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Pemeriksaan Immunoglobulin A (IgA) dengan metode Radial Immunodiffusion Ferdy,dr/ Endang Retnowati, dr, MS, SpPK(K) TUTOR IMUNOLOGI 1
2 subklas:  IgA1 dan IgA2 Antibodi penting pada imunitas mukosa. IgA 3-5 gram/hari disekresi ke lumen intestinal 75% Ig total dihasilkan tubuh 2 Pendahuluan
Struktur Ig A Gambar 1: Struktur Ig A (Sumber: Siemel, 2006) 3
Pendahuluan 4
Arti klinis Ig A menurun: Selective IgA deficiency Infeksi Neisseria gonorrhœae 5
IgA meningkat Ig A nephropathy Penyakit kronis seperti hepatitis Sirosis hepatis Myeloma Penyakit autoimun seperti SLE 6
Pemeriksaan Immunoglobulin A Metode : Radial immunodifusion Radioimmunoassay Nefelometri ELISA 7
Presipitasi 8
9
Kurva Presipitasi Gambar2. KurvaPresipitasi. (Sheehan, 1990) 10
Gambar3. Kompleks Ag – Ab.  (Sheehan, 1990) 11
RADIAL IMMUNODIFFUSION (RID) 12 Antisera/ Ab Gel agarosa fase cair (50oC) 50 – 80 jam   cincin presipitasi Serum standar 1 Tes serum 2 8 Antisera  dalam agar Tes serum 3 dinginkan pd suhu kamar 7 Tes serum 4 Ukur diameter cincin presipitasi, tempatkan pd kurva standar  menentukan kadar imunoglobulin secara kuantitatif 6 5 Tes serum Tes serum (Handojo, 2003)
KECEPATAN DIFUSI Ukuran molekul Interaksi  matriks gel & reaktan FAKTOR Viskositas & Hidrasi gel Berat  molekul  Ag Temperatur 13 (Sheehan, 1990)
CARA PEMBACAAN RID  METODE Mancini, endpoint diffusion. Fahey, kinetic diffusion. 14 (Sheehan, 1990)
CARA PEMBACAAN RID Fahey Mancini ,[object Object]
Grafiksemilogaritmik; sumbuy:kadarygdianalisa;
sumbu x :diameter cincinpresipitasi (termasuksumuran)
Ag dibiarkanberdifusimencapaipresipitasimaks
IgA; inkubasi 48 jam
Grafik; sumbu y:kadar Ag & sumbux: cincinpresipitasi. 15 (Sheehan, 1990)
Gambar 4. Grafik Fahey (kinetic diffusion method) 16 (Sheehan, 1990)
Gambar 5. Grafik Mancini (endpoint method)  17 (Sheehan, 1990)
18 HASIL Hasil dikali  faktor  pengenceran Hasil kurang  dari standar (Sheehan, 1990)
mengisi sumuran terlalu sedikit/ banyak  menumpahkan serum pasien ke dalam gel agarosa   hasiltdkakurat perludiulang melubangi  samping  sumuran  Waktu&suhu  Inkubasi tdk tepat  KESALAHAN PEMBACAAN RID (Sheehan, 1990) 19
Gambar6. PolacincinpresipitasipadaRID(Sheehan, 1990) 20
Pemeriksaan IgA di Instalasi Patologi Klinik RSUD.dr.Soetomo Prinsip pemeriksaan (RID): Ig A serum/plasma manusia membentuk kompleks imun dengan antibodi spesifik pada gel agarose pada cakram Partigen Diameter cincin presipitasi berbanding lurus dengan konsentrasi Ig A dalam sampel Hasil dikonversi ke dalam konsentrasi Ig A menggunakan tabel nilai referensi. 21
Reagen:  Tiap plate NOR partigen mempunyai 12 sumuran imunodifusi untuk menentukan Ig A serum/plasma. Komposisi: Cakram NOR partigen mengandung lapisan agarose gel yang mengandung antiserum monospesifik terhadap Ig A dalam plasma.  Antisera diperoleh dari kelinci, domba, kuda, kambing yang diimunisasi dari manusia. Cakram NOR Partigen juga mengandung sodium azide (<1 g/l) dan sodium timerfonate (<0.1 g/l) sebagai pengawet. 22
23 Gambar7. NOR Partigen Plate (Lab. PK, 2011)
Penyimpanan dan stabilitas NOR Patigen sebaiknya disimpan pada suhu 2-8°C pada pembungkus yang belum dibuka, stabil sesuai masa berlaku Kontrol Sebaiknya digunakan kontrol sampel yang tersedia komersial 24
Spesimen serum maupun plasma segar (24 jam sesudah pengambilan) bila sampel tidak langsung dikerjakan sebaiknya disimpan pada suhu 2-8°C maksimum 8 hari  Penyimpanan pada suhu -20°C dapat bertahan hingga 1 tahun. 25
Prosedur 5 µl sampel 1 Tes serum 2 8 Antisera  dalam agar Tes serum 3 7 UKUR DIAMETER CINCIN PRESIPITASI SETELAH 48 JAM Tes serum 4 6 5 Tes serum Tes serum (Handojo, 2003)  26
Gambar8. Meβ Projector, Lab. PK. Dr. Soetomo, 2011 27
28 Gambar9. TabelReferensi (Lab. PK, 2010)
Nilai normal IgA	: 70 – 350 mg/ dL 29
TERIMAKASIH 30
we defined IgA deficiency as plasma samples with less than 100 ngIgA per mL, decreased level of IgA as plasma samples with 100 ng to600 mg IgA per mL,  and normal level of IgA as plasmasamples with more than 0.6 mg per mL as determined by the IgA-ELISA 31
IgA measurement by nephelometry Beckman Coulter, Inc. (Fullerton,Calif.). IgA levels were considered deficient when they were less than thelower limit of established age-dependent reference intervals.  These limits: 70 mg/dl for teenagers and adults,  23 mg/dl for children 3 to 12 years old,  17mg/dl for children ,3 years old. 32
Grafik Semilogaritmik 33
34 NEFELOMETRI  Nephelometri :  carapengukurankadarzatdenganmengukurpendarancahaya (scattered) yang mengenaipartikeldalamlarutan. Dasarpemeriksaan : reaksipresipitasi antigen-antibodi. Metodeinidigunakanuntukmengetahuikuantitas protein spesifiksecaralebihakuratdanprecise.
35 PRINSIP NEPHELOMETRI (1) Prinsip nephelometri : 		1. pendaran cahaya, 		2. kinetik cairan pada fase presipitasi.
36 PRINSIP NEPHELOMETRI (2) PENDARAN CAHAYA Pendarancahayadipengaruhi :  		a. diameter partikel, 	   b. panjanggelombang. Contoh : a. partikelkecil (albumin, Ig.G,Ig.M) : 		- pendarancahaya Rayleigh semetrisantaraforwarddanbackward. 		- pendarancahaya minimum dilihatpada90º darisumbercahaya.
37 PRINSIP NEPHELOMETRI (3) 	b. partikellebihbesar (komplek Ag-Ab) : 		- menghasilkanpendarancahayaRaeyleigh-Debye 		- pendarandilihatdariforward (sudutmendekatinol)      intensitascahayalebihbesar. Sudutuntukmendeteksipendarancahayaikutmenentukansensitivitas, dimanaintensitassumbercahayamakinkuatmakasudut yang dihasilkanmakinkecil
38 PRINSIP NEPHELOMETRI (4)
39 PRINSIP NEPHELOMETRI (5)

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IgA Measurement Methods Comparison

  • 1. Pemeriksaan Immunoglobulin A (IgA) dengan metode Radial Immunodiffusion Ferdy,dr/ Endang Retnowati, dr, MS, SpPK(K) TUTOR IMUNOLOGI 1
  • 2. 2 subklas: IgA1 dan IgA2 Antibodi penting pada imunitas mukosa. IgA 3-5 gram/hari disekresi ke lumen intestinal 75% Ig total dihasilkan tubuh 2 Pendahuluan
  • 3. Struktur Ig A Gambar 1: Struktur Ig A (Sumber: Siemel, 2006) 3
  • 5. Arti klinis Ig A menurun: Selective IgA deficiency Infeksi Neisseria gonorrhœae 5
  • 6. IgA meningkat Ig A nephropathy Penyakit kronis seperti hepatitis Sirosis hepatis Myeloma Penyakit autoimun seperti SLE 6
  • 7. Pemeriksaan Immunoglobulin A Metode : Radial immunodifusion Radioimmunoassay Nefelometri ELISA 7
  • 9. 9
  • 10. Kurva Presipitasi Gambar2. KurvaPresipitasi. (Sheehan, 1990) 10
  • 11. Gambar3. Kompleks Ag – Ab. (Sheehan, 1990) 11
  • 12. RADIAL IMMUNODIFFUSION (RID) 12 Antisera/ Ab Gel agarosa fase cair (50oC) 50 – 80 jam  cincin presipitasi Serum standar 1 Tes serum 2 8 Antisera dalam agar Tes serum 3 dinginkan pd suhu kamar 7 Tes serum 4 Ukur diameter cincin presipitasi, tempatkan pd kurva standar  menentukan kadar imunoglobulin secara kuantitatif 6 5 Tes serum Tes serum (Handojo, 2003)
  • 13. KECEPATAN DIFUSI Ukuran molekul Interaksi matriks gel & reaktan FAKTOR Viskositas & Hidrasi gel Berat molekul Ag Temperatur 13 (Sheehan, 1990)
  • 14. CARA PEMBACAAN RID METODE Mancini, endpoint diffusion. Fahey, kinetic diffusion. 14 (Sheehan, 1990)
  • 15.
  • 17. sumbu x :diameter cincinpresipitasi (termasuksumuran)
  • 20. Grafik; sumbu y:kadar Ag & sumbux: cincinpresipitasi. 15 (Sheehan, 1990)
  • 21. Gambar 4. Grafik Fahey (kinetic diffusion method) 16 (Sheehan, 1990)
  • 22. Gambar 5. Grafik Mancini (endpoint method) 17 (Sheehan, 1990)
  • 23. 18 HASIL Hasil dikali faktor pengenceran Hasil kurang dari standar (Sheehan, 1990)
  • 24. mengisi sumuran terlalu sedikit/ banyak menumpahkan serum pasien ke dalam gel agarosa hasiltdkakurat perludiulang melubangi samping sumuran Waktu&suhu Inkubasi tdk tepat KESALAHAN PEMBACAAN RID (Sheehan, 1990) 19
  • 26. Pemeriksaan IgA di Instalasi Patologi Klinik RSUD.dr.Soetomo Prinsip pemeriksaan (RID): Ig A serum/plasma manusia membentuk kompleks imun dengan antibodi spesifik pada gel agarose pada cakram Partigen Diameter cincin presipitasi berbanding lurus dengan konsentrasi Ig A dalam sampel Hasil dikonversi ke dalam konsentrasi Ig A menggunakan tabel nilai referensi. 21
  • 27. Reagen: Tiap plate NOR partigen mempunyai 12 sumuran imunodifusi untuk menentukan Ig A serum/plasma. Komposisi: Cakram NOR partigen mengandung lapisan agarose gel yang mengandung antiserum monospesifik terhadap Ig A dalam plasma. Antisera diperoleh dari kelinci, domba, kuda, kambing yang diimunisasi dari manusia. Cakram NOR Partigen juga mengandung sodium azide (<1 g/l) dan sodium timerfonate (<0.1 g/l) sebagai pengawet. 22
  • 28. 23 Gambar7. NOR Partigen Plate (Lab. PK, 2011)
  • 29. Penyimpanan dan stabilitas NOR Patigen sebaiknya disimpan pada suhu 2-8°C pada pembungkus yang belum dibuka, stabil sesuai masa berlaku Kontrol Sebaiknya digunakan kontrol sampel yang tersedia komersial 24
  • 30. Spesimen serum maupun plasma segar (24 jam sesudah pengambilan) bila sampel tidak langsung dikerjakan sebaiknya disimpan pada suhu 2-8°C maksimum 8 hari Penyimpanan pada suhu -20°C dapat bertahan hingga 1 tahun. 25
  • 31. Prosedur 5 µl sampel 1 Tes serum 2 8 Antisera dalam agar Tes serum 3 7 UKUR DIAMETER CINCIN PRESIPITASI SETELAH 48 JAM Tes serum 4 6 5 Tes serum Tes serum (Handojo, 2003) 26
  • 32. Gambar8. Meβ Projector, Lab. PK. Dr. Soetomo, 2011 27
  • 33. 28 Gambar9. TabelReferensi (Lab. PK, 2010)
  • 34. Nilai normal IgA : 70 – 350 mg/ dL 29
  • 36. we defined IgA deficiency as plasma samples with less than 100 ngIgA per mL, decreased level of IgA as plasma samples with 100 ng to600 mg IgA per mL, and normal level of IgA as plasmasamples with more than 0.6 mg per mL as determined by the IgA-ELISA 31
  • 37. IgA measurement by nephelometry Beckman Coulter, Inc. (Fullerton,Calif.). IgA levels were considered deficient when they were less than thelower limit of established age-dependent reference intervals. These limits: 70 mg/dl for teenagers and adults, 23 mg/dl for children 3 to 12 years old, 17mg/dl for children ,3 years old. 32
  • 39. 34 NEFELOMETRI Nephelometri : carapengukurankadarzatdenganmengukurpendarancahaya (scattered) yang mengenaipartikeldalamlarutan. Dasarpemeriksaan : reaksipresipitasi antigen-antibodi. Metodeinidigunakanuntukmengetahuikuantitas protein spesifiksecaralebihakuratdanprecise.
  • 40. 35 PRINSIP NEPHELOMETRI (1) Prinsip nephelometri : 1. pendaran cahaya, 2. kinetik cairan pada fase presipitasi.
  • 41. 36 PRINSIP NEPHELOMETRI (2) PENDARAN CAHAYA Pendarancahayadipengaruhi : a. diameter partikel, b. panjanggelombang. Contoh : a. partikelkecil (albumin, Ig.G,Ig.M) : - pendarancahaya Rayleigh semetrisantaraforwarddanbackward. - pendarancahaya minimum dilihatpada90º darisumbercahaya.
  • 42. 37 PRINSIP NEPHELOMETRI (3) b. partikellebihbesar (komplek Ag-Ab) : - menghasilkanpendarancahayaRaeyleigh-Debye - pendarandilihatdariforward (sudutmendekatinol) intensitascahayalebihbesar. Sudutuntukmendeteksipendarancahayaikutmenentukansensitivitas, dimanaintensitassumbercahayamakinkuatmakasudut yang dihasilkanmakinkecil
  • 45. 40 PRINSIP NEPHELOMETRI (6) 2. KINETIK CAIRAN PADA FASE PRESIPITASI Sinartampak (visible) yang mengenaibentukanlatticependarancahaya Rayleigh-Debye. Latticemaksimalzonaekivalen. Nephelometrimengukurkonsentrasi antigen padakeadaankelebihanantibodi, dimanaterdapatjumlahlattice pendarancahaya yang terukursesuaijumlah antigen sampel.
  • 46. Low values Ig A Some people are born with low or absent levels of IgA antibodies. Low levels of IgA occur in some types of leukemia, kidney damage (nephrotic syndrome) a problem with the intestines (enteropathy) a rare inherited disease that affects muscle coordination (ataxia-telangiectasia). 41
  • 47. High values IgA monoclonal gammopathy of unknown significance (MGUS) or multiple myeloma autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), and in liver diseases, such as cirrhosis and long-term (chronic) hepatitis. 42
  • 48. 3 most commonly used methods for the quantitative determination of serum IgA concentration: nephelometry (or turbidimetry), passive hemagglutination inhibition assay (PHAI), and enzyme-linked immunosorbent assay (ELISA). The basic principle of nephelometric and turbidimetric assays for quantifying IgA concentration is light scatter at various angles by immune complexes formed between IgA (antigen) in the patient's serum and anti-IgA (antibody) antiserum added to the patient's serum. Passive hemagglutination techniques use hemagglutination as the end point of the antigen-antibody reaction, while ELISA methods use a capture antibody bound to the well of a microtiter plate to "capture" antigen (eg, IgA) and a signal antibody, containing an enzyme "signal", that recognizes a different epitope on the antigen than the capture antibody. The addition of substrate specific for the enzyme attached to the signal antibody results in the production of a color whose intensity is directly proportional to the amount of antigen present. 43
  • 49. Among these methods for the quantitation of IgA concentration, ELISA methods provide the best analytical sensitivity [ie, lower limit of detection (LLD) <0.005 mg/dL] ( Table 2 ). Nephelometric or turbidimetric methods are used routinely in most clinical laboratories for the quantitation of serum IgA concentration because these methods are rapid, easy to use, and are amenable to automation. According to the data in the College of American Pathologists (CAP) 2004 Diagnostic Immunology Survey Set-B Summary, 93.6% (712 of 760) of participating laboratories used nephelometric or turbidimetric methods for the quantitation of IgA, with the majority (55.2%) of these laboratories reporting the use of nephelometry over turbidimetry.[2] 44