Preparation and staining of peripheral blood smear(PS)|A Step-by-Step Guide: Peripheral Blood Smear Preparation and Staining

Introduction: Peripheral blood smear preparation and staining is a fundamental technique used in clinical laboratories to examine the cellular components of blood. It involves spreading a thin layer of blood on a glass slide, fixing the cells, and applying appropriate stains to enhance the visibility of different blood cell types. This article will guide the preparation and staining process, highlighting key considerations and best practices.

Step 1: Collection of Blood Sample for peripheral blood smear

The first step in preparing a peripheral blood smear is to collect a blood sample using appropriate aseptic techniques. A venipuncture is typically performed on the patient’s arm using a sterile needle and evacuated tubes containing anticoagulants, such as ethylenediaminetetraacetic acid (EDTA). Heparinized blood should be avoided because it imparts a bluish tinge in staining

Blood films can be prepared from fresh blood from a finger prick using a lancet or needle.

Step 2: Preparing the Smear:

To create a peripheral blood smear, follow these steps:

  • Obtain a clean glass,slide should wipe clean to remove any dust
  • Place a small drop of well-mixed blood about 1cm from  one end of the slide
  • without delay, Using a spreader slide, place it against the blood droplet at a 45-degree angle.
  • Slowly and smoothly pull the spreader slide backward, allowing the blood to spread along the edge of the slide. Maintain a steady speed to achieve an even distribution of blood across the slide(refer video
  • The spreader slide should always have a smooth edge that results smooth smear formation without irregularity on tail
  • Lift the spreader slide away from the main slide when the blood has spread across its entire width.
  • Allow the smear to air dry completely before proceeding to the next step.

characteristics of a good smear

    • Good smear is tongue shaped with smooth tail
    • Has both thin and thick areas
    • No lines or holes
    • Should occupy 2/3 of the total slide
    • Should not touch any edge of slide
    • Should be Margin free except point of application

Step 3: Fixation:

Fixation helps preserve cellular morphology and prevents artifacts during staining.  Dip the air-dried smear into a container of absolute methanol for 1-2 minutes. This fixes the cells by denaturing proteins. If you are using leishman stain fixation is not required

Step 4 : Staining 

  • Romanowsky stains are routinely used. These stains contain the combinations of acidic and basic dyes that produces subtle distinction between staining of cell and staining of granules differentially Granules in neutrophils are stained by Azure complexes, eosinophilic granules by acidic components of dye and basophilic granules which contain acid heparin has affinity for basic component of dye
  • Different types of Romanowsky  stains
    • Leishman’s stain
    • Wright’s stain
    • Giemsa’s stain
    • Jenner’s stain
    • Field’s stain
    • May Grunwald Giemsa
Leishman Staining:

Use commercially available stain or prepare stain as follows

1.Add 1.5 grams of leishman powder to 500 ml methanol ,add few glass beads

2.shake well, then incubate at 37 overnight

3.  Filter the stain if u see particles

Method of staining:

  • Place the air-dried smear on a staining rack or staining dish.
  • Cover the smear completely with the Leishman stain solution.
  • leave for 30 seconds to 1 min to fix
  • add twice as much as buffered water (pH 7.2)
  • leave for 8-10 min ,don’t do staining under fan it will cause drying and precipitation, A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate.
  • wash off stain under tap water or distilled water
  • Clean the backside of slide with tissue paper or cloth
  • Allow slide to dry at room temperature, slide is now ready for microscopic examination
wright staining 
  1. Staining Solution
    Wright’s stain powder = 1.0 gm
    Water free methanol = 400 ml
  2. Phosphate buffer (0.15M, ph 6.5/6.8)
    Potassium dihydrogen phosphate, anhydrous = 0.663 gm
    Disodium hydrogen phosphate, anhydrous = 0.256 gm
    Distilled water = 100 ml

Method of staining:

  • Place the air-dried smear on the slide staining rack, smear side facing upwards.
  • Cover the blood film with undiluted staining solution. The undiluted stain fixes and partially stains the smear.
  • Let stand for 2-3 minutes.
  • Add approximately equal amount of buffered water (pH 6.5). The diluted stain shouldn’t overflow. Mix by gentle blowing. A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate.
  • Leave for 5 minutes.
  • Without disturbing the slide, flood the distilled water and wash until the thinner parts of the film are pinkish red.
  • Allow the slide to dry at room temperature and examine under microscope.

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