Blood Smear Preparation and Staining

Procedure for Blood Smear Preparation and Staining in Haematological Labs

Blood smears are a cornerstone of haematological diagnostics, allowing for the visualization and evaluation of blood cell morphology, identification of abnormalities, and detection of blood-borne pathogens. Proper preparation and staining of blood smears are crucial for accurate and reliable microscopic analysis.

Requirements for Blood Smear Preparation

To ensure high-quality blood smear preparation and staining, the following materials and equipment are needed:

1. Materials

• Glass slides: Clean, grease-free microscope slides.
• Spreader slide: A second slide is used to spread the blood.
• Capillary tubes or micropipettes: For blood collection (with EDTA if using an anticoagulated sample).
• Marker or pencil: To label the slides.
• Immersion oil: For high-resolution microscopy.

2. Reagents

• Anticoagulated blood sample: Collected using EDTA to prevent clotting. Ideally, the smear should be prepared within 2 hours to avoid morphological changes.
• Romanowsky stains: Includes:
  • Wright’s stain
  • Leishman’s stain
  • Giemsa stain
  • May-Grünwald stain

3. Staining Equipment

• Staining jars: For dipping slides into stain and buffer solutions.
• Buffered distilled water: Typically, pH 6.8 for optimal staining.

4. Microscope

   

• Light microscope with an oil immersion objective (100x magnification) for detailed examination.

 

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Blood Smear Preparation Procedure

The quality of the blood smear depends heavily on the technique used. Below is a step-by-step guide to creating a well-prepared smear:

1. Labelling the Slide

• Use a pencil or marker to label one end of the glass slide with patient identification information, as some stains (e.g., Wright’s) can wash off ink.

2. Blood Sample Collection

• Collect 2-3 µL of blood using a capillary tube or micropipette.
  • Mix the sample using anticoagulated blood by gently inverting the collection tube to prevent clumping.

3. Applying the Blood to the Slide

• Place the drop of blood about 1-2 cm from one end of the slide. This should be a small, well-formed drop—if too large, it will spread unevenly.

4. Spreading the Blood

• Hold the spreader slide at a 30-45° angle.
• Pull the spreader slide backwards to touch the blood drop, allowing the blood to spread along the edge of the spreader slide.
• Push the spreader slide forward in a quick, smooth motion to create an even smear.
  • The smear should thin out towards the other end of the slide, forming a feathered edge where cells are spread in a monolayer.

5. Air Drying

• Allow the slide to air dry completely. Avoid blowing on the slide or using heat, which can distort the cells.

 

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Staining Procedure for Blood Smears

Staining is critical for differentiating various blood cells, as the dyes interact with cell components based on their chemical properties. The most commonly used stains are Romanowsky-type stains, such as Leishman, Wright, and Giemsa stains.

1. Leishman Staining Procedure

Leishman’s stain is commonly used in haematology for blood smears due to its simplicity and quick application.

Steps:

1. Fixation:
   • Leishman stain contains methanol, which acts as a fixative.
   • Flood the slide with Leishman stain and allow it to stand for 2 minutes for fixation.
2. Staining:
   • Add double the volume of buffered water (pH 6.8) to the stain on the slide and gently mix by tilting the slide.
   • For complete staining, allow the stain to remain on the slide for 8-10 minutes.
3. Washing:
   • Wash the slide under a gently running tap or distilled water to remove excess stains.
   • Allow the slide to air dry in an upright position.

 

2. Wright’s Staining Procedure

Wright’s stain is a quick and effective stain used in most haematology labs for routine blood smear staining.

Steps:

1. Fixation:
   • Flood the slide with Wright’s stain and let it stand for 1-3 minutes for fixation (the methanol in the stain acts as the fixative).
2. Staining:
   • Add an equal volume of buffered distilled water (pH 6.8) to the stain.
   • Mix gently by tilting the slide and allow the stain to act for 4-5 minutes.
3. Washing:
   • Rinse the slide gently with buffered water or distilled water.
   • Allow the slide to air dry before microscopic examination.

 

3. Giemsa Staining Procedure

Giemsa stain is often used for more detailed cellular and parasitic studies, including malaria detection.

Steps:

1. Fixation:
   • Fix the smear by dipping the slide into methanol for 1-2 minutes.
   • Allow the slide to air dry completely before staining.
2. Staining:
   • Flood the slide with diluted Giemsa stain (1:20 dilution in buffered water with pH 6.8).
   • Let the stain act for 20-30 minutes.
3. Washing:
   • Wash the slide under running water or use distilled water.
   • Allow the slide to air dry in an upright position.

 

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Microscopic Examination of the Blood Smear

Once the blood smear is prepared and stained, it is ready for microscopic examination. The following steps outline how to examine the smear properly:

1. Low Power Scanning (10x or 40x objective)

• Begin with low magnification to identify the area where the cells are evenly distributed (the feathered edge of the smear).
• Ensure the cells are well-spread and not overlapping. This area provides the most reliable data for differential counts.

2. High Power Examination (100x Oil Immersion)

• Apply a small drop of immersion oil to the slide and switch to the 100x oil immersion objective.
• Examine the blood cells closely, paying attention to:
  • Red blood cells (RBCs): Evaluate their shape, size, colour (central pallor), and any inclusions or deformities (e.g., anisocytosis, poikilocytosis).
  • White blood cells (WBCs): Perform a differential count, noting the proportion of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Check for abnormal or immature forms of WBCs (e.g., blasts).
  • Platelets: Assess their number, size, and distribution.

 

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Common Problems and Solutions in Blood Smear Preparation and Staining

1. Thick or Thin Smear:
   • Problem: If the smear is too thick or too thin, the cells may overlap, or there may not be enough cells to observe.
   • Solution: Ensure the proper size of the blood drop and maintain a 30-45° angle while spreading.
2. Uneven Smear:
   • Problem: Uneven smears lead to poorly distributed cells, making differential counts inaccurate.
   • Solution: Ensure the spreader slide moves smoothly and quickly across the slide in one motion.
3. Artifacts in Stain:
   • Problem: Artifacts can appear as unwanted deposits or distortions, making it difficult to differentiate cellular structures.
   • Solution: Use clean, grease-free slides and freshly prepared stain solutions. Also, avoid air bubbles or contaminations during the staining process.
4. Understaining or Overstaining:
   • Problem: Understained smears make it difficult to differentiate cells, while overstained smears can obscure cell morphology.
   • Solution: Follow the appropriate staining times for each type of stain and ensure the pH of the buffer is correct (typically 6.8).