Flow cytometry leukocyte differential : a critical appraisal

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1 Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department University Hospital of Bordeaux, Pessac, France 2008 HORIBA ABX, All rights reserved.

2 Current reference method for leukocyte differential Until now morphology method based on the famous NCCLS document Reference leukocyte differential count (proportional) and evaluation of instrumental methods. NCCLS Approved Standard H20-A, 1992 But Numerous criticisms of morphology method were put forward and published

3 Limitations and errors of morphology method in leukocyte differential routine Slide distribution errors and bad choice of the differential enumeration area Statistical leukocyte count errors ( < 200 counted cells -see Rümke table-) Observer errors Poor efficiency and time consuming method Need for a new reference method and a new routine method

4 Main historical features of FCM leukocyte differential Toward a new reference method for the leukocyte five-part Differential Hübl et al (Cytometry, 30:72-84, 1997) Detection and quantification of blast cells and immature granulocytes in peripheral blood samples F. Lacombe (ISLH meeting Banff (1998)) Flow cytometric method for enumeration and classification of reactive immature granulocyte populations Fujimoto et al (Cytometry, 42: , 2000)

5 Hübl et al (Cytometry, 30:72-84, 1997) Combination of MoAb CD45-FITC CD16 HLA-DR PE CD2 CD14-PC5

6 Detection and quantification of blast cells and immature granulocytes in peripheral blood samples F. Lacombe (ISLH meeting Banff (1998)) ISLH Banff (1998) 450 blood samples IG Blast cells

7 Blast cells Immature Granulocytes CD45 CD45 + CD16

8 CD45 Gating FS Peripheral blood Bone marrow CD45

9 Normal sample FS CD14 CD16 CD45 Lymphocytes : 31.7% Monocytes : 6% Neutrophils : 59.2% IG : 0.5% Eosinophils : 0.8%

10 Basophiles, Eosinophiles and Monocytes FS CD9-CD14 CD16 CD45 Lymphocytes : 30.5% Monocytes : 11.1% Neutrophils : 38.2% IG : 1.36% Eosinophils : 10.3% Basophils : 3.1% CD9-CD14 CD16

11 Blast cells FS CD14 CD16 CD45 Lymphocytes : 8.1% Monocytes : 16.4% Neutrophils : 69.3% IG : 1.3% Blasts : 2.8% CD45

12 Immature Granulocytes(IG) FS CD14 CD16 CD45 Lymphocytes : 10.8% Monocytes : 7.9% IG Neutrophils : 52.3% IG : 27.9% Eosinophils : 1.8% CD16

13 Degranulated Immature granulocytes (IG) FS CD14 CD16 CD45 Lymphocytes : 3.3% Monocytes : 14.9% Neutrophils : 74.9% IG : 6.6% CD14 CD16

14 Immature Granulocytes(IG) + Blast cells FS CD14 CD16 CD45 Lymphocytes :10.8% Monocytes :24.9% Neutrophils : 19.1% IG : 6.1% Blasts : 37.7% CD16 CD45

15 CML FS CD9 CD16 CD45 Lymphocytes :1.4% Monocytes : 0.9% Neutrophils : 35.9% IG : 39.2% Blasts : 7.1% Eosinophils : 3.1% Basophils : 5.1% CD45

16 Automatic determination of IG and polymorphonuclears % PMN 20 Cell Number IG 5 S121 S CD16 S1 S31 S % IG % PMN PMN IG

17 Blast cells

18 Immature Granulocytes

19 Polymorphonuclears

20 Lymphocytes

21 Monocytes

22 Fujimoto et al (Cytometry, 42: ) (1) Combination of MoAb CD16-FITC CD11b-PE CD45-PerCP

23 Fujimoto et al (Cytometry, 42: ) (2)

24 FDA approved propositions

25 Genesis of the «magic tube» F. Lacombe and MC Béné

26 CD11b/CD14/CD45/CD16

27 CD11b/CD14/CD45/CD16 (Granulo)

28 CD11b/CD14/CD45/CD16 (Mono)

29 CD11b/CD14/CD45/CD16 (Lympho)

30 CD11b/CD14/CD45/CD16 (bermudes)

31 Bone marrow CD11b / CD14 / CD16 / CD45

32 Bone marrow CD11b / CD14 / CD16 / CD45

33 Myeloïd differentiation Synthesis

34 The problems of erythroblasts (NRBC) 1- What is the reference method? 2- Should they be included in FCM leukocyte differential? 3- If not, how to eliminate them?

35 The problems of erythroblasts (NRBC) 1- What is the reference method?

36 The problems of erythroblasts (NRBC) 1- What is the reference method? 2- Should they be included in FCM leukocyte differential? NRBC are well detected and counted by hematology analyzers. Thus, is it necessary to count again them in a FCM differential?

37 The problems of erythroblasts (NRBC) 1- What is the reference method? 2- Should they be included in FCM leukocyte differential? 3- If not, how to eliminate them?

38 Bone marrow CD36 / CD71 / CD11c / CD45 Erythroblasts Left Treshold of bermudes region

39 The current «magic tube» CD14/CD71/CD45/CD11b/CD16

40 CD14/CD71/CD45/CD11b/CD16

41 CD14/CD71/CD45/CD11b/CD16 (Lympho)

42 CD14/CD71/CD45/CD11b/CD16 (Mono)

43 CD14/CD71/CD45/CD11b/CD16 (Granulo)

44 CD14/CD71/CD45/CD11b/CD16 (bermudes)

45 CD71 + cells (C gate)

46 CD71 + cells (B gate)

47 CD71 + cells (B gate)

48 Introduction of CD263 (CRTH2) Positive identification of eosinophils Positive identification of basophils

49

50 Cytometry (Feuillard et al)

51 Cytometry (Feuillard et al)

52 Figure 2A WBC FSC C CD16 CD16+ low CD16+ high CD16- low CD16- high C CD45 C

53 Figure 2B CD16 CD16+ low CD16- low CD16+ high CD16- high CD2+CRTH2 Basophils Non cytotoxic T-cells CD36 CD19+ C CD45 CD19

54 Figure 2C 6 CD16neg Monocytes CD CD45 CD36 CD19+ 7 C B-blasts B-cells CD19 CD45 C My+ blasts CD45

55 Figure 2D 9 3 CD2 + CRTH2 Cytotox CD16pos Mono 11 CD16 CD16+ low CD16- low CD16+ high CD16- high 10 CD36 C Eos C Imm Gran CD2 + CRTH2 C CD45

56 Figure 3 Neutrophils low CD16pos CD36/CD2+CRTH2 CD16pos monocytes 9 CD16pos lymphocytes 1 C/FSC 2 Elimination of debris CD45/C 3 Selection of WBC C/CD16 10 CD45/C Immature myeloid cells CD16neg, Chigh Chigh CD45high 11 CD2+CRTH2/C low CD16neg CD45/CD2+CRTH2 CD2neg and CRTH2neg Eosinophils CD19/CD36 CD45/CD36CD45/C CD16neg monocytes 4 5 Blasts My+ Non cytotoxic T lymphocytes Basophils CD45/C B-cells B-blasts

57 Cytometry (Feuillard et al) AML 4 Splenic Lymphoma Villous l Iatrogenic infection

58 The Beckman Coulter approach The previous FCM concept is now commercialized by Beckman Coulter in the Hematoflow system with the same panel of MoAb but with a new automatized gating strategy essentially based on negative gating

59 The Beckman Coulter approach All Cells Previous less B Cell Previous less NonWBC Previous less Mature Ne Previous less Mono Previous less ImG&Eo Previous less CytoT NK Previous less nonct/nk Patient X (LMD ) Previous less Baso & BlastT

60 The Beckman Coulter approach From Baso&BlastT gate From Mono Gate From ImG&Eo From B Cell gate Patient X (LMD ) From B Cell less BlastB Ne = 75% Ly = 13% Mono = 7% Eos = 2% Baso = 1% ImGran = 1%

61 The Horiba approach (F Lacombe) Thiazole Orange CD45 TO FS TO CD45

62 The Horiba approach (F Lacombe) Blood subpopulation n r (XL/Manual) RI (XL/Manual) r (XL/Advia) RI (XL/Advia) PMN Eosinophiles Lymphocytes Monocytes Basophils NRBC IG (Hematol disease) IG (Inflammation)

63 The Horiba approach (F Lacombe) Normal sample

64 The Horiba approach (F Lacombe) AML2 sample with 10.7% blast cells

65 The swedish approach

66 The swedish approach

67 The swedish approach

68 The swedish approach

69 FCM Reference Methods (ICSH) The International Committee on Standardization in Hematology (ICSH) has initiated an effort to develop a new flow cytometric reference method for the enumeration of nucleated cell populations in peripheral blood. This is intended as a replacement for the method outlined in the CLSI H20-A2 document and is expected to address many of the deficiencies of the current reference method. B Davis Co-chairman

70 FCM Reference Methods (ICSH) The proposed method should have the ability to detect at a frequency of greater than 0.1% of the total nucleated cells (5 cells/ul) with a coefficient of variation (CV) of less than 5% and directly enumerate in cells/ul the following nucleated cell populations : Neutrophils, Monocytes, Lymphocytes, Eosinophils, Basophils, Immature myeloid cells, Blasts and Nucleated erythrocytes. If possible, the enumeration of B cells, T cells and NK cells is considered desirable, although not required. B Davis Co-chairman

71 FCM Reference Methods (ICSH) Panel FC 500 Fluorochrome French Tube 1 French Tube 2 Swedish Seattle Tube 1 Seattle Tube 2 FITC CD36 Syto 16 CD36 CD16 + CD19 Syto 16 PE CD2 + CRTH2 CD13 + CD20 CD203c +CD138 CD123 CD34 + CD117 ECD CD19 HLA-DR CD45 HLA-DR CD45 PE-Cy5 CD16 CD34 + CD117 CD16 + CD56 CD33 + CD64 CD33 + CD64 PE-Cy7 CD45 CD45 DRAQ5 CD45 CD38

72 FCM Reference Methods (ICSH) Canto Fluorochrome French Tube 1 French Tube 2 Swedish Seattle Tube 1 Seattle Tube 2 FITC CD36 Syto 16 CD36 CD16 + CD19 Syto 16 PE CD2 + CRTH2 CD13 + CD20 CD203c +CD138 CD123 CD34 + CD117 APC CD19 (APC) CD34 + CD117 DRAQ5 (APC-Cy7) HLA-DR (APC-Cy7) CD45 (APC-H7) PE-Cy5 CD16 HLA-DR (APC-Cy7) CD45 (PerCP-Cy5.5) CD33 + CD64 CD33 + CD64 PE-Cy7 CD45 CD45 CD16 + CD56 CD45 CD38

73 Multipotential stem cell Lymphoid progenitor Myeloid progenitor Bone Marrow Lymphoblast Prolymphocyte Monoblast Promonocyte Eosinophilic promyelocyte Eosinophilic myelocyte Myeloblast Neutrophilic promyelocyte Neutrophilic myelocyte Basophilic promyelocyte Basoophilic myelocyte Proerythroblast Basophilic erythroblast Polychromatic erythroblast Megakaryoblast Promegakaryocyte Eosinophilic metamyelocyte Neutrophilic metamyelocyte Basophilic metamyelocyte Megakaryocyte Acidophil erythroblast Eosinophilic Band cell Neutrophilic Band cell Basophilic Band cell Reticulocyte Reticulated Thrombocyte Blood B Lymphocyte T Lymphocyte Monocyte Eosinophil Neutrophil Basophil Erythrocyte Thrombocyte Plasma cell NK cell Tissue Dendritic cell Macrophage

74 Lymphoid progenitors Bone Marrow CD2 CD7 T lineage NK B lineage Monocytic lineage CD34 CD19 CD10 CD38 DR CD13 CD33 DR CD34 CD117 CD33 CD13 Myeloid progenitors CD34 DR CD117 Proerythroblast Megakaryoblast CD33 CD13 CD36 CD71 CD235 CD33 CD13 CD36 CD64 CD41 CD42 CD61 T lymphocytes NK Blymphocytes Monocytes Eosinophils Neutrophils Basophils Erythrocytes Platelets Blood CD2 CD5 CD7 CD3 CD4:CD8 CD2 CD16 CD56 CD19 CD22 CD24 CD20 CD21 CD11b DR sig DR CD13 CD33 CD38 CD36 CD11b CD11c CD14 CD64 CD294 CD13 CD10 CD11b CD16 CD24 CD15 CD65 CD294 Blood groups CD41 CD2 CD61 Tissues Dendritic cells Llin- CD1 CD11c CD123 CD14 Macrophages DR CD33 CD68

75 Conclusions Immunophenotyping is becoming the new reference method for leukocyte differential both in peripheral blood and in bone marrow A consensus is necessary for standardizing monoclonal antibody panels analysis strategy

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