Document 6456852

ARTHRITIS & RHEUMATISM
Vol. 63, No. 1, January 2011, pp 191–200
DOI 10.1002/art.30084
© 2011, American College of Rheumatology
Pattern on the Antinuclear Antibody–HEp-2 Test Is a Critical
Parameter for Discriminating Antinuclear Antibody–Positive
Healthy Individuals and Patients With
Autoimmune Rheumatic Diseases
Henrique A. Mariz, Emı́lia I. Sato, Silvia H. Barbosa, Silvia H. Rodrigues,
Alessandra Dellavance, and Luis E. C. Andrade
extractable nuclear antigen was present in 1 healthy
individual (anti-SSA/Ro) and in 52 patients with ARDs
(37.7%). None of the 40 reevaluated healthy individuals
developed ARDs, and 29 (72.5%) remained ANA positive. All healthy individuals who became ANA negative
had an ANA titer of 1:80 at baseline.
Conclusion. Our findings suggest that the titer,
and especially the pattern, on the ANA–HEp-2 test
strongly enhances our ability to discriminate ANApositive healthy individuals and patients with ARDs.
Objective. To identify features of antinuclear antibody (ANA)–HEp-2 test results that discriminate
ANA-positive healthy individuals and patients with autoimmune rheumatic diseases (ARDs).
Methods. We sequentially retrieved data on 918
healthy individuals and 153 patients with ARDs after
clinical assessment. ANA-positive healthy individuals
for whom data were available were reevaluated after
3.6–5.0 years. An ANA–HEp-2 test result was considered
positive when a clear ANA pattern was observed at 1:80
dilution in 2 distinct commercial HEp-2 slides by 2
blinded independent observers.
Results. ANAs were present in 118 healthy individuals (12.9%) and 138 patients with ARDs (90.2%).
The ANA titer was higher in patients with ARDs than in
healthy individuals (P < 0.001). The ANA pattern
profile was distinct in the 2 groups. Nuclear homogeneous, nuclear coarse speckled, and nuclear centromeric patterns appeared exclusively in patients with
ARDs. The nuclear dense fine speckled pattern occurred
only in healthy individuals. The most frequent ANA
pattern in both groups was the nuclear fine speckled
pattern, which occurred at lower titer in healthy individuals than in patients with ARDs (P < 0.001). Anti–
Antinuclear antibodies (ANAs) are considered a
hallmark of autoimmune rheumatic diseases (ARDs),
and the indirect immunofluorescence (IIF) assay on
HEp-2 cells (ANA–HEp-2 test) is the standard method
for ANA detection (1). However, a positive ANA–
HEp-2 test result at a 1:80 dilution has been reported in
up to 13.3% of healthy individuals (2–9). Therefore,
ANA–HEp-2 testing outside a proper clinical framework may yield a sizable portion of ANA-positive individuals with no consistent evidence of autoimmune
disease, causing some concern and anxiety in patients
and physicians. This becomes even more crucial with the
perception that autoantibodies may precede the clinical
onset of ARD for many years (10,11). Therefore, it
would be useful to identify intrinsic features of a positive
ANA–HEp-2 test result that would allow us to discriminate subjects with and those without autoimmune disease.
It is generally accepted that individuals without
autoimmune disease would present lower autoantibody
serum levels than those with autoimmune disease (12).
Accordingly, ANA titers are usually thought to be low in
subjects without autoimmune disease and with a positive
ANA–HEp-2 test result. Less emphasis has been given
Supported by the São Paulo Research Foundation (FAPESP
grants 2004/00102-9 and 04/00781-3) and the Brazilian Society of
Rheumatology.
Henrique A. Mariz, MD, Emı́lia I. Sato, MD, PhD, Silvia H.
Barbosa, BSc, Silvia H. Rodrigues, MSc, Alessandra Dellavance, MSc,
Luis E. C. Andrade, MD, PhD: Universidade Federal de São Paulo,
São Paulo, Brazil.
Address correspondence to Luis E. C. Andrade, MD, PhD,
Universidade Federal de São Paulo, Rua Botucatu 740, 3rd Floor,
Rheumatology Division, São Paulo 04023-062, Brazil. E-mail:
luis.andrade@unifesp.br.
Submitted for publication December 7, 2009; accepted in
revised form September 30, 2010.
191
192
MARIZ ET AL
to another intrinsic feature of the ANA–HEp-2 test,
namely, the IIF pattern. The IIF pattern reflects the
topographic distribution of the target autoantigens and
therefore may convey significant information about their
nature. In fact, several IIF patterns have been shown to
bear tight association with certain autoantibody specificities. This is the case for antibodies to proliferating cell
nuclear antigen (13), p80-coilin (14), nuclear mitotic
apparatus 1 (NuMA-1) and NuMA-2/HsEg5 (15,16),
CENP-F (17), DFS70/lens epithelium–derived growth
factor (LEDGF)–P75 (18,19), and DNA topoisomerase
I (20). Considering that not all autoantibody specificities
are tightly associated with autoimmunity, it would be
expected that subjects with and those without autoimmune disease and having a positive ANA–HEp-2 test
result would display different IIF pattern profiles. This
reasoning is supported by recent demonstrations that the
nuclear dense fine speckled pattern, which is correlated
with anti–DFS70/LEDGF-P75 autoantibody (18,19), is
preferentially displayed in samples from individuals with
no evidence of active systemic autoimmune disease
(8,19,21,22).
The aim of this study was to look for features
within positive ANA–HEp-2 test results that differentiate healthy individuals and patients with defined ARD.
Additionally, the prognostic significance of a positive
ANA–HEp-2 test result in apparently healthy individuals was assessed by reevaluating a series of healthy
individuals with positive ANA–HEp-2 test results after
an average time period of 4 years.
PATIENTS AND METHODS
Subjects. A total of 918 healthy individuals (634
women and 284 men, mean ⫾ SD age 32.2 ⫾ 10.3 years [range
18–66 years]) from São Paulo, Curitiba, and Foz do Iguaçu
(large cities in southeast Brazil) were included after being
considered healthy according to a clinical questionnaire that
was administered to investigate current or past ARD, serious
chronic infections, and neoplasia. All subjects fulfilled the
following inclusion criteria: 1) age ⱖ18 years; 2) negative
findings on serologic tests for infection with human immunodeficiency virus, hepatitis B virus, and hepatitis C virus; and
3) no regular use of glucocorticoids, immunosuppressive
agents, or antiinflammatory or antimicrobial drugs. Patients
with ARDs and healthy individuals belonged to the same
ethnic blended Brazilian background, composed mainly of
European, African, and American Indian ancestry. All participants signed the consent form approved by the Ethics Committee at Universidade Federal de São Paulo. From August
2002 to December 2003, all healthy individuals donated 10 ml
of peripheral blood. Serum was separated immediately and
frozen at –20°C until use. The control group included 153
patients with ARDs (87 with systemic lupus erythematosus
[SLE], 45 with systemic sclerosis [SSc], 11 with Sjögren’s
syndrome [SS], and 10 with idiopathic inflammatory myopathy) enrolled according to established criteria for those
diseases (23–26). All samples from patients with ARDs were
selected from patients regularly followed up at the Outpatient
Rheumatology Division.
After a 4-year period, the 66 blood donors living in São
Paulo who had a positive ANA–HEp-2 test result were invited
by letter and/or telephone for clinical reevaluation and for a
new blood draw. Twenty subjects had changed their address
and telephone number and could not be found, and 5 refused
to participate. Forty-one individuals (62.1%) agreed to participate in the reevaluation, and 40 of them agreed to have
additional blood drawn. The mean ⫾ SD time interval between
the 2 samples was 3.9 ⫾ 0.3 years (range 3.6–5.0 years).
ANA–HEp-2 testing and autoantibody determination.
Serum samples from all participants were subjected to the
ANA–HEp-2 test using 2 commercial HEp-2 cell slides (BioRad and Bion). Serum samples were diluted in 0.15M NaCl
and 10 mM phosphate buffered saline (PBS), pH 7.4, and
incubated with HEp-2 cells for 30 minutes at room temperature in a moist chamber. After washing twice in PBS for 10
minutes, cells were incubated with fluorescein isothiocyanate–
conjugated goat anti-human Ig (IgG heavy and light chains) for
another 30 minutes in the dark. After washing twice as before,
slides were assembled with buffered glycerol, pH 9.5, and
coverslips. ANA titer was determined by testing successive
2-fold dilutions of the serum up to 1:5,120. Analysis was
performed by 2 independent expert observers (SHB and
LECA) using an Olympus BX 50 microscope under 400⫻
magnification. In the first round, samples from the 918 healthy
individuals and 153 patients with ARDs were processed in a
nonblinded manner by both independent observers. Samples
were classified as ANA–HEp-2 positive if a well-defined IIF
pattern was identified at 1:80 dilution in both substrates and by
both observers. The requirement for a positive reaction in 2
commercial HEp-2 slides was a precaution to avoid a falsely
high frequency, since some HEp-2 brands are extremely sensitive. The requirement for agreement between both observers
aimed to minimize the influence of subjectivity.
In a second round, all samples from healthy individuals
and patients with ARDs with a positive ANA–HEp-2 test
result were processed simultaneously in a blinded manner for
definition of ANA titer and pattern. Discrepant cases were
segregated in a “waiting line” and were reprocessed for a
second blinded analysis by both observers. The observers were
aware that those samples had been discordant but did not
know the nature of the findings or how each sample had
previously been rated. Most samples became concordant in
the second round reading. There were 2 samples that remained discrepant (borderline positive), and these were considered negative. For the followup comparison, the 2 samples
from each patient (baseline and followup) were tested in the
same assay and were read in a blinded manner by the same
observers.
All sera positive on the ANA–HEp-2 test were
screened for antibodies against extractable nuclear antigens
(ENAs: Sm, U1 RNP, SSA/Ro, SSB/La) by double immunodiffusion against calf spleen extract as the antigen source,
according to the Ouchterlony technique as previously described (27). Secondary standards derived from the Centers for
DISCRIMINATORY CAPACITY OF ANA–HEp-2 TEST PROFILES
Disease Control and Prevention primary standards were used
for identification of the antigen specificity. All samples were
screened for anti–native DNA at a 1:10 dilution by IIF on
Crithidia luciliae as previously described (28). Samples with the
nuclear homogeneous and nuclear quasihomogeneous patterns were processed for antinucleosome and antihistone
antibodies according to the instructions of the manufacturers
(Inova Diagnostics and HUMAN, respectively).
Western blotting. Sera displaying the nuclear fine
speckled and the nuclear dense fine speckled IIF patterns at
titers ⱖ1:320 were analyzed by Western blotting with HEp-2
whole cell extract that was separated by 10% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis and transferred to
nitrocellulose as previously reported. (19). Briefly, serum
samples were tested at 1:50 dilution in 5% skim milk–0.05%
Tween 20 in PBS for 1 hour at room temperature, with
shaking. After two 15-minute washing steps in 0.05% Tween
20–PBS, nitrocellulose strips were incubated with horseradish
peroxidase–labeled goat anti-human IgG antibodies (Bio-Rad)
diluted to the ratio 1:1,500 in 5% skim milk–0.05% Tween 20
in PBS for 1 hour at room temperature in the dark, with
shaking. After washing with 0.05% Tween 20–PBS for 15
minutes and with PBS for 15 minutes, strips were incubated
with chromogenic solution (6 mg 4-chloro-1-naphthol in 2 ml
methanol, 10 ml PBS, and 20 ml 30% H2O2). The reaction was
stopped with water.
Statistical analysis. Categorical variables, such as sex,
age group, and ANA–HEp-2 IIF pattern, were analyzed by the
chi-square test. The Mann-Whitney U test was used to compare ANA–HEp-2 titers between groups. The ANA–HEp-2
titer in samples collected 4 years apart was analyzed by
Wilcoxon’s paired sample test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the accuracy of
the ANA–HEp-2 test in distinguishing healthy individuals and
patients with ARDs. All data were analyzed using Excel
Microsoft 2007 and SPSS for Windows 15.0. P values less than
0.05 were considered significant.
RESULTS
A positive ANA–HEp-2 test result was observed
in 118 healthy individuals (12.9%), with no difference in
193
ANA prevalence according to sex (13.8% in women
versus 10.5% in men; P ⫽ 0.2) or age (P ⫽ 0.43).
However, there was a trend toward higher frequency of
positive ANA–HEp-2 test results in individuals ages
51–66 years (20.8%; n ⫽ 48) compared with individuals
ages 18–30 years (13.4%; n ⫽ 461), individuals ages
31–40 years (11.0%; n ⫽ 236), and individuals ages
41–50 years (12.9%; n ⫽ 154). There was a preponderance of low-titer reactivity among the ANA–positive
healthy individuals. This was in frank contrast to samples
from patients with ARDs, which had a 90.2% frequency
of positive test results and a skewed distribution toward
high-titer ANAs (Figure 1A and Table 1). However, it
should be emphasized that high-titer ANA reactivity was
also observed in a sizable fraction of healthy individuals
(Table 1). The frequency of positive ANA–HEp-2 test
results was 96.5% in SLE, 88.8% in SSc, 70% in SS, and
63.6% in polymyositis/dermatomyositis.
ROC curve analysis showed good performance of
the ANA–HEp-2 test for discriminating patients with
ARDs and healthy individuals, with an area under the
curve of 0.923 (P ⬍ 0.001) (Figure 1C). As expected, the
ability to discriminate patients with ARDs and healthy
individuals varied according to the dilution cutoff level.
At a screening dilution of 1:80, the ANA–HEp-2 test
had sensitivity of 90.2% and specificity of 87.1% with a
high (98.1%) negative predictive value (NPV). At a
dilution of 1:160 (recommended by the ANA Subcommittee of the International Union of Immunological
Societies Standardization Committee [6]), the sensitivity
was 83.7% and the specificity was 93.0%. At the 1:5,120
dilution cutoff level, the test had sensitivity of 44.4% and
specificity of 99.2% with a moderate (90.6%) positive
predictive value (PPV). At an intermediate 1:1,280
dilution cutoff level, the test had sensitivity of 65.4% and
Table 1. Distribution of ANA-positive healthy individuals and ANA-positive patients with ARDs according to the titer and prevalent patterns on
the ANA–HEp-2 test*
P†
Healthy
individuals
(n ⫽ 54)
Patients with
ARDs
(n ⫽ 58)
P†
Nuclear dense fine
speckled
pattern (healthy
individuals only)
(n ⫽ 39)
⬍0.001
0.259
0.477
⬍0.001
0.002
0.507
⬍0.001
33 (61.1)
5 (9.3)
8 (14.8)
5 (9.3)
0 (0)
1 (1.9)
2 (3.7)
9 (15.5)
5 (8.6)
12 (20.7)
0 (0)
11 (19.0)
1 (1.7)
20 (34.5)
⬍0.001
0.831
0.573
0.056
0.02
0.515
⬍0.001
10 (25.6)
1 (2.6)
4 (10.3)
15 (38.5)
5 (12.8)
1 (2.6)
3 (7.7)
All patterns
Titer
Healthy
individuals
(n ⫽ 118)
Patients with
ARDs
(n ⫽ 138)
1:80
1:160
1:320
1:640
1:1,280
1:2,560
ⱖ1:5,120
54 (45.8)
9 (7.6)
15 (12.7)
21 (17.8)
9 (7.6)
3 (2.5)
7 (5.9)
10 (7.2)
5 (3.6)
23 (16.7)
0 (0)
31 (22.5)
1 (0.7)
68 (49.3)
Nuclear fine speckled pattern
* Values are the number (%) of subjects. ANA ⫽ antinuclear antibody; ARDs ⫽ autoimmune rheumatic diseases.
† By chi-square test.
194
MARIZ ET AL
Figure 1. A and B, Distribution of antinuclear antibody (ANA)–positive healthy individuals (HI) and patients with autoimmune rheumatic disease
(ARD) according to titer for all patterns combined on the ANA–HEp-2 test (A) and for the nuclear fine speckled pattern on the ANA–HEp-2 test
(B). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and
the lines outside the boxes represent the 10th and 90th percentiles. ⴙ indicates outliers. C, Receiver operating characteristic (ROC) curve analysis
of the ANA–HEp-2 test for discriminating 918 healthy individuals and 153 patients with ARDs. The area under the curve ⫽ 0.923 (P ⬍ 0.001).
specificity of 97.9% with a high (94.6%) NPV and a
moderate (84.9%) PPV.
The ANA pattern distribution among the 118
ANA-positive healthy individuals and 138 ANA-positive
Table 2. Distribution of 118 ANA-positive healthy individuals and 138 ANA-positive patients with
ARDs according to the pattern on the ANA–HEp-2 test*
Pattern on ANA–HEp-2 test
ANA-positive healthy
individuals†
ANA-positive patients
with ARDs†
P‡
Nuclear fine speckled
Nuclear dense fine speckled
Nuclear coarse speckled
Nuclear homogeneous
Nuclear centromeric
Nuclear quasihomogeneous speckled
Nucleolar
Cytoplasmic
Other
54 (45.8)
39 (33.1)
0 (0)
0 (0)
0 (0)
5 (4.2)
8 (6.8)
12 (10.2)§
8 (6.8)
58 (42.0)
0 (0)
36 (26.1)
10 (7.2)
11 (8.0)
19 (13.8)
18 (13.0)
5 (3.6)¶
7 (5.1)
0.636
⬍0.001
⬍0.001
0.008
0.005
0.017
0.148
0.065
0.604
* Values are the number (%) of subjects. See Table 1 for definitions.
† Total percentage in each column is ⬎100% because some samples had ⬎1 pattern.
‡ By chi-square test.
§ Exclusively cytoplasmic staining.
¶ One patient with exclusively cytoplasmic staining and 4 patients with additional patterns (2 with
nucleolar pattern, 1 with nuclear fine speckled pattern, and 1 with nuclear coarse speckled pattern).
DISCRIMINATORY CAPACITY OF ANA–HEp-2 TEST PROFILES
patients with ARDs is shown in Table 2. It should be
noted that some samples from both groups had more
than 1 pattern (multipattern). The morphologic characteristics of the most relevant nuclear patterns are shown
in Figure 2. The nuclear homogeneous pattern was
characterized by smooth texture staining of the whole
interphase nucleus and bright hyaline staining of the
metaphase chromatin plate. The nuclear quasihomogeneous pattern was defined by an extremely fine grainy
texture staining of the whole interphase nucleus with
similar staining of the metaphase chromatin plate. The
nuclear fine speckled pattern was defined by a variably
grainy texture staining of the interphase nucleus and no
staining of the metaphase chromatin plate, usually sparing the nucleolar domains. The nuclear coarse speckled
pattern was characterized by several larger and brighter
speckles standing out against a coarse grainy texture
staining of the whole interphase nucleus with no staining
of the metaphase chromatin plate. The nuclear dense
fine speckled pattern was characterized by tightly packed
speckled staining of the whole interphase nucleus with
heterogeneity in brightness of the several speckles and
similar staining pattern in the metaphase chromatin
plate. The centromere pattern was characterized by
numerous discrete speckles spread throughout the interphase nucleus and aligned in an orderly manner at the
metaphase chromatin plate.
The most frequent ANA patterns in healthy
individuals were the nuclear fine speckled pattern, which
was present in 54 subjects (45.8% of ANA-positive
healthy individuals), and the nuclear dense fine speckled
pattern, which was observed in 39 subjects (33.1% of
ANA-positive healthy individuals). Interestingly, the frequency of the nuclear fine speckled pattern was stable
across 4 age strata (43.5% in those ages 18–30 years,
38.5% in those ages 31–40 years, 50% in those ages
41–50 years, and 50% in those ages 51–65 years), but the
frequency of the nuclear dense fine speckled pattern
dropped significantly in healthy individuals ⬎50 years of
age (32.3%, 42.3%, 35.5%, and 10%, respectively). The
most frequent ANA pattern in patients with ARDs was
also the nuclear fine speckled pattern, which was present
in 58 subjects (42.0% of ANA-positive patients with
ARDs), followed by the nuclear coarse speckled pattern,
which was observed in 36 patients (26.1% of ANApositive patients with ARDs). No healthy individual had
the nuclear coarse speckled pattern, and no patient with
ARD had the nuclear dense fine speckled pattern. Other
patterns exclusively observed in patients with ARDs
were the nuclear homogeneous (7.2%), nuclear centro-
195
Figure 2. Morphologic characteristics of the most relevant nuclear patterns found on indirect immunofluorescence assay on HEp-2 cells,
performed on samples from healthy individuals and patients with autoimmune rheumatic diseases. Patterns are as follows: A, homogeneous; B,
quasihomogeneous; C, nuclear fine speckled; D, nuclear coarse speckled;
E, nuclear dense fine speckled; and F, nuclear centromeric. Sera were
diluted 1:80. Bars ⫽ 5 ␮m in A and B; 13 ␮m in C–F.
196
meric (8.0%), and cytoplasmic dense fine speckled
(2.8%) patterns.
The nuclear quasihomogeneous pattern (Figure
2) was predominantly observed in samples from patients
with ARDs. This pattern is distinct from the nuclear
homogeneous pattern in that its texture is finely grainy
as opposed to the plain smooth texture of the homogeneous pattern. This difference is particularly appreciated
at the chromatin mass in mitotic cells, where the homogeneous pattern assumes a hyaline appearance, in contrast to the quasihomogeneous pattern, which keeps its
finely grainy texture. In addition, samples with the
quasihomogeneous pattern had a different autoantibody
profile from the homogeneous pattern. No quasihomogeneous pattern sample from patients with ARDs or
from healthy individuals was reactive against native
DNA. Furthermore, no quasihomogeneous pattern sample from healthy individuals reacted with nucleosome or
histone, and only 60% of the quasihomogeneous samples from SLE patients reacted with nucleosome and/or
histone. In contrast, all 10 samples with the homogeneous pattern reacted with components of the chromatin
system. In fact, all reacted with nucleosome: 3 reacted
only with nucleosome, 4 reacted with nucleosome and
histone, 1 reacted with nucleosome and native DNA,
and 2 reacted with nucleosome, histone, and native
DNA.
Since the nuclear fine speckled pattern was the
most frequently observed pattern in healthy individuals
and in patients with ARDs, we decided to analyze the
titer of the nuclear fine speckled pattern in both groups.
Healthy individuals presented a significantly lower titer
nuclear fine speckled pattern than did patients with
ARDs (P ⬍ 0.01) (Figure 1B). On the other hand, the
nuclear dense fine speckled pattern was observed exclusively in healthy individuals and mostly at high titer
(Table 1). In fact, in healthy individuals, samples with
the nuclear dense fine speckled pattern had higher titers
than did samples with the nuclear fine speckled pattern.
The nuclear fine speckled pattern occurred at a titer
ⱕ1:320 in 46 of the 54 healthy individuals with that
pattern (85.2%), and the nuclear dense fine speckled
pattern occurred at a titer ⱖ1:640 in 24 of the 39 healthy
individuals with that pattern (61.5%) (P ⬍ 0.001). The
antigenic specificity of the 2 ANA patterns observed
most frequently in healthy individuals, the nuclear fine
speckled pattern and the nuclear dense fine speckled
pattern, was investigated by Western blotting. As expected, all samples displaying the nuclear dense fine
speckled pattern recognized a polypeptide with an estimated molecular weight of 75 kd (18,19,22) (Figure 3).
MARIZ ET AL
On the other hand, no specific band was observed
among the samples displaying the nuclear fine speckled
pattern (Figure 3).
Another distinctive feature differentiating ANApositive samples from healthy individuals and ANApositive samples from patients with ARDs was the
presence of anti-ENA antibodies in the samples from
patients with ARDs. Fifty-two samples from ANApositive patients with ARDs (37.7%) had anti-ENA
reactivity (35 with anti-SSA/Ro [25.4%], 13 with antiSSB/La [9.4%], 3 with anti-Sm [2.2%], and 20 with
anti–U1 RNP [14.5%]). As expected, some samples had
more than 1 antibody specificity. The frequency of
anti-ENA antibodies in each ARD was 42.5% in SLE,
13.3% in SSc, 50% in SS, and 36.3% in polymyositis/
dermatomyositis. In contrast, only 1 ANA-positive
healthy individual (0.85%) had anti-ENA antibody (antiSSA/Ro). As expected, anti-DNA antibody was absent in
healthy individuals and exclusively found in 12 SLE
patients.
We were able to reassess 41 of the ANA-positive
healthy individuals 3.6–5 years after the initial evalua-
Figure 3. Western blot analysis of antibody specificity associated with
nuclear dense fine speckled pattern and nuclear fine speckled pattern
in sera from healthy individuals. Whole HEp-2 cell extract was
separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with sera diluted at a ratio of 1:50. Lanes 1–18,
Samples that showed the nuclear dense fine speckled pattern on the
antinuclear antibody (ANA)–HEp-2 test; lane 19, anti-SSA/Ro control; lanes 20–29, samples that showed the nuclear fine speckled
pattern on the ANA–HEp-2 test; lane 30, negative control. Arrows
indicate proteins migrating with mobility compatible with 52 kd and
75 kd.
DISCRIMINATORY CAPACITY OF ANA–HEp-2 TEST PROFILES
Figure 4. Longitudinal findings from antinuclear antibody (ANA)–
HEp-2 testing in 40 ANA-positive healthy individuals at the first
evaluation and at reassessment within an average 4-year interval. Solid
lines indicate stable or increased titer; dashed lines indicate decreased
titer; Ø indicates negative.
tion. None had symptoms suggestive of an ARD, except
for 1 woman who presented with mild hand pallor after
exposure to cold; however, the clinical examination and
nailfold capillaroscopy performed at the reassessment
yielded normal findings. Among the 40 ANA-positive
healthy individuals who agreed to donate blood at the
reassessment, there was slight variation in titer in both
directions, as shown in Figure 4, with no significant
variation in ANA pattern between paired samples.
Twenty-nine healthy individuals (72.5%) remained
ANA positive without significant variation in ANA titer
(P ⫽ 0.795). All 11 ANA-positive healthy individuals
who became ANA negative had significantly lower initial
titer (1:80) than those who remained ANA positive
(P ⫽ 0.002).
DISCUSSION
Taking into consideration that the ANA–HEp-2
test yields positive results in a sizable portion of the
general population, it is critical for the accurate interpretation of the test that we uncover differential characteristics of the results in healthy individuals and in
patients with ARDs. In the present study, we were able
to identify distinctive ANA profiles in healthy individuals and patients with ARDs by analyzing samples from
197
918 healthy individuals and 153 patients with ARDs.
Positive ANA–HEp-2 test results in healthy individuals
involved predominantly low-to-moderate titers, and the
most common ANA patterns were the nuclear fine
speckled pattern and the nuclear dense fine speckled
pattern. In contrast, positive ANA–HEp-2 test results in
patients with ARDs involved predominantly moderateto-high titers and exhibited a distinct ANA profile
characterized mainly by the absence of the nuclear dense
fine speckled pattern and the exclusive occurrence of the
nuclear coarse speckled, nuclear homogeneous, and
nuclear centromeric patterns.
The frequency of positive ANA–HEp-2 test results in the present series of healthy individuals (12.9%)
was within the range established by previous studies
(3,6–9). Several studies reported in the literature found
a higher prevalence of ANAs in healthy women than in
healthy men (3–5,7,29). In fact, sex-related factors, such
as the hormone profile, fetal microchimerism, and strategic genes located at sex chromosomes, may be involved
in the female predominance in autoimmune diseases
(30). Interestingly, we could not find any difference in
the frequency of positive ANA–HEp-2 test results between women and men despite the substantial sample
size for both sexes. We observed a higher prevalence of
ANAs in elderly healthy individuals, as shown by previous studies in nonagenarians and centenarians (31,32).
However, the difference did not reach statistical significance, which may be related to the limited number of
healthy individuals older than 65 years in the present
study.
Our study indicates that ANA titers of 1:80 and
1:160 are suitable dilutions for screening for ARDs. The
high NPV for ARD diagnosis found in the present series
is consistent with findings of Slater et al, who evaluated
1,010 consecutive samples in which ANA–HEp-2 testing
was ordered (33). These results indicate that a negative
ANA–HEp-2 test result at 1:80 screening dilution is
unlikely in patients with ARDs, especially those with
SLE, SSc, and SS. The observed predominance of lowtiter ANAs (ⱕ1:160) among healthy individuals is also
consistent with previous reports (2,6–8). On the other
hand, we did find a substantial fraction of healthy
individuals with a moderate titer (1:320 and 1:640) and a
smaller fraction with a high titer (ⱖ1:1,280). ROC curve
analysis and traditional diagnostic performance parameters (sensitivity, specificity, and predictive values)
showed that in a scenario of indiscriminate requests for
ANA–HEp-2 tests, there was relevant gain in specificity
and PPV for discriminating healthy individuals and
patients with ARDs up to a titer of 1:1,280. Therefore,
198
although the 1:80 and 1:160 dilutions are appropriate for
screening, it is advisable that clinical laboratories proceed with testing up to a 1:1,280 dilution. Altogether,
these findings suggest that the titer of the ANA–HEp-2
reaction is a relevant but limited parameter in discriminating ANA-positive healthy individuals and patients
with ARDs.
This limitation may be partially overcome by the
analysis of the pattern on the ANA–HEp-2 test. In fact,
our findings suggest that this pattern is a critical parameter for the interpretation of a positive ANA–HEp-2
test result. In the present study, the pattern on the
ANA–HEp-2 test was comparatively scrutinized in samples from patients with ARDs and healthy individuals.
Some patterns, such as the nuclear coarse speckled,
nuclear homogeneous, and nuclear centromeric patterns, were observed solely in samples from patients with
ARDs. This is probably because these patterns are
typically associated with disease-restricted autoantibodies, such as anti-Sm/anti–U1 RNP, anti–double-stranded
DNA/antinucleosome, and anticentromere antibodies,
respectively (34–37). On the other hand, the nuclear
dense fine speckled pattern was not observed in any
sample from a patient with an ARD, but was found
exclusively in samples from healthy individuals. Confirming previous reports, all tested samples with a nuclear dense fine speckled pattern in this series reacted
with a 75-kd protein compatible with the LEDGF/p75
antigen (8,18,19).
There are not many studies focusing on the
pattern on the ANA–HEp-2 test in healthy individuals,
and the majority of them were performed before the
characterization of the nuclear dense fine speckled
pattern (2,4,5,38–40). Given the high prevalence of the
nuclear dense fine speckled pattern among healthy
individuals, it is reasonable to assume that this pattern
might have been mistaken for some other pattern in
those studies. In the present series, the nuclear dense
fine speckled pattern was the second most frequent
ANA pattern among healthy individuals. Of special
importance, the majority of samples with this pattern
had a high ANA titer, which might mislead the clinical
judgment of physicians not acquainted with the ANA–
HEp-2 test. Similar findings have been previously reported by Watanabe et al among 597 healthy hospital
workers in Japan (8) and by our group in a study with
13,641 samples determined to be positive by an ANA–
HEp-2 test performed in a Brazilian general clinical
laboratory that was certified on-site by the US College of
American Pathologists (19). However, it should be noted
that the nuclear dense fine speckled pattern and anti-
MARIZ ET AL
LEDGF/p75 antibodies have also been observed in
patients with a variety of unrelated conditions, such as
interstitial cystitis, asthma, Hashimoto thyroiditis, and
atopic dermatitis (18,19,21,22).
The combined analysis of pattern on the ANA–
HEp-2 test and ANA titer was especially relevant for
tests showing the nuclear fine speckled pattern. This was
the most frequent ANA pattern in healthy individuals
and in patients with ARDs, and our results indicated
that a low-titer nuclear fine speckled pattern was preferentially observed in healthy individuals, whereas a
high-titer nuclear fine speckled pattern was preferentially observed in patients with ARDs. Interestingly, the
samples with a nuclear fine speckled pattern from
healthy individuals failed to elicit any reactivity on
Western blotting, and this may indicate some peculiar
features of the autoantigen(s) associated with this pattern in healthy subjects. One other useful element in the
interpretation of a positive ANA–HEp-2 test result with
the nuclear fine speckled pattern is the presence of
antibodies to ENAs. In the present series, antibodies to
SSA/Ro and SSB/La were largely restricted to samples
with the nuclear fine speckled pattern from patients with
ARDs. In contrast, only 1 healthy individual had antiSSA/Ro antibody. In addition, antibodies to U1 RNP
and Sm, which are usually associated with the nuclear
coarse speckled pattern, were observed solely in samples
from patients with ARDs.
At the followup visit, none of the 41 ANApositive healthy individuals had developed signs and
symptoms of ARDs, and 72.5% of the 40 individuals who
agreed to donate blood at the reassessment remained
ANA positive after a mean followup period of 3.9 years.
Our findings also indicate that those with low-titer
ANAs on the ANA–HEp-2 test are the ones with the
highest probability of future normalization of the result.
The extended persistence of ANA positivity in healthy
individuals has been previously reported by Xavier et al,
who found that 78% of 23 elderly healthy individuals
continued to have a positive ANA–HEp-2 test result
after an average followup period of 4 years (40). In
another study, Dinser et al also found a low PPV for a
positive ANA–HEp-2 test result at titers ⬎1:320 for the
development of ARD after a 3-year followup period
(41). Therefore, a positive ANA–HEp-2 test result in
apparently healthy individuals seems to be a sustained
phenomenon in ⬃75% of the cases and appears to have
a low PPV for the development of a future ARD.
However, this assumption should be made with caution,
because the followup in the present study included only
samples (from healthy individuals) with patterns on the
DISCRIMINATORY CAPACITY OF ANA–HEp-2 TEST PROFILES
ANA–HEp-2 test shown not to be associated with ARD.
In the unusual scenario of a pattern on the ANA–HEp-2
test associated with ARD (e.g., nuclear homogeneous or
nuclear coarse speckled) in an apparently healthy individual, a careful followup is advised, since it has been
demonstrated that specific autoantibodies may antedate
signs and symptoms of SLE for years (10).
In conclusion, the ANA–HEp-2 assay offered
distinctive titer and pattern profiles for ANA-positive
healthy individuals and for patients with ARDs. The
ANA pattern on the test seemed to be more consistent
than the ANA titer for discriminating ANA-positive
healthy individuals and patients with ARDs. The nuclear
dense fine speckled pattern was observed exclusively in
healthy individuals and tended to appear at high titer
and to be stable over the years. Some other patterns on
the ANA–HEp-2 test (nuclear coarse speckled, nuclear
homogeneous, nuclear centromeric) were restricted to
patients with ARDs. The nuclear fine speckled pattern
was equally frequent in both groups and showed different titer distributions in healthy individuals (predominantly low titer) and patients with ARDs (predominantly high titer). ANA-positive healthy individuals
tended to keep ANA reactivity but did not develop
evidence of ARD after a 4-year followup period. Given
the critical role of the pattern on the ANA–HEp-2 test,
future efforts shall address the reproducibility of ANA–
HEp-2 test interpretation among different ANA experts
and among different ANA–HEp-2 test slide brands.
199
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
We acknowledge Gilda Ferreira, MD, PhD, for providing some of the serum samples for the group of patients
with ARDs, Valdecir Marvoulle, PhD, for expert advice with
statistics, and Edward Chan, PhD, and Eng M. Tan, MD, for
helpful comments.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Andrade had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Mariz, Sato, Andrade.
Acquisition of data. Mariz, Barbosa, Rodrigues, Dellavance, Andrade.
Analysis and interpretation of data. Mariz, Sato, Dellavance, Andrade.
13.
14.
15.
16.
17.
18.
REFERENCES
19.
1. Solomon DH, Kavanaugh AJ, Schur PH, and the American
College of Rheumatology Ad Hoc Committee on Immunologic
Testing Guidelines. Evidence-based guidelines for the use of
20.
immunologic tests: antinuclear antibody testing. Arthritis Rheum
2002;47:434–44.
Fritzler MJ, Pauls JD, Kinsella TD, Bowen TJ. Antinuclear,
anticytoplasmic, and anti-Sjögren’s syndrome antigen A (SS-A/
Ro) antibodies in female blood donors. Clin Immunol Immunopathol 1985;36:120–8.
Vrethem M, Skogh T, Berlin G, Ernerudh J. Autoantibodies
versus clinical symptoms in blood donors. J Rheumatol 1992;199:
1919–21.
De Vlam K, De Keyser F, Verbruggen M, Vandenbossche M,
Vanneuville B, D’Haese D, et al. Detection and identification of
antinuclear autoantibodies in the serum of normal blood donors.
Clin Exp Rheumatol 1993;11:393–7.
Forslid J, Heigl Z, Jonsson J, Scheynius A. The prevalence of
antinuclear antibodies in healthy young persons and adults, comparing rat liver tissue sections with HEp-2 cells as antigen substrate. Clin Exp Rheumatol 1994;12:137–41.
Tan EM, Feltkamp TE, Smolen JS, Butcher B, Dawkins R, Fritzler
MJ, et al. Range of antinuclear antibodies in “healthy” individuals.
Arthritis Rheum 1997;40:1601–11.
Fernandez SA, Lobo AZ, Prado de Oliveira ZN, Fukumori LM,
Perigo AM, Rivitti EA. Prevalence of antinuclear autoantibodies
in the serum of normal blood donors. Rev Hosp Clin Fac Med Sao
Paulo 2003;58:315–9.
Watanabe A, Kodera M, Sugiura K, Usuda T, Tan EM, Takasaki
Y, et al. Anti-DFS70 antibodies in 597 healthy hospital workers.
Arthritis Rheum 2004;50:892–900.
Al Jabri AA, Al Buloshi MS. Anticardiolipin and antinuclear
antibodies in the adult healthy Omani individuals. Saudi Med J
2004;25:313–7.
Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis
GJ, James JA, et al. Development of autoantibodies before the
clinical onset of systemic lupus erythematosus. N Engl J Med
2003;349:1526–33.
Nielen MM, van Schaardenburg D, Reesink HW, van de Stadt RJ,
van der Horst-Bruinsma IE, de Koning MH, et al. Specific
autoantibodies precede the symptoms of rheumatoid arthritis: a
study of serial measurements in blood donors. Arthritis Rheum
2004;50:380–6.
Lacroix-Desmazes S, Kaveri SV, Mouthon L, Ayouba A,
Malanchere E, Coutinho A, et al. Self-reactive antibodies (natural
autoantibodies) in healthy individuals. J Immunol Methods 1998;
216:117–37.
Takasaki Y, Fishwild D, Tan EM. Characterization of proliferating
cell nuclear antigen recognized by autoantibodies in lupus sera. J
Exp Med 1984;159:981–92.
Andrade LE, Chan EK, Raska I, Peebles CL, Roos G, Tan EM.
Human autoantibody to a novel protein of the nuclear coiled body:
immunological characterization and cDNA cloning of p80-coilin. J
Exp Med 1991;173:1407–19.
Andrade LE, Chan EK, Peebles CL, Tan EM. Two major
autoantigen–antibody systems of the mitotic spindle apparatus.
Arthritis Rheum 1996;39:1643–53.
Whitehead CM, Winkfein RJ, Fritzler MJ, Rattner JB. The
spindle kinesin-like protein HsEg5 is an autoantigen in systemic
lupus erythematosus. Arthritis Rheum 1996;39:1635–42.
Casiano CA, Humbel RL, Peebles C, Covini G, Tan EM. Autoimmunity to the cell cycle-dependent centromere protein p330d/
CENP-F in disorders associated with cell proliferation. J Autoimmun 1995;8:575–86.
Ochs RL, Stein TW Jr, Peebles CL, Gittes RF, Tan EM. Autoantibodies in interstitial cystitis. J Urol 1994;151:587–92.
Dellavance A, Viana VS, Leon EP, Bonfa ES, Andrade LE, Leser
PG. The clinical spectrum of antinuclear antibodies associated
with the nuclear dense fine speckled immunofluorescence pattern.
J Rheumatol 2005;32:2144–9.
Dellavance A, Gallindo C, Soares MG, da Silva NP, Mortara RA,
200
21.
22.
23.
24.
25.
26.
27.
28.
29.
MARIZ ET AL
Andrade LE. Redefining the Scl-70 indirect immunofluorescence
pattern: autoantibodies to DNA topoisomerase I yield a specific
compound immunofluorescence pattern. Rheumatology (Oxford)
2009;48:632–7.
Ayaki M, Ohoguro N, Azuma N, Majima Y, Yata K, Ibaraki N, et
al. Detection of cytotoxic anti-LEDGF autoantibodies in atopic
dermatitis. Autoimmunity 2002;35:319–27.
Ochs RL, Muro Y, Si Y, Ge H, Chan EK, Tan EM. Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis
and other conditions. J Allergy Clin Immunol 2000;105:1211–20.
Hochberg MC, for the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology. Updating the
American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis
Rheum 1997;40:1725.
Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis
(scleroderma). Arthritis Rheum 1980;23:581–90.
Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al, and the European Study Group on
Classification Criteria for Sjögren’s Syndrome. Classification criteria for Sjögren’s syndrome: a revised version of the European
criteria proposed by the American–European Consensus Group.
Ann Rheum Dis 2002;61:554–8.
Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two
parts). N Engl J Med 1975;292:344–7.
Nakamura RM, Peebles CL, Rubin RL, Molden DP, Tan EM.
Autoantibodies to nuclear antigens (ANA): advances in laboratory
tests and significance in systemic rheumatic diseases. 2nd ed.
Chicago: American Society of Clinical Pathology Press; 1985. p.
109–40.
Aarden LA, de Groot ER, Feltkamp TE. Immunology of DNA.
III. Crithidia luciliae, a simple substrate for the determination of
anti-dsDNA with the immunofluorescence technique. Ann N Y
Acad Sci 1975;254:505–15.
Hayashi N, Koshiba M, Nishimura K, Sugiyama D, Nakamura T,
Morinobu S, et al. Prevalence of disease-specific antinuclear
antibodies in general population: estimates from annual physical
examinations of residents of a small town over a 5-year period.
Mod Rheumatol 2008;18:153–60.
30. Lleo A, Battezzati PM, Selmi C, Gershi ME, Podda M. Is
autoimmunity a matter of sex? Autoimmun Rev 2008;7:626–30.
31. Candore G, Di Lorenzo G, Mansueto P, Melluso M, Frada G, Li
Vecchi M, et al. Prevalence of organ-specific and non organspecific autoantibodies in healthy centenarians. Mech Ageing Dev
1997;94:183–90.
32. Hurme M, Korkki S, Lehtimaki T, Karhunen PJ, Jylha M,
Hervonen A, et al. Autoimmunity and longevity: presence of
antinuclear antibodies is not associated with the rate of inflammation or mortality in nonagenarians. Mech Ageing Dev 2007;128:
407–8.
33. Slater CA, Davis RB, Shmerling RH. Antinuclear antibody testing:
a study of clinical utility. Arch Intern Med 1996;156:1421–5.
34. Tan EM, Chan EK. Molecular biology of autoantigens and new
insights into autoimmunity. Clin Investig 1993;71:327–30.
35. Humbel RL. Detection of antinuclear antibodies by immunofluorescence. In: van Venrooij WJ, Maini RN, editors. Manual of
biological markers of disease. Dordrecht (The Netherlands): Kluwer Academic Publishers; 1993. A2: p. 1–16.
36. Wiik AS. Anti-nuclear autoantibodies: clinical utility for diagnosis,
prognosis, monitoring, and planning of treatment strategy in
systemic immunoinflammatory diseases. Scand J Rheumatol 2005;
34:260–8.
37. Lyons R, Narain S, Nichols C, Satoh M, Reeves WH. Effective use
of autoantibody tests in the diagnosis of systemic autoimmune
disease. Ann N Y Acad Sci 2005;1050:217–28.
38. Manoussakis MN, Tzioufas AG, Silis MP, Pange PJ, Goudevenos
J, Moutsopoulos HM. High prevalence of anti-cardiolipin and
other autoantibodies in a healthy elderly population. Clin Exp
Immunol 1987;69:557–65.
39. Ruffati A, Rossi L, Calligaro A, Del Ross T, Lagni M, Marson P,
et al. Autoantibodies of systemic rheumatic diseases in the healthy
elderly. Gerontology 1990;36:104–11.
40. Xavier RM, Yamauchi Y, Nakamura M, Tanigawa Y, Ishikura H,
Tsunematsu T, et al. Antinuclear antibodies in healthy aging
people: a prospective study. Mech Ageing Dev 1995;78:145–54.
41. Dinser R, Braun A, Jendro MC, Engel A. Increased titres of
anti-nuclear antibodies do not predict the development of associated disease in the absence of initial suggestive signs and symptoms. Scand J Rheumatol 2007;36:448–51.