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Measurement of increases in anti-double-stranded dna antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus.

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634
MEASUREMENT OF INCREASES IN
ANTI-DOUBLE-STRANDED DNA ANTIBODY LEVELS
AS A PREDICTOR OF DISEASE EXACERBATION IN
SYSTEMIC LUPUS ERYTHEMATOSUS
A Long-Term, Prospective Study
E. J.
TER
BORG. G. HORST, E. J. HUMMEL, P. C. LIMBURG. and C. G. M. KALLENBERG
To evaluate the predictive power of changes in
levels of antibodies to double-stranded DNA (antidsDNA) as a predictor of disease exacerbations in
systemic lupus erythematosus (SLE), we performed a
prospective study on 72 unselected patients with SLE
(mean duration of study 18.5 months, range 6-35
months). Patients were seen at least once every 3
months, and disease activity was scored according to a
specific protocol. Plasma samples were obtained at least
once every month and were assessed for anti-dsDNA
antibody (by the Crifhidiu luciliue assay, an enzymelinked immunosorbent assay [ELISA], and the Farr
assay) and for complement components C3 and C4.
Twenty-seven of 33 disease exacerbations observed during the study period were accompanied by a positive test
result for anti-dsDNA antibody (27 by the Farr assay, 19
by the C luciliae assay, and 23 by the ELISA). Twentyfour of these exacerbations were preceded by a significant increase in anti-dsDNA antibody levels (23 by the
Farr assay, 12 by the C luciliae assay, and 17 by the
ELISA). The first observance of a significant increase in
anti-dsDNA antibody levels preceded the exacerbation
by 8-10 weeks. Significant increases in anti-dsDNA
antibody levels not followed by an exacerbation were
observed in 5 cases by the Farr assay, in 7 cases by the
C luciliue assay, and in 3 cases by the ELISA; however,
From the Department of Internal Medicine, Divisions of
Clinical Immunology and Rheumatology. University Hospital,
Groningen. The Netherlands.
Supported by grant 87/CW64 from the Dutch Foundation
Against Rheumatism.
E. J. ter Borg. MD; G. Horst; E. J. Hummel; P. C.
Limburg, PhD; C. G. M. Kallenberg. MD, PhD.
Address reprint requests to E. J. ter Borg, MD, Department
of Internal Medicine, University Hospital, Oostersingel 59. PO Box
30.001, Groningen 9700 EZ. The Netherlands.
Submitted for publication June 26. 1989; accepted in revised form December 14. 1989.
Arthritis and Rheumatism, Vol. 33, No. 5 (May 19w)
in 3 cases, 2 cases, and 1 case, respectively, these
increases were followed by a n increase in disease activity
that did not fulfill the criteria for an exacerbation. Serial
measurement of anti-dsDNA antibody levels was more
sensitive for predicting exacerbations than was measurement of C3 a n d o r C4 levels (P < 0.03). Serial assessment of anti-dsDNA antibody levels, especially by the
Farr assay, is a sensitive and reasonably specific method
for predicting disease exacerbations in SLE.
Systemic lupus erythematosus (SLE) is characterized by protean clinical manifestations and the
occurrence of a variety of autoantibodies. Among
these, anti-double-stranded DNA (anti-dsDNA) antibodies are highly specific for SLE and are detected at
a high frequency (75-95%) in untreated patients with
active disease (1). Several reports have claimed a
correlation between disease activity and levels of
anti-dsDNA antibodies in SLE (2-1 I ) , especially in
patients with renal involvement (3,4). Other studies,
however, did not find such a correlation (12). Asymptomatic patients with persistently high levels of antidsDNA antibody have been described (13). This antibody is thought to play an important role in the
pathogenesis of SLE by forming immune complexes
with DNA (14).
Anti-dsDNA antibodies constitute a heterogeneous family with respect to immunoglobulin class,
avidity, complement-fixing ability, antigenic specificity, and cross-reactive patterns. Their detection
depends. in part, on the assay used. AntidsDNA
antibodies with different characteristics seem to be
associated with different disease manifestations. It has
been stated, for example, that the Farr assay predominantly detects high-avidity anti-dsDNA antibodies
associated with renal involvement, whereas enzyme-
PREDICTIVE VALUE OF ANTI-dsDNA IN SLE
Table 1. Cumulative characteristics of 72 systemic lupus erythematosus patients at the start of the study
Number
(%I of patients
Malar rash
Discoid rash
Photosensitivity
Oralhasal ulcerations
Arthritis
Serositis
Renal disorder
Neurologic disorder
Hemolytic anemia
Leukopenia
Thromboc ytopenia
Anti-double-stranded DNA antibodies
Anti-Sm antibodies
Antinuclear antibodies
25 (35)
9 (13)
40 (56)
28 (39)
45 (63)
32 (44)
42 ( 5 8 )
6 (8)
14 (19)
49 (68)
26 (36)
53 (74)
4 (6)
72 ( 100)
linked immunosorbent assays (ELISA) also detect
low-avidity anti-dsDNA antibodies associated with
cerebral involvement (IS).
Changes in anti-dsDSA antibody levels may be
more relevant in relation to disease activity than are
their absolute values. Several reports suggest that
increases in anti-dsDNA antibody levels are followed
by flares of disease activity, especially when the flare
is characterized by renal involvement (5,7,16-19).
Most of these studies, however, have been retrospective and have been hampered by the fact that blood
samples were not obtained on a frequent basis.
In view of these earlier findings, we prospectively evaluated the predictive value of changes in
anti-dsDNA antibody levels in predicting changes in
disease activity and exacerbations in SLE. For reasons mentioned above, 3 different assays for measuring anti-dsDNA antibodies (Crirkidia luciliae, ELISA,
and Fan) were used. Since measurement of complement profiles is a widely accepted means of assessing
disease activity in SLE: (10.18.20), we compared
changes in anti-dsDNA antibody levels with changes
in levels of complement components C3 and C4.
Patients were screened for signs of disease activity at
regular intervals. and disease activity was scored
according to a standard protocol. Blood samples were
obtained at least once every month. We addressed the
following questions: First, what is the significance of
changes in levels of anti-dsDNA antibodies for assessing disease activity and for predicting disease exacerbations? Second, how do the 3 anti-dsDNA antibody
assays compare in their predictive power? The results
show that quantitation of anti-dsDNA antibodies is
very useful for predicting disease exacerbations in
SLE patients.
635
PATIENTS AN11 METHODS
Patients. Seventy-two patients (61 women and 1 1
men) who fulfilled the American Rheumatism Association
( A M ) revised criteria for S1.E (21) were studied. Their
Table 2.
Systemic lupus erythematosus disease activity index*
Point st
Kidneys
Proteinuria
Newly developed (>0.5 g d d n y ) or
doubling within 4 months
(baseline proteinuria >0.5 g d d a y )
Eryt hrocyturia
Newly developed (I5 erythrocytedhigh power
field) or doubling
Erythrocyte and/or granular casts
Presence
Newly developed
Creatinine clearance
Decrease of >25% (within 4 months)
Central nervous system$
Cerebrovascular accident
Seizure
Psychosis
Choreoathetosis
Transverse myelitis
Motor nerve palsy
Skinlmucosa
AlopeciaS
Active discoid rash
Malar rash
Other active rash
Active oral and/or nasal ulcerations
Blood
Hemolytic anemia
Hgb < I00 gmlliter
Hgb 4 0 @liter
Leukopenia
<4.0 x 109/liter
<3.0 x 109/liter
Thrombocytopenia
<loo x lO9/liter
<50 x 109/liter
<25 x 109/liter
Musculoskeletal system
Anhralgia and/or myalgia$
Arthritis ( > 2 joints) and/or tendinitis
Serosa
Pleural and/or pericardial pain$
Pleural and/or pericardial rub
Abnormalities on chest radiograph. EKG.
andlor echocardiogram
Vessels
Minor vasculitis (purpura. periungual infarction)
Major vasculitis (ulcerations. mononeuritis)
Miscellaneous
Uveitis andlor chorioretinitis
Myositis
Increasing CPK > I50 units/liter
Increasing CPK >500 units/liter
2
I
I
2
2
4
4
4
4
4
4
1
I
2
1
2
I
2
I
2
I
2
3
I
I
I
2
2
2
4
3
2
3
* Hgb = hemoglobin; EKG = electrocardiogram; CPK = creatine
phosphokinase.
t When it is likely that an item is related to medication or to an
unrelated condition. no points are given.
$ Scored only when occumng within 2 weeks of the time of the
outpatient visit or hospital admission under consideration.
636
Table 3.
TER BORG ET AL
Criteria for major exacerbation*
1. Severe renal disease
Recent renal biopsy showing active proliferative lupus nephritis (>50% of glomeruli affected),and/or decrease of creatinine
clearance of >25% within 4 months, accompanied by an active sediment ( > 5 erythrocyteslhigh power field andor casts)
and by proteinuria of >O.S gm/day
2. Severe central nervous system disease
Seizures, cerebrovascular accident. coma, transverse myelitis,
psychosis. choreoathetosis, central nerve palsy
3. Hematologic disease
Hemolytic anemia (Hgb <60 gm/liter) and/or
thrombocytopenia (<SO x 109/liter)
Severe serositis
Pericarditis with (impending) tamponade and/or massive
pleural effusion
5 . Uveitis andlor retinitis
6. Myocarditis with arrhythmia and/or congestive heart failure
7. Severe myositis with proximal muscle weakness
8. Lung involvement with hemoptysis
9. Major vasculitis
With ulcerations and/or mononeuritis multiplex
4.
10. Miscellaneous
Fever (>38"C rectally), serositis. hemolytic anemia (Hgb >60
gditer). or thrombocytopenia (>SO x I@/liter).all without
improvement after prednisolone therapy at a maximum dosage
of 30 mg/day for at least 1 week
* A patient was considered to have a major exacerbation if he or she
fulfilled 1 or more of these criteria. Only features present within 2
weeks of the outpatient visit or hospital admission under consideration were considered. Hgb = hemoglobin.
mean age was 37.5 years (range 18-73). The mean disease
duration before the start of the study was 7.2 years (range
0-25 years). The mean observation time during this study
was 18.5 patient months (range 6-35 months). Cumulative
patient characteristics, based on the revised A R A criteria
(21). at the start of the study are given in Table I . All patients
were seen at least once every 3 months at an outpatient
clinic. During periods of active disease. the patients were
seen more frequently, according to the clinician's judgment.
At every outpatient clinic visit, and at least once every week
during inpatient hospitalization, a medical history was obtained using a standardized protocol, and a physical examination and standard laboratory procedures were performed.
Treatment decisions were based on clinical symptoms and
results of standard laboratory procedures, without the clinician's knowledge of the levels of anti-dsDNA antibodies or
complement.
Assessment of disease activity. A disease activity
index (Table 2) was calculated at every outpatient clinic visit
and at least once a week during inpatient hospitalization by
a physician (EJtB) who was not informed about the results of
the tests for anti-dsDNA antibodies and complement. Our
disease activity index contained objective items and results
of standard laboratory examinations only, and was calculated without prior knowledge of the study parameters. The
index had been validated by comparing its scores with the
scores on a 10-cm visual analog scale, as assessed by
clinicians experienced in the management of lupus patients (r
= 0.67, P < 0.001). As such, this index fulfilled the desired
properties of a measure of SLE activity as formulated
recently by Liang et a1 (22). Major and minor exacerbations
were defined according to previously reported criteria (23).
with minor modifications (Tables 3 and 4, respectively).
Blood sampling. At least once a month and more
frequently during active disease, venous blood samples were
drawn into tubes containing [IIITA. Plasma was stored at
- 20°C.
Study design. Plasma samples were assessed for
anti-dsDNA antibodies by the C luciliae assay and an
ELISA. Tests were performed at study entry, throughout
the study period at 6-month intervals, and during each
exacerbation. If a patient tested positive for anti-dsDNA
antibodies at one assessment, antibody levels found at all
other assessments were noted. in order to detect changes in
these levels over time. To compare the C luciliae assay and
the ELISA with the Farr assay in terms of a potential to
detect (changes in) anti-dsDNA antibody levels, the Farr
assay was performed on samples obtained at study entry and
during each exacerbation, on all serial samples from patients
showing a significant rise (see criteria) in anti-dsDNA antibody levels determined by the C luciliae assay a n d o r the
ELISA, and on all serial samples from patients who, during
an exacerbation, tested positive for anti-dsDNA antibodies
by the Fan- assay only. Complement C3 and C4 levels were
measured in serial plasma samples at least once a month
from 6 months prior to each exacerbation until 2 months
afterward, and in samples from patients who had a significant increase in anti-dsDNA antibody levels in one or more
of the assays.
Assays for anti-&DNA antibodies. C luciliae assay.
This method used C luciliae as a substrate and fluorescein
isothiocyanate-labeled anti-Ig (Central Laboratory of the
Red Cross Blood Transfusion Service, Amsterdam, The
Netherlands) as a conjugated antibody (24). Values were
expressed in titers of 2-fold dilutions, starting at a dilution of
1: 10.
ELISA. l h i s techniquc used calf thymus DNA (Sigma, St. Louis. MO) as a substrate. To achieve efficient
coating of test plates with DNA, test plates were precoated
with protamine sulfate. 500 &ml distilled water, and incubated for 45 minutes at 4°C. DNA (10 pghl in 10 mM Tris.
pH 8.0, 0.15M NaCI) was added to the plates, which were
incubated overnight at 4°C. Plasma was incubated for 1 hour
at 37°C and for 2 hours at 4°C in 0.01M Tris HCI, pH 8.0.
0.15M NaCI. 0.05% Tween 20. 1% bovine serum albumin.
Horseradish peroxidase-labeled goat anti-human IgG (Kallestad, Austin. TX) was used as a conjugate antibody
Table 4.
Criteria for minor exacerbation*
-.
I . Increase of activity index by 2-2 points within 6 months, with a
minimum disease activity index of 3 points, accompanied by:
2. The clinical necessity of beginning a regimen of prednisolone at
a dosage of at least 10 rnglday, or of increasing the
prednisolone dosage by 2 5 @day, or of starting a regimen of
antimalarial or immunosuppressive drugs, and
3. Not fulfilling the criteria for a major exacerbation.
* A patient was considered to have a minor exacerbation if he or she
fulfilled all of these criteria.
637
PREDICTIVE VALUE OF ANTI-dsDNA IN SLE
(1:2,500. 30-minute incubation at 37°C). Normal values of
anti-dsDNA antibodies determined by this ELISA in our
laboratory are 580 IU/ml. Both intraassay and interassay
coefficients of variation by the ELISA were 10%.
Farr assay. This method used '251-labeled recombinant dsDNA (Diagnostic Products, Los Angeles. CA), which
is free from contamination with single-stranded DNA. The
assay was performed according to the manufacturer's instructions. To obtain measurements within the range of the
assay, positive samples were tested at different dilutions.
Complement component C l q does not interfere with this
assay. Normal values of this assay in our laboratory are 126
IU/ml. Both intraassay and interassay coefficients of variation were <lo%. Results of ELISA and Farr assays were
expressed in IU/ml. using Wo/80 antibody as the ultimate
standard (25).
Complement assay. Complement C3 and C4 levels
were measured by nephelometry (normal range 0.64-1.20
g d i t e r and 0.11-0.40 gm/liter. respectively). Intraassay and
interassay coefficients of variation for both C3 and C4 were
-=
< 10%.
Criteria for significant changes in levels of anti-dsDNA
antibodies and complement C3 and C4. Only changes with a
consistent pattern within 6 months prior to an exacerbation
were considered to be related to this exacerbation. The
criteria for significant changes were based partly on values of
intraassay and interassay variation for the tests used. A
significant increase in anti-dsDNA antibody levels was thus
defined as an increase of (a) 2 2 titers (24-fold increase) by
the C lrrciliue test (initial value 21:lo), (b) 225% and at least
100 IU/ml by the ELISA, and (c) 225% and at least 30 1U/ml
by the Farr assay. This increase in anti-dsDNA antibody
levels had to occur within a period of 4 months. A significant
decrease in complement levels was defined as a decrease in
C3 of 225% and at least 0.10 gmlliter and as a decrease in C4
of 225% and at least 0.05 gdliter. These decreases had to
occur within a period of 4 months.
Statistical analysis. Pearson's correlation coefficient
was calculated for detecting possible correlations between
study parameters. Chi-square analysis was used for comparison of differences in prevalence between groups. Pitman's
test was used for comparison of the different assays within 1
group. P values less than 0.05 were considered significant.
RESULTS
SLE exacerbations. Of the 72 patients participating in the study, 17 experienced 33 exacerbations (13
major and 20 minor) during the study period. The
incidence of exacerbation was 1 exacerbation per 40.4
patient months. Of the 33 exacerbations, 13 were
characterized by renal involvement as the only or
predominant manifestation, 3 were characterized by
central nervous system disease. and the remaining 17
involved miscellaneous manifestations. The disease
manifestations and anti-dsDNA antibody findings during the exacerbations are shown in Table 5. The
disease activity index score rose from a mean of 0.5
Table 5. Prevalence of disease manifestations and positive antidouble-stranded DNA antibody test results in systemic lupus
erythematosus patients during 33 disease exacerbations, at the time
of maximal disease activity*
Number (%) of manifestations
accompanied by positive
antibody test results
Number
(%)of At least
C
Manifestation
patients I assay luciliae ELISA Farr
Reual disease
Central nervous system
disease
Musculoskeletal discase
Rash
Alopecia
Vasculitis (minorhajor)
Oral ulcerdtions
Serositis
Uveitis
Hematologic disease
18
3
18 ( 5 5 ) 18 (100)t
4 (12) 3 (75)
14
I
17
1
15 (45)
I I (33)
2 (6)
10 (30)
2 (6)
12 (36)
I (3)
18 ( 5 5 )
10
11
II
8
2
8
9
2
10
2
8
10
2
0
11
1
13
I 1 (73)
10 (91)
2(100)
10 (100)
2 (100)
8 (67)
I (100)
13 (72)
1
8
0
8
10
2
8
* A manifestation is considered present when it fulfills criteria of the
disease activity index (see Table 2). C luciliae = Crirhidia luciliae
assay; ELISA = enzyme-linked immunosorbent assay.
t P < 0.05 versus serositis and versus hematologic disease.
prior to exacerbation (median 0, range 0-2) to a mean
of 6 at the time of maximal disease activity during
exacerbation (median 5, range 3-15) (Table 6).
Prevalence of positive test results for anti-dsDNA
antibodies at study outset and throughout the study
period. At the start of the study, 49 patients (68%) had
at least 1 positive anti-dsDNA antibody test result,
whereas 51 patients (71%) had positive test results for
anti-dsDNA antibodies at any time during the study.
Of the latter 51 patients, 42 tested positive for antidsDNA antibodies by the C luciliue test, versus 34
patients by the ELISA ( P < 0.02). and 47 patients
tested positive by Fan- assay ( P < 0.001 versus the
ELISA).
Levels of anti-dsDNA antibodies in relation to
disease exacerbations. Twenty-seven exacerbations occurred in 14 of the 51 patients who had at least 1
positive anti-dsDNA antibody assay result during the
study period, whereas only 6 exacerbations occurred
in 3 of 21 patients without any positive anti-dsDNA
antibody test result during the study period ( P <
0.0005). Exacerbations with renal disease (18 exacerbations) occurred only in patients with positive antidsDNA antibody test results. During 27 (82%) of the
33 exacerbations, at least I anti-dsDNA antibody
assay gave positive results. with the F a n assay giving
positive results in all 27 cases, the ELISA in 23 cases,
and the C luciliue assay in 19 cases (Tablc 6). Each
638
TER BORG ET AL
exacerbation with symptoms of active renal disease
(18 exacerbations) andor vasculitis ( 13 exacerbations)
was accompanied by positive test results for antidsDNA antibodies. During exacerbations with renal
disease the Farr assay gave positive results in all
cases, the ELISA in I7 cases, and the C luciliae assay
in 14 cases (Table 5).
Twenty-four (89%) of the 27 exacerbations that
were accompanied by positive anti-dsDNA findings,
including all 13 in which renal involvement was the
only or the predominant manifestation, were preceded
by a significant increase in anti-dsDNA antibody levels
as determined by at least 1 of the assays (Table 7). This
Frequency of exacerbations preceded by a significant
increase in anti-double-stranded DNA antibody levels'
Table 7.
Number of cases with an increase
in antibody levels assessed by
Type of exacerbation
All accompanied by positive
antibody test results
(n = 27)
With predominantly renal
involvement (n = 13)
With predominantly central
nervous system
involvement (n = 3)
At least
1 assay
luciliue
ELISA
Farr
24
12
17
23t
13
9
II
12
2
0
0
2
C
* See Table 5 for definitions.
t P < 0.03 versus the C luciliae assay.
Table 6. Anti-double-stranded DNA (anti-dsDNA) antibody levels in 17 systemic lupus erythematosus patients during 33 disease
exacerbations,
Significant increase in
Antibody levels at the
antibody levels prior
Disease peak of the exacerbationt to the exacerbation
activity
index
C
C
Patient score luciliae ELISA Farr luciliae ELISA Farr
7
I
80
11
200
t
+
I
3
160
24
640
t
+
2
640
6
600
1,730
t
+
3
1,280
106
610
5
+
58
40
4
15
179
t
+
5
300
5
2.560
390
L
f
5
5
1.280
22
488
+
+
5
5
70
880
2,560
t
+
5
4
50
470
1.280
+
+
5
4,160
6
500
20.480
t
+
144
6
640
1,580
13
t
+
640
6
49
1.290
6
t
+
6
3 20
31
615
9
t
+
7
6
42
980
160
t
+
23
6
320
7
580
+
+
7
135
1.480
640
3
i
+
7
5
3 10
1.170
148
+
14
8
4
80
I80
5
41
98
9
20
-I
5
64
9
30
+
49
17
9
9
t
+
10
5
141
58
II
11
174
9
+
4
12
123
+
12
86
3
+
13
82
6
+
14
173
6
I5
5
16
5
17
9
17
3
4
17
17
5
' See Table 5 for definitions.
t Normal values were negative (reciprocal titer) for the C
luciliae
assay, 5 8 0 IUlml for the ELISA. and 5 2 6 IUlml for the F a n assay.
was observed by the C luciline assay in 12 cases, by
the ELISA in 17 cases, and by the Farr assay in 23
cases ( P < 0.0002. Farr versus C luciliae). When the
occurrence of an increase in the anti-dsDNA level was
related to the presence of a positive anti-dsDNA result
at the time of the exacerbation, there was a significant
increase detected by the C luciliae assay for 12 (63%)
of the 19 C luciliae-positive exacerbations, a significant increase detected by ELISA for 17 (74%) of the 23
ELISA-positive exacerbations, and a significant increase detected by the Farr assay for 23 (85%) of the
Farr-positive exacerbations (Table 6). The first observation of a significant increase preceded the exacerbation by a mean period of 8 weeks, 9 weeks, and 10
weeks for the C luciliae, ELISA, and Farr assays,
respectively.
Specificity of changes in anti-dsDNA antibody
levels for predicting an exacerbation. Nine periods with
a significant increase in anti-dsDNA antibodies. as
assessed by at least 1 assay, were not followed by an
exacerbation (7 increases seen by the C litciliae assay,
3 by the ELISA. and 5 by the Farr assay). However, in
2 cases, 1 case, and 3 cases, respectively, the increase
in the anti-dsDNA level was followed by some increase in disease activity, although the criteria for
exacerbation were not fulfilled.
Patterns of change in anti-dsDNA antibody levels. All measurements of anti-dsDNA antibodies were
plotted as a function of time. Fluctuations in levels of
anti-dsI>NA antibodies generally showed a parallel
course as assessed by the 3 assays (Figure 1 ) . A
number of increases in antibody levels prior to exacerbation asscssed by the EI.ISA (9 instances) and/or
the Farr assay (6 instances) had a biphasic pattern.
PREDICTIVE V A L U E OF ANTI-dsDNA IN SLE
:t1
during other exacerbations (77% versus 35%; P <
0.05). Seventeen of the 33 exacerbations were preceded by a significant decrease in the level of C3 (16
cases) and/or C4 (13 cases). These decreases preceded
15 of the 27 exacerbations that were accompanied by
the presence of anti-dsDNA and 2 of the 6 during
which anti-dsDNA was not detected. For the former
27 exacerbations, serial measurements of C3 and/or C4
proved to be less sensitive for predicting the exacerbation than serial measurements of anti-dsDNA antibody ( P < 0.03; Table 8). When exacerbations were
preceded by a significant change in both anti-dsDNA
and C3 and/or C4 levels, these changes generally
1400
- 1280
- 320
800 800 Farr
I*I-U- Ol m l I
639
--a
onti-ds
Bo crithidio
luciltoe
l r e c titer1
IIUlrnl]
.20
'86 '87
121 2 3 4 5 6
Ymr
month
'ea
7 8 91011121
-0
diseose
octlvlty
index lpotntsl
0 0 0 0
6
3
Figure 1. Anfiaouble-stranded DNA (anti-dsDNA) antibody levels. determined by an enzyme-linked immunosorbent assay
(ELISA). the Farr assay, and the Crirliidia luciliae assay. in relation
to disease activity in a patient H ith systemic lupus erythematosus.
i.v. = intravenous; rec. = reciprocal.
130-OL5
1.10..035
c
.
wherein the first phase showed a gradual increase in
antibody levels, and the second phase showed a rapid
increase in anti-dsDNA antibody levels starting a few
months to weeks prior to the exacerbation (Figures 2
and 3). In 13 instances, antibody levels as measured by
ELISA doubled within a period of 3 months; in all but
1 case, an exacerbation occurred within a period of 3
weeks after antibody levels had doubled. Antibody
levels assessed by the Farr assay doubled within a
period of 4 months in 15 cases, and in all these cases,
an exacerbation occurred within a period of 5 weeks
after this doubling. Just prior to the exacerbation.
before immunosuppressive treatment was started, a
moderate decrease in antibody levels, after an initial
(significant) increase, was detected by El.ISA in 2 of
the 14 and by the Farr assay in 6 of the 14 cases in
which a plasma sample taken within 4 weeks prior to
the exacerbation was available (Figure 3).
C3 and C4 levels during exacerbations. During
17 of 33 exacerbations, decreased C3 and/or C4 levels
were observed (17 instances and 7 instances, respectively). Decreased C3 and/or C4 levels occurred more
frequently during the 13 exacerbations in which renal
involvement was the predominant manifestation than
'3
(glll
0.90. .0.25
0.70. .0.15
0.50-0.05
16001200-
c
.
I9G m.
ontl-ds
DNA
ELISA 800IIUlml)
600Loo-
2ool 4
OJ
disease
achvity
indn (po~nts)
0
0
0 2 2 2 2
3 c 5
0 0
Figure 2. Relationship of complement C3 and C4 and antidouble-stranded DNA (anti-dsDNA) antibody levels to disease
activity in a patient with systemic lupus erythematosus. ELISA =
enzyme-linked immunosorbent assay.
640
TER BORG ET AL
year
month
‘86
‘87
7 8 9 1011 12 1 2 3 4
~
240
‘
“
“
‘
.
~
‘88
8 9 1011 12 1 2 3
5 6 7
~
~
~
.
.
l
.
.
l
.
,
.
prednisolone
60 mg a day
-
200 exacerbation
-renal hilure
,
*--a 160-
Farr
I
I
(IU/rnl)
-pleurisy
-thmmbocytopenia
-hemolytic anemia
-oral ulcerations
12080
-
b
40 -
0disease
activity
index (points)
0 0
2
2 2
1
3
13
Figure 3. Anti-double-stranded DNA antibody levels determined by the F a n assay. in relation to disease activity in a patient with
systemic lupus erythematosus.
showed an antiparallel course (Figure 2). The decrease
in C3 and C4 generally started simultaneously with the
increase in anti-dsDNA antibodies (Figure 2).
DISCUSSION
We performed a detailed prospective study
(including the period prior to the exacerbation) on the
predictive value of changes in anti-dsDNA antibody
levels with respect to disease exacerbations in SLE,
using 3 different assays for antibody detection. Disease activity as scored according to a validated disease
activity index showed a good correlation with the
clinician’s impression of the grade of disease activity.
We took blood samples at a minimum of I-month
intervals. The risk of not detecting an increase in
antibody levels within this interval is negligible (17).
Plasma was used instead of serum, to avoid a possible
bias caused by binding of antibodies to DNA from
disrupted blood cells (26).
Our study population comprised a generally unselected group of SLE patients. The cumulative preva-
lence of anti-dsPNA antibody of 74% in the patient
population at the start of the study is comparable with
prevalence previously reported in large p o p
the 60-7W0
ulations (21.27). The low frequency of anti-Sm antibody
(6%) may well be explained by the high preponderance
of white individuals (88%) in our SLE population; whites
have been shown to have a lower prevalence of anti-Sm
Table 8.
Changes in anti-double-stranded DNA (anti-dsDNA) antibody levels and complement C3 and/or C4 levels in 27 systemic
lupus erythematosus disease exacerbations accompanied by positive test results for anti-dsDNA antibody
Significant increase in
C3 and/or C4
No signiiicant increase
in C3 and/or C4
Total
* Assessed by at least
Significant
increase in
anti-dsDNA
an ti body *
No significant
increase in
anti-dsDNA
antibody’
Total
I5
1
16
9
2
II
24
3
27
I of the 3 assays used.
PREDICTIVE VALUE OF ANTI-dsDNA I N SLE
than other races (28). The cumulative prevalences of
neurologic disease and serositis were somewhat lower
than previously reported (27). but the prevalence of renal
disease was comparable.
In this study, the Farr assay, using '2SI-labeled
recombinant dsDNA, proved to be the most sensitive
assay for anti-dsDNA antibody, which is in accordance with a recent report (29). Exacerbations, especially those with renal involvement, occurred more
frequently in the group of patients who were positive
for anti-dsDNA antibodies. which has also been reported previously (17). During 82% of the exacerbations, at least 1 of the anti-dsDNA antibody assays
showed positive results. with the Farr assay yielding
positive results in all these cases.
The Farr assay has been reported to detect
predominantly high-avidity anti-dsDNA antibodies,
the C luciliae assay antibodies of intermediate avidity,
and the ELISA both high- and low-avidity antibodies
(15). High-avidity anti-dsDNA antibody has been presumed by some investigators to be associated with
renal disease and low-avidity anti-dsDNA antibody
with cerebral involvement (15). These data have not
been confirmed by others, however (30). In the present
study, we also did not find an association between
clinical symptoms and anti-dsDNA antibody findings
according to the specific tests. The higher number of
positive results with the Farr assay in comparison with
the C luciliae assay and the ELISA, both throughout
the study period and specifically during exacerbations,
might have been due to the codetection by the Farr
assay of IgM anti-dsDNA antibodies, which are usually associated with mild disease (31). However, this
could be substantiated in only a minority of the cases
in which there were discrepant results for anti-dsDNA
antibody (data not shown).
Twenty-four of the exacerbations accompanied
by a positive test result for anti-dsDNA antibody were
preceded by a significant increase in antibody levels
detected by at least 1 of the assays. These increases
generally occurred many weeks prior to the exacerbation. An exacerbation could be expected particularly
after a rapid increase in anti-dsDNA antibody levels.
These data suggest that the immune process preceding
an exacerbation is biphasic, with an insidious process
of B cell activity followed by a sharp increase in
immune activation. The triggers underlying these processes are still largely unknown.
Comparing the 3 assays with respect to their
ability to predict an exacerbation, the Farr assay had
the highest sensitivity and may be considered the
64 1
assay of choice for predicting exacerbations of SLE.
The ELISA may be an alternative to the Farr assay,
especially for hospitals lacking the equipment to perform the Farr assay. Although our findings confirm
earlier reports on the predictive value of increases in
anti-dsDNA antibodies with respect to exacerbations
of SLE (5,7,16-18), all but 1 of these studies (18) were
performed retrospectively and may therefore be biased. The 1 prospective study was based on measurements of anti-dsDNA antibodies by the Farr assay
only (18). In that study, which dealt only with major
flares, all exacerbations were preceded by an increase
in anti-dsDNA antibodies followed by a sharp decrease just before the exacerbation. The latter observation has never been confirmed by others, however.
In the present study, a moderate decrease in antidsDNA antibody levels just prior to exacerbation,
which may be explained by complex formation between antibody and dsDNA, was found in a limited
number of cases, usually those with renal involvement. The close temporal relationship between
changes in anti-dsDNA antibody levels and ensuing
exacerbations might suggest a pathophysiologic role
for these antibodies.
No correlation was observed, however, between the scores on the disease activity index and the
absolute values of anti-dsDNA antibodies. High, but
usually stable, anti-dsDNA antibody levels were detected without significant disease activity, while major
exacerbations occurred in some cases in the absence
of these antibodies, in accordance with previous reports (13,321. Increases in antibody levels seem to be
of more importance for reflecting disease activity in
SLE than are their absohte levels. One might argue
that qualitative changes in anti-dsDNA, such as the
development of anti-dsDNA antibodies that are crossreactive with glomerular basement membrane constituents (33). may underlie certain disease exacerbations.
However, using 3 qualitatively different assays, we did
not observe qualitative changes in these antibodies
prior to exacerbations. Thus, our data provide evidence against a direct pathophysiologic role of antidsDNA antibodies with respect to disease activity in
SLE, and more strongly indicate that these antibodies
are an epiphenomenon, as part of an immunologic
process leading to an exacerbation of SLE.
Measurement of complement factors is widely
used to assess disease activity in SLE (10.18,20).
Decreased C3 levels (17,34). as well as C4 levels (35).
have been reported to be associated with active (especially renal) disease in SLE. In 1 of the few studies in
TER BORG ET AL
642
which levels of complement were followed prospectively, a decrease in Clq, C3, and C4 was observed
prior t o all but 1 renal exacerbation (18). T h e present
study showed that only half of the exacerbations were
preceded by a significant decrease in C3 and/or C4.
Thus, serial measurements of anti-dsDNA antibodies
had a higher sensitivity for predicting an exacerbation
than did serial measurements of C3 and/or C4. When
anti-dsDNA antibody was assessed serially, serial
measurements of C3 and/or C4 did not contribute t o
predicting an exacerbation.
We conclude that in SLE patients with antidsDNA antibodies, exacerbations usually can be predicted by serial measurement? of these antibodies. A
significant increase in antibody levels frequently precedes an exacerbation by many weeks. Measurement
of these antibodies at one or several time points, in
contrast t o serial testing. however, is of no value for
assessing or predicting disease activity in SLE. Perhaps, in order to prevent or t o mitigate disease exacerbations, SLE patients who demonstrate a significant
increase in anti-dsDNA antibody levels should be
treated with immunosuppressive agents, even in the
absence of disease activity.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
We are indebted to Drs. P. M. Houtman, Department
of Rheumatology, Medisch Centrum Leeuwarden, S. van
der Burg, Department of Rheumatology. Diaconnessenhuis
Groningen, and F. Speerstra, Department of Rheumatology,
Wilhelmina Ziekenhuis Assen for allowing us to study some
of their patients.
13.
14.
15.
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