3. Flow cytometry Presented By Prof.Dr.Mohammed Abd El Hakim Sorour

6. Flow Cytometry - Principles Target: Evaluation of single cells Solution: hydrodynamic Focusing

8. Cytology

11. Instrument Parameters Y Laser Side Scatter (SSC) 90° deflection ~ Cell structures Forward Scatter (FSC) < 10° detection ~ Cell size Fluorescence Intensity Antigen Density

12. scatter detector iris blocker Optics for forward scatter Laser beam

13. 488 nm laser + - Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector

14. Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Flow Chamber ++ -- Signal direction Signal direction

15. SMALL BEAD LARGE BEAD Frequency Histogram SMALL BEAD LARGE BEAD Sample in Sheath Sheath in Laser beam Stream Charge +2KV -2KV Waste SORT RIGHT SORT LEFT SORT DECISIONS Piezoelectric crystal oscillator Last attached droplet LEFT RIGHT Sensors Sensor

22. Fluorochromes Y Y

23. Common Fluorochromes for 488nm Excitation FITC = Fluorescein Isothiocyanate PE = Phycoerythrin (RD1) ECD = Energy Coupled Dye PI = Propidium Iodide PC5 = Phycoerythrin Cyanin 5 (PC5) FITC 520 PE 575 ECD 615 PC5 665 LASER 488 PI 620 <390   400-450   450-500 500-570   570-590  590-620   620-750  >750   ultra- violet blue gr een yellow orange red infra- violet red 

28. Immunophenotyping of Leukemia and Lymphoma

29. Hematological diagnostics - Blood count - Differential blood count - Cytology - Cytochemistry - Flow cytometry - Molecular diagnostic

31. Hematopoetic System

32. Acute Leukemia’s Acute leukemia's ALL AML T-ALL B-ALL pre-T-ALL Common-T-ALL early T-ALL mature T-ALL pro-B-ALL C-ALL pre-B-ALL B-ALL AUL M1 M2 M3 M4 M5 M6 M7

34. Model of Leukemia/Lymphoma Maturation stop Proliferation Leukemia/ Lymphoma Morphology Lymphoblast Plasma cell Immunology Pro-B-cell Plasma cell Y Y Y Y

35. B Cell Leukemia's TdT TdT 24 24 CD19 24 CD34 Pro B-ALL (BI) TdT CD22 CD79a CD19 CD10 CD22 Common B-ALL (BII) CD79a Y IgM CD19 CD20 CD22 CD79a CD24 B-ALL (BIV) CD19 CD10 CD22 CD79a Pre B-ALL (BIII)   

37. AML Cds 34 13 33 14 15 Glyc A 61 41 MPO TDT M0/M1 + + - - - - - - + -/+ M2 + + + - - - - - + - M3 + + + - - - - - + - M4/M5 + - - + + - - - - - M6 + - - - - + - - - - M7 + - - - - - + + - -

38. AUL M0 Blast resmble L2 but –ve for lymphoid marker CD3 - /CD19 - CD33+/MPO+ blasts of a AML FAB M0

39. Biphenotypic hybrid Definition Case with typical lymphoblast or myeloblast that unexpectedly express two or more Ags of the opposit lineage on the same cells MPO CD13 CD33 CD22 CD79a CD34

40. Biphenotypic hybrid It is nessesary to score more than 2 point from 2 separate lineage Point B.Lineage markers B.lineage markers Meyloid markers 2 CD 79a Cd22 µ chaine CD 3 MPO 1 CD 19 CD 10 CD2 CD5 CD 13 CD 33 0.5 TdT TdT CD 7 CD 11 b/c CD 14/15

41. Mixed or bilinear Definition The cells show separate population of lymphoblast and meyloblast . CD 13 CD19 CD22 CD79a CD34 TdT CD33 CD34

42. Antigen Pattern I Definition Cells are clearly defined by several pan-lineage antigens and co-express an antigen of a different lineage e.g. CD65 - myeloid antigen - expressed on a immature B-cell (CD19 + , CD34 + , cyCD22 + , cyCD79a + ) CD65 CD19 CD22 CD79a CD34 TdT aberrant

43. Antigen PatternII Definition The differentiation stage of the cells are clearly defined by several antigens e.g. antigens of mature B-cells, but co-express an antigen of immature B-cells like CD34. CD34 Y IgM CD19 CD20 CD22 CD79a CD24 asynchronous

45. B CLL & lymphoma Cds 19 20 22 23 5 10 11c 25 103 79a 38 HLDR BCLL + + - + + - - + - - - + BPLL + + + - - - - + - + - + MCL FMC7 + + + - + - - - + + - + FCL + + + + - + - + - + - + HCL + + + - - - + + + + - + PLCL - - - - - - - - - - + -

46. B CLL & lYMPHOMA CDs CLL F C L M C L CD 5 + - + CD 10 - + - CD23 + + - FMC7 - - +

48. HCL CD103-PE+ cells of a HCL

49. DNA ANALYSIS

51. CELL CYCLE

52. The Cell Cycle G 1 M G 2 S G 0 Quiescent cells

57. Diploid : Only one G0/G1 peak is observed. Aneuploid : at least 2 separate G0/G1 peaks are demonstrated. - Hyperdiploid : abnormal peak above diploid peak. - Hypodiploid : abnormal peak below diploid peak. - Multiploid : Two or more abnormal peaks. - Tetraploid : Presence of abnormal peak at G2/M position.

60. Stem Cells - Definition Stem cells self renewal capacity pluripotent differentiation

61. Stem Cells - Pluripotent - Reconstitution of bone marrow - 2 % in bone marrow - CD34 + - Mobilization by chemo- and factor- therapy P eripheral B lood S tem C ell T ransplantation ( PBSCT )

62. PBSCT-Principle 2 x 10 6 CD34 + - Cells/kg BW

66. RBCs

69. PLATELETS

70. Hemostasis

73. Shape Changes in Platelets

74. Normal Person Thrombasthenia Glanzmann Down Regulation of Receptors Platelets

75. CD62p Assay - Pre-activated Platelets

76. Immuno- deficiencies

77. Immune system cellular immune response humor immune response T B G M

80. Immunodeficiencies primary = congenital secondary = acquired

85. HLA-Typing Rapid and easy Detection of HLA-B27 by flow cytometry $ Clinical relevance 1. T r ansplantation 2. Proof of paternity 3. Autoimmune diseases Correlation of HLA-subtypes and incidence classifying rheumatoid diseases

86. Apoptosis $$ programmed cell death = apoptosis

91. Thank You

92. Thank You