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Flow cytometry Presented By Prof.Dr.Mohammed Abd El Hakim Sorour
Flow Cytometry - Definition ,[object Object],[object Object],[object Object],[object Object]
 
Flow Cytometry - Principles Target:  Evaluation of single cells Solution:  hydrodynamic Focusing
Flow Cytometry - Principles ,[object Object],[object Object],[object Object],[object Object],[object Object]
Cytology
Immune Cytology ,[object Object],[object Object],[object Object],Y
Multiparametric Analysis ,[object Object],[object Object],[object Object],[object Object],Y
Instrument Parameters Y Laser Side Scatter (SSC) 90° deflection ~ Cell structures Forward Scatter (FSC) < 10° detection ~  Cell size Fluorescence Intensity Antigen Density
scatter detector iris blocker Optics for forward scatter Laser beam
488 nm laser + - Fluorescence Activated  Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector
Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Flow Chamber ++ -- Signal direction Signal direction
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
Signal Analysis ,[object Object],[object Object],[object Object],0  Channel Number  1024 Number of cells             cells      0  Channel Number  1024 Cell counts mean fluorescence intensity %  positive cells %  negative cells
Signal Analysis ,[object Object],Scatter Diagram: Morphology Coexpression  of 2 antigens
Cluster Differentation ,[object Object],[object Object],[object Object],[object Object],[object Object]
Stainings - Techniques ,[object Object],[object Object],[object Object],[object Object],[object Object],Y mab1  FITC Y mab2   PE
Monoclonal Antibodies ,[object Object],[object Object],[object Object],Y
Common Fluorochromes for 488nm and 633nm Excitation ,[object Object],[object Object],[object Object],[object Object]
Fluorochromes Y Y
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 
Flow Cytometry - Applications ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Flow Cytometry ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Flow Cytometric Samples ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Prerequisite Single Cell Suspension
Applications in Flow Cytometry  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Immunophenotyping of  Leukemia and Lymphoma
Hematological diagnostics - Blood count -  Differential blood count -  Cytology -  Cytochemistry -  Flow cytometry -  Molecular diagnostic
Flow Cytometry Discovers & Diagnoses ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Hematopoetic System
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
Screening for Leukemia ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Model of Leukemia/Lymphoma Maturation stop Proliferation Leukemia/ Lymphoma Morphology  Lymphoblast  Plasma cell Immunology  Pro-B-cell    Plasma cell Y Y Y Y
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)   
T  ALL ,[object Object],[object Object],[object Object],[object Object]
AML Cds 34 13 33 14 15 Glyc A 61 41 MPO TDT M0/M1 + + - - - - - - + -/+ M2 + + + - - - - - + - M3 + + + - - - - - + - M4/M5 + - - + + - - - - - M6 + - - - - + - - - - M7 + - - - - - + + - -
AUL  M0 Blast resmble L2 but –ve for lymphoid marker CD3 - /CD19 - CD33+/MPO+ blasts of a AML FAB M0
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
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
Mixed or bilinear Definition The cells show separate population of lymphoblast and meyloblast . CD 13 CD19 CD22 CD79a CD34 TdT CD33 CD34
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
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
Diagnosis of NHL’s NHL Y > 20 % CD19 CD19 B-NHL  ,[object Object],[object Object],[object Object],[object Object],[object Object],Y    or   CD5 CD23 < 20% Y CD19 CD19 Y    and   B-cell
B CLL & lymphoma   Cds 19 20 22 23 5 10 11c 25 103 79a 38 HLDR BCLL + + - + + - - + - - - + BPLL + + + - - - - + - + - + MCL  FMC7 + + + - + - - - + + - + FCL + + + + - + - + - + - + HCL + + + - - - + + + + - + PLCL - - - - - - - - - - + -
B  CLL & lYMPHOMA   CDs CLL F C L M C L CD 5 + - + CD 10 - + - CD23 + + - FMC7 - - +
CLL ,[object Object],[object Object]
HCL CD103-PE+ cells of a HCL
DNA  ANALYSIS
[object Object],cell cycle   ,[object Object],[object Object],[object Object],[object Object],[object Object]
CELL CYCLE
The Cell Cycle G 1 M G 2 S G 0 Quiescent cells
Definitions & Terms ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],Cancer is a disease where regulation of the cell cycle goes away and normal cell growth and behavior is lost.
[object Object],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.  ,[object Object]
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.
[object Object]
ALL ploidy  ,[object Object],Hyperploidy:  (50-56 ch  ) has better  prognosis Near tetraploidy: rare in childhood , 5% in adult  Near haploidy  (24 -34 ch) Interactable leukemia
Stem Cells - Definition Stem cells  self renewal capacity pluripotent differentiation
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 )
PBSCT-Principle 2  x 10  6  CD34 + - Cells/kg BW
PBSCT ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
MINIMAL  RESIDUAL DISEASE  ,[object Object],[object Object]
MRD  detection  by FCM ,[object Object],[object Object],[object Object],[object Object],[object Object]
RBCs
P N H  ,[object Object],FCM can be used to detect absence of MIRL   (CD59-ve ) cell population
OTHER APPLICATION OF FCM IN EVALUTION OF RBCs ,[object Object],[object Object],[object Object]
PLATELETS
Hemostasis
Platelets and their Receptors ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Thrombinreceptor CD62p
Context of Platelet Analysis ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Shape Changes in Platelets
Normal Person  Thrombasthenia Glanzmann Down Regulation of Receptors Platelets
CD62p Assay -  Pre-activated Platelets
Immuno-  deficiencies
Immune system cellular immune response humor immune response T B G M
Lymphocytes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
T Cells ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Immunodeficiencies primary =  congenital secondary =  acquired
Immune deficiencies ,[object Object],[object Object],[object Object],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!
Normal Ranges ,[object Object],[object Object],[object Object]
Immune Deficiencies ,[object Object],Attacks and depletes CD4 + -T-helper cells
HIV Management ,[object Object],[object Object],[object Object]
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
Apoptosis $$  programmed cell death = apoptosis
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Apoptosis & Diseases
Cell death / Apoptosis  ,[object Object],[object Object]
Methods of Measuring Apoptosis ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Flow cytometric Methodes for detection of apoptosis ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Thank You
Thank You

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Prof.dr.moh'd sorour bch

  • 1.  
  • 2.  
  • 3. Flow cytometry Presented By Prof.Dr.Mohammed Abd El Hakim Sorour
  • 4.
  • 5.  
  • 6. Flow Cytometry - Principles Target: Evaluation of single cells Solution: hydrodynamic Focusing
  • 7.
  • 9.
  • 10.
  • 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
  • 16.
  • 17.
  • 18.
  • 19.
  • 20.
  • 21.
  • 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 
  • 24.
  • 25.
  • 26.
  • 27.
  • 28. Immunophenotyping of Leukemia and Lymphoma
  • 29. Hematological diagnostics - Blood count - Differential blood count - Cytology - Cytochemistry - Flow cytometry - Molecular diagnostic
  • 30.
  • 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
  • 33.
  • 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)   
  • 36.
  • 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
  • 44.
  • 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 - - +
  • 47.
  • 50.
  • 52. The Cell Cycle G 1 M G 2 S G 0 Quiescent cells
  • 53.
  • 54.
  • 55.
  • 56.
  • 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.
  • 58.
  • 59.
  • 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
  • 63.
  • 64.
  • 65.
  • 66. RBCs
  • 67.
  • 68.
  • 71.
  • 72.
  • 73. Shape Changes in Platelets
  • 74. Normal Person Thrombasthenia Glanzmann Down Regulation of Receptors Platelets
  • 75. CD62p Assay - Pre-activated Platelets
  • 77. Immune system cellular immune response humor immune response T B G M
  • 78.
  • 79.
  • 80. Immunodeficiencies primary = congenital secondary = acquired
  • 81.
  • 82.
  • 83.
  • 84.
  • 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
  • 87.
  • 88.
  • 89.
  • 90.