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Presented By Dr.Mohammed Attia Director of flow cytometry Facility Tanta University.

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Presentation on theme: "Presented By Dr.Mohammed Attia Director of flow cytometry Facility Tanta University."— Presentation transcript:

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2 Presented By Dr.Mohammed Attia Director of flow cytometry Facility Tanta University

3 FCM is a technique for counting, examining and sorting microscopic particles suspended in a fluidFCM is a technique for counting, examining and sorting microscopic particles suspended in a fluid It allows simultanous analysis of the physical and /or chemical charactaristics of the examined particles It allows simultanous analysis of the physical and /or chemical charactaristics of the examined particles

4 FCM involves the analysis of flourescence and light scatter properties of single particle ( e.g. Cell, nuclei, chromosome, bacteria, bead ect) during its passage within a liquid streamFCM involves the analysis of flourescence and light scatter properties of single particle ( e.g. Cell, nuclei, chromosome, bacteria, bead ect) during its passage within a liquid stream

5 Light scatter

6  Neutrophils  Lymphocytes  Monocytes  Eosinophils  Basophils

7 Light scatter

8 To Distinguish Between 2 Cell types A. Size Has To Be Different OR B. Internal Complexity If These Two Parameters Are The Same, Then No Distinction Can Be Made Limitations of Light Scatter

9 Immuno Cytology membranous /intracellular structures dye-coupled monoclonal antibody (mab) Y

10 Simultaneous Detection of up to 20 Parameters  Cell size  Cell structure  Dyes up to 18 colors Y

11 Data display

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14 Advantages Analysis of high cell numbers (10 7 cells) Short time of analysis (sec) Gating allows detection of cell sub-populations Measurement of rare events Objective measurement of fluorescence intensity Simultaneous detection of many parameters per cell Advantages Analysis of high cell numbers (10 7 cells) Short time of analysis (sec) Gating allows detection of cell sub-populations Measurement of rare events Objective measurement of fluorescence intensity Simultaneous detection of many parameters per cell

15 Disadvantages Relatively expensive technology Test cell in suspension Tissue architecture is lost Disadvantages Relatively expensive technology Test cell in suspension Tissue architecture is lost

16  Blood  Bone marrow  BAL  C S F  Effusions  Cell culture  Sperms  Tissue cells e.g. tumor Prerequisite Single Cell Suspension Prerequisite Single Cell Suspension

17 Research Laboratories Clinical Laboratories

18 Research Laboratories  Immune function studies  Hematopoietic stem cell research and regenerative medicine  Apoptosis  Cell cycle and cell Kinetics  Intracellular cytokine production  Intracellular signaling  Sperms and IVF  Flow and FISH  Microbiology and virology

19 Clinical Laboratories  Haematology  Haemato-oncology  Clinical immunology  Organ transplantation and Stem cell therapy  Cancer  Rare event applications  Application in clinical microbiology

20 RBCs

21  Autoantibodies in H A  Fetal RBCs in fetomaternal hge  Blood groups  Membrane proteins  Fragility of RBCs ( OFT)  PNH

22 PNH Principle of FCM: Absent or markedly diminished expression of glycosyl phosphatidylinositol-anchored protein (GPI-AP) on red cells and/or white cells in the appropriate clinical setting. GPI-AP - CD59 (MIRL) & CD55 (DAF) Use two GPI-AP for confirmation

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24 Blood transfusion Quality control of blood products * Enumerate number of residual WBC contaminating 1- Leucodepleted RBCs unit 2- Platelets unit * Enumerate number of residual WBC, RBC & platelets in FFP units

25 Haematology

26 GPIIb-IIIaCD41/CD61 GPIb-IXCD42b/CD42a GPIa-IIaCD49b/CD29 GPIc-IIaCD49e/CD29 GPIc-IIaCD49f/CD29 GPIIaCD31 GPIVCD36 GP53CD63 Vitronectinreceptor  CD51 Vitronectinreceptor  CD61 Thrombinreceptor Thrombospondin Thrombinreceptor CD62p

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28 Haemato-oncology # Acute leukemia # LPD

29 Acute leukemia's ALL AML M0 M1 M2 M3 M4 M5 M6 M7 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 BAL Haemato-oncology

30 # CLL # NHL ( FCL, MCL, SLVL, LPL etc) # Hairy cell leukaemia

31 Diagnosis and classification of L&L  Detect malignant cells  Define lineage (Myliod, T or B)  Sub-typing and classification  Prognosis (DNA Ploidy, CD38 & ZAP 70)  Follow up and therapy monitoring (MRD)

32 Acute Leukaemia Panel

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34 Case study n Seven (7) year old, male patient, referred with Clinical history of Mediastinal Mass. n P B Examination showed high Leukocyte Count with 26% Blasts.

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36 Looking at this case display you can tell that there is an abnormal population (green) that is v.bright CD45 “brighter than normal lymphocytes” and has a high side scatter. There is almost no normal lymphocyte in this bone marrow specimen.

37 This is a very informative slide and probably gave you the diagnosis already. Looking at the CD3/CD4/CD8 patterns we can see that the majority of the gated cells are dual positive for CD4/CD8 and negative for surface CD3. This pattern is very uncommon in bone marrow and indicate the immature T-cell nature of the blasts. Among the abnormal cells you can recognize the normal lymphocytes pattern (see red arrow).

38 Looking at this slide we learn more about the nature of these cells. We find that they are negative for CD10,CD19,CD34,CD33,CD13 and positive for CD7. The later antibody “CD7” confirm the T cell lineage of the abnormal cells.

39 The abnormal cells are positive for CD2 and CD11b and negative for HLA-DR. Usually in most cases of the T- cells they tend to be negative for HLA-DR. The positivity of CD11b is also atypical of this kind of malignancy.

40 Again the abnormal cells continue to confirm its T-cell lineage; they are positive for CD56, CD5, partially CD1a and negative for CD117 and CD64. Usually the expression of CD56 in leukemias is associated with worse prognosis.

41 The cytoplasmic staining of TdT and CD3 confirm the immature nature of the blasts.

42 Antigen profile: Positive for CD4, CD8, CD7, CD2, CD5, CD56, partially CD1a, cCD3 and negative for HLA-DR. n Diagnosis: Acute T-cell Leukemia

43 # The lower level of disease (residual malignant cells) detectable in patients after therapy. # Aberrant phenotypic pattern by FCM and Leukemic cell DNA OR RNA by PCR are the most widely used as they have the most sensitive and specific level

44  ALL It requires the identification of specific pattern of Ags at presentation which then sought at follow up  T-ALL (TdT and CD3) reliable up to 1 in 10 4 since normal BM doesnt contain it  B-ALL (19, 34, 10 &TdT, 20,38,58,45 )

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46 Immunology

47 #Lymphocyte Sub-setting: T,B & NK cells (CD3/CD4/CD8/CD19/CD16+56) * HIV patient monitoring * SCID patients * Immuno compromised patients

48 #T cell function assay FCM can assess lymphocyte activation of * T helper CD4 cells * T cytotoxic CD8 cells

49 Applying Gates for sub-population analysis Simple gating stratagies … Whole blood light scatter Gate on lymphocytes (light scatter) Assess T-cell population (fluorescence)

50 …to more complex!

51 #Neutrophil Functional assay Number of ready to use kits are available assess Neutrophil * Migratest (measure chemotactic & adhesion) * Phagotest (determine phagocytic activity) * Oxidative Burstest (Quantify oxidative burst activity)

52 Organ transplantation and Stem cell therapy

53 Organ transplantation and Stem cell therapy  Stem cell enumeration -HSC Banking -HSC Transplantation  HLA cross matching  Pre & Post Transplantation monitoring of immune system

54 Stem Cell enumeration

55 Cancer

56  DNA Ploidy and cell cycle kinetics  MDR  Circulating Tumor cells ( CTCs)

57 DNA analysis: Aneuploidy and/or elevated S-phase fraction have been shown to be prognostic indicators in breast, colon, rectal, prostate, ALL and bladder tumors.

58 Cell Cycle Analysis Note the cell volume (size) and DNA concentration change as the cell progresses through the cell cycle

59 Cell Cycle Analysis DNA probes DAPI} Hoechst}UV Propidium iodide (PI)} 7-AAD}488 TOPRO-3} DRAQ5}633 These dyes are stoichiometric – number of bound molecules are equivalent to the number of DNA molecules present The cell cycle

60 l Typical DNA histogram Stoichiometric DNA probe binding Cell Cycle Analysis

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62 Assessing cell proliferation using FCM

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64 Apoptosis Gene directed cell death An event that occurs during development and a response to trauma or disease Cancer cells develop a strategy to evade apoptosis Apoptosis results in a number of cellular events that can be analysed by FACS: Fragmentation of DNA (subG1 assay, Hoechst dyes) Membrane structure and integrity Annexin-V, PI) Mitochondrial function (Mitotracker Red) Caspase activity (antibodies assay)

65 Sub G1 apoptosis assay Sub-G1 peak DNA fragmentation allows apoptosis to be quickly assessed with eg. PI Can be seen as a population of small peaks to the left of G1 in a histogram Quick and easy way to determine if apoptosis is occurring Apoptosis

66 Apoptosis detection using viability dye uptake Changes in membrane permeability due to apoptosis allow intracellular dyes to stain unfixed cells 7-AAD (DNA) Live cells exclude dye Apoptotic cells stain 7-AAD dim Dead cells stain 7-AAD bright Apoptosis

67 Annexin-V/PI assay for apoptosis:  PS normally on inside of cellular membrane  AnnV can bind to externalised PS highlighting cells that are apoptotic  PI will only go into cells with compromised membranes – dead (necrotic) cells AnnV-FITC PS XXXXXX XXXXXX PI Apoptosis

68 Membrane potential of the organelle reduced Mitochondrial activity appears to change in parallel with cytoplasmic and plasma membrane events Dyes that accumulate in mitochondria can therefore play role in detecting apoptosis -Mitotracker Red CMXRos -JC-1 -DiOC 2 (3) -Laser Dye Styryl-751 (LDS-751) Reagent combinations can provide a window on intracellular processes not available with the much used pairing of annexin V and propidium iodide Apoptosis – Organelle Analysis Apoptosis

69 Live/Dead assay Utilise the properties of dyes that are impermeable to intact cell membranes: Propidium iodide DAPI TOPRO-3 +ve fluorescence indicates compromised cell membranes and therefore dead cells Yeast cells + TOPRO-3 Dead cells show more granularity and reduced size Live cells retain their morphology and appear larger in size and less granular Apoptosis

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71  Cell sorting  Allows rare populations to be isolated from heterogenous populations (cell culture, blood samples, etc)  Can isolate sub cellular particles (e.g. endosomes, nucleus, chromosomes)  Can produce purity >95%

72 Thank You


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