Turkish AI Guide

OTOANTĠKORLARIN LABORATUVAR TANISI
BÖLÜM 3 - ANA Saptanmasında IIF Yöntemi ve Standardizasyon
CHAPTER 3
INDIRECT
IMMUNOFLUORESCENCE (IIF)
METHOD AND
STANDARDIZATION IN THE
DETECTION OF ANTI-NUCLEAR
ANTIBODY (ANA)
Introduction
Anti-nuclear antibody (ANA) test results may vary depending on the method used. Today,
different methods are used for the detection of ANA. These methods can be listed as
bidirectional immunodiffusion, radioimmunoassay (RIA), chemiluminescent immunoassay
(CLIA), enzyme labeled immunosorbent assay (ELISA), immunodot, immunoblot, microarray,
multiplex laser fluorometry and indirect immunofluorescence (IIF) method. The accepted gold
standard in the detection of ANA is the IIF method, in which HEp-2 (HEp-2000) cells are used as
substrate. The reliability of the results obtained by this method is of great importance for the
diagnosis of the disease. At this point, the two most important factors are reliable HEp-2 cell
line and reliable microscopic evaluation. The parameters affecting the test result in the ANA
IIF test can be summarized as follows (Table 3).
Tablo 3. IIF yönteminde test sonucunu etkileyen faktörler
Pre-analytical factors analytical factors Post-analytical factors
− Selection of suitable patients − Method and kit to be studied − Reporting process

− Sample collection parameters − Characteristics of the cell − Determination of the limit
− Specimen storage and series used value
processing procedures − Determining the
− Transport conditions − Scan dilution
consistency of the results
− Factors originating from − Microscopic evaluation
with the clinical
the patient − Quality control
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ANA Saptanmasında IIF Yöntemi ve Standardizasyon - BÖLÜM 3
Sample acceptance, ANA IIF method and kit to be studied

Sample acceptance/rejection criteria
For example, serum or plasma (EDTA, heparin or citrate) is used. Patient name-surname, patient
number/protocol number etc. Samples without patient-identifying information, such as Test requests
that are not made in accordance with the test request period specified in the rational test request
procedure may be rejected. Hemolyzed and lipemic specimens should not be used as they may affect
test results. (For patient samples storage and working conditions, see Working procedure)
ANA IIF method

The IIF method is an in-vitro diagnostic method in which cell or tissue sections are used as solid
phase and specific antibodies are investigated in patient serum. It is the most commonly used
method in the detection of ANA. The method is based on the binding of the antibody in the
patient's serum to the antigen and the binding of the fluorescently labeled anti-human
antibody to this complex. In this method, slide (slide) is used as solid phase, and various cells or
tissues are fixed on these slides. Patient serum is dripped onto these commercially prepared
slides and incubated in accordance with the recommendations of each manufacturer. After
washing with buffer solution, FITC-labeled anti-human antibody (conjugate) is added and
incubation and washing are performed again. Then, the slide is covered with a coverslip by
dripping glycerol and examined under a fluorescent microscope.
Cells and tissues used in the ANA IIF method
HEp-2 cells recommended as the gold standard in the ANA IIF method are human laryngeal
epithelial carcinoma cells. Since HEp-2 cells have similar characteristics in each series, they
provide a more standard substrate for the detection of ANA compared to other tissues. Since
the SS-A antigen is generally low-expressed in HEp-2 cell lines, "false-negative" results may be
encountered. To overcome this problem, the HEp-2000 cell line was developed in which HEp-2
cells were transfected with SS-A antigen. Evaluation of HEp-2 cells with the IIF method allows
the detection of autoantibodies that can develop against the nuclear, cytoplasmic and
mitotic structures of the cell.
Additional textures may be required to differentiate some patterns. For example, monkey liver tissue is
particularly useful in determining the presence of nuclear membrane and distinguishing nuclear dot-
centromere patterns. For this purpose, in doubtful cases, kits in which liver and liver tissue are provided
together with HEp-2 cells or kits in which liver tissue is provided separately can be used.
Advantages of HEp-2 cell lines in the detection of ANA: Large nuclei in HEp-2 cell lines, easy
visibility of cell structures, homogeneous and monolayer cell collection, expression of antigens
belonging to all phases of the cell cycle (ability to capture more than 100 autoantigens), new
It has advantages such as allowing the discovery of autoantibodies.
Disadvantages of HEp-2 cell lines in the detection of ANA: Depending on the use of HEp-2 cell
lines, problems in detecting anti-SS-A, anti-tRNA synthetase (Jo-1) and anti-ribosomal P (anti-
Rib-P) antibodies may occur. . Because these antigens are expressed at very low levels in
HEp-2 cells or can be denatured in tissues during fixation procedures, "false negativity" may
occur. Therefore, the most suitable HEp-2 cell lines are acetone-fixed cells; Fixation with
ethanol or methanol may cause loss of SS-A antigen. Laboratories using the ANA HEp-2
method should definitely investigate the presence of anti-SS-A with additional tests. In order to
partially overcome this problem experienced in HEp-2 cells, HEp-2000 cells that highly express
the SS-A/Ro60 antigen can be used.
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ANA IIF test kit selection

Most ANA are of the IgG isotype. Searching for ANA IgM and/or IgA isotypes does not
increase the sensitivity as well as decrease the specificity. Therefore, only ANA IgG is sufficient
for diagnosis. It is important to consider the following points when selecting the ANA IIF assay
to run in the laboratory (see this Section, Quality control).
 Homogeneity of cell density and distribution on the slide

 Adequate number of mitotic cells (3-5 metaphases/fieldX200 magnification)
 Cell morphology is intact
 Absence of background fluorescence
 Adequate expression of specific antigens in the cell
 The conjugate used must be a fluorochrome (FITC or others) labeled anti-human IgG
specific secondary antibody. The use of polyvalent antibodies may result in a higher rate
of “false positive” reactions. It is recommended that the FITC/protein ratio be
approximately 3.0; a high ratio increases non-specific binding. The ratio of antibody to
protein in the conjugate should be ≥0.1,
 The use of acetone-fixed slides is recommended; ethanol or methanol fixation can lead
to loss of SS-A antigen.
 The ready-to-use solutions reduce the potential for error.
 Having different slide formats according to the number of different patients can be
preferred in terms of ease of application and cost.
 Providing the material in kit form simplifies the working process and reduces the margin of error.
 The expiration period of the kit should be long and the stability of the kit content should be
maintained during this period.
In particular, the verification process to be performed using patient sera with a clinical diagnosis during
the test setup phase is important for quality control (see this Section, Quality control). The points to be
considered during the verification studies of the ANA IIF tests are as follows:
 High reproducibility of the test: Consistency of the results obtained within the study and
between the studies during the experiment.
 Proof of accuracy in the study with positive and negative samples for which clinical
information is available (high sensitivity and specificity values)
Working procedure
Considerations in the study of the ANA IIF test
The sample studied for the ANA IIF test is serum and/or plasma. It is recommended to store the
samples at +2-8°C for up to 3-14 days in line with the recommendation of the kit
manufacturer, and at -20°C if longer storage is required. Thawed samples should not be
frozen and thawed again. Test kits should be stored as recommended by the manufacturer, in
an appropriate environment and temperature, protected from moisture. Out-of-date kits
should not be used. Care should be taken to ensure that all samples and reagents are
brought to room temperature prior to testing. The calibrations of the automatic pipettes used
should be checked at regular intervals, and the routine maintenance of the devices should
be done regularly. Positive and negative controls must be used in each run and should be run
in accordance with quality control guidelines (see this Section, Quality control).
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Scan dilution
One of the important parameters affecting the ANA IIF study is the screening dilution. It is
recommended that each laboratory set its own breakpoint based on the 95th percentile.
However, since this application may not be valid for every laboratory, the recommended
screening dilution for adults is 1/160. Although there is no definitive screening dilution for
pediatric patients, 1/160 dilution is recommended for this age group.
Although 1/160 is recommended in the international literature regarding ANA screening
dilution, the common practice in our country is to scan with 1/100 dilution. As a result of the
collection of our national data, it is expected that a final decision will be made on ANA
screening with 1/160 dilution. At this stage, the initial dilution recommended by the
manufacturer should not be ignored. A scan dilution of 1/160 will reduce low-positive ANA
results compared to screening with 1/100, and thus unnecessary reflex (monospecific) test
requests will be avoided to some extent. Low positive (1/100) ANA positivity accounts for
approximately 70-75% of ANA positive results. These results are considered as "ANA positive"
unnecessarily by some clinicians, reflex testing is requested and increases the patient cost. On
the other hand, depending on the characteristics of the HEp-2 cell line used, the capture of
some patterns (see Chapter 5, ANA patterns, Fine spotted pattern (Anti-SS-A/SS-B-like staining;
AC-4) may vary depending on the dilution coefficient, for example HEp. It is possible to detect
the SS-A, SS-B pattern with 1/100 dilution, which cannot be detected with 1/160 dilution in -2
cells.After this situation is supported by our local data, the ANA screening dilution that will be
valid for our country will be determined.
Microscopic evaluation
The HEp-2 cell used in the ANA IIF evaluation and some of the structures in it that may cause
different staining patterns are schematized in Figure 2.
Figure 2. HEp-2 cell fine structure
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During the microscopic evaluation of the ANA IIF test, it is possible to detect antibodies
against antigens released in the patient serum at different stages of the cell cycle. In order to
make a detailed and accurate ANA IIF evaluation, it is necessary to know the cell cycle
stages. The cell cycle stages and the positive chromosome bands that can be seen in these
stages are shown in Figure 3.
Figure 3. Mitosis stages and positive chromosome bands seen in these stages
(Adapted from http://pulpbits.net/wp-content/uploads/2013/12/mitosis-process-illustration.jpeg)
The events that take place during the cell cycle phases can be summarized as follows:
Interphase: It is the stage of preparation for division. ATP synthesis accelerates, the amount of
organelles and proteins increases, DNA replicates itself.
Prophase: Chromatid threads become chromosomes, centrioles go to opposite poles, spindle
fibers begin to form.
Metaphase: Chromosomes line up on the equatorial plane of the cell.
Anaphase: Sister chromatids in each chromosome are pulled to
opposite poles, spindle fibers become prominent in the middle In the
Telophase: Nuclear membrane forms, nucleolus emerges, evaluation of ANA
chromosomes turn into chromatin threads again, spindle fibers IIF, cell staining
disappear, cytoplasm division begins.
patterns should be
In the ANA IIF examination, especially the cell staining patterns
in the interphase and metaphase stages should be carefully
carefully examined,
examined (Figure 4 and Figure 5). Since the interphase stage is especially in the
the fastest in cell metabolism and the most antigen expression, interphase and
many different antigens can be released. The metaphase
stage is another stage that should be carefully examined in
metaphase stages.
order to distinguish many patterns, especially homogeneous
and mottled patterns of the nucleus.
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KLIMUD
Figure 4. Examples of chromosome areas seen in interphase and different stages of mitosis.
Figure 5. Examples of the types of staining seen in the nuclei of HEp-2 and liver cells in the interphase phase.
Specific nuclear and cytoplasmic antigens with proven association with fluorescent staining
patterns are given in Table 4.
In microscopic examination, firstly, positivity and negativity are evaluated with 10 or 20
magnification. Positive samples are then examined for fluorescent staining pattern at 40-60x
magnification. patterns on ANA IIF slides; nuclear, cytoplasmic and mitotic patterns should be
studied and reported separately (see Chapter 5, Anti-nuclear antibody patterns). See Table 5
to decide on the ANA pattern according to the staining characteristics of cells in the
interphase and metaphase stages in the ANA IIF examination.
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Tablo 4. ANA IIF paternleri ve ilgili antijenler

Anti-Cell
ANA Paterni İlgili Antijenler
Kodu
Nükleer Paternler
AC-1 Homojen dsDNA, histon, kromatin/nükleozom
AC-2 Yoğun ince benekli (DFS) DFS70/LEDGF-P75
AC-4 Ġnce benekli SS-A/Ro, SS-B/La, Mi-2, Ku, TIF1, TIF1 P
U1RNP, Sm (U2-nRNP), RNA polimeraz III, Nükleer matriks
AC-5 Büyük/Kaba benekli
proteini
AC-29 Topo-I benzeri DNA Topoizomeraz 1
AC-3 Sentromer CENP-A/B (C)
AC-6 Çok sayıda nükleer noktalar Sp-100, PML proteinleri, MJ/NKP-2
AC-7 Az sayıda nükleer noktalar p80-coilin, SMN
Nükleolar (Homojen / Kümeli / PM/Scl-75, PM/Scl-100, To/Th, nükleofosmin, nükleolin,
AC-8,9,10
Noktalı) fibrillarin (U3-nRNP), RNA polimeraz 1, NOR-90
Lamin A, B, C, gp210, nükleoporin p62, nükleer por
AC-11,12 Nükleer membran (Düz/Noktalı)
kompleks antijenleri
AC-13 Pleomorfik PCNA PCNA
AC-14 Pleomorfik CENP-F CENP-F
Sitoplazmik Paternler
Fibriler (Lineer / Filamentöz / Aktin, kas dıĢı miyozin, sitokeratin, vimentin, tropomiyozin,
AC-15,16,17
Segmental) -aktin, vinkülin
Benekli {Ayrık noktalar/Yoğun GW 182, lizozom, Ribozomal P proteinleri, PL-7, PL-12, Jo-
AC-18,19,20
ince benekli / Ġnce benekli) 1/ histidil-tRNA sentetaz
Retiküler (Anti Mitokondriyal PDC-E2/M2, BCOADC-E2, OGDC-E2, Ela PDC,
AC-21
Antikor-AMA) E3BP/protein X
AC-22 Polar/Golgi benzeri Golgi proteinleri
AC-2 3 Çubuk ve halkalar IMPDH2
Mitotik paternler
AC-24 Sentrozom Pericentrin, ninein, Cep250, CepllO
AC-25,26 Ġğsi iplikçikler/NuMA benzeri NuMA
AC-27 Hücreler arası köprü Yok
AC-28 Mitotik kromozomal Modifiye histon H3, MCA-1
Because microscopic evaluation is subjective, it is difficult to achieve inter-laboratory and

intra-laboratory standardization. However, evaluations with well-defined clinical specimens or
standard sera may provide a more objective interpretation (see this Section, Quality control).
An important factor in the interpretation of fluorescence intensity is the light intensity and
lifetime of the fluorescent microscope. In microscopes in which mercury lamps are used as
photon source, the light intensity decreases as the usage time increases, causing lower titers
to be reported. For this reason, it is recommended to replace mercury lamps after 100-200
hours and position them correctly. Fluorescent microscopes with LED light source are more
reliable as they do not have a heating problem and decrease in light intensity over time. In
addition, the lamp focus settings are not impaired in LED lamps (see this Chapter, Quality
control).
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Tablo 5. ANA IIF HEp-2 interfaz ve metafaz hücrelerin boyanma özelliklerine göre patern karar tablosu
İNTERFAZ BOYANMA METAFAZ BOYANMA SİTOPLAZMİK BOYANMA

Çubuk
Homojen Benekli Nükleolar Noktalı Homojen Benekli Noktalı Fibriler Benekli Retiküler Golgi
ve halka
PATERN İLGİLİ ANTİJENLER REFLEKS TEST
BOYANMA YOK BOYANMA YOK BOYANMA YOK Negatif (AC-0) YOK YOK
dsDNA, histon
VAR VAR Homojen (AC-1) dsDNA, ENA
nükleosom
Yoğun ince
DFS70/LEDGF
VAR VAR benekli (DFS) ENA
-P75
(AC-2)
SS-A, SS-B, Mi2, Ku, RNP,
Benekli ENA
VAR Sm, RNA polimeraz III,
(AC-4,5,29) Miyozit panel
Topo I
PM/Scl, To/Th,
Nükleolar nükleofosmin, nükleolin,
VAR ENA
(AC-8,9,10) fibrillarin, RNA polimeraz
I, NOR-90
Sentromer ENA
VAR VAR CENP-A,B (C)
(AC-3) Sentromer
Nükleer noktalar Sp-100, PML, Karaciğer

VAR
(AC-6,7) p80 koilin paneli
Sitoplazma ASMA
Aktin, vimentin,
VAR fibriler Karaciğer
tropomiyozin, vinkulin
(AC-15,16,17) paneli
Sitoplazma
Lizozom, PL-7, PL11, ENA
VAR benekli
Rib P, Jo-1 Miyozit panel
(AC-18,19,20)
Sitoplazma
PDC-E2/M2, BCOADC- Karaciğer
VAR retiküler
E2, OGDC-E2, E1 PDC paneli
(AMA) (AC-21)
Polar/Golgi
VAR Golgi proteinleri Yok
(AC-22)
Çubuk ve
VAR halkalar IMPDH2 HCV antikorları
(AC-23)
Reporting process
It is of great importance that the ANA IIF results are reported in the appropriate format. First of all, the
ANA test working method should be stated in each report (IIF, solid phase methods-ELISA etc).
In the reporting, after the result is stated as negative or positive considering the limit value determined
by the laboratory, the fluorescent staining pattern and intensity must be included in the positive ANA
reports. Although the term ANA does not include extranuclear antibodies, it was decided to preserve
this term. In case of cytoplasmic or mitotic reactivity, staining patterns should be added to the ANA IIF
test result. When reporting patterns, they should be specified as nuclear, cytoplasmic or mitotic
patterns, and the AC code should be added. In addition to these patterns, the nucleus should also
be reported when the pattern, which is defined as “dense fine speckled” and abbreviated as DFS 70
(dense fine speckled), is observed (see Chapter 5, Spotted patterns (speckled, granular; AC-2,4, for
detailed information). 5.29)). The DFS pattern is mostly due to the presence of antibodies against the
DFS70 antigen, also known as LEDGF/p75 (lens epithelium-derived growth factor p75), and is a rare
pattern in ANA-related rheumatic diseases. It should also be reported separately when the disease is
seen with associated patterns.
The fluorescence intensity observed on ANA IIF slides should be specified

as qualitative (weak positive, +, ++, +++, ++++) or semi-quantitative (titer). Information that
Examples of standard reports recommended in the ANA IIF tests are
must be included in
provided under each pattern in the relevant sections of this Guide. ANA reports:
Reporting in samples with positive ANA screening results can be  Titre
done in two ways:
 Fluorescent
1. Qualitative (weak positive, +, ++, +++, ++++): Giving qualitative staining pattern
results saves time and kit usage. Results are evaluated as weak
positive, +, ++, +++, ++++ according to the brightness of the
image in the scan dilution. Since the interpretation of ANA <1/40
fluorescence intensity in the microscope is subjective, a report
should be considered
like this can be preferred:
negative!
When scanning with 1/100 (see Table 6):
Titers of ANA ≥1/40 to
ANA: Positive (+++) <1/160 should be
considered low positive. In
Note: “(+++) result indicates positivity at titers between
the absence of specific
1/1000-1/3200; If a precise titer result is desired, it is appropriate
symptoms, further
to request a new dilution test.”
diagnostic testing should
When scanning with 1/160 (see Table 7): not be performed, but the
patient should be
ANA: Positive (+++) monitored!
Note: “(+++) result indicates positivity at titers between
ANA ≥1/160 positivity
1/640-1/1280; If a precise titer result is desired, it is appropriate
are significant
to request a new dilution test.”
breakpoints for
2. Semi-quantitative (titer): In the samples found positive by systemic autoimmune
screening dilution, further dilution is made and the final dilution diseases, further
that is positive is determined as the titer value. Images of an diagnostic tests are
example that has been serially diluted in this way are given in recommended!
Figure 6. Quantitative results are more successful in
distinguishing between low and high titer ANA, but there is also
subjectivity in this method.
25
KLIMUD
Table 6. ANA positivity titers when 1/100 is used as the screening dilution
Titre <1/100 1/100 >1/100 - <1/320 ≥1/320-<1/1000 ≥1/1000 -<1/3200 > 1/3200
Fluorescent intensity - (+) + ++ +++ ++++
Result Negative weak positive Positive Positive strong positive very strong positive
Tablo 7. Tarama dilüsyonu olarak 1/160'ın kullanıldığı durumda ANA pozitiflik titreleri
Titre <1/160 1/160 >1/160 - <1/320 >1/320-<1/640 >1/640-<1/1280 > 1/1280
Fluorescent intensity - (+) + ++ +++ ++++
Result Negative weak positive Positive Positive strong positive very strong positive
Figure 6. Example: IIF images obtained as a result of serial dilution of serum from a patient with ANA test speckled
pattern
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In the report, it should be stated

that ANA positivity can be detected in
healthy controls at a titer of 1/40,
10-15% of 1/80 and 5% of ≥1/160!
ANA results should never be
reported simply as "positive". The
initial dilution studied and the
fluorescence intensity detected at that
dilution must be defined!
The patterns of nuclear,
cytoplasmic and mitotic apparatus
should be specified separately in the
report. The AC code of the pattern must
be added to the report! If detected, the DFS pattern, also
defined as "dense fine speckled",
should also be noted in the report!
Quality control
As in all laboratory tests, it is necessary to work in accordance with laboratory quality
standards in ANA IIF tests for the reliability of test results.
Standardization in the ANA IIF method is often problematic, and the ANA pattern and titer are
the most problematic. The main reasons for the problems experienced in standardization are
that the substrate used and the fixation process differ according to the kits, the microscopes
used differ between laboratories, and the subjectivity of the interpretation of IIF images. The
low analytical and clinical sensitivity for some autoantigens is another limitation of the ANA IIF
method. In particular, HEp-2 cells have low analytical and therefore low clinical sensitivity in
detecting anti-Rib-P, anti-SS-A and anti-Jo-1 antibodies.
Different test methods have been developed to standardize and facilitate ANA tests. These
methods include ELISA, immunoblot, and multiplex solid-phase immunoassay. The most
commonly used method is ELISA (see Chapter 4, ELISA, immunoblot tests and standardization
in the detection of autoantibodies).
Efforts are being made to ANA titer calibration and quality control to achieve a world-class
standardization of ANA IIF assays. For this purpose, the World Health Organization (WHO), the
Center for Disease Control and Prevention (CDC), the International Union of Immunology
Societies (IUIS), the European Autoantibody Standardization Initiative (EASI) and some
national initiatives are working towards the creation and distribution of standard reference
serums worldwide.
Systematic evaluation of these reference materials is still ongoing. In order to solve the
standardization problem related to ANA to some extent, there is a reference serum with the
WHO-IRP 66/233 code prepared by WHO and standard sera prepared by the CDC. However,
since it is not easy to provide these serums continuously, it would be a useful approach in
terms of internal quality control for each laboratory to prepare and use well-defined patient
sera as a standard.
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KLIMUD
In order to ensure in-laboratory quality control in ANA IIF tests, the negative and positive
control included in the kit must be studied in every test study, and the positive control should
be diluted in series in line with the manufacturer's recommendations and used as a model for
determining the titer. In addition, as an internal quality control, it can be prepared as a
collection of 20-30 serums that are clinically well-defined (serum samples from patients with a
definite diagnosis of disease as a result of clinical findings and other laboratory examinations)
and stored in aliquots at at least -20°C. It is a useful approach in terms of internal quality
control to include control samples prepared in this way in the test series at every kit or lot
change and at least once a month when working with the same kit and lot (or as often as the
laboratory will determine).
Especially in cases where mercury lamps are used, the linearity of the lamp fluorescence
intensity should be measured at regular intervals.
In addition to internal quality control studies in ANA IIF tests, participation in an external quality
evaluation program is also important for the laboratory to evaluate itself within all laboratories
and among users of the same kit.
It is important to provide two-way education and communication between the laboratory
and the clinician in order to ensure the reliability of the results in the ANA IIF test. Feedback
from clinicians is important for the laboratory to evaluate itself and organize the necessary
corrective-preventive actions.
General recommendations regarding the use of the ANA

test
ANA should only be investigated in individuals with signs of systemic autoimmune disease.
Weak ANA positivity can also be observed in healthy individuals and in many different non-
rheumatic diseases. The relationship between ANA IIF titers and disease course is weak. The
only exceptions to this situation are anti-PCNA and anti-chromatin (nuclear homogeneous
pattern) antibodies, since in both these cases a decrease in titer or a disappearance of the
antibody indicates treatment efficacy.
Kaynaklar
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Testing for Antinuclear Antibodies.
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Herold M, Klotz W, Cruvinel WM, Mimori T, von Muhlen C, Satoh M, Chan EK. Clinical relevance of HEp-2 indirect
immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis
2019 Mar 12. From doi: 10.1136/annrheumdis-2018-214436.
17. Damoiseaux J. Autoimmune highlights. 2020 Feb 6;11(1):4. doi: 10.1186/s13317-020-0127-3.
29
DIAGNOSIS OF AUTOANTIBODIES
CHAPTER 9 - Systemic Vasculitides and Diagnostic
Autoantibodies
CHAPTER 9
SYSTEMIC VASCULITIES
AND AUTOANTIBODIES
USED IN DIAGNOSIS
Systemic vasculitides
Vasculitis is a disease that occurs with the development of malnutrition in the tissues and
organs fed by the relevant vessel as a result of inflammation of the blood vessels. Vasculitides
are basically divided into two as rare primary vasculitides and more common secondary (due
to drug, infection, collagen tissue diseases and malignancies) vasculitides. Although the
cause is unknown in most primary vasculitides, autoimmune events are often described as the
underlying mechanism. Primary vasculitides are classified as large, medium and small vessel
vasculitides according to the vessel diameter they involve.
a. Large vessel vasculitides: Temporal arteritis, Takayasu arteritis, isolated aortitis.
b. Moderate vessel vasculitides: Kawasaki disease, polyarteritis nodosa.
c. Small vessel vasculitides: They are divided into anti-neutrophil cytoplasmic antibody (anti-
neutrophil cytoplasmic anti-tibodies, ANCA) associated vasculitides and immune
complex-mediated small vessel vasculitides.
Three diseases included in ANCA-associated small vessel vasculitides are polyangiitis granulomatosis
(GPA), formerly known as Wegener's granuloma-dust (WG), microscopic polyangiitis (MPA), and
eosinophilic polyangiitis granulomatosis (EGPA), formerly known as Churg-Strauss. ANCA tests are used
to diagnose small vessel vasculitides and to monitor inflammatory activity.
Immune complex mediated small vessel vasculitides include diseases such as anti-glomerular
basement membrane (GBM) disease, cryoglobulinemic vasculitis, and IgA vasculitis. These
diseases are also important in differential diagnosis because they have similarities with ANCA-
related vasculitides. It is known that some laboratories test ANCA and anti-GBM
autoantibodies simultaneously to aid in differential diagnosis. As can be seen, ANCA is an
important autoantibody in terms of giving a name to a disease group.
The idea that ANCA might be important in vasculitis first started in 1982, when Davies et al.
reported the presence of “anti-neutrophil autoantibodies” in the sera of patients with
necrotizing glomerulonephritis and that they might be associated with viral infections. In 1985,
a group of researchers showed that the anti-neutrophil antibody, called anti-cytoplasmic
autoantibody, is strongly associated with a different form of vasculitis (GPA=WG). In
subsequent studies, it has been shown that anti-cytoplasmic autoantibodies cause
involvement in the cytoplasmic pattern (c-ANCA) for GPA and in the perinuclear pattern (p-
ANCA) for MPA. Then, the target antigens causing this different image were determined and
it was shown that the target antigen was proteinase 3 (PR3) for c-ANCA appearance, and
myeloperoxidase (MPO) was the target antigen for p-ANCA appearance.
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Sistemik Vaskülitler ve Tanıda Otoantikorlar - BÖLÜM 9
Pathogenesis
ANCA is not only an autoantibody used in the diagnosis of ANCA-associated vasculitides, it is
also important in pathogenesis in that it activates neutrophils and subsequently causes
endothelial cell damage and vasculitis.
Neutrophils contain four different types of granules. These are primary (azurophilic granules), secondary
(specific granules), gelatinase granules and secretory granules. The enzymes found in the azurophilic
granules are MPO, neutrophil elastase, cathepsin G, PR3, azurosidin, bactericidal/permiability
enhancing protein (BPI) and defensins. Specific granules contain enzymes such as lactoferrin, lysozyme,
collagenase, etc. Although many antigenic targets have been identified in ANCA, those that are
clinically relevant are often those against PR3 and MPO. PR3 is a 29 kD serine protease. It breaks down
the tissue and allows the neutrophils to pass into the inflammatory focus. It is also involved in neutrophil
maturation. MPO is an enzyme that causes the formation of hypochlorite and reactive oxygen
molecules and has a bactericidal effect.
ANCA target antigens are located "in a shield" in the cytoplasmic granules of resting
neutrophils. However, in neutrophils triggered by cytokines and microbial products, these
granule contents migrate to the outer region of the neutrophil plasma membrane and
become possible for ANCA to bind. Cytokine-mediated expression of granule proteins (eg,
PR3) on the surface of neutrophils and monocytes leads to the interaction of ANCA with
surface antigens. As a result of this interaction, neutrophils are activated (eg, respiratory burst,
degranulation, etc.), neutrophil actin skeletal structure is disrupted, capillary sequestration is
observed by distorting the cell shape, and endothelial damage occurs as a result of
interaction with the endothelium. As a result of the interaction of PR3-expressing apoptotic
neutrophils with ANCA, the clearance process by macrophages increases and causes a
proinflammatory response in the form of interleukin (IL)-1, IL-8 and tumor necrosis factor-alpha
(TNF-α) release. However, it is not yet clear what triggers ANCA production at the end of this
information. Activation of neutrophils by ANCA is central to the pathogenesis of ANCA-
associated vasculitis.
ANCA-associated vasculitides
PR3 ANCA titer ANCA-associated vasculitides are necrotizing vasculitides
monitoring is associated with MPO-ANCA or PR3-ANCA, with little or no
important in immune complex deposition, predominantly affecting small
determining disease vessels (capillaries, venules, arterioles, and small arteries).
activity. Microscopic polyangiitis (MPA) is a small vessel vasculitis
involving the kidneys and less frequently the lungs. Unlike other
It has been reported ANCA-related vasculitides, granulomatous inflammation is not
that ANCA becomes observed. The disease peaks at 65-75 years of age. According
positive or titer to studies from Europe, the total annual incidence is 2-8 cases/
increases before million. It is more common in men than in women. It is rare in
relapses. childhood.
In polyangiitis granulomatosis (GPA), there is necrotizing
ANCA titer increases granulomatous inflammation affecting the upper and lower
have predictive respiratory tracts. Arteritis involving large, medium and small vessels is
observed. Necrotizing glomerulonephritis is also common. Its etiology is
value, especially in unknown. According to British sources, its annual incidence is reported
vasculitides with as 8.5 per million, and its annual prevalence is reported as 30 per
renal involvement. million in the USA. It is observed with equal frequency in men and
women. The age at the time of diagnosis is usually 40-50 years.
Eosinophilic granulomatosis with polyangiitis (EGPA) often presents with eosinophil-rich
necrotizing granulomatous inflammation involving the respiratory tract. Necrotizing vasculitis,
asthma and eosinophilia are observed predominantly affecting small and medium-sized
vessels. Nasal polyps are common. Its incidence is reported as 0.9-4.0 per million.
Clinically, ANCA-associated vasculitides have common features and it is difficult to
distinguish between them.
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BÖLÜM 9 – Sistemik Vaskülitler ve Tanıda Otoantikorlar
Diseases in which ANCA positivity can be observed other than vasculitis

ANCA positivity can be observed in glomerulonephritis tables other than the diseases
mentioned above. ANCA positivity is reported at a rate of 30% in anti-GBM disease.
Determination of anti-GBM antibodies is important in the differential diagnosis of ANCA-
associated vasculitides. Diseases in which ANCA can be detected positive and the rates of
seropositivity are given in Table 12.
Table 12. Target antigens of cANCA and pANCA, associated diseases and seropositivity rates
IIF Gray-positive
antigens Diseases
patern (%)
cANCA PR3 Granulomatosis with polyangiitis (GPA, WG) 80-90

Microscopic polyangiitis 20-40
Primary pauciimmune crescent glomerulonephritis 20-40
Granulomatosis with eosinophilic polyangiitis (EGPA, 35
Churg-Strauss syndrome)
pANCA MPO Microscopic polyangiitis 42-70
Primary pauciimmune crescent glomerulonephritis 50
Granulomatosis with eosinophilic polyangiitis (EGPA, 18-60
Churg-Strauss syndrome)
catalase ulcerative colitis 50-70
Alpha enolaseCrohn's disease 10-30
actin active rheumatoid arthritis 30
Lactoferrin drug-induced vasculitis ?
Elastase primary sclerosing cholangitis 87
Cathepsin G Type 1 autoimmune hepatitis 70-90
Defensin Primary biliary cirrhosis 30
Atipik BPI drug-induced vasculitis
ANCA
MPO Inflammatory bowel diseases
cathepsin G rheumatoid arthritis
It has been reported that the evaluation of ANCA and anti-Saccharomyces cerevisiae
antibodies (ASCA) is useful in distinguishing between inflammatory bowel diseases ulcerative
colitis and Crohn's disease. Patients with ulcerative colitis are often pANCA positive and ASCA
negative, while Crohn's patients are more likely to be pANCA negative and ASCA positive.
Methods used to determine ANCA

ANCA is important in the diagnosis or exclusion of the above-mentioned diseases, especially
vasculitides, and in determining disease progression. In the guide published in 2003 on ANCA
evaluation in non-vasculitis diseases, quality control conditions, comments and
recommendations are generally included in ANCA evaluations. The first step in ANCA
evaluation is to screen for the presence of ANCA in normal peripheral blood neutrophils with
the IIF method, and then to specifically identify PR3-ANCA or MPO-ANCAs with the ELISA
method. There are ELISA kits that can detect mono-specifically PR3-ANCA or MPO-ANCA, as
well as ANCA profile ELISA kits that can simultaneously detect PR3, MPO, BPI, elastase,
cathepsin G, lysozyme, and lactoferrin.
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KLIMUD
With the help of some IIF kits developed in recent years, the presence of ANCA in the patient
can be detected simultaneously, while the presence of PR3-ANCA or MPO-ANCA on a
different substrate can be determined in the same serum sample.
ANCA assessment with II F test

The first step in ANCA evaluation is to screen the substrate prepared from peripheral blood
neutrophils with the IIF method, the first substrate to be evaluated is ethanol-fixed
granulocytes. Because the separation of perinuclear and cytoplasmic ANCA can only be
made on this substrate. The serum dilution to be used is 1/10, the conjugate used should be
the IgG type. After working on this substrate, essentially two different patterns are detected:
c-ANCA: In the cytoplasmic type, there is diffuse granular involvement spread to the
neutrophil cytoplasm and central intralobular enhancement in fluorescent staining. The cell
nucleus is not involved.
p-ANCA: In the perinuclear type, a smooth fluorescence is observed that wraps around the
cell nucleus in the form of a ribbon and extends to the nuclear region (Figure 27).
Figure 27. ANCA positivity in

ethanol-fixed granulocytes.
The reason for this different staining is that if they are not placed in a fixative such as formalin
that fixes the proteins in place before fixation with alcohol, that is, they are fixed only with
ethanol, soluble granule elements such as MPO, lactoferrin, elastase, cathepsin G and basic
proteins migrate towards the nucleus. They are ligated and cause perinuclear staining. The
electrical attraction of the positively charged granule contents by the negatively charged
nuclear membrane under test conditions is thought to play a role in this.
Figure 28. ANCA positivity in

ethanol and formalin-fixed
granulocytes
Source: Hoffman GS, Specks
U: Antineutrophil
cytoplasmic antibodies.
Arthritis Rheum 1998;
Adapted from 41:1521-1537.
114
Apart from these two main patterns, there is also a-ANCA pattern defined as “atypical ANCA”. In this
less common pattern, cytoplasmic and perinuclear patterns are observed together. There is no central
intralobular enhancement usually seen in cANCA, or the cytoplasm shows diffuse rather than granular
staining. The target antigen has been shown to be more commonly BPI. Atypical ANCA is thought to be
associated with drug (most often propyl thiouracil, hydralazine) vasculitides, chronic diseases,
inflammatory bowel diseases, or rheumatoid arthritis.
After perinuclear-cytoplasmic separation is made in the ethanol-fixed substrate, the formalin-fixed

granulocyte substrate is evaluated. Fluorescent staining is only scattered in the cytoplasm, as formalin
does not allow the migration of the granules and fixes them where they are in the cytoplasm. However,
it was stated that some ANCA types were not detected in formalin-fixed granulocytes due to their
sensitivity to formalin (ANCA positive-formalin-sensitive); such as eg, BPI, lactoferrin, cathepsin G.
Therefore, when both substrates are examined together, both perinuclear and cytoplasmic
discrimination can be made, as well as formalin-sensitive-resistant discrimination in identifying target
antigen.
An important point to be considered in the evaluation of ANCA
with the IIF method is the presence of accompanying ANA in
patient serum. Considering that the dilution in ANCA study is If accompanying
1/10, ANA positivity in this dilution, which can mask pANCA, can ANA is thought due to
cause problems in evaluations. For this reason, it is
intranuclear
recommended to study MPO-ANCA and PR3-ANCA ELISA in
serum samples with ANA positivity. It is known that ANA, which
involvement in the
exists alone, is lost in the formalin-fixed granulocyte substrate or ethanol-fixed
continues only with nuclear staining. However, in order to granulocyte substrate,
reveal the masked pANCA in the presence of ANA HEp-2 substrate should
accompanying pANCA, some manufacturers added HEp-2 be evaluated and ANA
substrate containing scattered granulocytes next to ethanol- IIF should be studied in
fixed granulocyte and formalin-fixed granulocyte substrates. In
the patient report. (It
the presence of ANA accompanying ANCA, HEp-2 cell nuclei
on this substrate fluoresce. However, granulocyte nuclei should be kept in mind
appear brighter than HEp-2 cells as they take up more that the screening
fluorescent dyes due to both ANA and pANCA. dilution is 1/10 in the
ANCA test!)
According to the consensus report of 2017, up-to-date information regarding ANCA

tests:
If there is
pANCA  A strategy based on clinical findings should be followed when ordering the test.
together with  Antigen-specific tests for high-quality PR3-ANCA and MPO-ANCA should be used
ANA in the as the initial screening method. (Not yet used in routine practice).
serum, the  If the result is negative for both PR3-ANCA and MPO-ANCA and there is still
fluorescence strong clinical suspicion for small vessel vasculitides; use of other
immunoassays and/or IIF or referral to an experienced laboratory is
intensity of recommended. Application of a second test or IIF will significantly increase
granulocytes specificity in cases with low positive test results.
scattered on  In the case of a negative PR3-ANCA and MPO-ANCA result, the diagnosis of
the HEp-2 ANCA-associated vasculitis cannot be excluded.
substrate is  A positive PR3-ANCA and MPO-ANCA result is helpful only in the diagnosis of
ANCA-associated vasculitis, not on its own.
higher than
 Consideration of antibody levels improves clinical interpretation.
that of HEp-2
 These renewed consensus recommendations are particularly valid for the
cells alone. diagnosis of vasculitides such as GPA and MPA, but are not valid as an
adjunctive test for the diagnosis of IBD, OIH, and drug-associated
autoimmunity.
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KLIMUD
pANCA formalin resistant(MPO ANCA)
Figure 160. Ethanol-fixed granulocytes (x40) Figure 161. Formalin-fixed granulocytes (x40)
Figure 162. HEp-2 cells containing granulocytes (x40)
microscopic view Ethanol-fixed granulocytes: Ribbon-like fluorescent staining is seen

around the nuclear membrane.
Formalin-fixed granulocytes: Fluorescent staining in the form of scattered
granules is seen in the cytoplasm.
Since there is no ANA positivity, there is no nuclear staining in HEp-2 cells. In
scattered granulocytes, there is staining around the nuclei.
Related antigens Myeloperoxidase (MPO)
Associated diseases Microscopic polyangiitis (MPA), granulomatosis with eosinophilic polyangiitis (EGPA,
Churg-Strauss syndrome), primary “pauciimmune” crescent glomerulonephritis
REPORT EXAMPLE
Result ANCA: Positive (++)
Pattern: pANCA formalin resistant
Titer: (++) result indicates positivity at titers between 1/32 and 1/100; If a
precise titer result is desired, it is appropriate to request a new dilution test.
Working method IIF

Cell/tissue studied Ethanol-fixed granulocytes, formalin-fixed granulocytes, HEp-2 cells
containing granulocytes*
reference value <1/10 titer - Negative
Description/suggestion It is recommended to run the MPO ANCA ELISA test.
* If HEp-2 cells containing granulocytes are used, they can be specified in the cell/tissue section studied.
116
cANCA formalin resistant(PR3 ANCA)
microscopic view Ethanol-fixed granulocytes: Fluorescent staining is seen in the form of

scattered granules in the cytoplasm and central intralobular enhancement.
Formalin-fixed granulocytes: Fluorescent staining in the form of scattered
Since there is no ANA positivity, there is no nuclear staining in HEp-2 cells.
There is cytoplasmic staining in scattered granulocytes.
Related antigens Proteinase 3 (PR3)
Associated diseases Granulomatosis with polyangiitis (GPA, WG), microscopic polyangiitis,
primary "pauci immune" crescent glomerulonephritis, granulomatosis with
eosinophilic polyangiitis (EGPA, Churg-Strauss syndrome)
REPORT EXAMPLE
Result ANCA: Pozitif (+++)
Patern: cANCA formalin dirençli
Titre: (+++) sonuç 1/100 - 1/320 arasındaki titrelerde pozitifliği iĢaret eder; kesin
bir titre sonucu isteniyorsa yeni dilüsyonlu test istemi yapılması uygundur.
Working method IIF

containing granulocytes
Description/suggestion It is recommended to run the PR3 ANCA ELISA test.
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KLIMUD
pANCA formalin sensitive
microscopic view Etanolle fikse granülositler: Nükleus membranı çevresini kurdele tarzında saran
floresan boyanma olur.
Formalin-fixed granulocytes: No fluorescent staining is observed. HEp-2
cells should also be examined to distinguish the fluorescent appearance
in ethanol-fixed granulocytes from ANA-nuclear membrane positivity. If
there is no accompanying ANA, no staining is observed in the nuclei.
Fluorescent staining is seen around the nuclear membrane in scattered
granulocytes.
Related antigens
Associated diseases Ulcerative colitis, Crohn's disease, primary sclerosing cholangitis, SLE,
rheumatoid arthritis (lactoferrin)
REPORT EXAMPLE
Result ANCA: Pozitif (++)
Patern: pANCA formalin duyarlı
Titre: (++) sonuç 1/32-1/100 arasındaki titrelerde pozitifliği iĢaret eder; kesin bir
titre sonucu isteniyorsa yeni dilüsyonlu test istemi yapılması uygundur.
Working method IIF
Description/suggestion If it is desired to determine the target antigen, it is recommended to run the ANCA profile
ELISA test.
118
cANCA formalin sensitive
microscopic view Ethanol-fixed granulocytes: Fluorescent staining in the form of scattered

Formalin-fixed granulocytes: No fluorescent staining is observed. Since the
presence of accompanying ANA in HEp-2 is not considered, no staining is
observed in nuclei. Fluorescent staining occurs in the form of granules in the
cytoplasm in the scattered granulocytes.
Related antigens Bactericidal/permiability enhancing protein (BPI)
Associated diseases Primary sclerosing cholangitis, ulcerative colitis, Crohn's disease
REPORT EXAMPLE
Result ANCA: Positive (+++)
Pattern: cANCA formalin sensitive
Titer: (+++) result indicates positivity at titers between 1/100 - 1/320; If a
Working method IIF
Description/suggestion If it is desired to determine the target antigen, it is recommended to run the ANCA
profile ELISA test.
119
KLIMUD
pANCA- ANA togetherness
microscopic view Ethanol-fixed granulocytes: Fluorescent staining is observed in the nucleus. Due
to the presence of pANCA, the fluorescence intensity around the nuclear
membrane is more intense than in the interior of the nucleus.
Formalin-fixed granulocytes: If formalin-resistant, fluorescent staining is seen in
the cytoplasm as scattered granules. If formalin-sensitive, no fluorescence is
detected in the cytoplasm of formalin-fixed granulocytes.
Fluorescent staining is observed in nuclei due to the presence of concomitant
ANA in HEp-2. Fluorescent staining is seen both in the nucleus and around the
nucleus in the scattered granulocytes. However, the distinguishing point here is
that the fluorescence intensity in granulocytes is significantly higher than in
HEp-2 cells.
Related antigens MPO (see Chapter 5, antigens that may cause the presence of ANA)
Associated diseases Microscopic polyangiitis (MPA), granulomatosis with eosinophilic

polyangiitis (EGPA, Churg-Strauss syndrome), primary “pauci immune”
crescent glomerulonephritis
120
REPORT EXAMPLE
Result ANCA: Positive (+++)
Pattern: pANCA formalin resistant
Titer: (+++) result indicates positivity at titers between 1/100 - 1/320; If a
Working method IIF
Çalışılan hücre/doku Ethanol-fixed granulocytes, formalin-fixed granulocytes, HEp-2 cells

reference value <1/10 titre - Negative
Description/suggestion It is recommended to study the ANA IIF to assess the presence of ANA. It is
recommended to run the MPO ANCA ELISA test.
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KLIMUD
ANCA- cytoplasmic staining association
microscopic view Ethanol-fixed granulocytes: Fluorescent staining in the form of scattered

granules in the cytoplasm is observed in the cANCA pattern. In the pANCA
pattern, there is involvement around the nuclei.
Formalin-fixed granulocytes: With ANCA resistant or susceptible to
formalin, cytoplasmic staining may or may not be seen because staining
may persist on the formalin substrate due to some concomitant
cytoplasmic autoantibodies other than ANCA.
If the accompanying cytoplasmic staining is due to AMA, granular staining
is observed in the cytoplasm of HEp-2 cells. The intensity of fluorescent
staining in granulocytes is higher than the intensity of fluorescent staining in
HEp-2 cells.
Related antigens see. Antigens causing the pANCA and cANCA pattern, Table 12
If the accompanying cytoplasmic staining is of AMA, see Supplementary
Fig. Chapter 7, Diagnosis of autoimmune liver diseases
Associated diseases Granulomatosis with polyangiitis (GPA, WG), microscopic polyangiitis (MPA),
Granulomatosis with eosinophilic polyangiitis (EGPA, Churg-Strauss
syndrome), primary
“pauciimmune” crescent glomerulonephritis, inflammatory bowel diseases,
autoimmune liver diseases
122
REPORT EXAMPLE
Result ANCA: Positive (++) (Denoted as pANCA or cANCA, depending on
involvement - may not be interpreted as formalin resistant/susceptible).
Titer: (++) result indicates positivity at titers between 1/32-1/100; If a precise
titer result is desired, it is appropriate to request a new dilution test.
Working method IIF

reference value <1/10 titre - Negatif
Description/suggestion It is recommended to run the ANCA profile ELISA test. It is

recommended to study AMA IIF for cytoplasmic involvement.
123
KLIMUD
Examples of conditions that can be confused with ANCA

positivity
ANA (homogeneous pattern) positivity
microscopic view Ethanol-fixed granulocytes: Fluorescent staining is observed, including the

inside of the nuclei, due to ANA positivity.
Formalin-fixed granulocytes: No fluorescent staining is observed.
Due to the presence of ANA, HEp-2 cells have fluorescent uptake in the
nucleus. Due to ANA, fluorescent staining is observed in granulocytes
scattered in this substrate, similar to the fluorescence intensity in HEp-2 cells.
Related antigens It can be any antigen that can cause ANA positivity. However, it should be
kept in mind that the dilution studied here is 1/10 and that ANA can be
found at this titer in the healthy population.
Associated diseases (As it is ANCA negative, the associated diseases were not specified.)
REPORT EXAMPLE
Result ANCA: Negative
Working method IIF
reference value <1/10 titre - Negatif
Description/suggestion It is recommended to study the ANA IIF test.
124
ANA (nuclear membrane) positivity
microscopic view Ethanol-fixed granulocytes: Fluorescent staining is observed around the

nuclei due to ANA nuclear membrane positivity. This image should not be
confused with the panCA image. In order to differentiate this from pANCA,
HEp-2 cells must be evaluated.
Formalin-fixed granulocytes: No fluorescent staining.
Due to ANA positivity in the nuclear membrane pattern, HEp-2 cells have
fluorescent uptake around the nuclei. Due to the ANA nuclear membrane in
granulocytes scattered in this substrate, fluorescent staining is observed
around the nuclei, similar to the fluorescence intensity in HEp-2 cells.
Related antigens cf. Chapter 5, ANA Nuclear membrane pattern (AC-11,12)

Associated diseases Since it is ANCA negative, the associated diseases were not specified.
REPORT EXAMPLE
Result ANCA: Negatif
Working method IIF
Description/suggestion It is recommended to study the ANA IIF.
125
KLIMUD
ANA (anti-lysosome) positivity
microscopic view Ethanol-fixed granulocytes: Due to autoantibodies against lysosomal

antigens, diffuse fluorescence uptake is observed in the cytoplasm.
Formalin-fixed granulocytes: Scattered fluorescence uptake is seen in the
cytoplasm. In HEp-2 cells, scattered fluorescent uptakes of various sizes are
observed in the cytoplasm. Fluorescent staining is seen in the cytoplasm,
similar to the fluorescence intensity in HEp-2 cells, in granulocytes scattered
in this substrate.
Related antigens Lysosomal antigens
Associated diseases Since it is ANCA negative, the associated diseases were not specified.
REPORT EXAMPLE
Working method IIF

Description/suggestion Its clinical relevance is unknown. It may not be reported.
126
ANCA negative sample
microscopic view Ethanol-fixed granulocytes: No fluorescent staining is observed.

Formalin-fixed granulocytes: No fluorescent staining is observed.
Related antigens -
Associated diseases -
REPORT EXAMPLE
Working method IIF

reference value <1/10 titre- Negative
Description/suggestion
127
KLIMUD
Algorithm used in the diagnosis of systemic vasculitides
IIF with granulocyte substrate fixed in ethanol
Perinuclear staining cytoplasmic staining Staining in the nucleus Negative
Examine formalin-fixed
Examine formalin-fixed Examine formalin-fixed Examine formalin-fixed
granulocyte and HEp-2
granulocyte substrate granulocyte substrate granulocyte substrate
substrates
There is granular If there is no If there is no

cytoplasmic staining cytoplasmic staining Negative
uptake in granular uptake granular uptake
formalin in the formalin in the formalin
substrate and substrate, but substrate and “ANCA
nuclei are the nuclei are the nuclei are negative”
Available N/A Available Available stained in HEp-2 stained on the stained on the
HEp-2 substrate, HEp-2 substrate, report as
substrate, if the
fluorescence the the
fluorescence fluorescence If ulcerative
report as report as report as report as intensity in
granulocytes is intensity in the intensity in the colitis or
pANCA pANCA cANCA cANCA
formalin formalin higher than granulocytes is granulocytes is Crohn's
formalin formalin
HEp-2 higher than that the same as disease is
resistant sensitive resistant sensitive
of HEp-2. HEp-2 considered,
study ASCA
with the IIF
method.
Report as Report as
In case of pANCA positivity, if Report as
ulcerative colitis or Crohn's disease “pANCA “ANCA
is suspected, study ASCA with the “pANCA
positive negative,
IIF method. positive
formalin ANA IIF is
formalin
sensitive, ANA appropriate
resistant, Positive Negative
IIF study is to study”
ANA IIF
appropriate,
Positive Negative suitable to
ANCA profile
be studied” Think in
ELISA study is
favor of
Think in favor recommended
Crohn's
of ulcerative ”
disease
colitis
Figure 29. Algorithm to be used for ANCA determination.
If ulcerative colitis or Crohn's disease is suspected, an ASCA IIF is recommended.
Saccharomyces cerevisiae x40 ASCA positive Saccharomyces cerevisiae x40 ASCA negative
128
If anti-glomerular basement membrane antibody IIF was used in the differential diagnosis of vasculitis;
Primate kidney x20 Primate kidney x40 Primate kidney x40

anti GBM positive anti GBM positive anti GBM
negative
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OTOANTĠKORLARIN LABORATUVAR TANISI

BÖLÜM 3 - ANA Saptanmasında IIF Yöntemi ve Standardizasyon

Tablo 3. IIF yönteminde test sonucunu etkileyen faktörler

Pre-analytical factors analytical factors Post-analytical factors

− Selection of suitable patients − Method and kit to be studied − Reporting process

Sample acceptance, ANA IIF method and kit to be studied

ANA IIF method

ANA IIF test kit selection

 Homogeneity of cell density and distribution on the slide

Figure 2. HEp-2 cell fine structure

Tablo 4. ANA IIF paternleri ve ilgili antijenler

Because microscopic evaluation is subjective, it is difficult to achieve inter-laboratory and

BÖLÜM 3 - ANA Saptanmasında IIF Yöntemi ve Standardizasyon

PATERN İLGİLİ ANTİJENLER REFLEKS TEST

Nükleer noktalar Sp-100, PML, Karaciğer

The fluorescence intensity observed on ANA IIF slides should be specified

Fluorescent intensity - (+) + ++ +++ ++++

Titre <1/160 1/160 >1/160 - <1/320 >1/320-<1/640 >1/640-<1/1280 > 1/1280

Fluorescent intensity - (+) + ++ +++ ++++

In the report, it should be stated

General recommendations regarding the use of the ANA

Diseases in which ANCA positivity can be observed other than vasculitis

cANCA PR3 Granulomatosis with polyangiitis (GPA, WG) 80-90

Methods used to determine ANCA

ANCA assessment with II F test

Figure 27. ANCA positivity in

Figure 28. ANCA positivity in

After perinuclear-cytoplasmic separation is made in the ethanol-fixed substrate, the formalin-fixed

According to the consensus report of 2017, up-to-date information regarding ANCA

pANCA formalin resistant(MPO ANCA)

Figure 162. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Ribbon-like fluorescent staining is seen

Working method IIF

cANCA formalin resistant(PR3 ANCA)

Figure 165. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Fluorescent staining is seen in the form of

Working method IIF

pANCA formalin sensitive

Figure 168. HEp-2 cells containing granulocytes (x40)

cANCA formalin sensitive

Figure 171. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Fluorescent staining in the form of scattered

pANCA- ANA togetherness

Figure 174. HEp-2 cells containing granulocytes (x40)

Associated diseases Microscopic polyangiitis (MPA), granulomatosis with eosinophilic

Working method IIF

Çalışılan hücre/doku Ethanol-fixed granulocytes, formalin-fixed granulocytes, HEp-2 cells

reference value <1/10 titre - Negative

ANCA- cytoplasmic staining association

Figure 177. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Fluorescent staining in the form of scattered

Working method IIF

Cell/tissue studied Ethanol-fixed granulocytes, formalin-fixed granulocytes, HEp-2 cells

reference value <1/10 titre - Negatif

Description/suggestion It is recommended to run the ANCA profile ELISA test. It is

Examples of conditions that can be confused with ANCA

ANA (homogeneous pattern) positivity

Figure 180. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Fluorescent staining is observed, including the

ANA (nuclear membrane) positivity

Figure 183. HEp-2 cells containing granulocytes (x40)

microscopic view Ethanol-fixed granulocytes: Fluorescent staining is observed around the