Vox Sang. 34: 4 3 4 5 (1978)
1251-Anti-ImmunoglobulinTest : A New Tool for the Detection of Drug-Allergic Platelet Antibodies C . Mueller-Eckhardt, G . Schulz, C . Dienst, I . Mahn and Bettina Mayser Department of Clinical Immunology and Blood Transfusion, University of Giessen, Giessen
Abstract. It is shown that the l Z 5I-anti-immunoglobulin test with platelets is as sensitive and reproducible for the detection of quinidine-associated antibodies as platelet complement fixation. Because of its independence from complement activation, this test might prove most valuable for the demonstration of non-complement-fixing, drug-associated antibodies.
Since Ackroyd’s classical experiments the immunological nature of some cases of drug-associated thrombocytopenic purpura has clearly been established [for a review, see Miescher and Pepper, 31. Unfortunately, the serological detection of the causative agent is still the exception rather than the rule. One possible reason for the low incidence of antibody findings is probably the lack of a sensitive method which would allow not only the demonstration of complete, i.e., complement-fixing, but also of ’incomplete’ (blocking?) antibodies. We therefore report the results of a study aimed to demonstrate drug-induced platelet antibodies by a newly developed radioimmune antiglobulin test with platelets (PRAT) and to compare it to conventional platelet complement fixation (PCFT). 1 Supported by the ‘Deutsche Forschungsgemeinschaft’ ( M u 277/7).
Materials and Methods Throughout this study, a strong complementfixing, quinidine-induced platelet antibody was used. The serum originated from a patient who developed an acute thrombocytopenic ptirpura after repeated ingestion of quinidine for cardiac arrhythmias. Test platelets were obtained by differential centrifugation of citrated platelet-rich plasma of HLA-typed donors of blood group 0, and washed 3 times in a modified albumin-tyrode buffer (ATB) containing prostaglandin [l]. After washing, the platelets were suspended in ATB without prostaglandin, adjusted to a concentration of 1,000,000 platelets/!tl and stored at 4 O C for several weeks. 0.1% sodium azide was added as a preservative. The preparation of reagents and the test procedure itself were similar to those recently described by Soitlier et ( I / . [ 5 ] , with certain modifications. Details of the method will be given elsewhere [4]. In brief, human IgG was fractionated from pooled human serum by ion exchange chromatography using QAE-A50 gel (Pharmacia). The purified human IgG was then coupled to CNBr-activated Sepharose 4B (Pharmacia) and used for irnmuno-
44
Mueller-Eckhardt/Schulz/Dienst/Mahn/Mayser
r
16
05
- 14 04
- 12
-E 0 3 -c
- 10
-8
W
m 4
0
-6
02
c c d
8-4 p
01
D
r2
Serum dilution
s
i
+
2
2 5 0 5 0 2 5 005 0025 0005 0003 00005 Final quinidine concentration, rnM
Fig. 1. Demonstration o f quinidinc-induced plaFig. 2. Dependence of antibody detection on and PCFT (VV) the concentration of quinidine (signs as in figure 1). telet antibodies by PRAT (00) in the presence ( 0 7 )and absence (OV) of the drug.
absorption of a commercially available antiserum against human IgG produccd in rabbits (Behringwerkc). The spccific IgC antibodies bound to the IgG-Sepharose were eluted by a HCI-glycin buffer (0.1 M, pH 2.5). and the fractions containing the anti-IgC were neutralized, pooled and concentrated by ultrafiltration. The activity of the anti-lgG was dcterminetl against human red blood cells (blood group 0, Rh-positive) sensitizcd by incomplete anti-D anribodies. The purified anti-IgC was stored in small aliquots at -60 OC until labeling. For labeling of anti-IgG with 1351 [sodium iodide-(lrsl), spec. act. 8-15 Ci l25l/mg 1; Behringwerke]. the iodine monochloride procedure of McFnrlane [2] was followed. A specific activity o f approximately 100 :tCi/mg anti-TgC was achieved. 'The protein-boiind radioactivity was over 98.5%. The labeled anti-lgC was kept frozen at d O 0 C in small aliquots until use. The test itsclf is essentially an indirect antiglobulin reatiori. 50 x 108 test platelets were incubated for 30 min at 37 O C with 50,ul of the heat-inactivated serum to be tested and 50yl of quinidine sulphate (Mcrck) in buffered saline. The platelets were then washed with 0.5% albumin buffer (pH 7.4) and thereafter incubated with 100 p l of the Irsl-anti-lgG appropriately diluted to give a total of approximately 10,000 cprn per test reaction. After incubation (20 min at room tempera-
ture) the platelets were washed again and thc radioactivity of the platelet pellet was determined. 'Ihc fixed radioactivity was expressed in percent of total radioactivity. All tests were performed in duplicate. An antiserum known to contain complement-fixing HLA-antibodies (positive control) and sera of young male blood donors without a history of previous blood transfusions and without detectable platelet antibodies (negative controls) were run in parallel for each test. Quantitative complement fixation with platelets was performed as described by Svejguctrd and Kissniryer-Nielsen 161, with minor modifications.
Results The sensitivity of the PRAT for thc detection of quinidine-induced antibodies was equal to that of the PCFT (fig. 1). The dependence of both tests on various quinidinc concentrations for positive reactions is demonstrated in figure 2. With either method, the minimal quinidine concentration nccessary for antibody fixation on test platelets in vitro was between 0.05 and 0.025 mM.
Platelet Radioactive Antiimmunoglobulin Test
Conclusions
Our results show that the P R A T is a new and useful technique for the detection of drug-induced platelet antibodies. T h e advantages it shares with the P C F T are its considerable sensitivity, the high degree of reproducibility, and the usability of stored platelets of known antigenicity. Thus, the test is independent of viable platelets, a prerequisite for all immunological methods measuring changes of platelet function such as lac-serotonin release, aggregation or platelet factor 3 release. As n o activation of complement is needed, P R A T seems also to be superior to P C F T and, at least theoretically, might permit the serological determination of ‘incomplete’, non-complement-fixing, drug-associated antibodies. Further studies are necessary to verify this assumption.
References I Heinrich, D.; Stephinger, U., and Mueller-Eckhardt, C.: Specific interaction of H1.A antibodies (eluates) with washed platelets. Br. J. Haemat. 3.5: 443 (1977).
45
2 McFarlane, A. S.: I n vivo behaviour of 1311fibrinogen. J. din. Invest. 42: 346 (1963). 3 Miescher, P. A. and Pepper, J. J.: Drug-induced immunologic blood dyscrasias; in Miescher arid Miiller-Eherhnrd Textbook of immunopathology; 2nd ed., vol. 1, p. 421 (Grime & Stratton, Ncw York 1976). 4 Mueller-Eckhardt, C.; Schulz, G.; Sauer, K. H.; Dienst, C., and Mahn, I.: Studies on the platelet radioactive anti-immunoglobulin test. J. immunol. Methods (in press). 5 Soulier, J. P.; Patereau, C., and Drouet, I.: Platelet indirect radioactive Coonibs test. Its utilization for Pla, grouping. Vox Sang. 29: 253 (1975). 6 Svejgaard, A. and Kissmeyer-Nielsen, F.: Coniplenient-fixing platelet isoantibodies. I. A quantitative technique for their detection. Vox Sang. 14: 106 (1968).
Received: February 25, 1977 Accepted: March 24, 1977 Prof. Dr. C. Mueller-Eckhardt, Department of Clinical Immunology and Blood Transfusion, University of Giessen, Langhansstrasse 7, D-6300 Giessen (FRG)
1251-Anti-ImmunoglobulinTest : A New Tool for the Detection of Drug-Allergic Platelet Antibodies C . Mueller-Eckhardt, G . Schulz, C . Dienst, I . Mahn and Bettina Mayser Department of Clinical Immunology and Blood Transfusion, University of Giessen, Giessen
Abstract. It is shown that the l Z 5I-anti-immunoglobulin test with platelets is as sensitive and reproducible for the detection of quinidine-associated antibodies as platelet complement fixation. Because of its independence from complement activation, this test might prove most valuable for the demonstration of non-complement-fixing, drug-associated antibodies.
Since Ackroyd’s classical experiments the immunological nature of some cases of drug-associated thrombocytopenic purpura has clearly been established [for a review, see Miescher and Pepper, 31. Unfortunately, the serological detection of the causative agent is still the exception rather than the rule. One possible reason for the low incidence of antibody findings is probably the lack of a sensitive method which would allow not only the demonstration of complete, i.e., complement-fixing, but also of ’incomplete’ (blocking?) antibodies. We therefore report the results of a study aimed to demonstrate drug-induced platelet antibodies by a newly developed radioimmune antiglobulin test with platelets (PRAT) and to compare it to conventional platelet complement fixation (PCFT). 1 Supported by the ‘Deutsche Forschungsgemeinschaft’ ( M u 277/7).
Materials and Methods Throughout this study, a strong complementfixing, quinidine-induced platelet antibody was used. The serum originated from a patient who developed an acute thrombocytopenic ptirpura after repeated ingestion of quinidine for cardiac arrhythmias. Test platelets were obtained by differential centrifugation of citrated platelet-rich plasma of HLA-typed donors of blood group 0, and washed 3 times in a modified albumin-tyrode buffer (ATB) containing prostaglandin [l]. After washing, the platelets were suspended in ATB without prostaglandin, adjusted to a concentration of 1,000,000 platelets/!tl and stored at 4 O C for several weeks. 0.1% sodium azide was added as a preservative. The preparation of reagents and the test procedure itself were similar to those recently described by Soitlier et ( I / . [ 5 ] , with certain modifications. Details of the method will be given elsewhere [4]. In brief, human IgG was fractionated from pooled human serum by ion exchange chromatography using QAE-A50 gel (Pharmacia). The purified human IgG was then coupled to CNBr-activated Sepharose 4B (Pharmacia) and used for irnmuno-
44
Mueller-Eckhardt/Schulz/Dienst/Mahn/Mayser
r
16
05
- 14 04
- 12
-E 0 3 -c
- 10
-8
W
m 4
0
-6
02
c c d
8-4 p
01
D
r2
Serum dilution
s
i
+
2
2 5 0 5 0 2 5 005 0025 0005 0003 00005 Final quinidine concentration, rnM
Fig. 1. Demonstration o f quinidinc-induced plaFig. 2. Dependence of antibody detection on and PCFT (VV) the concentration of quinidine (signs as in figure 1). telet antibodies by PRAT (00) in the presence ( 0 7 )and absence (OV) of the drug.
absorption of a commercially available antiserum against human IgG produccd in rabbits (Behringwerkc). The spccific IgC antibodies bound to the IgG-Sepharose were eluted by a HCI-glycin buffer (0.1 M, pH 2.5). and the fractions containing the anti-IgC were neutralized, pooled and concentrated by ultrafiltration. The activity of the anti-lgG was dcterminetl against human red blood cells (blood group 0, Rh-positive) sensitizcd by incomplete anti-D anribodies. The purified anti-IgC was stored in small aliquots at -60 OC until labeling. For labeling of anti-IgG with 1351 [sodium iodide-(lrsl), spec. act. 8-15 Ci l25l/mg 1; Behringwerke]. the iodine monochloride procedure of McFnrlane [2] was followed. A specific activity o f approximately 100 :tCi/mg anti-TgC was achieved. 'The protein-boiind radioactivity was over 98.5%. The labeled anti-lgC was kept frozen at d O 0 C in small aliquots until use. The test itsclf is essentially an indirect antiglobulin reatiori. 50 x 108 test platelets were incubated for 30 min at 37 O C with 50,ul of the heat-inactivated serum to be tested and 50yl of quinidine sulphate (Mcrck) in buffered saline. The platelets were then washed with 0.5% albumin buffer (pH 7.4) and thereafter incubated with 100 p l of the Irsl-anti-lgG appropriately diluted to give a total of approximately 10,000 cprn per test reaction. After incubation (20 min at room tempera-
ture) the platelets were washed again and thc radioactivity of the platelet pellet was determined. 'Ihc fixed radioactivity was expressed in percent of total radioactivity. All tests were performed in duplicate. An antiserum known to contain complement-fixing HLA-antibodies (positive control) and sera of young male blood donors without a history of previous blood transfusions and without detectable platelet antibodies (negative controls) were run in parallel for each test. Quantitative complement fixation with platelets was performed as described by Svejguctrd and Kissniryer-Nielsen 161, with minor modifications.
Results The sensitivity of the PRAT for thc detection of quinidine-induced antibodies was equal to that of the PCFT (fig. 1). The dependence of both tests on various quinidinc concentrations for positive reactions is demonstrated in figure 2. With either method, the minimal quinidine concentration nccessary for antibody fixation on test platelets in vitro was between 0.05 and 0.025 mM.
Platelet Radioactive Antiimmunoglobulin Test
Conclusions
Our results show that the P R A T is a new and useful technique for the detection of drug-induced platelet antibodies. T h e advantages it shares with the P C F T are its considerable sensitivity, the high degree of reproducibility, and the usability of stored platelets of known antigenicity. Thus, the test is independent of viable platelets, a prerequisite for all immunological methods measuring changes of platelet function such as lac-serotonin release, aggregation or platelet factor 3 release. As n o activation of complement is needed, P R A T seems also to be superior to P C F T and, at least theoretically, might permit the serological determination of ‘incomplete’, non-complement-fixing, drug-associated antibodies. Further studies are necessary to verify this assumption.
References I Heinrich, D.; Stephinger, U., and Mueller-Eckhardt, C.: Specific interaction of H1.A antibodies (eluates) with washed platelets. Br. J. Haemat. 3.5: 443 (1977).
45
2 McFarlane, A. S.: I n vivo behaviour of 1311fibrinogen. J. din. Invest. 42: 346 (1963). 3 Miescher, P. A. and Pepper, J. J.: Drug-induced immunologic blood dyscrasias; in Miescher arid Miiller-Eherhnrd Textbook of immunopathology; 2nd ed., vol. 1, p. 421 (Grime & Stratton, Ncw York 1976). 4 Mueller-Eckhardt, C.; Schulz, G.; Sauer, K. H.; Dienst, C., and Mahn, I.: Studies on the platelet radioactive anti-immunoglobulin test. J. immunol. Methods (in press). 5 Soulier, J. P.; Patereau, C., and Drouet, I.: Platelet indirect radioactive Coonibs test. Its utilization for Pla, grouping. Vox Sang. 29: 253 (1975). 6 Svejgaard, A. and Kissmeyer-Nielsen, F.: Coniplenient-fixing platelet isoantibodies. I. A quantitative technique for their detection. Vox Sang. 14: 106 (1968).
Received: February 25, 1977 Accepted: March 24, 1977 Prof. Dr. C. Mueller-Eckhardt, Department of Clinical Immunology and Blood Transfusion, University of Giessen, Langhansstrasse 7, D-6300 Giessen (FRG)
