Arthritis and Rheumatism OficialJournal of the drnerican Rheumatismassociation VOL. 11, NO. 2 APRIL, 1959

Differentiation between Rheumatoid Arthritis and Systemic Lupus Erythematosus by Sheep

Cell Agglutination Tests

By ARNOLD BUCK, MILTON GOLDIN, ROBERT M. POSKE AND LEE MALMED

In the cold precipitation method (Svartz) the hemagglutinating factor of systemic lupus erythematosus is reported to re- main in the supernatant. Efforts to show clear-cut serologic differences between patients with rheumatoid arthritis, sys- temic lupus erythematosus and rheu- matoid arthritis with L.E. cells utilizing the euglobulin method (Ziff) and the cold precipitation method (Svartz) were at- tempted. While absolute differentiation was not possible, the authors believe that these methods may be of aid in diagnos- tic problems involving these diseases.

In le methodo a cryo-precipitation de Svartz, le factor hemagglutinatori de systemic lupus erythematose remane, reportatemente, in le liquid0 super- natante. Esseva facite le essayo de monstrar clar differentias serologic inter patientes con arthritis rheumatoide, systemic lupus erythematose, e arthritis rheumatoide con cellulas de lupus ery. thematose. In isto le methodo a euglo- bulina de Zif€ e a cryo-precipitation de Svartz esseva usate. Un differentiation absolute non esseva possibile, sed le autores crede que le methodos es pos- sibilemente de adjuta in problemas diagnostic que concerne le morbos mentionate.

HE CLINICAL DIFFERENTIATION between rheumatoid arthritis TW.1 and systemic lupus erythematosus (S.L.E.) offers no difficulties in the usual patient. However, certain cases of S.L.E. may exhibit a significant degree of arthritis, even to the extent of 'abnormal x-ray hdings.l Conversely, varying numbers of patients with R.A. (3 to 27 per cent) have been found to have L.E. cells in the periphefal blood on examination by the usual tech- nic~.*-~ Thus, situations occur in which some confusion may arise, especially from the standpoint of prognosis and therapy.

Our own experience with this problem began in 1955 when a group of patients with clinically overt R.A. were being studied for..'the incidence of L.E. cells. Prolonged and repeated examinations of many blood smears finally resulted in positive findings in 25 of 91 patients (27 per cent)." Almost all

From the Mount Sinai Hospital and Medical Research Foundah, and the Chfcugo Medical School, Chicago, Ill., Veterans Administration Hospital, Hines, 111. and Unitmsfty of lllinofs College of Medidne, Chicago, ZU.

Aided by a grant from the ZUfnois Chapter, Arthdtis and Rheumatism FoundatcOn.

Q9

100 BLACK, GOLDIN, POSKE AND MALMED

the positive cases had elevated titers when tested by the ziff modification of the sensitized sheep cell test (euglobulin method).6 When patients with S.L.E. were similarly tested, over 50 per cent demonstrated elevated sheep cell titers. Most of this latter group had some definite joint involvement, usually in the form of a synovitis. Serologic differentiation between R.A. and S.L.E. by the euglobulin procedure was thus not very satisfactory.

In 1953, Svartz and Schlossman7 described a technic in which the globulin fraction of the serum to be tested was precipitated out by the addition of cold distilled water. They subsequently reported" that, under these conditions, the R.A. factor was present in the precipitate while the hemagglutinating substance of S.L.E. (and other collagen diseases) remained in the supernatant.

In the present study, sheep cell agglutination tests were done on a group of cases by both the euglobulin and cold precipitation procedures. The objectives were: first, to determine whether it was possible to differentiate serologically between R.A. and S.L.E., and secondly, to evaluate a group of patients previously studied who had clinical R.A. and L.E. cells in the peripheral blood.

MATERIALS AND METHODS The technics of both procedures are well known and basically similar, with the ex-

ception of the globulin fraction to be tested. In the Ziff procedure,' the euglobulin frac- tion is separated out by dialysis of the absorbed serum against an acid buffer at 4" C. for 48 hours. The precipitate is redissolved and serial twofold dilutions, beginning with an initial dilution of 1:14, are tested. A titer of 1:28 is considered positive.

In the Svartz procedure,' the globulin is precipitated by adding 14 volumes of cold distilled water and allowing it to stand for 48 hours at 4" C. The precipitate is redissolved and serial twofold dilutions beginning with an initial dilution of 1:4 are te9ted. A titer of 1:16 is considered positive. Four groups of patients were studied. All clinical diagnoses were established inde-

pendently of the results of the serologic tests.

Group I. Rheumatoid arthritis: 35 cases. These fulfilled the diagnostic criteria of the American Rheumatism Association, including negative L.E. preparations. All had involvement of the peripheral joints; two also had spondylitis.

Group 11. Systemic lupus erythematosus: 28 cases. All had clinical findings usimlly considered compatible with this diagnosis, including L.E. cells in the peripheral blood.'

Group III. Rheumatoid arthritis with positive L.E. preparations: 17 cases. These pa- tients were essentially similar to those in Group I, except that the latter had disease that was of a longer duration and with a higher incidence of nodules.

Group IV. Normal healthy young adults: 30 cases.

RESULTS

The results are summarized in table 1. In Group I (R.A.) 10 cases were negative by the euglobulin method. Of these, two had spondylitis with peripheral joint disease. Seventeen were negative by the cold precipitation method. Those positive by this method (18 cases) were also positive by the euglobulin method.

'We are indebted to Dr. Victor Pollak for allowing us to include some of his cases in this study.

SHEEP CELL AGGLUTINATION TESTS IN R.A. AND S.L.E. 101

TABLE l.-ReSults of Sensitized Sheep Cell Agglutindion Tests in Four Goup of PatcentS

Eunlobulin (Zif?) Xethod Cold Precipitation (ha&) Method Titmheb Titar Lev&

Low Medium Low Medium TOW Poaitive 1:28 1:112 High Positive 1:lS 1:64 High

Clinical GrmD Cased Titen '1:66 1:224 1:448+ Titm 1:82 1:128 1:266+

I. Rheumatoid arthritis(R.A.) 35 25(71%) 6 8 11 l8(51%) 6 6 6

11. systemic lupus erythematosus (S.L.E.) 28 15(54%) 10 4 1 4(14%) 3 1 0

111. Rheumatoid arthritis with positive L.E. preparations 17 16(94%) 3 5 8 12(71%) 4 5 3

N.Normalcontrols 30 0 0

In Group I1 (S.L.E.) the cold precipitation technic was of considerable help. Only four cases (14 per cent) were positive, three such results occurring in the lowest titer (1:16). The other patient was a 35 year old woman with a two ye& history of fever, bilateral pleural effusion, hepatosplenomegaly and rheumatoid-like involvement of the fingers and wrists. Laboratory results included: positive L.E. phenomenon, total protein of 8.6 Gm. with a globulin of 6.1 Gm., and a thymol turbidity of 19.8 units. The cold precipitation titer was 1:64, while the euglobulin titer was 1:896.

In this same group, 15 patients had positive euglobulin tests. Five of these had definite synovitis, although there were five other patients with similar findings who had negative titers.

In Group I11 (R.A. with L.E. cells) the results were essentially the same as in Group I except that there was a higher incidence of positive results and in higher titers. Most of these patients had severe arthritis of long duration, and nine had subcutaneous nodules. There was never any clinical evidence of S.L.E. in any of these cases.

Both tests were negative in Group IV (normal controls).

DISCUSSION The basic purpose of this study was to evaluate a serologic differentiation

between R.A. and S.L.E. It was not in the realm of this project to evaluate the relative sensitivity of the tests used in R.A, Our percentages of positive results were generally lower than those reported by the origind authors,@JO but this may have been due to the selection of cases.

It became apparent that the cold precipitation test was practically always negative in cases of S.L.E., thus confirming the original author's concept of separation of the R.A. and L.E. hemagglutinating factors. In those few patients who exhibited positive results, the titers were always in the lowest range, with the one exception previously described. This patient appeared to have S.L.E. but in addition had a significant degree of arthritic changes in the hands and wrists, both clinically and on x-ray indistinguishable from

102 BLACK, GOLDIN, POSKE AND MALMED

R.A. This would indicate that the test was highly specific for R.A. (in con- trast with S.L.E.). However, the over-all results did not demonstrate adequate sensitivity in our series of patients. On the other hand the euglobulin method, while a more sensitive test for R.A., was not very specific since there were many “‘false positive” results in S.L.E. (54 per cent). This would suggest that both tests should be performed simultaneously in cases which present diagnostic problems.

It should be admitted, however, that there is a certain area in the spectrum of these diseases in which the overlapping of clinical findings is so great as to defy objective differentiation. The one case mentioned above might be included in this category, and only long-term observation together with repeated serologic testing may finally reveal a significant answer.

Finally, it is of interest to note that patients with clinical R.A. and L.E. cells in the peripheral blood react serologically exactly like comparable patients with R.A.

SUMMARY AND CONCLUSIONS 1. The euglobulin (Ziff) and the cold precipitation (Svartz) modifications

of the sheep cell agglutination tests for R.A. were simultaneously performed on four groups of cases. These included 35 patients with R.A., 28 patients with S.L.E., 17 patients with R.A. and L.E. cells in the peripheral blood, and 30 patients as normal controls.

2. The euglobulin test, while sensitive for R.A., was also found to be posi- tive in a majority of patients with S.L.E.

3. The cold precipitation test, though less sensitive for R.A., was highly specific for this disease and practically always negative in S.L.E. 4. Patients with R.A. and L.E. cells in the peripheral blood reacted

serologically exactly like comparable patients with R.A. 5. It is suggested that in diagnostic problems involving these diseases, both

the euglobulin and the cold precipitation tests be performed, the former for its sensitivity and the latter for its specificity.

REFERENCES 1. Harvey, A. M., Shulmm, L., Tumulty,

P., Conley, C. and Schoenrich, E.: Systemic lupus erythematosus. Medi- cine 33:291, 1954.

2. Ishmael, W. K., Owens, J. N., Jr. and Payne, R. W.: The incidence of posi- tive L.E. preparations in patients with rheumatoid arthritis. Ann.Rheumat. Dis. 14:415, 1955.

3. Kievits, J. H., Goslings, J., Schuit, H. R. E. and Hijmans, W.: Rheumatoid arthritis and the positive L.E. cell phenomenon. Ann.Rheumat.Dis. 15:

4. Weiss, R. S. and Swift, S.: The sig- . 211, 1956.

nificance of a positive L.E. phenome- non. A.M.A. Arch.Dermat. &Syph. 72: 103, 1955.

5. Friedman, I., Sickley, J., Poske, R., Black, A,, Bronsky, D., Hartz, W., Feldhake, C., Reeder, P. and Katz, E.: The L.E. phenomenon in rheu- matoid arthritis. Ann.Int.Med. 46: 1113, 1957.

6. Ziff, M., Brown, P., Badin, J. and Mc- Ewen, C.: A hemagglutination test for rheumatoid arthritis with enhanced sensitivity using the euglobulin frac- tion. Bu1I.Rheumat.Dis. 575, 1954.

7. Svartz, N. and Schlossmann, K.: Agglu-

SHEEP CELL AGGLUTRUTION TESTS IN R.A. AND S.L.E. 103

tination of sensitized sheep erythro- cytes by cold precipitable serum sub- stances in rheumatoid arthritis. Acta med.Scandinav. 146:313, 1953.

8. - and -: Cold precipitable hemagglu- tinating factor in serum from patients with rheumatoid arthritis. Ann.Rheu- mat.Dis. 14:191,, 1955.

9. Zil€, M., Brown, P., Lospalluto, J., Badin.

J. and McEwen, C.: Agglutination and inhibition by serum globulin in the sensitized sheep cell agglutiuation re- action in rheumatoid arthritis. Am.J. Med. 20:500, 1958.

rum cold precipitable hemaggluti- nating factor in rheumatoid arthritis. Acta med.Scandinav. 144:83, 1954.

10. Svartz, N. and S c h l ~ ~ ~ m m , K.: A ~ e -

Amold Black, M.D., Assistant Clinical Professor o f Medicine, Chicago Medical School, Chicago, Ill.; Attending Physician in Internal Medicine (Arthritis), U.S. Veterans Hospital,

Hines, Il l .

Milton Goldin, M.S., Chief Bacteriologist, Mt. Sinai Hospital and Mt. Sinai Medical Research Foundation, Chicago, Ill.;

Instructor in Microbiology, Chicago Medical School.

Robert M. Poske, M.D., Assistant to the Chief o f Medical Seruice, U S . Veterans Hospital, Hincs, Ill.; Clinical AssoCMte, Department of Medicine, University of Illinois College of

Medicine, Chicago, Ill.

Lee Malmed, B.S., Senior Medical Student, University of Illinois College of Medicine, Chicago, 111.