Maimun Z Arthamin Lab Patologi Klinik FKUB/RSSA Pemeriksaan Laboratorium Imunologi Maimun Z Arthamin Lab Patologi Klinik FKUB/RSSA

I. Based on a group of diseases that facilitate diagnosis Immunological laboratory diagnostic methods can be classified from several aspects: I. Based on a group of diseases that facilitate diagnosis Immunological profile tests for the detection of immunodefi ciency Hypersensitivity tests Autoimmunity tests

II. Based on availability of the methods Methods performed in a surgery Methods included in haematological or biochemical analysis, and histological or visualisation methods that provide valuable information for immunological diagnosis A group of basic methods conducted in a specialised immunological laboratory Advanced immunological methods above all, used in clinical research

Basic laboratory examinations Leukocyte count and differential leukocyte count, i.e. standard haematological parameters should be included in basic laboratory examinations, Cytology and histology of various samples obtained from biopsies are also of great value for immunological diagnosis. Preliminary methods selected for humoural immunity testing involve the assessment of total immunoglobulins using a simple precipitation method or serum electrophoresis. Inflammatory parameters: the C-reactive protein test

A non-specific immunological profile testing

Immunoassay Sistem pemeriksaan yang mempergunakan satu atau lebih produk atau reagen imunologik Prinsip dasar: ikatan antara molekul imunoglobulin (Ab) dengan antigen (Ag) Hasil interaksi Ag – Ab (kompleks imun) harus terlihat dan dapat diukur

Reaksi Ag dengan Ab spesifik Tujuan Dasar Reaksi Ag dengan Ab spesifik Tujuan Mendeteksi keberadaan Ag dalam serum memakai Ab spesifik Mendeteksi keberadaan Ab dalam serum memakai Ag yang sesuai

Manfaat Menentukan status imunitas Memperkirakan prevalensi penyakit Mengetahui adanya invasi mikroorganisme, jika isolasi kuman tidak dapat dilakukan Menunjang diagnosis penyakit

Macam : Uji kualitatif hasil + / - Uji kuantitatif hasil kadar Pengenceran tertinggi hasil + (uji semikuantitatif) Contoh : pengenceran 1 dalam 8: 1 vol serum +7 vol pengencer: titer 1/ 8 sampel diencerkan 8 X

Bahan pemeriksaan Serum, plasma, urin Plasma hanya untuk pemeriksaan tertentu saja. Puasa: untuk metode aglutinasi. Serum harus dihindarkan dari hemolisis, lipemik & kontaminasi bakteri (pengiriman < 2 jam) Disimpan dalam Suhu 2 – 8oC : 48 jam -20oC s/d - 70oC: > 48 jam Diberi label

Teknik Pemeriksaan Imunoserologi Imunopresipitasi Aglutinasi, flokulasi Fiksasi komplemen Radioimmunoassay (RIA) Enzyme immunoassay (EIA) atau Enzyme linked immunosorbent assay (ELISA) Immunofluorescent (IF) Immunochromatographic technique (ICT) Non Labelling Labelling

Interaksi Antigen-Antibodi Interaksi primer: Pengikatan Ag-Ab tingkat molekuler Memerlukan indikator/label (isotop, enzim, floresen) Sesuai utk pengukuran Ag/Ab dgn kadar yg rendah Interaksi sekunder: Reaksi Ag-Ab bisa secara langsung atau dgn bantuan komplemen Prinsip dasar : reaksi presipitasi/ aglutinasi Bila partikel Ag terikat latex atau eritrosit → aglutinasi

+ Primary immune phenomena Secondary immune phenomena Ag Ab Kompleks Imun Secondary immune phenomena

IMUNOASSAI NON LABEL

Interaksi sekunder → Ag-Ab komplek tdk larut (presipitat) 1. Imunopresipitasi Interaksi sekunder → Ag-Ab komplek tdk larut (presipitat) Media : cair atau semisolid (gel) Faktor yg mempengaruhi : Aviditas Ab → stabilitas komplek Ag-Ab Suhu (optimal 0-37o C) pH (netral = 6-7,5), pH < 6 ; >7,5 → mudah disosiasi Molaritas (molaritas < 0,15 M) ; >0,15 M → men-cegah presipitasi

ZE sempit → Ag bersifat mudah larut Imunopresipitasi… Pembentukkan presipitat terjadi apabila konsentrasi Ag dan Ab seimbang (zona ekivalen = ZE) Konsentrasi Ag berlebih → Komplek Ag-Ab yg terbentuk larut kembali disebut postzone effect Konsentrasi Ab berlebih → Komplek Ag-Ab yg terbentuk tetap larut disebut prozone effect ZE sempit → Ag bersifat mudah larut ZE lebar → Ag bersifat tdk mudah larut, BM besar, & multikomponen Ag

Postzone Prozone Ekses antibodi Seimbang Ekses antigen PRESIPITASI ANTIGEN-ANTIBODI KONSENTRASI ANTIGEN Postzone Prozone

Umumnya : Ag bentuk partikel + Ab spesifik → Aglutinasi Reaksi 2 tahap : Ab dgn salah satu antigen binding site (Fab) bereaksi dgn Ag Fab yg lainnya berikatan dgn Ag lain yg sudah berikatan dgn Ab gumpalan (lattice) Aglutinasi lebih mudah terjadi pada IgM o/k pentamer dibanding IgA dan IgG

Ag Ab K.I + Tahap I Tahap II Aglutinasi

Contoh-contoh pemeriksaan aglutinasi Aglutinasi direk Aglutinasi indirek (pasif) Aglutinasi pasif terbalik Hambatan aglutinasi

3. Fiksasi komplemen Interpretasi : tidak hemolisis hemolisis Tahap : Pengikatan sejumlah komplemen oleh kompleks Ag – Ab Penghancuran Eritrosit yg telah dilapisi hemolisin oleh komplemen Interpretasi : tidak hemolisis hemolisis Contoh : Deteksi tripanosoma, virus + _

+ + Tes Fiksasi Komplemen + c + + + + + c Ab Eritrosit Ag ? Ag Sistem hemolitik Eritrosit Ag + + c + + ? Ab Hemolisin tidak hemolisis Eritrosit + Ag + + + c Hemolisin C - Komplemen hemolisis

Imunoasai berlabel Imunoassai yang menggunakan indikator utk melacak Ag atau Ab dengan konsentrasi rendah Mampu melacak interaksi primer Ag-Ab (initial binding) Sensitifitas analitik lebih tinggi dibanding imunoassai non label Label yg digunakan : isotop : I125, H3, C14 non isotop : enzim (ALP, HRP), floresen (fluorescein, rhodamine), kemiluminesen Selain uji kuantitatif, dpt digunakan pada uji kualitatif (ANA tes, antitiroid Ab)

Imunoassai Berlabel Imunoassai berlabel homogen Sinyal kadar analit diperoleh langsung dari reaksi ikatan label dgn analit Tidak memerlukan separasi Ag terikat dan Ag bebas (B/F) Imunoassai berlabel heterogen Sinyal kadar analit diperoleh secara tdk langsung Memerlukan seperasi B/F Lebih sensitif dibandingkan imunoassai homogen

Imunoasai kompetitif Ab label dan analit direaksikan sekaligus terhadap Ag Imunoasai non kompetitif Ag analit yg diukur terikat antara Ab phase solid & label Ab Lebih sensitif dibandingkan metode kompetitif

Imunoasai Berlabel Radioimunoassai (RIA/IRMA) Imunofloresen (IF) Enzyme Imunoassay (EIA/ELISA) Imunokromatografi (ICT)

1. Radioimunoasai - β particle → H3 Immunoassay berlabel radioisotop  membedakan Ag yang terikat Ab dengan Ag bebas. Sensitif & spesifik untuk ttk kadar bahan yang amat rendah dalam serum Yang diukur : - γ-ray → I125 - β particle → H3 Radiolabel pada imunoassai dibagi 2 kelompok: Radioimmunoassay (RIA): radiolabelisasi pada Ag Immunoradiometric Assay (IRMA): radiolabelisasi pada Ab

Bahaya efek radiasi bahan radioaktif Kerugian: Bahaya efek radiasi bahan radioaktif Waktu paruh reagen singkat, γ dan β counter mahal Keuntungan: Presisi baik and high sensitivity Isotope conjugation lebih mudah Signal detection tanpa optimalisasi Lebih stabil terhadap interferensi environment (pH, suhu)

+ + Prinsip dasar RIA Radiaton counter cuci E E E E E E Ab pd fase padat Ag berlabel Ag Radiaton counter

2. Imunofloresen assay (IFA) Merupakan teknik untuk deteksi Ag/Ab pada cairan tubuh atau jaringan/sel Prinsip : Molekul yg mampu menyerap energi radiasi dan memancarkannya kembali dlm btk cahaya (floresensi) Memerlukan alat fluorometer/mikroskop Menggunakan label: Fluorescein → warna hijau Rhodamin → warna merah

Imunofloresen assay

Enzyme immunoassay Immunoassay dengan menggunakan label enzim Relatif murah, banyak tersedia, reagen bertahan lama, mudah diotomatisasi, peralatan yang relatif murah Enzim yang digunakan dipilih berdasakan jumlah molekul substrat yang dapat dirubah per satu molekul enzim, mudah dan cepat mendeteksi serta stabil. Dibaca dengan alat : Spektrofotometer ( λ = 492 m) → microELISA reader Fluorometer/Luminometer

Kerugian: Reaksi enzim lebih kompleks dari pada label isotop Masih dipengaruhi faktor environment (plasma constituents) Keuntungan: Mudah dikerjakan Relatif murah, simultan dgn pemeriksaan yg lain Bahaya radioaktif (-)

One-step sandwich EIA

Imunokromatografi Imunokromatografi Kerugian : Keuntungan : Lateral flow test Membacanya cukup dgn mata saja Tidak membutuhkan substrat Penggunaan colloidal gold waktu inkubasi pendek (<15 menit) Kerugian : Nitrocelulose membrane tdk stabil pada suhu ↑ Keuntungan : Prosedur cepat (<15 menit) dan praktis Nilai diagnostik baik Stabil untuk jangka panjang Relatif tidak mahal

Prinsip dasar ICT A. Melacak Analit (Ag) Reaksi langsung (Double Antibody Sandwich)/Asai Imunometrik  untuk melacak analit yang besar dan memiliki > 1 epitop (LH,hCG dan HIV) Reaksi kompetitif/Hambatan kompetitif (competitive Inhibition)  untuk melacak molekul kecil dengan epitop tunggal B. Melacak Ab  Indirect Assay

The Tuberculin test The Tuberculin test has been the traditional method for detection of infection with tubercle bacilli. In clinical practice, it is used to find out the presence or absence of tuberculous infection. This aids in the differential diagnosis of TB among children and to decide about administration of chemoprophylaxis.

The TST is performed by injecting 0 The TST is performed by injecting 0.1 ml of tuberculin purified protein derivative (PPD) into the inner surface of the forearm. The injection should be made with a tuberculin syringe, with the needle bevel facing upward. The TST is an intradermal injection. When placed correctly, the injection should produce a pale elevation of the skin (a wheal) 6 to 10 mm in diameter.

The skin test reaction should be read between 48 and 72 hours after administration. A patient who does not return within 72 hours will need to be rescheduled for another skin test. The reaction should be measured in millimeters of the induration (palpable, raised, hardened area or swelling). The reader should not measure erythema (redness). The diameter of the indurated area should be measured across the forearm (perpendicular to the long axis).

An induration of 5 or more millimeters is considered positive in: HIV-infected persons A recent contact of a person with TB disease Persons with fibrotic changes on chest radiograph consistent with prior TB Patients with organ transplants Persons who are immunosuppressed for other reasons (e.g., taking the equivalent of >15 mg/day of prednisone for 1 month or longer, taking TNF-a antagonists)

An induration of ≥ 10 mm is considered positive in: Recent immigrants (< 5 years) from high-prevalence countries Injection drug users Residents and employees of high-risk congregate settings Mycobacteriology laboratory personnel Persons with clinical conditions that place them at high risk Children < 4 years of age Infants, children, and adolescents exposed to adults in high-risk categories

An induration of 15 or more millimeters is considered positive in any person, including persons with no known risk factors for TB. However, targeted skin testing programs should only be conducted among high-risk groups.

Skin testing for allergies Skin testing for allergies is used to identify the substances that are causing your allergy symptoms. It is often performed by applying an extract of an allergen to your skin, scratching or pricking the skin to allow exposure, and then evaluating the skin's reaction. It may also be done by injecting the allergen under the skin, or by applying it to a patch that is worn on the skin for a specified period of time.

The three main types of skin tests are Scratch test (also known as a puncture or prick test). Areas on your skin are then marked with a pen to identify each allergen that will be tested. A drop of extract for each potential allergen is placed on the corresponding mark. A small disposable pricking device is then used so the extract can enter into the epidermis. Intradermal test Patch test. Another method is to apply an allergen to a patch which is then placed on the skin.

Autoantibodies assay Detection of autoantibodies is an important diagnostic tool for diagnosis of autoimmune diseases. Despite the fact that their occurrence is not quite specific for a respective disease, it may considerably facilitate the diagnosis.

Serologic Tests Useful in the Diagnosis of Systemic Lupus Erythematosus Fluorescent antinuclear antibody (ANA) ANA panel: anti-ds DNA*, anti-Sm, anti-U1RNP, anti- Ro/SSA, anti-La/SSB Serum complement level VDRL** Anticardiolipin antibodies Lupus anticoagulant Coombs test *dsDNA, double-stranded DNA **VDRL, Venereal Disease Research Laboratories

Anti Nuclear Antibodies Antibodies directed against cell nuclei Screening for Rheumatic Autoimmune Diseases, especially for SLE (a diagnostic criteria) Very sensitive but less specific 2 detection methods : IF and EIA Normal value: < 1/100 (IF), < 20 units (EIA) Positive result should be evaluated for more specific auto ab (anti-dsDNA, anti-ENA, etc)

Anti-dsDNA antibodies Autoantibodies which target the ribose phosphate backbone of native DNA Appear in approximately 75% of SLE patients SLE diagnostic test and disease activity monitoring (specificity 94%) Play an important role in the pathogenesis of SLE and strongly correlated with lupus nephritis Normal Value : < 25 IU/ml

Anti Phospholipid Antibodies Antibodies directed against anionic phospholipids membrane cell 2 laboratory test : ACA and LA Diagnosis of SLE and APS Strongly associated with thrombocytopenia, thrombosis and reccurrent abortion Normal value : ACA IgM / IgG < 12 U/ml

Complement Measurement Methods Normal Value : Plasma and cells surface protein 15 % globulin fraction, proenzyme or zymogen Measurement Methods Total Hemolytic Complement Assay Immunoassay Normal Value : C3 : 85 – 185 mg/L ; C4 : 10 - 50 mg/L Indication: Monitoring the disease activity

Rheumatoid Factor Autoantibodies reactive with epitopes in the Fc portion of IgG Diagnosis and prognosis of RA Sensitivity is 60- 90% and specificity 50-60% Correlation with disease activity and disease progression ??? Normal Value : < 1/8 (<8 IU/ml)

Rheumatoid Factor

Coombs test Agglutination of red blood cells is used in the Coombs test. Coombs test (also known as Coombs' test, antiglobulin test or AGT) refers to two clinical blood tests used in immunohematology and immunology. The two Coombs tests are: Direct Coombs test (also known as direct antiglobulin test or DAT). Indirect Coombs test (also known as indirect antiglobulin test or IAT).

The direct Coombs test Used to detect red blood cells sensitized with IgG alloantibody, IgG autoantibody, and complement proteins. It detects antibodies bound to the surface of red blood cells in vivo. The red blood cells (RBCs) are washed (removing the patient's own plasma) and then incubated with antihuman globulin (also known as "Coombs reagent"). If this produces agglutination of the RBCs, the direct Coombs test is positive. Ex: warm type AIHA, HDN

The indirect Coombs test Used in prenatal testing of pregnant women, and in testing blood prior to a blood transfusion. It detects antibodies against RBCs that are present unbound in the patient's serum. In this case, serum is extracted from the blood, and the serum is incubated with RBCs of known antigenicity. If agglutination occurs, the indirect Coombs test is positive

The immune complex test The immune complex test is a test designed to evaluate the status or proper functioning of the immune system. An immune complex is an association formed between large numbers of antigens and the corresponding antibodies. Normally, immune complexes are removed from the bloodstream by macrophage of the spleen and by other specialized cells located in the liver. if this clearance does not occur, the immune complexes will continue to circulate, and will become trapped in the kidneys, lung, skin, joints, or blood vessels.

Immune complexes can be detected by the application of special stains to tissue that has been obtained from a patient. The stains contain antibodies that bind to the complexes and this binding is highlighted by the presence of the staining agent. This test is useful because it directly detects the presence of the immune complexes. However, for routine clinical use, this method is cumbersome and invasive. This has stimulated the development of blood tests that indirectly detect the complexes in the blood serum.

There are several methods available There are several methods available. Immune complex tests include the Raji cell, C1q binding, conglutinin, and anti-C3 assays. The Raji cell assay, for example, detects the immune complexes following the binding of the complexes with complement. C1q binding assy dan Raji cell immune complex assay: Quantitative Flow Cytometry/Semi-Quantitative Enzyme-Linked Immunosorbent Assay A normal result in an immune complex test is a negative result.

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