Vox Sang. 31: 81-86 (1976)
Detection of Hepatitis B Antigen m Blood Donors by Radioimmunoassay and Three Reverse Passive Haemagglutination Techniques S. SEIDLand G. B. ZIEGLER Institute of Immunohaematology and Red Cross Donor Service, Hessen, University of Frankfurt/Main, Frankfurt
Abstract. I n a comparative study 5,569 sera from voluntary donors were tested simultaneously for hepatitis B antigen by three reverse passive haemagglutination (RPHA) techniques (Auscelle, Hepanosticone, Hepateste) and the radioimmunoassay (RIA) (Ausria 11). Among the three RPHA methods, no significant differences could be found as far as sensitivity is concerned. The percentage of positive sera detected by these RPHA methods varies from 0.43 to 0.56%. Eleven sera were detectable only by Ausria 11. Except for two, all these sera had low counts per minute in the RIA.
Zntroduction For the detection of hepatitis B surface antigen (HB,Ag) several immunologic techniques are available which vary considerably in specificity, sensitivity, and simplicity. The ‘phase’ 1 test (originally designated as ‘first-generation’) for HB,Ag detection was immunodiffusion. In the meantime this test has been replaced by tests of ‘phase’ 2 (i.e., counterimmunoelectrophoresis [CIEP], complement fixation). CIEP is still the most widely used technique for large-scale screening of blood donors. However, specimens which have been found negative for HB,Ag by CIEP may still be capable of transmitting hepatitis B [5, 101. Recently, two more sensitive assays have been developed including reverse passive haemagglutination (RPHA) and radioimmunoassay (RIA). Both are considered to be tests of the ‘phase’ 3. The development of a simple, direct, solid-phase RIA (Ausria 11; Abbott Laboratories) has encouraged its application in blood transfusion services. RIA is a rather costly procedure, which is also time-consuming and laborious. It is the purpose of Received: August 3, 1975; accepted: September 17,1975.
82
SEIDL/ZIEGLER
this study, therefore, to compare three commercially available RPHA reagents with the RIA in order to find out which of the ‘phase’ 3 tests would be the most suitable one for donor screening. Materials and Methods Sera Serum samples from blood donors were screened for HBsAg by RPHA methods and RIA. All sera had been inactivated (30min +56OC) and were investigated after 24 or 48h storage at +4OC. In all RPHA methods sera were screened at a dilution of 1:16. Except for this modification, Auscell (Abbott Laboratories) and Hepatest (Wellcome Reagents) were performed according to the instructions of the manufactures, Hepanosticon (Organon-Teknika) was used as micromethod, previously described by REESINKet al. [9].The dilrients used were saline (Hepanosticon), phosphate-buffered saline containing normal horse and human serum (Hepatest), and phosphate-buffered saline containing normal guinea pig as well as normal human serum and gelatin (Auscell). The NIH reference hepatitis B antigen panel No. 2 was investigated to assess the specificity and sensitivity of each technique. Techniques Auscell. In this RPHA technique human red cells are used which are coated with anti-HBs produced in guinea pigs: 0.025 ml 1:8 serum dilution and 0.025 ml test cells were placed in V-microtiter-plates (Linbro disposable V-plates, Abbott Laboratories). The results were read after 2 h incubation. Positive sera were neutralized using horse anti-HB8 delivered from the manufacturer, and then tested again. Hepanosticon. This test uses sheep red cells coated with HBsAb produced in sheep. In the micro-Hepanosticon 0.025 ml of 1:8 diluted serum is placed in U-microtiter-plates (Greiner, Niirtingen, FRG) to which 0.025 ml reagent is added (final dilution 1:16). The reaction pattern was observed after 3 h and overnight incubation, using a rack incorporating an angled mirror. Positive sera were then treated with an absorbent consisting of sheep red cells to which sheep immunoglobulin (not containing HBRAb) had been tagged. After overnight incubation the supernatant was pipetted off and the test repeated. Additionally, a neutralization test had been performed: 0.025 ml donor serum were mixed with 0.175 ml horse anti-HB8 serum which was previously diluted 1:2 by saline. After 2 h incubation at +37 O C the serum was tested again. Hepatest screening kit. The test uses turkey red cells coated with anti-HB8 produced in horses: 0.025 ml serum (1:8 diluted) is mixed with an equal volume of test cell suspension. Settling time is 30 min. Sera showing agglutination were absorbed with control cells (turkey red cells coated with equine immunoglobulin, without anti-HBS activity) and then titrated in serial dilutions (1:2-1512). The test was repeated using both test and control cells. Furthermore, all sera with positive reactions in the screening test were neutralized using the same scheme as described above. RIA. The Ausria I1 was carried out according to the instructions of the manuf acturer.
Detection of Hepatitis B Antigen
83
Results Sera obtained from 5,569 voluntary donors were screened by RPHA and RIA. The results of the comparative study are included in table I. In the first column those sera which gave a positive reaction in the screening test of the RPHA method concerned are listed. In Hepatest 1.63% reacted positively, 2.01 % in Auscell, whereas the micro-Hepanosticon resulted in 3.5% positive reactions. After absorption and/or neutralization only a small percentage were considered as ‘true positive’, i.e., those sera giving the appropriate reactions. The percentage varies from 0.43 to 0.56%. In Ausria I1 only 14 (0.25%) of these sera had a positive reaction, whereas the total number of sera which were detected and confirmed by Ausria I1 was 25 (0.45%). These 14 sera were found to be positive with all three RPHA techniques. None of the 11 sera which were detectable only by Ausria I1 were among those considered as positive in either the first or the second interpretation of RPHA techniques (table I). In figure 1 the results of the 25 RIA-positive sera are plotted against their detectability in the RPHA methods. Except for two, all sera giving high counts per minute in Ausria I1 were also demonstrable by haemagglutination in the three RPHA tests. A similarity existed when the results of the CIEP testing were compared with the strength of the reaction in RIA. In a comparative study these three RPHA methods and the Ausria I1 were tested against the NIH panel No. 2. The results are listed in table 11. The number of correctlydetected specimens was similar with all three RPHA methods. We observed 100% sensitivity and specificity with the Ausria 11, slightly less, 96%, was found with the RPHA techniques.
Discussion Our results confirm previous reports which showed that the RPHA techniques are slightly less sensitive than the RIA [la,6-8, 111. Among the three RPHA techniques no significant differences could be found as far as the detection of HB,Ag-positive blood donors were concerned. Eleven sera found to be positive by Ausria I1 were not detected by one of these RPHA methods. Discordant positive results by RPHA and RIA were also observed. In general, only half of the sera which reacted positive by the RPHA methods could be confirmed by Ausria 11.
a4
SEIDL/ZIEGLER
Table I. Results of blood donor screening (n = 5569) using three RPHA techniques and RIA Method
Positive in screening test
Positive after True positive absorption/neu- in RPHA tralization
Positive in RIA und RPHA
Auscell
112 (2.01%)
311
31 (0.56%)
14 (0.25%)
21 (0.48%) 24 (0.43%) 25 (0.45%)
14 (0.25%) 14 (0.25%)
MicroHepanosticon Hepatest Ausria I1
195 (3.5%) 91 (1.63%) 109 (1.96%)
21/21 272 30128 242 25 (0.45%)
No absorbent is available, the sera were only neutralized. Sera which reacted positive after absorption and negative after neutralization.
0 RIA+CIEP positive
0 0
0 00
00
0
Negative
Positive
RPHA
Fig. 1. Comparison of RIA-positive and CIEP-positive sera with three RPHA techniques.
Detection of Hepatitis B Antigen
85
Tuble ZZ. Comparison between three RPHA techniques and RIA using the NIH reference hepatitis B antigen panel No. 2 Method
Correctly detected, %
Ausria I1 Auscell Micro-Hepanosticon Hepatest
100
96 96 96
Sera No. 236 and 237 where not detected by all three RPHA techniques.
Similar observations have been reported by VANDERVELDE et al. [12] and REESINKet al. [9]. The high number of non-specific results observed in the screening test by all three RPHA techniques as well as by the Ausria I1 was a rather peculiar observation. As far as the RPHA techniques are concerned this may reflect the personal assessment of the haemagglutination and may be diminished by more experience. When the sera were re-tested after 2 weeks storage at -20 "C,the number of nonspecific positives was reduced to approximately 1% in all three RPHA methods. However, false-positive reactions are also eliminated by absorption and/or neutralization techniques. The combination of these two procedures further reduces the number of non-specific positive sera [3, 9, 131. It should also be noted that Ausria I1 has a high number of falsely positive results after screening which is mainly caused by insufficient washing. Also other factors such as cost and suitability have to be considered. Since RPHA methods are less expensive than the RIA, it has been suggested to perform donor screening by one of the RPHA methods, whereas the RIA should be reserved for confirmatory testing of those sera reacting positive in RPHA methods [12]. However, our results clearly indicate that out of the 25 RIA-positive sera, 11 were not detected by one of the RPHA methods. Although most of these sera have HB,Ag in low concentration, two exceptions with high counts per minute have been observed. It is still not clear whether sera containing HB,Ag in low concentration are of clinical importance or whether they should be eliminated. Since the RPHA techniques can be performed more rapidly than the RIA, they are advantageous in testing blood for urgent clinical situations.
86
SEIDL/~IEGLER
The turkey red cells used in Hepatest settle more rapidly because they are nucleated. Therefore, the results can be read after 30 min of incubation, whereas 2 or 3 h are needed for the other RPHA techniques. The micro-Hepanosticon further reduces the costs and can be considered as the most economical.
References 1 BARBARA, J.; DENNING,J. V.; CLEGHORN, T. E.; DANE, D. S., and BRIGGS, M.: Detection of HBsAg by passive haemagglutination test. Lancet i: 975 (1975). 2 CAYZER, I.; DANE,D. S . ; CAMERON, C. H., and DENNING,J. V.: A rapid haernagglutination test for hepatitis-B antigen. Lancet i: 7864 (1974). 3 CHICOT,D.; WRIGHT,R.; BROTMAN, B., and PRINCE,A. M.: Hepanosticon in screening for HBsAg. Lancet i: 345 (1975). I. L. ; ISLAM,M. N.; BANATVALA, J. E., and CAYZER, I. : Clinical evaluation 4 CHRYSTIE, of the turkeyerythrocyte passive-haemagglutination test for hepatitis-B surface antigen. Lancet i: 1193 (1974). 5 KORETZ,R. L.; KLAHS,D. R., RITMAN, S.; DAMUS, K. H., and GITNICK,G. L.: Posttransfusion hepatitis in recipients of blood screened by newer assays. Lancet ii: 694 (1973). 6 MEISSL,M. : Thesis, University of Frankfurt/Main (in preparation). 7 PETERSON, D. A.; FROESNER, G. G., and DEINHARDT, F. W.: Evaluation of passive haemaglutination, solid-phase radioimmunoassay, and immunoelectroosmophoresis for the detection of hepatitis B antigen. Microbiology 26: 376 (1973). 8 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 (1973). H. G. J.: 9 REESINK,H. W.; ELVEN,F. VAN;BOER,J. E. G. DE, and BRUMMELHUIS, Evaluation of different techniques for the demonstration of hepatitis B antigen, with special reference to a modified hepanosticon test. IABS Symposium on Viral Hepatitis, Milan 1974. 10 SCRIBA, B. und SEIDL,S.: Der Nachweis von Hepatitis B Antigen im Serum von Blutspendern durch die passive Haernagglutination. Transf. Immunhaemat. 2: 635 (1974). 11 SEIDL,S.; PADE,K. und KALTSCHMID, 0.: Der Nachweis von Hepatitis B Antigen bei Blutspendern durch die passive Haemagglutination. Klin. Wschr. 52: 599 (1974). E. M.; GOFFIN, C.; MEGSON,B.; MAHMOOD, N.; PORTER,A., and 12 VANDERVELDE, Y.E.: User’s guide to some new tests for hepatitis-B-antigen. Lancet ii: COSSART, 1066 (1974). 13 WOLTERS, G.; THAL,P.; SCHUURS, A. H. W. M., and KACAKI,J.: Hepanosticon in screening for HBsAg. Lancet i: 1193 (1975).
Prof. Dr. S. SEIDL,Blutspendedienst Hessen, SandhofstraRe 1, 0-6000FrankfurtlMain 73 (FRG)
Detection of Hepatitis B Antigen m Blood Donors by Radioimmunoassay and Three Reverse Passive Haemagglutination Techniques S. SEIDLand G. B. ZIEGLER Institute of Immunohaematology and Red Cross Donor Service, Hessen, University of Frankfurt/Main, Frankfurt
Abstract. I n a comparative study 5,569 sera from voluntary donors were tested simultaneously for hepatitis B antigen by three reverse passive haemagglutination (RPHA) techniques (Auscelle, Hepanosticone, Hepateste) and the radioimmunoassay (RIA) (Ausria 11). Among the three RPHA methods, no significant differences could be found as far as sensitivity is concerned. The percentage of positive sera detected by these RPHA methods varies from 0.43 to 0.56%. Eleven sera were detectable only by Ausria 11. Except for two, all these sera had low counts per minute in the RIA.
Zntroduction For the detection of hepatitis B surface antigen (HB,Ag) several immunologic techniques are available which vary considerably in specificity, sensitivity, and simplicity. The ‘phase’ 1 test (originally designated as ‘first-generation’) for HB,Ag detection was immunodiffusion. In the meantime this test has been replaced by tests of ‘phase’ 2 (i.e., counterimmunoelectrophoresis [CIEP], complement fixation). CIEP is still the most widely used technique for large-scale screening of blood donors. However, specimens which have been found negative for HB,Ag by CIEP may still be capable of transmitting hepatitis B [5, 101. Recently, two more sensitive assays have been developed including reverse passive haemagglutination (RPHA) and radioimmunoassay (RIA). Both are considered to be tests of the ‘phase’ 3. The development of a simple, direct, solid-phase RIA (Ausria 11; Abbott Laboratories) has encouraged its application in blood transfusion services. RIA is a rather costly procedure, which is also time-consuming and laborious. It is the purpose of Received: August 3, 1975; accepted: September 17,1975.
82
SEIDL/ZIEGLER
this study, therefore, to compare three commercially available RPHA reagents with the RIA in order to find out which of the ‘phase’ 3 tests would be the most suitable one for donor screening. Materials and Methods Sera Serum samples from blood donors were screened for HBsAg by RPHA methods and RIA. All sera had been inactivated (30min +56OC) and were investigated after 24 or 48h storage at +4OC. In all RPHA methods sera were screened at a dilution of 1:16. Except for this modification, Auscell (Abbott Laboratories) and Hepatest (Wellcome Reagents) were performed according to the instructions of the manufactures, Hepanosticon (Organon-Teknika) was used as micromethod, previously described by REESINKet al. [9].The dilrients used were saline (Hepanosticon), phosphate-buffered saline containing normal horse and human serum (Hepatest), and phosphate-buffered saline containing normal guinea pig as well as normal human serum and gelatin (Auscell). The NIH reference hepatitis B antigen panel No. 2 was investigated to assess the specificity and sensitivity of each technique. Techniques Auscell. In this RPHA technique human red cells are used which are coated with anti-HBs produced in guinea pigs: 0.025 ml 1:8 serum dilution and 0.025 ml test cells were placed in V-microtiter-plates (Linbro disposable V-plates, Abbott Laboratories). The results were read after 2 h incubation. Positive sera were neutralized using horse anti-HB8 delivered from the manufacturer, and then tested again. Hepanosticon. This test uses sheep red cells coated with HBsAb produced in sheep. In the micro-Hepanosticon 0.025 ml of 1:8 diluted serum is placed in U-microtiter-plates (Greiner, Niirtingen, FRG) to which 0.025 ml reagent is added (final dilution 1:16). The reaction pattern was observed after 3 h and overnight incubation, using a rack incorporating an angled mirror. Positive sera were then treated with an absorbent consisting of sheep red cells to which sheep immunoglobulin (not containing HBRAb) had been tagged. After overnight incubation the supernatant was pipetted off and the test repeated. Additionally, a neutralization test had been performed: 0.025 ml donor serum were mixed with 0.175 ml horse anti-HB8 serum which was previously diluted 1:2 by saline. After 2 h incubation at +37 O C the serum was tested again. Hepatest screening kit. The test uses turkey red cells coated with anti-HB8 produced in horses: 0.025 ml serum (1:8 diluted) is mixed with an equal volume of test cell suspension. Settling time is 30 min. Sera showing agglutination were absorbed with control cells (turkey red cells coated with equine immunoglobulin, without anti-HBS activity) and then titrated in serial dilutions (1:2-1512). The test was repeated using both test and control cells. Furthermore, all sera with positive reactions in the screening test were neutralized using the same scheme as described above. RIA. The Ausria I1 was carried out according to the instructions of the manuf acturer.
Detection of Hepatitis B Antigen
83
Results Sera obtained from 5,569 voluntary donors were screened by RPHA and RIA. The results of the comparative study are included in table I. In the first column those sera which gave a positive reaction in the screening test of the RPHA method concerned are listed. In Hepatest 1.63% reacted positively, 2.01 % in Auscell, whereas the micro-Hepanosticon resulted in 3.5% positive reactions. After absorption and/or neutralization only a small percentage were considered as ‘true positive’, i.e., those sera giving the appropriate reactions. The percentage varies from 0.43 to 0.56%. In Ausria I1 only 14 (0.25%) of these sera had a positive reaction, whereas the total number of sera which were detected and confirmed by Ausria I1 was 25 (0.45%). These 14 sera were found to be positive with all three RPHA techniques. None of the 11 sera which were detectable only by Ausria I1 were among those considered as positive in either the first or the second interpretation of RPHA techniques (table I). In figure 1 the results of the 25 RIA-positive sera are plotted against their detectability in the RPHA methods. Except for two, all sera giving high counts per minute in Ausria I1 were also demonstrable by haemagglutination in the three RPHA tests. A similarity existed when the results of the CIEP testing were compared with the strength of the reaction in RIA. In a comparative study these three RPHA methods and the Ausria I1 were tested against the NIH panel No. 2. The results are listed in table 11. The number of correctlydetected specimens was similar with all three RPHA methods. We observed 100% sensitivity and specificity with the Ausria 11, slightly less, 96%, was found with the RPHA techniques.
Discussion Our results confirm previous reports which showed that the RPHA techniques are slightly less sensitive than the RIA [la,6-8, 111. Among the three RPHA techniques no significant differences could be found as far as the detection of HB,Ag-positive blood donors were concerned. Eleven sera found to be positive by Ausria I1 were not detected by one of these RPHA methods. Discordant positive results by RPHA and RIA were also observed. In general, only half of the sera which reacted positive by the RPHA methods could be confirmed by Ausria 11.
a4
SEIDL/ZIEGLER
Table I. Results of blood donor screening (n = 5569) using three RPHA techniques and RIA Method
Positive in screening test
Positive after True positive absorption/neu- in RPHA tralization
Positive in RIA und RPHA
Auscell
112 (2.01%)
311
31 (0.56%)
14 (0.25%)
21 (0.48%) 24 (0.43%) 25 (0.45%)
14 (0.25%) 14 (0.25%)
MicroHepanosticon Hepatest Ausria I1
195 (3.5%) 91 (1.63%) 109 (1.96%)
21/21 272 30128 242 25 (0.45%)
No absorbent is available, the sera were only neutralized. Sera which reacted positive after absorption and negative after neutralization.
0 RIA+CIEP positive
0 0
0 00
00
0
Negative
Positive
RPHA
Fig. 1. Comparison of RIA-positive and CIEP-positive sera with three RPHA techniques.
Detection of Hepatitis B Antigen
85
Tuble ZZ. Comparison between three RPHA techniques and RIA using the NIH reference hepatitis B antigen panel No. 2 Method
Correctly detected, %
Ausria I1 Auscell Micro-Hepanosticon Hepatest
100
96 96 96
Sera No. 236 and 237 where not detected by all three RPHA techniques.
Similar observations have been reported by VANDERVELDE et al. [12] and REESINKet al. [9]. The high number of non-specific results observed in the screening test by all three RPHA techniques as well as by the Ausria I1 was a rather peculiar observation. As far as the RPHA techniques are concerned this may reflect the personal assessment of the haemagglutination and may be diminished by more experience. When the sera were re-tested after 2 weeks storage at -20 "C,the number of nonspecific positives was reduced to approximately 1% in all three RPHA methods. However, false-positive reactions are also eliminated by absorption and/or neutralization techniques. The combination of these two procedures further reduces the number of non-specific positive sera [3, 9, 131. It should also be noted that Ausria I1 has a high number of falsely positive results after screening which is mainly caused by insufficient washing. Also other factors such as cost and suitability have to be considered. Since RPHA methods are less expensive than the RIA, it has been suggested to perform donor screening by one of the RPHA methods, whereas the RIA should be reserved for confirmatory testing of those sera reacting positive in RPHA methods [12]. However, our results clearly indicate that out of the 25 RIA-positive sera, 11 were not detected by one of the RPHA methods. Although most of these sera have HB,Ag in low concentration, two exceptions with high counts per minute have been observed. It is still not clear whether sera containing HB,Ag in low concentration are of clinical importance or whether they should be eliminated. Since the RPHA techniques can be performed more rapidly than the RIA, they are advantageous in testing blood for urgent clinical situations.
86
SEIDL/~IEGLER
The turkey red cells used in Hepatest settle more rapidly because they are nucleated. Therefore, the results can be read after 30 min of incubation, whereas 2 or 3 h are needed for the other RPHA techniques. The micro-Hepanosticon further reduces the costs and can be considered as the most economical.
References 1 BARBARA, J.; DENNING,J. V.; CLEGHORN, T. E.; DANE, D. S., and BRIGGS, M.: Detection of HBsAg by passive haemagglutination test. Lancet i: 975 (1975). 2 CAYZER, I.; DANE,D. S . ; CAMERON, C. H., and DENNING,J. V.: A rapid haernagglutination test for hepatitis-B antigen. Lancet i: 7864 (1974). 3 CHICOT,D.; WRIGHT,R.; BROTMAN, B., and PRINCE,A. M.: Hepanosticon in screening for HBsAg. Lancet i: 345 (1975). I. L. ; ISLAM,M. N.; BANATVALA, J. E., and CAYZER, I. : Clinical evaluation 4 CHRYSTIE, of the turkeyerythrocyte passive-haemagglutination test for hepatitis-B surface antigen. Lancet i: 1193 (1974). 5 KORETZ,R. L.; KLAHS,D. R., RITMAN, S.; DAMUS, K. H., and GITNICK,G. L.: Posttransfusion hepatitis in recipients of blood screened by newer assays. Lancet ii: 694 (1973). 6 MEISSL,M. : Thesis, University of Frankfurt/Main (in preparation). 7 PETERSON, D. A.; FROESNER, G. G., and DEINHARDT, F. W.: Evaluation of passive haemaglutination, solid-phase radioimmunoassay, and immunoelectroosmophoresis for the detection of hepatitis B antigen. Microbiology 26: 376 (1973). 8 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 (1973). H. G. J.: 9 REESINK,H. W.; ELVEN,F. VAN;BOER,J. E. G. DE, and BRUMMELHUIS, Evaluation of different techniques for the demonstration of hepatitis B antigen, with special reference to a modified hepanosticon test. IABS Symposium on Viral Hepatitis, Milan 1974. 10 SCRIBA, B. und SEIDL,S.: Der Nachweis von Hepatitis B Antigen im Serum von Blutspendern durch die passive Haernagglutination. Transf. Immunhaemat. 2: 635 (1974). 11 SEIDL,S.; PADE,K. und KALTSCHMID, 0.: Der Nachweis von Hepatitis B Antigen bei Blutspendern durch die passive Haemagglutination. Klin. Wschr. 52: 599 (1974). E. M.; GOFFIN, C.; MEGSON,B.; MAHMOOD, N.; PORTER,A., and 12 VANDERVELDE, Y.E.: User’s guide to some new tests for hepatitis-B-antigen. Lancet ii: COSSART, 1066 (1974). 13 WOLTERS, G.; THAL,P.; SCHUURS, A. H. W. M., and KACAKI,J.: Hepanosticon in screening for HBsAg. Lancet i: 1193 (1975).
Prof. Dr. S. SEIDL,Blutspendedienst Hessen, SandhofstraRe 1, 0-6000FrankfurtlMain 73 (FRG)
