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SCREENING TUBES

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LUNCHEON WORKSHOP: SCREENING TUBES. 30th Annual meeting of the International Clinical Cytometry Society, 2015. Denver, CO USA

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SCREENING TUBES

  1. 1. Ten-Color, 14 Antibody Flow cytometry (FCM) Screening Tube for Lymphoproliferative Disorders and Myelodysplasia (MDS) – Related Changes in Bone Marrow Samples EMAIL: Amr.Rajab@uhn.ca Amr Rajab, BSc, MLT, QCYM (ASCP) Clinical Flow Cytometry Laboratory Laboratory Medicine Program Toronto General Hospital/University Health Network Ontario, Canada
  2. 2. TOPICS • Development • Validation for LL/AL screening (Phase 1)  Optimization of sample preparation  Titration of antibodies  Verification of cocktail stability  Instrument setup and compensation  Comparison study  Analysis  Cases • Validation for MDS Screening (Phase 2) • Current & Future Developments
  3. 3. GOAL  Develop a 10 Color, 14 antibody Flow cytometry (FCM) screening tube that correctly identifies lymphoproliferative disorders and myelodysplasia (MDS) – related changes in bone marrow samples
  4. 4. FLOW CYTOMETRY LAB - UHN Section A Section B Tests performed AL & LL CD34, CD4/CD8 & PNH Number of samples- 2014 Referred in samples 8143 40% 7174 40% Instruments Two 3L 10C Navios Two 1L 5C FC500 Number of Technologists 7 1 Total number of samples processed during 2014= 15317
  5. 5. Why the new screening tube (ST)? (1) 1. To be used for immunophenotyping of hypocellular samples, dry taps, FNAs, and body fluids (e.g. CSF). 2. To be used for “referred in” samples (mainly bone marrow aspirates and peripheral blood samples) not accompanied by sufficient clinical information, provisional diagnosis, or prepared smears (50% of referred samples)
  6. 6. Why the new ST? (2)
  7. 7. Why the new ST? (3) • These samples were usually screened using the UHN lymphoma protocol (2 tubes, 20 antibodies). • The outcome determined how much further investigation was needed. • 50% do not show any pathological population. Tube FITC PE ECD PC5.5 PC7 APC APC- AF700 APC- AF750 PB KO B-cell kappa lambda CD19 CD38 CD20 CD34 CD23 CD10 CD5 CD45 T-cell CD57 CD11c CD8 CD3 CD2 CD56 CD7 CD4 CD5 CD45
  8. 8. Similar approach has been applied by other groups in 4- color, 6-color, 8-color and 10-color settings – Focusing on lymphoid population • 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. 2009 Mar 20;27(9):1462-9. • Preijers FW…etc. B. OMIP-010: a new 10-color monoclonal antibody panel for polychromatic immunophenotyping of small hematopoietic cell samples. Cytometry A. 2012 Jun;81(6):453-5. • 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. 2006 Jul;20(7):1221-30 • van Dongen JJ,…etc. EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012 Sep;26(9):1908-75. • Hedley BD, Keeney M, Popma J, Chin-Yee I. Novel lymphocyte screening tube using dried monoclonal antibody reagents. Cytometry B Clin Cytom. 2015 May 4
  9. 9. SCREENING TUBE 10 COLORS, 14 ANTIBODIES • Enumerate Major Populations: • Blasts • B- lymphocytes, T- lymphocytes, • NK cells • Monocytes • Neutrophils • Enumerate Sub-populations: • T-Helper cells, T- Suppressor cells • CD34+/CD19+ B- cell progenitors • CD34+/CD33+ myeloid progenitors • CD33+/CD10+ mature granulocytes • B-cell clonality status: Kappa Lambda • B-cell maturation status CD20/CD10 expression.
  10. 10. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) • Optimization of sample preparation • Titration of antibodies • Verification of cocktail stability • Instrument setup and compensation • Comparison study
  11. 11. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Selection of lysing solution)
  12. 12. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) 1. Sample preparation • Wash cells x 2 with warm PBS • Resuspend cells in 1% BSA in PBS • Adjust cells to 5 x109 /L • Add Ab cocktail to 100ul (5 x105 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)
  13. 13. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Titration of antibodies) All antibodies titrated to determine the optimal concentrations Ab PN Titer (uL) FITC CD4 IM0448U 10 Kappa A64828 10 PE CD8 IM0452U 10 Lambda A64827 10 ECD CD3 IM2705U 5 CD14 IM2707U 3 PC5.5 CD33 A70198 3 PC7 CD20 IM3629U 5 CD56 A51078 10 APC CD34 IM2472U 5 A700 CD19 A78837 3 A750 CD10 A89310 5 PB CD5 A82790 5 KO CD45 A96416 5  Optimal signal-to-noise separation  Minimize background fluorescence
  14. 14. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Verification of cocktail stability)  Antibodies cross reactivity  Stability over two weeks
  15. 15. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (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 • FL10 was optimized using blood samples stained with CD45 KO
  16. 16. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Optimizing Instrument Compensation) • The compensation matrix was calculated using Kaluza software (BC) and list- mode files (LMD) from blood samples stained with single CD45 antibodies for each fluorescence channel . • Compensation for the screening tube was adjusted using normal BMA stained with the full screening panel.
  17. 17. • FlowCheck Pro (Beckman Coulter) • FlowSet Pro (Beckman Coulter) • Compensation verifier VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Instrument QC)
  18. 18. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) (Comparison study) Study Overview The validation was done using 32 patient samples The samples included 17 BMA, 11 blood samples (PB), 3 lymph node cell suspensions (LNB) and one body fluid (BF, pleural fluid). 16 female and 16 male patients, age 25-93(mean age 57) Diagnosis included 7 normal/reactive samples (3 BMA, 1 BF and 3 PB), 12 samples with B or T lymphoproliferative disorders (LPD), 1 Multiple myeloma (MM), 6 acute leukemias (AML/ALL), 5 MDS and one paroxysmal nocturnal hemoglobinuria (PNH).
  19. 19. KALUZA ANALYSIS TEMPLATE
  20. 20. NORMAL BM SAMPLE Living Cells = NOT Debris
  21. 21. NORMAL BM SAMPLE (BLAST ANALYSIS)
  22. 22. Dim CD45+CD45-ve NORMAL BM SAMPLE (CD45-/dim ANALYSIS)
  23. 23. NORMAL BM SAMPLE (B-CELL ANALYSIS)
  24. 24. NK = Lymphs AND (NOT CD19+) NORMAL BM SAMPLE (T-CELL/ NK ANALYSIS)
  25. 25. NORMAL BM SAMPLE (Grans & Monos Aanalysis)
  26. 26. KALUZA SELECTED STATISTICS
  27. 27. EXPORT KALUZA STATISTICS
  28. 28. SCREENING TUBE REPORT
  29. 29. FLOW CYTOMETRY LAB - UHN Paperless reporting All staff members have access to analysis software and share drive Shared drive storage/reporting
  30. 30. Lymphoproliferative neoplasm B- CLL FCL T-LPD
  31. 31. Plasma Cell Neoplasms ST MM
  32. 32. Paucicellular Cytologic Specimens (1) CSF WBC= 5 x106/L 1,315 events collected Reactive lymphoid T-cell population Rajab A, Boerner S, da Cunha Santos G, Geddie W, Ko HM, Porwit A. Ten-Color 14 antibody flow cytometry panel for immunophenotyping of paucicellular cytologic specimens. Cytometry Part B: Clinical Cytometry, 2013, Oct 25, 84, (6): 419 (abstract).
  33. 33. CSF WBC= 2 x106/L 3,262 events collected B-cell LPD Paucicellular Cytologic Specimens (2)
  34. 34. CSF WBC= 22 x106/L 6,560 events collected ATLL Paucicellular Cytologic Specimens (3)
  35. 35. Acute Leukemia AML B-ALL T-ALL
  36. 36. PNH -POSITIVE CD14 NEGATIVE PNH- MONOCYTE CLONE
  37. 37. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1- RESULTS) (The comparison study showed 100% concordance)
  38. 38. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) SUMMARY (1) • During August 2012 – December 2013 we have analyzed 1025 samples using ST. In 94% of the cases we could render a final FCM report and 6% of cases required further immunophenotyping. • The Screening Tube is capable of enumerating major blood and bone marrow cell populations. • It can detect aberrant antigen expression on B-cells (CD5, CD10, CD20, Kappa/Lambda) • Establish B-cell clonality status • Establish B-cell maturation status (CD10/CD20) • Detecting aberrant expression on T-cells (CD3, CD5, CD4, CD8, & CD10)
  39. 39. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1) SUMMARY (2) • The Screening Tube can detect abnormal Myeloid populations • The Screening Tube can reduce TAT • The Screening Tube reduces the need for Hem- Path consultation • The Screening Tube can reduce costs by up to 30% • Unable to detect minor clonal plasma cell population
  40. 40. Flow cytometry has recently been recommended as a significant parameter in the integrated diagnostics of patients with cytopenia and suspected MDS • Malcovati L, Hellstrom-Lindberg E, Bowen D, Ades L, Cermak J, del CC, et al. Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet. Blood 2013 Oct 24;122(17):2943-64. • Porwit A. Role of flow cytometry in diagnostics of myelodysplastic syndromes--beyond the WHO 2008 classification. Semin Diagn Pathol 2011 Nov;28(4):273-82. • Porwit A, van de Loosdrecht AA, Bettelheim P, Brodersen LE, Burbury K, Cremers E, et al. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Leukemia 2014 Jun 12. • Porwit A. Is There a Role for Flow Cytometry in the Evaluation of Patients With Myelodysplastic Syndromes? Curr Hematol Malig Rep. 2015 Sep;10(3):309-17 VALIDATION FOR MDS SCREENING (PHASE 2 background)
  41. 41. VALIDATION FOR MDS SCREENING (PHASE 2 background) Study enrolled patients from 6 institutions (Europe, USA, Japan) 417 low grade MDS (with <5% blasts) 380 pathological controls with non-clonal cytopenias FCM analysis was performed with various antibody combinations and various flow cytometers Haematologica 2012; 97(8)
  42. 42. VALIDATION (PHASE 2 background) FCM score > 2 was significantly associated with MDS diagnosis High values were associated with multilineage dysplasia, transfusion dependency and high-risk cytogenetics Della Porta et al. 2012
  43. 43. FCM- score for MDS using our screening tube (NORMAL BM) Gated Myeloblasts in all nucleated cells: 1.6% (If ≥2%, FCM score= 1) Gated B-cell progenitor in CD34+ cells: 5.3% (If ≤5%, FCM score=1) Lymphocyte to myeloblast CD45 MFI ratio= 6.4 (If ≤4 or ≥7.5, FCM score=1) Granulocyte to lymphocyte SSC mode ratio: 8 (If ≤6, FCM score=1) Total FCM score= 0
  44. 44. FCM- score for MDS (MDS case) Gated Myeloblasts in all nucleated cells: 3.3 (If ≥2, FCM score= 1) Gated B-cell progenitor in CD34+ cells: 0.4 (If ≤5, FCM score=1) Lymphocyte to myeloblast CD45 MFI ratio: 12.5 (If ≤4 or ≥7.5, FCM score=1) Granulocyte to lymphocyte SSC mode ratio: 4.7 (If ≤6, FCM score=1) Total FCM score= 4
  45. 45. VALIDATION (PHASE 2) • 741 BM samples were analyzed within 2013 • Results of MDS score were correlated with morphological and cytogenetic evaluation in a selected group of 440 bone marrow samples from patients with full clinical information:  8 normal BM samples  207 BM samples hospital controls*  18 B-cell or plasma cell neoplasm  33 post treatment for other malignancy  21 post BM transplant  47 MPN  106 samples from patients with MDS or MDS/MPN diagnosis (based on morphological, cytogenetic and clinical findings) * Patients with cytopenia(s) with no evidence of underlying malignancy, BM samples with reactive inflammatory changes or lymphoma staging specimens without lymphoma involvement.
  46. 46. RESULTS- VALIDATION (PHASE 2) Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):253-60 PPN= 96% NPV= 82% PPN= 75% NPV= 86%
  47. 47. RESULTS- VALIDATION (PHASE 2) Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):253-60
  48. 48. 10 color acute leukemia panel at University Health Network, Toronto AML 1 AML 2 AML 3 B-ALL T-ALL cytopl FITC CD65 CD36 CD71 CD58 CD7 nTdt PE CD13 CD64 CD11c CD22 CD1a cMPO ECD CD14 CD56 CD4 CD38 CD8 CD14 PC5.5 CD33 CD33 CD33 CD33 CD3 CD33 PC7 CD34 CD34 CD34 CD34 CD34 CD34 APC CD117 CD123 CD2 CD123 CD2 cCD79a APC_AlexaF700 CD7 CD19 CD10 CD10 CD10 cCD22 APC_AlexaF750 CD11b CD38 CD235a CD19 CD4 CD19 Pacific_BLUE CD16 HLA-DR CD15 CD20 CD5 cCD3 Krome Orange CD45 CD45 CD45 CD45 CD45 CD45
  49. 49. CONCLUSION • Developed 14 MAb 10-color FCM tube can be applied for a quick screening for aberrant lymphoid populations and myelodysplasia-related features. • MDS score 3 or 4 is highly indicative of MDS/MDS-MPN but can also be seen in MPN. • Score 2 in patients with differential diagnosis of MDS or MDS/MPN should prompt further investigation for myelodysplastic syndrome using comprehensive FCM panel. • MDS diagnosis should always be based on integrated diagnostics (morphology, FCM, cytogenetics)
  50. 50. CURRENT & FUTURE DEVELOPMENTS A. Improve the accuracy of MDS score B. Develop Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte population
  51. 51. Improve the accuracy of MDS score Porwit A, et al. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes- proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Leukemia 2014 Jun 12.
  52. 52. Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte population • 80% of LPD cases we diagnosed in 2014 in blood are CLL/MBL.
  53. 53. Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte population Ab FITC CD4 + Kappa PE CD8 + Lambda ECD CD3 + CD14 PC5.5 CD5 PC7 CD20 + CD56 APC CD10 APC-A700 CD19 APC-A750 CD200 PB CD57 +CD23 KO CD45 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
  54. 54. CLL
  55. 55. MCL
  56. 56. T-cell LGL
  57. 57. AITL
  58. 58. ACKNOWLEDGEMENTS • Anna Porwit • Jennifer Leung • Jessie Leung • Jordan Ngo • Josello Mandawi • Liz Valenzuela • May Ly • Sajid Dawji • Reza Jafari
  59. 59. Thank you for your attention

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