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
Lymfóm / Leukémia
ƒ 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)
ƒ 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)
ƒ 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
60
Years Ago
50
Years Ago
“Disease of the Blood”
40
Years Ago
Leukemia or Lymphoma
Chronic Leukemia
Acute Leukemia
Preleukemia
Today
5 year
Survival
~ 0%
Indolent Lymphoma
Aggressive Lymphoma
∼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)
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.
70%
Lymfóm / Leukémia
Imunofenotypizácia ako diagnostický a monitorovací nástroj
Imunofenotypizácia prietokovou cytometriou
– Jedinečný nástroj pre sledovanie ochorenia
– Počiatočná diagnóza
– Sub ‐ klasifikácia
– Monitorovanie pacienta
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
‐ 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
Štandardizácia – rok 2006 a 2008
2006 Bethesda International Consensus WHO 2008 Recommendations on the Classification of Immunophenotypic Analysis of Tumours of Hematolymphoid Neoplasia by Flow Hæmatopoietic and Cytometry
Lymphoid Tissues
Cytometry 2007 72B:S14‐22
4th Edition, 2008
IARC Press
Practical International Consensus Established
Conference Recommendations
Bethesda 2006
Consensus
Consensus
Markers
Combination
Strategy
‰
Initial Screening Approach
‰
Lineage‐based panels
‰ T‐Cell
‰ B‐Cell
‰ Myelo‐monocytic
‰ Plasma Cell ‰
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
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
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
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 BPrecursor
Common
CD2-/+
CD5-/+
CD2+
CD5+
CD2+
CD5+
TdT+
TdT+
sCD3-
Dual
CD4+/CD8+
CD4+ or
CD8+
CD10-
CD10+
sCD3-
sCD3+
clgsig-
clgsig-
CD4CD8-
Pre-B
CD10+
cig+
TdT+
sig-
Assessment of maturity with primary reagents
A
Require secondary panels for sub‐classification
B
sig+
CD10+/clg+/TdT-
Initial Classification of CLLs and Lymphomas
CLL and Non-Hodgkins Lymphoma
Plasma Cell
Leukemia/
Myeloma
CD38+
CD2-,
CD19-
CD16+ and/or
CD56+
CD2+, CD3-
CD19+
NK LGLL
CD2+
CD3+
T-Cell Lineage
B-Cell Lineage
CD5-
CD5+
B-CLL
or
Mantle Cell Lymphoma
CD10+
Follicular Cell
Or
Burkitt’s Lymphoma
Sub‐classification of Lymphomas Required
CD10-
SLVL
or
Marginal Zone Lymphoma
or
Hairy Cell Leukemia
Initial Classification of CLLs and Lymphomas
CLL and Non-Hodgkins Lymphoma
Plasma Cell
Leukemia/
Myeloma
CD38+
CD2-, CD19-
CD2+
CD3+
CD19+
CD16+ and/or
CD56+
CD2+, CD3-
B-Cell Lineage
T-Cell Lineage
CD4+
CD8+
CD7Sezary
Syndrome
T-Cell CLL
T-Cell Lymphoma
T-PLL
ATLL
(Adult T-Cell
Leukemia)
Sub‐classification of Lymphomas Required
T-Cell
LGLL
NK LGLL
Prietokové cytometre pre IVD
Cytomics FC 500
•
2 konfigurácie
5 color 1 Laser 5 color 2 Laser Navios
•
3 konfigurácie
6 color 2 Laser (5+1)
8 color 2 Laser (5+3)
10 color 3 Laser (5+3+2)
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
Beckman Coulter 5-Color Panel Solutions
• B‐Cell Kit
Kappa Lambda CD19 CD5
CD45
CD20
CD45
CD10
CD19 CD38
• T‐Cell Kit
CD2
CD56
CD7
CD5
CD45
CD8
CD4
⎯
CD3
CD45
CD15
CD11b
• Myeloid Kit
CD16 CD14
CD45
HLADR CD56
CD34 CD117
CD45
CD7
CD34 CD33
CD45
CD13
Aligned with Bethesda Recommendations
NaviosTM / FC500 and SOLASTRATM
Reagent Performance: Target Specifications
Myeloid Lineage Kit
• Specimen Types
‐ Peripheral Blood
FITC
PE
ECD
PC5.5
PC7
CD15
CD11b
CD16
CD14
CD45
HLADR
CD56
CD34
CD117
CD45
CD7
CD13
CD34
CD33
CD45
‐ Bone Marrow
‐ Lymphoid cell suspensions
• EDTA, Heparin T-Lineage Kit
FITC
PE
ECD
PC5.5
PC7
CD2
CD56
CD7
CD5
CD45
CD8
CD4
--
CD3
CD45
• 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 Kit
FITC
PE
ECD
PC5.5
PC7
Kappa
Lambda
CD19
CD5
CD45
CD20
CD10
CD19
CD38
CD45
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:
CD34+ 117+ Dim13+ 33+
15+ 11b-16- 14- 56- 2- 7+
Acute Leukocyte Leukemia - ALL (PB)
BL1:
Kappa/ Lambda / CD19/ CD5 /
CD45
ALL Phenotype:
CD10+ 19+ 34+ 20- sIg- 38+
BL2:
CD20/ CD10/ CD19 / CD38 /
CD45
Chronic Leukocyte Leukemia - CLL (PB)
BL1:
Kappa/ Lambda / CD19/
CD5 / CD45
BL2:
CD20/ CD10/ CD19 /
CD38 / CD45
CLL Phenotype:
CD5+ 10- 19+
Dim 20+
38variable/Dim Kappa+
Základný panel + ďalšie znaky
PB
KrOrange
FITC
PE
ECD
PC5.5
PC7
Kappa Lambda
CD19
CD20
CD10
CD19
CD38
CD5
CD45
CD2
CD8
CD45
CD45
CD56
CD4
CD7
⎯
CD15
CD11b CD16
HLADR CD56
CD34
CD7
CD13
CD34
CD5
CD3
CD14
CD45
CD117 CD45
CD33
CD45
APC APC-A700 APC-A750
Panel pre orientáciu
• Základné protilátky v jednej skúmavke
• 10 fluorescencií
• 15 monoklonálnych protilátok
lambda/CD7
CD10
CD4
CD8/CD15/CD20
CD45
kappa/CD4
lambda/CD56
CD7
CD117
CD56/CD117 CD3/CD33
CD10
CD34
A PCA F750
PC5.5
kappa/CD34
A PCA F700
ECD
CD45
A PC
PE
CD15/CD20
PC7
FIT C
633 Excitation
K rom e
O rang e
488 Excitation
Pacific
B lue
405 Excitation
CD8
CD19
CD19
CD3/CD33
Lymphoid Slide Ruler
www.coulterflow.com
www.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