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

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Page 1: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

© 2008 HORIBA ABX, All rights reserved.

Flow cytometry leukocyte differential :

a critical appraisal

Francis Lacombe

Flow cytometry departmentUniversity Hospital of Bordeaux, Pessac, France

[email protected]

Page 2: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Current reference methodfor leukocyte differential

Until now morphology method based onthe famous NCCLS document

Reference leukocyte differential count (proportional)and evaluation of instrumental methods. NCCLS Approved Standard H20-A, 1992

But

Numerous criticisms of morphology methodwere put forward and published

Page 3: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Limitations and errors of morphology methodin 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 methodand a new routine method

Page 4: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

• Detection and quantification of blast cells and immature granulocytes in peripheral blood samplesF. Lacombe (ISLH meeting Banff (1998))

• Flow cytometric method for enumeration and classification of reactive immature granulocyte populations Fujimoto et al (Cytometry, 42:371-378, 2000)

• Toward a new reference method for the leukocyte five-part DifferentialHübl et al (Cytometry, 30:72-84, 1997)

Main historical features of FCM leukocyte differential

Page 5: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Hübl et al (Cytometry, 30:72-84, 1997)

Combination of MoAb

CD45-FITCCD16HLA-DR PECD2CD14-PC5

Page 6: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

450 blood samples

IGBlast cells

ISLHBanff (1998)

Detection and quantification of blast cells and immature granulocytes in peripheral blood samples

F. Lacombe (ISLH meeting Banff (1998))

Page 7: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Blast cells Immature Granulocytes

CD45 CD45 + CD16

Page 8: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD45 Gating

SS

FSSS

CD45

Peripheral bloodBone marrow

Page 9: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Normal sample

Lymphocytes : 31.7%

Monocytes : 6%

Neutrophils : 59.2%

IG : 0.5%

Eosinophils : 0.8%

FS

SS

CD14CD14

SS

CD16

SS

CD45

SS

Page 10: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Basophiles, Eosinophiles and Monocytes

CD16

SS

Lymphocytes : 30.5%

Monocytes : 11.1%

Neutrophils : 38.2%

IG : 1.36%

Eosinophils : 10.3%

Basophils : 3.1%

CD9-CD14

SS

CD9-CD14 CD16 CD45

SS

FS

SSSSSS

Page 11: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Blast cells

CD45

SSLymphocytes : 8.1%

Monocytes : 16.4%

Neutrophils : 69.3%

IG : 1.3%

Blasts : 2.8%

SS SSSSSS

CD14 CD16 CD45FS

Page 12: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Immature Granulocytes(IG)

SSCD16

Lymphocytes : 10.8%

Monocytes : 7.9%

Neutrophils : 52.3%

IG : 27.9%

Eosinophils : 1.8%

SS SSSSSS

CD14 CD16 CD45FS

IG

Page 13: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

SS

CD14

SS

CD16

Lymphocytes : 3.3%

Monocytes : 14.9%

Neutrophils : 74.9%

IG : 6.6%

FS CD14 CD16 CD45

SSSS SS SS

Degranulated Immature granulocytes (IG)

Page 14: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

SS

CD45SS

CD16

Lymphocytes :10.8%

Monocytes :24.9%

Neutrophils : 19.1%

IG : 6.1%

Blasts : 37.7%

SS SSSSSS

CD14 CD16 CD45FS

Immature Granulocytes(IG) + Blast cells

Page 15: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CML

SS SSSSSS

CD9 CD16 CD45

SS

CD45

Lymphocytes :1.4%

Monocytes : 0.9%

Neutrophils : 35.9%

IG : 39.2%Blasts : 7.1%

Eosinophils : 3.1%

Basophils : 5.1%

FS

Page 16: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

1 11 21 31 41 51 61 71 81 91 101 111 121S1

S31

S61

S91

S121

0

5

10

15

20

25

30

CD16

SS

Cel

l Num

ber

PMN

IG

0

50

100

150

200

250

300

350

400

450

500

1 11 21 31 41 51 61 71 81 91 101 111 121

PMN

IG

34

10364

% IG % PMN

Automatic determination of IGand polymorphonuclears %

Page 17: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Blast cells

Page 18: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Immature Granulocytes

Page 19: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Polymorphonuclears

Page 20: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Lymphocytes

Page 21: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Monocytes

Page 22: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Fujimoto et al (Cytometry, 42:371-378) (1)

Combination of MoAb

CD16-FITCCD11b-PECD45-PerCP

Page 23: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Fujimoto et al (Cytometry, 42:371-378) (2)

Page 24: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

FDA approved propositions

Page 25: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Genesis of the « magic tube »

F. Lacombe and MC Béné

Page 26: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD11b/CD14/CD45/CD16

Page 27: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD11b/CD14/CD45/CD16 (Granulo)

Page 28: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD11b/CD14/CD45/CD16 (Mono)

Page 29: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD11b/CD14/CD45/CD16 (Lympho)

Page 30: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD11b/CD14/CD45/CD16 (bermudes)

Page 31: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Bone marrowCD11b / CD14 / CD16 / CD45

Page 32: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Bone marrowCD11b / CD14 / CD16 / CD45

Page 33: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

1 2

3 4 5

Myeloïd differentiationSynthesis

Page 34: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The problems of erythroblasts (NRBC)

2- Should they be included in FCM leukocyte differential ?

1- What is the reference method ?

3- If not, how to eliminate them ?

Page 35: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The problems of erythroblasts (NRBC)

1- What is the reference method ?

Page 36: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The problems of erythroblasts (NRBC)

2- Should they be included in FCM leukocyte differential ?

1- What is the reference method ?

NRBC are well detected and counted by hematology analyzers.Thus, is it necessary to count again them in a FCM differential?

Page 37: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The problems of erythroblasts (NRBC)

2- Should they be included in FCM leukocyte differential ?

1- What is the reference method ?

3- If not, how to eliminate them ?

Page 38: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Bone marrowCD36 / CD71 / CD11c / CD45

Erythroblasts

Left Treshold of bermudes region

Page 39: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The current « magic tube »

CD14/CD71/CD45/CD11b/CD16

Page 40: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD14/CD71/CD45/CD11b/CD16

Page 41: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

Page 42: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

Page 43: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

Page 44: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

Page 45: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD71 + cells (C gate)

Page 46: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD71 + cells (B gate)

Page 47: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD71 + cells (B gate)

Page 48: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Introduction of CD263 (CRTH2)

Positive identification of eosinophils

Positive identification of basophils

Page 49: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department
Page 50: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Cytometry (Feuillard et al)

Page 51: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Cytometry (Feuillard et al)

Page 52: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Figure 2A

1

SSC����

FS

C�� ��

WBC

2

CD45����

SS

C�� ��

CD16+SShighCD16+

SSlow

CD16-SShigh

3

SSC����

CD

16�� ��

CD16-SSlow

Page 53: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD16+SShighCD16+

SSlow

CD16-SShigh

3

SSC����

CD

16�� ��

CD16-SSlow

Basophils

4

Non cytotoxicT-cells

CD45����

CD

2+C

RT

H2 �� ��

Figure 2B

5

CD19+

CD19����

CD

36�� ��

Page 54: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD

36�� ��

SS

C�� �� My+

blasts

7

CD45����

5

CD19+

CD19����

CD

36�� ��

8

B-cellsB-blasts

Figure 2C

CD45����

SS

C�� ��

6CD16neg

Monocytes

CD45����

Page 55: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD16+SShighCD16+

SSlow

CD16-SShigh

3

SSC����

CD

16�� ��

CD16-SSlow

Eos

11

Figure 2D

CD16posMono

Cytotox

CD

2 +

CR

TH

2 �� ��

CD36 ����

CD2 + CRTH2 ����

SS

C�� ��

Imm Gran

10S

SC�� ��

CD45����

9

Page 56: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

SSC/FSC

CD45/SSC

Eliminationof debris

SSC/CD16

Selectionof WBC

SSlowCD16pos

CD36/CD2+CRTH2

CD16posmonocytes

CD16poslymphocytes

CD16neg,

SSChigh

CD45/SSC

Immaturemyeloid cells

SSChighCD45high

CD2+CRTH2/SSC Eosinophils

SSlowCD16neg

CD45/CD2+CRTH2

Non cytotoxicT lymphocytes

Basophils

CD19/CD36

CD45/SSCCD45/SSCCD45/CD36

B-cells

B-blasts

Blasts My+

CD16negmonocytes

Neutrophils

1

2

3

4

5

6 7 8

9

10

11

CD2neg andCRTH2neg

Figure 3

Page 57: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Cytometry (Feuillard et al)

AML 4

SplenicLymphomaVillous l

Iatrogenicinfection

Page 58: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

The Beckman Coulter approach

Page 59: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

All Cells Previous less B Cell

Previous less NonWBC Previous less Mature Ne

Previous less Mono Previous less ImG&Eo Previous less CytoTNK

Previous less nonCT/NK

Patient X (LMD 313422)

Previous less Baso & BlastT

The Beckman Coulter approach

Page 60: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

From Baso&BlastT gate From Mono Gate From ImG&Eo

From B Cell gate From B Cell less BlastB

Patient X (LMD 313422)

Ne = 75%

Ly = 13%

Mono = 7%

Eos = 2%

Baso = 1%

ImGran = 1%

The Beckman Coulter approach

Page 61: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

TO

FS

The Horiba approach (F Lacombe)

TO

SS

Thiazole Orange

SS

CD45

CD45

Page 62: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Blood subpopulation

n r (XL/Manual)

RI (XL/Manual)

r (XL/Advia)

RI (XL/Advia)

PMN + Eosinophiles

154 0.94 0.91 0.95 0.93

Lymphocytes 154 0.93 0.93 0.99 0.99 Monocytes 154 0.53 0.42 0.88 0.90 Basophils 100 0.94 0.97 0.89 0.94

NRBC 100 0.96 0.96 IG

(Hematol disease) 150 0.95 0.94

IG (Inflammation)

100 0.80 0.86

The Horiba approach (F Lacombe)

Page 63: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The Horiba approach (F Lacombe)

Normal sample

Page 64: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

AML2 samplewith 10.7% blast cells

The Horiba approach (F Lacombe)

Page 65: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The swedish approach

Page 66: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The swedish approach

Page 67: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The swedish approach

Page 68: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The swedish approach

Page 69: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

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

FCM Reference Methods (ICSH)

B Davis Co-chairman

Page 70: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

The proposed method should have the ability to detect at a frequencyof greater than 0.1% of the total nucleated cells (5 cells/ul)with a coefficient of variation (CV) of less than 5% and directly enumeratein 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 cellsis considered desirable, although not required.

FCM Reference Methods (ICSH)

B Davis Co-chairman

Page 71: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD38CD45DRAQ5CD45CD45PE-Cy7

CD33 + CD64CD33 + CD64CD16 + CD56CD34 + CD117CD16PE-Cy5

CD45HLA-DRCD45HLA-DRCD19ECD

CD34 + CD117CD123CD203c +CD138CD13 + CD20CD2 + CRTH2PE

Syto 16CD16 + CD19CD36Syto 16CD36FITC

SeattleTube 2

SeattleTube 1

SwedishFrenchTube 2

FrenchTube 1

Fluorochrome

Panel FC 500

FCM Reference Methods (ICSH)

Page 72: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

CD38CD45CD16 + CD56CD45CD45PE-Cy7

CD33 + CD64CD33 + CD64CD45 (PerCP-Cy5.5)HLA-DR (APC-Cy7)CD16PE-Cy5

CD45 (APC-H7)HLA-DR (APC-Cy7)DRAQ5 (APC-Cy7)CD34 + CD117CD19 (APC)APC

CD34 + CD117CD123CD203c +CD138CD13 + CD20CD2 + CRTH2PE

Syto 16CD16 + CD19CD36Syto 16CD36FITC

SeattleTube 2

SeattleTube 1

SwedishFrenchTube 2

FrenchTube 1

Fluorochrome

Canto

FCM Reference Methods (ICSH)

Page 73: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Neutrophilicmetamyelocyte

Neutrophilicmyelocyte

Neutrophilicpromyelocyte

Promegakaryocyte

Megakaryoblast

Megakaryocyte

ReticulatedThrombocyte

Basophilicerythroblast

Proerythroblast

Polychromaticerythroblast

Acidophilerythroblast

Reticulocyte

Erythrocyte Thrombocyte

Basophilicpromyelocyte

Basoophilicmyelocyte

Basophilicmetamyelocyte

Eosinophilicpromyelocyte

Eosinophilicmyelocyte

Eosinophilicmetamyelocyte

Myeloblast

NeutrophilicBand cell

BasophilicBand cell

EosinophilicBand cell

BasophilNeutrophilEosinophil

Monoblast

Promonocyte

Monocyte

Myeloid progenitor

Multipotential stem cell

Lymphoid progenitor

Lymphoblast

Prolymphocyte

B Lymphocyte T Lymphocyte

Plasma cell

Dendritic cell Macrophage

Bon

e M

arro

wB

lood

Tis

sue

NK cell

Page 74: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Dendritic cellsLlin-

CD1 CD11cCD123 CD14

MacrophagesDR CD33 CD68

Bon

e M

arro

wB

lood

Tis

sues

T lineage NK B lineage Monocytic lineage

CD2CD5CD7CD3

CD4:CD8

CD2 CD7

CD2CD16CD56

CD19CD22CD24CD20CD21CD11b

DRsIg

CD34

CD19CD10CD38

DRCD13CD33CD38CD36

CD11bCD11cCD14CD64

DRCD13CD33

DRCD34CD117CD33CD13

CD34DR

CD117

CD294CD294

CD33CD13

CD64

CD13CD10CD11bCD16CD24CD15CD65

Lymphoid progenitors

Myeloid progenitors

MegakaryoblastProerythroblast

Neutrophils BasophilsEosinophils Erythrocytes Platelets

CD41CD2

CD61

CD33CD13CD36

CD41CD42CD61

MonocytesT lymphocytes NK Blymphocytes

CD36CD71CD235

Bloodgroups

Page 75: Flow cytometry leukocyte differential : a critical appraisal Cytometry... · Flow cytometry leukocyte differential : a critical appraisal Francis Lacombe Flow cytometry department

Immunophenotyping is becoming the new reference method for leukocyte differentialboth in peripheral blood and in bone marrow

A consensus is necessary for standardizing• monoclonal antibody panels• analysis strategy

Conclusions