Prof.dr.moh'd sorour bch

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Prof.dr.moh'd sorour bch

  1. 3. Flow cytometry Presented By Prof.Dr.Mohammed Abd El Hakim Sorour
  2. 4. Flow Cytometry - Definition <ul><li>Flowcytometry (FCM) : </li></ul><ul><li>Is the measurement (-metry) of cellular (-cyto) properties ,as they are moving in a fluid stream (flow) by a stationary set of detectors </li></ul><ul><li>Evaluation of Single Cells </li></ul><ul><li>Multiparametric Analysis </li></ul>
  3. 6. Flow Cytometry - Principles Target: Evaluation of single cells Solution: hydrodynamic Focusing
  4. 7. Flow Cytometry - Principles <ul><li>Microscopy </li></ul><ul><li>Flow Cytometry </li></ul><ul><ul><li>Laser beam </li></ul></ul><ul><ul><li>Fluidics, Flow Cell and florochromes </li></ul></ul><ul><ul><li>Detectors/Computer </li></ul></ul>
  5. 8. Cytology
  6. 9. Immune Cytology <ul><li>membranous /intracellular </li></ul><ul><li>structures </li></ul><ul><li>dye-coupled monoclonal antibody (mab) </li></ul>Y
  7. 10. Multiparametric Analysis <ul><li>Simultaneous Detection of up to 12 Parameters </li></ul><ul><ul><li>Cell size </li></ul></ul><ul><ul><li>Cell structure </li></ul></ul><ul><ul><li>Dyes </li></ul></ul>Y
  8. 11. Instrument Parameters Y Laser Side Scatter (SSC) 90° deflection ~ Cell structures Forward Scatter (FSC) < 10° detection ~ Cell size Fluorescence Intensity Antigen Density
  9. 12. scatter detector iris blocker Optics for forward scatter Laser beam
  10. 13. 488 nm laser + - Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector
  11. 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
  12. 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
  13. 16. Signal Analysis <ul><li>Statistics </li></ul><ul><li>% Negative & % Positive Cells </li></ul><ul><li>Antigen density ~ Mean Fluorescence Intensity ~ Channel number </li></ul>0 Channel Number 1024 Number of cells           cells      0 Channel Number 1024 Cell counts mean fluorescence intensity % positive cells % negative cells
  14. 17. Signal Analysis <ul><li>Dual Parameter Correlation </li></ul>Scatter Diagram: Morphology Coexpression of 2 antigens
  15. 18. Cluster Differentation <ul><ul><li>All cells have membrane proteins on the surface </li></ul></ul><ul><ul><li>Appear or disappear at different stages of maturation </li></ul></ul><ul><ul><li>M onoclonal a nti b odies ( mab ) are used to “tag” these proteins </li></ul></ul><ul><ul><li>Mabs are in vitro produced antibodies specific to these proteins </li></ul></ul><ul><ul><li>Mabs with identical characteristics are grouped together in one cluster (CD) </li></ul></ul>
  16. 19. Stainings - Techniques <ul><ul><li>Direct immune fluorescence with fluorochrome coupled antigen with specific mabs </li></ul></ul><ul><ul><li>Simultaneous staining with up to 4 mabs </li></ul></ul><ul><ul><li>Using different dyes (FITC, PE, Per CP, PC5) </li></ul></ul><ul><ul><li> Detection of the co-expression up to 4 antigens on a single cell = </li></ul></ul><ul><ul><li>MULTIPARAMETER ANALYSIS </li></ul></ul>Y mab1 FITC Y mab2 PE
  17. 20. Monoclonal Antibodies <ul><li>CD = Cluster of Differentation </li></ul><ul><ul><li>A CD number comprises all antibody clones detecting the same human antigen - not epitope specific </li></ul></ul><ul><ul><ul><ul><li>e.g. CD34 </li></ul></ul></ul></ul>Y
  18. 21. Common Fluorochromes for 488nm and 633nm Excitation <ul><li>Fluorescein Isothiocyanate (FITC) </li></ul><ul><li>Phycoerythrin (PE) </li></ul><ul><li>Allophycocyanin (APC) </li></ul><ul><li>Peridinin-Chlorophyll Protein (PerCP) </li></ul>
  19. 22. Fluorochromes Y Y
  20. 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 
  21. 24. Flow Cytometry - Applications <ul><li>Detection/Quantification of </li></ul><ul><ul><li>membranous structures </li></ul></ul><ul><ul><ul><li>antigens/receptors </li></ul></ul></ul><ul><ul><li>bacteria/fungi/algae... </li></ul></ul><ul><li>intracellular structures </li></ul><ul><ul><ul><li>Cytokines </li></ul></ul></ul><ul><ul><ul><li>Enzymes </li></ul></ul></ul><ul><ul><ul><li>DNA/RNA ... </li></ul></ul></ul>
  22. 25. Flow Cytometry <ul><li>Advantages </li></ul><ul><li>Analysis of high cell numbers (10 7 cells) </li></ul><ul><li>Short time of analysis (sec) </li></ul><ul><li>Gating allows detection of cell sub-populations </li></ul><ul><li>Measurement of rare events </li></ul><ul><li>Objective measurement of fluorescence intensity </li></ul><ul><li>Simultaneous detection of many parameters per cell </li></ul>
  23. 26. Flow Cytometric Samples <ul><ul><li>Blood </li></ul></ul><ul><ul><li>Bone marrow </li></ul></ul><ul><ul><li>Liquor </li></ul></ul><ul><ul><li>BAL </li></ul></ul><ul><ul><li>C S F </li></ul></ul><ul><ul><li>Joint effusion </li></ul></ul><ul><ul><li>Pleura effusion </li></ul></ul><ul><ul><li>Ascitic Fluid </li></ul></ul><ul><ul><li>tissue cells </li></ul></ul><ul><ul><ul><li>e.g. tumor </li></ul></ul></ul>Prerequisite Single Cell Suspension
  24. 27. Applications in Flow Cytometry <ul><li>leukemia- and lymphoma typing </li></ul><ul><li>DNA-analysis and cell cycole </li></ul><ul><li>transplantation monitoring </li></ul><ul><li>therapy monitoring </li></ul><ul><li>Multi-Drug-Resistance </li></ul><ul><li>M R D </li></ul><ul><li>C S F </li></ul><ul><li>bronchio-alveolar lavage </li></ul><ul><li>PNH-diagnostics </li></ul><ul><li>reticulocytes </li></ul><ul><li>thrombocyte analysis </li></ul><ul><li>detection of enzymes and enzyme activities </li></ul><ul><li>autoimmune diseases </li></ul><ul><li>HLA-typing </li></ul><ul><li>immune deficiencies </li></ul><ul><ul><ul><ul><ul><li>Apoptosis </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>phagocytosis </li></ul></ul></ul></ul></ul>
  25. 28. Immunophenotyping of Leukemia and Lymphoma
  26. 29. Hematological diagnostics - Blood count - Differential blood count - Cytology - Cytochemistry - Flow cytometry - Molecular diagnostic
  27. 30. Flow Cytometry Discovers & Diagnoses <ul><li>If the specimen is flagged by the hematology analyzer, Flow Cytometry provides a tool to further study the malignant cells for: </li></ul><ul><ul><li>Cell Lineage </li></ul></ul><ul><ul><li>Cellular Differentiation </li></ul></ul><ul><ul><li>Proliferation Activities </li></ul></ul><ul><ul><li>Subtyping </li></ul></ul><ul><ul><li>Aberrant antigen expression </li></ul></ul><ul><ul><li> better patient management . </li></ul></ul>
  28. 31. Hematopoetic System
  29. 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
  30. 33. Screening for Leukemia <ul><li>The unique combination of technologies that make up the system provide the most accurate information on blast forms providing separate flags for: </li></ul><ul><ul><ul><ul><li>Myeloblasts </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Lymphoblasts </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Monoblasts </li></ul></ul></ul></ul><ul><li>This specific flagging leads you to testing by Flow Cytometry for </li></ul><ul><ul><ul><ul><li>cell lineage determination </li></ul></ul></ul></ul><ul><ul><ul><ul><li>subtyping </li></ul></ul></ul></ul><ul><ul><ul><ul><li>abberant antigen expression </li></ul></ul></ul></ul>
  31. 34. Model of Leukemia/Lymphoma Maturation stop Proliferation Leukemia/ Lymphoma Morphology Lymphoblast Plasma cell Immunology Pro-B-cell Plasma cell Y Y Y Y
  32. 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)   
  33. 36. T ALL <ul><li>Pre T ALL : </li></ul><ul><li>CD 3 + CD 7 +CD 5+ CD 2- CD1a- </li></ul><ul><li>T ALL : </li></ul><ul><li>CD 3 + CD 7+ CD 5+ CD 2+ CD 1a + </li></ul>
  34. 37. AML Cds 34 13 33 14 15 Glyc A 61 41 MPO TDT M0/M1 + + - - - - - - + -/+ M2 + + + - - - - - + - M3 + + + - - - - - + - M4/M5 + - - + + - - - - - M6 + - - - - + - - - - M7 + - - - - - + + - -
  35. 38. AUL M0 Blast resmble L2 but –ve for lymphoid marker CD3 - /CD19 - CD33+/MPO+ blasts of a AML FAB M0
  36. 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
  37. 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
  38. 41. Mixed or bilinear Definition The cells show separate population of lymphoblast and meyloblast . CD 13 CD19 CD22 CD79a CD34 TdT CD33 CD34
  39. 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
  40. 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
  41. 44. Diagnosis of NHL’s NHL Y > 20 % CD19 CD19 B-NHL <ul><li>Proliferation </li></ul><ul><li>Light chain restriction </li></ul><ul><li>Co-expression of </li></ul><ul><ul><li>immature antigens </li></ul></ul><ul><ul><li>activation antigens </li></ul></ul>Y  or  CD5 CD23 < 20% Y CD19 CD19 Y  and  B-cell
  42. 45. B CLL & lymphoma Cds 19 20 22 23 5 10 11c 25 103 79a 38 HLDR BCLL + + - + + - - + - - - + BPLL + + + - - - - + - + - + MCL FMC7 + + + - + - - - + + - + FCL + + + + - + - + - + - + HCL + + + - - - + + + + - + PLCL - - - - - - - - - - + -
  43. 46. B CLL & lYMPHOMA CDs CLL F C L M C L CD 5 + - + CD 10 - + - CD23 + + - FMC7 - - +
  44. 47. CLL <ul><li>CD5+/CD23+ </li></ul><ul><li>light chain restriction </li></ul>
  45. 48. HCL CD103-PE+ cells of a HCL
  46. 49. DNA ANALYSIS
  47. 50. <ul><li>The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. Non-dividing cells not considered to be in the cell cycle </li></ul>cell cycle <ul><li>Stages of the cell cycle </li></ul><ul><li>-The G1 stage stands for &quot;GAP 1&quot;. </li></ul><ul><li>-The S stage stands for &quot;Synthesis&quot;. This is the stage when DNA replication occurs. </li></ul><ul><li>-The G2 stage stands for &quot;GAP 2&quot;. </li></ul><ul><li>-The M stage stands for &quot;mitosis&quot;, and is when nuclear (chromosomes separate) and cytoplasmic (cytokinesis) division occur. </li></ul>
  48. 51. CELL CYCLE
  49. 52. The Cell Cycle G 1 M G 2 S G 0 Quiescent cells
  50. 53. Definitions & Terms <ul><li>Ploidy </li></ul><ul><ul><li>related to the number of chromosomes in a cell </li></ul></ul><ul><li>Haploid : Number of </li></ul><ul><li>chromosomes in a gamete (germ cell) is called the Haploid. </li></ul>
  51. 54. <ul><li>Hyperdiploid : greater than the normal 2n number of chromosomes </li></ul><ul><li>Hypodiploid : Less than the normal 2n number of chromosomes </li></ul><ul><li>DNA Tetraploidy : Containing double the number of chromosomes </li></ul><ul><li>DNA Index : The ratio between the mode of the relative DNA content of the test cells (in G0/G1phase) to the mode of the relative DNA content in normal G0/G1 diploid cells </li></ul>
  52. 55. <ul><li>In the simplest application DNA contents of cells reflects the stage of cell cycle. </li></ul><ul><li>Diploid somatic cell in G0/G1 have two copies of chromosomes (2N). </li></ul><ul><li>S is the phase defined by DNA synthesis; </li></ul><ul><li>during G2 the cells have duplicated their DNA amounts (4N), </li></ul><ul><li>and during M phase condensate on the chromosomes to two homologous pairs of chromosomes occur for cells to be divided </li></ul>Cancer is a disease where regulation of the cell cycle goes away and normal cell growth and behavior is lost.
  53. 56. <ul><li>Ki-67, which stains a proliferation-related nuclear antigen in human cells of all line ages, but not the nuclei of resting Go phase cells. </li></ul>Another approach to measuring proliferative activity tumors is to make use of antibodies against nuclear antigen newly expressed during various phases of the cell cycle. <ul><li>PCNA/cyclin appears in the cell nucleus during S-phase; </li></ul>
  54. 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.
  55. 58. <ul><li>DNA index : The ratio between the mode of the relative DNA content of the test cells (in G0/G1 phase) to the mode of the relative DNA content in normal G0/G1 diploid cells. </li></ul>
  56. 59. ALL ploidy <ul><li>. </li></ul>Hyperploidy: (50-56 ch ) has better prognosis Near tetraploidy: rare in childhood , 5% in adult Near haploidy (24 -34 ch) Interactable leukemia
  57. 60. Stem Cells - Definition Stem cells self renewal capacity pluripotent differentiation
  58. 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 )
  59. 62. PBSCT-Principle 2 x 10 6 CD34 + - Cells/kg BW
  60. 63. PBSCT <ul><ul><li>Treatment with combined factor- and chemotherapy (e.g. G-CSF) </li></ul></ul><ul><ul><li>WBC > 1000/  l start CD34 + monitoring </li></ul></ul><ul><ul><li>If CD34 + cells detectable start of harvest (Leukapheresis) </li></ul></ul><ul><ul><li>Harvest of minimal 2 x 10 6 CD34 + / kg body weight </li></ul></ul><ul><ul><li>Re-transfusion of CD34 + cells after myeloaplasia induced through high dose CT and/or radiation </li></ul></ul><ul><ul><li>Re-constitution of bone marrow after 12 days </li></ul></ul>
  61. 64. MINIMAL RESIDUAL DISEASE <ul><li>The lowe level of disease detectable in patients by method avialable. </li></ul><ul><li>Aberrent phynotypic pattern by FCM and LEUKEMIC CELL DNA OR RNA by PCR are the most widly used as the have the most sensitive and specific level </li></ul>
  62. 65. MRD detection by FCM <ul><li>ALL </li></ul><ul><li>It requeres the identification of specific pattern of Ags at presentation which then sought at follow up </li></ul><ul><li>In T ALL (TdT and CD3) reliable up to 1 in 10 4 since normal BM doesnt contain it </li></ul><ul><li>In B ALL (19, 34, 10 &TdT ) PB is 10 times more than Bm </li></ul><ul><li>AML (13 ,33 ,34 &177) </li></ul>
  63. 66. RBCs
  64. 67. P N H <ul><li>Acuired H A with hemolysis mainly I V IN ADDITION THE PT MAY HAVE thrombosis and pancytopenia due to partial (PNH II)or complete(PNH III) defeciency of GPI anchor required for many platelet to attach to the cell memb. * MIRL (CD 59) DAF (CD 55) </li></ul>FCM can be used to detect absence of MIRL (CD59-ve ) cell population
  65. 68. OTHER APPLICATION OF FCM IN EVALUTION OF RBCs <ul><li>Autoantibodies in H A </li></ul><ul><li>Fetal RBCs in fetomaternal hge </li></ul><ul><li>Blood groups </li></ul>
  66. 69. PLATELETS
  67. 70. Hemostasis
  68. 71. Platelets and their Receptors <ul><li>GPIIb-IIIa CD41/CD61 </li></ul><ul><li>GPIb-IX CD42b/CD42a </li></ul><ul><li>GPIa-IIa CD49b/CD29 </li></ul><ul><li>GPIc-IIa CD49e/CD29 </li></ul><ul><li>GPIc-IIa CD49f/CD29 </li></ul><ul><li>GPIIa CD31 </li></ul><ul><li>GPIV CD36 </li></ul><ul><li>GP53 CD63 </li></ul><ul><li>Vitronectinreceptor  CD51 </li></ul><ul><li>Vitronectinreceptor  CD61 </li></ul><ul><li>Thrombinreceptor </li></ul><ul><li>Thrombospondin </li></ul>Thrombinreceptor CD62p
  69. 72. Context of Platelet Analysis <ul><li>Hyperreactivity </li></ul><ul><ul><li>Prothrombotic syndromes </li></ul></ul><ul><ul><li>Vascular defects </li></ul></ul><ul><ul><li>Pre-eclampsia </li></ul></ul><ul><ul><li>Hematological malignancies </li></ul></ul><ul><li>Hyporeactivity </li></ul><ul><ul><li>Hereditary platelet membrane GPs defects (GT, BSS) </li></ul></ul><ul><ul><li>Hereditary granule defects (Gray, Dense granule Hermandsky-Pudlak syndrome) </li></ul></ul>
  70. 73. Shape Changes in Platelets
  71. 74. Normal Person Thrombasthenia Glanzmann Down Regulation of Receptors Platelets
  72. 75. CD62p Assay - Pre-activated Platelets
  73. 76. Immuno- deficiencies
  74. 77. Immune system cellular immune response humor immune response T B G M
  75. 78. Lymphocytes <ul><li>Cells that recognize antigens </li></ul><ul><li>Three types of lymphocytes </li></ul><ul><ul><li>Travel through the thymus </li></ul></ul><ul><ul><ul><li>T Cells - CD3 + </li></ul></ul></ul><ul><ul><li>Do not travel through the thymus </li></ul></ul><ul><ul><ul><li>B Cells - CD19 + </li></ul></ul></ul><ul><ul><li>Destroy antigens in circulation </li></ul></ul><ul><ul><ul><li>Natural Killer cells - CD56 + </li></ul></ul></ul>
  76. 79. T Cells <ul><li>Do not produce antibodies </li></ul><ul><li>Two basic populations </li></ul><ul><ul><li>Regulatory </li></ul></ul><ul><ul><ul><ul><li>T helper Cells CD3 + /CD4 + </li></ul></ul></ul></ul><ul><ul><ul><ul><li>T Suppresser Cells CD3 + /CD8 + </li></ul></ul></ul></ul><ul><ul><li>Cytotoxic </li></ul></ul><ul><ul><ul><ul><li>Natural Killer Cells (NK Cells) </li></ul></ul></ul></ul>
  77. 80. Immunodeficiencies primary = congenital secondary = acquired
  78. 81. Immune deficiencies <ul><li>1. Congenital </li></ul><ul><li>2. Immune suppression correlate to other diseases </li></ul><ul><li>2. Iatrogenic Immune suppression </li></ul>Trauma, Surgery,Chemotherapy, Plasmocytoma, Non-Hodgkin-Lymphoma, BMT, exsudative Enteropathia or Dermatitis, nephrotic Syndrome, M. Hodgkin,, chron. GvHD, Virus infections, steroids, radiation, anti-CD 3 ... RARE!
  79. 82. Normal Ranges <ul><li>T cells B cells LGL cells </li></ul><ul><li>PB 65-90 % 5-15 % 5-15 % </li></ul><ul><li>BM < 40 % < 40 % < 15 % </li></ul>
  80. 83. Immune Deficiencies <ul><li>Humane Immunodeficiency Virus </li></ul>Attacks and depletes CD4 + -T-helper cells
  81. 84. HIV Management <ul><ul><li>Patients are managed through CD4 + T lymphocyte count </li></ul></ul><ul><ul><li>Clinical complications are associated with depletion of CD4 + Cells </li></ul></ul><ul><ul><li>Qualification for treatment protocols dependent on the diagnosis , HIV and CD4 + count of less than 500 cells/  l </li></ul></ul>
  82. 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
  83. 86. Apoptosis $$ programmed cell death = apoptosis
  84. 87. <ul><ul><ul><li>Default of death </li></ul></ul></ul><ul><ul><ul><ul><li>Cancer </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Autoimmune diseases </li></ul></ul></ul></ul><ul><ul><ul><li>Excess death </li></ul></ul></ul><ul><ul><ul><ul><li>HIV infection </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Fulminant hepatitis </li></ul></ul></ul></ul>Apoptosis & Diseases
  85. 88. Cell death / Apoptosis <ul><ul><ul><li>Accidental death or necrosis </li></ul></ul></ul><ul><ul><ul><li>Programmed cell death or apoptosis </li></ul></ul></ul>
  86. 89. Methods of Measuring Apoptosis <ul><li>Morphology </li></ul><ul><li>Light scatter </li></ul><ul><li>Laddering gel </li></ul><ul><li>Flow cytometry </li></ul><ul><ul><ul><ul><li>PI incorporation </li></ul></ul></ul></ul><ul><ul><ul><ul><li>TdT Assay </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Annexin V </li></ul></ul></ul></ul><ul><ul><ul><ul><li>APO2.7 </li></ul></ul></ul></ul>
  87. 90. Flow cytometric Methodes for detection of apoptosis <ul><ul><ul><li>1. Sub G1 peak DNA distribution </li></ul></ul></ul><ul><ul><ul><li> PI staining </li></ul></ul></ul><ul><ul><ul><li>2. Plasma membrane (loss of assymetry)   Staining with Annexin V </li></ul></ul></ul><ul><li>3. Mitochondria (expression of specific, induced proteins)  </li></ul><ul><li>  Staining with APO2.7 antibody </li></ul><ul><li>4. Nucleus (DNA fragmentation)  </li></ul><ul><li>  TUNEL method </li></ul>
  88. 91. Thank You
  89. 92. Thank You

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