Cell sorting: A bridge between morphology and flow cytometry. Pr François Mullier, Pr Bernard Chatelain July 19th, 2013

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1 Cell sorting: A bridge between morphology and flow cytometry Pr François Mullier, Pr Bernard Chatelain July 19th,

2 CELL SORTING: PRINCIPLES D.Davies

3 CELL SORTING: PRINCIPLES Aims Efficiency Purity : Sort only the cells of interest Speed Sequential procedure : cell analysis then sorting At the time of the analysis, the cell is not inside a drop Decision to sort is based on a theorical drop at this time

4 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

5 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

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12 CD16 CD16 CD66 CD13

13 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias

14 CELL SORTING: MONOCYTIC MATURATION Unknown normal maturation patterns Sorting of monocytes not described in the literature Easy activation of monocytes Will they resist to the cell sorting?

15 CELL SORTING: MONOCYTIC MATURATION 15 Wood (2004) Methods Cell Biology 75:

16 SORTING OF NORMAL MONOCYTES (I) Whole blood No lysing solution (aggressive) Sorting of CD45+ CD14+ cells (scatter parameters may be influenced by vibration) Threshold: CD45 CCls: 1) Monocytes are resistant to the cell sorting (acceleration, electric charge and landing in the sorting device) 2) Presence of red blood cells (when a trigger is set, no physical elimination of other cells when coincidence between RBC and monocyte, RBC present in the sorting product)

17 SORTING OF NORMAL MONOCYTES (II) Whole blood FICOLL Sorting of CD45+ CD14+ cells (scatter parameters may be influenced by vibration) CD14 CD45 CD45 CCls: 1) Absence of red blood cells SSC 2) Cell activation is more marked (cytoplasmic prolongations)

18 SORTING OF NORMAL MONOCYTES (III) Do the sorted cells keep their enzymatic activity? YES (interesting in CMML) Myeloperoxydase Double esterase Mature monocytes: variable alpha naphtyl and weak to moderate peroxydase

19 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

20 MONOCYTIC MATURATION ANALYSIS (I) CMML or M4-M5 : important to differentiate Mature monocytes Immature monocytes: monoblasts or promonocytes Morphological and cytometric criteria unclear in the WHO Classification Difficult morphological differentiation between monocytes and promonocytes Study of the normal monocytic maturation before cell sorting to identify the important morphological criteria CD64 FITC CD56 PE HLA-DR PerCy5 CD34 PeCy7 CD117 APC CD11b APC H7 CD14 V450 CD45 V500

21 MONOCYTIC MATURATION ANALYSIS (II) Analysis of sorted and numerised cells (214 mature monocytes and 113 immature monocytes) following the followingcriteria: 1. Cell size 2. Cytoplasmic prolongations 3. Basophilia of cytoplasm 4. Presence or absence of azurophilic granulations in the cytoplasm 5. Presence of vacuoles in the cytoplasm 6. Nucleo-cytoplasmic ratio 7. Form of the nucleus 8. Aspect of the chromatin 9. Drawn nuclear membrane 10. Presence of a nucleole

22 MONOCYTIC MATURATION ANALYSIS (III) Invariable criteria according to the stage of maturation Drawn nuclear membrane Azurophilic granulations Criteria of maturity Numerous cytoplasmic prolongations More frequent vacuoles Low nucleo-cytoplasmic ratio (< 0,7) Absence of nucleole ou doubtful 100% 50% 0% 100% 80% 60% 40% 20% 0% CYTOPLASMIC PROLONGATIONS MURS absents MURS Vacuoles IMMATURES présents 0 1 à 3 > 3 IMMATURES Most frequent morphologic phenotype for the mature monocyte: cytoplasmic prolongations, grey cytoplasm, low nucleo-cytoplasmic ratio, highly notched nucleus, non fine chromatine

23 MONOCYTIC MATURATION ANALYSIS (IV) Criteria of immaturity Cytoplasm 100% Basophilia of the cytoplasm 80% High nucleo-cytoplasmic ratio(> 0,7) Less notched nucleus 60% 40% 20% Fine chromatine 0% MURS Well visible large nucleolus gris Aspect of the chromatine 100% IMMATURES basophile Nucleo-cytoplasmic ratio 100% 80% 80% 60% 60% 40% 40% 20% 0% 20% MURS IMMATURES chromatine fine chromatine non fine 0% MURS < 0,7 IMMATURES > 0,7

24 MONOCYTIC MATURATION ANALYSIS (V) J.E.Goasguen Haematologica 2009

25 MONOCYTIC MATURATION ANALYSIS (VI) Uncorrect criteria: 1) Immature monocyte: no mention in WHO Classification 2) Numerous cytoplasmic prolongations for the monocyte: not mentioned 3) nucleo-cytoplasmic ratio: not mentioned 4) The nuclear shape is more important than mentioned in the Goasguen classification J.E.Goasguen Haematologica 2009

26 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (I) AML M4 Whole blood Cell sorting CD14 high density (mature monocytes) CD14 low density and Bermudes (immature monocytes)

27 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (II) Mature monocytes Immature monocytes Cell sorting allows determination of morphological criteria to differentiate mature and immature monocytes Interest: standardization, education

28 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (III) Bone marrow (more difficult as monocytes monocytes are a rare event: 4-5%)

29 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (IV) Mature monocytes Immature monocytes Cell sorting allows determination of morphological criteria to differentiate mature and immature monocytes Interest: standardization, education

30 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (V) CD64 - CD56 - HLA-DR - CD34 - CD117 - CD11b + CD14 - CD45 + CD64 + (low d.) CD56 - HLA-DR + CD34 - CD117 - CD11b + CD14 + CD45 + CD64 - CD56 - HLA-DR - CD34 - CD117 - CD11b - CD14 - CD45 + CD64 - CD56 - HLA-DR + CD34 - CD117 + CD11b - CD14 - CD

31 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (VI) Cell sorting of the 4 populations Nozzle 70 micron Frequency 89.7 Amplitude 31.5 Drop Delay First Drop 158 Gap 8 Sheath Pressure 70 PSI Precision Purity Efficiency: 93% Efficiency: 95% Efficiency: 93% Efficiency: 94% 3 smears a. MGG staining b. Myeperoxydase c. Esterase 31 31

32 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (VII) Size? Small Small / Large Large Cytoplasmic Extensions? No Sometimes Yes Color of Cytoplasm? Basophilic Blue-Grey Grey Cytoplasmic azurophilic granules? No Sometimes Yes Vacuoles? No Yes (0-2) Yes (1-10) NCR? High (>0.7) Medium ( 0.7) Low (<0.7) Nucleus Shape? Round Irregular Irregular Kind Of Chromatin? Fine Diffuse Condensed Nucleus Membran"redrawn"? No No Yes Nucleole? Yes (>1) Yes (0-2) No 3 well defined populations Perox MGG Esterase 32

33 DISTINCTION BETWEEN MATURE AND IMMATURE MONOCYTES (VIII) CD64 - HLA-DR + CD117 + CD11b - CD14 - CD64 - HLA-DR - CD117 - CD11b + CD14 - CD64 + HLA-DR + CD117 - CD11b + CD14 + Perox MGG Monoblasts? Promonocytes? Monocytes? Esterase 33 Maturation

34 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

35 FOLLOW-UP OF ACUTE PROMYELOCYTIC LEUKEMIAS Peripheral blood at diagnosis

36 FOLLOW-UP OF ACUTE PROMYELOCYTIC LEUKEMIAS Bone marrow at diagnosis

37 FOLLOW-UP OF ACUTE PROMYELOCYTIC LEUKEMIAS CD34 CD13 CD33 CD15 CD7 HLADR One blastic population : 100 % Negative CD34-CD15 : 100 % Heterogenous positive CD13 : 100 % Negative HLADR : 91 % Homogeneous positive CD33 : 82 %

38 FOLLOW-UP OF ACUTE PROMYELOCYTIC LEUKEMIAS CD16+/CD33fd CD16 CD33 CD16-/CD33++ Important to determine the phenotype of normal neutrophils and malign neutrophils (ATRA induces maturation of leukemic cells) in order to classify the cells during the follow-up

39 FOLLOW-UP OF ACUTE PROMYELOCYTIC LEUKEMIAS Malign neutrophil

40 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

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43 Peroxydase

44 Immature cells are CD10+ CD19-?

45 CD79 positif B cells Intracytoplasmic

46 Cell sorting of CD10+/CD19- confirm the malign nature of these cells

47 DNA Analysis Aneuploid population CD10 + G0G1 Cell sorting Aneuploïdie index = 1.23 G2+m

48 CD19 control (different clone and different fluorochrome)

49 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

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55 Anti Glycophorine A Immunohistochemistry (without NH4Cl lysis): numerous erythroblasts

56 Anti A Immunohistochemistry (without NH4Cl lysis): numerous erythroblasts

57 Immunophenotype of immature cells : Immaturity CD38, CD71 positif CD45, HLADR, CD10, CD34 négatif Granulocytic antigens CD33, CD13, CD11b positifs CD117, MPO négatifs Monocyte antigens CD36, CD15 positif - CD14 négatifs Red blood cell antigens Glycophorine A négative Platelet antigens CD41, CD61, CD42 négatifs B Antigens ccd79a, CD19, CD20, skappa, slambda, cmu négatifs T antigens ccd3, CD4, CD8, CD2, WT31, Tδ1 négatifs Autres CD56 négatif

58 Cell sorting CD36/Glyco A GA+CD36- GA+CD36+ GA-CD36+

59 Cell sorting CD36+/Glyco A-

60 Cell sorting CD36+/Glyco A+

61 Cell sorting CD36-/Glyco A+

62 NH 4 Cl lysis Percoll separation (1.085) Proerythroblasts Proerythroblasts SIZE Granulocytes SIZE Granulocytes RBC and RBC debris Erythroblasts RBC, pycnotic Anti Polychromatophil erythroblasts Glycophorine A Glycophorine A Loss of erythroblasts due to NH4Cl lysis

63 Percoll separation Sample: blood, bone marrow Plasma Addition of the sample Centrifugation 400 g Nucleated cells Percoll RBC culot

64 Monocytes Proerythroblasts Basophilic erythroblasts Polychromatophils (maturation) Percoll polychromatophils pycnotic Erythroblasts CD36 CD36 polychromatophiles pycnotic Erythroblasts RBC Glycophorine A NH 4 Cl Glycophorine A

65 CELL SORTING: INTERESTS OF CELL SORTING Link between morphology, immunohistochemistry and flow cytometry: - Myeloid maturation - Monocytic maturation - Follow-up of acute monoblastic leukemias - Follow-up of acute promyelocytic leukemias - Diagnostic of acute lymphoblastic leukemias - Diagnosis of acute erythroblastic leukemias - Diagnostic of hereditary spherocytosis

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67 TAKE HOME MESSAGES (I) Interest of cell sorting: link between flow cytometry, immunohistochemistry and morphology A same immunological phenotype may correspond to different morphological phenotypes (heterogeneity) The study of monocytic maturation by flow cytometry and cell sorting will allows improving the standardization of the morphological reading and educating the less-experienced morphologists.

68 TAKE HOME MESSAGES (II) The cell sorting allows discriminating normal and malign neutrophils during the follow-up of acute promyelocytic leukemias The cell sorting may contribute to the diagnosis of acute lymphoblastic leukemias The cell sorting illustrates the elimination of erythroblasts by NH4Cl lysis which makes difficult the diagnosis of acute erythroblastic leukemias. The elimination of reticulocytes by cell sorting may facilitate the diagnosis of hereditary spherocytosis by SDS-PAGE

69 ACKNOWLEDGMENTS Pr Bernard Chatelain Ph.Laurence Miller Ph.Biol. Jean-François Classen Mr Nicolas Bailly

70 Thank you for your attention 70

71 Thank you for your attention 71

72 BIBLIOGRAPHY Goasgen JE, et al. Morphological evaluation of monocytes and their precursors. Haematologica 2009;94: Gorczyca W, et al. Immunophenotypic Pattern of Myeloid Populations by Flow Cytometry Analysis. Methods in Cell Biology. 2011;103: Grimaldi E, Carandente P, Scopacasa F, et al. Evaluation of the monocyte counting by two automated haematology analysers compared with flow cytometry. Clin Lab Haematol. 2005;27: Meintker L, et al. Comparison of Automated Differential Blood Cell Counts From Abbott Sapphire, Siemens Advia 120, Beckman Coulter DxH 800, and Sysmex XE in Normal and Pathologic Samples. Am J Clin Pathol 2013;139: Nguyen VTP, et al. Évaluation de l automate d hématologie Sysmex XN-2000 pour une utilisation en routine : comparaison avec l Advia 2120i. Immunol Biol Spec (2013)