immunophenotyping by flow cytometry...immunophenotyping by flow cytometry pr françois mullier pr...

Immunophenotyping by flow cytometry

Pr François Mullier

Pr Bernard Chatelain

BHS course, October 12th, 2013

INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS

INTRODUCTION: Principles

Tutorial: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Multiple parameters of

individual cells within a

heterogenous

preparation

http://images.google.be/imgres?imgurl=http://www.info.fundp.ac.be/~hto/image/logofundp2.jpg&imgrefurl=http://www.info.fundp.ac.be/~hto/pmwiki/index.php/Research/AboutMe&usg=__h-oSxDzomdEPP6rqMpBj1rfuFm4=&h=700&w=525&sz=65&hl=fr&start=4&sig2=9ggpas2Rr7Ecq0q4KHzK5g&tbnid=mYpvCI8fUtIqTM:&tbnh=140&tbnw=105&ei=X7i7SauCLYLI-AbX_aSCBQ&prev=/images?q=logo+FUNDP&gbv=2&hl=fr

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html


INTRODUCTION: Principles

More and more colors….so more

and more complex


SAMPLE PREPARATION:

QUALITY CONTROL CONCLUSION APPLICATIONS INTRODUCTION SAMPLE PREPARATION

• Individualized cells cell suspension

• Whole blood: Little preparation (already in suspension)

RBC lysis (NH4Cl, [RBC])

Enrichment (Ficoll, Percoll)

Anticoagulant: EDTA or heparin

• Washing sometimes required:

- cell concentration

- eliminate cell fragments

- mandatory if presence of molecules able to fix the detection

antibodies (ex:λκ)

:

Hematology


SAMPLE PREPARATION:


Bone marrow

27g needle

Ganglion

fragment

'

Saline

Glass rod

Ganglion fragment

:

Hematology

Bone marrow Ganglion


SAMPLE PREPARATION:


• Blood collection: - Careful

- Light tourniquet or not at all

- 21-G (or larger bore) needle

- Smooth draw (good flow)

- Discard the first 2 ml of blood drawn

- Processing within 30min

- Container: nonwettable surfaces: siliconized glass or

polypropylene to avoid initiation of clotting resulting from contact activation

• Anticoagulant

:

Hemostasis, preanalytic

LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10


SAMPLE PREPARATION:


• Sample handling: - Minimize time between drawing and preparation to

reduce spontaneous platelet activation

- Properly mix anticoagulant

- Avoid unnecessary agitation prior testing

- Transport: No pneumatic (platelet activaton)

No extreme temperatures

• Whole blood

:

Hemostasis, preanalytic


APPLICATIONS:


:

Current applications, Hematology

Cell blood Counting

Platelet quantification

Diagnostic, Prognosis and follow-up of hematological

diseases

Red Blood Cells pathologies diagnosis: PNH, HS

Primary and Acquired Immune defects (AIDS,…)

BCR-ABL qualification

Liquids

DNA Analysis


APPLICATIONS:


Current applications, Hemostasis

Platelet disorders diagnosis

Platelet activation

Antiaggregants follow-up (VASP)

Reticulated platelets


APPLICATIONS:


Current applications, Transfusion

Anti-platelet antibodies

Transplants, grafts: CD34 quantification

Transplants follow-up

Kleihauer


APPLICATIONS:


Acute leukemia


APPLICATIONS:


FCM

Promyelocytes

Nucleus

Cytoplasm

Auer rods

Peroxydase

t(15;17) t(15;17)

variant t(11;17)

ZBTB16

ATRA

Leucocytosis

Sensitive Sensitive Resistant

+++ +++ +++

Reg/Lob

Presents

Bilob Reg/ pelger

HyperG MicroG

Absents

HyperG

Absents

t(15;17) bcr3

transcript

CD34-/DR-

CD33+

CD15-

CD34-/DR-

CD33+

CD15-

CD34+/CD2+

CD33+/DR-

CD15-

CD34+/CD2+

DR-/CD33+

CD15-

Reg/Lob

Presents

HyperG

+++

Sensitive

t(5;17)

NPM1

HyperG++

MicroG+

Absents

Reg/Lob

Sensitive

CD34-/DR-

CD33+

CD15-

+++

CD56+ : bad prognosis

AML M3


APPLICATIONS:


Chronic Lymphoid Leukemia

Infinicyte


APPLICATIONS:


Chronic Lymphoid Leukemia

On CD19+ Ly Matutes 4/5 (CD22+ et CD79b+)

CLL cut-off:

5000 Monoclonal B

lymphocytes/µl


APPLICATIONS:


Mantle Lymphoma


APPLICATIONS:


Mantle Lymphoma

Other applications:

Multiple myeloma

Extrahematological invasions


APPLICATIONS:


T lymphomas

• Remains a challenge in 2013

• Clinical information required:

- skin rash, extensive lymphadenopathy, hypergammaglobulinemia AILT

- neutropenia, rheumatoid arthritis LGL

• >3000/μl with CD4/CD8 inversion morphology + serology

• CD4+CD8+: reported if >1%, TCR gene rerrangement study indicated

• Aberrant expression is suggestive for clonal T-cell proliferation but never the

proof

Philippe et al. Acta Clinica Belgica 2009


APPLICATIONS:


T lymphomas

Diffusion à 90°

CD

45

Pe

rCP

CD19 APC CD4 PE-Cy7

CD16/56 PE CD8 APC-Cy7

CD

4 P

E-C

y7

CD

3 F

ITC

CD

3 F

ITC

C

D3

FIT

C

Mullier F. and al. Hématologie 2009


APPLICATIONS:


T lymphomas

CD10 FITC

CD10 FITC CD56 PE-Cy7

CD4 APC

CD

3 P

erC

P

CD

4 A

PC

C

D4 A

PC

CD

56 P

E-C

y7

Mullier F. and al. Hématologie 2009


APPLICATIONS:


• Quantification: Morphology > FCM (Paiva et al. Haematologica 2009/ Rawtron et al.

Haematologica 2008)

- Heterogenous distribution of plasmocytes in BM

- Contamination by peripheral blood 1ml= pure BM (Batinic BMT 1990)

- Selection of area riched in plasmocytes by the cytologist

- Adhesion to lipids (Nadav et al. BJH 2006)

- Quantification on biopsy: probably more accurate but standardization needed

- Weak plasmocyte

* Loss in the needle?

* Loss during RBC lysis (Smock et al. APLM 2007):

* Lowered CD138 expression by lysis (Smock et al. APLM 2007)

* Loss during centrifugations (Smock et al. APLM 2007)

Multiple myeloma


APPLICATIONS:

INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 23

• Plasmocytes gating

- CD38: unspecific, more expressed

- CD-138 (BB-4): very specific

- Characteristic Light scatter

23

San Miguel. European Journal of Cancer 2006

Multiple myeloma


APPLICATIONS:


• Monoclonal gammapathies differentiation

- Myelomatous plasmocytes: CD38 ++, CD56+++, CD19- et CD45-

- Normal plasmocytes: CD38+++, CD56- CD19++ CD45+

24

San Miguel. European Journal of Cancer 2006

Myeloma diagnosis Myeloma post-transplantation

Multiple myeloma


APPLICATIONS:

INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 25 25

Paiva et al. Blood 2009

Multiple myeloma


APPLICATIONS:


Patient Day 100 post ACT

Patient in stable CR Several years

Rawstron et al Haematologica 2008;93:431

CD19-:MM

CD19+:PC

CD19-:PC

CD19+:PC

Multiple myeloma


APPLICATIONS:


Bruggeman et al Leukemia 2010;24(3):521-535

Minimal residual disease

Assessment of minimal residual disease (MRD) has acquired a prominent position

in European treatment protocols for patients with acute lymphoblastic leukemia

(ALL), on the basis of its high prognostic value for predicting outcome and the

possibilities for implementation of MRD diagnostics in treatment stratification.

Therefore, there is an increasing need for standardization of methodologies and

harmonization of terminology. For this purpose, a panel of representatives of all

major European study groups on childhood and adult ALL and of international

experts on PCR- and flow cytometry-based MRD assessment was built in the

context of the Second International Symposium on MRD assessment in Kiel,

Germany, 18-20 September 2008. The panel summarized the current state of MRD

diagnostics in ALL and developed recommendations on the minimal technical

requirements that should be fulfilled before implementation of MRD diagnostics into

clinical trials. Finally, a common terminology for a standard description of MRD

response and monitoring was established defining the terms 'complete MRD

response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD

terminology may allow a refined and standardized assessment of response to

treatment in adult and childhood ALL, and provides a sound basis for the

comparison of MRD results between different treatment protocols


APPLICATIONS:


Liquids: CSF

• Diagnosis leptomeningeal involvement: Great prognostic and therapeutic implications

(most common: myeloid and B lymphoid neoplasm)

• Immunopathogenesis of neuroinflammatory diseases (multiple

sclerosis,…)

• Cytology: High specificity but limited sensitivity (20 to 60%)

• FCM: Higher sensitivity

However: paucicellularity and rapidly decreasing viability

specific sample preparation protocols and analytical approaches

Kraan et al. Current protocols in Cytometry 2008; Unit 6.25:1-16

De Graaf et al. Cytometry Part B 2011; 80B:271–281


APPLICATIONS:


Liquids: CSF

• Critical parameters:

- Rapidly decreasing viability (Cytotoxic effect on WBC)

Solution: Analyse within 1h after sampling

- Blood contamination

- Removal of free Ig otherwise binding of anti-kappa or anti-lambda

reduced or absent reactivity with anti-Hu-Ig reagents

solution: diluting the sample with an equal amount of

buffer during the first centrifugation/concentration step

- Rare events CV: 100*√n/n

solution: count large number of cells

-Carry-over of cells in the cytometer from previous experiments

solution: extensive cleaning and washing

Kraan et al. Current protocols in Cytometry 2008; Unit 6.25:1-16


APPLICATIONS:


BCR-ABL Qualitative detection

Healthy subject CML


APPLICATIONS:


:

Paroxysmal Nocturn Hemoglobinuria

Red Blood Cells Granulocytes and Monocytes

Healthy subject


APPLICATIONS:


:

Paroxysmal Nocturn Hemoglobinuria

Red Blood Cells Granulocytes and Monocytes

Positive PNH


APPLICATIONS:


Paroxysmal Nocturn Hemoglobinuria, FLAER

Positive PNH FL

AE

R (

FIT

C)

Healthy subject

FL

AE

R (

FIT

C)

PNH clone

Ongoing standardization by the French Society of Flow Cytometry


APPLICATIONS:


:

Hereditary spherocytosis

Perrotta S and al. Lancet 2008; Vol 372:1411-1426 King MJ and al. BJH 2000; Vol 111(3):924-933

EMA binding


APPLICATIONS:


AIDS


APPLICATIONS:



Balduini C and al. Haematologica 2003 and 2004


APPLICATIONS:


Healthy

subject

Pauline

Subject Mean Fluorescence

Intensity

Number of GpIb

sites/cell

Normal range

Healthy subject 20825 33850 27000-49000

Pauline 27927 43450



APPLICATIONS:


Healthy

Subject

Pauline

Sujet Mean

Fluorescence

Intensity

Number of

GpIa sites/cell

Normal range Ratio

GpIb/GpIa

Sujet sain 2576 4805 2200-7800 7,0

Pauline 8873 14937 2,9



APPLICATIONS:


Healthy subject

Pauline

Subject Mean

Fluorescence

Intensity

Number of

GpIIIa sites

GpIIIa/cell

Normal range Ratio

GpIb/GpIIIa

Healthy

subject

27020 43173 41000-65000 0,8

Pauline 77614 112567 0,4


BSS

Heterozygous

GpIbβ:

Pro105Leu

Unpublished data (collaboration with KUL Kortrijk H. Deckmyn and K.Vanhoorelbeke)


APPLICATIONS:


Current applications, Reticulated platelets

Decreased formation: CIT and aplastic anemia

Enhanced destruction: ITP

Anti-platelet antibody

TPO

Spleen

Young, reticulated

platelet

Why? - Bone marrow megakaryocyte activity and platelet kinetics

- Differentiate low platelet production from enhanced consumption

- Decreases the invasive BM aspiration need and eliminates superfluous

platelet transfusions


APPLICATIONS:


Current applications, Reticulated platelets

Flow cytometry

Lack of standardisation

Flow cytometer and a cytometrist required

Not widespread as a daily routine test

Hematimetry (ex: XE 2100) Precise, automated, relatively inexpensive, non-ivasive

Good reproducibility and stability (48 hours)

Reference range:

1.1-6.1%

Autoimmune thrombocytopenia:

IPF=17-22%

Healthy subject ITP Aplastic Anemia


APPLICATIONS:


Mepacrine testing

Capture 16μM

Release 16μM


QUALITY CONTROL:


• Foundation: 2000

• Analysis on EDTA fresh samples:

- Leucocytosis (Absolute Number (AN)), Lymphocytes (%)

- CD3/CD4/CD8 (% and AN)

- CD19(%, AN), NK (%,AN)

- kappa/lambda/ratio (%)

- CD34 (blood)

• Perspectives

- CD34 (cytapheresis)

- Interpretation

:

Belgian quality control


QUALITY CONTROL:


89

10

11

12

13

Year

CV

(%

)

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

CD4

89

10

11

12

13

Year

CV

(%

)

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

CD4

10

12

14

16

18

Year

CV

(%

)

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

CD19

10

12

14

16

18

Year

CV

(%

)

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

CD19

:


With permission of ISP/WIV (M. Van Blerk)


QUALITY CONTROL:


:



2000/2009 Number of participants

CD3 CD4 CD8 CD19

1 colour 2/0 0/0 0/0 5/2

2 colours 12/2 15/2 15/2 15/1

3 colours 18/6 17/6 17/6 11/6

4 colours 6/20 6/20 6/20 5/19

5 colours 0/6 0/6 0/6 0/6

6 colours 0/15 0/15 0/15 0/15


QUALITY CONTROL:


:



Belgian consensus recommendations:

27.1% of the participants in 2000→86.0% in 2009

Gating strategy % in 2002 % in 2009

FSC/SSC 44.2 10.0

CD14/CD45 11.6 2.0

CD45/SSC 44.2 88.0


CONCLUSION:


• Numerous hematologic applications due to

technologic improvements since 1934

• Progress in quality control since 2000 however

lack of standardization remains one pitfall

• Perspectives: - Standardization

- New applications: MRD,…


References

1. Guidelines for an integrated diagnostic approach of chronic lymphoproliferative disorders in the routine laboratory of haematology in Belgium.

Philippé J, Nollet F, Bakkus M, Meeus P, Demanet C, Schaaf-Lafontaine N, Franke S, Chatelain B, Vermeulen K, Boone E, El Housni H, Heimann P, Husson B, Lambert F, Vannuffel P, Saussoy P, Maes B, Deschouwer P.

Acta Clin Belg. 2009 Nov-Dec;64(6):494-504. 2. Developments in the immunophenotypic analysis of haematological malignancies. Heel K, Tabone T, Röhrig KJ, Maslen PG, Meehan K, Grimwade LF, Erber WN. Blood Rev. 2013 Jul;27(4):193-207. doi: 10.1016/j.blre.2013.06.005. Epub 2013 Jul 8. 3. Flow cytometric immunophenotyping for hematologic neoplasms. Craig FE, Foon KA. Blood. 2008 Apr 15;111(8):3941-67. doi: 10.1182/blood-2007-11-120535. Epub 2008

Jan 15. Review.

http://www.ncbi.nlm.nih.gov/pubmed/20101872












immunophenotyping by flow cytometry...immunophenotyping by flow cytometry pr françois mullier pr...

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