Practical Application of Multi-dimensional Flow Cytometry in Hematological Disorders

李啟誠 , 朱崧肇 , 蔡喜修 , 許淑敏 , 謝馨慧 Michael R. Loken

Introduction

• Immunophenotyping has become a powerful

tool in the characterization of different

hematological disorders

• The key is to realize normal cellular

differentiation pathways on the flow

cytometric histographs and detect any

“deviation” from the normal patterns

Different in strategy of flow cytometric development

• rather than to design novel markers and apply more and more fluorescences, which are high-priced, time consuming, and only suitable for study of MRD but not the other hematological disorder such as MDS.

• For any suspicious cell population found on flow, the best way is to sort them and send for molecular confirmation.

Types of antigeneic abnormalities

• Lineage infidelity

• Maturational asynchrony

• Antigeneic absence

• Quantational abnormality

Wells DA, Benesch M, Loken MR et al, Blood, 2003, 102: 394-403

Sensitivity of MRD by flow cytometry

• Two variables: degree of phenotypic difference between target cells and remaining cells; number of cells can be analyzed

• 1 in 107 or more similar to PCR • Maximal sensitivity of 1 in 105 cells during analysis in

clinical sample• Consistent sensitivity: 1 in 104

Campana D, Acta haematologica, 2004, 112:8-15

Principle of flow cytometry

Forward Light Scatter

Laser

Red Orange Green Fluorescence Detectors

Right Angle Light

Structure of flow cytometry

Normal bone marrow with orderly myeloid maturation

Promye

Mye

Meta

Neu

Promye

Mye/Meta

Neu

I II III IV

B Lymphoid Maturation

CD 10

FMC 7

CD 34

CD 45

CD 19

CD 20

CD 22 TdT

CD 5

CD 23 Antigen E

xpre

ssio

n 103

102

101

Normal B lymphoid maturation

Normal B lymphoid maturation

CD19+

6 7

8 9

10 30 50 70 90 120

FSC -->

10

11

02

10

31

04

SS

C -

->

ML

101 102 103 104

SSC -->1

01

10

21

03

10

4C

D45 P

erC

P -

->

LymphMonoMyeloid

101 102 103 104

SSC -->

10

11

02

10

31

04

CD

19-A

PC

-->

CD19+

101 102 103 104

CD20 FITC -->

10

11

02

10

31

04

CD

45 P

erC

P -

->

101 102 103 104

CD10 PE -->

10

11

02

10

31

04

CD

45 P

erC

P -

->

101 102 103 104

CD20 FITC -->1

01

10

21

03

10

4C

D10 P

E -

->

III

III

IV

III

IIIIV

T cell

III

III, IVT

Normal B lymphoid maturation

CD19+

6 7

8 9

10 30 50 70 90 120

FSC -->

10

11

02

10

31

04

SS

C -

->

ML

101 102 103 104

SSC -->1

01

10

21

03

10

4C

D45 P

erC

P -

->

LymphMonoMyeloid

101 102 103 104

CD19-APC -->

10

11

02

10

31

04

CD

45 P

erC

P -

->101 102 103 104

CD22 FITC -->

10

11

02

10

31

04

CD

45 P

erC

P -

->

101 102 103 104

CD34 PE -->

10

11

02

10

31

04

CD

45 P

erC

P -

->

101 102 103 104

CD22 FITC -->1

01

10

21

03

10

4C

D34 P

E -

->

I

I

Antibodies panels used for myeloid assessment

Tube FITC PE PerCP

# 1 isotope isotope CD45

# 2 HLA-DR CD11b CD45

# 3 CD5 CD19 CD45

# 4 CD56 CD38 CD45

# 5 CD16 CD13 CD45

# 6 CD15 CD34 CD45

# 7 CD14 CD33 CD45

# 8 CD7 CD56 CD45

# 9 HLA-DR CD34 CD45

Antibodies panels used for B-lymphoid assessment

Tube FITC PE PerCP

# 1 isotope isotope CD45

# 2 HLA-DR CD11b CD45

# 3 CD5 CD19 CD45

# 4 CD56 CD38 CD45

# 5 CD20 CD10 CD45

# 6 CD22 CD34 CD45

# 7 kappa CD19 CD45

# 8 lambda CD19 CD45

# 9 FMC7 CD19 CD45

Examples of flow cytometric application in hematological disorders

• Minimal residual detection for AML

• Minimal residual detection for ALL

• Flow cytometric diagnosis of MDS

• Flow cytometric diagnosis of Lymphoma

• Flow cytometric diagnosis of multiple myeloma

• Flow cytometric analysis of tissue sample

• Flow cytometric diagnosis of non-malignant disorder

• Application of cell sorting for MRD confirmation

Minimal Residual Detection for AML

Diagnostic sample MRD 4.0%

Over-expression of CD34 & HLA-DR

Diagnostic sample MRD 0.1%

Aberrant expression of CD56 & CD7

Diagnostic sample MRD 0.6%

Aberrant expression of CD19

Minimal Residual Detection for ALL

Diagnostic sample MRD 0.03%

Precursor B-ALL, D15 s/p induction C/T

CALLA (-) precursor B-ALL

Relapse s/p BMT, MRD 0.3%

Flow Cytometric Diagnosis of MDS

56 y/o female, pancytopenia, blast 5.0%, CD13+, CD33+, aberrant CD15+, aberrant CD34-, asynchronous myeloid maturation on CD13 vs CD16 panel

Convex shape

CD15+ CD34-

53 y/o female, MDS, CD56+ on blast, monocyte & neutrophil

Flow Cytometric Diagnosis of Lymphoma

66 y/o man, Maltoma, lacrimal gland, for staging

HE staining of BM

7.0% clonal marginal zone lymphoma, lower CD45 as compared to lymphocyte, CD19+, CD20+, FMC7+, kappa+, lambda-

L26 staining

Flow Cytometric Diagnosis of Multiple myeloma

68 y/o female, multiple myeloma with cytoplasmic lambda restriction, (30.0%)

AML, M4eo, in remission; MGUS, 0.2% cyto-kappa+

Flow Cytometric Analysis of Tissue Sample

55 y/o man, left axillary LN: CD10+, CD20+, CD19/lambda+, diagnostic of follicular center B-cell lymphoma

Flow Cytometric Diagnosis of Non-malignant Disorder

27 y/o female, macrocytic anemia, Acid-Ham test (-) PB flow: PNH, heterozygous clone

Neutrophil: dim CD16

Monocyte: lose CD14

BM flow

Application of Cell Sorting for MRD Confirmation

Ab-Lymph

Lymph

10 11

12 13

E-7282

10 30 50 70 90 120

FSC -->

10

11

02

10

31

04

SS

C -

-> MLe-7282

101 102 103 104

SSC -->1

01

10

21

03

10

4C

D45 P

erC

P -

->

LymphMono

Myeloid

Blast

e-7282

101 102 103 104

CD5 FITC -->

10

11

02

10

31

04

CD

19 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

CD20 FITC -->

10

11

02

10

31

04

CD

10 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

CD20 FITC -->

10

11

02

10

31

04

CD

5 P

E -

->

Ab-LymphLymph

10 11

12 13

e-7282

101 102 103 104

CD23 FITC -->

10

11

02

10

31

04

CD

19 P

E -

->Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

CD25 FITC -->

10

11

02

10

31

04

CD

22 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

FMC7 FITC -->

10

11

02

10

31

04

CD

19 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

KAPPA FITC -->

10

11

02

10

31

04

CD

19 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

LAMBDA -->

10

11

02

10

31

04

CD

19 P

E -

->

Ab-Lymph

Lymph

10 11

12 13

e-7282

101 102 103 104

KAPPA FITC -->

10

11

02

10

31

04

CD

10 P

E -

->Ab-Lymph

Lymph

10 11

12 13

E-7282

101 102 103 104

LAMBDA FITC -->

10

11

02

10

31

04

CD

10 P

E -

->

53 y/o man, follicular center B-cell lymphoma

Monoclonal Peaks 345.7 bp for FR1 and 280.7 bp for FR2 in unsorted and sorted cell fraction (CD10+/CD45+) not in sorted control cell fraction (CD10-/CD45+) [blue=FR1; black=FR2; green=FR3; red=size standard]

Cell sorting for follicular B-cell lymphoma

Unsorted cells

Sorted cell: CD10+/CD45+ Sorted cell: CD10-/CD45+

Abn_Blasts

6 7

8 9

E-8409

1030507090 120

FSC -->

101

102

103

104

SS

C -

-> MLE-8409

101 102 103 104

SSC -->

101

102

103

104

CD

45 P

erC

P -

->

LymphMono

Myeloid

Blasts

Abn_Blasts

E-8409

101 102 103 104

CD19 PE -->

101

102

103

104

CD

45 P

erC

P -

->

Abn_Blasts_

E-8409

101 102 103 104

CD5 FITC -->

101

102

103

104

CD

19 P

E -

->

Abn_Blasts

6 7

8 9

E-8409

101 102 103 104

CD10 PE -->

101

102

103

104

CD

45 P

erC

P -

->

Abn_Blasts_

E-8409

101 102 103 104

CD20 FITC -->

101

102

103

104

CD

10 P

E -

->

Abn_Blasts

6 7

8 9

E-8409

101 102 103 104

CD34 PE -->

101

102

103

104

CD

45 P

erC

P -

->Abn_Blasts_

E-8409

101 102 103 104

CD22 FITC -->

101

102

103

104

CD

34 P

E -

->

19 y/o female, precursor B-ALL s/p BMT, 0.3% relapse

A)

B)

C)

Patient specific Tumor Clonality Profile

Monitoring Specimen (0.3 % tumor)

Tumor Cell Sort (Monitoring Specimen)

Cell sorting for precursor B-ALL

Conclusion

• Hierarchical approach to diagnostics• Integration of multiple techniques• Establish diagnosis and prognosis with

minimum of tests• Develop strategy for monitoring treatment• Requires close interaction between

hematologist/pathologist/hematological core Lab

Diagnostic Techniques

Morphology; IHC

Flow Cytometry Cytogenetics

FISH

Molecular Analysis;

DNA/RNA

Strategy of development at SCTC

Standard processing & instant report

• BM or PB or tissue sample come in

• Immediate morphology & flow screening (within 24 hours)

• Molecular study (sorted or unsorted) if needed (within 48 hours)

• Cytogenetics or FISH (sorted or unsorted) if needed (within 72 hours)

Member Recruitment & Training

• Technician for morphology & flow cytometry

• Technician for molecular analysis (include all hematological malignancies)

• Flow & sorting training: Hematologics, Seattle

• Honor Dr. Michael Loken by:

Inviting Hematologics affiliate to SCTC

Honor Dr. Loken to be distinguished professor at SCTC

Compassionate support to hematological society in Taiwan

• Cooperate with non-medical centers that can not perform standard hematological study

• Free charge for half a year since 2009

• Establish potential strategical alliances in the long run, including publication and patients referral

• Charge as government controlled insurance fee after solid relationship created

Thank You!