• Document: Flow Cytometry in the Diagnosis of Hematopoietic Neoplasia. Brent Wood MD, PhD Professor, Laboratory Medicine University of Washington, Seattle
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Flow Cytometry in the Diagnosis of Hematopoietic Neoplasia Brent Wood MD, PhD Professor, Laboratory Medicine University of Washington, Seattle 1 Flow Cytometer 2 The Power of Flow Cytometry • Single cell analysis • Multiparametric • Rapid • Quantitative • Flexible 3 Clinical Assays • Lymphocyte subset analysis • Immunodeficiency • Stem cell enumeration (CD34) • Paroxysmal nocturnal hemoglobinuria (PNH) • Reticulocytes • Fetal erythrocytes • HLA crossmatch • Leukemia and Lymphoma 4 Inferential reasoning 18% 66% 80% 0.4% • Insensitive • Misattribution if assumptions incorrect 07-086465 Direct Observation • Combination of reagents uniquely identifies cell type, lineage and maturational stage – Emphasize normal maturational patterns • Direct determination of immunophenotype without inference • Improvement in sensitivity and specificity • More simultaneous fluorochromes improves 6 Multiparametric Flow Cytometry • More accurate population identification - Greater informational content • Make better use of small specimens - Fewer cells, more information • Process fewer tubes - Save on reagents, tech and instrument time • Collect large number of events efficiently • Allow standardized reagent combinations 7 How Many Colors are Enough? • Ideal - Add all reagents of interest into single tube • Real - Too many hematopoietic disorders - Single comprehensive tube per cell lineage - Focus on screening tubes 8 Bethesda International Consensus Conference N = 35 9 Euroflow 10 Instrumentation Beckman-Coulter FC500 Becton-Dickinson FACSCanto 5 colors - 1 or 2 lasers I - 6 colors, 2 lasers II - 8 colors, 3 lasers Beckman-Coulter Gallios Becton-Dickinson LSRII 10 colors - 3 lasers ~20 color, up to 7 lasers 11 Panel Design PB FITC PE PE-TR PE55 PE7 A594 APC A700 APC7 B cells 45 κ λ 19 34 20 38 10 - 5 T cells 45 2 7 34 8 3 4 56 - 5 Blasts DR 15 33 19 117 13 38 34 71 45 Myeloid DR 64 123 4 14 13 38 34 16 45 12 9 Color Flow Cytometry 55 unique combinations 13 Abnormal population identification • Normal – Antigens expressed in consistent and reproducible patterns with maturation • Neoplastic – Increased or decreased normal antigens – Asynchronous maturational expression – Aberrant antigen expression – Homogeneous expression 14 Normal B cell Maturation Wood and Borowitz (2006) Henry’s Laboratory Medicine 15 Wood, Methods in Cell Biology, 200416 B cell clonality 10-10531 • Color kappa and lambda • Look for discrete abnormal B cell populations • Identify antigenic aberrancy in the context of clonality • Demonstration of clonality is not necessary 17 Follicle Center B cells Follicular Hyperplasia 08-03324 Follicular Lymphoma 08-01359 18 Plasma Cell Neoplasm 0.05% abnormal plasma cells 04-6716 19 ALL MRD 0.1% abnormal immature B cells 06-01469 20 Normal T cell Maturation

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