ELEKTROFORESIS PROTEIN SERUM<br />Siswanto armadi, dr, Sp.PK(K) / Nuzulul Quriyah, drD<br />6-16-2010<br />1<br />Tutor Im...
PENDAHULUAN<br />6-16-2010<br />2<br />
Elektroforesis<br />6-16-2010<br />3<br />Teknik pemisahan komponen/molekul bermuatan berdasarkan perbedaan tingkat migras...
Tutor Imun<br />PrinsipElektroforesis<br />by Nuzul<br />Muatan listrik molekul biologi (misal : protein) tergantung pd pH...
6-16-2010<br />5<br />Dengan elektroforesis, protein plasma dapat dipisahkan menjadi fraksi albumin serta α1, α2, β, dan γ...
ELEKTROFORESIS PROTEIN<br />6-16-2010<br />6<br />
Elektroforesis Protein<br />6-16-2010<br />Besarnya muatan<br />Media penyangga<br />Elektroendosmosis<br />Suhu<br />Ukur...
KomponenSistemElektroforesis<br />6-16-2010<br />8<br />Tutor Imun<br />by Nuzul<br />Power supply<br />Sampel<br />Bufer<...
Tutor Imun<br />by Nuzul<br />Yang sering dipakai : serum dan plasma<br />Cairan tubuh lain yang juga bisa dipakai : urine...
BUFER <br />6-16-2010<br />10<br />Tutor Imun<br />by Nuzul<br />Menjaga agar pH tetapkonstan<br />Mengalirkan (conduct) a...
SyaratBufer<br />6-16-2010<br />11<br />1<br />bufer tidak boleh bereaksi dengan sampel krn interaksi antara bufer dan sam...
MEDIA PENYANGGA<br />6-16-2010<br />12<br />Tutor Imun<br />by Nuzul<br />Agarose<br /> Gel<br />Poly-<br />acrylamide <br...
Endosmosis danWick Flow<br />6-16-2010<br />13<br />Tutor Imun<br />by Nuzul<br />Endosmosis (electroendosmosis)terjadiket...
Gambar 2. Endosmosis<br />6-16-2010<br />14<br />Tutor Imun<br />by Nuzul<br />
POWER SUPPLY<br />6-16-2010<br />15<br />Tutor Imun<br />by Nuzul<br />0-500 volt <br />0-100 mA<br />Jikaprosespemisahanb...
PEWARNAAN<br />6-16-2010<br />16<br />Ponceau <br />       S<br />Amido<br />Black<br />Oil Red O<br />Coomassie <br />Bri...
6-16-2010<br />17<br />PembacaanHasil<br />Tutor Imun<br />by Nuzul<br />Ada 2<br />Cara<br />Kuantitatif<br />Mengukur in...
ELEKTROFORESIS PROTEIN DI LABORATORIUM PK RSUD DR SOETOMO<br />6-16-2010<br />18<br />
SEBIA Hydragel Protein (E) K-20<br />6-16-2010<br />19<br />Tutor Imun<br />by Nuzul<br />Tabel 1. ReagendanBahandalam Kit...
Alat-Alat yang Dipakai<br />6-16-2010<br />20<br />Tutor Imun<br />by Nuzul<br />Gambar 3. Electrophoresis Chamber<br />Ga...
Alat-Alat yang Dipakai<br />6-16-2010<br />21<br />Tutor Imun<br />by Nuzul<br />Gambar 6. Aplikator<br />Gambar 7. Aplika...
Alat-Alat yang Dipakai<br />6-16-2010<br />22<br />Tutor Imun<br />by Nuzul<br />Scanner λ 570 nm atau filter kuning<br />
PROSEDUR SEBIA<br />6-16-2010<br />23<br />
TahapMigrasi<br />6-16-2010<br />24<br />Tutor Imun<br />by Nuzul<br />3<br />1<br />2<br />4<br />Tempatkanagarose gel pd...
TahapMigrasi (lanjutan)<br />6-16-2010<br />25<br />Tutor Imun<br />by Nuzul<br />Waktu 15`, 165 V, 7 ± 2 mA(set alat 60 m...
6-16-2010<br />26<br />Text<br />Text<br />Text<br />Fiksasidlm<br />fixation sol 15`<br />Keringkan<br />pd 80⁰ C, 10`<br...
6-16-2010<br />27<br />Text<br />Text<br />Text<br />Pewarnaan 4’<br />Destaining3x <br />sampaibersih<br />Scanning<br />...
Quality Control<br />6-16-2010<br />28<br /><ul><li>serum kontrol assayed Sebia PN 4785 / Agarose/CAPILLARYS Normal Contro...
HasilPemeriksaan<br />6-16-2010<br />29<br />Tutor Imun<br />by Nuzul<br />Tabel 2. Nilai Rujukan Protein Serum ( A Manual...
HasilPemeriksaan<br />6-16-2010<br />30<br />Tutor Imun<br />by Nuzul<br />Tabel 3. Nilai Rujukan Elektroforesis Protein S...
Pola NormalElektroforesis Protein Serum<br />6-16-2010<br />31<br />Tutor Imun<br />by Nuzul<br />Gambar 11. Electrophoreg...
PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />32<br />Tutor Imun<br />by Nuzul<br />
PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />33<br />Tutor Imun<br />by Nuzul<br />Tabel 4. PolaAbnorm...
PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />34<br />Tutor Imun<br />by Nuzul<br />Tabel 4. Pola Abnor...
Gambaran Abnormal Elektroforesis Protein Serum<br />6-16-2010<br />35<br />Tutor Imun<br />by Nuzul<br />
Gambaran Abnormal Elektroforesis Protein Serum<br />6-16-2010<br />36<br />Tutor Imun<br />by Nuzul<br />
Indikasi Elektroforesis Protein Serum<br />6-16-2010<br />37<br />Tutor Imun<br />by Nuzul<br />
6-16-2010<br />38<br />Ciri Monoclonal Gammopathy(protein M)<br />pita tajam, batas jelas (bisa karena heavy chain, atau k...
Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)<br />6-16-2010<br />39<br />Tutor Imun<br />by Nuzul<b...
Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)<br />6-16-2010<br />40<br />Tutor Imun<br />by Nuzul<b...
Atas perhatiannya<br />Terima kasih !<br />6-16-2010<br />41<br />
Kepustakaan<br />FW Sunderman, Jr. Recent Advances in Clinical Interpretation of Electrophoretic Fractionations of the Ser...
Key Term<br />Acute-Phase Reaction - The body's response to injury of inflammation, including fever, leukocytosis, and pro...
ANALYTICAL APPROACH<br />Serum protein electrophoresis: electrophoresis is a screening test which can be followed by quant...
GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS<br />Congenital Deficiency<br />	Immunodeficiency syndromes ...
GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS<br />Polyclonal Hyperimmunoglobulinemias<br />	-Chronic live...
6-16-2010<br />47<br />Example of identification of a monoclonal serum protein by immunofixation electrophoresis (Figure 1...
6-16-2010<br />48<br />Example of identification of a monoclonal serum protein by immunofixation electrophoresis (Figure 2...
A) Multiple myeloma<br />Multiple myeloma is a disease of older adults characterized by bone pain that is caused by a mali...
B) Waldenstrom’smacroglobulinemia<br />Waldenstrom’s macroglobulinemia is characterized by IgM monoclonal proteins which c...
C) Monoclonal gammopathy of undetermined significance<br />The term “monoclonal gammopathy of undetermined significance” (...
C) Monoclonal gammopathy of undetermined significance<br />In patients recently diagnosed with MGUS, serum protein electro...
6-16-2010<br />53<br />
Contents<br />6-16-2010<br />54<br />Click to add Title<br />Click to add Title<br />Click to add Title<br />Click to add ...
Contents<br />6-16-2010<br />55<br />2. Click to add Title<br />3. Click to add Title<br />4. Click to add Title<br />Clic...
Thank You !<br />www.themegallery.com<br />6-16-2010<br />56<br />
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  1. 1. ELEKTROFORESIS PROTEIN SERUM<br />Siswanto armadi, dr, Sp.PK(K) / Nuzulul Quriyah, drD<br />6-16-2010<br />1<br />Tutor Imunologi<br />
  2. 2. PENDAHULUAN<br />6-16-2010<br />2<br />
  3. 3. Elektroforesis<br />6-16-2010<br />3<br />Teknik pemisahan komponen/molekul bermuatan berdasarkan perbedaan tingkat migrasinya dalam sebuah medan listrik <br />Tutor Imun<br />by Nuzul<br />Pergerakan pd elektroforesis dijelaskan dengan gaya Lorentz :<br /> F = qE<br />F adalah gaya Lorentz, <br />q adalah muatan yang dibawa oleh obyek, <br />E adalah medan listrik <br />Posisimolekul yang terseparasipada gel dideteksidenganpewarnaan, ataudilakukankuantifikasidengan densitometer<br />
  4. 4. Tutor Imun<br />PrinsipElektroforesis<br />by Nuzul<br />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 <br />(bermuatan positif bila pH larutan lebih kecil daripada titik isoelektrik, bermuatan negatif bila pH larutan lebih besar daripada titik isoelektrik) <br />Dalam suatu medan listrik, molekul  biologi  yang  bermuatan  positif  (kation) bermigrasi ke elektroda negatif (katoda) dan sebaliknya,<br />yang bermuatan negatif (anion) bermigrasi ke elektroda positif (anoda)<br />6-16-2010<br />4<br />
  5. 5. 6-16-2010<br />5<br />Dengan elektroforesis, protein plasma dapat dipisahkan menjadi fraksi albumin serta α1, α2, β, dan γ-globulin <br />Protein dalam plasma memiliki konsentrasi sekitar 1 mmol/L <br />56% protein plasma merupakan fraksi albumin, 4% adalah α1-globulin, α2-globulin 10%, β-globulin 12%, dan 18% merupakan γ-globulin <br />Tutor Imun<br />Protein<br />by Nuzul<br />
  6. 6. ELEKTROFORESIS PROTEIN<br />6-16-2010<br />6<br />
  7. 7. Elektroforesis Protein<br />6-16-2010<br />Besarnya muatan<br />Media penyangga<br />Elektroendosmosis<br />Suhu<br />Ukuran molekul<br />Kekuatan medan listrik<br />Kekuatan ion dari bufer<br />Kecepatan migrasi molekul <br />Tutor Imun<br />by Nuzul<br />Click to add Title<br />7<br />
  8. 8. KomponenSistemElektroforesis<br />6-16-2010<br />8<br />Tutor Imun<br />by Nuzul<br />Power supply<br />Sampel<br />Bufer<br />Media penyangga<br />Power supply<br />Wicks<br />Pewarnaan<br />Gambar 1. Komponensistemelektroforesis<br />
  9. 9. Tutor Imun<br />by Nuzul<br />Yang sering dipakai : serum dan plasma<br />Cairan tubuh lain yang juga bisa dipakai : urine, cairan serebrospinal,<br />cairan dari pleura, perikardium, peritoneum, membran sinovial, dan air mata <br />Tidak ada persiapan khusus yang dilakukan pasien, tetapi keadaan puasa <br />lebih baik untuk menghindari hiperlipemia<br />Sampel yang diperlukan antara 0,3-25 μl tergantung dari konsentrasi analit <br />dan jenis media penyangga yang digunakan<br />Sampel diberi penanda warna untuk mengetahui kapan elektroforesis harus <br />dihentikan, pada bufer asam dipakai methylene green atau methylene blue, <br />sedangkan pada bufer basa dipakai bromphenol blue.<br />SAMPEL<br />6-16-2010<br />9<br />
  10. 10. BUFER <br />6-16-2010<br />10<br />Tutor Imun<br />by Nuzul<br />Menjaga agar pH tetapkonstan<br />Mengalirkan (conduct) arus listrik <br />
  11. 11. SyaratBufer<br />6-16-2010<br />11<br />1<br />bufer tidak boleh bereaksi dengan sampel krn interaksi antara bufer dan sampel dapat mempengaruhi gerakan molekul<br />Tutor Imun<br />by Nuzul<br />3<br />kekuatan ion dari larutan bufer harus dipertimbangkan kekuatan ionik rendah menghasilkan kecepatan migrasi lebih besar<br />2<br />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<br />
  12. 12. MEDIA PENYANGGA<br />6-16-2010<br />12<br />Tutor Imun<br />by Nuzul<br />Agarose<br /> Gel<br />Poly-<br />acrylamide <br />Gel<br />Starch <br />Gel<br />Cellulose <br />Acetate<br />Kertas<br />murah, tapi <br />menyerap banyak <br />protein <br />menghasilkan pita yang lebar,<br />resolusinya <br />jelek, waktu pemisahannya<br />lebih lama<br />granula yang <br />mengandung <br />amilosa dan <br />amilopektin, <br />bila suspensinya <br />dilarutkan dan <br />dipanaskan akan terbentuk larutan jernih dan menjadi gel pd pendinginan<br />sering dipakai, bebas dari ionizable groups pada buffer basa atau netral tidak terlalu terpengaruh oleh endosmosis,<br />resolusi lbh tajam, jernih<br />lebih kuat daripada kertas, dg ukuran pori-pori homogen,<br />pemisahan protein lebih cepat, absorpsi protein minimal <br /> hasil pita<br />lebih jelas dan <br />lebih tajam<br />masih terbatas <br />hanya untuk <br />pemisahan <br />isoenzim <br />dari alkali <br />fosfatase <br />
  13. 13. Endosmosis danWick Flow<br />6-16-2010<br />13<br />Tutor Imun<br />by Nuzul<br />Endosmosis (electroendosmosis)terjadiketika media penyangga jadi bermuatan negatif akibat adsorpsi ion hidroksil dari bufer muatan negatif menarik muatan positif dari bufer. <br />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 <br />Wick Flow<br />gerakankeatasdarilarutanbufermelaluikeduatepimembran yang terendamuntukmenggantikankelembaban yang hilangakibatpemanasan. Aliraninimemperlambatgerakanmolekulpada media penyangga<br />
  14. 14. Gambar 2. Endosmosis<br />6-16-2010<br />14<br />Tutor Imun<br />by Nuzul<br />
  15. 15. POWER SUPPLY<br />6-16-2010<br />15<br />Tutor Imun<br />by Nuzul<br />0-500 volt <br />0-100 mA<br />Jikaprosespemisahanbutuhwaktu lama, arus yang konstanlebihdianjurkanuntukmeminimalkankenaikansuhu<br />Jika waktu 30 menit atau kurang, maka voltase dinaikkan<br />
  16. 16. PEWARNAAN<br />6-16-2010<br />16<br />Ponceau <br /> S<br />Amido<br />Black<br />Oil Red O<br />Coomassie <br />Brilliant<br />Blue R 250<br />Tutor Imun<br />by Nuzul<br />Schiff’s<br />
  17. 17. 6-16-2010<br />17<br />PembacaanHasil<br />Tutor Imun<br />by Nuzul<br />Ada 2<br />Cara<br />Kuantitatif<br />Mengukur intensitas warna pada tiap pita.<br />Kualitatif<br />Membandingkan garis-garis fraksi dengan pola elektroforesis standar yang telah diketahui<br />Metode eluasi<br />Pita dipotong-potong beberapa bagian, pewarna kemudian dieluasikan dalam suatu larutan, warna yang larut tersebut dihitung dengan fotometer<br />Densitometri<br />-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<br />
  18. 18. ELEKTROFORESIS PROTEIN DI LABORATORIUM PK RSUD DR SOETOMO<br />6-16-2010<br />18<br />
  19. 19. SEBIA Hydragel Protein (E) K-20<br />6-16-2010<br />19<br />Tutor Imun<br />by Nuzul<br />Tabel 1. ReagendanBahandalam Kit Hydragel Protein (E) K20<br />
  20. 20. Alat-Alat yang Dipakai<br />6-16-2010<br />20<br />Tutor Imun<br />by Nuzul<br />Gambar 3. Electrophoresis Chamber<br />Gambar 5. Staining - Destaining solution<br />Gambar 4. Agarose gel<br />
  21. 21. Alat-Alat yang Dipakai<br />6-16-2010<br />21<br />Tutor Imun<br />by Nuzul<br />Gambar 6. Aplikator<br />Gambar 7. Aplikatorcarrier<br />Gambar 8. Microprocessor (power supply)<br />Gambar 9. Inkubator<br />
  22. 22. Alat-Alat yang Dipakai<br />6-16-2010<br />22<br />Tutor Imun<br />by Nuzul<br />Scanner λ 570 nm atau filter kuning<br />
  23. 23. PROSEDUR SEBIA<br />6-16-2010<br />23<br />
  24. 24. TahapMigrasi<br />6-16-2010<br />24<br />Tutor Imun<br />by Nuzul<br />3<br />1<br />2<br />4<br />Tempatkanagarose gel pd applicator carrier dg kutub + dibagianatas<br />Tempatkanapplicator ygberisi serum pd handle applicator carrier<br />Serum 10 μl pd sumuranapplicator<br />Tempatkanapplicator carrier pd tempatygdatar<br />
  25. 25. TahapMigrasi (lanjutan)<br />6-16-2010<br />25<br />Tutor Imun<br />by Nuzul<br />Waktu 15`, 165 V, 7 ± 2 mA(set alat 60 mA) <br />
  26. 26. 6-16-2010<br />26<br />Text<br />Text<br />Text<br />Fiksasidlm<br />fixation sol 15`<br />Keringkan<br />pd 80⁰ C, 10`<br />Pindahkan gel<br />Tutor Imun<br />TahapFiksasi<br />by Nuzul<br />
  27. 27. 6-16-2010<br />27<br />Text<br />Text<br />Text<br />Pewarnaan 4’<br />Destaining3x <br />sampaibersih<br />Scanning<br />Gel menghadap<br />kebawahλ570nm<br />Keringkan<br />TahapStaining-DestainingTahapScanning<br />Tutor Imun<br />by Nuzul<br />
  28. 28. Quality Control<br />6-16-2010<br />28<br /><ul><li>serum kontrol assayed Sebia PN 4785 / Agarose/CAPILLARYS Normal Controldan </li></ul>Sebia PN 4787 / Agarose/CAPILLARYS Hypergamma Control.<br />-liofilisat stabil, dipakai dalam jumlah kecil-kecil, dapat disimpan dalam lemari es selama kurang lebih 6 bulan<br />Tutor Imun<br />by Nuzul<br />Gambar 10. Hydragel B1+B2 15/30:Normal Control Pictured 1-15Hypergamma Control Pictured 16-30<br />
  29. 29. HasilPemeriksaan<br />6-16-2010<br />29<br />Tutor Imun<br />by Nuzul<br />Tabel 2. Nilai Rujukan Protein Serum ( A Manual of Laboratory and Diagnostic Test, 2000)<br />
  30. 30. HasilPemeriksaan<br />6-16-2010<br />30<br />Tutor Imun<br />by Nuzul<br />Tabel 3. Nilai Rujukan Elektroforesis Protein Serum<br />(Sheehan C, Clinical Immunology : Principles & Laboratory Diagnosis, 1990.<br />Roche, Reference Ranges for Adults & Chldren, 2004)<br />
  31. 31. Pola NormalElektroforesis Protein Serum<br />6-16-2010<br />31<br />Tutor Imun<br />by Nuzul<br />Gambar 11. Electrophoregram normal<br />
  32. 32. PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />32<br />Tutor Imun<br />by Nuzul<br />
  33. 33. PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />33<br />Tutor Imun<br />by Nuzul<br />Tabel 4. PolaAbnormalitasElektroforesis Protein Serum padaBerbagaiPenyakit<br />
  34. 34. PolaAbnormalitasElektroforesis Protein Serum<br />6-16-2010<br />34<br />Tutor Imun<br />by Nuzul<br />Tabel 4. Pola Abnormalitas Elektroforesis Protein Serum pada Berbagai Penyakit<br />
  35. 35. Gambaran Abnormal Elektroforesis Protein Serum<br />6-16-2010<br />35<br />Tutor Imun<br />by Nuzul<br />
  36. 36. Gambaran Abnormal Elektroforesis Protein Serum<br />6-16-2010<br />36<br />Tutor Imun<br />by Nuzul<br />
  37. 37. Indikasi Elektroforesis Protein Serum<br />6-16-2010<br />37<br />Tutor Imun<br />by Nuzul<br />
  38. 38. 6-16-2010<br />38<br />Ciri Monoclonal Gammopathy(protein M)<br />pita tajam, batas jelas (bisa karena heavy chain, atau karena ada kappa atau lambda light chain)<br />Ciri Polyclonal Gammopathy<br />pita diffuse dan lebar<br />Tutor Imun<br />Monoclonal GammopathyvsPolyclonal Gammopathy<br />by Nuzul<br />
  39. 39. Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)<br />6-16-2010<br />39<br />Tutor Imun<br />by Nuzul<br />
  40. 40. Electrophoregram penyakit Multiple Myeloma (dalam beberapa isotipe)<br />6-16-2010<br />40<br />Tutor Imun<br />by Nuzul<br />
  41. 41. Atas perhatiannya<br />Terima kasih !<br />6-16-2010<br />41<br />
  42. 42. Kepustakaan<br />FW Sunderman, Jr. Recent Advances in Clinical Interpretation of Electrophoretic Fractionations of the Serum Proteins. Philadelphia : Lippincott; 1964.<br />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.<br />Merck E. Clinical Laboratory. 11th ed. Germany; 1974. 145 - 60.<br />_______ . A Manual of Laboratory and Diagnostic Test; 2000.<br />Roche. Reference Ranges for Adults & Children; 2004. pp 62.<br />Rose RN, MD et al. Manual of Clinical Laboratory Immunology. 6th ed. Washington : American Society for Microbiology Press; 2002. pp 66 – 75. <br />Sebia. Instruction Manual. Sebia. 2002.<br />Sheehan C, MS, CLS, SI. Clinical Immunology : Principles and Laboratory Diagnosis. Philadelphia : J.B. Lippincott Company; 1990. pp 275 – 79. <br />Word KM, Lehmann CA, Leiken AM. Clinical Laboratory Instrumentation and Automation : Principles, Application and Selection.Philadelphia: WB Saunders Company; 1994. pp. 158-70.<br />ucsdlabmed.wikidot.com/chapter – 7 – laboratory - d<br />6-16-2010<br />42<br />
  43. 43. Key Term<br />Acute-Phase Reaction - The body's response to injury of inflammation, including fever, leukocytosis, and protein changes<br />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).<br />MGUS - monoclonal gammopathy of undetermined significance<br />Monoclonal immunoglobulinopathies - increased concentration of a single immunoglobin that originates from a single plasma cell clone; also known as M-proteins<br />Paraprotein - abnormal protein appearing in large quantities as a result of a pathological process/condition: also known as myeloma components (MCs)<br />Polyclonal hyperimmunoglobulinemias - increased concentration of immunoglobulins from many different plasma cell lines<br />Proteinuria - Protein in the urine<br />6-16-2010<br />43<br />
  44. 44. ANALYTICAL APPROACH<br />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<br />Identify abnormalities in immunoglobulin fraction by serum immunofixation<br />Follow specific protein abnormalities by quantitative analysis of the protein by immunoassay<br />6-16-2010<br />44<br />
  45. 45. GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS<br />Congenital Deficiency<br /> 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)<br />Secondary Hypogammaglobulinemias<br /> -Protein-losing conditions (gut, kidney)<br /> -Malignant lymphomas (marrow replacement and inhibition of immunoglobulin synthesis)<br /> -Leukemias<br /> -Multiple myeloma (monoclonal immunoglobulin increased while others decrease)<br />6-16-2010<br />45<br />
  46. 46. GENERAL COMMENTS ON CLINICAL ABNORMALITIES OF GAMMA-GLOBULINS<br />Polyclonal Hyperimmunoglobulinemias<br /> -Chronic liver disease (increase of IgA due to decreased catabolism)<br /> -Chronic infections (all are increased)<br /> -Malignancies<br /> -Autoimmune diseases (often IgM)<br /> -Miscellaneous conditions (any chronic inflammation)<br />Monoclonal Immunoglobulinopathies (M-Proteinemia)<br /> -Multiple myeloma (G>A>M>E,D)<br /> -Primary macroglobulinemia (Waldenstrøm’s, IgM)<br /> -Monoclonal gammopathy of undetermined significance (MGUS) (converts at 2% per year to myeloma)<br /> -Miscellaneous conditions (converts at 2% per year to myeloma)<br />6-16-2010<br />46<br />
  47. 47. 6-16-2010<br />47<br />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.<br />
  48. 48. 6-16-2010<br />48<br />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.<br />
  49. 49. A) Multiple myeloma<br />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.<br />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.<br />6-16-2010<br />49<br />
  50. 50. B) Waldenstrom’smacroglobulinemia<br />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.<br />6-16-2010<br />50<br />
  51. 51. C) Monoclonal gammopathy of undetermined significance<br />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.<br />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.<br />6-16-2010<br />51<br />
  52. 52. C) Monoclonal gammopathy of undetermined significance<br />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.<br />6-16-2010<br />52<br />
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