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Globulins resembling rheumatoid factor in serum of the aged
Globulins
Resembling in Serum
T
Rheumatoid
Facto-r
of the Aged*
rheumatoid factors are heat-stable $ot)ulins of high molecular \vcig;ht, prccipitablc 1)~ specially prepared aggregated gamma $ohulin, and detectable by a \.aricty of serologic tests. These tests require either cells sensitized with homologous antibody, or cells, lates or I,c:ntonite particles coated prefcrentiall) with human gamma globulin ( /,.?I. The incidence of the rheumatoid factors is highest 1)). far in persons with classic rheumatoid arthritis. Serllrn obtained from their as)mptomatic relatives frequent11 also contains rheumatoid factors, suggesting genetic transmission [.?I. In a considcra hlc numl)er of persons with the stigmata of rhwmatoid arthritis, however, the rheumatoid factors are either lacking or blocked by inhibitors so that their presence may only he inferred from inhibition tests [I.!]. Over the past few \wrs the presence of rheumatoid factors has also t1ec.n noted in diseases which bear no resemblance to rhcwmatoicl arthritis [z]. In addition to the seeming nonspecificity of the rheumatoid factors? evidence has also been furnished for chemical differences as well as for diflcrc-ncrs in serologic behavior. One type of rheumatoid factor was shown to react only with human gamma ,globulin (scropositi\.c in the latex lixation test) whereas the other type reacted also with rabbit gamma globulin (seropositive in latex fixation as ~vell as sensitized sheep cell test) [5,h‘]. \‘t:c wish to report on the high incidence of a cclnstituent of serum in persons o\‘er sixty-five y’rars of age which, when studied by a varict) of serologic and immunologic technics, resembles the. rheumatoid factors. HE
Xubjects. Serum specimens wcrr ohtainw-l from the following four groups of subjects: Grou/~7, a,~~~pww~: These subjects ranged in age from sixty-five to ninety-three years with an age distribution as indicated in Table I. There \verc: 101 female and twenty male subjects. Ninety-eight subjects lived in one old age home and nine in another. Fourteen subjects were hospitalized for various orthopedic disorders. ‘l‘he residents of old age homes MWX examined for evidence of joint and other disease. iz majority of the subjects suffered from ailments usually seen in this age group, hut persons \vith objective signs and symptoms of rheumatoid arthritis werr excluded from this group. Group .?, aged /wsons with active rhwnatnid arthritis: ‘l‘rn persons, who ranged in age from sixty-five to ninety-one years, were assigned to this group. ‘I‘hc duration of active disease was from five to twrnty-fivt )-ears. (&HI/I .3, prsons under sixty-Jive jears 01 age with a&r rheumatoid urthritis: Eight patients with high rheumatoid factor titers were selected as controls.
TABLE
I
* From the Philip II. \Yilson Research Foundation, Hospital for Special Surgery and the Dcpartmcnt of Medicine, Cornell L~niversity Medical College, New York, New York. This work was supported in part by research grant No. A-3592, Kational Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. Parts of this report have been prexnted at the 8th Interim Scientific Session of the American Rhrumatism Association, December 1961. Washinqton, D.C. Manuscript received August 21, 1962. t Present address: Srton Hall College of Medicinr, Jcrsry City, Nrw Jrrscy. VOL.
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AUGIJST
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175
176
Globulins
Resembling
Rheumatoid
TABLE II SEROLOGIC TFSTS PERFORMED ON SERUM SPECIMENS FROM AGED PERSONS
-
-
Test
Hyland RA Slide Test. Latex (0.8 p) fixation. Latex (0.2 p) fixation. SEA test.
-
NO. Tested
No. Positive
Positive
121 121 64 83
56 51 18 2
46 42 28 2
-
%
-i
Group 4, healthy subjects under sixty-five years of age:
This group consisted of fifty persons, mostly hospital personnel, who ranged in age from twenty-one to fifty-seven years. Serologic Tests for Rheumatoid Factors. Hyland slide test: Serum specimens, diluted 1:20 in glycine buffer
(pH 8.2, 0.1 M) and heated to 56”~. for ten minutes, were mixed with the Hyland RA reagent, and read after five minutes. Results were recorded on a scale of 0 to 4 plus. Latex jixation test: Performed by the method of Singer and Plotz [7], fixation of latex particles (0.8 EL) at serum dilutions of 1:20 or over was considered a positive test result. Fixation test with latex particles (0.2 p) precoated with human gamma globulin [8]: Latex particles (0.2 M)*
were incubated at 37”~. for one hour with a 1 per cent solution of gamma globu1in.t The mixture was then spun in a No. 40 rotor at 20,000 r.p.m. for fifteen minutes in a Spinco Ultracentrifuge, Model L. The sedimented latex particles were resuspended in glycine buffer and washed three times by ultracentrifugation and resuspension. The coated latex particles, stable over long periods of time, were kept as a stock suspension in the cold. A working standard in glycine buffer, reading 270 optical density units in the Klett Summerson photocolorimeter (64 filter), was prepared daily from the stock suspension. Aliquots of the reagent were added to serum, which had been diluted serially in glycine buffer. The procedure for incubation and reading of the test was similar to that of the standard latex fixation test. Fixation of latex particles at serum dilutions of 1 :20 or over was considered a positive test result. Sensitized sheep cell (SEA) test: Performed by the method of Heller et al. [9], agglutination of cells at serum dilutions of 1:28 or above was considered a positive test result. Prec$itin Tests. Serum specimens were heated to 56’~. for a half hour to eliminate a heat-labile 11 S globulin which also combines with aggregated gamma globulin [ 70,7 71. The serum samples were then stored * Latex particles were made available to us by Dr. Jacques M. Singer. t Human gamma globulin (fraction II) was a gift of the American
Red Cross.
Factor in Serum of the Aged-Heimer
et al.
in the cold for eighteen hours and centrifuged at 2,000 X g. Samples of 0.25 ml. each were pipetted into a series of conical test tubes, followed by quantities increasing from 1 to 5 mg. of human (fraction II) gamma globulin aggregated by the method of Christian [72]. The final volume in each tube was adjusted to 5 ml. with phosphate buffer (pH 8.0, 0.15 M). After cold storage for six days, the precipitates were washed three times in cold saline solution and analyzed for nitrogen with Nessler’s reagent [13]. Controls containing either gamma globulin or serum alone were also prepared for each set of experiments. The maximal amount of nitrogen precipitated was determined by plotting precipitin curves after correction for the appropriate controls. DEAE Cellulose Chromatography. Chromatography was performed in a column 1.5 X 25 cm., filled with DEAE cellulose (Brown Co.) and equilibrated with 0.02 M tris buffer at pH 7.4. The chromatography was carried out on 5 ml. of serum, dialyzed against starting buffer, by gradient elution with 0.02 M tris buffer at pH 7.4 and 0.3 M sodium chloride. Small aliquots of the eluate were collected by an automatic device, and analyzed for protein in a Beckman Spectrophotometer, Model DU at 280 m/*. The concentration of sodium chloride in each tube was determined by conductivity measurements. The samples were diluted serially and tested with the serologic reagents mentioned. RESUL’I’S Serologic Tests. ,4 summary of the results of the various serologic tests performed on serum samples obtained from aged persons (group 1) appears in Table II. Of the positive test results obtained by latex fixation, titers were below 1: 80 in 12 per cent, between 1: 160 and 1: 640 in 50 per cent, and exceeded 1: 640 in 37 per cent. Prozones, which could not be removed by preheating the serum to 56”~. for ten minutes, were seen in thirty per cent of the samples. Prozones were not seen in results of tests using 0.2 k latex particles precoated with gamma globulin. The reaction to this test, however, was somewhat less sensitive than to the standard latex fixation test. On arranging 116 subjects in group 1 by age (Table I), a trend toward a higher incidence of seropositivity was found in subjects between the ages of seventy-five and eighty-four. There was a decline in the incidence of seropositivity in those above eighty-five years of age. Fresh blood samples were obtained from eight persons three to six months after the initial testing. No change in the test response was observed on these occasions. AMERICAN
JOURNAL
OF
MEDICINE
Globulins
Resembling TABLE RESULTS
OF
Rheumatoid
Factor in Serum of the Aged-Heimer
TESTS
Rheumatoid (posItwe latex)
Subjects Tested tatcd
’
(kg.)
tated
b+Y.) .-Xh
Group 1, with latex fixation result. Group I, with latex lixation result. Group 3. with latex tixation result. Group 4, with latex fixation result.
negative test
b
‘\
‘b\ /AAged
3-19.5
‘\.,
7.3
(negative
latex)
positive test 7-20
1 12.4
~ 28-32
30.0
positiw test negative test o-9.5
~ I
3.0
The results of tests on persons over sixty-five years of age with active classic rheumatoid arthritis (group 2) were tabulated separately. Eight of ten such persons gave a strongly positive reaction to latex fixation tests. Positive reactions to SEA tests were observed in only two of the ten serum samples tested. Control studies on fifty healthy subjects under sixty-five years (group 4) gave an incidence of 4 per cent with the latex (0.8 p) fixation test, zero with the 0.2 p latex particles and 2 per cent with the SEA test. Efet of mercapoethanol: In common with serum from persons having rheumatoid arthritis, serum from the aged failed to fix latex particles after exposure to 0.1 M mercaptoethanol. .&tioity of euglobulins: The serologic activity resided in the euglohulin fraction of serum in groups 1, 2 and 3. Effect of heat: In most instances the titer of the latex fixation test on serum samples obtained from the aged remained unchanged after exposure to 56’~. for thirty minutes. Similar results were obtained with serum samples from persons with rheumatoid arthritis. There was no change in the titer of serum on heating to 6.1’~:. for ten minutes. At 68”~. the serologic activity, however, was destroyed rapidly. IZff~l of absorption with denatured human gamma globulin: The substance responsible for latex fixation was readily removed by absorption VOL. 35,
177
III
PRECIPITIN
1 No. of
Subjects
et al.
AUGUST
1963
7 N aggregated.
gammaglobulin
Frc. 1. Representative curves of quantitatice precipitin reactions between aggregated gamma globulin and 0.25 ml. of heated srrum of persons in groups 1. 3 and 4. with heat-denatured (72’c.), solid human gamma globulin. Serum from persons with rheumatoid arthritis also lost rheumatoid factor by such absorption. Precipitin Tests. Twenty-five serum samples were tested for their ability to precipitate in the presence of aggregated gamma globulin. Three samples with high titers for rheumatoid factors were from persons with classic rheumatoid arthritis; six samples, which gave a negative reaction to tests for rheumatoid factors, were from healthy persons under sixty-five years of age; and sixteen samples, eight of which gave a positive reaction to the latex fixation test, were from persons over sixty-five years of age. Table III indicates that the maximal amount of nitrogen precipitated by serum obtained from the aged was midway between that of healthy young adults and persons with rheumatoid arthritis. The difference in the amount of nitrogen precipitated by serum specimens (from the aged) with positive reactions to latex fixation tests and those with negative reactions was surprisingly small and overlapped in many instances. The shape of the precipitin cur\zes was the same for all subjects. (Fig. I.) The shape depended only on the type of gamma globulin preparation. With aggregated gamma globulin there was a well defined maximum, and no precipitation occurred in moderate excess of the
178
Globulins Resembling
Rheumatoid
reagent. When fraction II was used directly, larger amounts of the reagent were required to bring about maximum precipitation; there was only a moderate decline in the amount of precipitate formed with excess of fraction II. Little if any precipitate was formed with gamma globulin containing only trace amounts of aggregated gamma globulin. Fractionation of Serum by DEAE Cellulose Chromatograpr’Ly. Five serum samples (from aged persons) with moderately positive reactions to latex fixation tests were fractionated by DEAE cellulose chromatography. The results were compared with those obtained with two serum specimens with a negative reaction from persons in group 4 as well as with a number of serum specimens with a positive reaction from persons in group 3. All serum, regardless of donor, yielded fractions which gave positive reactions to latex fixation tests. The titers obtained were lowest with the two serum samples from healthy persons under sixty-five and highest with serum samples from persons with rheumatoid arthritis. Two seropositive fractions were recovered from the columns. One such fraction appeared after the emergence of serum albumin, requiring approximately 0.1 M sodium chloride. All serum tested contained this fraction. The second seropositive fraction appeared at a higher salt concentration, and was found in all but one serum specimen. This was from a subject in group 1. Apart from this discrepancy, it was not possible to detect qualitative differences between serum from the aged, serum from persons with rheumatoid arthritis or serum from healthy subjects under sixty-five years of age. As judged from their manner of elution, the active fractions most likely contained gamma macroglobulins, but because of the small amounts of serum employed in these experiments further tests by ultracentrifugation and electrophoresis were omitted. COMMENTS
Our studies indicate that approximately 40 per cent of the serum specimens from the aged contain unusual substances detectable by latex particles coated with human gamma globulin, but inactive when tested with sheep cells sensitized with rabbit gamma globulin. Similarly reacting serum components have been isolated previously from serum obtained from subjects with rheumatoid arthritis and hence identified as rheumatoid factors [5,6]. Whether occurring
Factor in Serum of the Aged-Heimer
et al.
in the serum of persons with rheumatoid arthritis or in the serum of aged persons (over sixty-five), these substances have the following characteristics. They are stable to 63Oc. on heating for ten minutes; they are euglobulins, the serologic activity of which is destroyed by mercaptoethanol. The substances yield precipitin-type curves in the presence of aggregated human gamma globulin, but remain in solution when unaggregated gamma globulin is used. The substances may be removed from serum by absorption with solid heat-denatured gamma globulin. Like other gamma macroglobulins, high salt concentrations are required for their elution from DEAE cellulose columns. Their manner of elution from such columns suggests heterogeneity with respect to physicochemical characteristics. These substances have been thought to be less specific for rheumatoid arthritis than other macroglobulins which react with human as well as with rabbit gamma globulin [Id]. The latter type of substances was detected in only two serum samples in group 1. Moreover, preliminary data suggest their somewhat diminished incidence in aged persons with rheumatoid arthritis. There are few diseases not related to rheumatoid arthritis in which the incidence of positive latex fixation test results equals or exceeds that found in old age. For example, the incidence of a positive latex fixation test result in liver diseases was 32.6 per cent [15] in one study, and 25 per cent in another [76]; in syphilis it was 11 per cent [77]; in lepromatous leprosy, 44 per cent [r8]; and in subacute bacterial endocarditis, 50 per cent [79]. In these diseases, as in old age, positive SEA test results were elicited only in a minority of the serum samples tested. The magnitude of the latex fixation titer in the aged population studied by us, while not equal to that of rheumatoid arthritis, hardly differs from that encountered in various diseases not related to rheumatoid arthritis. In our study, approximately 50 per cent of the positive latex fixation test results were found to equal or exceed titers of 1: 640. However, in an unusual number of the serum samples tested, prozones were encountered which could not be removed by heating. The occurrence of prozones in other diseases appears to be less frequent. There has been a growing awareness of the relationship between age and the incidence of rheumatoid factors. The comparative rarity of positive serologic reactions in juvenile AMERICAN
JOURNAL
OF
MEDICINE
Globulins
Resembling
Rheumatoid
Factor in Serum of the Aged-Heimer
rheumatoid arthritis has been commented upon frequently [I,?]. The increased incidence of positive latex fixation test results among the aged is suggested from the work of Arnon et al. 1161, \vho found a positive reaction to latex fixation tests in 17.6 per cent of 216 inhabitants over seventy y-ears of age in Tours, France. An age-dependent increase of incidence of positive latex fixation test results was also found by Jacqueline [lo]. Epidemiologic studies carried out in various countries also suggest an age trend with respect to rheumatoid serology [27]. Similar results were obtained in a study of residents of New Haven, Corm. [_??], where 4.2 per cent of persons over sixty-five years of age had a positive reaction to the latex fixation test contrasted to 0.47 per cent in persons betwcen the ages of fifteen to twenty-four. It is difficult to compare the results of this study with ours as no further breakdown by age was given of the group over sixty-five years. In studying the epidemiology of rheumatoid arthritis in northern Europe, Lawrence et al. 12.31 reported that the results of both the SEA and latex fixation tests show an increasing titer with age. Somewhat divergent results were obtained by Ball and Lawrence [24] with the SE.4 test in a study of an urban and a rural population. In the mining town of Leigh, 14.7 per cent of the men and 9.4 per cent of the women over 65 years of age gave a positive reaction to the SE.4 test. In FYensleydale, a rural community-, none of the sixty-seven men over sixty-live years of age had positive SEA test result, yet 6.3 per cent of 111 women in this community did. In the light of these findings, the question must be raised whether the unusually high incidence of positive latex fixation test results among the aged in New York City is a local phenomenon, is encountered in urban populations generally, or reflects biological processes unrelated to environment. :it the present time we cannot give a satisfactory explanation for the increased incidence of rheumatoid factor-like substances among the aged. Our aged population did not suffer from any of the diseases known to give rise to positive latex fixation test results. The most frequently encountered condition related to degenerative changes in joints; it was observed in seventy-five Hypertension and/or atherosclerosis persons. was found in sixty-nine persons. The incidence of positive latex fixation test results among persons falling into these two subgroups was VOL.
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AUGUST
1963
et ul.
179
similar to the subgroup in which these disorders lvere not clinically significant. The high incidence of positi\le latex fixation test results, therefore, is probably unrelated to recognized pathologic conditions among the agect. Although the cause of rheumatoid factor production is still unknown, the notion that the rheumatoid factors constitute antibodies against gamma globulin itself or that they are antiantibodies is currently favored by many ]1,.?5]. It has been possible to induce formation of rheumatoid factor-like substances in animals by prolonged injections with bacteria [lo], homologous soluble [_37] and insoluble 1281 immune complexes. The increased incidence of rheumatoid factorlike substances in old age may perhaps relate to a piling up of autoantibodies, as postulated in certain theories of aging [s]. Such theories suggest an age-dependent increase in the rate of somatic mutations of cellular clones, giving substances or causing failure rise to “foreign” of antibody forming cells to recognize “self” If the animal experiments from ‘inon-self.” cited are a guide, the consequence of such mutations may be the production of rheumatoid factors. Another possible explanation for the high incidence of positive reactions to latex fixation tests among the aged might be that these persons have decreased production of certain serum components which, because of their stabilizing effect on latex particles, block the serologic detection of rheumatoid factors. The net result of this decrease would be to facilitate detection of rheumatoid factors, which might very well be present in the serum of persons of all ages. Suggestive evidence for the presence of rheumatoid factors in the absence of positive serologic test results has been obtained by Lospalluto ancl Ziff [5] as well as by us. On chromatography, seronegative samples from healthy donors were found to yield fractions which gave positive reactions to latex fixation tests. Recent investigations on asymptomatic relatives of persons with rheumatoid arthritis have revealed a greater incidence of seropositivity than could be expected on a statistical basis [3]. The data were interpreted as evidence for a familial and probably genetic basis of production of rheumatoid factors. As it appears unlikely that asymptomatic relatives live longer than other persons, a more likely explanation of our
180
Globulins
Resembling
Rheumatoid
results might be that rheumatoid factor production, n addition to being a familial trait, can also be acquired. The acquired form may appear in persons of advanced age. Apart from the more obvious serologic differences, it may well be that the rheumatoid factors found in rheumatoid arthritis are governed by genetics peculiar to it, and that the rheumatoid factorlike globulins found in other diseases as well as in the aged are acquired rather than genetically transmitted. Regardless of the true cause of the increased incidence of positive reactions to latex fixation tests in serum samples from the aged, the hazards of using serologic tests as an aid in the diagnosis of rheumatoid arthritis in elderly persons should be recognized. In addition to the increased incidence of positive latex fixation test results in the aged population, attention is called to results obtained with precipitin tests with aggregated human gamma globulin. It was found that the precipitation of heated serum obtained from the aged fell midway between that of serum obtained from persons in groups 3 and 4. The amount of nitrogen precipitated in the serum from the aged correlated only moderately well with the ability to fix latex particles. In serum obtained from subjects in group 3, however, there appears to be a direct correlation between serologic titer and amount of nitrogen precipitated. A possible explanation for this difference may be that the serum obtained from the aged contains as yet unknown heat-stable proteins with afftnity for aggregated gamma globulin but which are inert in the latex test. Alternately, such serum might contain smaller amounts of stabilizer of latex particles. Experiments are under way to clarify these results. SUMMARY
One hundred and twenty-one serum samples from persons over sixty-five years of age were tested by the Hyland RA slide test and by the latex fixation test. It was found that 46 per cent of the samples gave a positive reaction to the Hyland RA slide test and only 42 per cent to the latex fixation test. By contrast, only 2 per cent of the samples gave a positive reaction to the sensitized sheep cell test. Attempts were made to characterize the unusual serum constituents responsible for serologic activity. It was found that the activity resided in a heat-stable euglobulin fraction and
Factor in Serum of the Aged-Heimer
et al.
that reactivity in the latex fixation test could be treatment with mercaptodestroyed upon ethanol. The behavior of these serum constituents was also studied by DEAE cellulose chromatography and by precipitation reactions with different preparations of human gammaglobulin. The results of the study indicate that these constituents are indistinguishable from the unusual serum components, the rheumatoid factors, which are present in the serum of persons suffering from rheumatoid arthritis. The incidence of globulins resembling the rheumatoid factors in serum of the aged was contrasted with the incidence of similar moieties in serum of persons with diseases other than rheumatoid arthritis. The possible significance of a relationship between age and incidence of these globulins is discussed. Acknowledgment: Il’e are greatly indebted to Dr. Ann Maloney and Sister Richard of the Josephine Baird Home for the Aged, and to Dr. Bernard Rogoff, who helped us obtain serum from persons F ree dman Home for thf?gEd.at the Andrew REFERENCES 1.
2. 3.
4.
5.
6.
7.
8.
9.
10.
J. II. Serum responses in rheumatoid arthritis. Am. J. Med., 26: 596, 1959. SINGER, J. M. The latex fixation test in rheumatic diseases. Am. J. Med., 31: 766, 1961. ZIFF, M., SCHMID, F. R., Lawn, A. J. and TANNER, M. Familial occurrence of the rheumatoid factor. Arthritis @ Rheumat., 1: 392, 1958. ZIFF, M., BROWN, P., LOSPALLUTO.J., BADIN.J. and MCEWEN, C. Agglutination and inhibition by serum globulin in the sensitized sheep cell agglutination reaction in rheumatoid arthritis. Am. J. Med., 20: 500, 1956. LOSPALLUTO, J. and ZIFF, M. Chromatographic studies of the rheumatoid factor. J. Expey. Med., 110: 169, 1959. HEWER, R. and SCHWARTZ, E. R. Isolation of rheumatoid factors. Arthritis & Rheumat., 4: 153, 1961. SINGER, J. M. and PLOTZ, C. M. The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med., 21: 888, 1956. HALBERSTAM,D., SINGER,J. M., ALLEN, E. G. and PLOTZ, C. M. Stability characteristics of .2 micron diameter polystyrene ‘latex particles. In: Atti de1 X Congr&so deila Lega Internationale contra ii Rheumatismo, vol. 2, P. 2854. Turin 1961. Minerva Medica. _ HELLER, G., JACOBSON,A. S. and KOLODNY, M. H. A modification of the hemagglutination test for rheumatoid arthritis. Proc. Sac. Exper. Biol. @ Med., 72: 316, 1949. TARANTA, A., WEISS, H. S. and FRANKLIN, E. C. VAUGHAN,
Precipitating
factor
AMERICAN
for JOURNAL
aggregated OF
gammaMEDICINE
Globulins
11.
12.
13.
14. 15.
16.
17.
18.
19.
VOL.
Resembling
Rheumatoid
Factor in Serum of the Aged-Heimer
globulins in normal human sera. Nature, London, 189: 4760, 1961. MUELLER-EBERAARD, H. J. and KUNKEI., H. G. Isolation of a heat labile serum component which interacts with gammaglobulin aggregates. Fed. I’rw.> 19: 76, 1960. CIIKISTIAN, C. L. Characterization of the “reactant” (gamma globulin factor) in the FII precipitin reaction and the FII tanned sheep cell agglutination test. J. Exper. Med., 108: 139, 1958. HAWK. P. B., OSER, B. L. and SUMMERSON, W. 1-I. Practical Physiological Chemistry, p. 878. New York, 1954. McGraw-Hill Book Co. ZIFF, M. The agglutination reaction in rheumatoid arthritis. J. Chron. Dis., 5: 644. 1957. ~II)WI 1960. AKNON, E.. VARGUES, R. and LECHEVALLIER, P. I,. The latex reaction. .4pplication to the study of 1.150 serums. pr)rrsseMM., 69: 54, 1961. Pxr:rr~:~. A. and CHRISTIAN? C. L. The presence of thr “rheumatoid factor” in sera from patients with syphilis. :lrt/zri/z.c3 Rheumnt.. 2: 1, 1959. CATIICAR.~, E. S., \VILLIAMS, R. C., Ross, SISTER HILI.ARV and CALKINS, E. The relationship of the latex fixation test to the clinical and serological manifestations of leprosy. ri?~. J. .Zfwf.. 31 : 758. 1061. \~II.I.I-\MS, 1~. C. and KUNKEI.. 11. G. Rheumatoid factor. complcmrnt and conglutinin aberrations
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1963
20. 21.
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et al.
181
in patients with subacute bacterial endocarditis. J. Clin. Inuest., 41: 666, 1962. JACQUELINE, F. Personal communication. VALKENBURG, A. and HIJMANS. W. Population studies on the rheumatoid factor. In: Atti de1 X Congresso della Lega Internationale contra il Rheumatismo, vol. 2, p. 789. l’urin, 1361. Minerva Medica. KISSI~K, W. L. Epidemiology of rheumatoid factor. Arthritis @ Rheumat., 4: 424, 1961. LAWRENCE. J. S., LANE, V. A. and DISGRAAF, R. The epidemiology of rheumatoid arthritis in northern Europe. Proc. Ro.7. SW. Mrd., 54: 454. 1961. BALL, J. and LAWRENCE, J. S. Epidemiology of the sheep cell agglutination test. Ann. Rhcumat. Dis.. 20: 235, 1961. KUNKEL, H. G. Immunologic aspects of rhcaumatoid arthritis. J. Chron. IX., 10: 418, 195’). SCHICHIKAWA,K., YAMAMOTO,T. and FUJIOKA, M. experimentale ud facteul Sur la production rheumatoide. In: Atti de1 X Congress0 della Lega Intrrnationale contra il Rheumatismo. vol. 2. p. 789. Turin, 1961. Minerva Medica. Ano, K. and WAGER, 0. Production of “anti-antibodies” in rabbits. Ann. Med. Exfw. P/ biol. Fenniap, 39: 79, 1961. ABRUZ~~, J. L. and CHRISTIAN. C. 1,. Induction of a rheumatoid factor-like substance in rabbits. J. &,brr. hfrd.. 114: 791, 1961. A.. The position of fundamental age COMFORT, studies. .‘lm. &art .I.. 62: 203, 1961.
Resembling in Serum
T
Rheumatoid
Facto-r
of the Aged*
rheumatoid factors are heat-stable $ot)ulins of high molecular \vcig;ht, prccipitablc 1)~ specially prepared aggregated gamma $ohulin, and detectable by a \.aricty of serologic tests. These tests require either cells sensitized with homologous antibody, or cells, lates or I,c:ntonite particles coated prefcrentiall) with human gamma globulin ( /,.?I. The incidence of the rheumatoid factors is highest 1)). far in persons with classic rheumatoid arthritis. Serllrn obtained from their as)mptomatic relatives frequent11 also contains rheumatoid factors, suggesting genetic transmission [.?I. In a considcra hlc numl)er of persons with the stigmata of rhwmatoid arthritis, however, the rheumatoid factors are either lacking or blocked by inhibitors so that their presence may only he inferred from inhibition tests [I.!]. Over the past few \wrs the presence of rheumatoid factors has also t1ec.n noted in diseases which bear no resemblance to rhcwmatoicl arthritis [z]. In addition to the seeming nonspecificity of the rheumatoid factors? evidence has also been furnished for chemical differences as well as for diflcrc-ncrs in serologic behavior. One type of rheumatoid factor was shown to react only with human gamma ,globulin (scropositi\.c in the latex lixation test) whereas the other type reacted also with rabbit gamma globulin (seropositive in latex fixation as ~vell as sensitized sheep cell test) [5,h‘]. \‘t:c wish to report on the high incidence of a cclnstituent of serum in persons o\‘er sixty-five y’rars of age which, when studied by a varict) of serologic and immunologic technics, resembles the. rheumatoid factors. HE
Xubjects. Serum specimens wcrr ohtainw-l from the following four groups of subjects: Grou/~7, a,~~~pww~: These subjects ranged in age from sixty-five to ninety-three years with an age distribution as indicated in Table I. There \verc: 101 female and twenty male subjects. Ninety-eight subjects lived in one old age home and nine in another. Fourteen subjects were hospitalized for various orthopedic disorders. ‘l‘he residents of old age homes MWX examined for evidence of joint and other disease. iz majority of the subjects suffered from ailments usually seen in this age group, hut persons \vith objective signs and symptoms of rheumatoid arthritis werr excluded from this group. Group .?, aged /wsons with active rhwnatnid arthritis: ‘l‘rn persons, who ranged in age from sixty-five to ninety-one years, were assigned to this group. ‘I‘hc duration of active disease was from five to twrnty-fivt )-ears. (&HI/I .3, prsons under sixty-Jive jears 01 age with a&r rheumatoid urthritis: Eight patients with high rheumatoid factor titers were selected as controls.
TABLE
I
* From the Philip II. \Yilson Research Foundation, Hospital for Special Surgery and the Dcpartmcnt of Medicine, Cornell L~niversity Medical College, New York, New York. This work was supported in part by research grant No. A-3592, Kational Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. Parts of this report have been prexnted at the 8th Interim Scientific Session of the American Rhrumatism Association, December 1961. Washinqton, D.C. Manuscript received August 21, 1962. t Present address: Srton Hall College of Medicinr, Jcrsry City, Nrw Jrrscy. VOL.
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176
Globulins
Resembling
Rheumatoid
TABLE II SEROLOGIC TFSTS PERFORMED ON SERUM SPECIMENS FROM AGED PERSONS
-
-
Test
Hyland RA Slide Test. Latex (0.8 p) fixation. Latex (0.2 p) fixation. SEA test.
-
NO. Tested
No. Positive
Positive
121 121 64 83
56 51 18 2
46 42 28 2
-
%
-i
Group 4, healthy subjects under sixty-five years of age:
This group consisted of fifty persons, mostly hospital personnel, who ranged in age from twenty-one to fifty-seven years. Serologic Tests for Rheumatoid Factors. Hyland slide test: Serum specimens, diluted 1:20 in glycine buffer
(pH 8.2, 0.1 M) and heated to 56”~. for ten minutes, were mixed with the Hyland RA reagent, and read after five minutes. Results were recorded on a scale of 0 to 4 plus. Latex jixation test: Performed by the method of Singer and Plotz [7], fixation of latex particles (0.8 EL) at serum dilutions of 1:20 or over was considered a positive test result. Fixation test with latex particles (0.2 p) precoated with human gamma globulin [8]: Latex particles (0.2 M)*
were incubated at 37”~. for one hour with a 1 per cent solution of gamma globu1in.t The mixture was then spun in a No. 40 rotor at 20,000 r.p.m. for fifteen minutes in a Spinco Ultracentrifuge, Model L. The sedimented latex particles were resuspended in glycine buffer and washed three times by ultracentrifugation and resuspension. The coated latex particles, stable over long periods of time, were kept as a stock suspension in the cold. A working standard in glycine buffer, reading 270 optical density units in the Klett Summerson photocolorimeter (64 filter), was prepared daily from the stock suspension. Aliquots of the reagent were added to serum, which had been diluted serially in glycine buffer. The procedure for incubation and reading of the test was similar to that of the standard latex fixation test. Fixation of latex particles at serum dilutions of 1 :20 or over was considered a positive test result. Sensitized sheep cell (SEA) test: Performed by the method of Heller et al. [9], agglutination of cells at serum dilutions of 1:28 or above was considered a positive test result. Prec$itin Tests. Serum specimens were heated to 56’~. for a half hour to eliminate a heat-labile 11 S globulin which also combines with aggregated gamma globulin [ 70,7 71. The serum samples were then stored * Latex particles were made available to us by Dr. Jacques M. Singer. t Human gamma globulin (fraction II) was a gift of the American
Red Cross.
Factor in Serum of the Aged-Heimer
et al.
in the cold for eighteen hours and centrifuged at 2,000 X g. Samples of 0.25 ml. each were pipetted into a series of conical test tubes, followed by quantities increasing from 1 to 5 mg. of human (fraction II) gamma globulin aggregated by the method of Christian [72]. The final volume in each tube was adjusted to 5 ml. with phosphate buffer (pH 8.0, 0.15 M). After cold storage for six days, the precipitates were washed three times in cold saline solution and analyzed for nitrogen with Nessler’s reagent [13]. Controls containing either gamma globulin or serum alone were also prepared for each set of experiments. The maximal amount of nitrogen precipitated was determined by plotting precipitin curves after correction for the appropriate controls. DEAE Cellulose Chromatography. Chromatography was performed in a column 1.5 X 25 cm., filled with DEAE cellulose (Brown Co.) and equilibrated with 0.02 M tris buffer at pH 7.4. The chromatography was carried out on 5 ml. of serum, dialyzed against starting buffer, by gradient elution with 0.02 M tris buffer at pH 7.4 and 0.3 M sodium chloride. Small aliquots of the eluate were collected by an automatic device, and analyzed for protein in a Beckman Spectrophotometer, Model DU at 280 m/*. The concentration of sodium chloride in each tube was determined by conductivity measurements. The samples were diluted serially and tested with the serologic reagents mentioned. RESUL’I’S Serologic Tests. ,4 summary of the results of the various serologic tests performed on serum samples obtained from aged persons (group 1) appears in Table II. Of the positive test results obtained by latex fixation, titers were below 1: 80 in 12 per cent, between 1: 160 and 1: 640 in 50 per cent, and exceeded 1: 640 in 37 per cent. Prozones, which could not be removed by preheating the serum to 56”~. for ten minutes, were seen in thirty per cent of the samples. Prozones were not seen in results of tests using 0.2 k latex particles precoated with gamma globulin. The reaction to this test, however, was somewhat less sensitive than to the standard latex fixation test. On arranging 116 subjects in group 1 by age (Table I), a trend toward a higher incidence of seropositivity was found in subjects between the ages of seventy-five and eighty-four. There was a decline in the incidence of seropositivity in those above eighty-five years of age. Fresh blood samples were obtained from eight persons three to six months after the initial testing. No change in the test response was observed on these occasions. AMERICAN
JOURNAL
OF
MEDICINE
Globulins
Resembling TABLE RESULTS
OF
Rheumatoid
Factor in Serum of the Aged-Heimer
TESTS
Rheumatoid (posItwe latex)
Subjects Tested tatcd
’
(kg.)
tated
b+Y.) .-Xh
Group 1, with latex fixation result. Group I, with latex lixation result. Group 3. with latex tixation result. Group 4, with latex fixation result.
negative test
b
‘\
‘b\ /AAged
3-19.5
‘\.,
7.3
(negative
latex)
positive test 7-20
1 12.4
~ 28-32
30.0
positiw test negative test o-9.5
~ I
3.0
The results of tests on persons over sixty-five years of age with active classic rheumatoid arthritis (group 2) were tabulated separately. Eight of ten such persons gave a strongly positive reaction to latex fixation tests. Positive reactions to SEA tests were observed in only two of the ten serum samples tested. Control studies on fifty healthy subjects under sixty-five years (group 4) gave an incidence of 4 per cent with the latex (0.8 p) fixation test, zero with the 0.2 p latex particles and 2 per cent with the SEA test. Efet of mercapoethanol: In common with serum from persons having rheumatoid arthritis, serum from the aged failed to fix latex particles after exposure to 0.1 M mercaptoethanol. .&tioity of euglobulins: The serologic activity resided in the euglohulin fraction of serum in groups 1, 2 and 3. Effect of heat: In most instances the titer of the latex fixation test on serum samples obtained from the aged remained unchanged after exposure to 56’~. for thirty minutes. Similar results were obtained with serum samples from persons with rheumatoid arthritis. There was no change in the titer of serum on heating to 6.1’~:. for ten minutes. At 68”~. the serologic activity, however, was destroyed rapidly. IZff~l of absorption with denatured human gamma globulin: The substance responsible for latex fixation was readily removed by absorption VOL. 35,
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PRECIPITIN
1 No. of
Subjects
et al.
AUGUST
1963
7 N aggregated.
gammaglobulin
Frc. 1. Representative curves of quantitatice precipitin reactions between aggregated gamma globulin and 0.25 ml. of heated srrum of persons in groups 1. 3 and 4. with heat-denatured (72’c.), solid human gamma globulin. Serum from persons with rheumatoid arthritis also lost rheumatoid factor by such absorption. Precipitin Tests. Twenty-five serum samples were tested for their ability to precipitate in the presence of aggregated gamma globulin. Three samples with high titers for rheumatoid factors were from persons with classic rheumatoid arthritis; six samples, which gave a negative reaction to tests for rheumatoid factors, were from healthy persons under sixty-five years of age; and sixteen samples, eight of which gave a positive reaction to the latex fixation test, were from persons over sixty-five years of age. Table III indicates that the maximal amount of nitrogen precipitated by serum obtained from the aged was midway between that of healthy young adults and persons with rheumatoid arthritis. The difference in the amount of nitrogen precipitated by serum specimens (from the aged) with positive reactions to latex fixation tests and those with negative reactions was surprisingly small and overlapped in many instances. The shape of the precipitin cur\zes was the same for all subjects. (Fig. I.) The shape depended only on the type of gamma globulin preparation. With aggregated gamma globulin there was a well defined maximum, and no precipitation occurred in moderate excess of the
178
Globulins Resembling
Rheumatoid
reagent. When fraction II was used directly, larger amounts of the reagent were required to bring about maximum precipitation; there was only a moderate decline in the amount of precipitate formed with excess of fraction II. Little if any precipitate was formed with gamma globulin containing only trace amounts of aggregated gamma globulin. Fractionation of Serum by DEAE Cellulose Chromatograpr’Ly. Five serum samples (from aged persons) with moderately positive reactions to latex fixation tests were fractionated by DEAE cellulose chromatography. The results were compared with those obtained with two serum specimens with a negative reaction from persons in group 4 as well as with a number of serum specimens with a positive reaction from persons in group 3. All serum, regardless of donor, yielded fractions which gave positive reactions to latex fixation tests. The titers obtained were lowest with the two serum samples from healthy persons under sixty-five and highest with serum samples from persons with rheumatoid arthritis. Two seropositive fractions were recovered from the columns. One such fraction appeared after the emergence of serum albumin, requiring approximately 0.1 M sodium chloride. All serum tested contained this fraction. The second seropositive fraction appeared at a higher salt concentration, and was found in all but one serum specimen. This was from a subject in group 1. Apart from this discrepancy, it was not possible to detect qualitative differences between serum from the aged, serum from persons with rheumatoid arthritis or serum from healthy subjects under sixty-five years of age. As judged from their manner of elution, the active fractions most likely contained gamma macroglobulins, but because of the small amounts of serum employed in these experiments further tests by ultracentrifugation and electrophoresis were omitted. COMMENTS
Our studies indicate that approximately 40 per cent of the serum specimens from the aged contain unusual substances detectable by latex particles coated with human gamma globulin, but inactive when tested with sheep cells sensitized with rabbit gamma globulin. Similarly reacting serum components have been isolated previously from serum obtained from subjects with rheumatoid arthritis and hence identified as rheumatoid factors [5,6]. Whether occurring
Factor in Serum of the Aged-Heimer
et al.
in the serum of persons with rheumatoid arthritis or in the serum of aged persons (over sixty-five), these substances have the following characteristics. They are stable to 63Oc. on heating for ten minutes; they are euglobulins, the serologic activity of which is destroyed by mercaptoethanol. The substances yield precipitin-type curves in the presence of aggregated human gamma globulin, but remain in solution when unaggregated gamma globulin is used. The substances may be removed from serum by absorption with solid heat-denatured gamma globulin. Like other gamma macroglobulins, high salt concentrations are required for their elution from DEAE cellulose columns. Their manner of elution from such columns suggests heterogeneity with respect to physicochemical characteristics. These substances have been thought to be less specific for rheumatoid arthritis than other macroglobulins which react with human as well as with rabbit gamma globulin [Id]. The latter type of substances was detected in only two serum samples in group 1. Moreover, preliminary data suggest their somewhat diminished incidence in aged persons with rheumatoid arthritis. There are few diseases not related to rheumatoid arthritis in which the incidence of positive latex fixation test results equals or exceeds that found in old age. For example, the incidence of a positive latex fixation test result in liver diseases was 32.6 per cent [15] in one study, and 25 per cent in another [76]; in syphilis it was 11 per cent [77]; in lepromatous leprosy, 44 per cent [r8]; and in subacute bacterial endocarditis, 50 per cent [79]. In these diseases, as in old age, positive SEA test results were elicited only in a minority of the serum samples tested. The magnitude of the latex fixation titer in the aged population studied by us, while not equal to that of rheumatoid arthritis, hardly differs from that encountered in various diseases not related to rheumatoid arthritis. In our study, approximately 50 per cent of the positive latex fixation test results were found to equal or exceed titers of 1: 640. However, in an unusual number of the serum samples tested, prozones were encountered which could not be removed by heating. The occurrence of prozones in other diseases appears to be less frequent. There has been a growing awareness of the relationship between age and the incidence of rheumatoid factors. The comparative rarity of positive serologic reactions in juvenile AMERICAN
JOURNAL
OF
MEDICINE
Globulins
Resembling
Rheumatoid
Factor in Serum of the Aged-Heimer
rheumatoid arthritis has been commented upon frequently [I,?]. The increased incidence of positive latex fixation test results among the aged is suggested from the work of Arnon et al. 1161, \vho found a positive reaction to latex fixation tests in 17.6 per cent of 216 inhabitants over seventy y-ears of age in Tours, France. An age-dependent increase of incidence of positive latex fixation test results was also found by Jacqueline [lo]. Epidemiologic studies carried out in various countries also suggest an age trend with respect to rheumatoid serology [27]. Similar results were obtained in a study of residents of New Haven, Corm. [_??], where 4.2 per cent of persons over sixty-five years of age had a positive reaction to the latex fixation test contrasted to 0.47 per cent in persons betwcen the ages of fifteen to twenty-four. It is difficult to compare the results of this study with ours as no further breakdown by age was given of the group over sixty-five years. In studying the epidemiology of rheumatoid arthritis in northern Europe, Lawrence et al. 12.31 reported that the results of both the SEA and latex fixation tests show an increasing titer with age. Somewhat divergent results were obtained by Ball and Lawrence [24] with the SE.4 test in a study of an urban and a rural population. In the mining town of Leigh, 14.7 per cent of the men and 9.4 per cent of the women over 65 years of age gave a positive reaction to the SE.4 test. In FYensleydale, a rural community-, none of the sixty-seven men over sixty-live years of age had positive SEA test result, yet 6.3 per cent of 111 women in this community did. In the light of these findings, the question must be raised whether the unusually high incidence of positive latex fixation test results among the aged in New York City is a local phenomenon, is encountered in urban populations generally, or reflects biological processes unrelated to environment. :it the present time we cannot give a satisfactory explanation for the increased incidence of rheumatoid factor-like substances among the aged. Our aged population did not suffer from any of the diseases known to give rise to positive latex fixation test results. The most frequently encountered condition related to degenerative changes in joints; it was observed in seventy-five Hypertension and/or atherosclerosis persons. was found in sixty-nine persons. The incidence of positive latex fixation test results among persons falling into these two subgroups was VOL.
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1963
et ul.
179
similar to the subgroup in which these disorders lvere not clinically significant. The high incidence of positi\le latex fixation test results, therefore, is probably unrelated to recognized pathologic conditions among the agect. Although the cause of rheumatoid factor production is still unknown, the notion that the rheumatoid factors constitute antibodies against gamma globulin itself or that they are antiantibodies is currently favored by many ]1,.?5]. It has been possible to induce formation of rheumatoid factor-like substances in animals by prolonged injections with bacteria [lo], homologous soluble [_37] and insoluble 1281 immune complexes. The increased incidence of rheumatoid factorlike substances in old age may perhaps relate to a piling up of autoantibodies, as postulated in certain theories of aging [s]. Such theories suggest an age-dependent increase in the rate of somatic mutations of cellular clones, giving substances or causing failure rise to “foreign” of antibody forming cells to recognize “self” If the animal experiments from ‘inon-self.” cited are a guide, the consequence of such mutations may be the production of rheumatoid factors. Another possible explanation for the high incidence of positive reactions to latex fixation tests among the aged might be that these persons have decreased production of certain serum components which, because of their stabilizing effect on latex particles, block the serologic detection of rheumatoid factors. The net result of this decrease would be to facilitate detection of rheumatoid factors, which might very well be present in the serum of persons of all ages. Suggestive evidence for the presence of rheumatoid factors in the absence of positive serologic test results has been obtained by Lospalluto ancl Ziff [5] as well as by us. On chromatography, seronegative samples from healthy donors were found to yield fractions which gave positive reactions to latex fixation tests. Recent investigations on asymptomatic relatives of persons with rheumatoid arthritis have revealed a greater incidence of seropositivity than could be expected on a statistical basis [3]. The data were interpreted as evidence for a familial and probably genetic basis of production of rheumatoid factors. As it appears unlikely that asymptomatic relatives live longer than other persons, a more likely explanation of our
180
Globulins
Resembling
Rheumatoid
results might be that rheumatoid factor production, n addition to being a familial trait, can also be acquired. The acquired form may appear in persons of advanced age. Apart from the more obvious serologic differences, it may well be that the rheumatoid factors found in rheumatoid arthritis are governed by genetics peculiar to it, and that the rheumatoid factorlike globulins found in other diseases as well as in the aged are acquired rather than genetically transmitted. Regardless of the true cause of the increased incidence of positive reactions to latex fixation tests in serum samples from the aged, the hazards of using serologic tests as an aid in the diagnosis of rheumatoid arthritis in elderly persons should be recognized. In addition to the increased incidence of positive latex fixation test results in the aged population, attention is called to results obtained with precipitin tests with aggregated human gamma globulin. It was found that the precipitation of heated serum obtained from the aged fell midway between that of serum obtained from persons in groups 3 and 4. The amount of nitrogen precipitated in the serum from the aged correlated only moderately well with the ability to fix latex particles. In serum obtained from subjects in group 3, however, there appears to be a direct correlation between serologic titer and amount of nitrogen precipitated. A possible explanation for this difference may be that the serum obtained from the aged contains as yet unknown heat-stable proteins with afftnity for aggregated gamma globulin but which are inert in the latex test. Alternately, such serum might contain smaller amounts of stabilizer of latex particles. Experiments are under way to clarify these results. SUMMARY
One hundred and twenty-one serum samples from persons over sixty-five years of age were tested by the Hyland RA slide test and by the latex fixation test. It was found that 46 per cent of the samples gave a positive reaction to the Hyland RA slide test and only 42 per cent to the latex fixation test. By contrast, only 2 per cent of the samples gave a positive reaction to the sensitized sheep cell test. Attempts were made to characterize the unusual serum constituents responsible for serologic activity. It was found that the activity resided in a heat-stable euglobulin fraction and
Factor in Serum of the Aged-Heimer
et al.
that reactivity in the latex fixation test could be treatment with mercaptodestroyed upon ethanol. The behavior of these serum constituents was also studied by DEAE cellulose chromatography and by precipitation reactions with different preparations of human gammaglobulin. The results of the study indicate that these constituents are indistinguishable from the unusual serum components, the rheumatoid factors, which are present in the serum of persons suffering from rheumatoid arthritis. The incidence of globulins resembling the rheumatoid factors in serum of the aged was contrasted with the incidence of similar moieties in serum of persons with diseases other than rheumatoid arthritis. The possible significance of a relationship between age and incidence of these globulins is discussed. Acknowledgment: Il’e are greatly indebted to Dr. Ann Maloney and Sister Richard of the Josephine Baird Home for the Aged, and to Dr. Bernard Rogoff, who helped us obtain serum from persons F ree dman Home for thf?gEd.at the Andrew REFERENCES 1.
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J. II. Serum responses in rheumatoid arthritis. Am. J. Med., 26: 596, 1959. SINGER, J. M. The latex fixation test in rheumatic diseases. Am. J. Med., 31: 766, 1961. ZIFF, M., SCHMID, F. R., Lawn, A. J. and TANNER, M. Familial occurrence of the rheumatoid factor. Arthritis @ Rheumat., 1: 392, 1958. ZIFF, M., BROWN, P., LOSPALLUTO.J., BADIN.J. and MCEWEN, C. Agglutination and inhibition by serum globulin in the sensitized sheep cell agglutination reaction in rheumatoid arthritis. Am. J. Med., 20: 500, 1956. LOSPALLUTO, J. and ZIFF, M. Chromatographic studies of the rheumatoid factor. J. Expey. Med., 110: 169, 1959. HEWER, R. and SCHWARTZ, E. R. Isolation of rheumatoid factors. Arthritis & Rheumat., 4: 153, 1961. SINGER, J. M. and PLOTZ, C. M. The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. Am. J. Med., 21: 888, 1956. HALBERSTAM,D., SINGER,J. M., ALLEN, E. G. and PLOTZ, C. M. Stability characteristics of .2 micron diameter polystyrene ‘latex particles. In: Atti de1 X Congr&so deila Lega Internationale contra ii Rheumatismo, vol. 2, P. 2854. Turin 1961. Minerva Medica. _ HELLER, G., JACOBSON,A. S. and KOLODNY, M. H. A modification of the hemagglutination test for rheumatoid arthritis. Proc. Sac. Exper. Biol. @ Med., 72: 316, 1949. TARANTA, A., WEISS, H. S. and FRANKLIN, E. C. VAUGHAN,
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AMERICAN
for JOURNAL
aggregated OF
gammaMEDICINE
Globulins
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Factor in Serum of the Aged-Heimer
globulins in normal human sera. Nature, London, 189: 4760, 1961. MUELLER-EBERAARD, H. J. and KUNKEI., H. G. Isolation of a heat labile serum component which interacts with gammaglobulin aggregates. Fed. I’rw.> 19: 76, 1960. CIIKISTIAN, C. L. Characterization of the “reactant” (gamma globulin factor) in the FII precipitin reaction and the FII tanned sheep cell agglutination test. J. Exper. Med., 108: 139, 1958. HAWK. P. B., OSER, B. L. and SUMMERSON, W. 1-I. Practical Physiological Chemistry, p. 878. New York, 1954. McGraw-Hill Book Co. ZIFF, M. The agglutination reaction in rheumatoid arthritis. J. Chron. Dis., 5: 644. 1957. ~II)WI
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in patients with subacute bacterial endocarditis. J. Clin. Inuest., 41: 666, 1962. JACQUELINE, F. Personal communication. VALKENBURG, A. and HIJMANS. W. Population studies on the rheumatoid factor. In: Atti de1 X Congresso della Lega Internationale contra il Rheumatismo, vol. 2, p. 789. l’urin, 1361. Minerva Medica. KISSI~K, W. L. Epidemiology of rheumatoid factor. Arthritis @ Rheumat., 4: 424, 1961. LAWRENCE. J. S., LANE, V. A. and DISGRAAF, R. The epidemiology of rheumatoid arthritis in northern Europe. Proc. Ro.7. SW. Mrd., 54: 454. 1961. BALL, J. and LAWRENCE, J. S. Epidemiology of the sheep cell agglutination test. Ann. Rhcumat. Dis.. 20: 235, 1961. KUNKEL, H. G. Immunologic aspects of rhcaumatoid arthritis. J. Chron. IX., 10: 418, 195’). SCHICHIKAWA,K., YAMAMOTO,T. and FUJIOKA, M. experimentale ud facteul Sur la production rheumatoide. In: Atti de1 X Congress0 della Lega Intrrnationale contra il Rheumatismo. vol. 2. p. 789. Turin, 1961. Minerva Medica. Ano, K. and WAGER, 0. Production of “anti-antibodies” in rabbits. Ann. Med. Exfw. P/ biol. Fenniap, 39: 79, 1961. ABRUZ~~, J. L. and CHRISTIAN. C. 1,. Induction of a rheumatoid factor-like substance in rabbits. J. &,brr. hfrd.. 114: 791, 1961. A.. The position of fundamental age COMFORT, studies. .‘lm. &art .I.. 62: 203, 1961.