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    Preparation and Staining of Peripheral Blood Film

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    Raja Som Raj
    A peripheral blood smear provides information about blood cells. It involves three steps: preparation of a blood smear using the wedge smear method, fixation of the smear, and staining. For routine work, venous blood is used to make a wedge smear by placing a drop of blood on a slide and spreading it using a spreader at a 45 degree angle. The smear is then fixed and stained using the Romanowsky method, such as Leishman's stain, which uses methylene blue and eosin dyes to stain cell components different colors for examination. Proper preparation is required to obtain an acceptable smear for accurate analysis of blood cells.

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    Preparation and Staining of Peripheral Blood Film

    Uploaded by

    Raja Som Raj
    186 views20 pages
    A peripheral blood smear provides information about blood cells. It involves three steps: preparation of a blood smear using the wedge smear method, fixation of the smear, and staining. For routine work, venous blood is used to make a wedge smear by placing a drop of blood on a slide and spreading it using a spreader at a 45 degree angle. The smear is then fixed and stained using the Romanowsky method, such as Leishman's stain, which uses methylene blue and eosin dyes to stain cell components different colors for examination. Proper preparation is required to obtain an acceptable smear for accurate analysis of blood cells.

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    PREPARATION AND STAINING OF PERIPHERAL BLOOD FILM

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    A peripheral blood smear provides information about blood cells. It involves three steps: preparation of a blood smear using the wedge smear method, fixation of the smear, and staining. For routine work, venous blood is used to make a wedge smear by placing a drop of blood on a slide and spreading it using a spreader at a 45 degree angle. The smear is then fixed and stained using the Romanowsky method, such as Leishman's stain, which uses methylene blue and eosin dyes to stain cell components different colors for examination. Proper preparation is required to obtain an acceptable smear for accurate analysis of blood cells.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    186 views20 pages

    Preparation and Staining of Peripheral Blood Film

    Uploaded by

    Raja Som Raj
    A peripheral blood smear provides information about blood cells. It involves three steps: preparation of a blood smear using the wedge smear method, fixation of the smear, and staining. For routine work, venous blood is used to make a wedge smear by placing a drop of blood on a slide and spreading it using a spreader at a 45 degree angle. The smear is then fixed and stained using the Romanowsky method, such as Leishman's stain, which uses methylene blue and eosin dyes to stain cell components different colors for examination. Proper preparation is required to obtain an acceptable smear for accurate analysis of blood cells.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 20

    PREPARATION AND STAINING OF

    PERIPHERAL BLOOD FILM


    Introduction

    • A blood smear is a blood test that gives information about the


    number and shape of blood cells

    • Examination of a fixed and stained blood film is an essential


    part of a haematological examination

    • To obtain maximum information, the films must be well


    spread, well stained and examined systematically
    Making of a blood film

    There are three basic steps in making of a blood film:

    • Preparation of blood smear


    • Fixation of blood smear
    • Staining of blood smear
    Blood smear preparation

    • Three methods may be used for preparation of blood smears:

    • The cover glass smear


    • The wedge smear
    • The spun smear
    The spun smear requires an automated slide spinner
    For routine laboratory work, we use wedge smear
    Wedge Blood Smear

    Specimen:
    • EDTA anticoagulated venous blood : Smears should be made
    within 1 hour of collection to avoid distortion of cell
    morphology

    • Capillary blood : Obtained by finger prick method

    Equipment:
    • Spreaders
    • Clean slides
    • Applicator stick wood
    • Blood capillary tube or micropipette
    Procedure

    • Fill a capillary tube three quarter full with anticoagulated


    specimen or a wooden stick

    • A drop of blood , about 2 mm in diameter an inch from the


    frosted area of the slide

    • Place the narrow edge of the spreader slide just in front of the
    drop of blood at 45 degree angle

    • Draw the spreader backwards so as to touch the drop and


    hold there till the blood runs along the full width of the
    spreader at the line of junction
    • The spreader is then moved slowly and smoothly to the other
    end of the slide maintaning an angle of 45 degree

    • Allow the slide to dry

    • The thickness of the spread is determined by


    - Angle of the spreader slide (Greater the angle, the thicker and
    shorter the smear)
    - Size of the blood drop
    - Speed of spreading
    Modifications in spreader angle in
    polycythemia and anemia
    Unacceptable peripheral blood
    films. Slide appearances associated
    with the most common errors are
    shown, but note that a
    combination of causes may be
    responsible for unacceptable
    films. A, Chipped or rough edge on
    spreader slide. B, Hesitation in
    forward motion of spreader
    slide. C, Spreader slide pushed too
    quickly. D, Drop of blood too
    small. E, Drop of blood not allowed
    to spread across the width of the
    slide. F, Dirt or grease on the slide;
    may also be due to elevated lipids
    in the blood specimen. G, Uneven
    pressure on the spreader
    slide. H, Time delay; drop of blood
    began to dry.
    Fixing and staining of blood films

    Romanowsky stains :
    • Romanowsky stains are universally employed for staining
    blood films
    • Examples include: May –Grunwald-Giemsa s
    Giemsa stain
    Wright’s stain
    Field’s stain
    Leishman’s stain
    Romanowsky Principle:

    • The main components of Romanowsky stains are:


    • A cationic or basic dye (Methylene blue or its oxidation
    products like azure B) which binds to anionic sites and gives a
    blue-grey colour to nucleic acids (DNA & RNA),
    nucleoproteins, granules of basophils and weakly to granules
    of neutrophils

    • An anionic or acidic dye such as eosin Y or eosin B which


    binds to cationic sites on proteins and gives a orange-red
    colour to haemoglobin and eosinophil granules
    Leishman’s Stain

    • It is a polychromatic stain
    • Methanol: fixes cells to slide
    • Methylene blue: Stains RNA, DNA (Blue grey colour)
    • Eosin stains hemoglobin, eosin granules (Orange red colour)
    • Sorensen’s phosphate buffer(pH-6.8)
    Staining Procedure
    The smear is air dried

    Flood the smear with the stain Leave for 2 mins

    Add double the volume of buffered water and stain for 5-7 mins

    Mix stain and water evenly by gentle blowing and intermittently


    with the help of a dropper

    If dilution of the stain is correct, the fluid will be covered by a


    thin, greenish scum Wash it in a stream of buffered water
    until it acquires a pinkish tinge (upto 2 mins)
    Thick smears are used for detection of malarial
    parasites while thin smears are used for species
    identification
    THANK YOU

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