Diagnosis of Acute Leukemia (AL) by Flow Cytometry (FCM) S. Oukid, S. Taoussi, M.T. Abad.

Department of Hematology, EHS CAC Blida, Algeria

ESH : International conference on AN UPDATE ON THE MANAGEMENT

OF HAEMATOLOGICAL MALIGNANCIES

Hammamet, Tunisia – October 28 – 30- 2010

Objectives

A major application of flow cytometry in hematology is to

characterize the malignant cells in acute leukemia for diagnosis, therapeutic decision and assessment of prognosis.

Introduction

                  Flow cytometry has been introduced in our laboratory since 03 years

we analyze :

            - Chronic lymphoproliferative disorders

            - Acute Leukemia

            - Non-Hodgkin Lymphoma : on fine needle aspiration of

              lymph nodes and trituration after biobsy

            - PHN

Acquisition and analysis by flow cytometry on a BD Facs Calibur 4 colors.

Study Methods

- We report results of 142 cases of AL.

   - Study of the cytologic blood smear and bone marrow.

                - Samples for CMF                                                   - Blood                                                   - Bone Marrow

               - Immunophenotyping using a panel of monoclonal Antibody                  broad targeting:                                            * Myeloid populations                                             * B Lymphoid populations

* T Lymphoid populations

Classification by EGIL score to determine the lines B, T, myeloid and biphenotypic

 

Materials : 142 cases

Sex /Age : Children : 44 cases (30,9%) Sex ratio : 0,83 (M= 20/ W=24).

average age : 08 years (03 days - 15 years).

Adults : 98 cases (69%) Sex ratio : 1,72 (M=62/ W=36).

average age : 43,1 years (16 – 82).

20

62

2436

0

20

40

60

80

Children Adults

Men

Women

Study materials           

               1 – Samples for FCM : - Blood sample : 54 cases (38%)                                                                           - Bone  marrow sample : 88 cases (62%)

    2 - Filtration of Bone Marrow

                3 - Count : GB : 94 397/mm3 (3000 - 518 000)

                4 - Dilution: If GB > 20 000/mm3

                5 - Sample preparation (FITC, PE, PerCP Cy5.5, APC)

                6 - Acquisition

                7 - Analysis: - FSC/ SSC                                      - Determining the negative zone                                      - Gating : CD 45                                     - Analysis of various fluorochromes positivity and  intensity.

Results of FCM :

04 Groups : * ALL : 60 cases (42,3%)

* AML : 76 cases ( 53,5%)

* Biphénotypic AL : 06cases (4,2%)

* A L undifferentiated : 05 cas

Results of FCM Group 01 :

ALL : 60 cases

Children Adults Total

ALL B 16 (50%)

16 (50%) 32 (53,4%)

ALL T 13 (46,4%)

15 (53,6%)

28 (46,6%)

Total 29

(48,4%) 31 (51,6)

60

Children : ALL B : 16 cases

Results of FCM 1 : ALL T : 13 cases

ALL : 60 cases Adults : ALL B : 16 cases

AL L T : 15 cases

0

1

2

3

4

5

6

7

8

9

Pro B Pre B Common B Pro T Pre T T Cortical T Mature

1

5

9

1

3

1

7

4

1

9

6

0

1

4

3

5

Adults

Children

Comparison of frequencies of different ALL subtypes (%) with literature

Ref

Pro B % Common % Pre B % B % ALL T %

Children Adults Children Adults Children Adults Children Adults Children Adults

Ludwig 5 11 63 52 16 9 3 3 13 24

CPMC Alger 83

10 7 30 23,3 7,5 2,7 7,5 23 42,5 44

Blida 60 3,4 3,2 31,2 16,2 20,6 29 0 3,2 44,8 48,4

Percentage of ALL T in different countries

12,320

28

43,3746,67 48 50

60

0

10

20

30

40

50

60

70

Results FCM 2 : Children : 12 cases (16%)

AML : 76 cases Adults : 64 cases (84%)

0

2

4

6

8

10

12

14

0

2

4

01

4

10 0

4

2

12

1413

9

1 1

8

Children

Adults

AML :

AML3 : FISH : t (15 ; 17) observed in 03/04 studied

Children Adults

Blida (12) CPMC (12) Blida (64) CPMC (68)

LAM0 0 0 4 2

LAM1 2 3 2 17

LAM2 4 2 12 17

LAM3 0 1 14 6

LAM4 1 6 13 15

LAM5 4 0 9 8

LAM6 1 0 1 1

LAM7 0 0 1 0

LAM 0 0 8 2

Frequency of different markers in AML

Our series TUNISIA Dr E.GOUIDER BELHADJALI

Literature

MPO 73% 93% 75-85%

CD13 76% 80% 70-95%

CD33 89% 67% 70-90%

CD15 58% 42%

CD117 45% 39% 75%

HLA Dr 66% 68% 70-80%

CD34 62% 49% 60%

Résults of FCM 3

Biphénotypic LA : - 03 Children - 03 Adults

Cases N° 2 : karyotype and FISH : Philadelphia chromosome positive.

                  Cases N° 5 : karyotype and FISH : trisomy 21.

Cases Age Cytological FCM AM L ALL T ALL B

1 12 LAM4 /M5 LAM2 + LAL T 5,5 4,5

2 17 LA LAM + LAL B 4 4

3 30 LA LAM + LALT 3,5 6

4 65 MDS T LA LAL B + LAL T 4,5 4

5 3 J LA LAM + LAL T 6 6

6 03 LA LAM5 + LAL B 3 4

FCM : ALL Pre B

FCM : AL L T cortical

Conclusions

Recently introduced into our environment, FCM has been a major

        contribution to the diagnosis of AL and it usefully complements

the cytological and cytochemical traditionally existing in our

laboratory and was often decisive in the statement of the type of AL

and thus the therapeutic orientation.

 

References : 1 Flow Cytometry in Hematopathology A Visual Approach to Data Analysisand Interpretation Doyen

Nguyen, MD Lawrence W. Diamond, MD Raul C. Braylan, MD.

    2 British Committee for Standards in Haematology, Revised guidelines for immunophenotyping

of acute leukemia and chronic lymphoproliferative disorders, Clin. Lab. Haematol., 24, 1–13, 2002

3 Immunophénotypage des hémopathies malignes Bernard Husson Centres Hospitaliers Jolimont –

Lobbes Site de Jolimont Laboratoire de Biologie Clinique - Département Hématologie Unité de CMF

7100 – Haine-Saint-Paul.

4 Hematopathology : Farmarz Naeim et Col

5 Ludwig W. D ; Reiter. A, Loffler. H, and al

Immunophenotypic Features of childhood and Adult Acute lymphoblastic Leukemia ( ALL) :

Experience of the German Multicentre Trials ALL – BFM and GMALL

Leukemia and lymphoma , 1994, Vol 13, Supp 1 , pp 71 – 76.

6 Trabzi – Azeli Anissa

Application de l’immunophénotypage à la classification des leucémies aigues

These pour le diplôme de docteur en science médicales

Juillet 1999, Faculté de Médecine d’Alger

.