Welcome. Welcome. Emerging Technologies in Flow Cytometry

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1 Emerging Technologies in Flow Cytometry Dr. William Dittman December 11, 2012 You may download a copy of the handout by clicking on the handout icon, located in the upper right hand corner of your screen The phone lines will open 15 minutes prior to the start of the webinar Welcome Your Host: Karen Riba Handout is available by clicking on the handout icon in the upper right hand corner of your screen For technical difficulties please kchandler@pamlcom For questions you have during the presentation use the Q & A link at the top of your screen Questions will be answered at the end of the presentation Welcome P.A.C.E. credit may be obtained by submitting your completed evaluation form at the end of the webinar CE credit may be obtained by downloading the Certificate of Completion PAML employees will be able to receive one hour of continuing education. 1

2 Speaker Information Speaker Photo Speaker Image William A. Dittman, Jr., MD PAML Technical Director, Hematology Dr. Dittman directs the Hematology division at PAML and Providence Sacred Heart Medical Center and Children s Hospital. His specialties include blood hematology, coagulation and oncology. Learning Objectives At the end of this presentation participants will be able to: Outline an overview of clinical Flow Cytometry Review PAML s flow panels and how they are to be used Describe how to integrate Flow Cytometry with Histology, Cytogenetics and Molecular studies in Hematopathologty What is Flow Cytometry? Laboratory technology that is used primarily to characterize and measure cells in relationship to Leukemia and Lymphoma. It is commonly referred to as Immunophenotyping. 2

3 Flow Cytometry at PAML Coulter Epics PROFILE Three color Introduced to PAML in the late 1980s Flow Cytometry at PAML Coulter EPICS XL Five color To PAML in mid 1990s Identification of cell populations by expression of CD 45 (leukocyte common antigen) and side scatter Analysis of Normal and Leukemic Bone Marrow by CD45-side scatter analysis ) Jennings C D, Foon K A Blood 1997;90: by American Society of Hematology 3

4 2012 Flow Cytometry Technology BD Facs/Canto Eight color Blood Marrow Screening Panels Tubes for Immunophenotyipng Screen Panel FITC PE PerCP-Cy 5.5 PE-Cy7 APC APC-H7 V450 V500 T Cell CD7 CD56 CD5 CD3 CD8 CD4 CD2 CD45 B Cell CD103 Lambda CD5 CD10 Kappa CD19 CD20 CD45 Myeloid 1 CD41a CD38 CD33 CD13 CD71 HLADR CD16 CD45 Myeloid 2 CD34 CD64 CD33 CD117 CD11b CD14 CD15 CD45 Tissue Screening Panel Tubes for Immunophenotyipng Tissue Screen Panel FITC PE PerCP-Cy 5.5 PE-Cy7 APC APC-H7 V450 V500 T Cell CD7 CD56 CD5 CD3 CD8 CD4 CD2 CD45 B Cell CD103 Lambda CD5 CD10 Kappa CD19 CD20 CD45 Viability 7-AAD CD45 4

5 Disease/Diagnosis Specific Panels FITC PE PerCP-Cy 5.5 PE-Cy7 APC APC-H7 V450 V500 CLL czap-70 CD3 CD200 CD5 CD23 CD19 FMC7 CD45 Hairy Cell CD103 CD11c CD19 CD25 CD22 CD14 FMC7 CD45 Hematogones Immature lymphs CD49f CD58 CD34 CD10 CD22 CD19 CD38 CD45 NK Cell CD57 CD7 CD16 CD3 CD56 CD8 CD2 CD45 Disease/Diagnosis Specific Panels FITC PE PerCP-Cy 5.5 PE-Cy7 APC APC-H7 V450 V500 T Cell Receptor TCR a/b TCR g/d CD3 CD25 CD43 CD7 CD45 Plasma Cell CD38 clambda CD138 CD56 ckappa CD19 CD20 CD45 Cytoplasmic Acute ccd22 cmpo ccd79a ccd3 ctdt CD45 Cytoplasmic Lymphs cbcl-2 ccd3 CD34 CD10 ctdt CD19 CD20 CD45 Paroxysmal Nocturnal Hemoglobinuria (PNH) Panel FITC PE PerCP-Cy 5.5 PE-Cy7 APC APC-H7 V450 V500 PNH WBC FLAER CD24 CD64 CD33 CD14 CD15 CD45 PNH RBC Glyco A CD59 5

6 When to Order Bone Marrow usually helpful Anemia Thrombocytopenia Leukemia Lymphoma Myelodysplasia Plasma cell disorders Others When to Order Blood Leukocytosis - Especially Lymphocytosis Circulating immature cells Suspect PNH Tissue Suspect Lymphoma 6

7 What Test to Order What sample do you have? Blood Marrow CSF for Leukemia/Lymphoma Order IMMUNOPHENOTYPE SCREEN (REFLEXIVE) Test Code - IPSCRN What Test to Order What sample do you have? Anything else Order IMMUNOPHENOTYPE TISSUE SCREEN (REFLEXIVE) Test Code - IPTISS 7

8 When to Ask for Disease Specific Panel Rarely Almost always if an abnormal population is present we will find it and add appropriate tubes Plasma cell abnormalities Might miss on screen Consider ordering with plasma cell tube Please Provide History If we don t know what you are looking for or why you are doing the study we may not be looking for the right thing. Plasma cell disorder Plasma cells don t always survive processing and instrument May be abnormal despite normal numbers of cells Treated leukemia or lymphoma May be very small number of abnormal cells May alter numbers of normal cells 8

9 Analysis of Flow 9

10 Hematopathology Report 10

11 Paroxysmal Nocturnal Hemoglobinuria GPI Anchored Proteins Johnson and Hillmen,Mol Pathol,

12 Clinical Manifestations of PNH Indications for testing Highly variable and dependent upon the size of the abnormal clonal population in any individual Hemolysis Thrombosis Relative/absolute bone marrow failure Possibly myelodysplasia PH Testing - Normal Granulocytes Monocytes Red Blood Cells 12

13 PH Testing PH Present Granulocytes Monocytes Red Blood Cells PH Testing Myelodysplasia Present Granulocytes Monocytes Red Blood Cells Consultative Report Bring together all information for hematopathology; Morphology Immunophenotyping/flow cytometry Cytogenetics FISH Molecular 13

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15 In Summary PAML has new, state of the art Flow Cytometry instruments PAML s new reflexive flow panels are designed for rapid, correct result determination PAML s new flow evaluation software will allow virtual flow More report integration coming soon.... Questions 15

16 Thank-you for Attending P.A.C.E. credit may be obtained by submitting your completed evaluation form. You will find the form by clicking on the handouts icon in the upper right hand corner of your screen CE credit may be obtained by downloading the Certificate of Completion under the handouts icon PAML employees will be able to receive one hour of continuing education credit by submitting your attendance through CE Manager. Thank-you for Attending We will be leaving the webinar open for 15 minutes to allow you to download the handouts This webinar has been recorded and will be available by Tuesday, December 18 th, at PAML s next webinar will be in early Be watching for detailed information in January. Please send ideas for future webinar topics. 16