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    04 Sample Preparation and Processing

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    candiddreams
    Flow cytometric analysis involves three stages: preanalytical specimen processing, analytical running of samples through the flow cytometer, and postanalytical data analysis and interpretation. Samples can be liquid specimens like blood or body fluids, or solid tissues like lymph nodes. Proper sample collection and handling is important to ensure cell viability and allow for accurate analysis. Samples are stained with fluorescent antibodies before running through the flow cytometer, which detects the antibodies and categorizes cells based on their surface marker expression profiles. Data analysis identifies cell populations and abnormalities.

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    04 Sample Preparation and Processing

    Uploaded by

    candiddreams
    32 views6 pages
    Flow cytometric analysis involves three stages: preanalytical specimen processing, analytical running of samples through the flow cytometer, and postanalytical data analysis and interpretation. Samples can be liquid specimens like blood or body fluids, or solid tissues like lymph nodes. Proper sample collection and handling is important to ensure cell viability and allow for accurate analysis. Samples are stained with fluorescent antibodies before running through the flow cytometer, which detects the antibodies and categorizes cells based on their surface marker expression profiles. Data analysis identifies cell populations and abnormalities.

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    04 Sample Preparation and Processing

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    Flow cytometric analysis involves three stages: preanalytical specimen processing, analytical running of samples through the flow cytometer, and postanalytical data analysis and interpretation. Samples can be liquid specimens like blood or body fluids, or solid tissues like lymph nodes. Proper sample collection and handling is important to ensure cell viability and allow for accurate analysis. Samples are stained with fluorescent antibodies before running through the flow cytometer, which detects the antibodies and categorizes cells based on their surface marker expression profiles. Data analysis identifies cell populations and abnormalities.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    32 views6 pages

    04 Sample Preparation and Processing

    Uploaded by

    candiddreams
    Flow cytometric analysis involves three stages: preanalytical specimen processing, analytical running of samples through the flow cytometer, and postanalytical data analysis and interpretation. Samples can be liquid specimens like blood or body fluids, or solid tissues like lymph nodes. Proper sample collection and handling is important to ensure cell viability and allow for accurate analysis. Samples are stained with fluorescent antibodies before running through the flow cytometer, which detects the antibodies and categorizes cells based on their surface marker expression profiles. Data analysis identifies cell populations and abnormalities.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 6

    03-10-2012

    1
    Flow cytometric analysis involves 3 stages:
    Preanalytical - Specimen handling & processing,
    including Ab staining
    Analytical - Running sample through the flow
    cytometer & acquiring data
    Postanalytical - Data analysis & interpretation
    Dr.GarimaGoel,MAMC
    2 broad groups:
    Liquid specimens
    Peripheral blood
    Bone marrow aspirate (BMA)
    Body fluids
    Dr.GarimaGoel,MAMC
    2 broad groups:
    Solid tissue specimens
    Lymph nodes
    Bone marrow biopsy
    Tonsils/adenoids
    Spleen
    Dr.GarimaGoel,MAMC
    Peripheral blood & BMA samples:
    EDTA - anticoagulant of choice
    Hemogram
    Smears can be prepared
    Specimen processed within 24hrs
    Heparin
    Specimen processed within 48 hrs
    Dr.GarimaGoel,MAMC
    Solid tissue samples:
    Submitted as thin slices (<2mm thick) in sterile
    tissue culture media at 4

    C.
    Dr.GarimaGoel,MAMC
    03-10-2012
    2
    Ideally sample - processed within 48 hours of
    collection
    For tumors with high turnover rate - time
    window
    Dr.GarimaGoel,MAMC
    WBC count, differential & unstained blood
    film - accompany each specimen
    Specimens - 18

    -22

    C in a leak proof
    container
    Temp. <4C & >30C - avoided
    Dr.GarimaGoel,MAMC
    Poor sample collection major source of
    potential unsatisfactory FCM analysis
    Factors affecting viability of cells
    Time elapsed between sample collection &
    delivery to lab
    Environmental conditions during transport of
    sample to lab
    Dr.GarimaGoel,MAMC
    Samples unacceptable for analysis:
    Exposure to extreme temperature
    Presence of blood clots
    Grossly hemolysed sample
    Dr.GarimaGoel,MAMC
    Dr.GarimaGoel,MAMC
    Sample volume (peripheral blood & BMA) -
    0.5 to 1ml in each tube
    Concentration of nucleated cells in each tube
    for test - 0.5 to 2x10
    6
    /ml (MC - 1x10
    6
    /ml or
    1x10
    3
    /l)
    TLC of sample - <10
    4
    /l
    If more dilute the sample with PBS
    If less take more sample to decrease dilution
    Dr.GarimaGoel,MAMC
    03-10-2012
    3
    Lyses of RBCs
    Removal of lysed RBCs
    Staining with Abs
    Dr.GarimaGoel,MAMC
    Nucleated cells - separated from RBCs
    RBC lysis - before or after staining with Abs
    Lysing agents
    Can be with or without fixative
    E.g. - Ammonium chloride
    Dr.GarimaGoel,MAMC
    Failure of RBC lysis
    Reticulocytes in peripheral blood
    Lipids in serum
    Dr.GarimaGoel,MAMC
    Lysed RBCs
    Removed by multiple (at least 2) washings with
    isotonic fluid
    Isotonic fluid e.g. Phosphate Buffered Saline
    Dr.GarimaGoel,MAMC
    Staining with desired Abs -before or after RBC lysis
    2 types of antigens
    Cell surface antigen ( )
    e.g. CD19, CD10,
    CD13, CD33, CD117,
    CD38 etc.
    Intracellular antigens ( )
    e.g. TdT, cytoplasmic
    light chains, cCD3,
    cCD22, MPO, bcl-2 etc.
    Dr.GarimaGoel,MAMC
    Cell surface Ag
    Staining done on
    viable unfixed cells
    Intracellular Ag
    Staining requires cell
    fixation &
    permeabilization
    Dr.GarimaGoel,MAMC
    03-10-2012
    4
    To maintain structural integrity
    Fixative
    Preserve epitope of intracellular antigen
    Without aggregation of cell suspension
    E.g. formaldehyde
    Dr.GarimaGoel,MAMC
    To allow Abs to reach appropriate
    intracellular targets
    Permeabilizing agent - low concentration non
    ionic detergents like saponin
    Dr.GarimaGoel,MAMC
    Simultaneous detection of intracellular & cell
    surface Ag - sensitivity of diagnosis
    Ensure stability of AgAbfluorochrome
    complexes on cell surface
    Preservation of targeted intracellular
    antigens
    Dr.GarimaGoel,MAMC
    E.g.
    CD19 & 10 with TdT
    CD22 & cCD3
    Dr.GarimaGoel,MAMC
    For each sample analyzed one control tube
    is required
    Cells are mixed in the presence of an isotypic
    control
    Determines the level of non specific binding &
    autofluroscence
    Dr.GarimaGoel,MAMC
    100l sample + Abs
    Vortex +20mins incubation in dark
    500l lysing agent
    2 washes with isotonic fluid
    Resuspendin 1ml isotonic fluid
    Vortex +10mins incubation in dark
    Dr.GarimaGoel,MAMC
    03-10-2012
    5
    50 50l sample + surface Abs
    15mins incubation in dark
    100l fixative + vigourous vortex +15mins incubation
    1 wash with isotonic fluid +100l permeabilizing agent 1 wash with isotonic fluid +100l permeabilizing agent
    Resuspendin 0.5ml isotonic fluid
    DO NOT VORTEX +5mins incubation in dark DO NOT VORTEX +5mins incubation in dark
    2 washes with isotonic fluid
    Add Intracellular Abs +15mins incubation in dark Add Intracellular Abs +15mins incubation in dark
    Dr.GarimaGoel,MAMC
    Type of Ab monoclonal or polyclonal
    Ab isotype - IgG1, G2 or M
    Ab clone
    Dye conjugation
    Stoichiometry between Ab & fluorochrome
    Dr.GarimaGoel,MAMC
    Dyes- accept light energy at a given wavelength
    & re-emit it at a longer wavelength
    Different fluorochromes
    FITC Fluoresicein Isothiocyanate
    PE Phycoerythrin
    ECD PhycoerythrinTexas Red
    PC5 &7 - Phycoerythrin-Cyanin5 &7
    APC Allophycocyanin
    Dr.GarimaGoel,MAMC
    Depends on
    Fluorochrome brightness
    Ag density
    Background staining of Ab
    Amount of compensation required between
    conjugates
    Single or multiple laser
    Dr.GarimaGoel,MAMC
    Brighter fluorochrome used for
    Dull Ab
    Low Ag expression
    For staining cells with high autofluoroscent
    background
    E.g. PE is used with CD25
    Dr.GarimaGoel,MAMC
    Duller fluorochrome used for
    Bright Ab
    High Ag expression
    E.g. FITC with anti kappa, anti lambda, CD45
    Dr.GarimaGoel,MAMC
    03-10-2012
    6
    Simultaneous use of >1 flurochrome
    Saves time & sample
    Exponential in information
    Helps identify new/rare cell populations
    no. of tubes in Ab panel
    no. cells required for entire FCM analysis
    Provides internal controls
    Dr.GarimaGoel,MAMC
    Carefully choose combinations of
    flurochrome conjugates
    Non availability of all reagents in all colours
    Greater potential for errors in compensation
    Requirement of proper controls
    Dr.GarimaGoel,MAMC
    Conjugated antibodies
    Stored between 2 & 8

    C
    Protected from light
    Dr.GarimaGoel,MAMC
    Do not use reagent beyond expiry date
    Do not freeze reagent or sample
    Let reagent come down to room temp before
    use
    Dr.GarimaGoel,MAMC
    Minimize exposure to light
    Avoid microbial contamination of reagents
    Absolutions like anti kappa & anti lambda
    contain sodium azide& should be handled
    carefully
    Dr.GarimaGoel,MAMC Dr.GarimaGoel,MAMC

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