics)
revealed
a
sedimentation profile
12% suspension of this ISG
was given intraan amount similar chimpanzee, colony-born patients in the outbreak in which this lot had
U.S.
venously to that given to been implicated. Weekly to a
serum samples from this chimpanzee revealed the transmission of hepatitis B to the chimpanzee, with HBsAg (RIA) detectable from weeks 9 to 13 after inoculation. Anti-HBc (RIA) was first detected at week 12, and anti-HBs (RIA) at week 13. Serum aspartate and alanine aminotransferase levels were increased from weeks 12 to 16. Histopathological changes were detected at one of the monthly liver biopsies (week 13) and were characterised by lymphocytic infiltration, parenchymal cell angulation, and eosinophilic degeneration with acidophilic bodies. No other chimpanzees housed in this facility were inoculated or infected with hepatitis B virus during this time. This study documents the transmission of hepatitis B by ISG made from plasma collected before the U.S. requirement to test donor plasma for HBsAg by sensitive tests. Although the intravenous route may have enhanced the transmission of hepatitis B, this study confirms the presence of HBV in an ISG preparation which had been associated with a hepatitis B outbreak among humans receiving it by the intramuscular route. ISG fractionated by the Cohn-Oncley cold ethanol procedure is often thought to be unable to transmit hepatitis B because of the combination of Cohn-Oncley fractionation and neutral-’ isation of HBV by anti-HBs present in the ISG; however, the anti-HBs may not neutralise the HBV if high concentrations of HBV, low concentrations of anti-HBs, or both are present in the original plasma pool. Such ISG might become more prevalent as donors with high titre anti-HBs are excluded from the manufacture of hepatitis B immune globulin and their plasma is no longer used in preparing ISG; however, this does not seem to be happening in the U.S. probably because of the elimination of HBsAg-positive plasma from the preparation of ISG by sensitive test methods (unpublished).
RISK OF HEPATITIS B TRANSMISSION BY ISG
Morgado AF, da Fonte JG. An outbreak of hepatitis attributable to inoculation with contaminated gamma globulin. Bull Pan Am Health Org 1979;
13: 177-86. FL, Crovari P, de Flora S. Hepatitis B in subjects treated with a drug containing immunoglobulins. J Infect Dis 1977; 135: 252-58. 3. Nakamura S, Sato T. Acute hepatitis B after administration of gammaglobu2. Petrilli
lin. Lancet 1976; i: 478. 4. John JT, Ninan GT, Rajagopalan MS, et al. Epidemic hepatitis B caused by commercial human immunoglobulin. Lancet 1979; 1: 1074. 5. de Silva LC, Sette H, Antonacio F, Lopes JD. Commercial gammaglobulin (CGG) as a possible vehicle of transmission of HBsAg in familial clustering. Rev Inst Med Trop Sao Paulo 1977; 19: 352-54. 6. Meneghetti F, Pornaro E, Bertaggia A, Ongaro G. HBsAg investigation in commercial preparations containing gamma globulins. G Mal Infett 1977; 29: 1027-30. 7. Dioguardi N, de Franchis R. Hepatitis-B surface antigen in commercial gamma-globulin. Lancet 1975; ii: 816. 8. Murray R, Diefenbach WCL, Geller H, Leone NC, Ratner F. The problem of reducing the danger of serum hepatitis from blood and blood products. NY State J Med 1955; 55: 1145-50. 9. Hoofnagle JH, Gerety RJ, Barker LF. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-13. 10. Holland PV, Alter HJ, Purcell RH, Lander JT, Sgouris JT, Schmidt PJ. Hepatitis B antigen (HBAg) and antibody (anti-HBAg) in cold ethanol fractions of human plasma. Transfusion 1972; 12: 363-70. 11. Trepo C, Hantz O, Jacquier MF, Nemoz G, Cappel R, Trepo D. Different fates of hepatitis B virus markers dunng plasma fractionation. Vox Sang
HBsAg-positive lots of ISG are rare. In a study of 1278 lots of ISG prepared by nineteen U.S. manufacturers between 1962 and 1974, only 10 lots (0-8%) contained HBsAg, and all of these were produced by two manufacturers. 13 No lot of ISG submitted to the Bureau of Biologics for release since 1973 has been HBsAg-positive9 (and unpublished); all have had detectable anti-HBs. The chances of an HBsAg-positive lot of ISG occurring are even less likely now, since the 1975 requirement that all donor plasma be tested for HBsAg by third generation methods. Thus, U.S. regulations requiring testing of donor plasma for HBsAg by sensitive methods appear to have prevented the manufacture of "high risk" ISG such as that described here (table). Hepatitis Branch, Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20205, U.S.A.
EDWARD TABOR ROBERT J. GERETY
1978; 35: 143-48. 12.
Barker LF. The prevalence of hepatitis B surface antigen in commercially prepared plasma products. J Lab Clin Med 1976; 88: 102-13.
Hoofnagle JH, Gerety RJ, Thiel J,
13.
Barker LF. Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-13.
Hoofnagle JH, Gerety RJ,
’
