1. Tutor Imunologi Putaran 1 PEMERIKSAAN ANTIBODI ANTINUKLEARDENGAN METODE ELISA PADA PENDERITA SLE Betty A Tambunan Selasa, 1 Juli 2011

2. MANIFES BERMACAM-MACAM AUTOIMUN DISEASE PENDAHULUAN Systemic Lupus Erythematosus

3. 3 KriteriaAMERICAN COLLEGE OF RHEUMATOLOGY

5. PrinsipPemeriksaan ANA ELISA 5

6. PrinsipPemeriksaan ANA ELISA 6

7. Sampel Pemeriksaan ANA ELISA Serum Sampel yang terkontaminasi mikroba, mengandung partikel yang nyata, hemolisis dan lipemik tidak dapat digunakan untuk pemeriksaan ini. Penyimpanan sampel : Maksimal 48 jam pada suhu 2-80C > 48 jam pada suhu -200C. 7

10. Kontrolnegatif ELISA serummanusia yang tidakmengandungantiboditerhadap antigen intidansitoplasma.Bahan dan Reagen Quanta Lite ANA ELISA

11. GAMBAR BAHAN DAN REAGEN 9 Microwell Plate ELISA High Positive Control ELISA Low Positive Control ELISA Negative Control

13. TMB chromogen

14. HRP Stop Solution,berisi 0,344 M asam sulfur.

15. ANA Sample Diluent :Tris BufferedSaline, Tween 20, stabilisator protein.

16. HRP Wash Concentrate : TrisBufferedSalinedanTween 20Bahan dan Reagen Quanta Lite ANA ELISA

17. GAMBAR BAHAN DAN REAGEN 11 TMB Chromogen HRP Ig Conjugation HRP Stop Solution ANA Sample Diluent HRP Wash Concentrate

18. CHEMWELL 12

19. Indirect ELISA 13

20. Pemeriksaan Quanta Lite ANA ELISA dengan alat Chemwell 1. Sampeldanreagendiletakkan padasuhu kamar(20-260C) 2. MengencerkanHRP Wash Concentrate (1:40) 3. Mengencerkansampel(1:41) 4. Menentukanletaksampeldankontrol padamesinChemwell 14

21. Prosedur Pemeriksaan ANA Menempatkanmicrowellplateyang dibutuhkanpadaholder Menambahkan100 mikroliterlarutan kontroldaninkubasipadasuhukamarselama 30 menit Pencucian Menambahkan 100 µLlarutan konjugat dan inkubasi selama 30 menit 15

22. 9. Mengulangi langkah ke 7 untuk pencucian 10. Menambahkan 100 µL TMB kromogen pada masing- masing well dan inkubasi 30 menit 16

23. 11. Menambahkan 100 µL HRP Stop Solution pada masing-masing well 12. Baca pada panjang gelombang 450 nm 17

24. Quality Control 1. Semua kontrol pada ANA ELISA harus diperiksa bersama sampel untuk memastikan semua reagen dan prosedur baik 2. Semua kontrol tidak perlu diencerkan karena sudah diencerkan sebelumnya 3. Hasil tes dianggap valid jika : OD kontrol (+) harus lebih tinggi dari OD kontrol rendah dan harus lebih tinggi dari OD kontrol (-) 18

25. Perhitungan Hasil OD sampel Hasil = --------------------------------- x kontrol positif rendah OD kontrol positif rendah (unit) Dikatakan : Negatif bila kurang dari 20 unit. Positif moderat bila nilai 20-60 unit. Positif kuat bila nilai lebih dari 60 unit. 19

26. Lembar hasil pemeriksaan 20

27. TERIMA KASIH 21

28. Epidemiologi 1kasus per 2500 orang Lebih banyak pada perempuan (9:1), terutama pada perempuanusiasubur Onset : dekadekeduaatauketigatetapidapat pula terjadi semua usia. 22

29. Quality Control 23 OD : harus lebih dari 0,250 bila kurang dari 0,250 hasilnya dapat positif OD high harus lebih dari 1 OD negatif harus kurang dari 0,15

30. 24 KriteriaAMERICAN COLLEGE OF RHEUMATOLOGY

32. Inkubasi dan cuci untuk menghilangkan antibodi yang berlebihan.

33. Menambahkan antibodi yang dikonjugasi dengan enzim. 25

35. Intensitas reaksi warna yang terjadi sebanding dengan jumlah antibodi di dalam serum.26

37. Murah.

38. Dapat digunakan untuk pemeriksaan jumlah sampel yang besar. 27

39. 28

40. Prinsip Capture ELISA 1. Antibodi Ig M pada serum diikatkan dengan antibodi IgM anti-human pada plat. 2.Mencuci plat dan menambahkan antigen dan dibiarkan agar berikatan dengan antibodi Ig M. 3. Setelah mencuci lagi kemudian menambahkan chromogen 29

41. AntibodiAntinuklear (ANA) Autoantibodiyang melawankomponennukleardansitoplasmasel Dapatmerusakjaringansecaralangsungataupundalambentukendapankompleksimun Tidakspesifikterhadap organ atauspesies. 4kategori: antiboditerhadapDNA antibody terhadaphiston antiboditerhadap protein nonhiston yang terikatpadaRNA antiboditerhadap antigen nukleolus 30

42. Ruammalar Eritematetap, datarataumeninggi, melebihitonjolanmalar, cenderungtidakmengenailipatannasolabialis. Ruamdiskoid Bercakeritematosamenonjoldenganskuamakeratosisdansumbatanfolikel; parutatrofidapatmunculpadalesi yang lebih lama. 31

43. Double Antibody Sandwich ELISA 1. Bahan pemeriksaan yang mengandung antigen direaksikan dengan antibodi pertama yang terikat pada fase padat. 2. Selanjutnya ditambahkan antibodi kedua berlabel enzim. 3. Ditambahkan substrat dari enzim tersebut. 32

44. False Negatif : Kelebihan antigen sehingga antigen binding site telah terisi Afinitas antibodi rendah, kadar antibodi yang berlabel suboptimal. 33

46. Prevalensi dapat lebih tinggi pada beberapa penderita transplantasi dan penderita karsinoma dengan terapi antibodi monoklonal.34

47. Persiapan Pemeriksaan ANA Sampeldanreagendiletakkan padasuhukamar (20-260C). Mengencerkan HRP Wash Concentrate (1:40) : mencampur 25 ml HRP Wash Concentrate dengan aqua destilata 975 ml. Mengencerkansampel(1:41) dengan mencampur10 mikrolitersampeldengan 400 mikroliterpengencersampel ANA. MenentukanletaksampeldankontrolpadamesinChemwell. 35

48. Pencucian : mengisitiapsumuran 200-300 µL buffer HRP Wash yang telah diencerkankemudiandiaspirasi dan dibuang.Mengulangiurutankerjatersebutdua kali Menambahkan 100 µLHS HRP Ig G konjugasi pada tiap-tiap sumuran dan inkubasi sumuran selama 30 menit. 36

50. Mendeteksi antibodi Ig M dengan menggunakan antibodi monoklonal anti-human Ig M yang dilabel enzim.

51. Menambahkan substrat cromogen. 37

52. Disease Autoantibody Systemic Lupus Erythematosus Rheumatoid Arthritis SjogrensSyndrom Systemic Sclerosis Polymyositis/Dermatomyositis Mixed Connective Tissue Disease Wegener’s Granulomatosus Anti-dsDNA, Anti-SM RF (IgM), Anti-RA33, Anti-CCP Anti-Ro(SS-A), Anti-La(SS-B) Anti-Scl-70, Anti-centromere Anti-Jo-1 Anti-U1-RMP C-ANCA

54. Clinical Features of SLE

55. QUANTA Lite TM ANA is an enzyme-linked immunosorbent Assay (ELISA) for the semi quantitative detection of anti-nuklear antibodies in human serum.The presence of ANA can be used in conjuntion with clinical findings and other laboratory test to aid in the diagnostic of rheumatic diseases such as systemic lupus erythematosus , Sjogren syndrom, scleroderma, and mixed connective tissue disease. 41

56. QUANTA Lite® ANA IVD-In Vitro Diagnostic Use CE markedTechnology: ELISACLIA Complexity: High Description: This assay is designed for the detection of anti-nuclear IgG antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of rheumatic diseases such as systemic lupus erythematosus, Sjögren's Syndrome, scleroderma, and mixed connective tissue disease. Antigen: native, recombinant, extracts from HEp-2 nuclei and nucleoli Packaging: 1 X 96 wells 42

57. QUANTA Lite TM ANA Detects G59chromatin (dsDNA and histones), Sm/RNP, SS-A SS-B, Scl-70, centromere, and PCNA. Shows cytoplasmic antigens such as Jo-1, mitochondria (M-2) and ribosomal-P protein.

59. Ro-52: positif

60. Ds-DNA: positiflemah

61. Nucleosomes: positif

62. Histones: positifkuat

63. Rib-P-Protein: positifkuatDx Lab : SLE, 3 minggukemudianmeninggal

64. Tes konfirmasi ANA ELISA 45

65. AUTOIMMUNE DISEASES (CMDT, 2011)Connective Tissue Diseases (CTD)1. Rheumatoid Arthritis 2. Systemic Lupus Erythematosus 3. Antiphospholipid Antibody Syndrome 4. Raynaud Phenomenon 5. Systemic Sclerosis ( Scleroderma ) 6. Idiopathic Inflammatory Myopathies (Polymyositis & Dermatomyositis) 7. Sjögren Syndrome 8. Wegner Granulomatosis

66. RA = Rheumatoid arthritis SLE = Systemic lupus erythematosus SjS = Sjögren’s syndrome DS = Diffuse scleroderma LS = Limited scleroderma (CREST syndrome ) PD = Polymyositis / dermatomyositis WG = Wegener’s granulomatosis

67. Radang sendi atau artritis reumatoid (bahasa Inggris: Rheumatoid Arthritis, RA) merupakan penyakit autoimun (penyakit yang terjadi pada saat tubuh diserang oleh sistem kekebalan tubuhnya sendiri) yang mengakibatkan peradangan dalam waktu lama pada sendi. Penyakit ini menyerang persendian, biasanya mengenai banyak sendi, yang ditandai dengan radang pada membran sinovial dan struktur-struktur sendi serta atrofi otot dan penipisan tulang. Berdasarkan studi, RA lebih banyak terjadi pada wanita dibandingkan pria dengan rasio kejadian 3 : 1. Umumnya penyakit ini menyerang pada sendi-sendi bagian jari, pergelangan tangan, bahu, lutut, dan kaki. Pada penderita stadium lanjut akan membuat si penderita tidak dapat melakukan aktivitas sehari-hari dan kualitas hidupnya menurun. Gejala yang lain yaitu berupa demam, nafsu makan menurun, berat badan menurun, lemah dan kurang darah. 48

68. Rheumatoid Arthritis Abnormal antibodies can be found in the blood of people with rheumatoid arthritis. An antibody called "rheumatoid factor" can be found in 80% of patients with rheumatoid arthritis. Patients who are felt to have rheumatoid arthritis and do not have positive rheumatoid factor testing are referred as having "seronegative rheumatoid arthritis." Citrulline antibody (also referred to as anticitrulline antibody, anticycliccitrullinated peptide antibody, and anti-CCP) is present in most people with rheumatoid arthritis. It is useful in the diagnosis of rheumatoid arthritis when evaluating cases of unexplained joint inflammation. A test for citrulline antibodies is most helpful in looking for the cause of previously undiagnosed inflammatory arthritis when the traditional blood test for rheumatoid arthritis, rheumatoid factor, is not present. Citrulline antibodies have been felt to represent the earlier stages of rheumatoid arthritis in this setting. Another antibody called the "antinuclear antibody" (ANA) is also frequently found in people with 49

69. Antiphospholipid syndrome (APS or APLS) or antiphospholipid antibody syndrome is a disorder of coagulation that causes blood clots (thrombosis) in both arteries and veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, or severe preeclampsia. The syndrome occurs due to the autoimmune production of antibodies against phospholipid (aPL), a cell membrane substance. In particular, the disease is characterised by antibodies against cardiolipin (anti-cardiolipin antibodies) and β2 glycoprotein I. 50

70. Antiphospholipid antibody syndrome Physicians use a combination of clinical symptoms (see above) and laboratory tests to diagnose APS. The common blood tests for antiphospholipid antibodies are as follows: Anticardiolipin antibodies (IgG, IgM, and IgA) Lupus anticoagulant – a panel of blood clotting tests that may include the dilute Russel Viper venom time (dRVVT), lupus aPTT, mixing studies, and hex phase phospholipid test, platelet neutralization procedure Antibodies to b2-glycoprotein I (IgG, IgM, IgA) Panels of tests for antibodies to phospholipids other than cardiolipin are available but have not undergone the rigorous international standardization efforts applied to anticardiolipin assays. A number of experts in the field question the usefulness of these panels, which may be quite expensive. 51

71. Scleroderma is a chronicsystemic autoimmune disease (primarily of the skin) characterized by fibrosis (orhardening), vascular alterations, and autoantibodies. 52

72. Scleroderma The diagnosis of the scleroderma syndrome is based on the finding of the clinical features of the illnesses. In addition, nearly all patients with scleroderma have blood tests that suggest autoimmunity, because antinuclear antibodies (ANAs) are usually detectable. A particular antibody, the anticentromere antibody, is found almost exclusively in the limited, or CREST, form of scleroderma. Anti-Scl 70 antibody (antitopoisomerase I antibody) is most often seen in patients with the diffuse form of scleroderma. Other tests are used to evaluate the presence or extent of any internal disease. These may include upper and lower gastrointestinal tests to evaluate the bowels, chest X-rays, lung-function testing (pulmonary function test), and CAT scanning to examine the lungs, EKG and echocardiograms, and sometimes heart catheterization to evaluate the pressure in the arteries of the heart and lungs. 53

73. Sjogren's syndrome is an autoimmune disease characterized by dryness of the mouth and eyes. Autoimmune diseases feature the abnormal production of extra antibodies in the blood that are directed against various tissues of the body. This particular autoimmune illness features inflammation in glands of the body that are responsible for producing tears and saliva. Inflammation of the glands that produce tears (lacrimal glands) leads to decreased water production for tears and dry eyes. Inflammation of the glands that produce the saliva in the mouth (salivary glands, including the parotid glands) leads to dry mouth and dry lips. 54

74. Sjogren Syndrome Patients with Sjogren's syndrome typically produce a variety of extra antibodies against body tissues (autoantibodies). These can be detected through blood testing and include antinuclear antibodies (ANA), which are present in nearly all patients. Typical antibodies that are found in most, but not all patients, are SS-A and SS-B antibodies (Sjogren's syndrome A and B antibodies), rheumatoid factor, thyroid antibodies, and others. Low red blood count (anemia) and abnormal blood levels of markers of inflammation (sedimentation rate) are seen. 55

75. Wegener's granulomatosis (WG), more recently granulomatosis with polyangiitis, is a form of vasculitis(inflammation of blood vessels) that affects the nose, lungs, kidneys and other organs. Due to its end-organ damage, it is life-threatening and requires long-term immunosuppression.[1] Five-year survival is up to 87%, with some of the mortality due to toxicity of treatment. It is named after Dr. Friedrich Wegener, who described the disease in 1936.[2] In 2011, three professional bodies proposed a more descriptive name.[3] Wegener's granulomatosis is part of a larger group of vasculitic syndromes, all of which feature an autoimmune attack by an abnormal type of circulating antibody termed ANCAs (antineutrophilcytoplasmic antibodies) against small and medium-size blood vessels. Apart from Wegener's, this category includes Churg-Strauss syndrome and microscopic polyangiitis.[1] Although Wegener's granulomatosis affects small and medium-sized vessels,[4] it is formally classified as one of the small vessel vasculitides in the Chapel Hill system.[5] 56

76. Wegener Syndrome Blood tests that detect the abnormal inflammation include the sedimentation rate (sed rate) and C-reactive protein. A more specific blood test used to diagnose and monitor Wegener's granulomatosis is the antineutrophilcytoplasmic antibody (ANCA test), which is commonly elevated when the disease is active. The diagnosis of Wegener's granulomatosis is confirmed by detecting both abnormal cellular formations, called granulomas, and vasculitis in a biopsy of tissue involved with the inflammatory process. For examples, an open lung biopsy or a kidney biopsy are commonly used in making a diagnosis of Wegener's granulomatosis. 57

77. Polymyositis (PM)is a type of chronic[1]inflammatory myopathy related to dermatomyositis and inclusion body myositis. Polymyositis means 'many muscle inflammation'. 58

78. Polimyositis Blood testing usually (but not always) reveals abnormally high levels of muscle enzymes, CPK or creatininephosphokinase, aldolase, SGOT, SGPT, and LDH. These enzymes are released into the blood by muscle that is being damaged by inflammation. They can also be used as measures of the activity of the inflammation. Other routine blood and urine tests can also look for internal organ abnormalities. Chest X-rays,mammograms,PAP smears, and other screening tests might be considered. 59

79. Raynaud's phenomenon (RP) is a condition resulting in a particular series of discolorations of the fingers and/or the toes after exposure to changes in temperature (cold or hot) or emotional events. 60

81. Prosedur Pemeriksaan dengan Mesin Chemwell Pengenceran sampel (1:40) 400 µl sample diluent + 10 µl serum sampel dihomogenkan ambil 100 µl larutan (dalam tabung Eppendorf) 62

82. Prosedur Pemeriksaan dengan Mesin Chemwell Nyalakan program komputer Klik ‘Start of Day’ 63

83. Prosedur Pemeriksaan dengan Mesin Chemwell 64 Menjalankan program komputer Klik ‘Run’  klik ‘New Job’

84. Prosedur Pemeriksaan dengan Mesin Chemwell 65 Memasukkan nomor sampel Klik ‘OK’

85. Prosedur Pemeriksaan dengan Mesin Chemwell 66 Menjalankan program Assay Klik ‘Assay’

86. Prosedur Pemeriksaan dengan Mesin Chemwell 67 Menyiapkan tempat reagen pada mesin Pada layar komputer Pada mesin Chemwell

87. Prosedur Pemeriksaan dengan Mesin Chemwell 68 Menyiapkan tempat sampel pada mesin Pada layar komputer Pada mesin Chemwell

88. Prosedur Pemeriksaan dengan Mesin Chemwell 69 Menyiapkan well pada mesin Pada layar komputer Pada mesin Chemwell

89. Prosedur Pemeriksaan dengan Mesin Chemwell 70 Mengecek ulang tempat reagen, rak sampel dan well

90. Prosedur Pemeriksaan dengan Mesin Chemwell 71 Mengecek ulang tempat reagen, rak sampel dan well

91. Prosedur Pemeriksaan dengan Mesin Chemwell 72 Menjalankan proses pemeriksaan Klik ‘RUN’  klik ‘Start”

92. Prosedur Pemeriksaan dengan Mesin Chemwell 73 Hasil pemeriksaan

93. 74

94. 75

95. 76

96. 77

97. 78

98. HRP 79