Immunophenotyping by flow cytometry. Pr François Mullier Pr Bernard Chatelain BHS course, October 12th, 2013

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1 Immunophenotyping by flow cytometry Pr François Mullier Pr Bernard Chatelain BHS course, October 12th, 2013

2 INTRODUCTION: Principles Multiple parameters of individual cells within a heterogenous preparation Tutorial:

3 INTRODUCTION: Principles More and more colors.so more and more complex

4 SAMPLE PREPARATION: : Hematology 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) INTRODUCTION SAMPLE PREPARATION APPLICATIONS QUALITY CONTROL CONCLUSION

5 SAMPLE PREPARATION: : Hematology Bone marrow Ganglion Bone marrow 27g needle Ganglion fragment Glass rod Saline Ganglion fragment ' INTRODUCTION SAMPLE PREPARATION APPLICATIONS QUALITY CONTROL CONCLUSION

6 Blood collection: - Careful SAMPLE PREPARATION: : Hemostasis, preanalytic - 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 LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10 INTRODUCTION SAMPLE PREPARATION APPLICATIONS QUALITY CONTROL CONCLUSION

7 SAMPLE PREPARATION: : Hemostasis, preanalytic 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) Whole blood No extreme temperatures INTRODUCTION SAMPLE PREPARATION APPLICATIONS QUALITY CONTROL CONCLUSION

8 : 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

9 Current applications, Hemostasis Platelet disorders diagnosis Platelet activation Antiaggregants follow-up (VASP) Reticulated platelets

10 Current applications, Transfusion Anti-platelet antibodies Transplants, grafts: CD34 quantification Transplants follow-up Kleihauer

11 Acute leukemia

12 Acute leukemia

13 Acute leukemia

14 AML M3 t(15;17) Promyelocytes t(15;17) variant t(11;17) ZBTB16 t(15;17) bcr3 transcript t(5;17) NPM1 Nucleus Cytoplasm Auer rods Reg/Lob HyperG Presents Bilob MicroG Absents Reg/ pelger HyperG Absents Reg/Lob HyperG Presents Reg/Lob HyperG++ MicroG+ Absents Leucocytosis Peroxydase FCM CD34-/DR- CD33+ CD CD34+/CD2+ CD56+ CD34-/DR- : bad prognosis CD34+/CD2+ DR-/CD33+ CD15- CD33+ CD15- CD33+/DR- CD CD34-/DR- CD33+ CD15- ATRA Sensitive Sensitive Resistant Sensitive Sensitive

15 Chronic Lymphoid Leukemia Infinicyte

16 Chronic Lymphoid Leukemia On CD19+ Ly Matutes 4/5 (CD22+ et CD79b+) CLL cut-off: 5000 Monoclonal B lymphocytes/µl

17 Mantle Lymphoma

18 Mantle Lymphoma Other applications: Multiple myeloma Extrahematological invasions

19 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

20 CD45 PerCP CD4 PE-Cy7 CD3 FITC CD3 FITC CD3 FITC APPLICATIONS: T lymphomas CD19 APC CD4 PE-Cy7 Diffusion à 90 Mullier F. and al. Hématologie 2009 CD8 APC-Cy7 CD16/56 PE

21 CD56 PE-Cy7 CD4 APC CD3 PerCP CD4 APC APPLICATIONS: T lymphomas CD4 APC CD10 FITC CD10 FITC CD56 PE-Cy7 Mullier F. and al. Hématologie 2009

22 Multiple myeloma 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)

23 Multiple myeloma Plasmocytes gating - CD38: unspecific, more expressed - CD-138 (BB-4): very specific - Characteristic Light scatter San Miguel. European Journal of Cancer

24 Multiple myeloma Monoclonal gammapathies differentiation - Myelomatous plasmocytes: CD38 ++, CD56+++, CD19- et CD45- - Normal plasmocytes: CD38+++, CD56- CD19++ CD45+ Myeloma diagnosis Myeloma post-transplantation San Miguel. European Journal of Cancer

25 Multiple myeloma Paiva et al. Blood

26 Multiple myeloma CD19+:PC CD19-:MM Patient Day 100 post ACT CD19+:PC CD19-:PC Patient in stable CR Several years Rawstron et al Haematologica 2008;93:431

27 Bruggeman et al Leukemia 2010;24(3): APPLICATIONS: 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, September 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

28 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:

29 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

30 BCR-ABL Qualitative detection Healthy subject CML

31 : Paroxysmal Nocturn Hemoglobinuria Healthy subject Red Blood Cells Granulocytes and Monocytes

32 : Paroxysmal Nocturn Hemoglobinuria Positive PNH Red Blood Cells Granulocytes and Monocytes

33 Paroxysmal Nocturn Hemoglobinuria, FLAER Healthy subject FLAER (FITC) PNH clone Positive PNH FLAER (FITC) Ongoing standardization by the French Society of Flow Cytometry

34 : Hereditary spherocytosis EMA binding Perrotta S and al. Lancet 2008; Vol 372: King MJ and al. BJH 2000; Vol 111(3):

35 AIDS

36 Platelet disorders diagnosis Balduini C and al. Haematologica 2003 and

37 Platelet disorders diagnosis Healthy subject Pauline Subject Mean Fluorescence Intensity Number of GpIb sites/cell Normal range Healthy subject Pauline

38 Platelet disorders diagnosis Healthy Subject Pauline Sujet Mean Fluorescence Intensity Number of GpIa sites/cell Normal range Ratio GpIb/GpIa Sujet sain ,0 Pauline ,9 38

39 Platelet disorders diagnosis Healthy subject Pauline BSS Heterozygous GpIbβ: Pro105 Leu Subject Healthy subject Mean Fluorescence Intensity Number of GpIIIa sites GpIIIa/cell Normal range Ratio GpIb/GpIIIa ,8 Pauline ,4 Unpublished data (collaboration with KUL Kortrijk H. Deckmyn and K.Vanhoorelbeke) 39

40 Current applications, Reticulated platelets Enhanced destruction: ITP Decreased formation: CIT and aplastic anemia TPO Antiplatelet antibody 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

41 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: % Autoimmune thrombocytopenia: IPF=17-22% Healthy subject ITP Aplastic Anemia

42 Mepacrine testing Release 16μM Capture 16μM

43 QUALITY CONTROL: : Belgian 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

44 QUALITY CONTROL: : Belgian quality control CV (%) CD CV (%) CD Year Year With permission of ISP/WIV (M. Van Blerk)

45 QUALITY CONTROL: : Belgian 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 With permission of ISP/WIV (M. Van Blerk)

46 QUALITY CONTROL: : Belgian quality control Belgian consensus recommendations: 27.1% of the participants in % in 2009 Gating strategy % in 2002 % in 2009 FSC/SSC CD14/CD CD45/SSC With permission of ISP/WIV (M. Van Blerk)

47 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,

48 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 Nov-Dec;64(6): Developments in the immunophenotypic analysis of haematological malignancies. Heel K, Tabone T, Röhrig KJ, Maslen PG, Meehan K, Grimwade LF, Erber WN. Blood Rev Jul;27(4): doi: /j.blre Epub 2013 Jul Flow cytometric immunophenotyping for hematologic neoplasms. Craig FE, Foon KA. Blood Apr 15;111(8): doi: /blood Epub 2008 Jan 15. Review.