Jh4

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Jh4

  1. 1. Bastiana/Yetti Hernaningsih<br />J Clin Exp Hematopathol, Vol. 48, No.1, Apr 2008<br />1<br />Prediction of Clinical Outcome in Patients with Idiopathic Thrombocytopenic Purpura by Evaluating Bone Marrow Clot CD20+ B Lymphocytes and Morphological Changes of Megakaryocytes<br />Departemen Patologi Klinik Universitas Airlangga/RSUD Dr. Soetomo<br />23 Februari 2010<br />
  2. 2. Pendahuluan<br />Journal Reading Bastiana, 2010<br />2<br />1<br />2<br />3<br />Idiopathic Thrombocytopenic Purpura<br />Gangguan yang diperantarai imun <br />Trombosit diopsonisasi oleh autoantibodi<br />dan dihancurkan dini oleh RES <br />Clinical outcome bervariasi<br />25 - 30% kronis dan menjadi refrakter terhadap kortikosteroid, <br />splenoktomi, dan terapi lain<br />Clinical <br />outcome<br />Add Your Text<br />Faktor Prediktor<br />belum diketahui<br />
  3. 3. Pendahuluan<br />Journal Reading Bastiana, 2010<br />3<br />Dalam penelitian ini<br />Penelitian ITP sebelumnya<br />Limfosit B mempunyai <br />peran penting dalam<br />etiologi.<br /><ul><li>Mengevaluasi temuan patologis clot sutul (sumsum tulang) dengan metode imunohistokimia
  4. 4. Menentukan faktor mana yang dapat memprediksi outcome pada pasien ITP.</li></ul>Folikel hiperplastik dlm white pulp pd zona marginal, yang dibentuk oleh limfosit B  gambaran patologis utama pada limpa<br />Ditemukan ekspansi klonal sel B & Ab monoklonal anti CD20 berguna pada px ITP refrakter<br />Gambaran patologis limfosit B masih belum jelas<br />
  5. 5. Journal Reading Bastiana, 2010<br />4<br />Prognostik hasil terapi 13 minggu setelah terapi lengkap atau 6 bulan setelah diagnosis<br />Perubahan patologis pada clot sumsum tulang (sutul)<br />Tujuan Penelitian<br />Pengaruh??<br />
  6. 6. Journal Reading Bastiana, 2010<br />5<br />73 Pasien ITP<br /> (Trombosit <50x109/L)<br />Jan 1997-Juni 2005<br />Clot / biopsi sutul<br />BMA pd saat Dx<br />Biopsi Sutul <br />jika bahan BMA (-)<br />Metode Penelitian<br />Pemeriksaan Imuno-<br />histokimia<br />Dievaluasi blind oleh seorang ahli Patologi<br />Dihubungkan dengan respon terapi<br />Px dengan Myelodisplastic Syndrome pd sutul dieksklusi dari penelitian<br />
  7. 7. Journal Reading Bastiana, 2010<br />6<br />Definisi respon terapi<br />Metode Penelitian<br />Complete response (CR)<br />Jumlah trombosit minimal 150 x 109/L dalam 13 minggu setelah terapi lengkap.<br />Relaps<br />Jumlah trombosit yang menurun <50x109/L sesudah mencapai CR atau PR<br />Partial response (PR)<br />Peningkatan jumlah dua kali dari jumlah trombosit awal atau jumlah trombosit di atas 50 x 109/L.<br />Refrakter<br />Pasien yang tidak dapat mencapai CR atau PR dengan terapi pertama dan atau terapi kedua<br />
  8. 8. Journal Reading Bastiana, 2010<br />7<br />Metode Penelitian<br />Eradikasi Helicobacter pylori<br />1-3 Bulan<br />7 Hari<br />Penilaian Eradikasi<br />13 Curea breath test 1-3 bulan sesudah terapi lengkap.<br />Terapi H.pylori (selama 7 hari)<br /><ul><li>Lansoprazole </li></ul> (30 mg 2x sehari)<br /><ul><li> Clarithromycin </li></ul> (200 mg 2x sehari)<br /><ul><li> Amoxicillin </li></ul> (750 mg 2x sehari) <br />Infeksi Helicobacterpylori(H. pylori) diketahui dengan melakukan 13 C urea breath test<br />
  9. 9. Metode Penelitian<br />Journal Reading Bastiana, 2010<br />8<br />Pemeriksaan Imunohistokimia<br />Clot sumsum tulang  difiksasi formalin diletakkan dalam parafin<br />Pewarnaan dengan hematoxylineosin<br />1<br />3a<br />2000.10 Add Your Text<br />2000.10 Add Your Text<br />2000.10 Add Your Text<br />3b<br />2<br />Pewarnaan dobel dengan metode naphthol-ASD chloro-acetate esterase plus Giemsa (ASD-Giemsa method). <br />2003.10 Add Your Text<br />2003.10 Add Your Text<br />2003.10 Add Your Text<br />Pemotongan serial<br />2001.10 Add Your Text<br />2001.10 Add Your Text<br />2001.10 Add Your Text<br />
  10. 10. Studi Imunohistokimia: metode streptavidin-biotin.<br />Antibodi monoklonal tikus, termasuk CD20 (Dako, Glostrup, Denmark)  sebagai antibodi primer.<br />Antigen retrieval dilakukan dengan 0.5 % trypsin digestion pada 37°C selama 30 menit atau dengan microwave dalam 0.1 M citrate buffer pada pH 6.0.<br />Aktivitas peroksidase endogen dihambat melalui inkubasi dengan0.3% H2O2 dalam methanol.<br />Reaksi dibuat visibel dengan 3,3’diaminobenzidinedalam 0.005% H2O2sebagai chromogen, diikuti dengan counterstaining dengan Mayer hematoxylin.<br />Journal Reading Bastiana, 2010<br />9<br />Metode Penelitian<br />Metode Streptavidin-Biotin<br />
  11. 11. Fisher’s exact: analisis data kategori. <br />One-way Anova: Analisis distribusi variabel kontinyu: analisis satu arah (one-way analysis)<br />Kruskal-Wallis test (jika variasi tidak homogen)<br />Multivariate logistic regression models: (menghitung odds ratio faktor resiko potensial dari outcome<br />Journal Reading Bastiana, 2010<br />10<br />Analisis Statistik<br />Nilai p < 0.05 ada perbedaansignifikan<br />Semua analisis dilakukan menggunakanSPSS 11 <br />
  12. 12. Clot sutul yang diperoleh saat diagnosis berisi sel berinti yang memadai untuk pemeriksaan patologis pada 56 dari 73 pasien.<br />Median umur 56 pasien: 66 tahun (16-86 tahun). 25 laki-laki dan 31 perempuan. <br />Median durasi dari follow-up: 15,6 bulan (0,03-139,8 bulan).<br />Karakteristik pasien: tabel 1.<br />Temuan patologis: gambar 1. <br />Journal Reading Bastiana, 2010<br />11<br />Hasil: Karakteristik Pasien<br />
  13. 13. Journal Reading Bastiana, 2010<br />12<br />Median umur: 66 thn<br />T<br />Mean durasi follow up= 15,6 bulan<br />
  14. 14. Hasil: Klasifikasi Pasien penelitian <br />Journal Reading Bastiana, 2010<br />13<br />C<br />A<br />B<br /><ul><li> tidak ada perubahan morfologi megakariosit
  15. 15. tidak ada peningkatan CD20+ limfosit
  16. 16. ada perubahan patologis megakariosit
  17. 17. peningkatan CD20+ limfosit (≥ 1% dari sel berinti);
  18. 18. ada perubahan patologis megakariosit
  19. 19. tidak ada peningkatan CD20+ limfosit (< 1% dari sel berinti) </li></ul>Berdasarkan temuan patologis pada clot sutul, peneliti menggolongkan pasien dalam 3 kelompok: <br />
  20. 20. Journal Reading Bastiana, 2010<br />14<br />Gambar 1  Temuan patologi pada ITP<br />3 kelompok Pasien (tabel 1):<br /> Kelompok A (n = 21); B (n = 21) dan C (n = 14)<br />
  21. 21. Journal Reading Bastiana, 2010<br />15<br />Gambar 1  Temuan patologi pada ITP<br />Gambar (1a)<br />Temuan histopatologis sutul dari kasus ITP. Peningkatan jumlah megakariosit secara random terdistribusi pada partikel sutul. (clot sutul, pengecatan HE , x20).<br />Gambar (1b) <br />Megakariosit dari kasus ITP. Megakariosit matur menunjukkan pinggiran sitoplasma yang tebal, proyeksi sitoplasma seperti “corona” dengan perlekatan neutrofil pada puncak proyeksi. Tidak ada temuan displastik. (clot sutul, clot, pengecatan HE, x400).<br />Immunohistological analysis of bone marrow clots<br />
  22. 22. Journal Reading Bastiana, 2010<br />16<br />Gambar 1  Temuan patologi pada kasus ITP<br />Gambar (1c) <br />Temuan sutul pada kasus ITP. Sekitar 5% sel yang diperiksa dengan lapangan pandang besar adalah CD20+ B-cells (clot sutul, CD20 immunostaining, x100).<br />Gambar (1d) <br />Temuan sutul dari kontrol normal. Kurang dari 1 % sel adalah CD20+ B-cells. (clot sutul, CD20 immunostaining, x200).<br />
  23. 23. Journal Reading Bastiana, 2010<br />17<br />Hasil Penelitian: Terapi ITP  ditunjukkan Tabel 1<br /><ul><li>Terapi primer berisi prednisolon pada 39 dari 56 </li></ul> pasien. <br /><ul><li>CR dicapai pada 25 pasien, dan PR dicapai pada 14</li></ul> pasien setelah terapi primer.<br /><ul><li>CR dan PR spontan tanpa terapi terjadi masing-</li></ul> masing pada 3 dan 4 pasien.<br /><ul><li>26 pasien menjalani 13 C urea breath test.
  24. 24. Hasil dari 13C urea breath test dan eradikasi </li></ul> H. pyloria ditunjukkan pada tabel 1.<br />
  25. 25. Journal Reading Bastiana, 2010<br />18<br />CR= 25 ; PR= 14<br />13C UBT= 26 px<br />
  26. 26. Hasil Penelitian: Temuan Patologis dan Respon TerapiHasil analisis multivarian ditunjukkan pada tabel 2.<br />Temuan patologis merupakan faktor prognostik yang signifikan terhadap respon terapi. <br />Dibandingkan dengan kelompok A, kelompok B merupakan faktor prognostik yang signifikan untuk mencapai CR, (p = 0.04; odds ratio, 6.65; 95% confidence interval, 1,09 - 40,54) <br />Status kelompok C merupakan faktor prognostik yang signifikan untuk respon terapi apapun (p = 0.04; odds ratio, 14.26; 95% confidence interval, 1,08 – 188,02).<br />Journal Reading Bastiana, 2010<br />19<br />
  27. 27. Journal Reading Bastiana, 2010<br />20<br />
  28. 28. 3 pasien yang refrakter terhadap terapi apapun, semuanya termasuk kelompok A.<br />Satu dari 3 pasien meninggal karena pneumonia cytomegalovirus 18 bulan setelah diagnosis.<br />Dua pasien lainnya diobati secara suportif dan masing-masing bertahan hidup selama 36 dan 90 bulan setelah diagnosis.<br />Journal Reading Bastiana, 2010<br />21<br />
  29. 29. Diskusi<br /><ul><li>Peneliti telah membagi pasien ITP dalam 3 kelompok dengan clinical outcome yang berbeda berdasarkan pemeriksaan imunohistopatologis dari clot sutul.
  30. 30. Pasien kelompok A (termasuk semua pasien refrakter) menunjukkan treatment outcome paling jelek.
  31. 31. Hal tsb menunjukkan adanya CD20+ limfosit pada sutul berhubungan dengan outcome yang jelek .</li></ul>Journal Reading Bastiana, 2010<br />22<br />
  32. 32. Diskusi<br />Peneliti menekankan pentingnya investigasi dari B cell clones. Penelitian sebelumnya menunjukkan B-cell clones pada ITP mungkin memproduksi antibodi anti platelet dan terlibat dalam aktivasi fungsi reseptor Fc makrofag dan klirens trombosit yang ter-coated IgG.<br />Namun peran B cells masih kontroversial.<br /> Peningkatan B-cell clones mungkin merupakan hasil dari disregulasi sel B. <br />Penelitian lebih lanjut peran clones tersebut dapat memungkinkan interpretasi lebih baik dari temuan ini.<br />Journal Reading Bastiana, 2010<br />23<br />
  33. 33. <ul><li>Perubahan morfologis megakariosit merupakan faktor penting untuk outcome pasien. Namun patofisiologi perubahan ini belum diketahui karena temuan morfologi tsb belum pernah diteliti sebelumnya.
  34. 34. Perlekatan antibodi anti platelet dan komplemen ke megakariosit dapat meginduksi perubahan tersebut.
  35. 35. Diperlukan penelitian lebih lanjut untuk menentukan patofisiologi yang tepat.</li></ul>Journal Reading Bastiana, 2010<br />24<br />
  36. 36. Median umur tinggi dan dominasi pasien bukan perempuan  tidak konsisten dengan penelitian ITP sebelumnya pada orang dewasa.<br />Latar belakang pasien mungkin mempengaruhi hasil, sehingga perlu penelitian skala besar.<br />Journal Reading Bastiana, 2010<br />25<br />
  37. 37. Penelitian terbaru menunjukkan ada hubungan antara infeksi H. pylori dengan ITP.<br />Pada penelitian ini, adanya infeksi H. pylori infection hampir sama pada semua kelompok. Namun jumlah pasien yang menjalani 13 C urea breath test kecil. <br />Journal Reading Bastiana, 2010<br />26<br />
  38. 38. Penelitian ini memberikan informasi terbaru (novel) pengembangan stratetegi berbasis resiko (risk based strategy) dalam mengobati ITP. <br />Penelitian ini memiliki keterbatasan: <br /> - jumlah pasien sedikit<br /> - retrospektif: ada bias mempengaruhi hasil.<br /> - Heterogeneitas antara spesimen berbeda dari <br /> pasien yang sama, tidak diteliti.<br /> - Observer sampel sutul hanya satu variasi <br /> inter observer tidak diketahui.<br />Journal Reading Bastiana, 2010<br />27<br />
  39. 39. <ul><li>Penelitian ini penelitian pertama yang menunjukkan evaluasi patologis CD 20+ limfosit dan perubahan morfologi megakariosit yang dapat bermanfaat sebagai faktor prognostik terhadap outcomes pasien.
  40. 40. Perlu penelitian dengan skala besar.
  41. 41. Penelitian hubungan infeksi H.pylori dengan temuan imunohistopatologis clot sutul juga diperlukan.</li></ul>Journal Reading Bastiana, 2010<br />28<br />
  42. 42. Pasien dengan peningkatan CD20+ lymphocytes dan perubahan morfologi megakaryocytes, lebih muda usianya daripada pasien dengan perubahan patologis yang lebih sedikit. <br />Hal tsb. menunjukkan bahwa etiologi dan manejemen optimal ITP mungkin berbeda antara pasien yang lebih muda dengan yang lebih tua.<br />Perlu penelitian multisentris dengan skala besar untuk mendapatkan interpretasi yang akurat.<br />Journal Reading Bastiana, 2010<br />29<br />
  43. 43. Kesimpulan<br />Pendekatan berdasarkan pemeriksaan patologis tampaknya berguna untuk menentukan strategi berbasis resiko, dalam pengobatan pasien ITP.<br /> (Pasien ITP dapat diklasifikasi dengan clinical outcome yang berbeda berdasarkan pemeriksaan imununohistopatologis clot sumsum tulang).<br />Journal Reading Bastiana, 2010<br />30<br />
  44. 44. 31<br />Thank You !<br />Journal Reading Hematology II,<br />23 Februari 2010<br /> Bastiana/Yetti Hernaningsih<br />
  45. 45. How might H pylori infection contribute to the development of thrombocytopenia?<br />H pylori expresses Lewis (Le) antigens in a strain-specific manner; Le antigens adsorb to platelets and might serve as targets for anti-Le antibodies in patients with an appropriate genetic background.<br />In addition, both H pylori infection and ITP are associated with a T helper 1 (Th1)–type immune response characterized by increased levels of interferon   and interleukin-2; hence, H pylori–induced alterations in cytokine profiles might promote development of immune thrombocytopenia<br />Journal Reading Bastiana, 2010<br />32<br />
  46. 46. How might H pylori infection contribute to the development of thrombocytopenia?<br /><ul><li>Some strains of H pylori bind von Willebrand factor (VWF) and induce glycoprotein Ib (GPIb) and Fc  RIIa dependent platelet aggregation in the presence of H pylori antibodies.
  47. 47. This interaction might contribute to the association of H pylori with cardiovascular disease and promote platelet consumption.
  48. 48. Finally, direct antigen mimicry between H pylori and platelet glycoproteins must be considered.</li></ul>Journal Reading Bastiana, 2010<br />33<br />
  49. 49. Standard Immunohistochemistry Staining MethodLabeled Streptavidin Biotin (LSAB) Method<br />Paraffin section or frozen section to water and rinse in PBS-Tween 20 for 2x2 min.<br />Antigen Retrieval: perform antigen retrieval using IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) or  IHC-TekTM Proteinase K Solution (Cat# IW-1101)if necessary.<br />Serum Blocking: incubate sections in normal serum – species same as secondary antibody. Note: since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type.<br />Primary Antibody: incubate sections with primary antibody at appropriate dilution in IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) for 1 hour at room temperature or overnight. No serum blocking is needed if antibody diluent is used. <br />Rinse in PBS-Tween 20 for 3x2 min. <br />Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Note: For acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in methanol prior to primary antibody incubation to avoid tissue distruction. <br />Rinse in PBS-Tween 20 for 3x2 min. <br />Secondary Antibody: incubate sections in Biotinylated secondary antibody in PBS for 30 minutes at room temperature. <br />Rinse in PBS-Tween 20 for 3x2 min. <br />Detection: incubate sections in HRP-Streptavidin solution for 30 minutes at room temperature. <br />Rinse in PBS-Tween 20 for 3x2 min. <br />Chromagen/Substrate: incubate sections in peroxidase substrate solution. <br />Rinse in PBS-Tween 20 for 3x2 min. <br />Counterstain with counterstain solution if desired. <br />Rinse in running tap water for 2-5 minutes. <br />Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2x3min. <br />Clear in xylene for 2x5min. <br />Coverslip with mounting medium. <br />   <br />Journal Reading Bastiana, 2010<br />34<br />
  50. 50. Blood. 2001; 98:952-957<br />Novel approaches to treatment of chronic refractory ITP are based on a better understanding of the immunologic abnormalities associated with this disorder, as well as the availability of sophisticated biotechnology products.<br />One such approach involves the use of monoclonal antibodies directed against the CD20 antigen, a transmembrane protein found on the surface of normal and malignant B cells, but not on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues. The CD20 molecule appears particularly suitable for targeted therapy because it does not shed from the cell surface and does not internalize upon antibody binding.<br />Journal Reading Bastiana, 2010<br />35<br />
  51. 51. Blood. 2001; 98:952-957<br />Rituximab is a genetically engineered chimeric murine/human anti-CD20 monoclonal antibody currently used for the treatment of non-Hodgkin lymphoma. The antibody is an IgG1 kappa immunoglobulin containing murine light- and heavy-chain variable region sequences and human constant region sequences.<br />The Fab domain of rituximab binds to the CD20 antigen on B lymphocytes, and the Fc domain recruits immune effector functions to mediate B-cell lysis in vitro. <br />Possible mechanisms of cell lysis include complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and induction of apoptosis. Rituximab is highly effective for in vivo depletion of B cells, and the rationale for its use in chronic ITP rests on the attempt to eliminate autoreactive B-cell clones.<br />Journal Reading Bastiana, 2010<br />36<br />
  52. 52. Platelets: In General <br />normal platelet count in adults ranges from 150,000-450,000/microL<br />Surgical bleeding usually does not occur until the platelet count is less than 50,000, and spontaneous bleeding does not occur until the platelet count is less than 10,000-20,000<br />Journal Reading Bastiana, 2010<br />37<br />
  53. 53. Platelets: In General<br />Platelets are produced in bone marrow from Megakaryocytes<br />An estimated 1000-5000 platelets are produced from each Megakaryocyte<br />In normal adults platelet production is ~35,000-50,000/microL of whole blood per day. This value can be increased 8-fold during times of increased demand<br />Journal Reading Bastiana, 2010<br />38<br />www.UpToDate.com 2005<br />
  54. 54. Mechanisms of Thrombocytopenia<br />Decreased platelet production<br />Increased platelet destruction<br />Dilutional Thrombocytopenia<br />Splenomegaly or splenic sequestration<br />Pseudothrombocytopenia<br />Journal Reading Bastiana, 2010<br />39<br />
  55. 55. Decreased Platelet Production<br />Usually some offense that causes bone marrow suppression or damage<br />Viral illness<br />HIV (direct damage to Megakaryocytes)<br />Chemo- or radiation therapy<br />Congenital or acquired bone marrow aplasia or hypoplasia<br />Direct EtOH toxicity<br />Vit. B12 or Folate deficiency<br />Journal Reading Bastiana, 2010<br />40<br />
  56. 56. Increased Platelet Destruction<br />Idiopathic (Immune) Thrombocytopenic Purpura<br />Alloimmune destruction—Posttransfusion, Post-transplantation<br />Disseminated Intravascular Coagulation<br />Thrombotic Thrombocytopenic Purpura<br />Antiphospholipid Antibody Syndrome<br />Certain drugs—Heparin, quinidine, valproate<br />Journal Reading Bastiana, 2010<br />41<br />
  57. 57. Splenic Sequestration<br />Normally, ~1/3 of platelets are sequestered in the spleen in any given time<br />In extreme splenomegaly, up to 90% of platelets can be trapped in the spleen<br />Platelet size and life span is usually not affected<br />Cirrhosis, Portal HTN, splenomegaly can all present with apparent thrombocytopenia, although these pts are not usually at risk for clinical bleeding<br />Journal Reading Bastiana, 2010<br />42<br />
  58. 58. Clinical Presentation<br />Asymptomatic<br />Mucosal or cutaneous bleeding<br />Petechiae<br />Purpura<br />Ecchymoses<br />Journal Reading Bastiana, 2010<br />43<br />www.UpToDate.com 2005<br />
  59. 59. Common Presentations <br />Journal Reading Bastiana, 2010<br />44<br />
  60. 60. Initial Approach<br />H&P<br />Bleeding Hx<br />Drug Ingestion<br />CBC and peripheral smear—”gold standard”<br />Bone Marrow aspiration and biopsy—indicated if no other etiology obvious unless pt is <60yrs old. The presumptive Dx in that case is usually ITP.<br />Idiopathic Thrombocytopenic Purpura (ITP) is a Dx of exclusion when H&P,Drug ingestion,CBC, and peripheral smear do not suggest other etiologies<br />Journal Reading Bastiana, 2010<br />45<br />
  61. 61. DDx of ITP<br />Journal Reading Bastiana, 2010<br />46<br />
  62. 62. IMUNOHISTOCHEMISTRY<br />Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps: <br />1)primary antibody binds to specific antigen; <br />2) antibody-antigen complex is bound by a secondary, enzyme-conjugated, antibody; <br />3) in the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen binding. <br />Journal Reading Bastiana, 2010<br />47<br />
  63. 63. Humoral Immunity refers to the production of antibody molecules in response to an antigen (def). These antibody molecules circulate in the blood and enter the tissue via inflammation. Humoral immunity is most effective against bacteria, bacterial toxins, and viruses prior to these agents entering cells. <br />Antibodies or immunoglobulins (def) are specific glycoprotein configurations produced by B-lymphocytes and plasma cells in response to a specific antigen and capable of reacting with that antigen. <br />Journal Reading Bastiana, 2010<br />48<br />
  64. 64. The antibodies produced during humoral immunity ultimately defend the body through a variety of different means <br />These include:1. Opsonization2. MAC Cytolysis3. Antibody-dependent Cellular Cytotoxicity (ADCC) by NK Cells4. Neutralization of Exotoxins5. Neutralization of Viruses6. Preventing Bacterial Adherence to Host Cells7. Agglutination of Microorganisms8. Immobilization of Bacteria and Protozoans.<br />Journal Reading Bastiana, 2010<br />49<br />
  65. 65. Antibody-Dependent Cellular Cytotoxicity (ADCC) by NK cells<br />NK cells are capable of antibody-dependent cellular cytotoxicity or ADCC. NK cells (def) have receptors on their surface for the Fc portion of IgG. When the antibody (def) IgG is made against epitopes (def) on "foreign" membrane-bound cells, e.g., virus-infected cells and cancer cells, the Fab portions (def) of the antibodies react with the "foreign" cell. The NK cells then bind to the Fc portion (def) of the antibody (see Fig. 1).<br />Journal Reading Bastiana, 2010<br />50<br />
  66. 66. The NK cell then releases pore-forming proteins called perforins, proteolytic enzymes called granzymes, and chemokines<br />Granzymes pass through the pores and activate the enzymes that lead to apoptosis (def) of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation (see Fig. 1A and Fig. 2). <br />As a result, the cell breaks into fragments that are subsequently removed by phagocytes. Perforins can also sometimes result in cell lysis. (When NK cells are carrying out ADCC, they are sometimes also referred to as killer cells.)<br />Journal Reading Bastiana, 2010<br />51<br />
  67. 67. Naphtyl AS-D Chloroacetate esterase<br />Principle<br /><ul><li>The aim of this stain is to demonstrate the presence of granulocytes. Granulocyte lysozomes contain a rather specific hydrolase that can use the Naphtol AS-D Chloroacetate as substrate. The liberated naphtol reacts with the diazonium salt "Fast Red Violet LB", forming red depots.</li></ul>Reagents<br />Sigma: Kit num. 91C Naphtol AS-D Chloroacetate Esterase<br />Working reagent preparation<br /><ul><li>Mix 0,1 ml of the sodium nitrite solution0,1 ml of the Fast Red Violet LB solution
  68. 68. Let stand for 2 minutes (diazo preparation)
  69. 69. Add 4 ml H2O at 37°C. Mix
  70. 70. Add 0,5 ml of Trizmal buffer pH 6,3 . Mix
  71. 71. Add 0,1 ml of Naphtol AS-D Chloroacetate reagent. Mix
  72. 72. Solution will become slightly red. If the solution forms a precipitate, filter or centrifuge </li></ul>Journal Reading Bastiana, 2010<br />52<br />
  73. 73. Naphtyl AS-D Chloroacetate esterase <br />Staining Procedure<br /><ul><li>In a test tube, add 100 à 200 ul of sediment
  74. 74. Add to this tube 2 ml of working reagent
  75. 75. Incubate at 37°C for 15 minutes
  76. 76. Centrifuge and read the results
  77. 77. Nucleus can be stained by adding the methylene blue. </li></ul>Results<br /><ul><li> Polynuclear cells stain deep red, while the rest of cells remain unstained, or take a light pink to orange red color due to non-specific adsorbtion. Mastocytes and some macrophages are stained with this method.</li></ul>Journal Reading Bastiana, 2010<br />53<br />
  78. 78. Hematoxylin and Eosin (H&E) staining<br />•Place slides containing paraffin sections in a slide holder (glass or metal) <br />•Deparaffinize and rehydrate sections: 3 x 3´ Xylene (blot excess xylene before going into ethanol) 3 x 3´ 100% ethanol 1 x 3´ 95% ethanol 1 x 3´ 80% ethanol 1 x 5´ deionized H2O •While sections are in water, skim surface of hematoxalin with a Kimwipe to remove oxidized particles. Blot excess water from slide holder before going into hematoxalin. <br /> •Hematoxalin staining: 1 x 3´ Hematoxalin Rinse deionized water 1 x 5´ Tap water (to allow stain to develop) Dip 8-12x (fast) Acid ethanol (to destain) Rinse 2 x 1´ Tap water Rinse 1 x 2´ Deionized water (can leave overnight at this stage)<br /> •Blot excess water from slide holder before going into eosin. •Eosin staining and dehydration:1 x 30 sec Eosin (up to 45 seconds for an older batch of eosin) 3 x 5´ 95% ethanol3 x 5´ 100% ethanol (blot excess ethanol before going into xylene)<br /> 3 x 15´ Xylene <br />•You can leave slides in xylene overnight to get good clearing of any water. <br />•Coverslip slides using Permount (xylene based). <br />•Place a drop of Permount on the slide using a glass rod, taking care to leave no bubbles.<br /> •Angle the coverslip and let fall gently onto the slide. Allow the Permount to spread beneath the coverslip, covering all the tissue. •Dry overnight in the hood. <br />Journal Reading Bastiana, 2010<br />54<br />
  79. 79. Journal Reading Bastiana, 2010<br />55<br />
  80. 80. ITP<br />100 cases per 1 milion persons per year. <br />About half of these cases occur in children. <br />Primary vs Secondary . <br />Acute vs Chronic ( >6 months). <br />Journal Reading Bastiana, 2010<br />56<br />
  81. 81. The etiology is still unknown and the pathogenesis is complex and possibly depends on disturbed antigen presentation, T cell activation and signaling, disregulated B cell stimulation and antibodies, unbalanced activation / suppression of complement.<br />Journal Reading Bastiana, 2010<br />57<br />
  82. 82. Affected children are young (peak age ~ 5yrs) and previously healthy, and they typically present with the sudden onset of petechiae or purpura a few days or weeks after an infectious illness.<br />Boys and girls are equally affected.<br />Journal Reading Bastiana, 2010<br />58<br />
  83. 83. <ul><li>In more than 70% of children, the illness resolves within six months, irrespective of whether they receive therapy.
  84. 84. By contrast, ITP in adults is generally chronic.
  85. 85. The bone marrow in patients with ITP contains normal or increased numbers of megakaryocytes</li></ul>Journal Reading Bastiana, 2010<br />59<br />
  86. 86. Journal Reading Bastiana, 2010<br />60<br />
  87. 87. Pathophysiology<br />ITP is mediated by IgG autoantibodies.<br />Glycoprotein IIb/IIa, Ib/Ix, Ia/IIa, IV and V ...<br />Accelerated clearance through Fcү receptors that are expressed by tissue macrophages (spleen & liver). <br />Journal Reading Bastiana, 2010<br />61<br />
  88. 88. Journal Reading Bastiana, 2010<br />62<br />
  89. 89. Genetics<br />Monozygotic twins.<br />An increased prevalence of HLA-DRw2 and DRB1*0410 alleles.<br />HLA-DR4 and DRB1*0410 alleles have been associated with unfavorable and favorable response to corticosteroids, respectively.<br />Journal Reading Bastiana, 2010<br />63<br />
  90. 90. Diagnosis<br />The diagnosis of ITP remains one of exclusion. <br />Secondary forms of the disease occur in association with SLE, the antiphospholipid syndrome, immunodeficiency states (IgA deficiency and common variable hypogammaglobulinemia), Lymphoproliferative disorders (CLL, Large granular lymphocytic leukemia, and lymphoma), infection with HIV and hepatitis c virus, and therapy with drugs such as heparin and quinidine. <br />Journal Reading Bastiana, 2010<br />64<br />
  91. 91. The guidelines of the American Society of Hematology state that a bone marrow examination is not required in adults younger than 60 yrs of age if the presentation is typical but is appropriate before splenectomy is performed.<br />Journal Reading Bastiana, 2010<br />65<br />
  92. 92. Marrow examination is necessary in the presence of atypical features (e.g., those with additional cytopenias, protracted fever, bone or joint pain, unexplained macrocytosis ), or in patients who do not have a brisk or robust response to therapy.<br />Journal Reading Bastiana, 2010<br />66<br />
  93. 93. There is consensus, that bone marrow examination is not necessary in children if management involves observation or IVIG.<br />Although it is not mandatory, many pediatric hematologists recommend that an aspiration be performed before starting corticosteroids to rule out the rare case of acute leukemia.<br />Journal Reading Bastiana, 2010<br />67<br />
  94. 94. The direct assay for the measurement of platelet-bound AB’s has an estimated sensitivity of 49-66%, an estimated specificity of 78-92%, and an estimated PPV of 80-83%.<br />Journal Reading Bastiana, 2010<br />68<br />
  95. 95. Journal Reading Bastiana, 2010<br />69<br />
  96. 96. Journal Reading Bastiana, 2010<br />70<br />
  97. 97. Treatment<br />Watch & Wait strategy.<br />Corticosteroids (high, standard or low dose).<br />IVIG (high or low dose, 2 day or 1 day).<br />IV anti-D immunoglobulin in Rh(D) positive patients (high or low dose).<br />Journal Reading Bastiana, 2010<br />71<br />
  98. 98. Journal Reading Bastiana, 2010<br />72<br />

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