1. ELEKTROFORESIS PROTEIN SERUM Siswanto armadi, dr, Sp.PK(K) / Nuzulul Quriyah, drD 6-16-2010 1 Tutor Imunologi

2. PENDAHULUAN 6-16-2010 2

3. Elektroforesis 6-16-2010 3 Teknik pemisahan komponen/molekul bermuatan berdasarkan perbedaan tingkat migrasinya dalam sebuah medan listrik Tutor Imun by Nuzul Pergerakan pd elektroforesis dijelaskan dengan gaya Lorentz : F = qE F adalah gaya Lorentz, q adalah muatan yang dibawa oleh obyek, E adalah medan listrik Posisimolekul yang terseparasipada gel dideteksidenganpewarnaan, ataudilakukankuantifikasidengan densitometer

4. Tutor Imun PrinsipElektroforesis by Nuzul Muatan listrik molekul biologi (misal : protein) tergantung pd pH dan komposisi medium dimana molekul biologi tersebut terlarut bersifat amfoter yaitu bisa bermuatan positif atau negatif (bermuatan positif bila pH larutan lebih kecil daripada titik isoelektrik, bermuatan negatif bila pH larutan lebih besar daripada titik isoelektrik) Dalam suatu medan listrik, molekul biologi yang bermuatan positif (kation) bermigrasi ke elektroda negatif (katoda) dan sebaliknya, yang bermuatan negatif (anion) bermigrasi ke elektroda positif (anoda) 6-16-2010 4

5. 6-16-2010 5 Dengan elektroforesis, protein plasma dapat dipisahkan menjadi fraksi albumin serta α1, α2, β, dan γ-globulin Protein dalam plasma memiliki konsentrasi sekitar 1 mmol/L 56% protein plasma merupakan fraksi albumin, 4% adalah α1-globulin, α2-globulin 10%, β-globulin 12%, dan 18% merupakan γ-globulin Tutor Imun Protein by Nuzul

6. ELEKTROFORESIS PROTEIN 6-16-2010 6

7. Elektroforesis Protein 6-16-2010 Besarnya muatan Media penyangga Elektroendosmosis Suhu Ukuran molekul Kekuatan medan listrik Kekuatan ion dari bufer Kecepatan migrasi molekul Tutor Imun by Nuzul Click to add Title 7

8. KomponenSistemElektroforesis 6-16-2010 8 Tutor Imun by Nuzul Power supply Sampel Bufer Media penyangga Power supply Wicks Pewarnaan Gambar 1. Komponensistemelektroforesis

9. Tutor Imun by Nuzul Yang sering dipakai : serum dan plasma Cairan tubuh lain yang juga bisa dipakai : urine, cairan serebrospinal, cairan dari pleura, perikardium, peritoneum, membran sinovial, dan air mata Tidak ada persiapan khusus yang dilakukan pasien, tetapi keadaan puasa lebih baik untuk menghindari hiperlipemia Sampel yang diperlukan antara 0,3-25 μl tergantung dari konsentrasi analit dan jenis media penyangga yang digunakan Sampel diberi penanda warna untuk mengetahui kapan elektroforesis harus dihentikan, pada bufer asam dipakai methylene green atau methylene blue, sedangkan pada bufer basa dipakai bromphenol blue. SAMPEL 6-16-2010 9

10. BUFER 6-16-2010 10 Tutor Imun by Nuzul Menjaga agar pH tetapkonstan Mengalirkan (conduct) arus listrik

11. SyaratBufer 6-16-2010 11 1 bufer tidak boleh bereaksi dengan sampel krn interaksi antara bufer dan sampel dapat mempengaruhi gerakan molekul Tutor Imun by Nuzul 3 kekuatan ion dari larutan bufer harus dipertimbangkan kekuatan ionik rendah menghasilkan kecepatan migrasi lebih besar 2 pH yang dipilih harus memberi muatan tapi tidak menyebabkan denaturasi protein dari molekul yang diperiksa (protein dipisahkan pada suasana basa, pH 8 – 9) protein bermuatan negatif

12. MEDIA PENYANGGA 6-16-2010 12 Tutor Imun by Nuzul Agarose Gel Poly- acrylamide Gel Starch Gel Cellulose Acetate Kertas murah, tapi menyerap banyak protein menghasilkan pita yang lebar, resolusinya jelek, waktu pemisahannya lebih lama granula yang mengandung amilosa dan amilopektin, bila suspensinya dilarutkan dan dipanaskan akan terbentuk larutan jernih dan menjadi gel pd pendinginan sering dipakai, bebas dari ionizable groups pada buffer basa atau netral tidak terlalu terpengaruh oleh endosmosis, resolusi lbh tajam, jernih lebih kuat daripada kertas, dg ukuran pori-pori homogen, pemisahan protein lebih cepat, absorpsi protein minimal hasil pita lebih jelas dan lebih tajam masih terbatas hanya untuk pemisahan isoenzim dari alkali fosfatase

13. Endosmosis danWick Flow 6-16-2010 13 Tutor Imun by Nuzul Endosmosis (electroendosmosis)terjadiketika media penyangga jadi bermuatan negatif akibat adsorpsi ion hidroksil dari bufer muatan negatif menarik muatan positif dari bufer. Saat dialiri listrik ion positif bergerak ke arah katoda, sedang gerakan muatan negatif (misal : protein) ke arah anoda. Krn endosmosis gerakan menjadi lebih lambat, tidak bergerak atau terdorong kearah katoda Wick Flow gerakankeatasdarilarutanbufermelaluikeduatepimembran yang terendamuntukmenggantikankelembaban yang hilangakibatpemanasan. Aliraninimemperlambatgerakanmolekulpada media penyangga

14. Gambar 2. Endosmosis 6-16-2010 14 Tutor Imun by Nuzul

15. POWER SUPPLY 6-16-2010 15 Tutor Imun by Nuzul 0-500 volt 0-100 mA Jikaprosespemisahanbutuhwaktu lama, arus yang konstanlebihdianjurkanuntukmeminimalkankenaikansuhu Jika waktu 30 menit atau kurang, maka voltase dinaikkan

16. PEWARNAAN 6-16-2010 16 Ponceau S Amido Black Oil Red O Coomassie Brilliant Blue R 250 Tutor Imun by Nuzul Schiff’s

17. 6-16-2010 17 PembacaanHasil Tutor Imun by Nuzul Ada 2 Cara Kuantitatif Mengukur intensitas warna pada tiap pita. Kualitatif Membandingkan garis-garis fraksi dengan pola elektroforesis standar yang telah diketahui Metode eluasi Pita dipotong-potong beberapa bagian, pewarna kemudian dieluasikan dalam suatu larutan, warna yang larut tersebut dihitung dengan fotometer Densitometri -Intensitas warna tiap pita diukur langsung pada medianya dalam suatu sistem optik yang mirip dengan fotometer. -Jika medianya tidak transparan, maka harus dijernihkan lebih dulu

18. ELEKTROFORESIS PROTEIN DI LABORATORIUM PK RSUD DR SOETOMO 6-16-2010 18

19. SEBIA Hydragel Protein (E) K-20 6-16-2010 19 Tutor Imun by Nuzul Tabel 1. ReagendanBahandalam Kit Hydragel Protein (E) K20

20. Alat-Alat yang Dipakai 6-16-2010 20 Tutor Imun by Nuzul Gambar 3. Electrophoresis Chamber Gambar 5. Staining - Destaining solution Gambar 4. Agarose gel

21. Alat-Alat yang Dipakai 6-16-2010 21 Tutor Imun by Nuzul Gambar 6. Aplikator Gambar 7. Aplikatorcarrier Gambar 8. Microprocessor (power supply) Gambar 9. Inkubator

22. Alat-Alat yang Dipakai 6-16-2010 22 Tutor Imun by Nuzul Scanner λ 570 nm atau filter kuning

23. PROSEDUR SEBIA 6-16-2010 23

24. TahapMigrasi 6-16-2010 24 Tutor Imun by Nuzul 3 1 2 4 Tempatkanagarose gel pd applicator carrier dg kutub + dibagianatas Tempatkanapplicator ygberisi serum pd handle applicator carrier Serum 10 μl pd sumuranapplicator Tempatkanapplicator carrier pd tempatygdatar

25. TahapMigrasi (lanjutan) 6-16-2010 25 Tutor Imun by Nuzul Waktu 15`, 165 V, 7 ± 2 mA(set alat 60 mA)

26. 6-16-2010 26 Text Text Text Fiksasidlm fixation sol 15` Keringkan pd 80⁰ C, 10` Pindahkan gel Tutor Imun TahapFiksasi by Nuzul

27. 6-16-2010 27 Text Text Text Pewarnaan 4’ Destaining3x sampaibersih Scanning Gel menghadap kebawahλ570nm Keringkan TahapStaining-DestainingTahapScanning Tutor Imun by Nuzul

29. HasilPemeriksaan 6-16-2010 29 Tutor Imun by Nuzul Tabel 2. Nilai Rujukan Protein Serum ( A Manual of Laboratory and Diagnostic Test, 2000)

30. HasilPemeriksaan 6-16-2010 30 Tutor Imun by Nuzul Tabel 3. Nilai Rujukan Elektroforesis Protein Serum (Sheehan C, Clinical Immunology : Principles & Laboratory Diagnosis, 1990. Roche, Reference Ranges for Adults & Chldren, 2004)

31. Pola NormalElektroforesis Protein Serum 6-16-2010 31 Tutor Imun by Nuzul Gambar 11. Electrophoregram normal

32. PolaAbnormalitasElektroforesis Protein Serum 6-16-2010 32 Tutor Imun by Nuzul

33. PolaAbnormalitasElektroforesis Protein Serum 6-16-2010 33 Tutor Imun by Nuzul Tabel 4. PolaAbnormalitasElektroforesis Protein Serum padaBerbagaiPenyakit

34. PolaAbnormalitasElektroforesis Protein Serum 6-16-2010 34 Tutor Imun by Nuzul Tabel 4. Pola Abnormalitas Elektroforesis Protein Serum pada Berbagai Penyakit

35. Gambaran Abnormal Elektroforesis Protein Serum 6-16-2010 35 Tutor Imun by Nuzul

36. Gambaran Abnormal Elektroforesis Protein Serum 6-16-2010 36 Tutor Imun by Nuzul

37. Indikasi Elektroforesis Protein Serum 6-16-2010 37 Tutor Imun by Nuzul

38. 6-16-2010 38 Ciri Monoclonal Gammopathy(protein M) pita tajam, batas jelas (bisa karena heavy chain, atau karena ada kappa atau lambda light chain) Ciri Polyclonal Gammopathy pita diffuse dan lebar Tutor Imun Monoclonal GammopathyvsPolyclonal Gammopathy by Nuzul

39. Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe) 6-16-2010 39 Tutor Imun by Nuzul

40. Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe) 6-16-2010 40 Tutor Imun by Nuzul

41. Atas perhatiannya Terima kasih ! 6-16-2010 41

42. Kepustakaan FW Sunderman, Jr. Recent Advances in Clinical Interpretation of Electrophoretic Fractionations of the Serum Proteins. Philadelphia : Lippincott; 1964. O’Connell Theodore X, M.D., Timothy J. Horita, M.D., Barsam Kasravi, M.D. Understanding and Interpreting Serum Protein Electrophoresis. American Family Physician. www.aafp.org/afp. January 1, 2005,Volume 71, Number 1. pp 105 – 12. Merck E. Clinical Laboratory. 11th ed. Germany; 1974. 145 - 60. _______ . A Manual of Laboratory and Diagnostic Test; 2000. Roche. Reference Ranges for Adults & Children; 2004. pp 62. Rose RN, MD et al. Manual of Clinical Laboratory Immunology. 6th ed. Washington : American Society for Microbiology Press; 2002. pp 66 – 75. Sebia. Instruction Manual. Sebia. 2002. Sheehan C, MS, CLS, SI. Clinical Immunology : Principles and Laboratory Diagnosis. Philadelphia : J.B. Lippincott Company; 1990. pp 275 – 79. Word KM, Lehmann CA, Leiken AM. Clinical Laboratory Instrumentation and Automation : Principles, Application and Selection.Philadelphia: WB Saunders Company; 1994. pp. 158-70. ucsdlabmed.wikidot.com/chapter – 7 – laboratory - d 6-16-2010 42

43. Key Term Acute-Phase Reaction - The body's response to injury of inflammation, including fever, leukocytosis, and protein changes Bence Jones protein - free light chains; An abnormal plasma or urinary protein, consisting of monoclonal immunoglobulin light chains, excreted in some neoplastic diseases and characterized by its unusual solubility properties as it precipitates on heating at 56 to 60° C and redissolves at 90 to 100° C. It is measured by immunofixation electrophoresis (IFE). MGUS - monoclonal gammopathy of undetermined significance Monoclonal immunoglobulinopathies - increased concentration of a single immunoglobin that originates from a single plasma cell clone; also known as M-proteins Paraprotein - abnormal protein appearing in large quantities as a result of a pathological process/condition: also known as myeloma components (MCs) Polyclonal hyperimmunoglobulinemias - increased concentration of immunoglobulins from many different plasma cell lines Proteinuria - Protein in the urine 6-16-2010 43

44. ANALYTICAL APPROACH Serum protein electrophoresis: electrophoresis is a screening test which can be followed by quantitative assay of specific proteins; independent measurement of total protein is required for conversion to absolute amounts Identify abnormalities in immunoglobulin fraction by serum immunofixation Follow specific protein abnormalities by quantitative analysis of the protein by immunoassay 6-16-2010 44

45. GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS Congenital Deficiency Immunodeficiency syndromes that are present at birth: Selective IgA deficiency, X-linked agammaglobulinemia, hyper-IgM with decreased IgG (CD40L deficiency), SCID (X-linked and autosomal) Secondary Hypogammaglobulinemias -Protein-losing conditions (gut, kidney) -Malignant lymphomas (marrow replacement and inhibition of immunoglobulin synthesis) -Leukemias -Multiple myeloma (monoclonal immunoglobulin increased while others decrease) 6-16-2010 45

46. GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS Polyclonal Hyperimmunoglobulinemias -Chronic liver disease (increase of IgA due to decreased catabolism) -Chronic infections (all are increased) -Malignancies -Autoimmune diseases (often IgM) -Miscellaneous conditions (any chronic inflammation) Monoclonal Immunoglobulinopathies (M-Proteinemia) -Multiple myeloma (G>A>M>E,D) -Primary macroglobulinemia (Waldenstrøm’s, IgM) -Monoclonal gammopathy of undetermined significance (MGUS) (converts at 2% per year to myeloma) -Miscellaneous conditions (converts at 2% per year to myeloma) 6-16-2010 46

47. 6-16-2010 47 Example of identification of a monoclonal serum protein by immunofixation electrophoresis (Figure 1). The first track is serum protein electrophoresis (ELP). The next tracks are IgG (G), IgA (A), IgM (M), kappa light chains (K) and lambda light chains (L). The arrow indicates the position of the monoclonal protein. The second track identifies the protein as IgG after reaction with IgG antibody and protein staining. The sixth track identifies the light chain asλ.

48. 6-16-2010 48 Example of identification of a monoclonal serum protein by immunofixation electrophoresis (Figure 2). The first track is serum protein electrophoresis (ELP). The next tracks are IgG (G), IgA (A), IgM (M), kappa light chains (K) and lambda light chains (L). The arrow indicates the position of the monoclonal protein. The third track identifies the protein as IgM after reaction with IgM antibody and protein staining. The sixth track identifies the light chain as λ.

49. A) Multiple myeloma Multiple myeloma is a disease of older adults characterized by bone pain that is caused by a malignancy of plasma cells. In multiple myeloma bone marrow cells become replaced by plasma cells which often produce unregulated amounts of a monoclonal antibody that can be detected in serum. On bone biopsy plasma cells >30%, 3 or more lytic lesions would define the patient as having multiple myeloma. As bone marrow is replaced, patients become anemic and eventually have bone marrow failure. The bone marrow lesions can be visualize on x-ray as characteristic “punched out lesions” which leads to bone pain, osteoporosis, pathologic fractures, and hypercalcemia. Immunofixation studies indicate that the abnormal protein is IgG in over half of the cases, one-fourth IgA, less than 10% IgM, much less than 1% IgD; and IgE is found rarely. Bence-Jones protein (kappa and lambda light chains) is present in the urine of over 50% of the patients. In about one-fourth of patients only light chains are produced by the abnormal plasma cells, and therefore monoclonal peaks may not be found in the serum since the light chains easily pass through the kidney. Hypogammaglobulinemia is often seen because immunoglobulin production by non-malignant plasma cells is also greatly reduced and catabolism is increased. 6-16-2010 49

50. B) Waldenstrom’smacroglobulinemia Waldenstrom’s macroglobulinemia is characterized by IgM monoclonal proteins which cause hyperviscosity of the patients serum. Typically there is an absence of lytic bone disease and no bone tenderness. The abnormal protein is of the IgM type with a molecular weight of about 1,000,000 and a sedimentation constant of 18S (Svedberg unit). Bence-Jones proteinuria occurs in about 10% of these patients. The blood may be very viscous, because these large molecules do not readily leave the plasma for the extra cellular space. The very viscous blood leads to blindness, hypertension, and priapism. 6-16-2010 50

51. C) Monoclonal gammopathy of undetermined significance The term “monoclonal gammopathy of undetermined significance” (MGUS) denotes the presence of an M-protein in persons without clinical evidence of multiple myeloma (MM), macroglobulinemia, primary amyloidosis (AL) or other related disorders. This occurs in 50-60% of patients presenting with M-proteins. The term “benign monoclonal gammopathy” is misleading because one does not know if the process will remain stable and benign or will develop into symptomatic MM, macroglobulinemia, or a related disorder. The frequency of monoclonal gammopathies increases with advancing age. The prevalence of MGUS is 1% of patients older than 50 years, 3% of those older than 70 years, and increases up to 10% in persons over the age of 80 years. The incidence of MGUS is higher in African-Americans and lower in elderly Japanese. MGUS is characterized by: the presence of less than 3 g/dL M-protein in serum; fewer than 10% plasma cells in the bone marrow; lack of, or only small amounts of M-protein in the urine, and a low plasma-cell labeling index (PCLI). PCLI measures the synthesis of DNA and when elevated is good evidence that the patient has MM or will soon develop it. In addition, evidence of bone lesions, anemia, hypercalcemia or renal insufficiency must be absent. Most importantly, the M-protein must remain stable and no other abnormalities develop. 6-16-2010 51

52. C) Monoclonal gammopathy of undetermined significance In patients recently diagnosed with MGUS, serum protein electrophoresis should be stable. The test should be repeated in 3 to 6 months. If the M-protein is constant, the test should be repeated in 6 to 12 months. If the M-protein remains constant, electrophoresis and clinical evaluation should be performed annually thereafter. Patients should be told that the actuarial risk of malignant transformation is 17% at 10 years and 33% at 20 years. The rate of development of serious disease does not differ whether the M- protein is IgG, IgA, or IgM. However, patients should also be aware that evolution from MGUS to MM can be abrupt; and therefore, they should be advised to seek medical evaluation if symptoms develop. 6-16-2010 52

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