Diagnostika onkohematologických ochorení pomocou prietokovej … · 29. máj 2013, KLINICKÁ BIOCHÉMIA, s.r.o. Žilina, Medicínske laboratórium Poprad Jozef Smolka Beckman Coulter

29. máj 2013, KLINICKÁ BIOCHÉMIA, s.r.o. Žilina, Medicínske laboratórium Poprad

Jozef Smolka

Beckman Coulter Slovenská republika s.r.o.

Diagnostika onkohematologických ochorení pomocou prietokovej cytometrie

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)

Lymfóm / Leukémia - rozdelenie

AML, B‐CLL a lymfómy sú najčastejšie v západnej časti sveta

Ref: NCI 1998, ACS, 2008

Disease Knowledge Evolution

50 Years Ago

Leukemia or Lymphoma40 Years Ago

Chronic LeukemiaAcute LeukemiaPreleukemia

Indolent LymphomaAggressive Lymphoma

60 Years Ago

“Disease of the Blood”

Today

∼38 Leukemia types identified:Acute myeloid leukemia (∼12 types)Acute lymphoblastic leukemia (2 types)Acute promyelocytic leukemia (2 types)Acute monocytic leukemia (2 types)Acute erythroid leukemia (2 types)Acute megakaryoblastic leukemiaAcute myelomonocytic leukemia (2 types)Chronic myeloid leukemiaChronic myeloproliferative disorders (5 types)Myelodysplastic syndromes (6 types)Mixed myeloproliferative/myelodysplastic syndromes (3 types)

∼51 Lymphomas identified:Mature B-cell lymphomas (∼14 types)Mature T-cell lymphomas (15 types)Plasma cell neoplasm (3 types)Immature (precursor) lymphomas (2 types)Hodgkin’s lymphoma (5 types)Immunodeficiency associated lymphomas (∼5 types)Other hematolymphoid neoplasms (∼7 types)

5 yearSurvival

~ 0%

70%

Ries LAG, Eisner MP, Kosary CL, Hankey BF, Miller BA, Clegg L, Mariotto A, Feuer EJ, Edwards BK (eds). SEER Cancer Statistics Review, 1975-2002, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2002/, based on Nov 2004 SEER data, posted to the SEER web site 2005.

Imunofenotypizácia prietokovou cytometriou

– Jedinečný nástroj pre sledovanie ochorenia

– Počiatočná diagnóza– Sub ‐ klasifikácia– Monitorovanie pacienta

Imunofenotypizácia ako diagnostický a monitorovací nástrojLymfóm / Leukémia

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

L / L klasifikačný systém• FAB, REAL, WHO klasifikácia

Úloha pre prietokovú cytometriu: Štandardizácia, harmonizácia, klasifikačný systém

‐ 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)

Štandardizácia – rok 2006 a 2008

2006 Bethesda International Consensus Recommendations on the

Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow

Cytometry

Cytometry 2007 72B:S14‐22

Practical International Consensus Established

WHO 2008 Classification of Tumours of

Hæmatopoietic and Lymphoid Tissues

4th Edition, 2008IARC Press

Conference Recommendations

CombinationStrategy

ConsensusMarkers

Bethesda 2006Consensus

Initial Screening ApproachLineage‐based panels

T‐CellB‐CellMyelo‐monocyticPlasma Cell

Not Defined

Highly expressed antigenson dimmer dyesCD45 as common marker

# of colors per tube (1, 2, 3, 4, 5, …colors)Sample prep & data analysis

Pathway to Standardization in L&L Phenotyping

Markers- descriptionCD2 T cells and most of the NK cellsCD3 Mature T cell (cytoplasmic expression in immature T cells)CD4 Helper/inducer T lymphocytes, monocytes, Immature myeloid cellsCD5 Thymocytes, mature T cells, subpopulation of B cellsCD7 T cells, NK cells, subpopulation of immature myeloid cellsCD8 Cytotoxic/suppressor T cells, subpopulation of NK cellsCD10 Common acute leukemia antigen (CALLA), lymphatic precursor cells, neutrophils,

subpopulation of mature B cellsCD11b Monocytes, macrophages, neutrophils, NK cellsCD13 Myeloid cellsCD14 Monocytes, weak expression on neutrophilsCD15 Neutrophils, weak expression on monocytesCD16 NK cells, neutrophils, subpopulation of monocytesCD19 Precursor and mature B cellsCD20 B cells and a subpopulation of B cell precursorCD33 Monocytes, myeloid precursor cells, weak expression on neutrophilsCD34 Myeloid and lymphoid precursor cellsCD38 Activated lymphocytes, subpopulation of B cells, plasma cellsCD45 All leukocytesCD56 NK cells, subpopulation of T cellsCD117 Myeloid precursor cellsKappa Kappa light chainLambda Lambda light chainHLA‐DR B lymphocytes, activated T lymphocytes, monocytes, precursor cells

Primary Panels – Aid in Initial ClassificationAcute Leukemia – Blasts Present >20%

Myeloid Lineage

CD13+ and/or CD33+ CD7+ and/or CD5+ CD19+

T-Lineage B-Lineage

Myeloid, no or minimal

maturation

CD34+/‐CD117+/‐HLADR+CD56+CD15‐/+

CD11b‐, CD14-

Promyeloid

CD15+/‐CD117+/‐CD34‐HLADR‐

CD11b‐, CD14‐

CD14+CD4+/‐

HLADR+CD15+

CD11b+

Myelomonocytic & Monocytic

Megakaryocytic and erythroid lineages require secondary panels

Acute Leukemias: Initial Lymphocytic Classification

Acute Leukemia – Blasts Present >20%

CD7+ and/or CD5+ CD19+

T-Lineage B-Lineage

CommonThymocyte

MatureThymocyte

CD2+CD5+Dual

CD4+/CD8+sCD3-

CD2+CD5+

CD4+ or CD8+sCD3+

CD2-/+CD5-/+sCD3-CD4-CD8-

TdT+CD10-clg-sig-

Pre-B

CD10+cig+TdT+sig-

B

sig+CD10+/-clg+/-TdT-

Common

TdT+CD10+

clg-sig-

EarlyThymocyte

AAssessment of maturity with primary reagentsRequire secondary panels for sub‐classification

Early B-Precursor

Initial Classification of CLLs and Lymphomas

CLL and Non-Hodgkins Lymphoma

CD2+CD3+

T-Cell Lineage

CD38+CD2-, CD19-

Plasma CellLeukemia/ Myeloma

B-Cell Lineage

CD19+

CD16+ and/or CD56+

CD2+, CD3-

CD5-

CD10-CD10+

Follicular Cell Or

Burkitt’s Lymphoma

SLVLor

Marginal Zone Lymphomaor

Hairy Cell Leukemia

CD5+

B-CLLor

Mantle Cell Lymphoma

Sub‐classification of Lymphomas Required

NK LGLL

Initial Classification of CLLs and Lymphomas

CLL and Non-Hodgkins Lymphoma

CD2+CD3+

T-Cell Lineage

CD38+CD2-, CD19-

Plasma CellLeukemia/ Myeloma

B-Cell Lineage

CD19+

CD16+ and/or CD56+

CD2+, CD3-

CD4+

T-Cell CLLT-Cell Lymphoma

T-PLL

ATLL(Adult T-CellLeukemia)

T-Cell LGLL

CD8+

CD7-

Sezary Syndrome

Sub‐classification of Lymphomas Required

NK LGLL

6 color 2 Laser (5+1)8 color 2 Laser (5+3)10 color 3 Laser (5+3+2)

Prietokové cytometre pre IVD

Cytomics FC 500• 2 konfigurácie

Navios• 3 konfigurácie

5 color 1 Laser 5 color 2 Laser

Navios Flow Cytometer

405nm

638nm

488nm

Available in 3 configurations6 color 2 Laser (5+1)8 color 2 Laser (5+3)

10 color 3 Laser (5+3+2)

Beckman Coulter 5-Color Panel Solutions

• B‐Cell Kit

• T‐Cell Kit

• Myeloid Kit

Aligned with Bethesda Recommendations

Kappa Lambda CD19 CD5 CD45

CD20 CD10 CD19 CD38 CD45


CD8 CD4 ⎯ CD3 CD45

CD15 CD11b CD16 CD14 CD45

HLADR CD56 CD34 CD117 CD45


NaviosTM / FC500 and SOLASTRATM

Reagent Performance: Target Specifications

• Specimen Types

‐ Peripheral Blood

‐ Bone Marrow

‐ Lymphoid cell suspensions

• EDTA, Heparin

• Specimen age: 24 – 48 hours post‐draw

• Target sensitivity: 1% when 50,000 events are collected

• Working WBC Range: 1000 – 50,000 cells/µL

• Sample preparation using VersaLyse + Fixative Lytic System

B-Lineage KitFITC PE ECD PC5.5 PC7

Kappa Lambda CD19 CD5 CD45


Myeloid Lineage KitFITC PE ECD PC5.5 PC7

CD15 CD11b CD16 CD14 CD45

HLADR CD56 CD34 CD117 CD45


T-Lineage KitFITC PE ECD PC5.5 PC7


CD8 CD4 -- CD3 CD45

Myelomonocytic Lineage Tube 1:

CD15 / CD11b / CD16 / CD14 / CD45


HLA-DR / CD56 / CD34 / CD117 / CD45


CD7/ CD13 / CD34 / CD33 / CD45

AML Phenotype:CD34+ 117+ Dim13+ 33+ 15+ 11b-16- 14- 56- 2- 7+

Acute Myeloid Leukemia - AML (BM)

BL1:

Kappa/ Lambda / CD19/ CD5 / CD45

BL2:

CD20/ CD10/ CD19 / CD38 / CD45

Acute Leukocyte Leukemia - ALL (PB)

ALL Phenotype:CD10+ 19+ 34+ 20- sIg- 38+

CLL Phenotype:CD5+ 10- 19+ Dim 20+ 38variable/-Dim Kappa+

Chronic Leukocyte Leukemia - CLL (PB)

BL1:

Kappa/ Lambda / CD19/

CD5 / CD45

BL2:

CD20/ CD10/ CD19 /

CD38 / CD45

Základný panel + ďalšie znaky

PB KrOrange FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750

Kappa Lambda CD19 CD5CD20 CD10 CD19 CD38 CD45

CD2 CD56 CD7 CD5 CD45CD8 CD4 ⎯ CD3 CD45

CD15 CD11b CD16 CD14 CD45HLADR CD56 CD34 CD117 CD45CD7 CD13 CD34 CD33 CD45

Panel pre orientáciu

405 Excitation 488 Excitation 633 Excitation

Paci

fic

Blue

Kro

me

Ora

nge

FITC

PE ECD

PC5.

5

PC7

APC

APC-

AF7

00

APC-

AF7

50

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

• Základné protilátky v jednej skúmavke• 10 fluorescencií• 15 monoklonálnych protilátok

Lymphoid Slide Ruler

www.coulterflow.comwww.beckmancoulter.com

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

Suggested Reading

• Consensus Conference Publications‐ 2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis

of Hematolymphoid Neoplasia by Flow Cytometry, Cytometry 2007 72B: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 8:3941‐3967, 2008.‐ J.W. Vardiman, N.L. Harris, R.D. Brunning, The World Health Organization (WHO) Classification

of the Myeloid Neoplasms, Blood, Volume 100, Number 7:2292‐2302, 2002.

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Diagnostika onkohematologických ochorení pomocou prietokovej … · 29. máj 2013, KLINICKÁ BIOCHÉMIA, s.r.o. Žilina, Medicínske laboratórium Poprad Jozef Smolka Beckman Coulter