Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte Population

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1 Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte Population Amr Rajab BSc, MLT, QCYM (ASCP) Flow cytometry Technical Specialist LifeLabs 100 International Blvd. Toronto, ON M9W 6J6 T Ext F C E Amr.Rajab@LifeLabs.com

2 Disclosure Have no conflict of interest to disclose for this presentation

3 Topics Development & validation of the of lymphoproliferative disorder screening tube (LPD-ST) Technology Transfer Using Harmonemia Concept Pattern recognition and empty spaces analysis

4 GOAL To developed a LPD-ST with the aim to provide comprehensive immunophenotyping of lymphocyte subsets with minimal need for additional testing

5 Why the LPD-ST? 10-color lymphoproliferative disorder (LPD) panel University Health Network, Toronto, Canada Mahdi T, Rajab A, Padmore R, Porwit A. Characteristics of lymphoproliferative disorders with more than one aberrant cell population as detected by 10-color flow cytometry. Cytometry Part B, Clinical cytometry

6 Why the LPD-ST? ~ 5000 samples (PB, BMA, Lymph node cell suspensions and cytology) per year analyzed using LPD panel 80% of LPD cases we diagnosed in 2014 in blood are CLL/MBL.

7 MYELOID/ LYMPHOID SCREENING TUBE 10 COLORS, 14 ANTIBODIES Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasiarelated features with one 10-color 14-antibody screening tube. Cytometry Part B, Clinical cytometry. 2015;88(4):

8 Similar approach has been applied by other groups in 4- color, 6-color, 8-color and 10-color settings Quijano S, etc. Spanish Group for the Study of CNS Disease in NHL.Identification of leptomeningeal disease in aggressive B-cell non-hodgkin's lymphoma: improved sensitivity of flow cytometry. J Clin Oncol Mar 20;27(9): Preijers FW etc. B. OMIP-010: a new 10-color monoclonal antibody panel for polychromatic immunophenotyping of small hematopoietic cell samples. Cytometry A Jun;81(6): Costa ES, etc. A new automated flow cytometry data analysis approach for the diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with absolute lymphocytosis. Leukemia Jul;20(7): van Dongen JJ, etc. EuroFlow Consortium (EU-FP6, LSHB-CT ). EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia Sep;26(9): Hedley BD, Keeney M, Popma J, Chin-Yee I. Novel lymphocyte screening tube using dried monoclonal antibody reagents. Cytometry B Clin Cytom May 4

9 Materials and Methods Development of the LPD screening tube (LPD-ST) Ab LPD Screening Tube ver. I FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 PB KO CD4 + Kappa CD8 + Lambda CD3 + CD14 CD5 CD20 + CD56 CD10 CD19 CD200 CD57 +CD23 CD45

10 Materials and Methods Development of the LPD screening tube (LPD-ST) Pilot study: Blood (n=10), BMA (n=3) and tissue samples (n=2). 3 normal specimens, one reactive blood sample with increased γδ T- cells, 5 CLL, 1 CD5-/CD10- B-cell LPD, 1 large B-cell lymphoma, 1 Follicular lymphoma, 1 hairy cell leukemia (HCL), 1 T-cell Large granular lymphocytosis (LGL) and 1 angioimmunoblastic T-cell lymphoma (AITL).

11 Materials and Methods Development of the LPD screening tube (LPD-ST) Results: Final diagnosis could be rendered in 10 cases (67%) including 3 normal cases, 5 CLL, 1 large B-cell and 1 follicular lymphoma. 5 cases (33%) required further investigation including a T-cell LGL, a CD5-/CD10- B-cell LPD, AITL, HCL and one case with increased γδ T-cells. ICCS 2015, Abstract #88, Poster Session 2 on Monday, October 12, 2015 Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte population Amr Rajab, Graeme Quest, Jennifer Leung, Anna Porwit Department of Laboratory Hematology, University Health Network Toronto, ON, Canada

12 Caveats Do we need two CLL markers in the tube (i.e. CD23 & CD200)? Dim CD200 APC-A750 expression in CLL cases

13 KO CD45 Materials and Methods Development of the LPD screening tube (LPD-ST) Ab LPD Screening Tube ver. II FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 PB CD4 + Kappa CD8 + Lambda CD3 + CD14 CD5 CD20 + CD56 CD10 CD19 CD38 CD57 +CD23

14 Caveats CD38 APC-A750(ST) expression is dim in comparison to CD38 PC5.5 (LPD) ST- v2 10C UHN-LPD

15 January 2016 Materials and Methods Development of the LPD screening tube (LPD-ST) LPD Screening Tube ver. III

16 Materials and Methods Sample preparation Wash cells x 2 with warm PBS Re-suspend cells in 1% BSA in PBS Add Ab cocktail to 100ul (5 x cells) of sample Incubate for 15 minutes in dark at RT. Lyse with 1mL VersaLyse (BC ref. IM3648) plus 25 ul IOTest 3 Fixative (BC ref. IM3515) Wash and suspend in 1 ml PBS and 12.5 ul IOTest 3 Fixative (BC ref. IM3515) 30-50,000 events were acquired on the Navios Flow cytometer In paucicellular samples, all available cells were acquired

17 Materials and Methods Instrument setup FSC/SSC settings were optimized using a whole blood sample lysed with VersaLyse FL1-FL9 were optimized using blood samples stained with CD4, CD5 or CD3 FL10 was optimized using blood samples stained with CD45 KO

18 Materials and Methods Optimizing instrument compensation The compensation matrix was calculated using Kaluza software (BC) and list-mode files (LMD) from Versacomp (BC) beads stained with the antibodies of the panel. Compensation for the LPD screening tube was adjusted using normal PB stained with the full LPD screening panel.

19 Materials and Methods Instrument QC FlowCheck Pro (Beckman Coulter) FlowSet Pro (Beckman Coulter) Compensation verifier

20 Sample analysis and reporting

21 Sample analysis and reporting

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25 Paucicellular Cytologic Specimens (1) CSF WBC= 5 x10 6 /L 1,315 events collected Reactive lymphoid T-cell population

26 Paucicellular Cytologic Specimens (2) CSF WBC= 2 x10 6 /L 3,262 events collected B-cell LPD

27 Paucicellular Cytologic Specimens (3) CSF WBC= 22 x10 6 /L 6,560 events collected ATLL

28 Validation cohorts for the LPD screening tube Data LPD-ST vs 10C UHN-LPD Toronto LPD-ST vs 8C LUH-LPD Lund No of samples Blood Bone marrow Lymph node suspension 6 5 FNA 4 9 Body fluids 3 3 Other 1* 0 Diagnosis Normal/reactive 9 15 CLL/SLL/MBL 14 8 MCL 2 2 Germinal center NHL 10 4 HCL 3 2 Other B lymphoma 9 8 Aberrant T-cell population 8 0 Other 5** 4*** Comparison results with routine LPD panel Concordant 100% 98% Discordant 0% 2% Require additional phenotyping 22 (37%) 10 (23%) This study was considered as validation of a new protocol and as such satisfied requirements of the Research Ethics Board of the University Health Network and Lund University Hospital.

29 Validation cohorts for the LPD screening tube Frequency of additional testing after LPD-ST and EuroFlow LST tubes included in validation study in Toronto Data LPD-ST EuroFlow LST Diagnosis Cases requiring additional phenotyping Normal/reactive (n= 9) 0 0 CLL/SLL/MBL (n=14)# 0 14 MCL (n= 2) 0 2 Germinal center NHL (n=10) 0 10 HCL (n=3) 3 3 Other B lymphoma (n=9) 9 9 Aberrant T- cell population (n=8) 5 8 Other (n=5 ) 5 5 Total (n=60) 22 (37%) 51 (85%) Van Dongen JJ, Lhermitte L, Bottcher S, Almeida J, van der Velden VH, Flores-Montero J, Rawstron A, Asnafi V, Lecrevisse Q, Lucio P, Mejstrikova E, Szczepanski T, Kalina T, de Tute R, Bruggemann M, Sedek L, Cullen M, Langerak AW, Mendonca A, Macintyre E, Martin-Ayuso M, Hrusak O, Vidriales MB, Orfao A, EuroFlow C. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012;26(9):

30 Implementation of LPD-LST in Lund, Sweden (Oct Dec 2016) Samples N=649 No aberrant population N= 425 Monoclonal B-cell population N= 204 Aberrant T-cell population N= 20 No extra testing B1 (1) B2 (2) B1 and B2 Extra T (3) (1) B1 : lambda, kappa, CD123, C11c, CD20, CD103, CD19, CD25, IgM, CD45 (2) B2 : CD81, CD79b, CD23, CD11c, CD5, CD200, CD19, CD43, CD22, CD45 (3) Extra T : TCRab, TCRgd, CD3, CD4, CD2, CD26, CD7, CD16, CD8, CD45 Or CD52, TCRab, CD3, CD4, CD2, CD30, CD7, CD25, CD8, CD45 -

31 Technology Transfer Using Harmonemia Concept Compensation LifeLabs Lacombe F,et al. Harmonemia: a universal strategy for flow cytometry immunophenotyping. A European LeukemiaNet WP10 study. Leukemia. 2016, 30(8):

32 Technology Transfer Using Harmonemia Concept Red events: UHN Blue events: LifeLabs

33 Pattern Recognition and Empty Spaces Analysis

34 Pattern Recognition and Empty Spaces Analysis

35 B-cell population CD20/CD38 Normal patterns BMA PB LNB

36 B-cell population CD20/CD38 Abnormal patterns CLL- PB CLL- BM MCL- BM DLBCL- BM FCL- PB HCL- PB CLL & MZL- PB LPL- BM

37 B-cell population CD10 Patterns Normal BM FCL- BM staging

38 Plasma cells CD45 v CD20/CD56 Normal BM ST-MGUS MM- panel

39 Take Home Messages The developed LPD-ST makes it possible to render a final flow cytometry report in more than ~75% of the samples submitted for LPD immunophenotyping. The LPD-ST can reduce TAT The LPD-ST can reduce costs by up to 30% Pattern recognition analysis approach improve analysis sensitivity Following the Harmonemia concept allowed an easy transfer of technology between laboratories

40 ACKNOWLEDGEMENTS Anna Porwit, MD, PhD Lund University, Lund, Sweden Olof Axler, MD, PhD Region Skåne, Klinisk Patologi, Lund, Sweden Miranda Wozniak MD FRCPC LifeLabs, Toronto, Canada Jennifer Leung University Health Network, Toronto, Canada

41 ACKNOWLEDGEMENTS Hematopathology/ Cytopathology Staff at UHN Dr. David Barth, Dr. Hong Chang, Dr. Jan Delabie, Dr. Rumina Musani, Dr. Anne Tierens, Dr. Emina Torlakovic, Dr. Graeme Quest, Dr. Scott Boerner, Dr. William Geddie, Dr. Gilda Santos and Dr. Hyang Ko Hematopathology/ Cytopathology Staff at the Department of Pathology, Lund University Hospital Dr. Mats Ehinger, Dr. Sigmund Dawiskiba, Dr.Henryk Domanski, Dr. Ann Hultquist, Dr. Anna Kwiecinska and Dr. Despoina Violidaki Hematopathology Staff at the Department of Pathology, LifeLabs Dr. Afaf Erfaei Flow cytometry laboratory staff at UHN Jessie Leung, Jordan Ngo, Josello Mandawe, Liz Valenzuela, and Sajid Dewji Flow cytometry laboratory staff at LUH Filipa Bild, Ulla Fält, Anette Ingemansson, Jasmina Orlovac, Nina Pries, Melisa Santa, Emma Skoog, Kerstin Torikka

42 Thank you for your attention