© 2008 HORIBA ABX, All rights reserved.

Flow cytometry leukocyte differential :

a critical appraisal

Francis Lacombe

Flow cytometry departmentUniversity Hospital of Bordeaux, Pessac, France

francis.lacombe@chu-bordeaux.fr

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

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

• 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

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

Combination of MoAb

CD45-FITCCD16HLA-DR PECD2CD14-PC5

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))

Blast cells Immature Granulocytes

CD45 CD45 + CD16

CD45 Gating

SS

FSSS

CD45

Peripheral bloodBone marrow

Normal sample

Lymphocytes : 31.7%

Monocytes : 6%

Neutrophils : 59.2%

IG : 0.5%

Eosinophils : 0.8%

FS

SS

CD14CD14

SS

CD16

SS

CD45

SS

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

Blast cells

CD45

SSLymphocytes : 8.1%

Monocytes : 16.4%

Neutrophils : 69.3%

IG : 1.3%

Blasts : 2.8%

SS SSSSSS

CD14 CD16 CD45FS

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

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)

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

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

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 %

Blast cells

Immature Granulocytes

Polymorphonuclears

Lymphocytes

Monocytes

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

Combination of MoAb

CD16-FITCCD11b-PECD45-PerCP

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

FDA approved propositions

Genesis of the « magic tube »

F. Lacombe and MC Béné

CD11b/CD14/CD45/CD16

CD11b/CD14/CD45/CD16 (Granulo)

CD11b/CD14/CD45/CD16 (Mono)

CD11b/CD14/CD45/CD16 (Lympho)

CD11b/CD14/CD45/CD16 (bermudes)

Bone marrowCD11b / CD14 / CD16 / CD45

Bone marrowCD11b / CD14 / CD16 / CD45

1 2

3 4 5

Myeloïd differentiationSynthesis

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 ?

The problems of erythroblasts (NRBC)

1- What is the reference method ?

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?

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 ?

Bone marrowCD36 / CD71 / CD11c / CD45

Erythroblasts

Left Treshold of bermudes region

The current « magic tube »

CD14/CD71/CD45/CD11b/CD16

CD14/CD71/CD45/CD11b/CD16

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

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

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

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

CD71 + cells (C gate)

CD71 + cells (B gate)

CD71 + cells (B gate)

Introduction of CD263 (CRTH2)

Positive identification of eosinophils

Positive identification of basophils

Cytometry (Feuillard et al)

Cytometry (Feuillard et al)

Figure 2A

1

SSC����

FS

C�� ��

WBC

2

CD45����

SS

C�� ��

CD16+SShighCD16+

SSlow

CD16-SShigh

3

SSC����

CD

16�� ��

CD16-SSlow

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�� ��

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����

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

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

Cytometry (Feuillard et al)

AML 4

SplenicLymphomaVillous l

Iatrogenicinfection

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

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

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

TO

FS

The Horiba approach (F Lacombe)

TO

SS

Thiazole Orange

SS

CD45

CD45

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)

The Horiba approach (F Lacombe)

Normal sample

AML2 samplewith 10.7% blast cells

The Horiba approach (F Lacombe)

The swedish approach

The swedish approach

The swedish approach

The swedish approach

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

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

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)

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)

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

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

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