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The influence of serum dilution on findings of antiperinuclear factor prevalence in rheumatoid arthritis.

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The influence of serum dilution on findings of
antiperinuclear factor prevalence in
rheumatoid arthritis
To the Edirur:
Detection of autoantibodies contributes to the diagnosis of many autoimmune diseases. The detection of rheumatoid factor in rheumatoid arthritis (RA) and antidouble-stranded DNA antibodies in systemic lupus
erythematosus are the best known examples. Both antibodies are included in the American Rheumatism Association
diagnostic criteria for these diseases (1.2).
Another antibody with an apparent specificity for RA
is the antiperinuclear factor (APF). It is reactive with cytoplasmic granules in human buccal mucosa cells and is
present in 49-86% of RA patient sera and up to 10% of
normal control sera (3-9). Its presence is related to disease
seventy (lo), and the antibody is probably elicited by
Epstein-Barr virus-induced antigens ( I 1).
While reviewing the literature on APF in RA. we
identified a striking phenomenon-the percentage of APFpositive sera in RA patients is closely related to the dilution
of the serum used in the immunofluorescence assay (Table
I ) . There seems to be a profound “prozone effect” in this
test. The techniques used in the studies listed in Table 1 were
quite similar. Five of the studies used fresh unfixed cells
(3-5.7,9). and the other 2 studies used unfixed stored cell
preparations (6,8). A 90-minute serum incubation period was
used in the majority of the studies (3,5.6,9), and in 1 study,
the substrate was incubated with serum for 45 minutes (4); 2
reports (7.8) did not mention incubation time, but the
Proportion of antiperinuclear factor ( APFkpositive sera
according to serum dilution used, in 7 studies of rheumatoid arthritis
Table 1.
No. of
% APF-
Author (ref.). year
Nienhuis and Mandema (3). 1964
Marmont et al (4). 1965
Sondag-Tschrootset a1 (5). 1979
Johnson et al (6). 1981
Youinou et al (7), 1983
Cassani et al (8). 1983
Janssens et al (9). 1987
81 (92)’
* Percentage in parentheses includes weakly positive sera.
I :5
I :5
method of “Sondag-Tschroots” was used, as described
elsewhere (5). Six of the studies used an antiserum against
human immunoglobulins (3-79). and the remaining study
used an anti-human F(ab’), antiserum (8). We always used
anti-human immunoglobulin antiserum and fresh unfixed
cells with a 90-minute serum incubation period. One report
described patients with “established RA” (6); the other
reports described classic or definite RA.
In our own experience, the differences in technique,
as discussed above, are not responsible for the differences in
percentages of positive sera. For example. storage of cell
preparations for prolonged periods of time (20 days) does not
alter the quality of the assay. In our study, we used a very
limited number of cell donors (5 donors). The number of
donors that can be used for the APF assay is limited because
we require donors whose antigen is of a consistent quality.
We assume that other groups had the same quality control
for their assays.
Differences in sensitivity are probably not a consequence of the technical matters discussed above. However,
the serum dilution used might be an exception. To investigate this hypothesis, we screened a group of 123 serum
samples from previously described RA patients (10); samples were used both undiluted and at a 1 : l O dilution. The
patient group was composed of patients with definite or
classic KA (I). with a disease duration of at least 3 years
(range 3-32 years). With the use of diluted samples, the
percentage of APF-positive sera increased from 41% to 70%.
Only 3 sera changed from weakly positive in the assay of
undiluted samples to negative in the assay of diluted samples. A second group of 58 sera sent to us for routine
determination of APF were also tested in undiluted solutions
and at 1: 10 dilutions. Only 1 of these sera was positive in the
assay of undiluted samples: however. in the assay of diluted
samples. an additional 27 samples were positive. The medical records of these 58 patients revealed that 18 had diagnoses of definite or classic RA. The 1 sample that was
positive in the assay of undiluted solutions and 17 of the 28
samples that were positive in the assay of diluted solutions
were from RA patients. The sensitivity of the assay thus
increased from 6% to 94%. and the specificity decreased
from 100% to 73%. with the use of diluted samples.
One factor known to influence antigen-antibody interactions is the pH of the incubation mixture (12). It is well
known that carbon dioxide (a component of thc major buffer
system in human serum) evaporates from serum samples and
causes an increase in the pH of the fluid. Some sera that
were negative in undiluted solution assays became positive
when tested after dialysis with a phosphate buffer at pH 7.4.
Preliminary experiments suggest that the increased sensitivity of the dilution assay is not simply a result of decreased
background staining obtained with diluted serum.
The results of our dialysis experiments could also
mean that a low molecular weight inhibitor of antigenantibody interaction is removed from the serum. Other
factors that might influence these results are high concentrations of low-affinity antibodies competing with specific highaffinity antibodies and high concentrations of a specific
isotype of APF antibody that was poorly recognized by our
second antibody (fluorescein isothiocyanate conjugate).
The sensitivity of APF for rheumatoid arthritis and
its close association with disease seventy suggest that this
antibody assay should be included in the diagnostic assessment of patients with inflammatory joint disease. especially
rheumatoid factor-negative RA patients (10). However, in
view of the findings discussed here, it is imperative that
clinicians are aware of the technical details of the assay
performed before interpreting the results. To overcome
these problems, standardization of the A P F assay is necessary. A recent workshop held in Ghent, Belgium (June
23-24, 1989) has provided an impetus for this. The first step
toward achieving an assay protocol should consist of a n
exchange of existing protocols and standard sera, including
making available a World Health Organization reference
Antonius A. A. Westgeest, MD, PhD
Agnes M. Th . Boerbooms, MD. PhD
Levinus B. A. van d e Putte, MD, PhD
University Hospital St. Radboud
Nijmegen, The Netherlands
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factors, dilution, antiperinuclear, arthritis, serum, findings, prevalence, influence, rheumatoid
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