Hepatitis B Antigen and Antibody in Active Chronic Hepatitis and Other Liver Diseases in Australia A Multicenter Collaborative Study W.G.E. Cooksley, MB, FRACP, L.W. Powell, MD, FRACP, S.P. Mistilis, MB, FRACP, I.R. Mackay, MD, FRACP, and L.F. Barker, MD
In a multicenter cooperative study, sera from 85 patients with active chronic hepatitis (ACH) were examined for the presence of hepatitis B (Australia) antigen (HBAg) by radioimmunoassay (RIA) and antibody to HBAg (anti-HBAg) by RIA and passive hemagglutination (PHA), the most sensitive currently available techniques. In addition, sera from 83 patients with other liver diseases, 98 other hospital patients, and 67 healthy controls were tested. HBAg was detected in 3 of the 85 patients (four percent) with ACH. In a further 3 patients (four percent) anti-HBAg was detected. Thus, 6 patients with ACH (seven percent) had evidence of present or prior infection with the hepatitis B virus (HBV). HBAg was also detected in 7 of the patients with other liver diseases, 2 of the other hospital patients, and none of the healthy controls. Anti-HBAg was detected in 17 of the non-ACH subjects. These results indicate that neither persistent nor prior self-limited infection with HBV is a major factor in the pathogenesis of ACH in Australia. T h e virus of serum hepatitis, HBV, is now suspected to be an important etioIogicat factor in A C H , since a high prevalence of H B A g has been found in a number of studies (1-6). In contrast to the high prevalence reported in parts of the United States (22-31~ and many countries of Europe (34-62%) (5, 6) the prevalence as judged by immunodiffusion has been as
From the Department of Medicine, University of Queensland, Royal Brisbane Hospital, Brisbane, Australia; A.W. Morrow Department of Gastroenterology, Royal Prince Alfred Hospital, Sydney, Australia; Clinical Research Unit, Walter & Eliza Hall Institute, Royal Melbourne Hospital, Melbourne, Australia; and Division of Virology, Bureau of Biologics,Food and Drug Administration, Rockville,Maryland. W.G.E. Cooksley and IR. Mackay were both in receipt of grants from the National Health and Medical Research Council of Australia. We thank Dr. Senga Whittingham for assistance with stored sera. Address for reprint requests: Dr. W.G.E. Cooksley,University Department of Medicine, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
110
low as 0-4% in England (7), Scandinavia (8), and Australia (9), suggesting geographical differences in either the frequency of chronic hepatitis due to viral hepatitis or the prevalence of H B V infection. In Australia (10) H B A g was detected in only 4 of 130 patients with A C H , using a variety of immunological techniques, immunodiffusion, immunoelectroosmophoresis (IEOP), and complement fixation, but not RIA. W i t h the introduction of the highly sensitive R I A for H B A g and anti-HBAg, application of this technique in a country claiming a low incidence of H B A g in A C H would allow further evaluation of the possibility that persistent HBAg, albeit in low titer, is involved in the pathogenesis of A C H . Therefore we have examined the sera of 85 patients with A C H , as well as sera from patients with other liver diseases, hospital inpatients with no apparent liver disease, and healthy controls, from three different geographical areas in Australia. In addition, P H A and R I A methods were employed to test
Digestive Diseases, Vol. 20, No. 2 (February 1975)
HBAg AND HBAb IN ACH Table 1. Prevalence of HBAg and Anti-HBAg in Patients with ACH and Other Diseases in Australia
Subjects studied
Center"
Patients tested (N)
HBAg positive (N)
Anti-HBAg positive (N)
ACH
M B S Total
38 29 18 85
2 1 0 3 (4%)
1 2 0 3 (4%)
Other liver diseases
M B Total
12 71 83
0 7 7 (8%)
1 4 5 (6%)
Other hospital patients
M B Total
72126 98
2 0 2
8 0 8 (8%)
B B Total
19 48 67
0 0 0
0 4 (8%) 4 (6%)
Controls Students and laboratory staff Voluntary blood donors
*M indicates Melbourne, B, Brisbane, S, Sydney. 1-Comprising systemic lupus erythematosis (22), pernicious anemia (14), rheumatoid arthritis (12), and multiple sclerosis (4), all negative, and miscellaneous (20, with 2 positive: a migrant woman with myotonic dystrophy and a woman with rheumatic carditis).
the same sera for a n t i - H B A g to detect evidence of past infection with H B V .
MATERIALS
AND METHODS
The criterion for the diagnosis of ACH was the presence of hepatitis of unknown cause that was biochemically active, as judged by raised plasma levels of transaminase enzymes for a period longer than 6 months and histological features of a destructive periportal hepatitis with or without cirrhosis (11-13). Patients contributed by the three centers are shown in Table 1. Patients with drug-induced liver disease were not included. Relevant clinical details and results of immunofluorescent testing for the serological antibodies, antinuclear factor, smooth-muscle antibody and antimitoehondrial antibody have been recently reported (10). Sera were assayed for HBAg by RIA (14, 15) at the National Institutes of Health, Bethesda, Maryland. All sera scored as positive were shown to be specific for HBAg by inhibition with excess antibody. Sera were also assayed "for anti-HBAg by RIA (14) and PHA (16) and those positive were similarly shown to be specific by inhibition with excess antigen. Sera were sent from Australia coded. Included were sera from 85 patients with ACH, from 83 patients with other liver diseases (acute and chronic liver disease), from 98 other hospital patients, and from 9 medical stuDigestive Diseases, Vol. 20, No. 2 (February 1975)
dents, 10 laboratory staff, and 48 volunteer blood donors (Tables 1 and 2). RESULTS
H B A g was detected in 3 of the 85 patients with A C H - - a prevalence of 4% ( T a b l e 1). T h e sensitivity of R I A can be seen in T a b l e s 3 and 4, where the results are c o m p a r e d with those of a previously reported study (10). A n t i - H B A g was detected in a further 3 patients (4%). T h u s , 6 patients (7%) had evidence of present or previous H B V infection. O f the 248 other subjects tested 9 were found to have H B A g in the serum (Table 1 ). O f the 83 patients with other liver diseases H B A g was detected in the sera of 7 (6 patients with acute viral hepatitis and 1 with alcoholic cirrhosis) (Table 2). A n t i - H B A g was detected in the sera of 6% of the patients with other liver diseases, 8% of the other hospital patients, and 6% of the healthy controls ( T a b l e 1). 111
COOKSLEY ET AL Table 2. Prevalence of HBAg in Patients with Liver Diseases Other Than ACH in Australia
Type of liver disease
Patients tested (N)
Acute liver disease Cholecystitis Acute viral hepatitis Alcoholic hepatitis Total
3 28 9 40
Chronic liver disease Alcoholic cirrhosis Hemochromatosis Cryptogenic cirrhosis Posthepatitic cirrhosis Primary biliary cirrhosis Wilson's disease Persistent hepatitis Chronic obstructive jaundice Cholangio carcinoma Total
14 9 10 1 3 1 1 3 1 43
Anti-HBAg positive (N)
HBAg positive (N)
6 6
1*
2 1t
1 1:1: 5
1
*Repeat testing with excess antibody could not be performed. tPatient was from Yugoslavia and had cryptogenic cirrhosis complicated by primary liver-cell cancer. :[:Patient had noncirrhotic liver.
Table 3. Patients with ACH Found Positive for HBAg Using Different Methods Method
Patients tested (N)
HBAg positive (N)
HBAg positive (%)
130 94 94 85
3 3 4 3
2 3 4 4
Immunodiffusion* Immunoelectroosmophoresis* Complement fixation* Radioimmunoassay *Results in a previously reported study (10).
Table 4. Results of Testing for HBAg in Positive Patients in Table 3 Patient Method Immunodiffusion Immu noelectroosmophoresis Complement fixation Radioimmunoassay
1
2
3
4
5
+ + + --*
+ + + --*
+ + + +
--+ +
---+
*Shown to be nonspecific by failure of inhibition with excess antibody (21).
112
Digestive Diseases, Vol. 20, No. 2 (February 1975)
HBAg AND HBAb IN ACH DISCUSSION
T h e low prevalence of H B A g detected by the most sensitive currently available techniques in patients with A C H confirms previous reports from Australia (9, 10). These results indicate that persistent H B V infection may be relevant to the pathogenesis of only a small minority of cases of A C H in Australia. T h e prevalence of a n t i - H B A g was lower in the group of A C H patients than in three of the four groups with which they were compared. These results suggest that prior self-limited H B V infection also is not a major factor in the pathogenesis of A C H in Australia, since most patients convalescent from H B V infection have a n t i - H B A g detectable by P H A a n d / o r R I A in their sera after recovery (17). T h e low prevalence of H B A g in A C H in Australia corresponds with the low incidence of acute infection with H B V in this country (18). T h e frequency of antigenemia detected by I E O P among blood donors including institutionalized donors (0.07 to 0 . 1 1 % ) ( 1 9 , 2 0 ) is among the lowest in the world. T h e incidence of other liver diseases believed to be possible sequelae of chronic H B V infection, such as cryptogenic cirrhosis and primary liver-cell cancer, is also very low compared with areas such as Africa and Asia. Thus, although persistence of H B V may be associated with A C H in certain circumstances, this finding is uncommon in Australia.
REFERENCES
1. Prince AM: Role of serum hepatitis virus in chronic liver disease. Gastroenterology 60:913921, 1971 2. Bulkley BH, Heizer WD, Goldfinger SE, Isselbacher K J, Shulman NR: Distinctions in chronic active hepatitis based on circulating hepatitis-associated antigen. Lancet 2:13231326, 1970 3. Wright R, McCollum RW, Klatskin G: Australia antigen in acute and chronic liver disease. Lancet 2:117-121, 1969 Digestive Diseases, Vol. 20, No. 2 (February 1975)
4. Vernace S, Paronetto F, Schaffner F: Immune reactions and Australia (HAA) antigen in chronic aggressive hepatitis. Gastroenterology 60:182-183, 1971 5. Naccarato R, Fagiolo U, Farini R: L'antigene Australia in alcune epatopatie acute e cronische. G Clin Med 51:170-179, 1970 6. Wewalka F, Gnan F, Krassnitzky O, Pesendorfer F: Au-SH-antigen in liver disease. Vox Sang 19:311-317, 1970 7. Fox RA, Niazi SP, Sherlock S: Hepatitis-associated antigen in chronic liver disease. Lancet 2:609-612, 1969 8. Reinicke V, Nordenfelt E: Australia-SH-antigen and diseases of the liver. Preliminary investigations of Danish drug addicts and patients with chronic liver diseases. Seand J Gastroenterot (Suppl) 7:85-88, 1970 9. Mathews JD, Mackay IR: Australia antigen in chronic hepatitis in Australia. Br Med J 1:259261, 1970 10. Cooksley WGE, Powell LW, Mistilis SP, Olsen G, Mathews JD, Mackay IR: Australia antigen in active chronic hepatitis in Australia: Results in 130 patients from three centres. Aust NZ J Med 2:261-265, 1972 11. Goldstein G, Mackay IR: Lupoid hepatitis: Computer analysis defining "hepatitis" and "cirrhosis" phases and relationships between hepatocellular damage and immune reactions in the liver. Aust Ann Med 16:62-69, 1967 12. Mistilis SP, Skyring AP, Blackburn CRB: Natural history of active chronic hepatitis. I: Clinical features, course, diagnostic criteria, morbidity, mortality and survival. Aust Ann Med 17:214-223, 1968 I3. Mistilis SP: Natural history of active chronic hepatitis. II: Pathology, pathogenesis and clinico-pathological correlation. Aust Ann Med 17:277-288, 1968 14. Peterson MR, Barker LF, Schade DS: Detection of antibody to hepatitis-associated antigen in hemophilia patients and in voluntary blood donors. Vox Sang 24:66-75, 1973 15. Ling CM, Overby LR: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 12SI-antibody. J Immunol 109:834841, 1972 16. Vyas GN, Shulman NR: Hemagglutination as113
COOKSLEY ET AL
say for antigen and antibody associated with viral hepatitis. Science 170:332 333, 1970 17. Barker LF, Peterson MR. Shulman NR, Murray R: Antibody responses in viral hepatitis, type B. JAMA 223:1005-1008, 1973 18. Cooksley WGE: Australia antigen: A review with emphasis on its clinical relevance in Australia. Med J Aust 2:477-487, 1972 19. Mason EC, Shaw AE, Harding M J, Witney K J: Higb-vohage immunoelectroosmophoresis
114
in Australia antigen screening of blood donors. Med J Aust 1:1020-1023, 1972 20. Nelson M, Cooke B: The incidence of Australia antigen in blood donors and in certain high-risk patient populations. Med J Aust 1:950-954, 1971 21. Prince AM, Brotman B, Jass D, Ikram H: Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis-B antigen. Lancet 1:1346-1350, 1973
Digestive Diseases, Vol. 20, No. 2 (February 1975)
In a multicenter cooperative study, sera from 85 patients with active chronic hepatitis (ACH) were examined for the presence of hepatitis B (Australia) antigen (HBAg) by radioimmunoassay (RIA) and antibody to HBAg (anti-HBAg) by RIA and passive hemagglutination (PHA), the most sensitive currently available techniques. In addition, sera from 83 patients with other liver diseases, 98 other hospital patients, and 67 healthy controls were tested. HBAg was detected in 3 of the 85 patients (four percent) with ACH. In a further 3 patients (four percent) anti-HBAg was detected. Thus, 6 patients with ACH (seven percent) had evidence of present or prior infection with the hepatitis B virus (HBV). HBAg was also detected in 7 of the patients with other liver diseases, 2 of the other hospital patients, and none of the healthy controls. Anti-HBAg was detected in 17 of the non-ACH subjects. These results indicate that neither persistent nor prior self-limited infection with HBV is a major factor in the pathogenesis of ACH in Australia. T h e virus of serum hepatitis, HBV, is now suspected to be an important etioIogicat factor in A C H , since a high prevalence of H B A g has been found in a number of studies (1-6). In contrast to the high prevalence reported in parts of the United States (22-31~ and many countries of Europe (34-62%) (5, 6) the prevalence as judged by immunodiffusion has been as
From the Department of Medicine, University of Queensland, Royal Brisbane Hospital, Brisbane, Australia; A.W. Morrow Department of Gastroenterology, Royal Prince Alfred Hospital, Sydney, Australia; Clinical Research Unit, Walter & Eliza Hall Institute, Royal Melbourne Hospital, Melbourne, Australia; and Division of Virology, Bureau of Biologics,Food and Drug Administration, Rockville,Maryland. W.G.E. Cooksley and IR. Mackay were both in receipt of grants from the National Health and Medical Research Council of Australia. We thank Dr. Senga Whittingham for assistance with stored sera. Address for reprint requests: Dr. W.G.E. Cooksley,University Department of Medicine, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
110
low as 0-4% in England (7), Scandinavia (8), and Australia (9), suggesting geographical differences in either the frequency of chronic hepatitis due to viral hepatitis or the prevalence of H B V infection. In Australia (10) H B A g was detected in only 4 of 130 patients with A C H , using a variety of immunological techniques, immunodiffusion, immunoelectroosmophoresis (IEOP), and complement fixation, but not RIA. W i t h the introduction of the highly sensitive R I A for H B A g and anti-HBAg, application of this technique in a country claiming a low incidence of H B A g in A C H would allow further evaluation of the possibility that persistent HBAg, albeit in low titer, is involved in the pathogenesis of A C H . Therefore we have examined the sera of 85 patients with A C H , as well as sera from patients with other liver diseases, hospital inpatients with no apparent liver disease, and healthy controls, from three different geographical areas in Australia. In addition, P H A and R I A methods were employed to test
Digestive Diseases, Vol. 20, No. 2 (February 1975)
HBAg AND HBAb IN ACH Table 1. Prevalence of HBAg and Anti-HBAg in Patients with ACH and Other Diseases in Australia
Subjects studied
Center"
Patients tested (N)
HBAg positive (N)
Anti-HBAg positive (N)
ACH
M B S Total
38 29 18 85
2 1 0 3 (4%)
1 2 0 3 (4%)
Other liver diseases
M B Total
12 71 83
0 7 7 (8%)
1 4 5 (6%)
Other hospital patients
M B Total
72126 98
2 0 2
8 0 8 (8%)
B B Total
19 48 67
0 0 0
0 4 (8%) 4 (6%)
Controls Students and laboratory staff Voluntary blood donors
*M indicates Melbourne, B, Brisbane, S, Sydney. 1-Comprising systemic lupus erythematosis (22), pernicious anemia (14), rheumatoid arthritis (12), and multiple sclerosis (4), all negative, and miscellaneous (20, with 2 positive: a migrant woman with myotonic dystrophy and a woman with rheumatic carditis).
the same sera for a n t i - H B A g to detect evidence of past infection with H B V .
MATERIALS
AND METHODS
The criterion for the diagnosis of ACH was the presence of hepatitis of unknown cause that was biochemically active, as judged by raised plasma levels of transaminase enzymes for a period longer than 6 months and histological features of a destructive periportal hepatitis with or without cirrhosis (11-13). Patients contributed by the three centers are shown in Table 1. Patients with drug-induced liver disease were not included. Relevant clinical details and results of immunofluorescent testing for the serological antibodies, antinuclear factor, smooth-muscle antibody and antimitoehondrial antibody have been recently reported (10). Sera were assayed for HBAg by RIA (14, 15) at the National Institutes of Health, Bethesda, Maryland. All sera scored as positive were shown to be specific for HBAg by inhibition with excess antibody. Sera were also assayed "for anti-HBAg by RIA (14) and PHA (16) and those positive were similarly shown to be specific by inhibition with excess antigen. Sera were sent from Australia coded. Included were sera from 85 patients with ACH, from 83 patients with other liver diseases (acute and chronic liver disease), from 98 other hospital patients, and from 9 medical stuDigestive Diseases, Vol. 20, No. 2 (February 1975)
dents, 10 laboratory staff, and 48 volunteer blood donors (Tables 1 and 2). RESULTS
H B A g was detected in 3 of the 85 patients with A C H - - a prevalence of 4% ( T a b l e 1). T h e sensitivity of R I A can be seen in T a b l e s 3 and 4, where the results are c o m p a r e d with those of a previously reported study (10). A n t i - H B A g was detected in a further 3 patients (4%). T h u s , 6 patients (7%) had evidence of present or previous H B V infection. O f the 248 other subjects tested 9 were found to have H B A g in the serum (Table 1 ). O f the 83 patients with other liver diseases H B A g was detected in the sera of 7 (6 patients with acute viral hepatitis and 1 with alcoholic cirrhosis) (Table 2). A n t i - H B A g was detected in the sera of 6% of the patients with other liver diseases, 8% of the other hospital patients, and 6% of the healthy controls ( T a b l e 1). 111
COOKSLEY ET AL Table 2. Prevalence of HBAg in Patients with Liver Diseases Other Than ACH in Australia
Type of liver disease
Patients tested (N)
Acute liver disease Cholecystitis Acute viral hepatitis Alcoholic hepatitis Total
3 28 9 40
Chronic liver disease Alcoholic cirrhosis Hemochromatosis Cryptogenic cirrhosis Posthepatitic cirrhosis Primary biliary cirrhosis Wilson's disease Persistent hepatitis Chronic obstructive jaundice Cholangio carcinoma Total
14 9 10 1 3 1 1 3 1 43
Anti-HBAg positive (N)
HBAg positive (N)
6 6
1*
2 1t
1 1:1: 5
1
*Repeat testing with excess antibody could not be performed. tPatient was from Yugoslavia and had cryptogenic cirrhosis complicated by primary liver-cell cancer. :[:Patient had noncirrhotic liver.
Table 3. Patients with ACH Found Positive for HBAg Using Different Methods Method
Patients tested (N)
HBAg positive (N)
HBAg positive (%)
130 94 94 85
3 3 4 3
2 3 4 4
Immunodiffusion* Immunoelectroosmophoresis* Complement fixation* Radioimmunoassay *Results in a previously reported study (10).
Table 4. Results of Testing for HBAg in Positive Patients in Table 3 Patient Method Immunodiffusion Immu noelectroosmophoresis Complement fixation Radioimmunoassay
1
2
3
4
5
+ + + --*
+ + + --*
+ + + +
--+ +
---+
*Shown to be nonspecific by failure of inhibition with excess antibody (21).
112
Digestive Diseases, Vol. 20, No. 2 (February 1975)
HBAg AND HBAb IN ACH DISCUSSION
T h e low prevalence of H B A g detected by the most sensitive currently available techniques in patients with A C H confirms previous reports from Australia (9, 10). These results indicate that persistent H B V infection may be relevant to the pathogenesis of only a small minority of cases of A C H in Australia. T h e prevalence of a n t i - H B A g was lower in the group of A C H patients than in three of the four groups with which they were compared. These results suggest that prior self-limited H B V infection also is not a major factor in the pathogenesis of A C H in Australia, since most patients convalescent from H B V infection have a n t i - H B A g detectable by P H A a n d / o r R I A in their sera after recovery (17). T h e low prevalence of H B A g in A C H in Australia corresponds with the low incidence of acute infection with H B V in this country (18). T h e frequency of antigenemia detected by I E O P among blood donors including institutionalized donors (0.07 to 0 . 1 1 % ) ( 1 9 , 2 0 ) is among the lowest in the world. T h e incidence of other liver diseases believed to be possible sequelae of chronic H B V infection, such as cryptogenic cirrhosis and primary liver-cell cancer, is also very low compared with areas such as Africa and Asia. Thus, although persistence of H B V may be associated with A C H in certain circumstances, this finding is uncommon in Australia.
REFERENCES
1. Prince AM: Role of serum hepatitis virus in chronic liver disease. Gastroenterology 60:913921, 1971 2. Bulkley BH, Heizer WD, Goldfinger SE, Isselbacher K J, Shulman NR: Distinctions in chronic active hepatitis based on circulating hepatitis-associated antigen. Lancet 2:13231326, 1970 3. Wright R, McCollum RW, Klatskin G: Australia antigen in acute and chronic liver disease. Lancet 2:117-121, 1969 Digestive Diseases, Vol. 20, No. 2 (February 1975)
4. Vernace S, Paronetto F, Schaffner F: Immune reactions and Australia (HAA) antigen in chronic aggressive hepatitis. Gastroenterology 60:182-183, 1971 5. Naccarato R, Fagiolo U, Farini R: L'antigene Australia in alcune epatopatie acute e cronische. G Clin Med 51:170-179, 1970 6. Wewalka F, Gnan F, Krassnitzky O, Pesendorfer F: Au-SH-antigen in liver disease. Vox Sang 19:311-317, 1970 7. Fox RA, Niazi SP, Sherlock S: Hepatitis-associated antigen in chronic liver disease. Lancet 2:609-612, 1969 8. Reinicke V, Nordenfelt E: Australia-SH-antigen and diseases of the liver. Preliminary investigations of Danish drug addicts and patients with chronic liver diseases. Seand J Gastroenterot (Suppl) 7:85-88, 1970 9. Mathews JD, Mackay IR: Australia antigen in chronic hepatitis in Australia. Br Med J 1:259261, 1970 10. Cooksley WGE, Powell LW, Mistilis SP, Olsen G, Mathews JD, Mackay IR: Australia antigen in active chronic hepatitis in Australia: Results in 130 patients from three centres. Aust NZ J Med 2:261-265, 1972 11. Goldstein G, Mackay IR: Lupoid hepatitis: Computer analysis defining "hepatitis" and "cirrhosis" phases and relationships between hepatocellular damage and immune reactions in the liver. Aust Ann Med 16:62-69, 1967 12. Mistilis SP, Skyring AP, Blackburn CRB: Natural history of active chronic hepatitis. I: Clinical features, course, diagnostic criteria, morbidity, mortality and survival. Aust Ann Med 17:214-223, 1968 I3. Mistilis SP: Natural history of active chronic hepatitis. II: Pathology, pathogenesis and clinico-pathological correlation. Aust Ann Med 17:277-288, 1968 14. Peterson MR, Barker LF, Schade DS: Detection of antibody to hepatitis-associated antigen in hemophilia patients and in voluntary blood donors. Vox Sang 24:66-75, 1973 15. Ling CM, Overby LR: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 12SI-antibody. J Immunol 109:834841, 1972 16. Vyas GN, Shulman NR: Hemagglutination as113
COOKSLEY ET AL
say for antigen and antibody associated with viral hepatitis. Science 170:332 333, 1970 17. Barker LF, Peterson MR. Shulman NR, Murray R: Antibody responses in viral hepatitis, type B. JAMA 223:1005-1008, 1973 18. Cooksley WGE: Australia antigen: A review with emphasis on its clinical relevance in Australia. Med J Aust 2:477-487, 1972 19. Mason EC, Shaw AE, Harding M J, Witney K J: Higb-vohage immunoelectroosmophoresis
114
in Australia antigen screening of blood donors. Med J Aust 1:1020-1023, 1972 20. Nelson M, Cooke B: The incidence of Australia antigen in blood donors and in certain high-risk patient populations. Med J Aust 1:950-954, 1971 21. Prince AM, Brotman B, Jass D, Ikram H: Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis-B antigen. Lancet 1:1346-1350, 1973
Digestive Diseases, Vol. 20, No. 2 (February 1975)
