Vox Sang. 31: 32-36 (1976)
Detection of Hepatitis-B Surface Antigen in Plasma Samples by Reversed Passive Haemagglutination Tests B. S. COMBRIDGE and BARBARA J. KENNEDY Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree
Abstract. A comparison of two commercial reversed passive haemagglutination tests with a counter-immunoelectrophoretic test, used to detect hepatitis-B surface antigen in reconstituted freeze-dried plasma, showed an increase in the number of positive results by the haemagglutination tests. No difficulty was encountered with the appearance of non-specific agglutination provided the plasma was heated at 60 OC for 30 min, frozen overnight at -35 O C , thawed and centrifuged before testing.
Introduction
The routine screening of serum for the detection of hepatitis-B surface antigen (HB,Ag), using two recently developed commercial reversed passive haemagglutination tests, has been evaluated [l, 2, 5, 7, 81. Both tests were shown to be more sensitive and detected more positive samples than counter-immunoelectrophoretic techniques. Testing plasma samples may be associated with difficulties however. VANDERVELDE et al. [8] observed that non-specific agglutination could occur in either of the haemagglutination tests when plasma was used and that this effect was not satisfactorily removed by heating at 56 "C. We describe the testing of reconstituted pools of freeze-dried citrated plasma. The donors whose plasma was contained in these pools had not previously been screened for HB,Ag.
Received: July 16; accepted: September 4, 1975.
33
Materials and Methods Plasma samples. Ten-donor pools of freeze-dried citrated plasma were r e constituted with distilled water to the pre-drying volume. A 1-ml sample of each pool was heated for 30 min in a water bath maintained at 60 O C . After cooling the samples were frozen overnight at -35OC. After thawing all samples were centrifuged at 3,500g for 10min. The clear supernatant, free of the upper lipid layer which separates on centrifugation, was removed and used in the tests. Hepatest (Wellcome Reagents Ltd) uses turkey erythrocytes coated with purified horse antibody to HB,Ag [l].The test was carried out according to the manufacturer’s instructions, adding 25 pl of erythrocyte suspension to 25 pl 1:8 dilution of the test sample. Hepanosticon (Organon Scientific Development Group) uses sheep erythrocytes coated with purified sheep antibody to HBsAg [6]. The test was carried out according to the manufacturer’s instructions, adding 10 pl of the test sample to 500 pl of erythrocyte suspension. In the agglutination tests the distinction between weak positive and negative patterns was made easier if the erythrocyte suspensions were mixed with the test samples on a vibrator (Luckham Ltd). Discontinuous counter-immunoelectrophoresis (DCIE) was carried out as described previously 131, but using plasma which had been concentrated 10-fold with Lyphogel (Gelman). The detector antibody was an immunoglobulin prepared for clinical use by the method of KISTLERand NITSCHMANN [4] from a pool of plasma consisting of about 250 donations containing anti-HBsAg and was used at a concentration of 40 mg proteidml. Radioirnmunoassay (RIA). All samples found to be positive for HB8Ag in other tests were checked using Ausria-2 (Abbott Laboratories) and confirmed by neutralisation with anti-HBs.
Results Table I shows the results of testing 5,971 ten-donor plasma pools for the presence of HI3,Ag with Hepatest and DCIE. This haemagglutination test detected 16 additional positive pools compared with DCIE. Table I1 shows the results of testing 4,486 tendonor plasma pools with the two haemagglutination tests (Hepatest and Hepanosticon) and DCIE. Additional positive pools were detected by both haemagglutination tests. In general it was observed that plasma samples showing a weak positive reaction at a 1:8 dilution by Hepatest were usually negative by Hepanosticon. This agrees with the findings of SHAT~OCK and SMITH 171 who obtained increased titres with Hepatest. Plasmas giving a positive reaction at a dilution of 1:8 by Hepatest were usually detected by DCIE
COMBRIDGJLKENNEDY
34
Table 1. Detection of HBsAg in plasma pools ~
Test
Number of plasma pools tested
~~~~
Equivalent number of donations
~
HBsAgpositive
~~
Hepatest 5,971
59,710
DCIE
44
All positives were confirmed by RIA and neutralized by anti-HBs.
Table ZZ. Comparison of three tests for the detection of HBsAg in plasma pools
Test
Hepatest Hepanosticon DCIE
Number of plasma pools tested
Equivalent number of donations
HBaAgpositive
4,486
44,860
49 39 34
All positives were confirmed by RIA. One Hepanosticon-positive (Hepatest- and DCIE-negative) was a weak positive by RIA but diIuted out by neutralization. All other positives were neutralized by anti-HBsAg.
provided the plasma was concentrated 10-fold, and the gels were washed and stained to reveal weak precipitin lines. No difficulty was experienced with Hepatest due to non-specific agglutination provided the plasma was treated in the manner described. In 10 pools (RIA-negative for HB,Ag) a delayed settling was observed which eventually formed a negative pattern after 2 h. 4 of these were shown to contain anti-HEI, by RIA. With Hepanosticon 13 pools (RIA-negative for HB,Ag) gave abnormal settling patterns forming poorly defined large rings which could be interpreted as weak positives. All these samples became negative after treatment with the Hepanosticon absorbent. Five of these were shown to contain anti-HB, by RIA.
Hepatitis-B Antigen in Plasma
35
Discussion
Provided plasma samples were heated at 60 O C for 30 min, frozen and then centrifuged in the manner described, we have shown that the two commercially available reverse passive haemagglutination tests for the detection of HB,Ag detected an increased number of antigen-positive plasma samples compared with DCIE. This improvement in the antigen detection rate agrees with the results of other laboratories where serum was the test material used in the routine screening of blood donors. The small number of samples giving non-specific agglutination in our series could possibly be related to heating samples at 60 instead of 56 "C, and to centrifugation before testing. It is unlikely that it is connected with the freeze-drying of the pools, since we have observed that plasma which has been stored in the frozen state at -25 OC can be tested in a similar manner. On the other hand the possibility exists that the process of pooling the plasma donations initially might have diluted out or permitted the cross-absorption of substances capable of causing non-specific agglutination Of the 23 pools giving abnormal settling patterns in the haemagglutination tests 9 were shown by RIA to contain anti-HB,. No attempt was made to determine the antigen frequency rate in this series of tests, since the plasma pools were obtained over a 2-year period, and it is likely that many of the individual donors involved contributed more than one donation of blood during that period,
Acknowledgements We should like to thank Dr. W. D'A. MAYCQCK for his interest in this work, and also Professor A. J. ZUCKERMAN (London School of Hygiene and Tropical Medicine) who carried out the radioimmunoassay and neutralization tests. We thank Dr. B. HURN (Wellcome Physiological Research Laboratories) for generously providing Hepatest kits.
References 1 CAYZER, I. ;DAM, D. S. ;CAMERON, C. H., and DENNING, J. V. :A rapid haemagglutination test for hepatitis-B antigen. Lancet i: 947-949 (1974). 2 CHRYSTE, I. L.;ISLAM, M. N.; BANATVALA, J. E., and CAYZER, I.: clinical evaluation of the turkeyerythrocytepassive- haemagglutination test for hepatitis-B surface antigen. Lancet i: 1193-1194 (1974).
COMBRIDGEXENNEDY
36
COMBRIDGE, B. S. and SHAW,C. : Imrnunoelectroosmophoresis using discontinuous buffer system to detect Australia antigen in pooled human plasma. Lancet ii: 1184-1 185 (1971).
KISTLER, P. and NITSCHMANN, H. : Large-scale production of human plasma fractions. Vox Sang. 7: 414-424 (1962). REESINK,H. W.; DUIMEL,W. J., and BRUMMELHUIS, H. G. J.: Evaluation of a new haemagglutination technique for the demonstration of hepatitis-B antigen. Lancet ii: 1351-1353 (1973).
SCHUURS, A. H. W. M. and KAEAKI,J.: Reversed haemagglutination test for the detection of hepatitis-B antigen. Vox Sang. 27: 97-114 (1974). SHAITOCK, A. G. and SMITH,A. N.: Commercialhaemagglutination tests for hepatitis-B antigen. Lancet i: 227-228 (1975). VANDERVELDE,E. M.; MAHMOOD, N.; GOFFIN,C.; PORTER,A.; MEGSON,B., and COSSART,Y. E.: User’s guide to some new tests for hepatitisB antigen. Lancet ii: 1066-1068 (1974).
B. S. COMBRIDGE, Blood Products Laboratory, Lister Institute of Preventive Medicine, Elsiree, Herts (England)
Detection of Hepatitis-B Surface Antigen in Plasma Samples by Reversed Passive Haemagglutination Tests B. S. COMBRIDGE and BARBARA J. KENNEDY Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree
Abstract. A comparison of two commercial reversed passive haemagglutination tests with a counter-immunoelectrophoretic test, used to detect hepatitis-B surface antigen in reconstituted freeze-dried plasma, showed an increase in the number of positive results by the haemagglutination tests. No difficulty was encountered with the appearance of non-specific agglutination provided the plasma was heated at 60 OC for 30 min, frozen overnight at -35 O C , thawed and centrifuged before testing.
Introduction
The routine screening of serum for the detection of hepatitis-B surface antigen (HB,Ag), using two recently developed commercial reversed passive haemagglutination tests, has been evaluated [l, 2, 5, 7, 81. Both tests were shown to be more sensitive and detected more positive samples than counter-immunoelectrophoretic techniques. Testing plasma samples may be associated with difficulties however. VANDERVELDE et al. [8] observed that non-specific agglutination could occur in either of the haemagglutination tests when plasma was used and that this effect was not satisfactorily removed by heating at 56 "C. We describe the testing of reconstituted pools of freeze-dried citrated plasma. The donors whose plasma was contained in these pools had not previously been screened for HB,Ag.
Received: July 16; accepted: September 4, 1975.
33
Materials and Methods Plasma samples. Ten-donor pools of freeze-dried citrated plasma were r e constituted with distilled water to the pre-drying volume. A 1-ml sample of each pool was heated for 30 min in a water bath maintained at 60 O C . After cooling the samples were frozen overnight at -35OC. After thawing all samples were centrifuged at 3,500g for 10min. The clear supernatant, free of the upper lipid layer which separates on centrifugation, was removed and used in the tests. Hepatest (Wellcome Reagents Ltd) uses turkey erythrocytes coated with purified horse antibody to HB,Ag [l].The test was carried out according to the manufacturer’s instructions, adding 25 pl of erythrocyte suspension to 25 pl 1:8 dilution of the test sample. Hepanosticon (Organon Scientific Development Group) uses sheep erythrocytes coated with purified sheep antibody to HBsAg [6]. The test was carried out according to the manufacturer’s instructions, adding 10 pl of the test sample to 500 pl of erythrocyte suspension. In the agglutination tests the distinction between weak positive and negative patterns was made easier if the erythrocyte suspensions were mixed with the test samples on a vibrator (Luckham Ltd). Discontinuous counter-immunoelectrophoresis (DCIE) was carried out as described previously 131, but using plasma which had been concentrated 10-fold with Lyphogel (Gelman). The detector antibody was an immunoglobulin prepared for clinical use by the method of KISTLERand NITSCHMANN [4] from a pool of plasma consisting of about 250 donations containing anti-HBsAg and was used at a concentration of 40 mg proteidml. Radioirnmunoassay (RIA). All samples found to be positive for HB8Ag in other tests were checked using Ausria-2 (Abbott Laboratories) and confirmed by neutralisation with anti-HBs.
Results Table I shows the results of testing 5,971 ten-donor plasma pools for the presence of HI3,Ag with Hepatest and DCIE. This haemagglutination test detected 16 additional positive pools compared with DCIE. Table I1 shows the results of testing 4,486 tendonor plasma pools with the two haemagglutination tests (Hepatest and Hepanosticon) and DCIE. Additional positive pools were detected by both haemagglutination tests. In general it was observed that plasma samples showing a weak positive reaction at a 1:8 dilution by Hepatest were usually negative by Hepanosticon. This agrees with the findings of SHAT~OCK and SMITH 171 who obtained increased titres with Hepatest. Plasmas giving a positive reaction at a dilution of 1:8 by Hepatest were usually detected by DCIE
COMBRIDGJLKENNEDY
34
Table 1. Detection of HBsAg in plasma pools ~
Test
Number of plasma pools tested
~~~~
Equivalent number of donations
~
HBsAgpositive
~~
Hepatest 5,971
59,710
DCIE
44
All positives were confirmed by RIA and neutralized by anti-HBs.
Table ZZ. Comparison of three tests for the detection of HBsAg in plasma pools
Test
Hepatest Hepanosticon DCIE
Number of plasma pools tested
Equivalent number of donations
HBaAgpositive
4,486
44,860
49 39 34
All positives were confirmed by RIA. One Hepanosticon-positive (Hepatest- and DCIE-negative) was a weak positive by RIA but diIuted out by neutralization. All other positives were neutralized by anti-HBsAg.
provided the plasma was concentrated 10-fold, and the gels were washed and stained to reveal weak precipitin lines. No difficulty was experienced with Hepatest due to non-specific agglutination provided the plasma was treated in the manner described. In 10 pools (RIA-negative for HB,Ag) a delayed settling was observed which eventually formed a negative pattern after 2 h. 4 of these were shown to contain anti-HEI, by RIA. With Hepanosticon 13 pools (RIA-negative for HB,Ag) gave abnormal settling patterns forming poorly defined large rings which could be interpreted as weak positives. All these samples became negative after treatment with the Hepanosticon absorbent. Five of these were shown to contain anti-HB, by RIA.
Hepatitis-B Antigen in Plasma
35
Discussion
Provided plasma samples were heated at 60 O C for 30 min, frozen and then centrifuged in the manner described, we have shown that the two commercially available reverse passive haemagglutination tests for the detection of HB,Ag detected an increased number of antigen-positive plasma samples compared with DCIE. This improvement in the antigen detection rate agrees with the results of other laboratories where serum was the test material used in the routine screening of blood donors. The small number of samples giving non-specific agglutination in our series could possibly be related to heating samples at 60 instead of 56 "C, and to centrifugation before testing. It is unlikely that it is connected with the freeze-drying of the pools, since we have observed that plasma which has been stored in the frozen state at -25 OC can be tested in a similar manner. On the other hand the possibility exists that the process of pooling the plasma donations initially might have diluted out or permitted the cross-absorption of substances capable of causing non-specific agglutination Of the 23 pools giving abnormal settling patterns in the haemagglutination tests 9 were shown by RIA to contain anti-HB,. No attempt was made to determine the antigen frequency rate in this series of tests, since the plasma pools were obtained over a 2-year period, and it is likely that many of the individual donors involved contributed more than one donation of blood during that period,
Acknowledgements We should like to thank Dr. W. D'A. MAYCQCK for his interest in this work, and also Professor A. J. ZUCKERMAN (London School of Hygiene and Tropical Medicine) who carried out the radioimmunoassay and neutralization tests. We thank Dr. B. HURN (Wellcome Physiological Research Laboratories) for generously providing Hepatest kits.
References 1 CAYZER, I. ;DAM, D. S. ;CAMERON, C. H., and DENNING, J. V. :A rapid haemagglutination test for hepatitis-B antigen. Lancet i: 947-949 (1974). 2 CHRYSTE, I. L.;ISLAM, M. N.; BANATVALA, J. E., and CAYZER, I.: clinical evaluation of the turkeyerythrocytepassive- haemagglutination test for hepatitis-B surface antigen. Lancet i: 1193-1194 (1974).
COMBRIDGEXENNEDY
36
COMBRIDGE, B. S. and SHAW,C. : Imrnunoelectroosmophoresis using discontinuous buffer system to detect Australia antigen in pooled human plasma. Lancet ii: 1184-1 185 (1971).
KISTLER, P. and NITSCHMANN, H. : Large-scale production of human plasma fractions. Vox Sang. 7: 414-424 (1962). REESINK,H. W.; DUIMEL,W. J., and BRUMMELHUIS, H. G. J.: Evaluation of a new haemagglutination technique for the demonstration of hepatitis-B antigen. Lancet ii: 1351-1353 (1973).
SCHUURS, A. H. W. M. and KAEAKI,J.: Reversed haemagglutination test for the detection of hepatitis-B antigen. Vox Sang. 27: 97-114 (1974). SHAITOCK, A. G. and SMITH,A. N.: Commercialhaemagglutination tests for hepatitis-B antigen. Lancet i: 227-228 (1975). VANDERVELDE,E. M.; MAHMOOD, N.; GOFFIN,C.; PORTER,A.; MEGSON,B., and COSSART,Y. E.: User’s guide to some new tests for hepatitisB antigen. Lancet ii: 1066-1068 (1974).
B. S. COMBRIDGE, Blood Products Laboratory, Lister Institute of Preventive Medicine, Elsiree, Herts (England)
