CD20 /CD34 /CD10 / CD22 /CD45 /CD19 CD7 /CD5 /CD13 /CD33 /CD45 /CD19 25

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1 J China Pediatr Blood Cancer June 2012 Vol 17 No. 3 B B B-ALL LAIP CD20 /CD34 /CD10 / CD22 /CD45 /CD19 CD7 /CD5 /CD13 /CD33 /CD45 /CD19 25 B-ALL FSC /SSC CD45 /SSC CD19 /SSC CD CD19 strong CD10 strong CD34 strong CD22 strong CD19 + /CD45 -/dim + CD19 + /CD34 + /CD38 - CD19 + /CD13 + CD19 + /CD15 + CD19 + /CD LAIP LAIP 1 LAIP 4 2 LAIP 8 3 LAIP 5 4 LAIP 3 5 LAIP 3 10 CD10 strong CD34 strong CD22 strong CD45 -/dim + CD19 + /CD13 + CD19 + /CD33 + CD19 + /CD34 + / CD20 + CD19 + /CD34 + /CD /CD34 + /CD10 dim /CD45 -/dim + /CD34 -/dim /CD10 - / CD20 - /CD34-11 LAIP LAIP 1 LAIP 4 2 LAIP 5 3 LAIP 3 4 LAIP 6 5 LAIP 5 B-ALL LAIP B-ALL CR MRD FSC /SSC SSC /CD45 SSC /CD19 CD19 + LAIP 6 CR MRD B The detection of leukemia-associated immune phenotypes by six-color flow cytometry in pediatric B-lineage acute lymphoblastic leukemia LIU Guangming WU Shaoqing SUN Xin. Guangzhou Women and Children's Medical Center Guangzhou China Corresponding author SUN Xin doctorsunxin@ hotmail. com Abstract Objective To compare the differences between three-color and six-color flow cytometry FCM on detecting leukemia-associated immune phenotype LAIP in pediatric B-lineage acute lymphoblastic leukemia B-ALL. Methods Twenty-five patients diagnosed as B-ALL by three-color FCM were detected by two groups of six-color antibody combinations CD20 /CD34 /CD10 /CD22 /CD45 / CD19 and CD7 /CD5 /CD13 /CD33 /CD45 /CD19. Naive cell populations and CD19 + cell populations were analyzed on multiparametric two-dimensional point maps through FSC /SSC CD45 /SSC and CD19 / YB B doctorsunxin@ hotmail. com

2 J China Pediatr Blood Cancer June 2012 Vol 17 No SSC gating. Results Nine LAIPs were detected using 23 antibodies by three-color FCM including CD19 strong CD10 strong CD34 strong CD22 strong CD45 dim + / - CD19 + /CD34 + /CD38 - CD19 + /CD13 + CD19 + /CD15 + and CD19 + /CD33 +. Twenty-three cases 23 /25 had LAIPs detected of which 4 cases had 1 LAIP 8 cases had 2 LAIPs 4 cases had 3 LAIPs 3 cases had 4 LAIPs and 3 had 5 LAIPs. By contrast 11 LAIPs were detected using 10 antibodies by six-color FCM including CD10 strong CD34 strong CD22 strong CD45 - /dim CD19 + /CD13 + CD19 + /CD33 + CD19 + /CD34 + /CD20 + CD19 + /CD34 + /CD /CD34 + /CD10 dim /CD45 - /dim + /CD34 - /dim + and 19 + /CD10 - / CD20 - /CD34 -. Twenty-three cases 23 /25 had LAIPs detected of which 4 cases had 1 LAIP 5 cases had 2 LAIPs 3 cases had 3 LAIPs 6 cases had 4 LAIPs and 5 had 5 LAIPs. Conclusions High proportions of LAIPs can be detected by FCM in B-ALL patients. Though fewer number of antibodies was used more LAIPs were detected by six-color FCM than those by three-color FCM. Besides six-color FCM can detect six antibodies at the same time in one tube and adjust the antibody combination flexibly which makes MRD detection more reasonable and convenient. Key words Six-color flow cytometry Leukemia-associated immunophenotypes Leukemia acute B-cell permeabilizing solution BD acute lymphoblastic leukemia ALL 1 complete remission CR minimal residual disease MRD flow cytometry FCM CR MRD B B-ALL FCM leukemia associated immunophenotype LAIP FCM B-ALL LAIP MRD B-ALL ~ FCM FACScantoⅡ FCM 1 2 lysing solution FITC PE PerCP 1 γ1 γ2 CD45 2 CD7 CD5 CD45 3 CD4 CD8 CD45 4 CD20 CD22 CD45 5 CD10 CD19 CD45 6 HLA - DR CD13 CD45 7 CD14 CD64 CD45 8 CD15 CD33 CD45 9 CD38 CD34 CD45 10 SIgM GlyA CD45 11 MPO * cycd79a * CD45 12 TdT * - CD45 13 cycd3 * - CD45 FITC PE PerCP * - 2 FITC PE PE-Cy7 APC PerCP APC-Cy7 1 γ1 γ2 γ3 γ4 γ5 γ6 2 CD20 CD34 CD10 CD22 CD45 CD19 3 CD7 CD5 CD13 CD33 CD45 CD19 APC Cy7 7

3 FCM 1 CD10 Ⅳ CD22 20 μledta-k2 CD19 CD45 CD20 PBS 10 6 /ml50 μl 4 1 D G J L O Q 2 E H J 4 20 min 2 L N P R T V Y Z 3 PBS 1 2 FCM 25 B-ALL 20 μledta-k2 PBS 23 LAIP 1 LAIP 10 6 /ml50 μl 4 2 LAIP 8 3 LAIP min 2 LAIP 3 25 CD μl 10 min 4 20 μl 15 min 5 PBS 5 CD34 6 CD22 24 h 3. 2 FCM CD38-1 IgG1-PE-Cy7 IgG1-APC-Cy7 CD10- PE-Cy7 CD22-APC CD19-APC-Cy7 CD13-PE-Cy7 CD19 strong CD10 strong CD34 strong CD22 strong CD45 - /dim CD19 + /CD34 + /CD38 - CD19 + /CD13 + CD19 + / CD33-APC 5 μl CD15 + CD19 + /CD μl 4 FACScantoⅡ 1 BD BD FACSdiva 3 FCM LAIP FSC /SSC CD45 /SSC 1 LAIP 4 2 LAIP 5 3 LAIP 3 4 CD45 LAIP 6 5 LAIP 5 FCM SSC / FCM CD10 6 CD34 FSC /SSC CD45 /SSC / 2 CD22 15 CD45 CD19 /SSC CD CD J China Pediatr Blood Cancer June 2012 Vol 17 No. 3 Ⅲ CD20 CD45 9 CD33 8 CD15 3 CD10 13 CD19 2 CD45 15 CD34 + / 9 LAIP FCM LAIP 3 LAIP CD33 5 CD45 /SSC CD19 /SSC CD % 10% 6 CD19 + /CD34 + /CD CD19 + /CD34 + /CD /CD34 + / 10-4 B-ALL CD10 dim /CD45 - /dim + /CD34 - /dim + 1 LAIP 19 + /CD10 - /CD20 - /CD34 - FCM CD10 strong CD34 strong CD22 strong CD45 - /dim CD19 + /CD B CD19 + /CD33 + CD19 + /CD34 + /CD20 + CD19 + / B 4 Ⅰ CD34 CD34 + /CD CD19 + /CD34 + /CD10 dim + CD19 + / HLA-DR TDT CD10 CD19 CD45 CD22 CD45 - /dim + /CD34 - /dim + CD19 + /CD10 - /CD20 - /CD34 - Ⅱ CD19 CD45 CD10 11 LAIP FCM LAIP 4 CD34 TDT CD20 CD22 LAIP 2

4 J China Pediatr Blood Cancer June 2012 Vol 17 No

5 FCM LAIP % LAIP % CD19 strong* 5 20 CD10 strong* CD34 strong* 6 24 CD22 strong* 2 8 CD45 - /dim# CD38 - # 1 4 CD19 + /CD CD19 + /CD CD19 + /CD * # 4 LAIP FCM LAIP % % CD10 strong* CD34 strong* 6 24 CD22 strong* 2 8 CD45 - /dim# CD19 + /CD CD19 + /CD CD19 + /CD34 + /CD CD19 + /CD34 + /CD CD19 + /CD34 + /CD10 dim CD19 + /CD45 - /dim + /CD34 - /dim CD19 + /CD10 - /CD20 - /CD * # 4 CD22 B-ALL CD45 CD19 CD10 CD34 CD22 CD20 B- ALL LAIP 8 B-ALL CR MRD CD7 /CD5 /CD13 /CD33 / LAIP LAIP CD45 /CD19 B-ALL B-ALL CD13 CD33 13 T 5-6 CD7 CD5 7 FCM MRD CD13 CD33 CD7 CD5 CD19 CD45 LAIP B-ALL LAIP CR MRD J China Pediatr Blood Cancer June 2012 Vol 17 No MRD LAIP 3 3 LAIP 44% FCM 25 B-ALL MRD LAIP 3 3 LAIP 56% FCM 3 3 LAIP FCM 16% FCM FCM CD45 -/dim + CD10 strong CD34 strong CD22 strong 7-10 CD19 + /CD13 + CD19 + /CD33 + CD19 + CD19 + /CD34 + /CD20 + CD19 + /CD34 + /CD CD19 + /CD34 + /CD10 dim + CD19 + /CD45 - /dim /CD34 - /dim CD19 + /CD10 - / CD20 - /CD34-5 FCM FCM CD19 FCM FCM CD19 APC-cy7 FCM CD19 CD7 B-M B-T B-M-T FCM CD20 /CD34 / CD10 /CD22 /CD45 /CD19 B-ALL LAIP CD19 CD10 CD20 CR MRD FCM B-ALL CD15 CD33

6 J China Pediatr Blood Cancer June 2012 Vol 17 No CD13 CD13 CD15 CD7 CD5 CD34 + /CD38 - CD7 CD5 CD34 CD38 MRD 6. Szezepanski T FCM 14 Lab Med 2006 FCM 7. MRD 15 FCM LAIP MRD FCM LAIP MRD CD20 /CD34 /CD10 /CD22 /CD45 / immunophenotypes LAIP are observed in 90% CD19 CD7 /CD5 /CD13 /CD33 /CD45 /CD19 25 B-ALL LAIP FCM FCM LAIP CR MRD FCM FCM CD45 CD CR MRD FCM FSC /SSC CD45 /SSC CD19 /SSC CD19 + LAIP CR FCM MRD FCM 6 CR MRD 1. Mejstríková E Fronková E Kalina T et al. Detection of residual b precursor lymphoblastic leukemia by uniform gating flow cytometry. Pediatr Blood Cancer Irving J Jesson J Virgo P et al. Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. Haematologica Bene MC Castoldi G Knapp W et al. Proposals for the immunological classification of acute leukemia. European Group for the Immunological Characterization of Leukemia EGIL. Leukemia Stewart CC Stewart SJ. Immunophenotyping. Curr Protoc Cytom 2001 Chapter 6 Unit Campana D. Determination of minimal residual disease in leukemia patients. Br J Haematol van der Velden VH van Dongen JM. Flowcytometric immunophenotyping of normal and malignant lymphocytes. Clin Chem B-ALL B Griesinger F Pirò-Noack M Kaib N et al. Leukaemia-associated of adult and childhood acute lymphoblastic leukaemia detection in remission marrow predicts outcome. Br J Haematol Dworzak MN Fröschl G Printz D et al. Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood Garc a Vela JA Monteserin MC Delgado I et al. Aberrant immunophenotypes detected by flow cytometry in acute lymphoblastic leukemia. Leuk Lymphoma Ciudad J San Miguel JF L pez-berges MC et al. Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol Camapana D Coustan-Smith E. Minimal residual disease studies by flow cytometry in acute leukemia. Acta Haematol Robillard N Cav H M chinaud F et al. Four-color flow cytometry bypasses limitations of Ig / TCR polymerase chain reaction for minimal residual disease detection in certain subsets of children with acute lymphoblastic leukemia. Haematologica Wehner S Soerensen J Schwabe D et al. 10-Parameter flow cytometry as a new tool to improve diagnosis and mrd follow-up of acute leukemias. Klin Padiatr Olaru D Campos L Flandrin P et al. Multiparametric analysis of normal and postchemotherapy bone marrow Implication for the detection of leukemia-associated immunophenotypes. Cytometry B Clin Cytom Björklund E Gruber A Mazur J et al. CD34 + cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections application for MRD detection in leukemia patients. Int J Hematol B B

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