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Immunophenotyping

Diagnostic and prognostic tools in haematological malignancies in 2010 (Part II) Flow cytometry. Immunophenotyping. Identification of molecular protein characteristics of abnormal cells Labelling with fluorescent monospecific monoclonal antibodies Multiparametric flow cytometry.

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Immunophenotyping

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  1. Diagnostic and prognostic tools in haematologicalmalignancies in 2010 (Part II)Flow cytometry

  2. Immunophenotyping • Identification of molecular protein characteristics of abnormal cells • Labelling with fluorescent monospecific monoclonal antibodies • Multiparametric flow cytometry

  3. The objectives of immunophenotyping in hematological disorders • Lineageassignment • Lymphoid acute/chronic • Myeloid acute/chronic • Erythroid • Megakaryocytic • Classification /Scoring • Detectionof MPAL • Maturation anomalies • Identification of Minimal ResidualDisease patterns

  4. Before....

  5. Now..... Harrogate 7th HLDA : 250 CD Adelaide 2004 8th HLDA : 339 CD Barcelona 2010 : >360 CD

  6. Immunophenotype of lymphoid cells • Well established maturation sequence • Staged and controlled to prepare long lived antigen-specific cells • Identified by studying leukemias • Validated on normal cells • Numerous similarities between B and T cells: • Lineage committment • Gene rearrangements for antigen recetpors • Selection

  7. CD21 CD22 CD24 P COOH CD20 CD19 or BCR CD79 ITAM ITAM ITAM ITAM ITAM CD23 ITIM NH2 ITIM ITAM ITIM ITIM B lineage associated markers

  8. Immunocyte Stem Cell Pro-B Cell Immunoblast Early-B Cell Pre-B Cell Naive B cell H0L0 H0L0 HxL0 HxL0 HRL0 HRLR CD34 DR cCD79 cCD22 CD19 cCD79 CD22 CD19 CD21 CD10 cCD79 CD22 CD19 CD20 CD21 CD10 cCD79 CD22 CD19 CD20 CD21 cµ sCD79 CD22 CD19 CD20 CD21 sµ/sd Plasma cells BONE MARROW TISSUES ACTIVATION & CLONAL PROLIFERATION Memory B cells

  9. B-I (pro-B) + - - - cCD79/CD19/c or sCD22 CD10 c-µ sIg B-II (common) + + - - B-III (pre-B) + + + - B-IV (mature) + + + + Classification of B- ALL (EGIL)

  10. CD1 CD5 CD2 CD7 TCR CD3 d e g e zz/zh ITAM ITAM ITAM ITAM ITAM ITAM ITAM ITAM ITAM ITAM CD4 CD8 T lineage associated markers

  11. sCD3 CD7 CD2 CD5 CD8 Cortico- thymocyte Stem cell Pro-T cell Immunocyte Immunoblast g0d0 a0b0 g0d0 a0b0 gXdX a0b0 gXdX aXbX gXdX aRbR CD34 DR cCD3 CD7 CD2 CD5 cCD3 CD7 CD2 CD5 CD1 cCD3 CD7 CD2 CD5 CD4 CD8 sCD3 CD7 CD2 CD5 CD4 Effector T cells TISSUES THYMUS BONE MARROW ACTIVATION & CLONAL PROLIFERATION Medullary Thymocyte Naïve T cell Memory T cells

  12. T-I (pro-T) + + - - - cCD3 CD7 CD2 CD5 CD8 CD1a+ sCD3+/ CD1a- T-II (pre-T) + + + - - T-III (cortical T) + + + + - T-IV (mature T) + + + - + Classification of T-ALL (EGIL)

  13. Lymphoproliferative disorders T LYMPHOMAS SEZARY) LARGE CELLS ANAPLASTIC PERIPHERAL ANGIOCENTRIC INTESTINAL MARGINAL ZONE NODAL MALT HCL MANTLE ZONE BURKITT FOLLICULAR INTESTINAL OR LUNG MALT Follicular, mantle zone, marginal zone WALDENSTROM CENTROBLASTIC or IMMUNOBLASTIC Large cell Lymphomas MYELOMA SLVL Circulating marginal zone cells

  14. Matutes’ scoring of CLL

  15. Comparative immunophenotypes

  16. Immunophenotype of myeloid cells • Known maturation sequence • Massive production of cells with no specificity • Early and late differentiation markers • Important lineage promiscuity

  17. COOH CD36 E E CD11b MPO CD117 NH2 CD13 Mg L L Mg CD33 L Mg CD35 L L L L L K L CD15 CD65 L P 3-fucosyl-N-acetyl-lactosamine céramide dodecasaccharide CD14 Myeloid lineage markers

  18. DR CD4 CD19 CD11B CD14 CD36 CD15 CD65 CD16 CD32 CD64 BONE MARRROW PERIPHERAL BLOOD TISSUES CD34 DR CD117 CD13 CD33 MPO CD7 Stem cell Immature precursors Differentiated cells

  19. Evolution immunohénotypique au cours de la maturation granuleuse (B Husson) Metamyelocyte Granulocyte Myelocyte Myeloblast CFU-GM Promyelocyte Band CD15 CD66 HLA-DR CD11b CD16 CD117 CD34 CD33 CD13 CD13 CD11c CD11c CD33 CD11b CD15 CD66 CD16

  20. Detection of BAL : EGIL’scoring system • NOT to be used for lineage assignment • Scoring based on the lineage specificity of critical differentiation antigens • Calculation of each lineage “score” • In BAL, at least two lineages have scores HIGHER than 2 • In “variant” AL coexpression with a score <2 is not rare

  21. B-LINEAGE T-LINEAGE MYELOID LINEAGE 2 points CD79 cµ cCD22 CD3 TCR MPO (lysozyme) 1 point CD19 CD10 CD20 CD2 CD5 CD8 CD10 CD13 CD33 CDw65 CD117 0.5 point TdT CD24 TdT CD7 CD1a CD14 CD15 CD64 Detection of BAL : EGIL ’scoring system

  22. WHO’s new classification Extensive immunophenotype Others Incl NK AUL Acute undifferentiatedleukemia MPAL Mixed Phenotype Acute leukemia NOS With t(9;22) With t(n;11q23)

  23. WHO’s new criteria of MPAL • Myeloid lineage • MPO • Or strong monocytic engagement • NSE • CD14, CD11c, CD36, CD64, Lysozyme • B-lineage • Bright CD19 + another B marker • Low CD19 + two other B markers • T-lineage : cCD3 (strong, PE or APC)

  24. One word on MDS

  25. Proposition de score Wells, 2003 0 1 2 3

  26. Working conference 2008M Loken, A van de Loosdrecht, K Ogata, A Orfao, D Wells • Classical blasts  enumeration • DR+/11b- • CD34 • CD117 • Anomalies of the CD13/CD16 pathway • Abnormal expression anormale of DR on monocytes

  27. Stachurski, 2008 • Blasts • CD34, CD117 • Abnormal CD2, CD5, CD7, CD56 • Increased CD117 • Granulocytes • Deganulation • Abnormal expression of CD33, CD13, CD11b, CD16, CD15, CD64, CD10, CD14, DR • Monocytes • Anomalies of CD33, CD13, CD11b, CD15, CD64, CD14, DR

  28. In practice, for hematological malignancies • Depending on • Previous clinical and morphological information • Sample volume • Monoclonal antibodies availability

  29. Consensual European Panel, European LeukemiaNet (2005) For quick orientation or paucicellular samples • cCD3, MPO, cCD79a, TdT • CD7, CD2, CD10, CD19, CD22 (s or c), sIg, CD13, CD33, CD34 • CD45 for gating purposes Sublineage classification and definition of clinical entities (also with adapted gating strategy) • DR, CD1a, CD4, CD5, CD8, CD3 (m), IgM (c), CD14, CD117, CD56, CD65, CD41 or CD61, RBC marker such as glycophorin A Complementary panel of useful referenced markers

  30. Acute leukemia Diagnosis PanelOther useful markers (>20) • MPO/LF (lactoferrin) (c): (i) Identification of late neutrophil granulocyte compartment (Lactoferrin positive) (25,52,54); (ii) for refined detection/quantification of MPO+ early myeloid cells in combination with CD14 (25,49) • LZ (lysozyme) (c): (i) for myeloblastic leukaemia; (ii) to discriminate pDCs and myeloid cells (53); (iii) to positively identify early monocytic cells (48) • K/L : (i) on surface for clonality, (ii) in the cytoplasm for rare B-IV cases • CD11b, CD11c : negative in APL (14) • CD15 : for myeloblastic leukemia (42) • CD16 : to discriminate mature PMNs (9) • CD35/36 : for GEIL´s AML classification (11), for RBC after excluding monocytes (10) • CD58 : to distinguish between normal regenerating B cells and B-cell blasts (59) • CD64 : for AML (9) • CD68 (c): (i) for AML (bright) and subset of B-ALL (weak) (53); (ii) for positive identification of normal pDCs (bright) (55) • CD71 : for cell proliferation/activation and/or RBC (22,41) • CD86 : prognostic factor in AML (34) • CD99 : to differentiate between blasts and non blastic T-cells (19) • CD123 : IL-3 R, for pDC and AML, some NK (24) • TCR chains for T-ALL, c and/or s (50) • Therapeutic targets: CD20 (40), CD52 (40), CD45 (39), CD33 (31), CD123 (3), CD87 (46), CD44 (20), uPAR(CD87)/uPACD116 (1)

  31. Chronic lymphoproliferative diseases. Mandatory panel (20 Abs) • Samples : peripheral blood, bone marrow, LN suspensions… (Fine needle aspiration for primary screeening to avoid unnecessary biopsies) • Gating markers CD19, CD3, CD56* • B oriented panel gated on CD19 CD5, CD20, CD23, CD103, CD10, K, L, Ig, CD25, CD79b, CD38 • T oriented panel gated on CD3 or other T-lineage marker CD2, CD3, CD4, CD5, CD8, CD7,

  32. Lymphoproliferative diseases: additional useful markers (< 15) • B lineage • CD2, CD7, CD123, FMC7, CD138, DR, CD24*, CD43 • (G, A, M, D), CD81* • Cytoplasmic : Bcl2, Zap70 (relative to internal control) • T lineage • TCRs*, CD30, CD10 • NK panel excluding CD19 and CD3 cells/ CD56 • CD57, CD16, CD94, perforin, granzyme B *CD81/CD22 useful for CLLfollow-up based on dim co-expression level *V-beta panel and imunophenotype *Absence of CD24 on marginal zone and HCL • Note : CD52 if alemtuzumab considered

  33. Chemosensitivity • Minimal residual disease • Quick assessment of response to therapy

  34. Why look for minimal residual disease?

  35. San Miguel, 1997 Campana, 1999 Kern, 2004 The founders

  36. MRD in ALL

  37. CD34 CD19 MRD B-II ALL 1.5 x 10-2

  38. MRD in AML

  39. CD117 CD34 MRD - AML 2.1 10-3

  40. AML peripheral blast cells decrease

  41. A SSC SSC b a FSC CD45 C B SSC SSC c d CD14 CD16 CD16 E CD11b D f CD45 e CD45 5.3% Blasts CD16 CD16 100 h g CD11b CD11b D0

  42. 0.2% 3.9% Blasts CD16 CD16 CD16 CD16 Blasts 3.6 73.7 h g CD11b CD11b b a CD11b CD11b CD16 1.1% Blasts CD16 CD16 e CD11b 21.2 D4 D1 D2 D3 c d CD11b CD11b 0.5% Blasts CD16 9.4 f CD11b

  43. Slope < -25 • Slope -25 < > -15 • Slope > -15

  44. Conclusions • Consensual approach • Rapid and informative diagnostic tool • Precise definition of the disease at diagnosis • Therapeutic indications • Aberrant immunophenotypes useful for follow up • Search for new markers: • New monoclonals • Microarrays

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