“Cellular phenotyping and application of cytometry for diagnostics purposes”

Part I

dr n. med. Karolina Bukowska-Straková

Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice. A common variation is to physically sort particles based on their properties, so as to purify populations of interest.

Flow cytometry - wikipedia definition ;-)

Forward scatter - FSC

Forward scatter - FSC

Forward scatter - FSC

Forward scatter - FSC

Side scatter - SSC

Side scatter - SSC

FSC versus SSC

Monoclonal antibodies – Monoclonal antibodies – conjugated with fluorochromeconjugated with fluorochrome

Monoclonal antibodies (mAbs or moAbs)Monoclonal antibodies (mAbs or moAbs)

Monoclonal antibodies (mAbs or moAbs):Monoclonal antibodies (mAbs or moAbs):

- made by identical immune cells that are all clones of a unique parent cell

- monospecific → monovalent affinity bind to the same epitope.

José M. Casasnovas, Mykol Larvie and Thilo StehleThe EMBO Journal (1999) 18, 2911 - 2922

Monoclonal antibodies (mAbs or moAbs):Monoclonal antibodies (mAbs or moAbs):

The same: isotype, allotype, idiotype Idiotype -differences in Ag recognition

Isotype -different heavy chains:µ, δ, γ, ε, αor light chains:κ, λ

Allotype- Individual

differences in amino acid sequence

(polymorphisms)

Fluorochrome excitationFluorochrome excitation

Flow cytometry

Lasers / fluorochroms

Fluorochrome chooseFluorochrome choose

Mean fluorescence intensity

Data presentation

Our instrument – LSR II 10 colors

Immunophenotyping of leukocytes

• Flow cytometric immunophenotyping with (monoclonal) antibodies

allows the recognition of leukocyte subsets.

• The CD nomenclature has created clarity in the field of membrane

bound leukocyte antigens (but not for intracellular markers)

• These CD antibodies recognise many different types of markers

(lineage specific, differentiation stage specific, etc) and thereby allow

the recognition of many different (immature and mature) leukocyte

subsets.

• Usage of multiparameter analyses allow the dissection of

differentiation and maturation pathways as well as the detecion of

specific proteins (see: Van Lochem et al. Cytometry 2004;60B:1-13).

Immature markers T-cell markers

CD34: precursor marker CD1: common thymocyte marker

TdT: terminal deoxnucleotidyl transferase CD2: pan-T-cell marker

CD117: myeloid precursor marker CD4: helper T-cell marker

HLA-DR: precursor cells (and APC cell) CD8: cytotoxic T-cell marker

CD3: mature T-cell marker

B-cell markers TCR: T-cell receptor

CD10: immature B-cell marker

CD19: pan-B-cell marker Myeloid markers

CD20: mature B-cell marker CD13 and CD33: pan-myeloid

SmIg: membrane bound Ig CD14: monocytic marker

CyIg: cytoplasmic Ig CD15: granulocytic marker

Classical leukocyte markers

Hematopoiesis

Physiologicalbalance of hematopoiesis

Its regulation depends on:cytokines, celluler interactions, transcription and metabolic factors

Hematopoietic cells may be missing or do not function properly

Immuno-deficiencies

Flow cytometry in primary immunodeficiency

Flow cytometric immunphenotyping and functional studies of blood / bone marrow / other material

Targeted analysis of immune cells: 1) are all cells present in normal frequencies;2) do they have all relevant proteins expressed;3) is their function normal?

Primary immunodeficiencies (PID)

Combined T and B-cell defectsPredominantly antibody deficiencies

Diseases of immune dysregulaton

Other well defined immunodeficiency syndromes

Congenital defects in fagocyte number or/and function

Defects in innate immunityDNA repair defects

Complement deficiencies

Alternative complement pathway defects

Regulatoty proteins of complement

Periodic fever syndromes

Peripheral blood lymphocyte subpopulations

Enumeration of lymphocyte subsets in PB healthy control

Enumeration of lymphocyte subsets in PB SCID T-B+NK- (common γ chain)

David Vetter – bubble boy

Enumeration of lymphocyte subsets in PB SCID T-B-NK- (ADA deficiency)

Enumeration of lymphocyte subsets in PB XLA patient

CSFE, 10 min, 37C

wash free CSFE

Culture with Ag/mitogen

Lymphocytes

Proliferation assay

culture

Signal from unstimulated cells

CSFE, 10 min, 37C

wash free CSFE

Culture with Ag/mitogen

Lymphocytes

Proliferation assay

Chronic granulomatous disease → NADPH oxidase

Medical Immunology 2006 5:4

CGD anti-gp91phox

Healthy

X-linked CGD

+

Reactive oxygen species – FCM measurement(superoxide anion)

Reactive oxygen species – FC measurement(hydrogen peroxide)

Hematopoietic cells may undergo malignant transformation into an abnormally proliferating

leukemic cells

LeukemiaLymphoma

Leukemias and Lymphomas

Leukemias (acute or chronic) and lymphomas can be regarded as malignant counterparts of

normal hematopoietic cells in different maturation stages.

B-ALL

T-ALL

M0 minimally differentiated acute myeloblastic leukemia M1 acute myeloblastic leukemia, without maturationM2 acute myeloblastic leukemia, with granulocytic maturation M3 promyelocytic, or acute promyelocytic leukemia (APL) M4 acute myelomonocytic leukemia

M4eo myelomonocytic together with bone marrow eosinophilia M5 (M5a) acute monoblastic leukemia

(M5b) acute monocytic leukemia M6 (M6a) acute erythroid leukemias, including erythroleukemia

(M6b) very rare pure erythroid leukemia M7 acute megakaryoblastic leukemia

AML - AML - Acute myeloid leukemia

ALLALL -Acute lymphoblastic leukemiaB-ALL from B lineageT-ALL from T lineage

Flow cytometry in hematology

Immunophenotyping plays an important role in establishing the diagnosis and classifying hematopoietic malignancies and is a basic

investigation, which precisely defines the lineage and stage of differentiation of malignantly transformed

hematopoietic cells.

CD45 expressionPB vs normal BM

Pro-B-cell

CD34TdT

CD22

Pre-B-I-cell(Pre-pre-B-cell)CD34TdTCD10bright

CD19

CD22CD45dim

Pre-B-II-cell(Pre-B-cell)

(TdT)CD10CD19CD20-/dim

CD22CD45CyIgµ

Immature B-cell(tr. Pre-B-cell)

CD10dim

CD19CD20CD22CD45bright

SmIgµ

Mature B-cell

CD19CD20CD22bright

CD45bright

SmIgµ

Plasma cell

CD19

CD45Cy/SmIg

Pro-B-cell

CD34TdT

CD22

Pre-B-I-cell(Pre-pre-B-cell)CD34TdTCD10bright

CD19

CD22CD45dim

Pre-B-II-cell(Pre-B-cell)

(TdT)CD10CD19CD20-/dim

CD22CD45CyIgµ

Immature B-cell(tr. Pre-B-cell)

CD10dim

CD19CD20CD22CD45bright

SmIgµ

Mature B-cell

CD19CD20CD22bright

CD45bright

SmIgµ

Plasma cell

CD19

CD45Cy/SmIg

Pro-B-cell Pre-B-I-cell(Pre-pre-B-cell)

Pre-B-II-cell(Pre-B-cell)

Immature B-cell(tr. Pre-B-cell)

Mature B-cell Plasma cell

Normal B-celldevelopment

Regenerating B cell precursor in bone marrowafter cytotoxic treatment

Physiological shift in B-cell compartment

Age-related shifts inB-cell compartment of elderly individuals

Lymphoid differentiation

1.

2.

3. 4. 5. 6. 7. 8. 9. 10.1. AUL2. proB ALL3. cALL4. preB ALL5. Trans. preB6. B-CLL7. HCL8. BL, DLBL, MALT9. LPL10. MM

11. Immature T-ALL12. c T-ALL13. Mature T-ALL14. T-LGL15. NK-LGL

11. 12.

13. 14.

15.

Myeloid differentiation

A. Plesa, 2008

Myelo/monoblastCD34CD117HLA-DRCD13CD33

Pro-monocyte

HLA-DRCD13high

CD33high

CD11bCD15dim

Monocyte

HLA-DRCD13high

CD33high

CD11bCD15dim

CD14

Macrophage

HLA-DRCD13CD33CD11b

CD14

Myelo/monoblastCD34CD117HLA-DRCD13CD33

Promyelocyte

CD117

CD13high

CD33high

CD15

Myelocyte

CD13dim

CD33dim

CD15CD11b

Neutrophil

CD13high

CD33CD15CD11bhigh

CD16high

Metamyelocyte

CD13CD33dim

CD15CD11bCD16

Myeloid differentiation

0.

0. AML-M01. AML-M12. AML-M23. AML-M34. AML-M45. AML-M56. AML-M67. AML-M7

1.

2. 3.

4. 5.

6.

7.

Thank you for your attention =)