Journal of Immunological Methods 387 (2013) 312–316

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Technical note

Reconstructing a 3-dimensional image of the results of antinuclear antibody

testing by indirect immunofluorescence
Ryosei Murai a, b, Koji Yamada b, Maki Tanaka a, b, Kageaki Kuribayashi a, b, Daisuke Kobayashi a, b,
Naoki Tsuji a, Naoki Watanabe a, b,⁎
a
Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, Japan
b
Division of Laboratory Medicine, Sapporo Medical University Hospital, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Indirect immunofluorescence anti-nuclear antibody testing (IIF-ANAT) is an essential screening
Received 23 May 2012 tool in the diagnosis of various autoimmune disorders. ANA titer quantification and interpretation
Received in revised form 5 October 2012 of immunofluorescence patterns are determined subjectively, which is problematic.
Accepted 8 October 2012 First, we determined the examination conditions under which IIF-ANAT fluorescence intensities
Available online 13 October 2012
are quantified. Next, IIF-ANAT was performed using homogeneous, discrete speckled, and mixed
serum samples. Images were obtained using Bio Zero BZ-8000, and 3-dimensional images were
Keywords: reconstructed using the BZ analyzer software. In the 2-dimensional analysis, homogeneous ANAs
ANA hid the discrete speckled pattern, resulting in a diagnosis of homogeneous immunofluorescence.
Autoimmune diseases
However, 3-dimensional analysis of the same sample showed discrete speckled-type ANA in the
Laboratory technique
homogeneous background.
3D imaging
This study strengthened the current IIF-ANAT method by providing a new approach to quantify
the fluorescence intensity and enhance the resolution of IIF-ANAT fluorescence patterns.
Reconstructed 3-dimensional imaging of IIF-ANAT can be a powerful tool for routine laboratory
examination.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction laboratory, but ANA titration and discrimination of ANA

Indirect immunofluorescence antinuclear antibody testing
(IIF-ANAT) is essential for diagnosing autoimmune diseases.
According to the guidelines from the National Committee for
Clinical Laboratory Standards, ANA testing should provide ANA
titers and fluorescence patterns (Kavanaugh et al., 2000).
IIF-ANAT has improved since its introduction into the clinical
Abbreviations: 2D, 2-dimensional; 3D, 3-dimensional; IIF-ANAT, indirect
immunofluorescence antinuclear antibody testing; MBL, Medical & Biolog-
ical Laboratories; PBS, phosphate-buffered saline.
⁎ Corresponding author at: Department of Clinical Laboratory Medicine,
Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543,
Japan. Tel.: +81 11 611 2111x3639; fax: +81 11 622 8502.
E-mail address: watanabn@sapmed.ac.jp (N. Watanabe).
fluorescence patterns remain problematic.
ANA titer is clinically important because high ANA titers are
commonly observed in patients with autoimmune diseases. In
addition, some of ANAs, including anti-dsDNA and anti-
centromere antibodies, are reported to correlate with disease
activity (Satoh et al., 2009). ANA titer is determined by
repeating a positive test with serial dilution until the test
becomes negative; the maximum dilution that tests positive is
the reported titer. Because positivity is determined by labo-
ratory technicians, the assay is dependent on subjective
interpretation, resulting in inter-laboratory, inter-observer,
and inter-examination variability (Satoh et al., 2007).
Interpretation of immunofluorescence patterns presents
another problem. Fluorescence patterns develop because of
ANA recognition of specific nuclear antigens; for example, a
speckled pattern is associated with anti-Smith and anti-snRNP
antibodies and a homogeneous pattern is associated with

0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jim.2012.10.004
 R. Murai et al. / Journal of Immunological Methods 387 (2013) 312–316
antibodies to DNA, histones, and nucleosomes. Discriminating
ANA immunofluorescence patterns is important because each
pattern is correlated with a different autoimmune disease,
sometimes in terms of pathogenesis and sometimes in associa-
tion with a disease-specific immune abnormality (Rokeach and
Hoch, 1992; Gonzalez et al., 2004; Migliorini et al., 2005).
Discrimination of immunofluorescence patterns is often difficult
because serum samples frequently contain more than one ANA
type (Sanchez-Guerrero et al., 1996).
To make IIF-ANAT more objective, we addressed these
issues by (i) quantifying ANA immunofluorescence intensities
and by (ii) reconstructing 3-dimensional (3D) images of IIF-
ANAT results to distinguish between ANA immunofluorescence
patterns derived from more than one ANA.
2. Materials and methods
2.1. Reagents and serum samples
ANA reference sera containing homogeneous or discrete
speckled ANAs (HEPASERA-1) and Hep-2 slides were pur-
chased from Medical & Biological Laboratories (MBL; Nagoya,
Japan). The other serum samples were collected from patients
at our hospital who provided their written informed consent.
2.2. Quantification of fluorescence intensity of indirect
immunofluorescence ANA testing
IIF-ANAT was performed according to a previously de-
scribed method (Dellavance et al., 2009). Briefly, Hep-2 slides
were incubated with HEPASERA-1 diluted 1:640 with
phosphate-buffered saline (PBS) for 30 min at room temper-
ature (RT). The slides were washed with PBS for 15 min,
incubated with fluorescein isothiocyanate-conjugated anti-
human IgG antibody (MBL) for 1 h at 37 °C, washed with PBS
for 15 min, and covered with Fluoromount/Plus mounting
reagent (Japan Tanner Corporation, Osaka, Japan). Images were
obtained with Bio Zero BZ-8000 (Keyence, Osaka, Japan). The
nuclear fluorescence intensity (130 μm2/nucleus) was mea-
sured using BZ analyzer software (Keyence) according to the
manufacturer's instructions.
2.3. 3D reconstruction of the results of indirect immunofluores-
cence ANA testing
For 3D reconstruction, IIF-ANAT was performed as described
above, except that HEp-2 slides were created using the cells
obtained from RIKEN BioResource Center (Tsukuba, Japan). The
HEp-2 cells were cultured on 2-chamber polystyrene vessel
tissue culture-treated glass slides (Becton Dickinson, Franklin
Lakes, NJ), washed with PBS, and fixed with methanol/acetone
(1:1) for 3 min at −20 °C. The slides were subjected to IIF-ANAT,
and 3-dimensional images were reconstructed using BZ Analyzer
software (Keyence) according to the manufacturer's instructions.
Briefly, 30–40 2-dimensional images were captured every
0.2 μm in the vertical direction using ×40 oil immersion
objective lens. The images were reconstructed to a 3D image
using real time 3D module of BZ Analyzer software. Fluorescence
patterns were analyzed by rotating the reconstructed 3D image.
313
2.4. Statistical analysis
Statistical analysis was performed in Microsoft Excel®.
Statistical significance was evaluated with the Student's t-test.
3. Results
3.1. Determination of assay condition for measuring fluorescence
intensity
We first examined the cell-to-cell variability in fluores-
cence intensity. We measured the fluorescence intensities of
20 cells from 4 different fields and compared the values. As
shown in Table 1, the average intensity of a single cell in 4
different fields did not differ significantly when evaluated by
Student's t-test, indicating that differences in nuclear protein
expression do not affect the examination, and random fields
can be used for immunofluorescence quantification. Thus, we
measured 20 cells per slide to determine the fluorescence
intensity.
Next, we optimized the exposure time. First, we determined
whether exposure time affects the quantification limit of
IIF-ANAT by testing ANA-negative and -positive samples; a
sample containing homogeneous ANA titer of 1:40 was used as
ANA-positive control. Six exposure times between 0.5 and 3 s
were examined. In all conditions, the ANA-positive sample
yielded greater fluorescence; the shorter the exposure time,
the greater the signal-to-noise ratio (Table 2). Next, we sought
exposure time that can quantify samples containing different
ANA titers. Discrimination of high and low ANA titer samples
was difficult with longer (1.5, 2, and 3 s) and shorter (0.5 s)
exposure times, respectively (Fig. 1). We concluded that
exposure times between 1 and 2/3 s are appropriate to
quantify the fluorescence intensity of IIF-ANAT.
3.2. Three-dimensional reconstruction of the results of indirect
immunofluorescence antinuclear antibody testing using ANA
reference sera
We tried to identify a discrete speckled pattern in a
homogeneous background, since this is often difficult in
routine examination. First, ANA-reference sera were used in
the assay. Two-dimensional (2D) analysis helped discrimi-
nate homogeneous (Fig. 2A) from discrete speckled (Fig. 2B)
patterns created by control serum samples, each containing a
single ANA type; however, when the homogeneous and
discrete speckled sera were combined, homogeneous ANAs
hid the discrete speckled pattern in 2D analysis (Fig. 2C). To
resolve the image in Fig. 2C, the same nucleus in each sample
was subjected to 3D reconstruction. For the control, 3D
images of a single ANA type were analyzed; the homoge-
neous pattern was maintained in the 3D analysis (Fig. 2D)
and discrete speckled serum yielded a comet-like appearance
Table 1
Variability of ANA fluorescence intensities in 4 different fields.
Field 1 2 3 4
Number of cells measured 20 20 20 20
Average intensity 89.45 95.40 90.15 91.65
Standard deviation 18.60 10.34 9.77 11.80
314 R. Murai et al. / Journal of Immunological Methods 387 (2013) 312–316

Table 2 clinical samples. Discrete speckled serum yielded a comet-like

Effect of exposure time on signal to noise ratio (S:N) using ANA-negative appearance (Fig. S1H) and homogeneous pattern was main-
and -positive samples.
tained (Fig. S1N) also in the 3D analysis of clinical samples. We
Exposure time (s) S:N P value next mixed serum samples containing homogeneous and
discrete-speckled ANAs at different ratios and examined
0.5 5.81 1.02 × 10−35
2/3 3.84 1.32 × 10−30 whether discrete speckled ANA can be identified on a diffuse
1 3.07 1.67 × 10−30 background. Blinded analysis was carried out for this purpose;
1.5 2.71 3.08 × 10−35 sets of 2D- and 3D-IIF-ANAT results (Fig. S1) were analyzed by 5
2 2.47 7.34 × 10−36
different laboratory technicians. In 2D analysis, 20%, 40%, 40%,
3 2.07 2.82 × 10−34
(n = 40) 40%, and 20% of the observers could find discrete speckled ANA
in Fig. S1B, C, D, E, and F, respectively. In 3D analysis, 80%, 100%,
Statistical significance between values of signal (nuclear fluorescence intensities
derived from homogeneous ANA) and noise (nuclear fluorescence intensities
100%, 80%, and 20% of the observers could find comet sign in Fig.
derived from ANA-negative serum) were evaluated by Student's t-test and the P S1I, J, K, L, and M, respectively. These results show that 3D
values are shown in the table. analysis is easier than 2D analysis to discriminate fluorescence
patterns of IIF-ANAT.
(Fig. 2E). As shown in Fig. 2F, the sample that was recognized as
homogeneous in 2D analysis (Fig. 2C) appeared as a comet sign
4. Discussion
on a diffuse background in 3D analysis (Fig. 2F), indicating the
presence of homogeneous- and discrete speckled-type ANAs.
We strengthened IIF-ANAT by quantifying immunofluo-
rescence intensities and enhancing the resolution of fluores-
3.3. Three-dimensional reconstruction of the results of indirect cence patterns to promote test standardization.
immunofluorescence antinuclear antibody testing using clinical Quantification of ANA titers currently requires additional
samples examinations, including additional IIF-ANAT using serially
diluted samples or enzyme-linked immunosorbent assays of
Next, we examined whether 3D analysis can discriminate whole nuclear extracts or specific nuclear protein antigens.
discrete speckled pattern in a homogeneous background using By creating reference ANA titers from standard reference

Fig. 1. Effect of exposure time on discrimination of ANA-negative and -positive samples. ANA-negative sample and ANA reference serum (HEPASERA-1) diluted
with PBS are used in the assay. Nuclear fluorescence intensities of 20 cells were measured using different exposure times and the mean ± SEM are shown in the
figure. Statistical significance was evaluated by Student's t-test; *, P b 0.01; **, P > 0.01. A.U., arbitrary units. HEp-2 slides (MBL) were used in this experiment.
 R. Murai et al. / Journal of Immunological Methods 387 (2013) 312–316 315

Fig. 2. 3D reconstruction of IIF-ANAT using ANA reference sera. Indirect immunofluorescence anti-nuclear antibody testing was carried out using cultured HEp-2
cells as described in the Materials and methods. ANA reference sera containing homogeneous ANA (A, D), discrete-speckled ANA (B, E), and the mixture (C, F)
were used in the assay. (A–C) 2-Dimensional analysis, and (D–F) 3-dimensional analysis.

sera, an ANA titer can be determined in a single IIF-ANAT to
minimize examination time and sample volume. This tech-
nique is especially advantageous when sample volume is
limited.
To the best of our knowledge, this is the first study to
demonstrate 3D image reconstruction of IIF-ANAT. The 3D
images in this study were constructed from approximately 40
2D images per nucleus; thus, a single 3D image provides more
information and higher resolution than a single 2D image. It
takes only a few minutes to obtain and reconstruct a single 3D
image. Thus, 3D analysis can be a powerful laboratory
technique for differentiating between homogeneous and
nonhomogeneous ANAs in a single sample. Given that 3D
images are more informative than 2D images, we anticipate a
reduction in the inter-observer variability in determining ANA
fluorescence patterns. This procedure can be used with 2D
analysis in samples with a homogeneous immunofluorescence
pattern.
The results of this study lead us to question the frequency at
which nonhomogeneous ANAs are present in samples previ-
ously diagnosed as homogeneous by 2D analysis. Rigorous 3D
examination of clinical samples diagnosed as having homoge-
neous immunofluorescence patterns by 2D analysis will answer
this question.
One hurdle must be overcome before 3D reconstruction can
be brought into routine examination. In this study, we used
cultured HEp-2 cells instead of commercially available ones to
create slides in 3D analysis because the nuclei of commercial
cells are too thin for the imaging. The expression levels of
nuclear proteins are affected by various factors during cell
culture, resulting in fluctuating ANA titers even in the reference
serum. To avoid this problem, we must develop slides with
HEp-2 cells that have thick nuclei and stable nuclear protein
expression.
Since Hargraves et al. discovered that identification of LE
cell in the bone marrow supports the diagnosis of SLE,
substantial efforts have been made to improve diagnostic
testing for autoimmune diseases (Kavanaugh et al., 2000). This
study provides a future direction for IIF-ANAT.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jim.2012.10.004.
Acknowledgment
All authors have no potential conflict of interest to disclose.
NT and NW were supported by Charitable Trust laboratory
Medicine Research Foundation of Japan.
316 R. Murai et al. / Journal of Immunological Methods 387 (2013) 312–316
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