Maimun Z Arthamin Lab Patologi Klinik FKUB/RSSA

Maimun Z ArthaminLab Patologi Klinik FKUB/RSSA

Immunological laboratory diagnostic methodscan 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

6

• Dasar Reaksi Ag dengan Ab spesifik• Tujuan

Mendeteksi keberadaan Ag dalam serum memakai Ab spesifikMendeteksi keberadaan Ab dalam serum memakai Ag yang sesuai

• Manfaat 1. Menentukan status imunitas2. Memperkirakan prevalensi

penyakit3. Mengetahui adanya invasi

mikroorganisme, jika isolasi kuman tidak dapat dilakukan

4. Menunjang diagnosis penyakit8

Macam :

1. Uji kualitatif hasil + / -2. 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, urinPlasma 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

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Teknik Pemeriksaan Imunoserologi

1. Imunopresipitasi2. Aglutinasi, flokulasi3. Fiksasi komplemen4. Radioimmunoassay (RIA)5. Enzyme immunoassay (EIA) atau

Enzyme linked immunosorbent assay (ELISA)

6. Immunofluorescent (IF)7. Immunochromatographic

technique (ICT)

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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

IMUNOASSAI NON LABEL

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

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 larutZE lebar → Ag bersifat tdk mudah larut, BM

besar, & multikomponen Ag

Imunopresipitasi…

PR

ES

IPIT

AS

I A

NT

IGE

N-A

NT

IBO

DI

Prozone KONSENTRASI ANTIGEN Postzone

Ekses antibodi Seimbang Ekses antigen

2. Aglutinasi

– Umumnya : Ag bentuk partikel + Ab spesifik → Aglutinasi

– Reaksi 2 tahap :1. Ab dgn salah satu antigen binding site

(Fab) bereaksi dgn Ag2. 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

+Tahap I

Ag Ab K.I

AglutinasiTahap II

Contoh-contoh pemeriksaan aglutinasi

1. Aglutinasi direk

2. Aglutinasi indirek (pasif)

3. Aglutinasi pasif terbalik

4. Hambatan aglutinasi

3. Fiksasi komplemen

Tahap :

1. Pengikatan sejumlah komplemen oleh kompleks Ag – Ab

2. Penghancuran Eritrosit yg telah dilapisi hemolisin oleh komplemen

Interpretasi :

tidak hemolisis

hemolisis Contoh : Deteksi tripanosoma, virus

+

_

Tes Fiksasi Komplemen

++

c

Ag

Ab

+ +

+c

Ag

++

+

Fiksasi komplemen

Eritrosit

Sistem hemolitik

Hemolisin tidak hemolisis?

Hemolisin

hemolisis C - Komplemen

Eritrosit

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)

Imunoasai berlabel

Imunoassai Berlabel

Imunoassai berlabel homogenSinyal kadar analit diperoleh langsung dari reaksi ikatan label dgn analitTidak memerlukan separasi Ag terikat dan Ag bebas (B/F)

Imunoassai berlabel heterogenSinyal kadar analit diperoleh secara tdk langsungMemerlukan seperasi B/FLebih 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

1. Radioimunoassai (RIA/IRMA)

2. Imunofloresen (IF)

3. Enzyme Imunoassay (EIA/ELISA)

4. Imunokromatografi (ICT)

1. Radioimunoasai

● 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:

a.Radioimmunoassay (RIA): radiolabelisasi pada Ag

b.Immunoradiometric Assay (IRMA): radiolabelisasi pada Ab

● 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

+E

E +E

E

E

E EE

Radiaton counter

cuciAb pd fase

padatAg

berlabel Ag

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 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)a. Reaksi langsung (Double Antibody

Sandwich)/Asai Imunometrik untuk melacak analit yang besar dan memiliki > 1 epitop (LH,hCG dan HIV)

b. 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 0.1 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.

– 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.

The three main types of skin tests are

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.

1. Fluorescent antinuclear antibody (ANA)

2. ANA panel: anti-ds DNA*, anti-Sm, anti-U1RNP, anti-Ro/SSA, anti-La/SSB

3. Serum complement level

4. VDRL**

5. Anticardiolipin antibodies

6. Lupus anticoagulant

7. Coombs test*dsDNA, double-stranded DNA**VDRL, Venereal Disease Research Laboratories

Serologic Tests Useful in the Diagnosis of Systemic Lupus Erythematosus

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

• 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

Complement

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

• 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).

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 direct Coombs test

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. 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.