Diagnostika onkohematologických ochorení pomocou prietokovej cytometrie Jozef Smolka Beckman Coulter Slovenská republika s.r.o.

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1 Diagnostika onkohematologických ochorení pomocou prietokovej cytometrie Jozef Smolka Beckman Coulter Slovenská republika s.r.o. 29. máj 2013, KLINICKÁ BIOCHÉMIA, s.r.o. Žilina, Medicínske laboratórium Poprad

2 Lymfóm / Leukémia Leukémia, najrozšírenejší typ rakoviny krvi Rakovinové leukocyty sa objavujú v krvi a v kostnej dreni Lymfatická leukémia Myeloi(cká)dná leukémia Môžu byť akútne alebo chronické (ALL, AML, CLL, CML) Lymfóm: rakovina lymfatického systému Kancerózne bunky majú tendenciu agregovať a vytvárať solídne tumory v lymfatických tkanivách Hodgkin`s lymfóm (6 podtypov) NonHodgkin`s lymfóm (30 podtypov, indolentné, agresívne) Myelóm Tumor spôsobený proliferáciou plazmatických buniek Zvyčajne veľmi vysoká koncentrácia proteínov, spôsobená produkciou protilátok (plazmatické bunky)

3 Lymfóm / Leukémia - rozdelenie AML, B CLL a lymfómy sú najčastejšie v západnej časti sveta Ref: NCI 1998, ACS, 2008

4 60 Years Ago 50 Years Ago Disease Knowledge Evolution Disease of the Blood 5 year Survival ~ 0% 40 Years Ago Leukemia or Lymphoma Chronic Leukemia Acute Leukemia Preleukemia Indolent Lymphoma Aggressive Lymphoma Today 38 Leukemia types identified: 51 Lymphomas identified: Acute myeloid leukemia ( 12 types) Mature B-cell lymphomas ( 14 types) Acute lymphoblastic leukemia (2 types) Mature T-cell lymphomas (15 types) Acute promyelocytic leukemia (2 types) Plasma cell neoplasm (3 types) Acute monocytic leukemia (2 types) Immature (precursor) lymphomas (2 types) Acute erythroid leukemia (2 types) Hodgkin s lymphoma (5 types) Acute megakaryoblastic leukemia Immunodeficiency associated lymphomas ( 5 types) Acute myelomonocytic leukemia (2 types) Other hematolymphoid neoplasms ( 7 types) Chronic myeloid leukemia Chronic myeloproliferative disorders (5 types) Myelodysplastic syndromes (6 types) Mixed myeloproliferative/myelodysplastic syndromes (3 types) 70% Ries LAG, Eisner MP, Kosary CL, Hankey BF, Miller BA, Clegg L, Mariotto A, Feuer EJ, Edwards BK (eds). SEER Cancer Statistics Review, , National Cancer Institute. Bethesda, MD, based on Nov 2004 SEER data, posted to the SEER web site 2005.

5 Lymfóm / Leukémia Imunofenotypizácia ako diagnostický a monitorovací nástroj Imunofenotypizácia prietokovou cytometriou Jedinečný nástroj pre sledovanie ochorenia Počiatočná diagnóza Sub klasifikácia Monitorovanie pacienta

6 Immunophenotyping in L&L CHARACTERIZE the leukemic population by describing the antigens expressed DIFFERENTIATE LEUKEMIC FROM NORMAL CELLS: Normal Antigens expressed in consistent and reproducible patterns with maturation Myeloid Maturation: Bone Marrow Peripheral Blood Lymphoid Maturation: Thymus Peripheral Blood Lymph Node Neoplastic L&L Increased or decreased normal antigens Dysynchronous maturational expression Aberrant antigen expression

7 L / L klasifikačný systém FAB, REAL, WHO klasifikácia Cytometrická diagnostika sa vykonáva v mnohých laboratóriách Vysoký počet protokolov a panelov pre typizáciu lymfómov a leukémií Potrebná harmonizácia metód Stanovenie diagnózy v rôznych laboratóriách Prognóza a terapia Vývoj a monitoring u nových liekov na základe výsledkov z rôznych pracovísk (multi centre studies) Úloha pre prietokovú cytometriu: Štandardizácia, harmonizácia, klasifikačný systém

8 Štandardizácia rok 2006 a Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry Cytometry B:S14 22 WHO 2008 Classification of Tumours of Hæmatopoietic and Lymphoid Tissues 4 th Edition, 2008 IARC Press Practical International Consensus Established

9 Conference Recommendations Bethesda 2006 Consensus Consensus Markers Initial Screening Approach Lineage based panels T Cell B Cell Myelo monocytic Plasma Cell Combination Strategy Highly expressed antigens on dimmer dyes CD45 as common marker Not Defined # of colors per tube (1, 2, 3, 4, 5, colors) Sample prep & data analysis Pathway to Standardization in L&L Phenotyping

10 Markers- description CD2 CD3 CD4 CD5 CD7 CD8 CD10 CD11b CD13 CD14 CD15 CD16 CD19 CD20 CD33 CD34 CD38 CD45 CD56 CD117 Kappa Lambda HLA DR T cells and most of the NK cells Mature T cell (cytoplasmic expression in immature T cells) Helper/inducer T lymphocytes, monocytes, Immature myeloid cells Thymocytes, mature T cells, subpopulation of B cells T cells, NK cells, subpopulation of immature myeloid cells Cytotoxic/suppressor T cells, subpopulation of NK cells Common acute leukemia antigen (CALLA), lymphatic precursor cells, neutrophils, subpopulation of mature B cells Monocytes, macrophages, neutrophils, NK cells Myeloid cells Monocytes, weak expression on neutrophils Neutrophils, weak expression on monocytes NK cells, neutrophils, subpopulation of monocytes Precursor and mature B cells B cells and a subpopulation of B cell precursor Monocytes, myeloid precursor cells, weak expression on neutrophils Myeloid and lymphoid precursor cells Activated lymphocytes, subpopulation of B cells, plasma cells All leukocytes NK cells, subpopulation of T cells Myeloid precursor cells Kappa light chain Lambda light chain B lymphocytes, activated T lymphocytes, monocytes, precursor cells

11 Primary Panels Aid in Initial Classification Acute Leukemia Blasts Present >20% CD13+ and/or CD33+ CD7+ and/or CD5+ CD19+ Myeloid Lineage T-Lineage B-Lineage Myeloid, no or minimal maturation CD34+/ CD117+/ HLADR+ CD56+ CD15 /+ CD11b, CD14- Promyeloid CD15+/ CD117+/ CD34 HLADR CD11b, CD14 Myelomonocytic & Monocytic CD14+ CD4+/ HLADR+ CD15+ CD11b+ Megakaryocytic and erythroid lineages require secondary panels

12 Acute Leukemias: Initial Lymphocytic Classification Acute Leukemia Blasts Present >20% CD7+ and/or CD5+ CD19+ T-Lineage B-Lineage Early Thymocyte Common Thymocyte Mature Thymocyte Early B- Precursor Common Pre-B B CD2+ CD5+ CD4+ or CD8+ scd3+ CD2+ CD5+ Dual CD4+/CD8+ scd3- CD2-/+ CD5-/+ scd3- CD4- CD8- TdT+ CD10- clg- sig- sig+ CD10+/- clg+/- TdT- TdT+ CD10+ clg- sig- CD10+ cig+ TdT+ sig- Assessment of maturity with A primary reagents Require secondary panels for sub classification

13 Initial Classification of CLLs and Lymphomas CLL and Non-Hodgkins Lymphoma Plasma Cell Leukemia/ Myeloma CD38+ CD2-, CD19- B-Cell Lineage CD19+ CD2+ CD3+ CD16+ and/or CD56+ CD2+, CD3- T-Cell Lineage NK LGLL CD5+ CD5- B-CLL or Mantle Cell Lymphoma CD10+ Follicular Cell Or Burkitt s Lymphoma CD10- SLVL or Marginal Zone Lymphoma or Hairy Cell Leukemia Sub classification of Lymphomas Required

14 Initial Classification of CLLs and Lymphomas CLL and Non-Hodgkins Lymphoma Plasma Cell Leukemia/ Myeloma CD38+ CD2-, CD19- B-Cell Lineage CD19+ CD2+ CD3+ CD16+ and/or CD56+ CD2+, CD3- NK LGLL T-Cell Lineage CD4+ CD8+ CD7- Sezary Syndrome T-Cell CLL T-Cell Lymphoma T-PLL ATLL (Adult T-Cell Leukemia) T-Cell LGLL Sub classification of Lymphomas Required

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16 Prietokové cytometre pre IVD Cytomics FC konfigurácie Navios 3 konfigurácie 5 color 1Laser 5 color 2 Laser 6 color 2 Laser (5+1) 8 color 2 Laser (5+3) 10 color 3 Laser (5+3+2)

17 Navios Flow Cytometer Available in 3 configurations 6 color 2 Laser (5+1) 8 color 2 Laser (5+3) 10 color 3 Laser (5+3+2) 638nm 488nm 405nm

18 Beckman Coulter 5-Color Panel Solutions B Cell Kit Kappa Lambda CD19 CD5 CD45 CD20 CD10 CD19 CD38 CD45 T Cell Kit CD2 CD56 CD7 CD5 CD45 CD8 CD4 CD3 CD45 Myeloid Kit CD15 CD11b CD16 CD14 CD45 HLADR CD56 CD34 CD117 CD45 CD7 CD13 CD34 CD33 CD45 Aligned with Bethesda Recommendations

19 Navios TM / FC500 and SOLASTRA TM

20 Reagent Performance: Target Specifications Specimen Types Peripheral Blood Bone Marrow Lymphoid cell suspensions EDTA, Heparin Specimen age: hours post draw Myeloid Lineage Kit FITC PE ECD PC5.5 PC7 CD15 CD11b CD16 CD14 CD45 HLADR CD56 CD34 CD117 CD45 CD7 CD13 CD34 CD33 CD45 T-Lineage Kit FITC PE ECD PC5.5 PC7 CD2 CD56 CD7 CD5 CD45 CD8 CD4 -- CD3 CD45 Target sensitivity: 1% when 50,000 events are collected Working WBC Range: ,000 cells/µl Sample preparation using VersaLyse + Fixative Lytic System B-Lineage Kit FITC PE ECD PC5.5 PC7 Kappa Lambda CD19 CD5 CD45 CD20 CD10 CD19 CD38 CD45

21 Acute Myeloid Leukemia - AML (BM) Myelomonocytic Lineage Tube 1: CD15 / CD11b / CD16 / CD14 / CD45 Myelomonocytic Lineage Tube 2: HLA-DR / CD56 / CD34 / CD117 / CD45 Myelomonocytic Lineage Tube 3: CD7/ CD13 / CD34 / CD33 / CD45 AML Phenotype: CD Dim b

22 Acute Leukocyte Leukemia - ALL (PB) BL1: Kappa/ Lambda / CD19/ CD5 / CD45 ALL Phenotype: CD sig- 38+ BL2: CD20/ CD10/ CD19 / CD38 / CD45

23 Chronic Leukocyte Leukemia - CLL (PB) BL1: Kappa/ Lambda / CD19/ CD5 / CD45 BL2: CD20/ CD10/ CD19 / CD38 / CD45 CLL Phenotype: CD Dim variable/- Dim Kappa+

24 Základný panel + ďalšie znaky PB KrOrange FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 Kappa Lambda CD19 CD5 CD20 CD10 CD19 CD38 CD45 CD2 CD56 CD7 CD5 CD45 CD8 CD4 CD3 CD45 CD15 CD11b CD16 CD14 CD45 HLADR CD56 CD34 CD117 CD45 CD7 CD13 CD34 CD33 CD45

25 Panel pre orientáciu Základné protilátky v jednej skúmavke 10 fluorescencií 15 monoklonálnych protilátok 405 Excitation 488 Excitation 633 Excitation Pacific Blue Krome Orange FITC PE ECD PC5.5 PC7 APC APC- AF700 APC- AF750 CD15/CD20 CD45 kappa/cd34 lambda/cd7 CD10 CD4 CD56/CD117 CD3/CD33 CD8 CD19 CD8/CD15/CD20 CD45 kappa/cd4 lambda/cd56 CD7 CD117 CD10 CD34 CD19 CD3/CD33

26 Lymphoid Slide Ruler

27 Summary Role of Flow Correlation of phenotype expression pattern to disease Differentiate leukemic population from normal Bethesda Consensus Conference 2006 for Phenotyping L&L Lineage Based panels for primary screening Testing driven by patient presentation of medical indications Beckman Coulter Solution Lineage Reagent Kits Aligned with Bethesda Phenotyping Strategy 5 Color Combinations for multi platform compatibility Initial characterization as primary screen Product Flexibility for sub classification

28 Suggested Reading Consensus Conference Publications 2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry, Cytometry B:S14 22 WHO 2008 Classification of Tumours of Hæmatopoietic and Lymphoid Tissues, 4th Edition, 2008, IARC Press Review Articles F.E. Craig & K.A. Foon, Flow Cytometric Immunophenotyping for Hematologic Neoplasms, Blood Volume 111, Number 8: , J.W. Vardiman, N.L. Harris, R.D. Brunning, The World Health Organization (WHO) Classification of the Myeloid Neoplasms, Blood, Volume 100, Number 7: , 2002.

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