Hepatitis B Antigen Card Test III. Sensitivity and Specificity

Transcription

1 Hepatitis B Antigen Card Test III Sensitivity and Specificity ROY W. STEVENS, PH.D., GERALDINE MCQUILLAN, M.S., DAVID DENCE, M.T.(ASCP), AND JAMES KELLY, M.D. Division of Laboratories and Research, New York State Department of Health, and Northeastern New Y Blood Center, The American National Red Cross, Albany, New York ABSTRACT Stevens, Roy W., McQuillan, Geraldine, Dence, David, and Kelly, James: Hepatitis B antigen card test III. Sensitivity and specificity. Am J Clin Pathol 66: 59-64, A simple, rapid hepatitis B antigen card test (card test III) with third-generation sensitivity has been developed. This test is a modification of the charcoal-particle agglutination-inhibition procedure. Sera from voluntary blood donors and hospital patients were tested by the new procedure, by the original inhibition technic, by reversed passive hemagglutination, and by radioimmunoassay. This comparison showed that CT III has a specificity equal to that of radioimmunoassay and is almost as sensitive. (Key words: Hepatitis; Hepatitis B antigen; Charcoal particle test; Agglutination-inhibition.) VARIOUS SEROLOGIC TESTS have been adapted for the detection and quantitation of hepatitis B antigen (HB s Ag). Immunodiffusion, counterelectrophoresis (CEP), reversed passive hemagglutination (RPHA), and radioimmunoassay (RIA) have been frequently used as clinical laboratory tests. 1 The effectivenesses of these tests have been measured by the numbers of HB s Ag-positive specimens detected in a population and by dilution studies of positive sera. As a result of these evaluations, immunodiffusion, the earliest and least sensitive test, has been largely replaced by CEP, which is about ten times more sensitive. The most recent adaptations, RPHA 6-19 and RIA, 9-10 are 100 times more sensitive than CEP, 2 ' 3 but each Received August 21, 1975; accepted for publication September 17, Address reprint requests to Dr. Stevens: Division of Laboratories and Research, New York State Department of Health, New Scotland Avenue, Albany, New York of these third-generation procedures presents some problems of specificity, technical complexity, or reagent instability. 1,21 The original charcoal-particle test developed here is a specific and simple technic for detecting and quantitating hepatitis B antigen and antibody. 18 We have now modified the technic to increase the sensitivity for HB s Ag to a third-generation level. The new procedure, card test III (CT III), has been compared with the original card test (OCT), RPHA, and RIA. The results of the comparisons and evi- ABBREVIATIONS HB s Ag = hepatitis B surface antigen CEP = counterelectrophoresis RPHA = reversed passive hemagglutination RIA = radioimmunoassay OCT = original card test CT III = card test III Anti-HB S = antibody to HB s Ag RIA I Ausria 125 test RIA II = Ausria test

2 60 STEVENS ET AL. A.J.CP. Vol. 66 / / S*. V FIG. 1. Agglutination and inhibition test control patterns. Card circle 1 contained HB s Ag-negative serum plus reference antibody; circle 2, positive serum and antibody. The added charcoal-hb s Ag reagent was aggregated by the antibody available in circle 1 only. dence of the sensitivity and specificity of CT III are presented in this report. Materials and Methods Charcoal Particle Tests Charcoal particle-hb s Ag suspensions and human reference antisera (anti-hb s ) were obtained from the hepatitis B card test (Hynson, Westcott and Dunning), an investigational new assay. Two sets of reagents were used. One set was stored in sealed vials and one in open vials, both at 5 C; titrations were made repeatedly to check the stability of the reagents as well as the reproducibility of the procedures. The particle- H B s Ag suspension was visibly aggregated by the reference antiserum; inhibition of this aggregation, indicating the presence of HB s Ag in serum, appeared as a uniform particle dispersion with a homogeneous gray background (Fig. 1). For the original test, 0.03 ml of undiluted serum specimen was mixed on a card with an equal volume of reference anti-hb s at one fourth the agglutinin titer. This inhibition mixture was held at room temperature for 2 min. CTIII inhibition was conducted in a tube at 45 C for one hour with 0.1 ml of specimen and an equal volume of anti- HB S at half the agglutinin titer. This mixture (0.06 ml) was then spread on a card. For each procedure one drop (0.016 ml) of charcoal-hb s Ag suspension was added, and the card was mechanically rotated at 100 rpm for 8 min, hand-tilted for further mixing, and examined for agglutination after standing an additional 12 min at room temperature. A negative serum and dilutions of standardized positive control sera were included with each test. To compare the sensitivities of the particle tests, 15 HB s Ag-positive sera diluted in a negative serum pool were examined. The reciprocal of the highest dilution that prevented particle aggregation was taken as the HB s Ag titer. To show the capacity of the tests to detect HB s Ag in the Bureau of Biologies reference panel, that panel was examined with each of the reagent sets by OCT and CT III. Hemagglutination The Auscell RPHA test (Abbott Laboratories), based on agglutination of guinea pig anti-hb s -sensitized erythrocytes by HB s Ag, was compared with CT 111. For this, 37 HB s Ag-positive sera were serially diluted with a negative serum pool. The reciprocal of the highest dilution that agglutinated the erythrocytes (RPHA) or inhibited particle aggregation (CT III) was taken as the HB s Ag titer. Radioimmunoassay The RIA I (Ausria 125; Abbott Laboratories) tube test with a 16-hour specimen incubation followed by a 90-min [ 125 I]- guinea pig anti-hb s incubation, both at

3 July 1976 HEPATITIS B CARD TEST III 61 room temperature, was compared with the OCT. Sera from 2,120 voluntary blood donors and 11 hospital patients with hepatic disorders tested by RIA were coded and re-examined by OCT. Portions of the positive specimens were referred to the Red Cross Blood Program laboratories for confirmation with standardized human anti-hb s. The newer RIA II (Ausria II-125; Abbott Laboratories) bead technic with 2-hour specimen and 1-hour [ 125 I]human anti-hb s incubation times at 45 C was compared with CTIII. Coded sera from 1,193 donors and 148 patients were tested. Results Specific reactivity and reproducibility of the particle tests, measured by control titrations with the reagent sets, did not change during this 5-month study. One reagent set was in use after storage in the original vials for as long as 12 months, the other after storage for as long as 18 months. Stability checks were satisfactory 5 and 8 months, respectively, after these vials were first opened. CT III titers of a series of standardized positive control sera were consistently 16 times those obtained with OCT. These levels of sensitivity were confirmed by results with 15 random sera (Fig. 2). In that specimen series, the maximum serum dilution positive by CT III was 4 to 256 times that detected by OCT. Findings obtained with the Bureau of Biologies reference panel are summarized in Table 1. The OCT detected antigen in two of the three samples with "C" level HB s Ag concentrations. The CT III found HB s Ag in all "C" samples and in one less concentrated "(C)" specimen. There was no measurable difference between the test sensitivities to ad and ay antigen serotypes. Quantitative CT III and RPHA test comparisons (Fig. 3) show that the dilution 10" ORIGINAL CARD TEST M CARD TEST III 103 FIG. 2. OCT (while bars) and CT III (shaded bars) test Liters of 15 HB s Ag-positive serum specimens. Z> a: UJ CO B 9 10 SERUM NUMBER 12 13

4 62 STEVENS ETAL. A.J.C.P. Vol. 66 IQ240 - >,?560 - f / i p - / < / I 10 - UNDIL ?,560 IQ240 CT III TITER FIG. 3. CT III and RPHA test titers of 37 HB s Agpositive sera. Each point represents one serum. of HB s Ag sera positive by CT III tends to be equal to, or greater than, that detected by RPHA. Of the 37 specimens tested, 23 had higher CT III titers, seven had the same titer in both tests, and seven had higher RPHA titers. The results of the card test-ria evaluations are summarized in Table 2. In the first study, only one of the group of 2,210 donors had a positive result in the OCT; that serum and one other were positive by RIA I. The RIA confirmatory test of the second specimen, however, was reported as nonspecific positive, as was one of the ten samples from the hospitalized group of II patients. One of these was OCT falselynegative. There was 100% agreement between RIA II and CT III tests on the second volunteer donor group; one specimen in the hospital-patient group was CT III falsely-negative. Discussion In sensitivity the new charcoal-particle technic equals the other third-generation tests. CT III is about 16 times more sensitive than the OCT, which was about eight times more sensitive than CEP 18 and thus has more than 100 times the sensitivity of the second-generation technology. A second-generation test is one capable of detecting HB s Ag in the reference panel sera designated "A", "(A)", and "B." Since the panel is arranged in order of decreasing antigen concentration, a thirdgeneration procedure is one that detects HB s Ag in the "A" and "B" plus the "(B)" and "C" samples. 3 The CT III found HB s Ag in all the required samples (Table 1); it therefore satisfies that definition of third-generation. The minimum concentrations of antigen detected by CT III and the third-generation RPHA test (Fig. 3) are essentially the same; thus, CT III, like RPHA, approaches RIA in sensitivity. 2 ' 3,11 Finally, in one specimen series, CT III results were similar to those from RIA II, a reference standard for evaluating the sensitivity of third-generation tests. 8,11 The agreement between the RIA II and CT III tests on sera of healthy and Table 1. National Institutes of Health Bureau of Biologies HB s Ag Reference Panel No. 2 Number Number Positive Description of Antigen Panel Speci- CT Subtype Designation mens OCT III ad A (A) B (B) C ay A (A) B 4 4 3* (B) C Not avail- C able (C) Nt * One specimen not available for CT111 test. t Negative for HU s Ag.

5 July 1976 HEPATITIS B CARD TEST III 63 hospitalized people also shows that the new procedure is specific. This is partly due to the use of the human rather than animal reference antiserum and the use of antigen rather than antibody on the indicator particle. The use of human antiserum precludes the formation of animal antiserum-human serum complexes, which have been responsible for false-positive RIA I results with [ 125 I] guinea pig anti-hb 12 s ' 14 and for nonspecific agglutination of guinea piganti-hb s -coated erythrocytes. 11 Antibody globulins on particles such as erythrocytes, 5 latex, 15 or charcoal 16 are reagents for the detection of rheumatoid factors (RF) or hyperglobulinemias. 20 Thus, in addition to agglutination with HB s Ag, these particles with anti-hb s affixed may show nonspecific aggregation with RF or elevated levels of specimen globulin. Such false-positive reactions have been observed with one RPHA reagent 13 and with latex-anti-hb s reagents. 4,7 In contrast, the charcoal agglutinationinhibition procedure, with antigen on the indicator particle, is unaffected by either qualitative or quantitative specimen protein abnormalities. 17 In this evaluation of 1,341 specimens, CT III showed specific aggregation of the charcoal particles or specific inhibition of the reference antibody by HB s Ag. CT III is thus sensitive to, and specific for, HB s Ag. In addition, the procedure has the unique advantages of reagent stability, technical simplicity, and speed. In contrast to the 2-to-4-week stability of fixed erythrocyte or radiolabeled reagents, working charcoal-hb s Ag suspensions and reference antibody were stable for at least 5 months. Particle agglutination tests require little skill or training; no special equipment or licensure is necessary, and the time required to set up, incubate, and read the test is less than 90 min. Results are clear and unequivocal (Fig. 1). CT III Specimen Group Table 2. Qualitative Card Test and Radioimmunoassay Results 1 Donor Patient II Donor Patient Number Tested 2, , OCT 1 8 Number Positive by* RIA I 2t 10t CT III RIA II * OCT = original card test: RIA I = Ausria 125 radioimmunoassay: CT III = card lest III: RIA II = Ausria radioimmunoassay. t Includes one nonspecific positive as determined by RIA confirmatory test. is thus a suitable procedure for the accurate screening or confirmatory testing of one, a few, or several hundred specimens at a time. Acknowledgments. Red Cross Blood Program Laboratories, Washington, D.C., provided the confirmatory RIA results. Dr. Robert Laffin, Albany Medical College, donated sera. Dr. Joseph Portnoy of Hynson. Westcott and Dunning, provided helpful advice, and technical assistance was provided by Katherine Moczulski and Kathy Gombel. References 1. Aschavai M, Peters RL: Manual for Hepatitis B Antigen Testing. Philadelphia, W. B. Saunders, 1973, pp Department of Health, Education and Welfare: Test for hepatitis associated (Australia) antigen; notice of proposed rule making. Fed Reg 39: , Department of Health, Education and Welfare: Test for hepatitis B surface antigen. Fed Reg 40: , Gratten MJ, Entwistle SW, Walker JA, et al: Latex-agglutination test for hepatitis-associated antigen. Lancet 1:485, Heller G, Jacobson AS, Kolodny MH, et al: The hemagglutination test for rheumatoid arthritis. II. The influence of human plasma fraction II (gamma globulin) on the reaction. J Immunol 72:66-78, Hirata AA, Emerick AJ, Boley WF: Hepatitis B virus antigen detection by reverse passive hemagglutination. Proc Soc Exp Biol Med 143: , Hopkins R, Das PC: Latex agglutination test for detection of Australia antigen (HB-Ag) among blood donors and patients. J Clin Pathol 27:40-44, Ling CM, Irace H, Decker R, et al: Hepatitis B virus antigen: Validation and immunologic

6 64 STEVENS ET AL. A.J.C.P. Vol. 66 characterization of low-titer serums with 125 I-antibody. Science 180: , Ling CM, Overby LR: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 125 I-antibody. J. Immunol 109: , Overby LR, Miller JP, Smith ID, et al: Radioimmunoassay of hepatitis B virus-associated (Australia) antigen employing 125 I-antibody. Vox Sang suppl 24: , Peterson DA, Froesner GG, Deinhardt FW: Evaluation of passive hemagglutination, solid-phase radioimmunoassay, and immunoelectroosmophoresis for the detection of hepatitis B antigen. Appl Microbiol 26: , Prince AM, Brotman B, Jass D, et al: Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis-b antigen. Lancet 1: , Reesink HW, Duimel WJ, Brummelhuis HGJ: Evaluation of a new hemagglutination technique for the demonstration of hepatitis-b antigen. Lancet 2: , Sgouris JT: Limitations of the radioimmunoassay for hepatitis B antigen. N Engl J Med 288: , Singer JM, Plotz CM: The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Am J Med 21: , Stevens RW, Stroebel E, Gaafer H: An automated test for rheumatoid factor. Arthritis Rheum 13: , Stevens RW, Ziegler FD, Almy RE, et al: Evaluation of charcoal particle agglutination tests for serum hepatitis (SH) antibody and antigen. Abstr Ann Meet Am Soc Microbiol 1972, p Stevens RW, Ziegler FD, Kelly JH: Detection of serum-hepatitis antigen by charcoal particle agglutination-inhibition. J Immunol 108: , Sultan WW, Whittington RO, Armstrong AS, et al: A reversed passive hemagglutination test system for the detection of hepatitis associated antigen. Abstr Ann Meet Am Soc Microbiol 1972, p Waller M: Present status of the rheumatoid factor. Crit Rev Clin Lab Sci 2: , World Health Organization: Viral Hepatitis. Report of a WHO Scientific Group. WHO Tech Rep Ser No 512:22-28, 1973