Clin Chem Lab Med 2021; 59(7): 1247–1255

Lieve Van Hoovels*, Xavier Bossuyt, Mariangela Manfredi, Valentina Grossi,

Maurizio Benucci, Stefanie Van Den Bremt, Heidi De Baere, Daria Franceschi, Emiliano Tosi,
Marco Meoni, Nicola Bizzaro and Maria Infantino

Integrating quality assurance in autoimmunity:

the changing face of the automated ANA IIF test
https://doi.org/10.1515/cclm-2020-1669
Received November 6, 2020; accepted February 5, 2021;
published online February 17, 2021
Abstract
Objectives: Currently available computer-aided diagnosis
(CAD) systems for the detection of anti-nuclear antibodies
(ANA) by indirect immunofluorescence (IIF) assay enable a
standardized measurement of system-specific fluorescent
intensity (FI) measures. We aimed to evaluate an internal
quality control (iQC) program that controls the total ANA
IIF process in routine practice.
Methods: In addition to the kit iQC materials, supple-
mental quality indicators were integrated in a total quality
assurance (QA) program: patient-derived iQC’s samples
(negative, 1/160 fine speckled and 1/160 homogeneous),
median sample FI per run and percentage of ANA IIF
positive samples per run. Analytical rejection criteria were
based on the imprecision of the positivity index (PI) mea-
sure of the Zenit PRO system (Menarini). Clinical rejection
criteria were based on changes in FI that correspond to a
change in ANA IIF titer of ≥2. To evaluate the QA program,
different artificial errors were introduced during the ANA
*Corresponding author: Lieve Van Hoovels, Department of Laboratory
Medicine, OLV Hospital, Aalst, Belgium; and Department of
Microbiology and Immunology, KU Leuven, Leuven, Belgium,
E-mail: Lieve.Van.Hoovels@olvz-aalst.be
Xavier Bossuyt, Department of Microbiology and Immunology, KU
Leuven, Leuven, Belgium; Department of Laboratory Medicine,
University Hospital Leuven, Leuven, Belgium
Mariangela Manfredi, Valentina Grossi and Maria Infantino,
Immunology and Allergy Laboratory Unit, San Giovanni di Dio
Hospital, Florence, Italy
Maurizio Benucci, Rheumatology Unit, S. Giovanni di Dio Hospital,
Florence, Italy
Stefanie Van Den Bremt, Department of Laboratory Medicine, OLV
Hospital, Aalst, Belgium
Heidi De Baere, Daria Franceschi and Emiliano Tosi, A. Menarini
Diagnostics, Florence, Italy
Marco Meoni, Visia Lab Srl, Arezzo, Italy
Nicola Bizzaro, Laboratorio di Patologia Clinica, Ospedale San
Antonio, Tolmezzo, Italy
IIF process. After every run, quality indicators were eval-
uated and compared to the pre-set target values.
Results: Rescanning the ANA IIF slides five times, using
an old conjugate and a needle obstruction resulted in
analytically and even clinically relevant errors in ANA IIF
results. All errors were correctly detected by the different
defined quality indicators. Traditional Westgard rules,
including analytically (and clinically) defined rejection
limits were useful in monitoring quality indicators.
Conclusions: The integration of a total process iQC pro-
gram in CAD systems, based on the specific FI measurands
and performance criteria of the system, adds value to QA.
Keywords: antinuclear antibodies; harmonization; indirect
immunofluorescence; quality assurance.
Introduction
Indirect immunofluorescence (IIF) on human epithelial
(HEp-2) cells is the most routinely used screening tech-
nique for the detection of anti-nuclear antibodies (ANA) [1].
The high sensitivity of the assay enables a wide range of
ANA to be detected, with distinct morphological patterns
guiding further identification [2–4]. Furthermore, the
higher the fluorescence titer, the higher the likelihood for a
systemic rheumatic disease [5–8]. Therefore, recent
guidelines issued by the International Committee on ANA
Patterns (ICAP) not only advocate to report the ANA
pattern, but also the titer of positivity as part of ANA IIF
result [3]. ANA IIF, however, has a number of disadvan-
tages. It is time consuming and requires considerable
expertise of the technicians [9, 10]. Moreover, visual
reading is subjective and suffers from intra- and inter-
laboratory variance [11–14]. The limited quality assurance
(QA) policy in manual ANA IIF analysis contributes to this
high variance. Current QA policies are restricted to the
analysis of qualitative or semi-quantitative interpretation
of a high positive and negative pre-diluted internal quality
control samples, included in commercially available kits.
Such policies, however, may overlook some analytical
problems. In many cases, troubleshooting in manual ANA
1248 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing
IIF analysis remains limited to the exclusion, by trial and
error, of only major pitfalls.
In order to harmonize ANA IIF results, recent stan-
dardization initiatives have focussed on the pre-analytical
phase, by automation of the processing of HEp-2 slides
[15, 16], the introduction of computer-aided diagnosis (CAD)
technologies to digitalize ANA IIF analysis [10, 14, 17–22], as
well as on the post-analytical phase, by introducing a
harmonized ICAP ANA IIF nomenclature [2, 3]. The main
goal of automation in the total ANA IIF process is to over-
come some of the disadvantages of manual ANA IIF. Every
CAD system allows automated image acquisition, data
analysis and electronic data transmission. Furthermore,
CAD systems offer a more standardized, automated quan-
titative reading of the fluorescence intensity (FI), translated
into system specific FI measures. This enables the classifi-
cation of samples as ANA IIF positive or negative, with
sensitivities close to 100% when compared to manual
reading [10, 23–25]. Next, most of CAD systems also have
their own algorithms for mathematical pattern recognition.
An overview of the characteristics of currently commercially
available CAD ANA IIF systems is presented in Table 1.
Nevertheless, also with CAD systems an important
variability in ANA IIF FI remains. Independent of the type
of CAD system, recent multicenter studies revealed
extreme FI outliers indicating unrecognized analytical
errors [26, 27]. Due to the high variability inherent to ANA
IIF analysis [28–30] there is a need for a continuous inter-
nal quality control (iQC) program that should cover the
total ANA IIF process, from pre-to post-analytical phase
[31]. The availability of CAD system-specific FI measurands
facilitates the integration of iQC FI results into traditional
iQC charts, assisting lab personnel in the technical vali-
dation of the automated ANA IIF system’s performance,
and in verifying the stability of the kits reagents and lot
variability [31–33]. Ideally, this iQC program is integrated
in a centralized middleware (Table 1) which enables the
supervision of all analytical steps of the different methods
used in autoimmune serology testing (IIF, enzyme-linked
immunosorbent assay, chemilumiscent assay, immuno-
blot assay, etc.). Such system also allows to track the real-
time status of samples and their workflow and enables a
harmonized reporting of results by applying codified rules
(Figure 1). By clarifying and formalizing its procedures, the
laboratory will also improve governance, process trans-
parency, diagnostic support and, importantly, overall
diagnostic quality [34]. Furthermore, integrating FI based
iQC charts into the routine ANA IIF workflow offers a
solution to current shortcomings of autoimmune labora-
tory testing in achieving ISO 15189 accreditation and could
bring this branch of autoimmunity closer to other immu-
nometric assays and their well-established rules [34–37].
The current study aimed to evaluate the integration of
an ISO 15189-compliant iQC program for a CAD ANA IIF
system in a clinical routine practice. We investigated how
errors (highlighted by the quality charts) assist in IIF
troubleshooting and promote corrective action.
Materials and methods
Automated IIF reading system
The Zenit PRO system (A. Menarini Diagnostics) is a fully automated
instrument for IIF that integrates an automated slide-processing
module with a reading unit [21]. The main characteristics of the CAD
system regarding ANA IIF analysis are summarized in Table 1.
Zenit PRO performs the evaluation of FI on every single identified
HEp-2 cell (A. Menarini Diagnostics ANA-HEp-2, Biosystems S.A.). An
average level of FI is calculated depending on the ANA IIF pattern and
expressed as a ‘positivity index’ (PI). Based on two PI thresholds, ANA
IIF results are classified into three different classes: ANA IIF negative
(PI<10.9), borderline (PI=10.9–19.6) and positive (PI>19.6). Further-
more, an ANA endpoint titer (EPT) suggestion is made based on a large
database of reference images used to train a state-of-art classifier.
A software interface presents the ANA IIF results for supervision
to the user, who can interpret, amend if needed, and validate ANA IIF
results.
Quality indicators
As recommended by the manufacturer, PI results of the positive and
negative kit iQC (further referred to as ‘kit iQC’) were integrated in the
iQC program. In addition, three patient serum pools were prepared as
additional iQC materials (further referred to as ‘sample iQC’): an ANA
IIF negative (AC-0); an ANA IIF positive pool with a fine speckled
(AC-4) pattern and an ANA IIF positive pool with a homogeneous
(AC-1) pattern. The sample iQC’s target a PI of 10–15 for the negative
pool and 20–25 for both positive pools, corresponding to an EPT of
respectively <1:80 and 1:80.
Furthermore, 22 routine ANA IIF samples were included, simu-
lating a real-life routine ANA IIF run in terms of positive/negative ratio
(14/22=63.6%) and distribution of the different PI-values and corre-
sponding EPT. For each of these samples a reference PI was obtained
during an ANA IIF run under perfectly controlled circumstances.
Median patient sample PI per run and percentage ANA IIF positive
patient samples per run were defined as additional ‘whole run’ quality
indicators [31, 32].
All serum samples were retrospectively selected from left-over
samples of routine screening for ANA and anonymized after selection.
No informed consent was needed for this retrospective study, but the
study was performed according to the Declaration of Helsinki and
approved by the local Ethical Committee of OLV Hospital Aalst (study
registration number B126201942365).
 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing 1249

Table : System characteristics of currently available computer-aided diagnosis systems for ANA IIF analysis.

System Aklides EUROPattern Image navigator Helios Nova view Zenit PRO
(Company) (Medipan) (Euroimmun) (Immoconcepts) (Aesku) (Inova) (A. Menarini)

Reagents
HEp- cell HEp- HEp- HEp- HEp- HEp- HEp-
substrate
Conjugate IgG heavy chain IgG heavy chain IgG heavy chain IgG heavy IgG heavy chain IgG heavy/Light
specific specific specific chain specific specific chain specific
DNA-binding DAPI Propidium iodide None None DAPI None
counter stain
iQC pos iQC (ready to pos iQC (ready to pos iQC (ready to pos iQC (ready pos iQC (ready to pos iQC (ready to
use) use) use) to use) use) use)
neg iQC (ready to neg iQC (ready to neg iQC (ready to neg iQC (ready neg iQC (ready to neg iQC (ready to
use) use) use) to use) use) use)
pos iQC (titratable) pos iQC pos iQC (titratable) pos iQC (titratable)
(titratable)
iQC source Human stabilized Human stabilized Human stabi- Human stabi- Human stabilized Human stabilized
serum serum lized serum lized serum serum serum
Slide processor
Instrument Akenomi IF-Sprinter Autoimmune fast Helios inte- QUANTA lyser Zenit PRO integrated
track grated slide slide processor
processor
Automated IIF microscope
Instrument Aklides EUROPattern Image Navigator Helios NOVA view Zenit PRO
Measure of Light intensity units Fluorescence Positivity value Fluorescence Light intensity units Positivity index
fluorescent intensity intensity
intensity
Screening pos/ Yes Yes Yes Yes Yes Yes
neg
ANA IIF pattern Homogeneous Homogeneous No No Homogeneous Homogeneous
recognitiona (AC-), centromere (AC-), dense fine (AC-), centromere (AC-), centromere
(AC-), speckled speckled (AC-), (AC-), speckled (AC-), speckled
(AC-,), nuclear centromere (AC-), (AC-,), nuclear (AC-,), nuclear
dots (AC-,), nucle- speckled (AC-,), dots (AC-,), nucle- dots (AC-,), nucle-
olar (AC-,,), nuclear dots olar (AC-,,), olar (AC-,,),
cytoplasmic, (AC-,), nucleolar cytoplasmic, cytoplasmic,
unrecognized (AC-,,), nuclear unrecognized mitochondrial-like
envelope (AC-,), (AC-)
cytoplasmic, mitotic
and negative (AC-)/
unspecific patterns
ANA IIF titer Yes Yes No No Yes Yes
prediction
ICAP nomen- Yes Yes Yes Yes Yes Yes
clature
integrationa
Middleware No, Aklides software Yes, EUROLabOffice No, Image Navi- Yes, Aeskulab Yes, QUANTA link Yes, Zenit HUB
gator software
Integrated total No Option No Yes No Yes
process QA
program
a
Reference to ICAP nomenclature (www.anapatterns.org []). IIF, indirect immunofluorescence; iQC, internal quality control; pos, positive; neg,
negative; QA, quality assurance.
1250 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing

Figure 1: Levey-Jennings quality charts of the positive patient pool iQC (AC-4) revealing.
(A) a conjugate washing step error (PI=20.7; ΔPI=−23.3%); (B) no error logging (PI=28; ΔPI= 3.7%); (C) needle obstruction error (PI=17.1; ΔPI=−36.7%).
Analytical performance criteria are highlighted in colored bars: warning limits (12CV% = ±20%) in orange and stop limits (13CV% = ±30%) in red.

Analytical and clinical rejection criteria
The total imprecision of the different iQC materials was preliminarily
evaluated on Zenit PRO in accordance to the CLSI EP5-A2 protocol
(CLSI, 2004) (Supplemental Data Table S1). Based on these experi-
mental data, we defined a target CV of 10% for the negative sample
iQC and of 10% for the positive sample iQC (Table 2). Westgard rules
12CV (a single control measurement exceeds the mean ± 2*CV%
target) and 13CV (a single control measurement exceeds the
mean ± 3*CV% target) were applied as, respectively, analytical
warning and stop limit.
Clinical quality rejection criteria were estimated based on the FI
error correlating with a change in ANA IIF titer of ≥2 [15] (Supplemental
Data Table S2). The higher limit of the stop range for the positive
sample iQC (AC-4 and AC-1) corresponded to a clinically relevant
change of >2 ANA IIF titers (target: 1:80; >3*CV%: >1:320). For a pos-
itive sample with PI around 25 with corresponding EPT of 1:80, a
decrease of >3*CV%, can result in a PI nearly below the negative cut-
off (PI=10.9) and a change in titer to <1:80.
An overview of the target values and rejection criteria for the
positive and negative iQC are given in Table 2. Of note, analytical
performance indicators will highlight an analytical issue before a
clinical inaccurate result can be reported.
Artificially induced errors
Artificial errors, mimicking plausible errors in routine ANA IIF prac-
tice, were introduced during the ANA IIF analytical process (one run
per error). A list of the artificial errors performed is shown in Table 3.
For each of these runs, the pattern, PI and EPT results of the iQC
materials and patient samples were recorded as generated by the Zenit
PRO system and after supervision by six experienced ANA IIF readers.
If they disagreed, a consensus was reached.
iQC results were assessed according to the Westgard rules and
patient results were compared to the pre-set target values and used to
calculate the quality performance indicator (Table 2).
Results
Figure 2 presents IIF pictures obtained after induction of
several artificial errors. In Table 4 the impact of all errors on
the different quality indicators is summarized.
Rescanning the slides five times reduced the PI value
of all samples. A relative change in PI of >20% (analytical
warning limit) was observed in 95.5% (n=21/22) of the
samples, and of >30% (analytical stop limit) in 31.8% (n=7/
22) of the samples. The inaccuracy was revealed by all five
quality indicators exceeding the analytical warning limit,
of which two (positive sample iQC AC-1 and the % of pos-
itive patient samples/run) even exceeded the analytical
stop limit (Tables 3 and 4).
The use of an old conjugate resulted in the most
extreme PI decrease, revealed by all quality indicators,
indicating an analytical (>30% decrease in PI) or clinical
(>60% decrease in PI) stop limit.
In case of needle obstruction, 86.4% (n=19/22) of the
samples had a relative change in PI that at least exceeded
the analytical stop limit of 30 and 63.6% (n=14/22) of the
 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing 1251

Table : Analytical and clinical rejection criteria for all quality performance indicators.

Target value and coefficient of variation (CV) Rejection criteria

Process control PI positive kit iQC – Pattern of initial ANA IIF analysis – No exact match of target
– Target PI (manufacturer): 20–25 (cutoff positivity; pattern
EPT 1:80) – Westgard rulesa
– Target CVanalytical: 10%
– Target CVclinical: 20%

PI negative kit iQC – Negative on 1/80 dilution – Positive

– Target CVanalytical: 20% – Westgard rulesa
– Target CVclinical: 30%

PI positive sample iQC AC- – Pattern of initial ANA IIF analysis – No exact match of target
PI positive sample iQC AC- – Target PI: ±25 (EPT 1:80) pattern
– Target CVanalytical: 10% – Westgard rulesa
– Target CVclinical: 20%

PI negative sample iQC – Negative on 1/80 dilution – Positive

– Target PI: ±10–15 (negative to low borderline – Westgard rulesa
positivity; EPT < 1:80)
– Target CVanalytical: 10%
– Target CVclinical: 20%

Monitoring of patient % Positive ANA IIF patient – Target value: Positive/negative ratio of a real-life – Westgard rulesa
results samples/run routine run
– Target CVanalytical: 10%
– Target CVclinical: 20%

Median patient sample LIU/ – Target value: Overall median PI of the 22 study – Westgard rulesa
run patient samplesb
– Target CVanalytical: 10%
– Target CVclinical: 20%
a
CV (a single control measurement exceeds the mean ±  CV% target) as a warning limit and CV (a single control measurement exceeds the
mean ±  CV% target) as stop limit. bdistribution of the PI-values and corresponding endpoint titers of a real-life routine run. iQC, internal quality
control; PI, positivity index; CV, coefficient of variation; AC, anti-cell; AC-, ANA IIF pattern negative (ICAP); AC-, ANA IIF pattern fine speckled
(ICAP); AC-, ANA IIF pattern homogeneous (ICAP); EPT, endpoint titer.

samples had a relative change in PI that exceeded the
clinical warning limit (>40%). Almost all sample-derived
quality indicators revealed an analytical stop limit.
Using an extra diluted PBS buffer did not result in a
consistent deviation of the quality indicators or in
exceeding analytical stop limits. Only an analytical warn-
ing limit was exceeded for the positive kit iQC and the
negative sample iQC. Supervision of the ANA IIF pictures
(provided by the CAD system), however, clearly indicated
the analytical artefact (Figure 2).

Table : Overview of all analytical errors artificially introduced in the ANA IIF process on Zenit PRO.

Pre-analytical problems

. Needle obstruction – 5 μL sample volume in 790 μL PBS-buffer, instead of 10 μL

Analytical problems

. PBS-buffer dilution – 1 bottle PBS-buffer in 2000 mL instead of 1 bottle in 1,000 mL

. Old conjugate – Use of conjugate stored at 30 °C for 72 h
. Sample wash step error – 1 wash cycle with 1 mL instead of 3 wash cycles with 2 mL
. Conjugate wash step error – 1 wash cycle with 1 mL instead of 3 wash cycles with 2 mL

Post-analytical problems

. Rescanning slide – 5x rescanning of same slide

1252 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing
Reduction of the wash cycle after the incubation of the
sample or the conjugate resulted in a decrease of the PI of
>20% for 45.5% (n=10/22) and 27.3% (n=6/22) of the sam-
ples, respectively. The analytical stop limit was only
violated for the positive sample iQC and kit iQC in the run
with wash cycle reduction after sample incubation. The
analytical warning limit was exceeded by almost all quality
indicators in the run with wash cycle reduction after con-
jugate incubation. A different trend was observed for the kit
iQC material compared to the sample derived quality in-
dicators in the latter experiment.
Discussion
For many years, high ANA IIF variability has been reported
and described as an intrinsic characteristics of the IIF method
[11, 13, 14, 28–30]. The variability of IIF is strongly influenced
by several issues inherent to the method, both biological (e.g.,
variability of cellular substrates) and non-biological [30].
Figure 2: ANA IIF troubleshooting.
(A) Reference run: pool speckled AC-4
(EPT 1/80); (B) old conjugate: loss in
reactivity of conjugate; (C) sample wash
error: possible presence of unbound
antibody, formation of immune complexes,
and unwanted background; (D) needle
obstruction: less/no volume of serum
dispensed; (E) PBS-buffer dilution:
problems related to the non-specific
binding; (F) conjugate wash error: higher
conjugate reactivity due to excess
secondary antibody and non-specific
antibody interactions.
Moreover, IIF is currently limited by a low level of standard-
ization and automation, subjective evaluation, intra and
inter-laboratory variability and the requirement of highly
skilled personnel [11, 14, 28, 38]. By the introduction of
automated ANA IIF systems in routine laboratory diagnostics,
manufacturers and lab specialists aimed to improve lean
ANA IIF analysis by reducing the hands-on time and by
providing a more objective way of quantifying FI. Different
CAD systems have been commercialized, each with specific FI
measurands (dependent on the measuring principle and
software algorithms) (Table 1). Previous reports on quality
assurance of automated ANA CAD systems revealed that
expanding the number of quality indicators based on FI
measurands improves quality assurance [31–33].
However, the choice of the quality indicators and the
definition of their quality goals is crucial.
First, a quality indicator is fit-for-purpose if it covers
the total analytical ANA IIF process. In the experiment
mimicking a needle obstruction, the kit iQC results are
not reflecting the analytical error seen when patient
 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing 1253

Table : Impact of each error on the different quality indicators expressed as % deviation from the reference run, performed in ideal analytical
circumstances.

Δ reference run (%) Rescanning x Old Needle Buffer Sample Conjugate

conjugate obstruction dilution wash wash
Scan  Scan  Scan  Scan 

PI positive kit iQC −. % −. % −. % −. % −. % . % . % −. % . %
PI negative kit iQC . % . % −. % −. % −. % . % . % . % . %
PI negative sample −. % −. % −. % −. % −. % −. % . % . % −. %
iQC AC-
PI positive sample −. % −. % −. % −. % −. % −. % −. % −. % −. %
iQC AC-
PI positive sample −. % −. % −. % −. % −. % −. % −. % −. % −. %
iQC AC-
% Positive patient −. % −. % −. % −. % −. % −. % . % −. % −. %
samples/run
Median patient −. % −. % −. % −. % −. % −. % . % −. % −. %
sample PI/run

Deviations exceeding analytical limits warning are highlighted in bold and analytical stop limits are highlighted in italic; deviations exceeding
clinical warning limits are marked in light gray and clinical stop limits are marked in dark gray. iQC, internal quality control; PI, positivity index;
CV, coefficient of variation; AC, anti-cell; AC-, ANA IIF pattern negative (ICAP); AC-, ANA IIF pattern fine speckled (ICAP); AC-, ANA IIF pattern
homogeneous (ICAP); EPT, endpoint ANA IIF titer.

samples are tested, since they are pre-diluted and not
subjected to the sample dilution step. Therefore, adding
a titratable negative and positive sample iQC as quality
indicators appeared to be mandatory, as they undergo
the same process steps as the patient samples. Addi-
tionally, to assure the clinical reproducibility of ANA IIF
results, conventionally accepted as a deviation within ≤2
ANA IIF titers [13], the quality goals of the positive
sample iQCs should be chosen around the clinical deci-
sion cut-off, e.g., an FI measurand that correlates with
an endpoint titer between 1/80 and 1/160. Additionally,
the higher the fluorescence intensity, the higher the
likelihood for antibody-associated rheumatic disease
(AARD) [5–8]. In this respect, it would be worthwhile to
monitor borderline positive and FI values with signifi-
cant correlation with AARD too. Preferably, the manu-
facturers of ANA IIF kits should provide titratable,
human stabilized iQC material targeting multiple
endpoint titers (e.g., <1/80, 1/80, 1/320). When unavai-
lable, lab specialists should consider implementing, in
parallel to the kit iQC materials, patient pool iQC
samples.
A good insight in the total imprecision of the FI
measurand is key for defining CAD system specific per-
formance criteria of the iQC samples. As there is a corre-
lation between the specific FI measurand and the ANA IIF
EPT it is important to define analytical warning and stop
limits as well as clinical warning and stop limits. The
advantage of defining analytical as well as clinical limits is
that in case analytical problems are revealed, consequent
corrective action can be undertaken before a clinical
inaccurate result is reported.
The deviation in PI caused by several analytical issues
resulted in the same trend (e.g., deviation in similar di-
rection) for the different quality indicators (% positive
samples/run, median PI/run, PI positive sample iQC, PI
negative sample iQC). One quality performance indicator
should never be interpreted on his own but always in
correlation with others because it is the same behavior of
different quality performance indicators that highlights an
analytical issue. A theoretical setting of analytical/clinical
warning and stop limits has to be thoroughly validated in
daily routine for feasibility.
The main goal of ISO 15189 accreditation is to ensure
that laboratories provide high-quality results. The rules to
obtain accreditation are mainly established for highly
standardized test systems, like e.g., clinical chemistry
automated platforms. Accreditation in autoimmunity lab-
oratories, however, is more challenging, mainly by the lack
of international standards for most antibody tests which
hampers method validation of novel tests and generation
of reference values [34]. Great initiatives to promote
harmonization in autoantibody testing are currently
ongoing [36, 37]. In parallel, we should focus on ISO 15189
norm elements that can be already implemented in routine
laboratory practice, and most importantly, use them for
clinical decision making. Our study results stimulate the
introduction of a thorough iQC program, based on the FI
measurand of CAD ANA IIF system, that covers all aspects
of ANA IIF testing and results in total process governance.
1254 Van Hoovels et al.: Integrating quality assurance in automated ANA IIF testing
Conventionally known Westgard rules are easily appli-
cable and guide lab technicians to analytically validate an
ANA IIF run.
CAD systems have the potential to reduce imprecision
in ANA IIF diagnostics. As these systems focus on cell
classification in terms of feature extraction representa-
tions [35], they are currently considered state-of-art to
increase diagnostic accuracy in ANA IIF analysis. How-
ever, notwithstanding the improved performance of these
systems compared to manual ANA IIF testing, a short-
coming of these learning systems is that they are closed,
e.g., devoted to one reference ANA IIF kit (HEp-2 slides,
conjugate, etc.) and to the system specific algorithms for
pattern recognition [12]. In contrast, artificial intelligence
deep learning systems, currently penalized by the
computational cost and the very large training dataset
required, promise to be an excellent prospect for the
future [39]. In the meantime, visual review of the images
by an experience lab technician remains important. In our
study, review of the ANA IIF pictures provided by the CAD
system had the potential to reveal an analytical artefact
(Figure 2). Current CAD systems only offer pattern recog-
nition for a limited set of common patterns, which still
need to be visually confirmed. Therefore, laboratories
should focus on training personnel and building image
libraries using guidance for assessing ANA, e.g., by the
recently launched ICAP training module (available on
www.anapatterns.org).
In conclusion, lab specialists and companies of auto-
mated ANA IIF systems should integrate QA programs,
based on analytical and clinical warning and stop limits,
based on the CAD system specific FI measurands and
performance criteria.
Acknowledgments: We gratefully acknowledge lab tech-
nicians of OLV Hospital Aalst for their practical support
and the colleagues of ASZ Aalst for the preliminary testing
of the ANA IIF samples on their Zenit PRO instrument.
Research funding: None declared.
Author contributions: All authors have accepted
responsibility for the entire content of this manuscript
and approved its submission.
Competing interests: Heidi de Baere, Daria Franceschi and
Emiliano Tosi are employees of A. Menarini Diagnostics;
Marco Meoni is an employee of Visia Lab Srl. All other
authors state no conflict of interest.
Ethical approval: The study was performed according to
the Declaration of Helsinki and approved by the local
ethical committee of OLV Hospital Aalst (study registration
number B126201942365).
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Supplementary Material: The online version of this article offers
supplementary material (https://doi.org/10.1515/cclm-2020-1669).