Failure to Confirm Specificity of Samples Containing a Very High Titer of Hepatitis B Surface Antigen (HBsAg) N. NATH,A. ARMBRUSTER A N D F. R. ELLIS From the Blood Research Laboratory, American Red Cross, Bethesda. Maryland, and rhe Missouri-Illinois Regional Red Cross Blood Program. St. Louis. Missouri
Two samples repeatedly positive for HBsAg by the AusRIA 11 screening procedure failed to be confirmed by neutralization with anti-HBs serum. Both samples could be effectively neutralized by the same anti-HBs sera if diluted l:lO, 150 and 1:lOO prior to testing. It is suggested that the inability to neutralize was due to a very high concentration of HBsAg in these samples.
THESEROLOGIC DIAGNOSIS of type B hepatitis is based on the demonstration of hepatitis B surface antigen (HBsAg) in serum. HBsAg is believed to be a component of the envelope (surface) of hepatitis B virus (HBV) and it is usually present in the blood of hepatitis B patients and carriers far in excess of complete HBV particles. Blood and blood products containing HBsAg, and presumably HBV, are known to transmit type B hepatitis when given to susceptible individuals. l v 2 It is now mandatory in the United States that all blood and blood products intended for human use must be tested for the presence of HBsAg with reagents that have been licensed by the Bureau of Biologics, Food and Drug Administration, U. S. Department of Health, Education and Welfare. The American Red Cross collects about five million units of blood from volunteer donors each year. Every unit of blood is tested by radioimmunoassay for the presence of HBsAg. All samples reactive for HBsAg are further tested to determine specificity and to confirm the presence of HBsAg. Samples failing to show at least 50 per cent reduction in net counts per minute following incubation with specific antibody Received for publication May 6, 1978; accepted June 13. 1978. Contribution No. 41 I from the American Red Cross.
to HBsAg are considered n o n s p e ~ i f i c We .~ recently encountered samples from two donors that, if tested only according to the recommended procedure, would have been mistaken to be nonspecific for HBsAg. Materials and Methods Serum from two male donors, 26 and 49 years of age respectively, denying prior illness suggesting hepatitis and appeared to be in excellent health at the time of donation, failed satisfactory verification by routine testing for the presence of HBsAg. Initial screening was performed with solid phase radioimmunoassay AusRIA 11-125 Diagnostic Test Kits (Abbott Laboratories, North Chicago, Illinois). Tests to prove specificity of reactive screening tests were performed with the AusRIA 11- 125 Confirmatory Neutralization Test (Abbott Laboratories kit #8310). Methods recommended in the manufacturer’s package instructions were followed explicitly in the initial tests performed in the Missouri- Illinois Regional Red Cross Blood Center. Aliquots of these samples or additional serum obtained later were submitted to the American Red Cross Blood Research Laboratory, Bethesda, Maryland, for further testing. The same screening procedures used in St. Louis were repeated along with the reverse passive hemagglutination, or RPHA ( AusCell, Abbott Laboratories). Neutralization tests used in St. Louis were also repeated using the same licensed commercial reagent as well as an additional high titer human anti-HBs serum prepared in the Bethesda Blood Research Laboratory.
Results Samples from both donor A and donor B were consistently found reactive for HBsAg in both laboratories by RIA and by RPHA in Bethesda, as shown in Table 1. Both samples were reactive at titers to when tested for HBsAg by RIA. DNA polymerase was detectable in both samples.
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Table 1. Results of Screening for HBsAg' Donor
RPHA (Auscell)
A B
Positive Positive
RIA (AusRIA II)
32.05 29.5
32.2 23.7
52.3 46.3
52.7 49.6
Figures are P/N ratios derived from net cpm values of samples and negative controls obtained by averaging repeated testings. Each column represents results of separate testing.
Confirmatory neutralization tests performed according to manufacturer's instructions showed both specimens to be nonspecific. Use of a pool of high titered anti-HBs human sera prepared in-house alongside the FDA licensed anti-HBs commercial reagent showed essentially the same inability to neutralize these donor samples to an acceptable level. The recommended 1:5 saline dilution of both anti-serums was made prior to their use. Table 2 summarized our findings. Undiluted, sample A could only be neutralized between 19 and 48 per cent: sample B was neutralized between 26 and 66 per cent. Dilution of the two samples from I : 10 to 1:lOO in saline prior to testing, however, permitted neutralization in excess of 90 per cent.
Discussion Blood samples from donors A and B could not be neutralized effectively using anti-HBs serum supplied with the neutralization kit and the recommended procedure for confirmation of HBsAg specificity, even though Table 2. Results of HBsAg Specificity Testing' Per Cent Neutralization by Anti-HBs Donor
Dilution ~~
A
Undiluted
1:lO 1 :50 1 :loo B
Undiluted
1:lO 1:15 1:lOO
Abbott Kit
BRL Pool
~
29.7 76.2 89.4 91.2
47.7 92.7 92.7 95.6
18.6 42.4 59.1 77.4 81.4 89.0 90.5
43.8 72.0 87.6 92.0
66.4 77.6 89.4 93.9
25.8 62.9 70.2 79.8 84.3 84.9 85.1
The titers of both anti-HBssera used in this study were similar, 32,000to 64.000,by passive hemagglutination assay.
Transfusion May-June 1979
both samples were repeatedly positive for HBsAg on screening by RIA. Donor B, the older man, has remained well more than 90 days since donating blood, while Donor A developed acute hepatitis nine days following his donation. During his acute illness, the RIA screening test remained positive and by about the third day of illness his recalcified plasma was neutralizable without prior dilution to between 48 and 80 per cent. It was considered unlikely that either of these samples was actually nonspecific positive as the simple interpretation of results indicated. Retesting the samples for HBsAg specificity after diluting them revealed that both samples were truly neutralizable. We suspect that the concentration of HBsAg in these samples may have been so high that sufficient HBsAg sites remained available to bind 1251-anti-HBsafter all available anti-HBs from neutralizing serum were satisfied. In other words, guinea pig antiHBs on the plastic bead in AusRIA I1 is capable of binding to HBsAg far in excess of that which is neutralizable by the anti-HBs in the neutralizing serum. In the HBsAg confirmatory test, samples containing extreme excess of antigen which are exposed to ordinary concentrations of neutralizing anti-HBs and then compared with the ability of normal human serum to bind radio-labeled reagent would be expected to appear as if no significant neutralization took place. We see this as analogous to prozone effect in serial dilution procedures. The data presented here support this view. It is also possible that the failure of high titer anti-HBs antibodies to neutralize HBsAg in these two samples could be due to the presence of some substance or substances that interferes with the antigen-antibody union. Nonspecific RIA has been attributed to such substance^.^ However, since the antigen-antibody reaction between our samples and antibody in the AusRIA I1 screening procedure is not affected, this possibility seems the less likely of the two explanations.
Volume 19 Number 3
SPECIFICITY TESTING HBsAg
Ling et al. ,4 using a different neutralization procedure and the older versions of AusRIA test kits (AusRIA-125, Abbott), found that high titered human anti-HBs serum was unable to neutralize a sample of the HBsAg if the HBsAg concentration in it was very high. They also reported that predilution of these samples resulted in significantly improved neutralization. We recommend that any sample giving repeatedly high counts (P/N ratios 3 20) on screening tests for HBsAg by AusRIA I1 should be tested for specificity confirmation not only undiluted, but after dilutions of 1: 10 and 1 5 0 with 0.9% sodium chloride solution as well. References I. Goldfield, M.: Some epidemiologicstudies of transfusion associated hepatitis. I n : Transmissible Disease and Blood Transfusion. T. J. Greenwalt and G. A. Jamieson, Eds. New York, Grune & Stratton, 1974, p. 141.
323
2. Hoofnagle, J. H., R. J. Gerety, J. Thiel, and L. F.
Barker: The prevalence of hepatitis B surface antigen in commerically prepared plasma products. J. Lab. Clin. Med. 88:102, 1976. 3. Irwin, G. R., A. M. Allen, W. H. Bancroft, M. Willhight, and P. K. Russell: Specificityand sensitivity of radioimmunoassay for hepatitis B antigen. Appl. Microbiol. 28:600, 1974. 4. Ling, C. M.. R. H. Decker, R. S. Foemmel, and L. R. Overby: Comparison of confirmation methods for hepatitis B antigen and the nature of false positives detected by '2sI-immunoglobulins. J. Lab. Clin. Med. 86:690. 1975.
N. Nath, Ph.D., Head, Hepatitis Section, Research Scientist, American Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014. A. P. Armbruster, M.L.T. (ASCP), Supervisor, Hepatitis Laboratory, Missouri-IllinoisRegional Red Cross Blood Service, 4050 Lindell Boulevard, St. Louis, Missouri 63108. F. R. Ellis, M.D., Medical Director, Missouri-Illinois Regional Red Cross Blood Service.
Two samples repeatedly positive for HBsAg by the AusRIA 11 screening procedure failed to be confirmed by neutralization with anti-HBs serum. Both samples could be effectively neutralized by the same anti-HBs sera if diluted l:lO, 150 and 1:lOO prior to testing. It is suggested that the inability to neutralize was due to a very high concentration of HBsAg in these samples.
THESEROLOGIC DIAGNOSIS of type B hepatitis is based on the demonstration of hepatitis B surface antigen (HBsAg) in serum. HBsAg is believed to be a component of the envelope (surface) of hepatitis B virus (HBV) and it is usually present in the blood of hepatitis B patients and carriers far in excess of complete HBV particles. Blood and blood products containing HBsAg, and presumably HBV, are known to transmit type B hepatitis when given to susceptible individuals. l v 2 It is now mandatory in the United States that all blood and blood products intended for human use must be tested for the presence of HBsAg with reagents that have been licensed by the Bureau of Biologics, Food and Drug Administration, U. S. Department of Health, Education and Welfare. The American Red Cross collects about five million units of blood from volunteer donors each year. Every unit of blood is tested by radioimmunoassay for the presence of HBsAg. All samples reactive for HBsAg are further tested to determine specificity and to confirm the presence of HBsAg. Samples failing to show at least 50 per cent reduction in net counts per minute following incubation with specific antibody Received for publication May 6, 1978; accepted June 13. 1978. Contribution No. 41 I from the American Red Cross.
to HBsAg are considered n o n s p e ~ i f i c We .~ recently encountered samples from two donors that, if tested only according to the recommended procedure, would have been mistaken to be nonspecific for HBsAg. Materials and Methods Serum from two male donors, 26 and 49 years of age respectively, denying prior illness suggesting hepatitis and appeared to be in excellent health at the time of donation, failed satisfactory verification by routine testing for the presence of HBsAg. Initial screening was performed with solid phase radioimmunoassay AusRIA 11-125 Diagnostic Test Kits (Abbott Laboratories, North Chicago, Illinois). Tests to prove specificity of reactive screening tests were performed with the AusRIA 11- 125 Confirmatory Neutralization Test (Abbott Laboratories kit #8310). Methods recommended in the manufacturer’s package instructions were followed explicitly in the initial tests performed in the Missouri- Illinois Regional Red Cross Blood Center. Aliquots of these samples or additional serum obtained later were submitted to the American Red Cross Blood Research Laboratory, Bethesda, Maryland, for further testing. The same screening procedures used in St. Louis were repeated along with the reverse passive hemagglutination, or RPHA ( AusCell, Abbott Laboratories). Neutralization tests used in St. Louis were also repeated using the same licensed commercial reagent as well as an additional high titer human anti-HBs serum prepared in the Bethesda Blood Research Laboratory.
Results Samples from both donor A and donor B were consistently found reactive for HBsAg in both laboratories by RIA and by RPHA in Bethesda, as shown in Table 1. Both samples were reactive at titers to when tested for HBsAg by RIA. DNA polymerase was detectable in both samples.
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Table 1. Results of Screening for HBsAg' Donor
RPHA (Auscell)
A B
Positive Positive
RIA (AusRIA II)
32.05 29.5
32.2 23.7
52.3 46.3
52.7 49.6
Figures are P/N ratios derived from net cpm values of samples and negative controls obtained by averaging repeated testings. Each column represents results of separate testing.
Confirmatory neutralization tests performed according to manufacturer's instructions showed both specimens to be nonspecific. Use of a pool of high titered anti-HBs human sera prepared in-house alongside the FDA licensed anti-HBs commercial reagent showed essentially the same inability to neutralize these donor samples to an acceptable level. The recommended 1:5 saline dilution of both anti-serums was made prior to their use. Table 2 summarized our findings. Undiluted, sample A could only be neutralized between 19 and 48 per cent: sample B was neutralized between 26 and 66 per cent. Dilution of the two samples from I : 10 to 1:lOO in saline prior to testing, however, permitted neutralization in excess of 90 per cent.
Discussion Blood samples from donors A and B could not be neutralized effectively using anti-HBs serum supplied with the neutralization kit and the recommended procedure for confirmation of HBsAg specificity, even though Table 2. Results of HBsAg Specificity Testing' Per Cent Neutralization by Anti-HBs Donor
Dilution ~~
A
Undiluted
1:lO 1 :50 1 :loo B
Undiluted
1:lO 1:15 1:lOO
Abbott Kit
BRL Pool
~
29.7 76.2 89.4 91.2
47.7 92.7 92.7 95.6
18.6 42.4 59.1 77.4 81.4 89.0 90.5
43.8 72.0 87.6 92.0
66.4 77.6 89.4 93.9
25.8 62.9 70.2 79.8 84.3 84.9 85.1
The titers of both anti-HBssera used in this study were similar, 32,000to 64.000,by passive hemagglutination assay.
Transfusion May-June 1979
both samples were repeatedly positive for HBsAg on screening by RIA. Donor B, the older man, has remained well more than 90 days since donating blood, while Donor A developed acute hepatitis nine days following his donation. During his acute illness, the RIA screening test remained positive and by about the third day of illness his recalcified plasma was neutralizable without prior dilution to between 48 and 80 per cent. It was considered unlikely that either of these samples was actually nonspecific positive as the simple interpretation of results indicated. Retesting the samples for HBsAg specificity after diluting them revealed that both samples were truly neutralizable. We suspect that the concentration of HBsAg in these samples may have been so high that sufficient HBsAg sites remained available to bind 1251-anti-HBsafter all available anti-HBs from neutralizing serum were satisfied. In other words, guinea pig antiHBs on the plastic bead in AusRIA I1 is capable of binding to HBsAg far in excess of that which is neutralizable by the anti-HBs in the neutralizing serum. In the HBsAg confirmatory test, samples containing extreme excess of antigen which are exposed to ordinary concentrations of neutralizing anti-HBs and then compared with the ability of normal human serum to bind radio-labeled reagent would be expected to appear as if no significant neutralization took place. We see this as analogous to prozone effect in serial dilution procedures. The data presented here support this view. It is also possible that the failure of high titer anti-HBs antibodies to neutralize HBsAg in these two samples could be due to the presence of some substance or substances that interferes with the antigen-antibody union. Nonspecific RIA has been attributed to such substance^.^ However, since the antigen-antibody reaction between our samples and antibody in the AusRIA I1 screening procedure is not affected, this possibility seems the less likely of the two explanations.
Volume 19 Number 3
SPECIFICITY TESTING HBsAg
Ling et al. ,4 using a different neutralization procedure and the older versions of AusRIA test kits (AusRIA-125, Abbott), found that high titered human anti-HBs serum was unable to neutralize a sample of the HBsAg if the HBsAg concentration in it was very high. They also reported that predilution of these samples resulted in significantly improved neutralization. We recommend that any sample giving repeatedly high counts (P/N ratios 3 20) on screening tests for HBsAg by AusRIA I1 should be tested for specificity confirmation not only undiluted, but after dilutions of 1: 10 and 1 5 0 with 0.9% sodium chloride solution as well. References I. Goldfield, M.: Some epidemiologicstudies of transfusion associated hepatitis. I n : Transmissible Disease and Blood Transfusion. T. J. Greenwalt and G. A. Jamieson, Eds. New York, Grune & Stratton, 1974, p. 141.
323
2. Hoofnagle, J. H., R. J. Gerety, J. Thiel, and L. F.
Barker: The prevalence of hepatitis B surface antigen in commerically prepared plasma products. J. Lab. Clin. Med. 88:102, 1976. 3. Irwin, G. R., A. M. Allen, W. H. Bancroft, M. Willhight, and P. K. Russell: Specificityand sensitivity of radioimmunoassay for hepatitis B antigen. Appl. Microbiol. 28:600, 1974. 4. Ling, C. M.. R. H. Decker, R. S. Foemmel, and L. R. Overby: Comparison of confirmation methods for hepatitis B antigen and the nature of false positives detected by '2sI-immunoglobulins. J. Lab. Clin. Med. 86:690. 1975.
N. Nath, Ph.D., Head, Hepatitis Section, Research Scientist, American Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, Maryland 20014. A. P. Armbruster, M.L.T. (ASCP), Supervisor, Hepatitis Laboratory, Missouri-IllinoisRegional Red Cross Blood Service, 4050 Lindell Boulevard, St. Louis, Missouri 63108. F. R. Ellis, M.D., Medical Director, Missouri-Illinois Regional Red Cross Blood Service.
