Arthritis and Rheumatism ~

01ciuljuurnul of the grnerican Rheumatismassociation

VOL. 11, NO. 1 FEBRUARY, 1959

The Presence of the ‘Rheumatoid Factor’ in Sera

from Patients with Syphilis
By ANDREPELTIERAND CHARLES
L. CHRISTIAN

Sera from patients having syphilis gave
positive tests for rheumatoid factor in
11 per cent of 147 cases studied when
the F I1 latex and tanned sheep cell pro-
cedures were used. The sensitized sheep
cell test was positive, however, in but
one serum giving positive results by the
F I1 tests. The factor in syphilis sera
responsible for positive rheumatoid re-
actions was separable from Wasserman
antibody.
Seros ab patientes con syphilis habeva
reactiones positive pro factor rheuma-
toide in 11 pro cento de un serie de
147 casos studiate per medio del tech-
nica a latex F I1 e a sensibilisate cellulas
ovin. Tamen, le test a cellulas ovin
esseva positive in solmente un caso de
positivitate per le test a F 11. Le factor
in seros syphilitic que es responsabile pro
le positive reactiones rheumatoide se
provava separabile ab le anticorpore de
Wassermann.

T HE SEROLOGIC TESTS for rheumatoid arthritis have been established

as reasonably specific. Positive reactions do, however, occur in diseases
other than rheumatoid arthritis. Positive serologic tests have been described
in patients with sarcoidosis, kala azar, cirrhosis of the liver,l hepatitis2 and
a wide variety of hemolytic and hepatic disorder^.^ The positive reactions
that occur in diseases other than rheumatoid arthritis are of particular inter-
est, since there may be factors in common which relate to the pathogenesis
of rheumatoid arthritis.
In the course of a study of the biologic false positive Wassermann reaction,
it was noted that some sera from patients having syphilis were positive in
the F I1 tanned sheep cell agglutination test ( F 11-TSCA). It is the purpose
of this report to describe studies of these sera.
MATERIALS
AND METHODS

1.) Blood was obtained from 147 patients with clinically or serologically detectable syphilis
who were followed in the Dermatology Clinic of the Presbyterian Hospital. Sera, when
not used immediately, were stored at -20°C.

From the Department of Medicine, Columbia Unkersity College of Physicians and

Surgeons, and the Edward Daniels Faulkner Arthritis Clinic of the Presbyterian Hospital,
N e w York, N.Y.
Supported in part by a grant from the U.S.P.H.S.
One of the authors (A. P.) is Trainee, National Institute of Arthritis and Metabolic
Diseases; the other (C. L. C.) is Fellow of the Arthritis and Rheumatism Foundation,
New York, N.Y.
1
2 ANDRE PELTIER AND CHARLES L. CHRISTIAN

2.) The F II-TSCA test was performed according to the technic described by Heller
et al.’ The test was considered 3 s positive when a strong agglutination pattern occurred
at a serum dilution of 1:56 or greater. 3 . ) The F I1 latex fixation test was performed
according to the technic described by Singer and Plotz.5 4.) F I1 precipitin was performed
as previously described.“ 5 . ) The amounts of precipitate were quantitated by the method
of Folin and Coicalteu, as described by Heidelberger and MacPherson.7 6. ) Sedimenta-
tion studies were performed in a Spinco Analytical Ultracentrifuge (Model E ) . 7.) The
sensitized sheep cell test (SSC) was performed according to the technic described by
Rose et al.” 8.) Cohn Fraction I1 ( F 11) was obtained from E. R. Squibb K Sons. 9.) Ab-
sorption of the “rheumatoid factor” from the F I1 positive. sera was accomplished by
adding increasing amounts of aggregated gamma globulin to constant aliquots of sera
(0.6 ml. ). The aggregated gamma globulin was concentrated from Cohn fraction I1 by
salt precipitation? After incubation for 30 minutes a t 37” C. and overnight a t 4” C., the
tubes were centrifuged and the supernatants studied in the F II-TSCA and VDRL tests.
10. ) Absorption of the “Wassermann antibody” was accomplished by adding increasing
volumes of undiluted cardiolipin antigen ( Mazzini flocculation antigen) to conTtant aliquots
of serum (0.5 ml.). The tubes were allowed to incubate at room temperature for a few
minutes and then centrifuged. The supernatants were tested as noted above.
11.) VDRL tests were performed according to the technic described in the Manual of
Serologic Tests for Syphilis.’’

RESULTS
1. Rheumatoid serologic tests ( F 11-TSCA, F 11 latex fixation and SSC).-Of
the 147 sera tested by the F 11-TSCA test, 16 (11 per cent) gave a positive
reaction and 131 were negative (89 per cent). This incidence of positive
tests is higher than the percentage encountered in a normal control popula-
tion (1to 3 per cent).4 The titers of positive sera ranged between 1:224 and
1:2800 (table 1 ) . The F II-TSCA tests were repeated at least once on each
serum. F I1 latex fixation tests confirmed these results, showing titers closely
parallel to the F II-TSCA titers. The study of the F I1 positive sera with sen-
sitized sheep cell tests (SSC) showed an interesting discrepancy: None of
the F I1 positive sera but one (Serum No. 501 with an F II-TSCA titer of
1:7000) demonstrated positive reactions in the SSC test.
2. Correlation between clinical and serologic data.-The analysis of the
clinical features of patients in this series did not reveal any significant differ-
ence between the F I1 positive and F I1 negative groups. The time of onset
of the syphilitic infection was only infrequently documented (five times in
the positive group and ten times in the F I1 negative group).
The clinical manifestations of syphilis were infrequent, most of the cases
being classified as late latent syphilis. The clinical manifestations, when
present, included tabes dorsalis, aortitis, aortic aneurysm, general paresis and
Argyll Robertson pupils. There were three patients with symptomatic syphilis
in the F I1 positive group and 13 in the F I1 negative group. The patients
were equally distributed in the F I1 positive group with regard to sex, but
there was a preponderance of females in the F I1 negative group (60 per
cent female).
Studies of the age of the patients and of the type of treatment revealed
no significant differences.
3. Examination of the patients.-Ten of the 16 F I1 positive patients were
examined. In none of them was there evidence of rheumatoid arthritis. Pa-
RHEUMATOID FACTOR I N SYPHILIS SERA 3

TABLE1.-Summary of Data on F I I Positive Sera from Patients Having Syphilis

~

F I1 Agslutina- Serologic Tests

tion Titers Clinical Data for Syphilis Treatment

9 ;
61 1/28000 >1/6000 86 M
nortitis with
+ - - + ? ? . ? ?

aneurysm
601 1/70oU 1/640 63 F L.L.S.t - + ++ ++ + + 4-+ 1947
61
31
1/7000
1/7000
1/6000
1/6000
79
62
M
Y
L.L.S.
L.L.S.
++ ++ ++
0
++ + + o +
0
0
0
1964
1964

+++ -++ -
41 1/3684 1/6000 48 F L.L.S.
601
301
1/1792
1/1792
l/ZSSO
1/640
46
a2
F
F
L.L.S.
L.L.S. - + +
not
0
0
+ 0
0 +
1966
1966
dune
71
11
1/1792
1/896
-
l/640
47
41
F
F
L.L.S.
L.L.S
- - c +
+ r
+-t- -
+
0
+
0
o
+
-
1962
801 1/896 1/640 47 F L.L.S. + f ++ 0 0 + 1967
21 1/448 1/320 78 Y L.L.S. + + + not
done
+ + o -
701 1/448 1/640 68 Y Tabesdorsalis -+- + + + 0 0 + 1966
81 1/224 - 69 Y Tabesplus + - - + 0 0 + 1962
optic nerve
atrophy
91 1/224 - 79 A¶ L.L.S. - + + + 0 0 - 1967
*Not included in the table are 2 syphilitic sera with F I1 TSCA titers of 1:3684 and 1:896 for which
clinical information was not available.
tL.L.S. = late latent syphilis.
tT.P.1. = Treponema immobilization test.

tient No. 61 (age 61) had experienced arthralgias of hands, left great toe
and ankles, associated with elevated uric acid levels. There was no objective
joint disease, and x-rays of the hands were normal. Patient No. 51 (age 85)
had an ankylosis of the left wrist due to tuberculous arthritis contracted 48
years previously but had exeperienced no other joint symptoms since that
time.
4. CoTrelution between the F I I tests and the serologic tests for syphilis
(STS).-In all of the F I1 positive patients, at least one of the clinical tests
(Kolmer, VDRL and Mazzini) was positive. In the group of F I1 negative
patients, the STS was positive in 85 of 107 and negative in 22. In the 22
cases where the STS was negative, the diagnosis could be confirmed by a
positive treponemal immobilization test (TPI) in 17 cases, by clinically ob-
vious syphilis in three cases and by a positive STS in the past in two cases.
The TPI test, when performed, was positive in all the patients of the F I1
positive group.
From the clinical and serologic data, it is possible to state that all of the
patients in this series had undoubted syphilis and that the F I1 positive
sera demonstrated persistent positive serologic tests for syphilis.
5. F ZZ precipitin studies.-The addition of pooled human Cohn fraction
I1 ( F 11) to some rheumatoid sera induces precipitate formation.6 Recent
work has demonstrated that the reactive part of F I1 in this reaction is ag-
gregated gamma globulin.gJ1 F I1 precipitin studies with sera from patients
with syphilis are illustrated in figure 1. A good correlation existed between
4 ANDRE PELTIER AND CHARLES L. CHRISTIAN

SERA FU TITER

7000

7.000

7000

3584
448

1792
1792
89 6
NEG.
2:.
240 600 1200 2400 4800
ADDED FII NITROGEN (MICROGRAMS)

FIG. 1.-F I1 precipitin studies with sera from patients having syphilis. The solid and
dotted lines represent F I1 positive and F I1 negative sera, respectively.

the titers of the sera in the F TI-TSCA test and precipitating reactivity, with
the exception of serum No. 701, which failed to demonstrate significant pre-
cipitation.
6. Ultracentrifugal study of the clissolued precipitate.-The precipitate that
forms when aggregated gamma globulin is added to rheumatoid serum can
be dissolved in an acid buffer or concentrated urea.ll Sedimentation studies
of dissolved precipitates demonstrate gamma globulin components with SX,
values of 7 and 19. The latter material (19 S ) possesses serologic activity.
Figure 2 illustrates sedimentation studies of syphilitic and rheumatoid pre-
cipitates that were dissolved in an acid buffer ( p H 3.2). Precipitations were
induced by the addition of aggregated gamma globulin to the sera. Both
RHEUMATOID FACTOR IN SYPHILIS SERA 5

I9

FIG.2.--Sedimentation studies of acid dissolved precipitates from A serum from a pa-

tient with syphilis (No. 51) and B rheumatoid arthritis serum. Tlie precipitates, which
were induced by aggregated gamma globulin, were dissolved in glycine-HC1 buffer
( p H 3.2). The photographs are 32 minute exposures at 47,660 RPM. The direction of
sedimentation is indicated. ( Sedimentation values above each component are approximate. )

TABLE2.-Absorption of Hheumatoid Factor with Aggregated Gamma Globulin

F 11-TSCA Titer ( Heller ) VDRL Reaction

Serum Added Aggregated Before After Before After
No. Gamma globulin* absorption absorption absorption absorption

61 250 1/7000 negative positive positive

31 125 1/3584 negative positive positive
41 12.5 1/3584 negative positive positive
60 1 12.5 1/1792 negative positive positive
21 12.5 1/224 negative weakly weakly
positive positive
"Micrograms of nitrogen added to 0.6 ml. serum.

TABLE
3.-Absorption of Wassermann Antibody with Cardiolipin Antigen (Mazzini Antigen)

VDRL Reaction F 11-TSCA Test (Heller)

Volume of added
Serum Mazzini antigen Before After Before After
h 0. ( ml. ) absorption absorption absorption absorption
61 .05 positive negative 1/7000 1/7000
31 .05 positive negative 1/7000 1/3584
41 .02 positive negative 1/3584 1/3584
601 .04 positive negative 1,/3584 1/1792
21 .05 weakly negative 1/448 11224
positive
6 ANDRE PELTIER AND CHARLES L. CHRISTIAN

studies demonstrate a major 19 S component and a slower sedimenting mate-

rial with an Sz0 value of approximately 7 .
The separation of the 19 S component of the dissolved syphilitic precipitate
from the bulk of the slower sedimenting gamma globulin was achieved in
a partition centrifuge cell. This heavy material had an F II-TSCA titer of
1:200,000 and was negative in the VDRL reaction.
7. Absorption experiments.-Since both the “rheumatoid factor” and the
Wassermann antibody are contained in the 19 S fraction of gamma globulin,
it was of interest to attempt differential absorption of the F I1 positive sera
from patients with syphilis. Absorption of the “rheumatoid factor” was ac-
complished by the addition of aggregated gamma globulin (see MATERIALS
AND METHODS). Absorption studies of this type are summarized in table 2.
The quantities of aggregated gamma globulin required for removal of the
F II-TSCA reactivity are noted. In all cases, the supernatants after absorp-
tion continued to demonstrate positive VDRL tests. (The strongly anticom-
plementary properties of aggregated gamma globulin precluded the use of
complement fixation procedures. )
Experiments employing cardiolipin for absorption of the Wassermann anti-
body (see MATERIALS AND METHODS)are summarized in table 3. Supernatants
after absorption were negative in the VDRL test, but retained reactivity in
the F II-TSCA test.
DISCUSSION
The present study demonstrates that some syphilitic sera react with F I1
gamma globulin in a manner similar to that of rheumatoid sera.* By the
technic employed in this study, this property of sera from patients with
syphilis was identical to that of rheumatoid sera. In both, the reactivity with
F I1 is dependent on the presence of a gamma globulin with an Sz0 value of 19.
One property of the F I1 positive sera from syphilic subjects which may
be important, was the failure of all but one in this series to react in the
SSC test. Although the SSC test is less sensitive than the test systems utilizing
human gamma globulin ( F I1 tests), rheumatoid sera with F I1 titers cor-
responding in magnitude to the sera of syphilis patients in this series are
almost invariably positive in the SSC procedure. Other situations where sera
have demonstrated positive F I1 tests and negative SSC tests include: (1) kala
azar and sarcoidosis sera studied by Kunkel et al.,l and ( 2 ) nonrheumatic
disease sera from Liberia.13 Since sensitization in the SSC test utilizes rabbit
gamma globulin as opposed to human gamma globulin in the F I1 tests, the
above sera-unlike rheumatoid sera-may react specifically with human gam-
ma globulin.
SUMMARY
Eleven per cent of 146 syphilitic sera were positive in the F II-TSCA and
F I1 latex fixation tests. The reactivity of positive sera with F I1 was further
demonstrated in the precipitin reaction. The factor present in the sera from

‘A study of Rothermich and Philips of the F I1 latex fixation test included two posi-
tively reacting sera from syphilis patients.l2
RHEUMATOID FACTOR IN SYPHILIS SERA 7

subjects with syphilis which was responsible for the F I1 reactions was a
gamma globulin with an S2,, value of 19. Absorption experiments with ag-
gregated gamma globulin and cardiolipin demonstrated that the F I1 reac-
tive factor in syphilitic sera was separate from the Wassermann antibody.
All but one of the F I1 positive syphilitic sera were negative in the sensitized
sheep cell test. This dissociation-positive in the F I1 tests and negative in
the sensitized sheep cell test-may be an important consideration in separating
rheumatoid sera from so-called false positive sera.
REFERENCES
1. Kunkel, H. G., Simon, H. J. and Fuden- 7. Heidelberger, M. and MacPherson, C.
berg, H.: Observations concerning F. C.: Quantitative micro-estimation
positive serologic reactions for rheu- of antibodies in the sera of man and
matoid factor in certain patients with other animals. Science 97~405,1943;
sarcoidosis and other hyperglobuline- 96:63, 1943.
mic sera. Arthritis and Rheumatism 8. Rose, H., Ragan, C., Pearce, E. and
1:289, 1958. Lipman, M. 0.: Differential agglu-
2. Ziff, M.: The agglutination reaction in tination of normal and sensitized
rheumatoid arthritis. J.Chron.Dis. 5: sheep erythrocytes by sera of patients
644, 1957. with rheumatoid arthritis. Proc.Soc.
3. Howell, D. S., Pike, R. and Malcolm, Exper.Biol.& Med. 68~1,1948.
J. M.: The nature of F I1 agglu- 9. Christian, C. L.: Characterization of
tinating factors in patients with non- the “reactant” (gamma globulin fac-
rheumatic diseases. Annual Meeting tor) in the F I1 precipitin reaction
of the American Association, San and the F I1 tanned sheep cell ag-
Francisco, June 23, 1958. glutination test. J.Exper.hfed. 108:
4. Heller, G., Jacobson, A. S., Kolodny, 138, 1958.
M. H. and Kammerer, W. H.: The 10. Manual of Serologic Tests for Syphilis
hemagglutination test for rheumatoid (Public Health Service Publication
arthritis. 11. The influence of human No. 411). Washington, D.C., U S .
plasma fraction I1 (gamma globulin) Department of Health, Education and
on the reaction. J.Immuno1. ?2:66, Welfare, 1955.
1954. 11. Franklin, E. C., Holman, H. R., Miiller-
5. Singer, J. M. and Plotz, C. M.: The Eberhard, H. J. and Kunkel, H. G.:
latex fixation test. Am.J.Med. 21:888, An unusual protein component of
1956. high molecular weight in the serum
6. Epstein, W., Johnson, A. and Ragan, of certain patients with rheumatoid
C.: Observations on a precipitin re- arthritis. J.Exper.Med. 105405, 1957.
action between serum of patients with 12. Hotherniich, N. 0. and Philips, V. K.:
rheumatoid arthritis and a prepara- The serological diagnosis of rheuma-
tion (Cohn Fraction 11) of human toid arthritis. J.A.M.A. 164:1939,
gamma globulin. Proc.Soc.Exper.Bio1. 1957.
& Med.
i 91:235, 1956. 13. Malawista, S.: Unpublished data.

Andre Peltier, M.D., Trainee, National Institute of Arthritis

and Metabolic Diseases, Columbia University College of
Physicians and Surgeons, New York, N.Y.

Charles L. Christian, M.D., Fellow of the Arthritis and

Rheumatism Foundation, New York, N.Y.; lnstructor in Medi-
cine, Columbia University College of Physicians and Surgeons.