ACTIVITY 2

SMEAR PREPARATION AND STAINING

Unit Intended Learning Outcomes

At the end of the experiment, the students (should be able/are expected to):

Disinfect properly the different areas of the laboratory.

Wear complete and proper personal protective equipment.
Execute correctly the different methods of preparing a blood smear.
Identify correctly the macroscopic and microscopic characteristics of a well-prepared
blood smear.
Determine correctly the factors contributing to a too thin or a too thick blood smear.
Stain properly the blood smear.
Determine correctly the factors contributing to a too basic or a too acidic stained
blood smear.
Perform correctly the proper waste segregation and disposal.

Pre-laboratory Discussion

The type of blood cells found in the peripheral blood smears may be of diagnostic and
prognostic importance. For this reason, proper preparation and staining of blood smear are
essential for the identification and study of the different blood cells.

PREPARATION OF BLOOD SMEAR

Either EDTA-anticoagulated whole blood or free-flowing capillary blood can be used. If EDTA is
used, smears must be prepared within 1 hour of collection. Before preparing the smear, store
the blood at 18°C to 25°C. Adequate mixing is necessary before blood smear preparation.

Types of Smear in Clinical Hematology Laboratory:

1. Thin smear (wedge smear, cover glass smear, spun smear) – used to evaluate the type
of anemia, to identify blood cell abnormalities, to screen presence of malarial
parasites, to perform manual differential count and white blood cell (WBC) count and
platelet count estimate.
2. Thick smear – to determine the species and the stage of the malarial parasite
3. Buffy coat smear – extremely low WBC count (<1.0 x 109/L)

For proper evaluation of blood cells, the well-made smear must be prepared.
Qualities of a well-made blood smear:
 1. Has gradual and even transition from thick to thin area.
2. Must be finger-shaped with a smooth tail.
3. Must occupy two-thirds to three-fourths of the glass slide.
4. Must not touch the outer borders of the slide or run off the sides or ends of the slide.
5. Must smooth surface free from ridges, waves, gaps and holes.
6. Must have feathery edge.

Factors that may be altered to produce a well-made blood smear:

THIN SMEAR THICK SMEAR

small drop of blood Large drop of blood

Slow spread Fast spread

Low angle (<30°) High angle (>45°)

Heavy pressure Light pressure

FIGURE 3. WELL-MADE BLOOD SMEAR

SOURCE: RODAK, B., ET AL., 2012

FIGURE 4. UNACCEPTABLE BLOOD SMEARS

SOURCE: RODAK, B., ET AL., 2012

Common causes of a poor blood smear:
1. Drop of blood too large or too small.
2. Spreader slide pushed across the slide in a jerky manner.
3. Failure to keep the entire edge of the spreader slide against the slide while making
the smear.
4. Failure to keep the spreader slide at a 30° angle with the slide.
5. Failure to push the spreader slide completely across the slide.
 6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty
slide
7. Holes in film: Slide contaminated with fat or grease
8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol
contaminated with water.

STAINING OF BLOOD SMEAR

To differentiate and identify cells, blood and other types of specimens can be stained using
Romanowsky-based stains (e.g. Giemsa stain, Wright stain, Leishman’s stain). These stains can
be prepared in the laboratory or purchased in a ready-to-use form. Either manual or
automated techniques can be used. Fixing of cells on the slide prior to staining is done using
methanol.

Components of Romanowsky stain:

1. eosin (acidic component) - stains acidophilic structures, e.g. hemoglobin, eosin
granules
2. methylene blue (basic component) - stains basophilic structures, e.g. nuclei (RNA and
DNA), ribosomes, GAGs

Causes of Too Acidic Smear

1. insufficient staining time
2. prolonged buffering or washing
3. old stain

Causes of Too Basic Smear

1. thick blood smear
2. prolonged staining
3. insufficient washing
4. alkaline pH of stain components

Teaching and Learning Activities

• Pre-Analytical Phase

1. Disinfect the working area using 10% sodium hypochlorite.

2. Wear complete and proper PPE.
• Analytical Phase

Reagents, Supplies and Equipment

1. EDTA anticoagulated blood
2. Glass slides (frosted or plain)
3. Spreader
4. Cover slip
5. Methanol
6. Wright stain or Wright-Giemsa stain (Romanowsky stain)
7. Buffer Solution or Aged Distilled Water
8. Coplin Jars
9. Transfer device (applicator sticks, pipets, or capillary tubes)
10. Slide holder
11. Paper towel
12. Personal Protective Equipment (PPE)

Procedure

PREPARATION OF THIN BLOOD SMEARS

A. Push Wedge Preparation/ Two Slide Method

1. Place a drop of blood (about 2 to 3 mm in diameter) to the stationary slide
about 0.25 inch (1 cm) from the end or near the frosted area.
2. Position the spreader (or a second slide) before the drop of blood at angle
between 30 to 45 degrees. Allow the drop of blood to spread evenly across the
width of the slide.
3. Quickly and smoothly push the spreader along the length of the slide with an
even and gentle pressure.
4. Allow the smear to air-dry.

FIGURE 5. PUSH WEDGE PREPARATION/ TWO SLIDE METHOD

SOURCE: RETRIEVED FROM: HTTP://WWW.KEYWORD-SUGGESTIONS.COM/YMXVB2QGC21LYXIGCHJVY2VKDXJL/

B. Two-Cover Slip/ Erlich’s Two-Coverglass Method

1. Get two coverglasses. Hold one coverglass on its adjacent corners with the
thumb and index finger.
2. Place one drop of blood in top of the held coverglass.
3. Position the other cover glass on top of the coverglass with blood so that the
two form a 16-sided figure. As this is done, the blood begins to spread.
4. Before the blood completely spreads, separate the two coverglasses by doing a
rapid, even, horizontal and lateral pull. Avoid squeezing the coverglasses
together.
5. Allow the smear to air-dry.
 FIGURE 6. TWO-COVER SLIP/ ERLICH’S TWO-COVERGLASS
METHOD

SOURCE: RETRIEVED FROM: HTTPS://WWW.STUDYBLUE.COM/NOTES/NOTE/N/

LEC-3-BLOOD-SMEAR-WRIGHT-STAIN-INTRO-TO-CELLS-OF-NORMAL-BLOOD/DECK/5364623

PREPARATION OF THICK BLOOD SMEAR

1. Place a small drop of blood (approximately 2 cm in diameter) on a clean slide.
2. Spread it with an applicator stick or the corner of another slide until small prints
are just visible through the blood smear.

STAINING OF PERIPHERAL BLOOD SMEAR

1. Ensure that the blood smears are completely air dried.

2. Prepare the staining solutions by placing them in separate coplin jars. Filter the
stains if necessary to avoid artifacts.
a. Solution 1 – methanol (fixative)
b. Solution 2 – eosin (acidic dye)
c. Solution 3 – methylene blue (basic dye)
3. Dip the blood smear in each solution for 30 seconds (or as stated by the
manufacturer).
4. Rinse off the excess stain by dipping the blood smear a few times in a buffer
solution or aged distilled water.
5. Allow the smear to air-dry.

• Post-Analytical Phase
1. Segregate and dispose all infectious and biohazardous waste in their proper waste
bins.
2. Disinfect the working area using 10% sodium hypochlorite.
3. Remove all used PPE after the activity.