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The problem of standardization in rheumatoid arthritis serology.

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CURRENT COMMENT
The Problem of Standardization in Rheumatoid
Arthritis Serology
By S. BOZS~KY
T
HE CLINICAL SIGNIFICANCE of the so-called rheumatoid factors is
not completely understood. Some of the contradictory data may be due
partly to the wide variety of serologic methods adapted at different laboratories.
In order to elucidate this problem, at the Xth International Congress of
Rheumatology in Rome a world-wide comparative study was initiated. Freezedried samples of a “positive” and a “negative” serum pool, collected from 50
rheumatoid arthritis patients and 50 normal subjects, respectively, were distributed among the participants.* About 30 serologists from 14 countries were
kind enough to take over the task of performing the comparative investigations. To date, 26 protocols have been returned, and have afforded the following information.
The sera were investigated by the sensitized sheep cell reaction (SSC test)
at 19 laboratories, and by the latex fixation reaction (LF-I1 test) in 14 cases.
Both reactions were simultaneously performed, however, in only nine instances. In addition to, or instead of these two methods, in some cases the
sensitized human cell reaction ( SHC test), tanned sheep cell reaction (SC-FI1 test ) , latex slide test, bentonite flocculation test, Streptococcus-L-agglutination and other procedures were carried out as well.
Table 1 shows the distribution of the different methods. Without entering
into details it can be stated that among the 26 Institutes participating in the
collaborative assay, in this respect not less than 12 variations occurred.
Similar lack of uniformity existed referring to the technologic prescriptions
as specified in the protocols. With the sensitized sheep cell reaction the sensitizing doses of hemolysin varied from 1/10 to 1/2 basic agglutination titer; in
some cases the titration had been performed on a hemolytic unit basis. With
the latex fixation test, latex particles of difFerent origin and size as well as
gamma globulins from different sources were used. The latter were either
liquid or in the freeze-dried state, mostly unfiltered and not heat-treated prior
to use. In some cases, however, heat treatment at 56 or 63 C., respectively, was
prescribed. No identity could be stated regarding the temperature and period
of incubation or the mode of reading the results. In other words, there existed
almost as many modifications as study groups.
This situation is self-evident when one considers that rheumatoid arthritis
serology works with reagents of biological nature and therefore of divergent
peculiarities. The agglutinability of red blood cells taken from different sheep
or even from the same animal but at different intervals may considerably
From the National Institute
of Rheumatism and Mediccil IIrJdrologv, Ihdapest, Hungary.
*We are indebted to Dr. K. Ujhelyi, State Institute of Hygime, Budapest, Hungary, for
freeze-drying the serum samples.
891
ARTHIWITSAND RHEUMATISM, VOL.6, No. 5 ((9cToxsm),1963
642
BOZS6KY
T a i l s perfomad
Immbar of oaaar
5 8 1 t e r t 4 Id-II-Caat
3
S8C-taat 4 ldmx r u d e t e a t
4
SW-teat 4 bP-XI-tart
SSC-teat
SlC-teat
+
letax e l i d e taat
+ aC-&eat + b#-XI-taat
+ L-Y-XI-tart
SBO-tent 4 tP-11-tent
+
SC-Y-XI-tent
SSC-tent b bentonite floooalution taat
950-tent
+ L-P-XI-teat + Stroptoooooua-L-rggl.
SSC-tent only
2
9
1
1
1
5
SIC-tent only
50-Y-XI-teat
2
1
only
1
e
bb’-IX-trnt only
rota1
a6
differ from each other,lV2 as well as many of the sensitizing capacities of individual hemolysin preparations-that is, rabbit immune sera being in reality
a mix‘ture of many different kinds of materials and also containing, among
others, hemolysin molecules again of different size and avidity.3 This is the
reason why each laboratory has to titrate its biological reagents by trial and
error when introducing the sensitized sheep cell test.
Although working with far more defined substances, the situation is just as
complicated with the latex fixation test. Our experience has shown that two
latex suspensicns of different sources may differ in quality from each other
much more markedly than any blood samples taken from individual sheep4
or supposedly even from individual alligators. In addition, F-I1 preparations
expected to replace hemolysin with the promise of greater uniformity introduced a number of new problems. Namely, during the course of manufacture,
gamma globulin molecules undergo changes like partial aggregation and
denaturdtion, resulting in marked variations of r e a ~ t i v i t y , ~while
.~
in the
case cf anti-sheep rabbit sera, the disturbing effect of their several components may be reduced by simply diluting as necessary to obtain the sensitizing
level.
On the ground of the aforementioned arguments, the lack of uniformity
ruling over the technologic prescriptions might eventually serve as a basis
for the comparability of the titration results.
Unfortunately, it proved not to be the case. As is seen in figure 1, the positive reference serum was found to exhibit titers between 1:32 and about
1:30,000 by the sensitized sheep cell reaction, those between 1:64 and about
1:20,000by the latex fixation test, and those between 1:64 and 1:1,280 by the
STANDARDIZATION IN RHEUMATOID ARTHRITIS SEROLOGY
643
0
me0
1oMO
-
0
.
51eO
6
2560
0
1880
640
.
am.
l60
80
.
,..
0.0
..
0.
Ob4
0
0
0
6.b
0
4 0 .
.
0..
..
.
ZO
- Fig. 1.-Titration results obtained by the SSC, LF-11 and SHC tests performed
at different laboratories.
sensitized human cell reaction. The negative serum pool was declared negative
by all of the reactions performed. With the sensitized sheep cell test a value
of 1:64 was also found, it is true. At the laboratory in question, however, the
titer of 1:128 was taken as the borderline value of positivity in this reaction.
Figure 2 shows the titration results of the positive serum pool as obtained
by the sensitized sheep cell test and the latex fixation reaction performed
simultaneously. Again a fair disagreement can be observed between the two
tests.
The titrations were performed at different points of time. According to
Robinson and his collaborators7 and Swahn and Grubb,s however, freeze-drying does nct influence the agglutinating capacity of the sera. When salt-concentrated fractions of rheumatoid factor were lyophilized, all activity was
lost. Nevertheless, whole plasma containing the active fraction may be kept
indefinitely when treated in this manner.
The data presented speak by themselves for the need of standardization in
rheumatoid arthritis serology. The question remains, however, if there is a
possibility of standardization.
One of the main difficulties seems to be due to the well-known complexity
of the so-called rheumatoid factors. LoSpalluto and Ziff,B Heimer and his collaborators,I0 Winblad,ll Harboe12 and others succeeded in demonstrating
the heterogeneity of these macroglobulins. It must be referred, however, to
real antibodies, where the complexity and heterogeneity of the molecules to
be measured is rather a rule than an e~cepti0n.l~
In spite of this complexity,
the determination of antibodies against diphtheria and tetanus toxins, strepto-
644
BOZS6KY
0
a
b
"i
10
Fig. %-Comparison of the titration results of the positive serum pool obtained
by the SSC and LF-I1 tests performed simultaneously.
lysin-0, typhoid agglutinogens, etc. may be controlled by means of proper
international standard sera.
Thus, differences in molecular weight and other physicochemical peculiarities do not exclude by themselves the possibility of standardization. The question is, whether there exist also some functional differences among the different
rheumatoid factors.
It is well-known that the rheumatoid factors can be divided into two
main groups: one group reacting both in the sensitized sheep cell and
latex F-I1 systems, the second one reacting only with human gamma globulin,
not with rabbit gamma globulin.9The amount of these two kinds of rheumatoid
factors is not proportional in individual sera. Therefore, in introducing an
eventual standard serum, one has to take into account this circumstance.
This problem is again not unfamiliar to those interested in the production of
bacterial agglutinating sera, where antibodies of differing or shared specificity
or of cross-reactivity may co-exist in the same serum. Standardization can .be
solved even in instances like this, the Standard Antityphoid Serum introduced
by Felix serving as an e~amp1e.l~
However, a more serious drawback in elaborating an international standard
rheumatoid serum would arise, if the existence of several functionally differing rheumatoid factor groups were demonstrated. According to Kunkel and
Fudenberg,15 at least eight different rheumatoid factors could be identified
on the basis of reactivity with different Rh antisera. This kind of iso-specificity,
however, could not be demonstrated with any of the other systems. Gamma
globulin aggregates from sera of different genotypes reacted without exception
645
STANDARDIZATION IN RHEUMATOID ARTHRITIS SEROLOGY
-roe.
02 -1.
M44
a
b44
-
v
I0
g
r
!
g
o(
g
g
8
b
g
Fig. 3.-Titration of agglutinating activity of test sera in parallel with standard
serum in the LF-I1 test.
with all rheumatoid factor preparations obtained from different sera by ultracentrifugation.
On the other hand, Butler and Vaughan,l6binding individual gamma globulin preparations with bis-diazotized benzidine bonds to human 0 erythrocytes,
tested these systems with different rheumatoid euglobulin preparations. Although all of the systems employed were agglutinated in a comparable manner
by a rabbit anti-human globulin serum, indicating that the degree of coating
was comparable, the same cells exhibited striking differences in their ability
to be agglutinated by individual rheumatoid factor preparations. This problem
awaits further elucidation.
The most valid argument for the possibility of establishing an international
standard rheumatoid arthritis serum is based upon the existence of local
reference sera, their use being recommended by Ball1' and Svartz and Schlossmann,ls early in the fifties. These authors stressed the advantages resulting
from the simultaneous testing of sera of known strength by routine work.
Thus, the introduction of an international standard serum, allowing calibration
of the reference sera in terms of the International Unit, would supposedly
meet the approval of major laboratories, especially since population studies on
a large scale have been initiated lately throughout the world. The establishment of a standard serum would be of particular significance on the field of
inhibition tests as well. One has to agree with Epstein,l9 that until a source of
reference sera is not available, results of inhibition tests cannot be accurately
compared,
Differences in the level of sensitivity with the same methods employed at
different laboratories or with different tests of the same laboratory would be
greatly reduced if titers were replaced by potency units, as proposed by
JerneZoand Miles,21 the unit of agglutinating potency being the specific
activity of an arbitrarily decided weight of a dried preparation of standard
serum. The potency, as the difference between logarithmic titers for the
serum under test and the standard serum examined in the same experiment,
646
BOZS~KY
can be calculated by dividing the reciprocal titer for the serum in question by
the titer of the standard serum.
Figure 3 shows the log-dose-response curves of two sera and that of a
reference serum tested with the latex fixation reaction, under the conditions
recommended by Singer and Plotz.22 It can be stated that the distance in
degree of dilution between two tubes of the individual series showing
agglutination amounted to 1280:320, i.e., 4:l in the case of serum-1, and to
80:320, i.e., 1:0.25 in the case of serum-2. If the potency of 100 units per ml.
had been arbitrarily assigned to the reference serum, the potency of test
serum-1 could be expressed as 4 x 100, i.e.,400 units per ml., that of test serum-2
as 0.25 x 100, i.e.,25 units per ml., irrespective of the sensitivity of the method
used for titration.
The unparallelism between the reaction curves for the reference serum and
test serum-2 reflects differences in avidity of the rheumatoid factor molecules
in question and means that in this case a generally valid relative potency
does not exist. The same conclusion would be drawn if the curves were parallel but showed different distances when different agglutinating systems were
used.
The problem of avidity was exhaustively dealt with some years ago by
I p ~ e nand
~ ~J e ~ - n ein~ ~
connection with different antibodies. Let us mention
that recently differences were found also by Spaun and his collaborators25
between the slopes of log-dose-response curves of some test sera and that
of the International Standard for Antistreptolysin-0, the differences being
most marked in the case of low titer sera. This circumstance, however, does
not invalidate the value of the antistreptolysin-0 standard serum for routine
work.
The data presented seem to add some practical evidence to the arguments
for an international rheumatoid arthritis standard serdm. While accepting all
of the difficulties that wculd be met on the way, it is encouraging to recall
the period when experts had very similar problems with the standardization
of serologic tests for syphilis. In this field a number of various reactions exists,
the mechanisms of which are neither identical nor completely understood, a
situation extremely similar to that we are facing. In spite of this, an International Standard for Human Syphilis Serum is already available.26 Similarly,
the establishment of an International Standard for Rheumatoid Arthritis Serum
would be one of the most important tasks for those interested in rheumatoid
arthritis research work.
++
APPENOIX
AUTHORS
AND LABOBATORIES
PARTICIPATING
IN THE COLLABORATIVE
ASSAY
Canada
D. K. Ford and H. S. Robinson
Department of Medicine, University of
British Columbia
Vancouver, B. C.
Czechoslovakia
V. Houba and Fr. Lenoch
V9zkumn.f Ustav Chorob Reiimatickj.ch
Prague
E. Kresinek and S. Sitay
V9zkumnp Ustav Reumatickfrch Chor6b
PieStany
Finland
0. Wager
Auroran Sairaala, Bakteriologinen laboratorio
Helsinki
STANDARDILATION IN RHEUMATOID ARTI-IBITJS SEROLOGY
France
A. I. Nesterov and V. I. Sachkov
Y. Badin
Centre d’Exploration Fonctionelle de la
Seine
Paris
M. F. Kahn and S. de Shze
H6pital LariboisiBre, Centre de Rhumatologie Viggo-Petersen
Paris
Germany
F. Harter
Medizinisch-Diagnostiisches Institct
Heidelherg
H. Seifert
Sachsisches Serumwerk A.G.
Dresden
Ef ungary
J. Bobory and Gy. PetrCnyi
OrvostudomCnyi Egyetem, 11. Belklinika
Debrecen
L. Gofman and A. HAmori
OrvostudomCnyi Egyetem, 11. Belklinika
National Institute of Rheumatism
Moscow
P&s
8. Schulhof and S. Bozs6ky
Orszigos Reuma 6s Furdougyi Int6zet
Budapest
Italy
A. Robecchi
Centro di Reumatologia, Ospedale Molinette
Torino
Xetherlunds
J. J. deBlkcourt
Department of Rheumatology, University
Hospital
Groningen
W. Hijmans
Department of Rheumatology, University
Hospital
Leiden
R~mnZa
I. Stoia
Centrul Metodologic de Reumatologie
Bucharest
soviet unim
647
Sweden
N. Svartz
Konung Gustaf V:s forskninginstitut
Stockholm
S. Winblad
Lunds Universitet, Institutionen fijr Klini ;k
Bakteriologi
hlalmij Allmanna Sjtikhus
Malmii
Sdtzerland
E. Meyer and G . H. Fallet
Centre $Etude des Maladies Rhuinatismales,
Policlinique
Universitaire de M6decine
Geneva
United Kingdom
J. J . R. Duthie
Rheumatic Unit, Northern General Hospital
Edinburgh, Scotland
United States of Arnerica
J. J . Bunim
National Institutes of Health, National Institute of Arthritis and Metabolic DiTeases
Bethesda, Md.
W. Epstein
University of California Medical Center
San Francisco, Calif.
C. McEwen and M. Tanner
Department of Medicine, New York University, Bellewe Medical Center
New York, N. Y.
Ch. M. PIotz
Arthritis Clinic of the Mount Sinai Hospital
New York, N. Y.
J. H. Vaughan
University of Rochester Medical Center
Rochester, N. Y.
M. Ziff
Department of Internal Medicine, Southwestern Medical School
Dallas, Texas
ACKNOWLEDGMENT
We are indebted to the authors for their worthy contribution and R. W. LamontHavers, Medical Director of the Arthritis and Rheumatism Foundation, New York, N. Y.,
for his valuable help in organizing the collaborative studies in the United States.
REFERENCES
1. Zarafonetis, C. J. D., and Oster, L. H.:
Heterophile agglutination variability
of erythrocytes from different sheep.
J . Lab. & Clin. Med. 34~1216,1946.
2. Keiper, T. W.: Pitfalls in the use of
sheep cells in (?-fixation and heterophilic antibody reactions. Am. J.
Clin. Path. 15:66, 1945.
648
BOZS~KY
3. Rockey, J. H., and Kunkel, H. G.:
tismo, Torino, Italy. Minerva med.
Studies of the rabbit antibodies which
1:298, 1961.
sensitize red blood cells for agglu- 12. Harboe, M.: Heterogeneity of the
tination by rheumatoid factor. Proc.
“rheumatoid factor.” Atti del X.
7th Interim Scientific Session of the
Congresso della Lega Internazionale
American Rheumatism Association,
contro il Reumatismo, Torino, Italy.
December 1960, Dallas, Texas.
Minerva med. 1:282, 1961.
4. Bozsbky, S.: Data on the mechanism 13. Schultze, H. E.: Immunologie und
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korperreaktionen. Zentralbl. Bakt. I.
della Lega Internazionale contro il
Orig. 184:324, 1962.
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standard antityphoid serum. Bull.
5. Singer, J. M., Altmann, G., GoldenWorld Health Organ. 10:911, 1954.
berg, A., and Plotz, Ch.M.: The 15. Kunkel, H.G., and Fudenberg, H. H.:
mechanism of particulate carrier reAuto- and iso-specificity of rheumaactions with rheumatoid sera. 11.
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Sensitizing capacity of various hudel X. Congresso della Lega Interman gamma globulins for latex
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6. Oreskes, J., Singer, J. M., and Plotz, 16. Butler, V. P., Jr., and Vaughan, J. H.:
Ch.M.: Studies of human gamma
Studies on the specificity of rheumaglobulin reactivity with rheumatoid
toid factor for various gamma globuarthritis sera. Atti del X. Congresso
lins. Atti del X. Congresso della Lega
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Internazionale contro il Reumatismo,
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7. Robinson, A. R., Stulberg, C. S., and 17. Ball, J.: Serum factor in rheumatoid
Kuyper, A. C.: Identification of the
arthritis
agglutinating
sensitized
substance active in sheep cell agglusheep red cells. Lancet 2520, 1950.
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Proc. SOC.Exper. Biol. & Med. 85:4,
hemagglutination test with sensitized
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sheep cells in rheumatoid arthritis
8. Swahn, B., and Grubb, R.: Purification
and some other diseases. Acta med.
of hemagglutinating factors in rheuscandinav. 142:420, 1952.
matoid arthritis sera by Cohn’s meth- 19. Epstein, V. W.:Inhibition test in the
od 10 and ultracentrifugation. Acta
diagnosis of rheumatoid arthritis.
path. et microbiol. scandinav. 42:
Arth. & Rheumat. 2347, 1959.
173, 1958.
20. Jerne, N. K.: The “unit” in preference
9. LoSpalluto, J., and Ziff, M.: Chromatoto the “titre” as a measure of aggraphic studies of the rheumatoid facglutinating activity. Bull. World
tor. J. Exper. Med. 110:169, 1959.
Health Organ. 10:937, 1954.
10. Heimer, R., Schwartz, E. R., and Frey- 21. Miles, A. A.: The case for unit natation
berg, R. H.: Different rheumatoid
in specifying the agglutinating pofactors in the serum of one patient
tency of standard antisera. Bull.
with rheumatoid arthritis. J. Lab. &
World Health Organ. 10:941, 1954.
Clin. Med. 57:16, 1961.
22. Singer, J. M., and Plotz, Ch.M.: The
11. Winblad, S.: The dependence of gamma
latex fixation test. Am. J. Med. 21:
globulin as reactant for demonstra888, 1956.
tion of rheumatoid arthritis serum 23. Ipsen, J.: A standard for antistreptolyfactor. Atti del X. Congresso della
sin-0 of human serum and its practiLega Internazionale contro il Reumacal application. Acta path. et micro-
STANDARDIZATION I N RHEUMATOID ARTHRITIS SEROLOGY
biol. scandinav. 21:203, 1944.
24. Jerne, N. K.: A study of avidity based
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toxin-antitoxin mixtures. Acta path.
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25. Spaun, J., Bentzon, M. W., Larsen, S.
649
O., and Hewitt, L. F.: International
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World Health Organ. 24:271, 1961.
26. Bentzon, M. W., and Gag, P.: T h e
International Standard for Human
Syphilitic Serum. Bull. World Health
Organ. 24557, 1961.
S . Bozsdky, M.D., Research Assmiate, N a $ b n d Imtitute of
R h m m h and Medical Hydrology, Budapest, Hungary;
a$ present, Research Fellow, Rheumatic D i s m . Study
~ ~ Group,
N e w Ywk University School of Medicine, B e l l m e Medical
C m m , N. Y.
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