1. TUTOR IMUNOLOGI MINICAPCapillary Electrophoresis SiswantoDarmadi, dr, SpPK(K) / RantiPermatasari, dr 1

2. PENDAHULUAN 2 ELEKTROFORESIS : carapemisahandanpengukurandarimakromolekul, seperti protein, lipoprotein, isoenzim, dan hemoglobin dalamberbagaimacamcairantubuh Capillary electrophoresis (CE) : elektroforesiskinerjatinggi yang dikerjakandalamtabungkapiler diameter 20-200 μm, denganatautanpapelapis, denganmenggunakanmedanlistrik.

3. KOMPONEN ALAT 3 Reagent Compartement Analysis Unit Rotating Sampler (carousel)

4. 4 ROTATING SAMPLER

5. 5 ANALYSIS UNIT

6. Reagent Compartment Reagent cups holder Buffer 1 Buffer 2 H2O Wash Loaded reagent cups Discarded reagent cupscontainer Waste bottle 6

7. PRINSIP KERJA 7 MINICAP menggunakansistem CE yang memisahkanmolekulberdasarkankecepatanelektroforesisnyadalambufer alkali dengan pH tertentudalamtabungkapilerdengan diameter 100 μm, yang dipengaruhioleh pH elektrolitdanelectroosmotic flow.

8. STEP 1 Back wash to clean capillary P Opened chamber Closed Chamber Anode Cathode Wash solution 8

9. STEP 2 Back wash to fill capillary with buffer P Opened chamber Closed Chamber Anode Cathode Buffer solution 9

10. STEP 3 Diluted sample injection:Vacuum (P) during a fixed period of time (t) P Sample micro cup Closed Chamber Anode Dilution Segment Cathode Buffer 10

11. STEP 4 Migration and Detection:High voltage applied (V) and Detection at 200 nm Detector V Opened Chamber - + Anode Buffer Cathode Buffer 11

12. STEP 4 Migration and Detection:High voltage applied (V) and Detection at 200 nm Detector V - + Anode Buffer Cathode Buffer 12

13. KEGUNAAN MINICAP 13

14. ELEKTROFORESIS PROTEIN Reagendanperlengkapan yang dibutuhkan : 14

17. LANGKAHOTOMATIS Pembacaan barcode padatabung (jikaada) Pengenceransampeldenganbuferdanpencucianprobe. Pencuciankapiler Sampel yang telahdiencerkandimasukkankedalamkapiler Prosesmigrasiberlangsungdengantegangan yang konstanselama 4 menit Protein dibacalangsungpadapanajnggelombang 200 nm danhasilnyamunculpadalayar LCD. 17

18. QUALITY CONTROL 18 Serum kontroldiperolehdari pool serum manusiadandikemasdalambentukliofilisasi. Serum kontroldapatdigunakanuntuk quality control elektroforesis protein, lipoprotein, kolesteroldanapolipoprotein

19. 19

20. 20

21. ELEKTROFORESIS HEMOGLOBIN Reagendanperlengkapan yang dibutuhkan : 21

22. 22

23. Sampel yang digunakanuntukelektroforesisHbyaitudarahantikoagulan. Antikoagulan yang dapatdigunakanyaitu EDTA, sitratatau heparin. Darahantikoagulandisentrifusdengankecepatan 5000rpm selama 5 menitkemudianbuangplasmanyadan vortex selama 5 detik. 23

24. PROSEDUR 24

25. 25

26. LANGKAH OTOMATIS Pembacaan barcode padatabung (jikaada) Pengenceransampeldenganbuferdanpencucianprobe. Pencuciankapiler Sampel yang telahdiencerkandimasukkankedalamkapiler Prosesmigrasiberlangsungdengantegangan yang konstanselama 8 menit Protein dibacalangsungpadapanjanggelombang 415 nm danhasilnyamunculpadalayar LCD. 26

27. QUALITY CONTROL 27 Kontrol normal HbA2 diperolehdari pool darahmanusiadandikemasdalambentukliofilisasi. Kontrol normal HbA2 dirancanguntukdapatdigunakanuntuk quality kontroljumlahdanposisi hemoglobin A2

28. 28

29. IMMUNOTYPING Reagendanperlengkapan yang dibutuhkan : 29

30. 30

33. Hemoglobin Capillary electrophoresis Protein Capillary electrophoresis 33

34. Perbandinganhasilelektroforesis protein hydrageldgnminicap (%) 34

35. Normal serum 35

36. Double Alpha 1->Heterozygousphenotype of alpha 1 antitrypsin 36

37. 37 Hb A Hb A=93.4% Hb F=1.1% HbA2=5.5% Hb A2 Hb F  ß-thalassemia- minor

38. 38 Hb A Hb A2 Hb F HbBart’s Hb H Hb H and Bart’s - Alpha-thalassemia

39. NILAI NORMAL Elektroforesis Protein Albumin : 53-65% α1- globulin : 2.5-5% α2 - globulin: 7-13% β - globulin : 8-14% γ - globulin : 12-22% (BISHOP 2005) Elektroforesis Hemoglobin Dewasa Hb A : 97% Hb A2:: 2-3.3% Hb F : 0.2-1% (DACIE and LEWIS 2006) 39

40. 40 ΤεЃιΜαΚαςιΗ

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42. 42

43. Hb identification for Capillarys/Minicap Z 15 : Hb H Z 14 : // Z 13 : Hb-J Rovigo, Hb N-Baltimore Z 12 : Hb Bart’s, Hb J-Providence, Hb J-Mexico, Hb J-baltimore…. Z 11 : DenaturatedHb A, HbKaoshiung Z 10 : Hb Hope, Hb M-Iwate Z 9 : Hb A,HbCamperdown, Hb Phnom Penh…… Z 8 : Acetylated Hb F, HbAltanta, Hb Athens-GA Z 7:Hb F,denaturatedHb S, Hb Porto-Alegre…….. Z 6 : Hb D-Punjab, denaturatedHb E, HbKorle-Bu, HbLepore, Hb Köln…. Z 5 : Hb S, HbHasharon, HbHandsworth, denaturatedHb O-Arab. Z 4 : Hb E, denaturatedHb C, Hb Köln, Hb A2 variants, M-Iwate Hb A2 variants Z 3 : Hb A2, Hb O-Arab Z 2 : Hb C, Hb Constant Spring, Setif HbA2 variant Z 1 : HbdA2 HbaA2, HasharonHb A2 variant, Winnipeg Hb A2 variant…… 43

44. Kriteriadx MM Mayor: BM-plasmacytosis >30% Histologidxplasmacytoma Serum paraproteinIgG>3,5g/dl atauIgA>2,0 g/dl Minor: BM-plasmacytosis 10-30% Serum paraprotein < dr mayor Osteoliticlession Hipogammaglobulinemia Waldenstormmacrogabulinemia : Tumor infiltrasi Circulating IgM Tissue IgM 44

45. Hemoglobin normal padadewasa Hb A (α2β2) 97% Hb F (α2γ2) 3-5% Hb A2 (α2β2) 1.5 – 3.5% 45