AUTREV-01214; No of Pages 4
Autoimmunity Reviews xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Autoimmunity Reviews
journal homepage: www.elsevier.com/locate/autrev
Review
A comparative study on the reliability of an automated system for the evaluation of
cell-based indirect immunofluorescence
Alessandra Melegari a,⁎, Chiara Bonaguri b, Annalisa Russo b, Battistelli Luisita b,
Tommaso Trenti a, Giuseppe Lippi b
a
b
Diagnostic Laboratory Department, NOSAE Hospital, Modena, Italy
Diagnostic Laboratory Department, Parma Hospital, Parma, Italy
a r t i c l e
i n f o
a b s t r a c t
Background: Automated interpretations systems for anti-nuclear antibody (ANA), anti-double stranded DNA
antibody (dsDNAab), and anti-neutrophil cytoplasmic antibody (ANCA) assessment by indirect
immunofluorescence (IIF) have been recently introduced. The aim of this study was to compare the
diagnostic performance of the automated IIF reading system AKLIDES with both traditional visual
interpretation of IIF by laboratory experts and confirmatory tests.
Methods: Visual and automated autoantibody interpretations of IIF findings using AKLIDES pattern recognition algorithms were performed for ANA on HEp-2 cells (n = 182), dsDNAab on Crithidia luciliae (n = 44)
and ANCA on human neutrophils (n = 46). All serum samples tested by IIF for ANCA and dsDNAab were
also assessed with the corresponding enzyme-linked immunosorbent assays (ELISAs). Out of the 182 sera
tested for ANA by IIF, 116 were also assessed for antibodies to extractable nuclear antigens (ENA) by ELISA
and dot immunoassay (DIA).
Results: ANA testing showed an excellent agreement between visual and AKLIDES reading (98.9%). The overall agreement of dsDNAab testing on C. luciliae substrate slides was 91.0%, whereas ANCA showed a concordance of 89.1%. There was a remarkable agreement of AKLIDES findings for dsDNAab with confirmatory tests.
Conclusion: Visual and automated interpretations of IIF findings for ANA, ANCA, and dsDNAab demonstrated a
good agreement when assessing patients with suspected autoimmune diseases. Automated interpretation
systems such AKLIDES may improve laboratory efficiency and support standardization of IIF in clinical
laboratories.
© 2012 Elsevier B.V. All rights reserved.
Article history:
Received 7 November 2011
Accepted 20 December 2011
Available online xxxx
Keywords:
Anti-nuclear antibody
Automated screening
Anti-neutrophile cytoplasmic antibody
Anti-dsDNA antibody
Crithidia luciliae indirect immunofluorescence test
HEp-2 cell
Contents
1.
2.
Introduction . . . . . .
Material and methods .
2.1.
Serum samples .
2.2.
ANA testing . . .
2.3.
ANCA testing . .
2.4.
dsDNAab testing.
3.
Results . . . . . . . .
3.1.
ANA testing . . .
3.2.
dsDNAab testing.
3.3.
ANCA testing . .
4.
Discussion . . . . . . .
Take-home message . . . .
References . . . . . . . . .
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⁎ Corresponding author. Tel.: + 39 059 3961791.
E-mail address: a.melegari@ausl.mo.it (A. Melegari).
1568-9972/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.autrev.2011.12.010
Please cite this article as: Melegari A, et al, A comparative study on the reliability of an automated system for the evaluation of cell-based
indirect immunofluorescence, Autoimmun Rev (2012), doi:10.1016/j.autrev.2011.12.010
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2
A. Melegari et al. / Autoimmunity Reviews xxx (2012) xxx–xxx
1. Introduction
Autoantibodies (AAB), in particular ANA, ANCA, and dsDNAab
play a pivotal role in the serological diagnosis of systemic rheumatic
and autoimmune liver diseases [1–5,22–26]. Autoantibody assessment may help monitor disease activity, sub-classify and predict
autoimmunity [6–8]. Indirect immunofluorescence tests are currently
recommended as screening tests for ANA, complimentary tests
for ANCA assessment, and confirmatory for dsDNAab detection
[2,9–13]. According to the recent recommendations of the American
College of Rheumatology (ACR) ANA Task Force, IIF ANA tests should
be considered the gold standard in ANA testing [10]. To meet
modern laboratory standards, high reproducibility and accuracy of
IIF reading are however required. Moreover, due to the growing
request of trained technicians, autoantibody detection is becoming
challenging, especially in large laboratories performing high volume
of testing.
The introduction of digital imaging of IIF along with the development of automated interpretation systems for the assessment of
AAB by IIF has paved the way to overcome important drawbacks
of IIF interpretation, such as the subjective visual interpretation
and the unsatisfactory reproducibility of results among different
clinical laboratories [14–16]. The automated IIF interpretation system AKLIDES has been evaluated for ANA detection on HEp-2
cells, dsDNAab on C. luciliae, and ANCA on human neutrophils
(i.e., AKLIDES), and was previously found to be reliable for the positive/negative differentiation of IIF patterns as well as for the pattern recognition of ANA and ANCA findings [15,17]. The aim of
the present study was to evaluate the performance of this fully automated IIF interpretation system in routine diagnostics and compare it with the visual expert interpretation of IIF.
2. Material and methods
2.1. Serum samples
A total number of 272 serum samples from patients with suspected autoimmune disease were collected from March to April
2011 for ANA (n = 182), dsDNAab (n = 44) and ANCA (n = 46) testing. The samples for ANA testing were either selected from random
routine samples (n = 66), or specifically from patient samples
previously known to be positive for autoantibodies (n = 116). All
samples were collected in the clinical laboratories of the Academic
Hospital of Parma and the General Hospital of Modena from
out-patients and patients hospitalized in different wards (e.g.,
nephrology, rheumatology, gastroenterology, internal medicine,
pediatric department).
2.2. ANA testing
Anti-nuclear antibodies were tested by HEp-2 AKLIDES cell IIF
assay (Medipan GmbH, Dahlewitz/Berlin, Germany). A positive ANA
was defined as a sample with a titer of 1:80 or higher. The same
serum samples were also tested by ELISA and DIA methods. For the
traditional interpretation of HEp-2 assays by experts, the detection
was carried out by ANA IIF assay using HEp-2 AKLIDES slides (Medipan GmbH, Dahlewitz/Berlin, Germany) according to the instructions
of the manufacturer [17]. The slides were read visually by two operators using a fluorescent microscope (Nikon Eclipse 50 i, Nikon Instruments Europe B.V., Italy).
For comparison, detection of ANA was carried out by the automated IIF interpretation system Aklides (Medipan GmbH, Dahlewitz/
Berlin, Germany). The software concept and the image acquisition for
the fully automated interpretation system have been described in detail
elsewhere [15,17]. Briefly, images of IIF patterns were automatically
captured using a fully motorized inverse microscope (Olympus IX81,
Olympus Corp., Tokyo, Japan) with a controllable motorized scanning
stage (Märzhäuser GmbH, Wetzlar, Germany)h 400 nm and 490 nm
light emitting diodes (LED) (CoolLED Ltd, Hampshire, UK), and a gray
level camera (Kappa opto-electronics GmbH, Göttingen, Germany).
The interpretation system is controlled by a specific AKLIDES software.
Algorithms were implemented in C++ programming language (Visual
Studio 2005; Microsoft Corp., Redmond, USA). A novel software-based
autofocus was developed employing Haralick's characterization of
image content by analyzing occurrences of gray level transitions. 4′,6diamidino-2-phenylindole (DAPI) staining was used for focusing of objects. The 116 positive samples were also tested for dsDNAab ELISA
(Phadia, Uppsala, Sweden), ENA ELISA (Phadia, Freiburg, Germany)
and ENA Dot Blot (Alphadia, Wavre, Belgium).
2.3. ANCA testing
ANCA were assessed on ethanol and methanol-fixed human granulocytes by AKLIDES C-ANCA and AKLIDES P-ANCA assays, respectively (Medipan GmbH, Dahlewitz/Berlin, Germany). Slides were
interpreted either visually or by the automated IIF interpretation system AKLIDES. A positive ANCA was defined as a sample with a titer of
1:20 or higher. For the traditional expert interpretation of ANCA
assay, the detection was carried out by ANCA IIF assay using CANCA and P-ANCA slides according to the instructions of the manufacturer. Processed slides were read visually by two operators using
a fluorescent microscope (Nikon Eclipse 50 i).
2.4. dsDNAab testing
Anti-dsDNA antibodies were determined by Crithidia luciliae immunofluorescence test (CLIFT) employing corresponding AKLIDES
slides (Medipan GmbH, Dahlewitz/Berlin, Germany) and interpreted
visually or by the automated IIF interpretation system AKLIDES. A
positive dsDNAab was defined as a sample with a titer of 1:20 or
higher.
3. Results
3.1. ANA testing
In total, 66 routine samples and 116 selected samples with known
AAB levels have been analyzed to assess the performances of the
AKLIDES system regarding positive/negative discrimination. Nine
out of the 66 routine samples showed a discrepant reading comparing
AKLIDES and visual interpretation by experts (Table 1). Eight of the
discrepant samples were classified as weak speckled positive by
AKLIDES and negative by visual examination. As such, only one case
was really discrepant, i.e., negative by AKLIDES reading and positive
by visual expert interpretation (weak speckled, ENA negative). Consequently, the total agreement was 98.5% (Table 1). Eight out of the
116 samples with known autoantibody reactivities demonstrated discrepant findings when comparing AKLIDES and expert results. Seven
of the discrepant samples were positive by AKLIDES reading, negative
by visual expert reading and negative for anti-ENA ELISA. Again, only
Table 1
ANA testing: agreement between EXPERT and AKLIDES interpretation on routine samples (n = 66).
AKLIDES
EXPERT
Positive
Negative
n
Positive
Negative
n
21
8
29
1
36
37
22
44
66
Please cite this article as: Melegari A, et al, A comparative study on the reliability of an automated system for the evaluation of cell-based
indirect immunofluorescence, Autoimmun Rev (2012), doi:10.1016/j.autrev.2011.12.010
A. Melegari et al. / Autoimmunity Reviews xxx (2012) xxx–xxx
Table 2
ANA testing: agreement between EXPERT and AKLIDES interpretation on samples with
highly defined autoantibodies presence (n = 116).
Table 4
ANCA testing: agreement between EXPERT and AKLIDES interpretation (n = 46).
AKLIDES
AKLIDES
EXPERT
Positive
Negative
n
Positive
Negative
n
74
7
81
1
34
35
75
41
116
3
EXPERT
Positive
Negative
n
Positive
Negative
n
10
1
11
5
30
35
15
31
46
4. Discussion
1 sample was really discrepantly negative by AKLIDES and recognized
Scl-70 by the expert and confirmed by ENA. The total agreement of visual expert and AKLIDES reading was 99.0% for these samples
(Table 2).
In summary, only 2 true discordant results were observed out of
the 182 samples investigated for ANA assessment, resulting in a
total agreement of 98.9% for ANA assessment.
3.2. dsDNAab testing
The agreement of visual expert and AKLIDES interpretation on the
44 serum samples that were tested for dsDNAab on slides with C. luciliae slides was 91.0% (Table 3). Differing results were obtained on 4
out of 44 samples. In 3 cases automated AKLIDES reading scored an
equivocal finding, whereas visual examination by experts defined
them as negative. These 3 samples were also weakly positive by
ELISA (cut-off >20 U/mL), showing dsDNAab concentrations from
29 to 48 U/mL. The remaining sample was reported as negative by
AKLIDES and ELISA with a dsDNAab level of 20 U/mL. This particular
patient had suffered from Systemic Lupus Erythematosus (LES), for
which he had been treated for years.
Remarkably, no samples were classified as negative by automated
AKLIDES reading and positive by expert examination; the 3 weak positive samples by AKLIDES interpretation were confirmed as positive
by ELISA.
3.3. ANCA testing
The overall agreement between visual expert reading and automated AKLIDES interpretation of the 46 samples that have been tested for ANCA was 87.0% (Table 4). Five out of 6 discordant samples
were classified as negative by both AKLIDES reading and ELISA,
whereas they were considered C-ANCA positive by expert interpretation. In the remaining case, the sample was positive by AKLIDES interpretation, but was instead negative by both the expert reading and
ELISA. A further ANA test employing HEp2 cells confirmed a positive
ANA with a speckled pattern. The fluorescent pattern on the granulocytes by IIF was correctly considered as non-significant regarding
ANCA assessment by visual expert reading. The 5 discordant cases
were instead in perfect agreement regarding AKLIDES interpretation
and anti-MPO as well as anti-PR3 ELISA, so that the final agreement
between visual expert and AKLIDES interpretation was considered
to be 89.1% (41/46).
Table 3
dsDNAab testing: Agreement between EXPERT and AKLIDES interpretation (n = 44).
AKLIDES
EXPERT
Positive
Negative
n
Positive
Negative
n
10
3
13
1
30
31
11
33
44
The assessment of AAB by IIF is essential for the serological diagnosis of autoimmune disorders, but is in general characterized by a
high degree of manual procedures, low throughput, and subjective interpretation [1,4,15]. Automation of autoantibody IIF reading including pattern recognition could therefore help reducing intra- and
inter-laboratory variability. In particular, it would meet the growing
demand for cost-effective assessment of large numbers of samples
(i.e., high throughput). The current study was designed to compare
automated and visual interpretation of IIF, and evaluate the usefulness of automated IIF reading by a novel interpretation system in routine laboratory diagnostics.
Common practice has shown that detection of ANA patterns using
the recommended standard substrate cell line HEp-2 is characterized
by high variability due to differing assay protocols and reagents. Furthermore, subjective interpretation depending for instance on differing experience and sensitivity of human eyes further amplifies this
problem. A concerning heterogeneity in description and classification
of staining patterns exists among different laboratories [3]. Taken together, all the aforementioned variables can contribute to a high degree of intra- and inter-laboratory variability. Experts who can
dedicate time and effort to perform and interpret manual HEp-2 cell
assays have become gradually unavailable for a variety of reasons including lack of education and economical constrains in clinical laboratories [18]. Conversely, the demand placed on AAB testing is growing
exponentially in the daily practice. It is hence increasingly challenging to meet the need of skilled personnel to perform and interpret
IIF reading for AAB detection appropriately.
The introduction of multiplexing technologies including ANA
ELISA, which are based on a limited number of autoantigenic targets
for AAB detection, seems a reliable alternative [19]. Meroni and
Schur showed in a recent review article that up to 35% of patients
with rheumatic diseases display AAB detectable by IIF, but not by
solid-phase immunoassays [10]. Accordingly, the task-force committee of the American College of Rheumatology recommended that IIF
should remain the gold standard for ANA testing.
The AKLIDES interpretation system that we have evaluated in this
study combines modern pattern recognition algorithms with automated image taking of cell-based IIF assays. Accumulating data, including that reported in this study, have shown a good agreement
between AKLIDES and visual IIF interpretation for ANA, ANCA, and
dsDNAab in patients with autoimmune diseases. The overestimation
of ANA findings by the AKLIDES system observed in this study might
require a reassessment of the threshold for the detection of weak
positive samples. As regards dsDNAab assessment by CLIFT, it is remarkable that 3 of the 4 discrepant positive samples by AKLIDES in
this study were confirmed as positive by ELISA. This seems to support the advantage of automated IIF reading compared with visual
interpretation, and strengthens the role of CLIFT as a confirmatory
test [20].
The single false positive ANCA finding by AKLIDES (i.e., additional
ANA reactivity in the sample) may be considered clinically negligible.
Positive findings need to be always confirmed by an expert before
being reported to clinicians.
The results of the present study suggest that automation of cellbased IIF testing may improve standardization of ANA, ANCA, and
Please cite this article as: Melegari A, et al, A comparative study on the reliability of an automated system for the evaluation of cell-based
indirect immunofluorescence, Autoimmun Rev (2012), doi:10.1016/j.autrev.2011.12.010
4
A. Melegari et al. / Autoimmunity Reviews xxx (2012) xxx–xxx
dsDNAab assessment as well as contribute to a greater harmonization
of this type of AAB testing. At present, this test system might be particularly helpful as a screening tool, although the associated performance
of slides and Aklides lecture needs to be further improved. Pattern recognition of immunofluorescent images by AKLIDES algorithms may
also represent a suitable novel approach for ANA multiplexing by
using bead-based assays [21].
In conclusion, the introduction of an automated IIF interpretation
system such as AKLIDES may offer new opportunities to the expert
in defining the appropriate setting of IIF within the autoimmunity
laboratory. The reliable and sensitive AAB screening of the AKLIDES
combined with the discrimination of the true positive samples
might provide the experts with the opportunity to save time and concentrate their activity on the true positive samples.
Take-home message
• The introduction of an automated IIF interpretation system in routine Lab may offer new skills to the expert to screen and to modify
the working strategy.
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Please cite this article as: Melegari A, et al, A comparative study on the reliability of an automated system for the evaluation of cell-based
indirect immunofluorescence, Autoimmun Rev (2012), doi:10.1016/j.autrev.2011.12.010