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  • ELEKTROFORESIS PROTEIN SERUM
    Siswanto armadi, dr, Sp.PK(K) / Nuzulul Quriyah, drD
    6-16-2010
    1
    Tutor Imunologi
  • PENDAHULUAN
    6-16-2010
    2
  • 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
  • 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
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    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
  • ELEKTROFORESIS PROTEIN
    6-16-2010
    6
  • 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
  • KomponenSistemElektroforesis
    6-16-2010
    8
    Tutor Imun
    by Nuzul
    Power supply
    Sampel
    Bufer
    Media penyangga
    Power supply
    Wicks
    Pewarnaan
    Gambar 1. Komponensistemelektroforesis
  • 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
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    9
  • BUFER
    6-16-2010
    10
    Tutor Imun
    by Nuzul
    Menjaga agar pH tetapkonstan
    Mengalirkan (conduct) arus listrik
  • 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
  • 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
  • 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
  • Gambar 2. Endosmosis
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    14
    Tutor Imun
    by Nuzul
  • 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
  • PEWARNAAN
    6-16-2010
    16
    Ponceau
    S
    Amido
    Black
    Oil Red O
    Coomassie
    Brilliant
    Blue R 250
    Tutor Imun
    by Nuzul
    Schiff’s
  • 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
  • ELEKTROFORESIS PROTEIN DI LABORATORIUM PK RSUD DR SOETOMO
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    18
  • SEBIA Hydragel Protein (E) K-20
    6-16-2010
    19
    Tutor Imun
    by Nuzul
    Tabel 1. ReagendanBahandalam Kit Hydragel Protein (E) K20
  • 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
  • Alat-Alat yang Dipakai
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    21
    Tutor Imun
    by Nuzul
    Gambar 6. Aplikator
    Gambar 7. Aplikatorcarrier
    Gambar 8. Microprocessor (power supply)
    Gambar 9. Inkubator
  • Alat-Alat yang Dipakai
    6-16-2010
    22
    Tutor Imun
    by Nuzul
    Scanner λ 570 nm atau filter kuning
  • PROSEDUR SEBIA
    6-16-2010
    23
  • 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
  • TahapMigrasi (lanjutan)
    6-16-2010
    25
    Tutor Imun
    by Nuzul
    Waktu 15`, 165 V, 7 ± 2 mA(set alat 60 mA)
  • 6-16-2010
    26
    Text
    Text
    Text
    Fiksasidlm
    fixation sol 15`
    Keringkan
    pd 80⁰ C, 10`
    Pindahkan gel
    Tutor Imun
    TahapFiksasi
    by Nuzul
  • 6-16-2010
    27
    Text
    Text
    Text
    Pewarnaan 4’
    Destaining3x
    sampaibersih
    Scanning
    Gel menghadap
    kebawahλ570nm
    Keringkan
    TahapStaining-DestainingTahapScanning
    Tutor Imun
    by Nuzul
  • Quality Control
    6-16-2010
    28
    • serum kontrol assayed Sebia PN 4785 / Agarose/CAPILLARYS Normal Controldan
    Sebia PN 4787 / Agarose/CAPILLARYS Hypergamma Control.
    -liofilisat stabil, dipakai dalam jumlah kecil-kecil, dapat disimpan dalam lemari es selama kurang lebih 6 bulan
    Tutor Imun
    by Nuzul
    Gambar 10. Hydragel B1+B2 15/30:Normal Control Pictured 1-15Hypergamma Control Pictured 16-30
  • HasilPemeriksaan
    6-16-2010
    29
    Tutor Imun
    by Nuzul
    Tabel 2. Nilai Rujukan Protein Serum ( A Manual of Laboratory and Diagnostic Test, 2000)
  • 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)
  • Pola NormalElektroforesis Protein Serum
    6-16-2010
    31
    Tutor Imun
    by Nuzul
    Gambar 11. Electrophoregram normal
  • PolaAbnormalitasElektroforesis Protein Serum
    6-16-2010
    32
    Tutor Imun
    by Nuzul
  • PolaAbnormalitasElektroforesis Protein Serum
    6-16-2010
    33
    Tutor Imun
    by Nuzul
    Tabel 4. PolaAbnormalitasElektroforesis Protein Serum padaBerbagaiPenyakit
  • PolaAbnormalitasElektroforesis Protein Serum
    6-16-2010
    34
    Tutor Imun
    by Nuzul
    Tabel 4. Pola Abnormalitas Elektroforesis Protein Serum pada Berbagai Penyakit
  • Gambaran Abnormal Elektroforesis Protein Serum
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    35
    Tutor Imun
    by Nuzul
  • Gambaran Abnormal Elektroforesis Protein Serum
    6-16-2010
    36
    Tutor Imun
    by Nuzul
  • Indikasi Elektroforesis Protein Serum
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    37
    Tutor Imun
    by Nuzul
  • 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
  • Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)
    6-16-2010
    39
    Tutor Imun
    by Nuzul
  • Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)
    6-16-2010
    40
    Tutor Imun
    by Nuzul
  • Atas perhatiannya
    Terima kasih !
    6-16-2010
    41
  • 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
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  • 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
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  • 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
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  • 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
  • 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
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  • 6-16-2010
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    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.
  • 6-16-2010
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    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.
  • 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
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  • 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
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  • 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
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  • 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.
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  • 6-16-2010
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  • Contents
    6-16-2010
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    6-16-2010
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