Making and Staining Blood Smears

Blood smears are an essential tool in hematology, allowing for the microscopic examination of blood cells. Proper preparation and staining of blood smears enable accurate identification and evaluation of red blood cells (RBCs), white blood cells (WBCs), platelets, and potential blood-borne parasites. This guide provides a comprehensive overview of the steps and best practices for making and staining effective blood smears.

1. Purpose of Blood Smears

Blood smears serve several diagnostic purposes:

• Evaluating the morphology of blood cells.
• Estimating white blood cell differential counts.
• Detecting blood-borne parasites like Plasmodium species (malaria).
• Assessing platelet number and morphology.
• Monitoring treatment in hematologic diseases.

2. Materials Needed

• Clean microscope slides (preferably labeled)
• Capillary tubes, pipettes, or an EDTA blood sample
• Spreaders (usually another clean slide or a dedicated spreader slide)
• Staining reagents (e.g., Wrights stain, Giemsa stain, or Diff-Quik)
• Buffered water or phosphate buffer (pH 6.87.2)
• Distilled water
• Gloves and personal protective equipment
• Methanol (for fixing the smear, depending on stain protocol)
• Microscope

3. Making the Blood Smear

3.1 Collecting the Sample

Blood is usually collected into an EDTA tube to prevent clotting. Using a capillary tube or pipette, place a small drop of blood near one end of a clean slide. The drop should be about 23 mm in diameter - too large will make smears difficult to manage; too small will produce insufficient areas for examination.

3.2 Preparation of the Smear

Place a second slide (the "spreader") at a 3045 angle touching the first slide just ahead of the drop of blood. The blood will spread along the edge of the spreader slide by capillary action.

Quickly and smoothly push the spreader slide forward to spread the blood across the first slide in a thin, even layer. The smear should start thick at the blood drop end and gradually become thinner to form a "feathered edge."

3.3 Features of a Good Blood Smear

• The smear should be a single layer of cells without clumps or overlapping.
• The blood cells will be evenly distributed, especially at the feathered edge.
• The smear should dry quickly to prevent cell distortion.
• A well-made smear is usually 23 cm in length.

Important: Avoid pressing down hard or pushing too slowly, as this can cause streaks, clumping, or uneven distribution.

4. Drying and Fixation

Once the smear is made, it should be air-dried rapidly by holding it horizontally or with gentle air flow. Do not blow on the slide as saliva contamination can occur.

Depending on the staining protocol, fixation may be necessary:

• Methanol Fixation: Immersion of the smear in methanol for 13 minutes is common before staining with Romanowsky-type stains like Wright's or Giemsa. This step preserves cellular morphology and helps stain adherence.
• Air-dried Smears: Some stains, such as Diff-Quik, stain air-dried smears directly without fixation.

5. Staining Procedures

Staining enhances visualization of cellular components by adding color contrast. The most common staining methods for blood smears are based on Romanowsky stains, which combine acidic and basic dyes.

5.1 Wrights Stain

Wrights stain contains eosin (acidic dye) and methylene blue (basic dye), which stain different parts of blood cells:

• Eosin stains hemoglobin and eosinophilic granules red or pink.
• Methylene blue stains nuclei and basophilic granules blue or purple.

Typical Procedure:

1. Fix smear in methanol briefly (1-3 minutes) and air dry.
2. Flood slide with Wrights stain solution for about 13 minutes.
3. Add an equal volume of buffered water or phosphate buffer (pH 6.87.2) to dilute the stain.
4. Let stand for an additional 510 minutes.
5. Rinse gently with buffered water.
6. Air dry and examine under a microscope.

5.2 Giemsa Stain

Giemsa stain is similar but usually more diluted and requires buffer for proper staining. It is especially valuable for detecting blood parasites and subtle morphological details.

1. Fix the air-dried smear in methanol (optional depending on protocol).
2. Immerse in a freshly prepared 5% Giemsa solution diluted in buffered water (pH 7.2) for 1530 minutes.
3. Rinse with buffered water gently and air dry.

5.3 Diff-Quik Stain

Diff-Quik is a rapid staining method widely used in clinical labs.

1. Air-dried smear does not require fixation.
2. Dip slide sequentially in fixative solution for 1020 seconds.
3. Dip in eosin solution (stains cytoplasm) for 1020 seconds.
4. Dip in methylene blue solution (stains nuclei) for 1020 seconds.
5. Rinse in distilled water and air dry.

6. Microscopic Examination

After staining, examine the smear using a light microscope. Begin by scanning under low power (10x or 20x objective) to locate suitable fields. Use oil immersion lens (100x objective) for detailed evaluation of cell morphology.

Features to assess include:

• Red blood cells: size, shape, color (hemoglobin content), arrangement, presence of inclusions or parasites.
• White blood cells: differential count, nucleus shape, cytoplasmic granules.
• Platelets: amount and morphology.

It is important to evaluate the feathered edge for platelet clumps or microfilaria, and the monolayer region for proper morphology assessment.

7. Troubleshooting Common Problems

• Thick smears: Result in overlapping cells, making morphology difficult. Correct by using a smaller drop of blood or a steeper angle of spreader slide.
• Thinner smears: Cells may be too sparse. Use a larger blood drop or lower the spreader angle.
• Streaks or ridges: Caused by uneven spreading. Push the spreader slide swiftly and smoothly.
• Uneven staining: Ensure proper pH of buffers and that stains are fresh and correctly diluted.
• Cells appear swollen or distorted: Overfixation or delayed drying may cause artifact. Air dry smears immediately.

8. Safety Considerations

Because blood specimens may contain infectious agents, always handle samples and blood smears using appropriate biosafety precautions:

• Wear gloves, lab coat, and eye protection.
• Dispose of sharps and biohazard materials properly.
• Work in well-ventilated areas or use fume hoods when working with methanol and staining reagents.
• Do not eat, drink, or apply cosmetics in the laboratory.

9. Summary

The preparation and staining of blood smears are fundamental skills in diagnostic hematology and parasitology. A well-made blood smear coupled with proper staining provides invaluable information for patient diagnosis and monitoring. Attention to detail in sample collection, smear preparation, drying, fixation, and staining ensures optimal results. Mastery of these techniques allows clinicians and laboratory technicians to identify diverse blood disorders and infectious agents with confidence.