Maimun Z Arthamin Lab Patologi Klinik FKUB/RSSA

1. PemeriksaanLaboratoriumImunologi Maimun Z Arthamin Lab Patologi Klinik FKUB/RSSA

2. 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

3. 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

4. Basic laboratory examinations • Leukocyte count and differential leukocyte count,i.e. standard haematological parameters shouldbe included in basic laboratory examinations, • Cytology and histology of varioussamples obtained from biopsies are also of greatvalue for immunological diagnosis. • Preliminarymethods selected for humoural immunity testinginvolve the assessment of total immunoglobulinsusing a simple precipitation method orserum electrophoresis. • Inflammatoryparameters: the C-reactive protein test

5. A non-specific immunological profile testing

6. 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

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

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

9. 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

10. 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

11. 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

12. Interaksi Antigen-Antibodi Interaksi primer: • Pengikatan Ag-Abtingkatmolekuler • Memerlukanindikator/label (isotop, enzim, floresen) • Sesuaiutkpengukuran Ag/Abdgnkadarygrendah Interaksisekunder: • Reaksi Ag-Abbisasecaralangsungataudgnbantuankomplemen • Prinsipdasar : reaksipresipitasi/ aglutinasi • Bilapartikel Ag terikat latex ataueritrosit→aglutinasi

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

14. IMUNOASSAI NON LABEL

15. 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

16. Imunopresipitasi… • Pembentukkanpresipitatterjadiapabilakonsentrasi Ag danAbseimbang (zonaekivalen = ZE) • Konsentrasi Ag berlebih→ Komplek Ag-Abygterbentuklarutkembalidisebutpostzone effect • KonsentrasiAbberlebih→ Komplek Ag-Abygterbentuktetaplarutdisebutprozone effect • ZE sempit→ Ag bersifatmudahlarut • ZE lebar→ Ag bersifattdkmudahlarut, BM besar, & multikomponen Ag

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

18. 2. Aglutinasi • 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

19. Ag Ab K.I + Tahap I Tahap II Aglutinasi

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

21. 3. Fiksasi komplemen • Tahap : • Pengikatansejumlahkomplemenolehkompleks Ag – Ab • PenghancuranEritrositygtelahdilapisihemolisinolehkomplemen • Interpretasi : • tidakhemolisis • hemolisis • Contoh : Deteksitripanosoma, virus + _

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

23. Imunoasai berlabel • Imunoassai yang menggunakanindikatorutkmelacak Ag atauAbdengankonsentrasirendah • Mampumelacakinteraksi primer Ag-Ab(initial binding) • Sensitifitasanalitiklebihtinggidibandingimunoassai non label • Label ygdigunakan : • isotop : I125, H3, C14 • non isotop : enzim (ALP, HRP), floresen (fluorescein, rhodamine), kemiluminesen • Selainujikuantitatif, dptdigunakanpadaujikualitatif (ANA tes, antitiroidAb)

24. Imunoassai Berlabel Imunoassaiberlabelhomogen • Sinyal kadaranalitdiperolehlangsungdarireaksiikatan label dgnanalit • Tidakmemerlukanseparasi Ag terikatdan Ag bebas (B/F) Imunoassaiberlabelheterogen • Sinyal kadaranalitdiperolehsecaratdklangsung • Memerlukanseperasi B/F • Lebihsensitifdibandingkanimunoassaihomogen

25. 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

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

27. 1. Radioimunoasai • Immunoassay berlabelradioisotop membedakan Ag yang terikatAbdengan Ag bebas. • Sensitif & spesifikuntukttkkadarbahan yang amatrendahdalam serum • Yang diukur : - γ-ray → I125 - βparticle → H3 Radiolabel padaimunoassaidibagi 2 kelompok: • Radioimmunoassay (RIA): radiolabelisasipada Ag • Immunoradiometric Assay (IRMA): radiolabelisasipadaAb

28. Kerugian: • Bahayaefekradiasibahanradioaktif • Waktuparuhreagensingkat, γdanβ counter mahal • Keuntungan: • Presisibaik and high sensitivity • Isotope conjugation lebihmudah • Signal detection tanpaoptimalisasi • Lebihstabilterhadapinterferensienvironment (pH, suhu)

29. Prinsip dasar RIA + E + E E E E E E E cuci Ab pd fasepadat Ag berlabel Ag Radiaton counter

30. 2. Imunofloresen assay (IFA) • Merupakanteknikuntukdeteksi Ag/Abpadacairantubuhataujaringan/sel • Prinsip : Molekulygmampumenyerapenergiradiasidanmemancarkannyakembalidlmbtkcahaya (floresensi) • Memerlukanalatfluorometer/mikroskop • Menggunakan label: • Fluorescein → warnahijau • Rhodamin → warnamerah

31. Imunofloresen assay

32. Enzyme immunoassay • Immunoassay denganmenggunakan label enzim • Relatifmurah, banyaktersedia, reagenbertahan lama, mudahdiotomatisasi, peralatan yang relatifmurah • Enzim yang digunakandipilihberdasakanjumlahmolekulsubstrat yang dapatdirubah per satumolekulenzim, mudahdancepatmendeteksisertastabil. • Dibacadenganalat : • Spektrofotometer ( λ= 492 m) → microELISA reader • Fluorometer/Luminometer

33. Kerugian: • Reaksienzimlebihkompleksdaripada label isotop • Masihdipengaruhifaktorenvironment (plasma constituents) • Keuntungan: • Mudahdikerjakan • Relatifmurah, simultandgnpemeriksaanyg lain • Bahayaradioaktif (-)

34. One-step sandwich EIA

35. Imunokromatografi Imunokromatografi • Lateral flow test • Membacanyacukupdgnmatasaja • Tidakmembutuhkansubstrat • Penggunaan colloidal gold waktuinkubasipendek (<15 menit) Kerugian : • Nitrocelulose membrane tdkstabilpadasuhu↑ Keuntungan : • Prosedurcepat (<15 menit) danpraktis • Nilaidiagnostikbaik • Stabiluntukjangkapanjang • Relatiftidakmahal

36. Prinsip dasar ICT A. MelacakAnalit (Ag) • Reaksilangsung (Double Antibody Sandwich)/AsaiImunometrikuntukmelacakanalit yang besardanmemiliki > 1 epitop (LH,hCGdan HIV) • Reaksikompetitif/Hambatankompetitif (competitive Inhibition)  untukmelacakmolekulkecildenganepitoptunggal B. MelacakAb Indirect Assay

39. The Tuberculin test • The Tuberculin test has been the traditionalmethod for detection of infection with tubercle bacilli. • In clinical practice, it is used to find out the presence or absenceof tuberculous infection. This aids in thedifferential diagnosis of TB among children andto decide about administration of chemoprophylaxis.

40. 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.

41. 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).

42. 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)

43. 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

44. 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.

47. 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.

48. 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.

50. Autoantibodiesassay • Detection of autoantibodies is an importantdiagnostic tool for diagnosis of autoimmunediseases. Despite the fact that their occurrenceis not quite specific for a respective disease, itmay considerably facilitate the diagnosis.