LOGOwww.themegallery.com 15 September 2014

Hindawi Publishing Corporation Journal of Immunology ResearchVolume 2014, Article ID 149316

Interpretation of ANA Indirect Immunofluorescence Test Outside the Darkroom Using NOVA View Compared to Manual MicroscopySusan S. Copple,1,2,3 Troy D. Jaskowski,4 Rashelle Giles,1 and Harry R. Hill1

Elvan, dr / Betty Agustina T, dr., Sp. PK

Jurnal Imunologi I

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Pendahuluan

2

** Anti Nuclear Antibodi (ANA) tes :

11 Uji Skrining untuk Autoimun dg deteksi Ab thd Ag inti & sitoplasma serta isinya

22 Metode standard pemeriksaan: IndirectImunofluoressens dg Substrat Hep-2 Cell

33 Interpretasi hasil: Manual (mikroskopFluoresens & instrumen otomatis)

** Berdasar Rekomendasi American College for Rheumathology (ACR)

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IIF Hep-2 Cell

Indirect Imunofluoressens (IIF): pola fluoressens inti (homogeneous,

spekled, nucleolar, centromere, periferal/rimmed, proliferating cel nuclear Antigen (PCNA)

Substrat: Hep-2 Cell (Human Epitelial Cell) mampu

deteksi lebih dari 100 autoantibodi

Hasil IIF: Semikuantitatif dan pola fluorossensi inti

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Click to edit headline

Kualitas IIF HEp-2 Cell

MIKROSKOP FLUORESSENS

TIPE LAMPU

MASA LAMPUKONJUGAT

SUBSTRAT HEp-2 Cell

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Interpretasi ANA tes IIF Hep-2 Cell

Manual

Mikroskop Imunofluoressens (kamar gelap)

Padat Karya

tenaga Ahli bersertifikat

Subjektif

Rentan terhadap pembacaan Bias

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Interpretasi ANA tes IIF Hep-2 Cell

Instrumen otomatis

Dikembangkan pertama kali oleh Perner et al pd tahun 2002

Berdasarkan intensitas cahaya Imunofluoressens / Light Intensity Unit

(LIU)

Dengan intensitas tertentu (cutoff) ditentukan nilai positif/negatif

(Semikuantitatif)

Pengenalan pola floresensi inti (homogeneous, speckled, centromere,

nucleolar, and nuclear dots.)

Untuk pola campuran tidak kenal, konfirmasi manual dg alat

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Interpretasi ANA IIF Hep-2 sel

Manual Mikroskop Otomatis

Tenaga ahli dan sertifikat

Rantan thd pembacaan Bias (subyektif)

Mikroskop fluoressens kamar gelap

Pengenalan pola Fluoresensi inti yg diarsipkkan

Cut off Ligh Intensity Unit (+/-)

Berdasar intensitas fluoresensi

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TUJUAN

Evaluasi Nova View terhadap Mikroskop fuoressens oleh tenaga ahli untk interpretasi tes ANA dengan Indirect Imunofluressens Hep-2 Cell

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Bahan dan MetodeSampel:Standard pola ImunofluoressensInstrumen dan reagen:Analisa data statistik:Kesesuaian manual & otomatis baik

sensitivitas maupun spesifitasi dg tabel kontigensi (tabel 2x2) dan CI untuk kappa statistic Korelasi antara manual & otomatis dengan spearman’s

correlation Kurva ROC dari interpretasi titer nova view

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Sampel

Clinical Samples (Diagnoses were established as previously described or according to the respective disease

classification criteria)CDC ANA Reference Panel (Biological Reference Reagents, NCID/SRP/BRR, Mailstop C-21, Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd. N.E., Atlanta, GA,

U.S.A).Consecutive Routine Samples (University of Utah Institutional Review Board-approved protocol number 7275 to meet the Health Information Portability and Accountability Act Patient Confidentiality Guidelines)

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Sampel n: 369

Klinis

Autoimun disease* n: 158

44 RA

50 SLE

35 SSc

19 SJs

10 PM

Sehatn: 99

CDCKontrol serum disease n: 12

ARUP Lab. Consecutive sampel n: 100

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Instrumen & Reagen

Manual: Mikroskop Nikon Eclipse 400 (ARUP Laboratories, Salt

Lake City, Utah) sumber cahaya LED.Otomatis:

NOVA View instrument with 1.0.2 softwareReagen:

NOVA Lite HEp-2 IgG ANA with DAPI kit Konjugat FITC fluorophor dg diamidino-2-phenylindole

(DAPI)

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HASIL

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Diskusi

Rekomendasikan ANA tes dengan IIF sbg metode rujukan

Keterbatatasan: Subyektifitas tinggi

Nova view meminimalisir subyektifitas

Teliti dalam memvalidasi alat otomatis

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Diskusi

Nova view, mempunyai instrumen gambar yg berkualitas tinggi sesuai dengan hasil IIF manual

Hasil Nova View dibanding IIF manual pada negatif dan positif sera, memiliki kesesuain sebesar 97%

Nova view mempunyai kesesuaian interpretasi gambar yg baik dengan hasil dari mikroskop

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Diskusi

Cutt off Nova view untuk hasil + / - : (+ ) : LIU ≥ 100 ( - ) : LIU < 100

- Namun, cutoff LIU ini tidak selalu berkorelasi dengan mikroskop manual untuk hasil positif rendah/ negatif rendah

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Kesimpulan

Adanya kesesuaian dalam interpretasi hasil

pemeriksaan ANA tes dengan metode Hep-2

cell, Indirect Imunofluoressens antara nova

view terhadap pemeriksaan manual dengan

mikroskop Imunofluoressens

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TERIMAKASIH

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Indirect Immunofluorescence

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ANA IIF Hep-2 Cell

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Principle of Fluorescence1. Energy is absorbed by the atom which becomes excited.2. The electron jumps to a higher energy level.3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing.

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Mikroskop fluorescence

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NOVA View

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NOVA VIEWvideo

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• In 2008, the American College of Rheumatology (ACR) initiated a task force to investigate and collect information from physicians to evaluate the extent of the problem.

• In August 2009, the ACR issued a statement declaring HEp-2 IFA as the preferred method for ANA screening.

The term "anti-nuclear antibodies" describes a variety of autoantibodies that react with constituents of cell nuclei including DNA, RNA and several proteins and ribonucleoproteins.1

These autoantibodies occur with high frequency in patients with connective tissue or rheumatic diseases, especially systemic lupus erythematosus (Tan EM: Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Advances in Immunology 33: 167-239, 1982 )

• *

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• Autoantibodi adalah ciri dari autoimunitas, yang antibodi anti-inti (ANA) telah mengambil tengah panggung selama 60 tahun terakhir. Istilah ANA sudah ketinggalan jaman dan bahkan membingungkan sebagai label sejarah ini telah datang untuk mencakup antibodi diarahkan pada berbagai kompartemen selular termasuk konstituen inti, komponen selubung nukleus, aparatus gelendong mitosis, sitosol, organel sitoplasma dan membran sel.

• Deteksi antibodi anti-seluler dari keluarga ANA adalah penting untuk diagnosis banyak autoimun diseases.1 2

• Selain itu, antibodi spesifik dari keluarga ANA dapat hadir tahun sebelum munculnya penyakit yang jelas, dan untuk beberapa kondisi pengujian serologi dapat memberikan informasi yang berguna pada kemungkinan klinis atau komplikasi (misalnya, miopati inflamasi, lupus eritematosus sistemik (SLE)). Oleh karena itu,

• penentuan ANA dapat memungkinkan prediksi, diagnosis dan aktivitas penentuan penyakit autoimun tertentu *

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• Antobodi anti-nuclear adalah antobodi yang tak biasa, dapat dideteksi di darah, memiliki kemampuan untuk mengikat struktur tertentu pada nukleus sel. Nukleus menganduk DNA, materi genetika. ANA ditemukan pada pasien dengan sistem imun yang telah terkan faktor predisposisi dan akhirnya terkena radang serta merusakan jaringan tubuh sendiri.

• Dengan ditemukannya titer ANA yang tinggi (1:160 misalnya), berbagai subtipe ANA dapat dibedakan.[1] Berikut adalah subtipe pada sel HEp-2, misalnya:

• Anti-ENA (Extractable nuclear antigen)• Anti-gp-210 (gp-210 pori nuklear)• Anti-p62 (Nucleoporin 62)• Anti-dsDNA (DNA rantai ganda)• Anti-Ro (SS-A)• Anti-La (SS-B)• Anti-Sm (Antigen Smith)• Anti-nRNP (ribonukleoprotein nukleus)• Anti Scl-70 (topoisomerase I)• Anti-centromere• Anti-Jo

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• The increased sensitivity of the HEp-2 ANA test compared with the ANA test performed on rodent tissue is associated with a lower specificity.

• Thus, more patients with diseases other than SLE as well as normal healthy persons have positive ANA test results.

• Some laboratories have attempted to adjust for this by using a higher titer of ANA as a cutoff for a positive result. For example, on rodent tissues, ANA titers of 1:20 or 1:40 or higher have been called positive, whereas on the HEp-2 substrate, titers of 1:80 or higher are usually called positive

Arthur Kavanaugh, et al.

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Recommendations of the American College of Rheumatology (ACR) Antinuclear Antibody (ANA) Task Force

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Common IF-ANA patterns associated with specific diseases

ANA pattern Antigen Associated diseases

Speckled ENA, RNP, Sm, SSA/Ro, SSB/La, Scl-70, Jo-1, ribosomal-P

SLE, Mixed CTD, SS, Primary Sjogren's syndrome, PM

Homogenous dsDNA, Histones SLE, Drug induced SLE

Peripheral (rim) RNP, Sm, SSA/Ro SLE, SS

NucleolarAnti-PM-Scl, anti-RNA polymerase I-III, anti-U3-RNP, To RNP

SS, PM

Centromere CENP A-E Limited SS

Kumar et al. Diagnostic Pathology 2009 4:1 doi:10.1186/1746-1596-4-1

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• Autoabs directed specifically against intra-nuclear antigens

• Most commonly (not always) detected by immunofluorecence on intact cells

• If an ANA is detected, the specific antigen may or may not be known (most ANA’s aren’t known – only detected by fluorescence inside of an intact nucleus)

• When an ANA screen is positive, one then uses more specific tests against known antigens to determine if that ANA is relevant to medical disease (Subserology)

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Mikroskop Fluorescence

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• The NOVA View measures light intensity in light intensity units (LIU). Calibration of the FITC light intensity standardizes the analytical sensitivity of the instrument relative to a master instrument, thereby minimizing instrument to instrument variation. This procedure measures the FITC output of the system in LIU, calculates a factor relative to the target value generated by the master instrument at manufacturing, and incorporates the factor into the software settings. The FITC light intensity should be calibrated at installation, during preventative maintenances, and at any major service or breakdown.

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NOVA Lite® HEp-2 ANA Kit with DAPI

• Indirect immunofluorescence is the reference method for ANA testing. Common substrates are thin sections of rodent organs or various types of cell lines. It is generally agreed that cell line substrates are preferable to organ sections since these rapidly dividing cells have higher levels of certain clinically relevant antigens, including centromere, SS-A(Ro), Scl-70 and PCNA/Cyclin.

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• Besides the type of substrate, three other factors are critical to the performance of an ANA test:

1) the fixative used in preparing the slide, 2) the fluorescein to protein (F/P) ratio and 3) the immunoglobulin subclass specificity of the conjugate.

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Ig G sbg konjugat

• Virtually all clinically significant autoantibodies exhibit IgG subclass specificity even in the presence of IgM and IgA specific ANA.4 In contrast, ANA found in healthy blood donors are generally of the IgM and IgA subclass only.5 Because of this, conjugates specific for IgG are more disease specific.

• In addition, the IgG conjugate specificity eliminates physiologic false positive results due to normally occurring low titer IgM autoantibodies, often found in older but otherwise healthy persons

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NOVA Lite® HEp-2 ANA

• the substrate chosen for NOVA Lite® HEp-2 ANA is optimally-fixed human epithelial (HEp-2) cell line and the conjugate is affinity purified anti-human IgG possessing a carefully selected F/P ratio

• HEp-2 (human epithelial cell) substrate slides; 12 wells/slide, with desiccant

• Anti-Human IgG Conjugate (Goat), fluorescein labeled in buffer containing DAPI and 0.09% sodium azide

• ANA Titratable Endpoint Pattern Control, 1 vial of buffer containing 0.09% sodium azide and human serum antibodies to HEp-2, prediluted

• IFA System Negative Control, 1 vial of buffer containing 0.09% sodium azide and no human serum antibodies to HEp-2, prediluted

• PBS II Concentrate (40x), sufficient for 2000 mL • Mounting Medium, 0.09% sodium azide • Coverslips 04/19/2023 66

Prosedur Indirect Immunofluorescene

• In the indirect immunofluorescence technique, samples are incubated with antigen substrate and unreacted antibodies are washed off.

• The substrate is incubated with specific fluorescein labeled conjugate and then unbound reagent is washed off.

• When viewed through a fluorescence microscope, autoantibody positive samples will exhibit an apple green fluorescence corresponding to areas of the cell or nuclei where autoantibody has bound.2

• Tan EM, et al.: The 1982 Revised criteria for the classification of systemic lupus erythematosus. Arthritis and Rheumatism 25: 1271-1277, 1982.

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Spesimen

• This procedure should be performed with a serum specimen. Addition of azide or other preservatives to the test samples may adversely affect the results. Microbially contaminated, heat-treated, or specimens containing visible particulates should not be used. Grossly hemolyzed or lipemic serum specimens should be avoided.

• Following collection, the serum should be separated from the clot. CLSI (NCCLS)

Document H18-A3 recommends the following storage conditions for samples: 1) Store samples at room temperature no longer than 8 hours. 2) If the assay will not be completed within 8 hours, refrigerate the sample at 2-8°C. 3) If the assay will not be completed within 48 hours, or for shipment of the sample, freeze at -20°C or lower. Frozen specimens must be mixed well after thawing and prior to testing.

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metode• Interpretasi hasil:• Negative Reaction. A sample is considered negative if specific staining is equal

to or less than the IFA System Negative Control. Samples can exhibit various degrees of background staining due to heterophile antibodies or low-level autoantibodies to cytoplasmic constituents such as contractile proteins.

• Positive Reaction. A sample is considered positive if specific staining is observed to be greater than the IFA System Negative Control.

Determine the fluorescence grade or intensity using these criteria: 4+ Brilliant apple green fluorescence 3+ Bright apple green fluorescence 2+ Clearly distinguishable positive fluorescence 1+ Lowest specific fluorescence that enables the nuclear and/or cytoplasmic

staining to be clearly differentiated from the background fluorescence.

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Pattern Interpretation. • A variety of patterns of nuclear and/or cytoplasmic staining can be exhibited depending on the types and relative amounts of autoantibodies present in the sample.

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• Homogeneous: A solid staining of the nucleus with or without apparent masking of the nucleoli.– Nuclear antigens present: dsDNA, ssDNA, histones– Disease association: High titers are suggestive of SLE; lower titers are suggestive of SLE or other

connective tissue diseases. • Peripheral: A solid staining, primarily around the outer region of the nucleus, with

weaker staining toward the center of the nucleus.– Nuclear antigens present: dsDNA, ssDNA, DNP, Histone – Disease association: High titers are suggestive of SLE; lower titers are suggestive of SLE or other

connective tissue diseases. • Speckled: A fine or grainy appearing staining of the nucleus, generally without

fluorescent staining of the nucleoli. – Nuclear antigens present: Sm, RNP, Scl-70, SS-A, SS-B, and other antigen/ antibody systems not

yet characterized. – Disease association: High titers suggestive of SLE (Sm antibody), mixed connective tissue disease

(RNP antibody), scleroderma (Scl-70 antibody), or Sjogren's syndrome-sicca complex (SS-B antibody); lower titers may be suggestive of other connective tissue diseases.

• Nucleolar: Large coarse speckled staining within the nucleus, generally less than 6 in number per cell, with or without occasional fine speckles. – Nuclear antigens present: 4-6S RNA and other unknown nuclear antigens. – Disease association: High titers are prevalent in scleroderma and Sjogren's

syndrome.

• Centromere: A discrete, speckled staining pattern. The nuclear speckles are very discrete and usually in some multiple of 46. – Nuclear antigens present: Chromosomal centromere (kinetochore). – Disease association: Highly suggestive of the CREST syndrome, a variant of

progressive systemic sclerosis (PSS). CREST is a form of PSS with prominent calcinosis, as well as Raynaud's phenomenon, esophageal dysmotility and limited involvement of the skin (often confined to the fingers or face), telanglectasia.

• Mitochondrial: A discrete speckling of the cytoplasm with relative sparing of the nuclear area. – Antigen present: Various types of mitochondrial antigens. – Disease association: High titers indicate primary biliary cirrhosis.

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Keterbatasan 1. High-titered ANA is suggestive of connective tissue disease but should not be considered diagnostic. The ANA result

should be considered in combination with other serological results as well as the overall clinical history of the

patient.

2. ANA patterns often change as the sample is titered out to endpoint. This phenomenon is due to lower titer

antibodies dropping below the sensitivity of the system as more dilute sample is tested.

3. A variety of external factors influence the test sensitivity including the type of fluorescence microscope used, the

bulb strength and age, the magnification used, the filter system and the observer.

4. If a band pass filter is used instead of a 515 barrier filter, increased artifactual staining may be observed.

5. Only pencil should be used to label the slides. Use of any other writing material may cause artifactual staining.

6. All coplin jars used for slide washing should be free from all dye residues. Use of coplin jars containing dye residue

may cause artifactual staining.

7. Results of this assay should be used in conjunction with clinical findings and other serological tests.

8. The assay performance characteristics have not been established for matrices other than serum. 04/19/2023 73

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Syarat tenaga ahli

• Extensive time is required to train a technologist to be competent in reading and interpreting ANA IIF testing.

• a board certified medical technologist was blinded to sample classification and has 5 years of reading IIF daily at ARUP laboratories.

• People who interpretHEp-2 ANA on clinical sera must be board certified

ANA screening: an old test withnew recommendations, P. L. Meroni et al. 2010

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Hep-2 cell

• Human epithelial type 2 (HEp-2) cells, considered to originate from a human laryngeal carcinoma

• allow recognition of over 30 different nuclear and cytoplasmic patterns that are given by upwards of 50 different autoantibodies are associated with various autoimmune conditions. Vast majority of these patterns will appear on this site. © University of Birmingham 2014

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Hep-2 Cell

• HEp-2 cells are of human origin, have a big cell nucleus and are, therefore, an ideal substrate to identify ANAs. HEp-2 cells (Aesku.Diagnostics, Wendelsheim, Germany) were

• cultured in Earle’s Minimum Essential Medium (EMEM, Biochrom AG, Berlin, Germany), 2 mM L-Glutamine (Biochrom AG, Berlin, Germany), and 10% FCS (PAA, C€olbe, Germany). Cells were

• seeded on glass slides (Menzel, Braunschweig, Germany) and cultured for 2 days at 37C and 5% CO2 in a humidified incubator (Nuaire, Integra Biosciences, Fernwald, Germany)

• Establishment of HEp-2 Cell Preparation for Automated Analysis of ANA Fluorescence Pattern. Daniel Hahm el al, 2006.

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IFA• It is well known that the IFA method is labor-intensive,

subjective, and prone to reader bias. Interpretation requires readers to be well trained and experienced in performing the assay to maintain competence.

• Other variables affecting IFA assay results are the type of microscope bulb used, the hours in use, and the microscope.

• Screening for IgG Antinuclear Autoantibodies by HEp-2 Indirect Fluorescent Antibody Assays and the Need for Standardization, Susan S. Copple, MS, MT(ASCP)SI et al. 2012

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• In three patients, women of 17, 25 and 15 years, suffering from arthralgia and in whom the existence of antinuclear antibodies (ANA) was established, the diagnosis 'systemic lupus erythematosus' was made or considered. Upon reevaluation, the ANA results could not be confirmed. The conclusion was drawn that in patients, who lack one or more clinical signs of a connective tissue disease (CTD), no indication is present for determination of ANA. Literature data indicate that the positive predictive value of ANA for the presence of CTD is 5-10%. Conversely, a negative ANA result does not rule out that CTD is present (negative predictive value: 98.5-99.3%). In addition, the question is raised if a hospital laboratory, lacking experience or routine in ANA assays could reliably perform these assays.

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standard

• The Task Force has reviewed the relevant literature published• to date and has concluded that solid phase immunaoassays may• not be appropriate at present for replacing IFA as a screening test• for the detection of ANA. The number of patients with AIDs• failing to be diagnosed because of a negative ANA ‘screen’ using• ANA ELISA or multiplex assay cannot be adequately ascertained• from the available literature, but may be as high as 35%• (table 1)• Ann Rheum Dis 2010;69:1420–1422.

doi:10.1136/ard.2009.127100 1421

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Automatic ANAtes interpretation

• Abstract. HEp-2 cells are used for the identification of antinuclear autoantibodies (ANA). They allow for recognition of over 30 different nuclear and cytoplasmic patterns, which are given by upwards of 100 different autoantibodies. The identification of the patterns has recently been done manually by a human inspecting the slides with a microscope.

• In this paper we present results on the analysis and classification of cells using image analysis and data mining techniques. Starting from a knowledge acquisition process with a human operator, we developed an image analysis and feature extraction algorithm.

• The collection of the data set was done based on an expert’s image reading and based on the automatic extracted features. A data set containing 132 features for each entry was set up and given to a data mining algorithm to find out the relevant features among this large feature set and to construct the classification knowledge. The classifier was evaluated by cross validation. The results gave the expert new insights into the necessary features and the classification knowledge and show the feasibility of an automated inspection system.

• Mining Knowledge for HEp-2 Cell Image Classification, P. Perner et al, 2002.

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Automati c + -

• This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems.

• Bizzaro N et al.

• Copyright © 2013 Elsevier B.V. All rights reserved.04/19/2023 83

Cont’

• Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells.

• Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians.

Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based mmunofluorescence tests, Karl Egerer1 etal. 2010.

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