Arthritis and Rheumatism

Transcription

1 ~ ~ ~~ Arthritis and Rheumatism VOL. VII, NO. 1 FEBRUARY, 1964 Modification of the Waaler-Rose Test Using Formalinized Sheep Erythrocytes r. By FELIX MILCROM, OLAV TONDER AND ULANA LOZA A slide modification of the Waaler- Rose test was developed using formalinized sheep erythrocytes. Good agreement of the results obtained with the standard procedure and the slide modification was observed. Esseva elaborate un lamina modification del test de Waaler-Rose, con le us0 de formalinisate erythrocytos ovin. Esseva notate un bon grado de concordantia con le methodo standard e le modification a lamina porta-objectos. EROLOGICAL TESTS for rheumatoid arthritis have been routinely used S for the last 10 years as valuable diagnostic tools. All these tests are based on the detection of a serum factor called the rheumatoid factor which has serological properties of an antibody against gamma g1obulin.l The first of these tests described by Waaler2 and by Rose et al.,3 employes sheep erythrocytes sensitized by subagglutinating doses of a rabbit antiserum; the sensitized red blood cells are agglutinated by the rheumatoid factor. The Waaler-Rose test is being replaced more and more by the much simpler latex test in which the rheumatoid factor agglutinates latex particles coated by human gamma gl~bulin.~ However, the Waaler-Rose test is still a valuable serodiagnostic procedure. It appears that this test is somewhat less sensitive, but more specific for rheumatcid arthritis than the latex test."8 Waller and her collaboratorsr found the Waaler-Rose test positive in 68 per cent of patients suffering from rheumatoid arthritis, whereas the latex test was positive in 86 per cent. In testing 340 apparently healthy blood donors, the same authors found the Waaler-Rose test positive in only one case (0.3 per cent) but the latex test was positive in 10 cases (3 per cent). A high prevalence of positive latex tests in older persons was reported by Heimer and Rudd.7 In testing 101 aged people the authors found the latex test positive in 50 persons. Only in one case the positive test was justified by a clinical diagnosis of rheumatoid arthritis. In the same material only two persons were positive by the Waaler-Rose test. This situation may be explained by the fact that anti-gamma giobulin fac- From the Department (:f Bacteriology and Immunology, Sfute [Jnicjcrvity of New York at Buffdo, Buffalo 14, New York. Airled by a grant from the Ortho Rmearch Foundation. I AHTHR~TIS AND RHEUhIAl.lbh1, \'Ol,. 7, NO. 1 (FEBHKJAHY), 1964

2 2 MILCROM, TONDER AND LOZA tors similar to the rheumatoid factor may accompany many diseases other than rheumatoid arthritis and they may also occur in normal human beings; apparently factors appearing in persons without rheumatoid arthritis would react more frequently with human gamma globulin (latex test) than with rabbit gamma globulin ( Waaler-Rose test). It seems that the application of both the Waaler-Rose test and the latex test would give most valuable information to a clinician. However, the standard Waaler-Rose test is rather laborious and may be performed only by well-trained laboratory workers. The purpose of the present study was to simplify the performance of the Waaler-Rose test by employing formalinized sheep erythrocytes and by performing the test on a slide. Source of sera MATERIALS AND METHODS Four hundred sera originated from patients of the Buffalo General Hospital. All these patients were to some extent suspected of having rheumatoid arthritis, inasmuch as the sera were sent to the laboratory with a specific request for rheumatoid arthritis serology. The sera were examined by the latex test and 109 sera were selected which gave positive reactions at this screening. In addition, 319 sera were tested which were sent to the serological laboratory of the Veterans Administration Hospital in Buffalo for syphilis serology. Preparation of formalinized sheep erythrocytes (FSE) The method described by Daniel et al.9 was employed. Sheep erythrocytes were washed three times in buffered saline, ph 7.2. To one volume of the erythrocyte sediment were added 8 volumes of a 3 per cent solution of formaldehyde in buffered saline. The mixture was left for 24 hours at 4 C.; continuous gentle agitation was provided by an clectric shaker. Thereafter, two more volumes of 40 per cent formaldehyde were added, and the mixture was again agitated in the cold for 24 hours. The FSE were then washed 10 times with buffered saline. Titratwn of rabbit anti-sheep erythrocyte smum Two-fold dilutions of the antiserum were prepared in 0.2 ml. volumes. In order to obtain a more precise titer, two titration rows were included, one starting with a 1:50 and the other with a 1:75 serum dilution. Two-tenths ml. of a 1 per cent suspension of FSE were added to each tube. The tubes were incubated at room temperature for two to three hours, and patterns of agglutination were read. The patterns were read again after the tubes were shaken and left overnight at 4 C. Sensitization of formahixed erythrocytes A 2 per cent suspension of FSE was mixed with an equal volume of rabbit anti-sheep erythrocyte serum (Cappel Laboratories) at a dilution four times lower than the highest dilution that gave definite pattern agglutination at the above described titration. Most experiments were performed with an antiserum that had a titer of 1:6400. Accordingly, this serum was used for sensitization of FSE at a dilution of 1:1600. After incubation for 30 minutes at 37 C. the sensitized FSE were washed three times with buffered saline and prepared as a 2 per cent suspension. Thimerosal was added to make a final concentration of 1:10,000, and the suspension was distributed to small bottles. It could be stored at 4 C. or frozen for at least two months without any appreciable loss of serological activity.

3 WAALER-ROSE TEST MODIFICATION 3 The sli& test The human serum to be tested was diluted 20 times in buffered saline. A suspension of sensitized FSE was vigorously shaken to obtain an even dispersion. One drop of the diluted serum was placed on a slide and mixed well with one drop of the suspension of sensitized FSE. The slides were left undisturbed for 5 to 10 minutes at room temperature, and the degree of macroscopically visible agglutination was read in indirect light against a dark background. Agglutination was graded from If to 3+. Identical results were obtained in tests performed with active sera and with sera inactivated for 30 minutes at 56 C. T': e Wader- Rose test This test was performed following a previously described procedure.10 A subagglutinating dose of rabbit anti-sheep erythrocyte sertun was employed for sensitization of red blood cells: the serum dilution used for sensitization was twice as high as the agglutinating titer. Two-tenths ml. of the 0.5 per cent suspension of sensitized erythrocytes were added to 0.2 ml. of two-fold serial dilutions of the human serum to be tested. The tubes were left overnight at 4 C. and the degree of agglutination was recorded. The titer of the test was expressed in tables as the reciprocal of the highest serum dilution that gave positive agglutination. The latex twt This test was performed on slides with a 1:20 diluted serum and a reagent purchased from the Hyland Laboratories, Los Angeles, Calif. RESULTS In developing the present method advantage was taken of a few features of FSE: 1. In spite of exposure to formaldehyde the red blood cells retain antigenic sites com bining with the rabbit antiserum against sheep erythrocytes. However, the FSE are not clumped in the form of true agglutination. The reaction with the corresponding rabbit antiserum may be recognized only as a pattern agglutination. Interestingly, the titer of pattern agglutination with FSE is very similar to the titer of the agglutination of fresh red blood cells.0 2. Natural antibodies against sheep erythrocytes present in practically all human sera combine with FSE, producing pattern agglutination but never a true agglutination. 3. FSE may be sensitized by rabbit anti-sheep erythrocyte serum at a dilution that elicits pattern agglutination. Such sensitized cells may be washed several times, and in spite of repeated centrifugation, they do not undergo true agglutination. Addition of a goat antiserum against rabbit gamma globulin or of a rheumatoid arthritis serum to the sensitized FSE results in the quick appearance of relatively large clumps. This can be demonstrated in the simplest way on a slide. Shaking would still result in dispersion of these clumps. 4. can be preserved at 4 C. or in a frozen state for at least two months, without any appreciable loss of activity. The major objective in the evaluation of the described slide modification was its comparison with the routine Waaler-Rose test. No attempt was made to obtain any precise information about the clinical value of the slide test,

4 4 MILCROM, TONDER AND LOZA inasmuch as this value of the Waaler-Rose test has been investigated on very extensive material by several groups of investigators.5-8j1 In order to compare the sensitivity of the two tests, 109 sera were selected which originated from patients suspected of having rheumatoid arthritis and which were positive in the latex test. In order to compare the specificity of the two tests, 319 sera sent for syphilis serology were tested that originated from patients suffering from various diseases and from healthy human beings. It was anticipated that only a few rheumatoid sera would be in this material and accordingly, it appeared proper for studies on specificity. As is to be seen in Table 1, very good agreement between the results obtained by the slide modification and by the standard Waaler-Rose test was observed in both populations that were studied. It was also of some interest to compare the results obtained by the described slide procedure using sensitized FSE with the results obtained by the latex test. Table 2A presents the data obtained by testing 109 sera previously selected for their latex positivity. The latex test gave positive reactions with 32 sera that were negative in the slide test with sensitized FSE. It was interesting to determine the clinical justification for the serological positivity in these discordant cases. Clinical data could be obtained only in 25 of these 32 cases. In seven patients there was a clinical diagnosis of rheumatoid arthritis, whereas in the remaining 18 patients, this diagnosis could not be established on the basis of clinical examinations. It should still be stressed that the 109 sera were preselected for the latex positivity and so it could not include sera that would be positive in the slide test with sensitized FSE and negative in the latex test. Table 2B brings results obtained by testing 319 sera sent for syphilis serology. Not a single serum was found that was negative by the latex test and positive by the slide test with sensitized FSE; however, a reverse situation was found with sera of 14 patients. Clinical information was obtained about 12 of these patients; only in one case was there any clinical indication for rheumatoid arthritis. DISCUSSION As already stressed, no attempt has been made to obtain a precise evaluation of the clinical usefulness of the described slide test with sensitized FSE. It appeared entirely sufficient to use an adequate number of sera in order to compare the results obtained by this test with those obtained by the routine Waaler-Rose test. This comparison gave very good agreement. The discrepancies between the two tests were indeed not larger than the discrepancies which might be encountered in testing a large number of sera twice by the Waaler- Rose procedure. Comparison of the results obtained by the slide test with sensitized FSE and by the latex test indicated higher specificity of the first test and higher sensitivity of the latter test. Very similar results have been reported in comparing the routine Waaler-Rose test with the latex test. The limitations of the practical application of the described test are very few. As already mentioned the naturaliy occurring heterophile antibodies do

5 ~~ WAALER-ROSE TEST MODIFICATIOS S Table 1.-Comparison of the Routine Waaler-Rose Test with the Slide Modification Employing Formalinized Sheep Erythrocytes A. Population of 109 sera that were positive by the latex test Total Titer of Waaler- 20 or less Rose test 40 and or more B. Population of 319 sera sent for syphilis serology. Total ,+++ Total Titer of Waaler- 20 or less Rose test 40 or or more Total Table 2.-Comparison of the Latex Test with the Slide Test Employing Formalinized Sheep Erythrocytes A. Population of 109 sera that were positive by the latex test ,+++ Total Results of latex test ++, a3 B. Popnlation of 319 sera sent for syphilis serology. Total _ Total Results of latex -,- test , Total not interfere with the performance of the slide test; none of the 500 sera tested at dilutions of 1:20 gave a positive reaction with non-sensitized FSE. Potent antibodies against sheep red blocd cells such as encountered in infectious mononucleosis and serum sickness may interfere with performing the test by giving false positive results due to the reaction with cell antigens rather than with the rabbit antibody globulin coating the cells. This, however, can be easily controlled by clinical information and in doubtful cases, a slide test may be performed using unsensitized FSE as a control. The test was developed as a con\-enient, simple and inexpcnsi1-e screening

6 6 MILGROM, TONDER AND LOZA procedure somewhat similar in its application to the latex test. The test may be useful for serological examinations performed in a physician s office and also in performing serological mass examinations. In both situations, parallel performance of this test and the latex test would appear advisable. The described slide test cannot replace the original Waaler-Rose test as a quantitative procedure which might be useful in the follow up of individual cases during the disease and treatment. It appears however, that FSE can also be employed for a quantitative test tube reaction quite analogous to the Waaler-Rose test. In preliminary experiments along these lines, a suspension of FSE sensitized by a subagglutinating dilution of a rabbit anti-sheep erythrocyte serum was employed to replace the fresh erythrocytes in the Waaler-Rose test. The results of the test were read as a pattern agglutination. The titers obtained in this procedure were identical with or very close to the titers obtained in the conventional Waaler-Rose test. Additional convenience in using FSE may be obtained by staining them. Preliminary experiments along these lines have been performed and encouraging results were obtained. SUMMARY A simple slide modification of the Waaler-Rose test for rheumatoid arthritis was developed using formalinized sheep erythrocytes (FSE ). Rather strong sensitization of erythrocytes could be achieved because FSE give only a pattern a:gglutination and not a true agglutination in the presence of rabbit anti-sheep erythrocyte serum. In the proper test, sensitized FSE undergo marked clumping when mixed on a slide with a rheumatoid serum at a 1:ZO dilution. Good agreement of results obtained by the routine Waaler-Rose test and by the slide modification was observed in testing 109 sera selected for a very high prevalence of rheumatoid arthritis and with 319 sera selected for a very low rrevalence cf this disease. Similarly as the routine Waaler-Rose test, the slide mcdification was less sensitive but more specific for rheumatoid arthritis than the latex test. The andicability of the described modification as a screening procedure in a physician s office and in mass examinations was discussed. ACKNOWLEDGMENT The authors are indebted to Mrs. Margaret Grybel for her technicai assistance. 1. Milgrom, F., and Witebsky, E.: The rheumatoid factor and its possible pathogenic role. In: Mechanism of celi and tissue damage produced by immune reactions. Grabar, P. and Miescher, P., eds. IInd Intern. Symposiuni on Immunopathology, Brook Lodge (Mich., USA), Benno Schwabe & Co., Basel 1962, pp Wader, E.: On the occurrence of a REFERENCES factor in human serum activating the specific agglutination of sheep blood corpuscles. Acta path. rnicrobiol. scand. 17:172, Rose, H. M., Ragan, C., Pearce, E., and Liprnan, M. 0.: Differential agglutination of normal and sensitized sheep erythrocytes by sera of patients with rheumatoid arthitis. Proc. SOC. Exper. Biol. & Med. 88:1, Singer, J. M., and Plotz, C. M.: The

7 WAALER-ROSE TEST MODIFICATION 7 latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med. 21:888, Ball, J., Bloch, K. J., Burch, T. A., Kellgren, J. H., Lawrence, J. S., and Tsigalidon, V.: Comparative studies of serological tests for rheumatoid disease. 11. A comparison of the bentonite flocculation test and the sensitized sheep cell agglutination test. Arth. & Rheumat. 5:61, , DeGraff, R., Valkenburg, H. A., and Boerma, F. W.: Comparative studies of serological tests for rheumatoid disease. I. A comparison of the latex test and two erythrocyte agglutination tests in random population sample. Arth. & Rheumat. 5:55, Heimer, R., and Rudd, E.: Globulins resembling the rheumatoid factors in the sera of the aged. Roc. Eighth Interim Scientific Session ARA, Arth. & Rheumat. 5:110, Waller, M. V., Decker, B., Toone, E. C., Jr., and Irby, R.: Evaluation of rheumatoid factor tests. Arth. & Rheumat. 4:579, Daniel, T. M., Weyand, J. G. M., and Stavitsky, A. B.: Micromethods for the study of proteins and antibodies. IV. Factors involved in the preparation and use of a stable preparation of formalinized, tannic acid-treated, protein-sensitized erythrocytes for detection of antigen and antibody. J. Immunol. 90:741, Tonder, 0.: Studies on the mechanism of the Waaler-Rose test. Univ. Bergen, aarbok, Norwegian Universities Press, 1962, pp Coke, H.: Laboratory investigations towards the diagnosis of the difficult and early cases of rheumatoid arthritis. Arch. interamer. Rheum. 2:391, Felix Milgrom, M.D., Associate Professor of Bacteriology and Immunology, State University of New YoTk at Buffalo. Olav Tonder, M. D., International Postdoctoral Research Fellow of the National Institutes of Health, State University of New York at Bufalo. Uhna Loza, M.D., Research Associate in Bacteriology and Immunology, State University of New York at Buflalo.