1. 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

2. 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.

3. 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.

4. 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

5. 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).

6. 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.

7. 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

8. 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

9. 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

10. Comparison of frequencies of different ALL subtypes (%) with literature Ref Pro B % Common % Pre B % B % ALL T % Children Adults Children AdultsChildren Adults Children Adults Children Adults Ludwig 5 11 63 52 16 9 3 3 13 24 CPMC Alger 83 10 7 3023,3 7,52,7 7,5 23 42,5 44 Blida 60 3,4 3,2 31,2 16,2 20,6 29 0 3,2 44,848,4

11. Percentage of ALL T in different countries

12. Results FCM 2 : Children : 12 cases (16%) AML : 76 cases Adults : 64 cases (84%)

13. AML : AML3 : FISH : t (15 ; 17) observed in 03/04 studied Children Adults Blida (12)CPMC (12) Blida (64)CPMC (68) LAM0 00 42 LAM1 23 217 LAM2 42 1217 LAM3 01 146 LAM4 16 1315 LAM5 40 98 LAM6 10 11 LAM7 00 10 LAM 00 82

14. 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%

15. 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. CasesAge Cytological FCM AM L ALL T ALL B 1 12 LAM4 /M5LAM2 + LAL T5,54,5 217 LALAM + LAL B44 330 LALAM + LALT3,56 465 MDS T LALAL B + LAL T4,54 53 J LALAM + LAL T66 603 LALAM5 + LAL B34

16. F CM : ALL Pre B

17. FCM : AL L T cortical

18. 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.

19. 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.