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Lesson 3-2
Preparation and Staining of Blood Smears

INTRODUCTION

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 films is essential for the identification and study of different kinds of leukocytes. The appearance of erythrocytes and thrombocytes will often give important clues that help distinguish between different types of diseases or other physical changes.

There are two basic methods for the preparation of blood smears: the cover slip and the slide methods. The cover slip method has certain advantages over the slide method; distribution of cells is like that of the in vivo circulation. The principal disadvantage of the latter method is that cover slips are very fragile and easily broken during processing.

The slides and cover glasses must be chemically clean and dry.

The foundation for the morphological study of blood was based on Ehrlich's investigations of the aniline dyes, dating back to 1877, while he was still a student. Originally, simple dyes were used in the clinical laboratory and tissues were stained successively if more than one color was desired. The majority of the aniline dyes are in the form of salts of acids and bases. During the process of staining, compounds are probably formed between the basic dyes and the acid nuclear substances of cells and between the acid dyes (so called "neutral" dyes). In this way, the staining principles of the original components were preserved; and, in addition, new staining properties dependent upon the union of the component dyes were developed. These were, therefore, termed polychromic dyes.

One modification of these polychromic stains is Wright's stain. This is the stain most used in Army laboratories today. Wright's stain is a methyl alcohol solution of an acid dye and a basic dye. The acid dye is known as eosin, which is red in color. The basic dye, methylene blue, is blue in color. The white cells are mostly identified by their preference for these dyes. In some cases the cells are even named for the dye that they prefer. For example, cells that prefer a mixture of the acid and basic dye are called neutrophils. In the staining process, a buffer solution is used to control the acid- base balance of the stain. This is a most important function. If the buffer solution is too acid it makes the acid dye too bright and the basic dye too faint. On the other hand, if the buffer solution is too basic it makes the basic dye too bright and the acid dye too faint. In either case, the result is a poorly stained slide. The acid-base balance of a solution is measured by its pH value. A buffer solution should have a pH value between 6.4 and 6.8. This allows the best color contrast between acid and basic dyes.

When optimal staining conditions exist, Wrights stain is very satisfactory and easily differentiates cells. The eosin component stains cell cytoplasm, and the methylene blue component stains nuclear material, granules, and inclusions. Both stains oxidize rapidly because they are in alkaline solution. Giemsa, a purified polychrome stain, is added to compensate for this defect by maintaining the azurophilic staining property of the mixture.


SLIDE METHOD FOR PREPARING BLOOD SMEARS

Principle. A small drop of blood is placed near one end of a clean glass slide. Using a second slide as the spreader, the blood is streaked into a thin film and allowed to dry. It is then fixed and stained with modified Wright's stain.

 Equipment.

(1) Venipuncture or finger puncture material.

(2) Clean glass slides.

Reagents.

(1) Methanol Fixative.

(2) Eosinate stain (orange).

(3) Polychrome stain (purple).

(4) Water, deionized.

SLIDE PREPARATION

Make a finger puncture or venipuncture in the usual manner.

Touch a drop of blood to a clean glass slide at a point midway between the sides of the slide and a short distance from one end. If a venipuncture is made, use a capillary tube to transfer a drop of blood from the tube to the slide. If a finger puncture is made, dispense the drop of blood from the puncture site after discarding the first drop.

NOTE: The drop of blood should be no larger than 1/8 to 3/16 inch in diameter (see figure 3-5, side method for preparation of blood films.

Lay the specimen slide on a flat surface and hold it securely. Place a smooth, clean edge of the spreader slide on the specimen slide at an angle of about 300 from the horizontal (see figure 3-5a).

Figure 3-5a. Side method for preparation of blood films:
Place spreader slide at an angle of about 300 from the horizontal.

Pull the spreader slide toward the drop of blood until contact is made within the acute angle formed by the two slides as shown in figure 3-5b.

Figure 3-5b. Side method for preparation of blood films:
Contact blood with spreader.

Allow the blood to spread toward the sides of the slide.

Push the spreader slide smoothly and lightly toward the opposite end of the specimen slide, drawing the blood behind it into a thin film (see figure 3-5c).

Figure 3-5c, Side method for preparation of blood films:
Finished slide.

Allow the blood film to air-dry completely. Do not blow on the slide in an effort to enhance drying.

Using a lead pencil, write the name (or identification) of the patient on the frosted end of the slide. Do not use a wax pencil or marker as it dissolves during the staining process.


SLIDE STAINING

Place stains in four separate containers in the following order:

(1) Methanol fixative.

(2) Eosinate stain (orange).

(3) Polychrome stain (purple).

(4) Water, deionized.

Dip the air dried blood smear in Methanol Fixative (up and down motion) for 30 seconds.

Dip the smear in orange Eosin stain (up and down motion) for 30 seconds.

Rinse slide with distilled or deionized water (tap water has chlorine which bleaches the stain.

Dip the smear in purple Polychrome stain (up and down motion) for 30 seconds.

Let slide air dry in the vertical position do not blot.

Cover solutions to prevent evaporation.

NOTE: Touch off excess liquid at the container edge to reduce carryover from one solution to another. - Stain smears within one hour of collection WBCs degrade in stored samples. Staining time vary between manufacturers

Discussion.

  1. A properly prepared blood smear is margin-free; has no lines, ridges, or holes; is placed centrally on the slide; has an adequate thin area; and has a uniform distribution of leukocytes.
  2. It is preferable that blood smears not be made from blood containing anticoagulants since the leukocytes change their staining characteristics, develop vacuoles, engulf oxalate crystals, and show nuclear deformities. However, satisfactory slides are made with blood anticoagulated with EDTA.
  3. Avoid the following errors:
  • Thick films made from an excess amount of blood placed on the slide.
  • Delay in transferring the blood to the slide.
  • A spreader slide that has damaged or unpolished ends.
  • The use of dirty, dusty, greasy, or scratched slides.
  1. All slides most by fixed in methanol 30 minutes before staining.
  2. In cases of marked leukopenia, smears can be prepared from the white cell layer ("huffy coat") obtained by centrifuging the blood slowly in a Wintrobe hematocrit tube at 500-800 rpm for 5 minutes.
  3. It is important that the blood film be completely dried before staining; otherwise the wet areas will wash off the slide.
  4. Protect blood slides from insects such as flies, cockroaches, etc. They can "clean" raw blood slides very rapidly.
  5. Protect slides from areas of high humidity. Excessive moisture tends to hemolyze red blood cells.
  6. Slides should be stained as soon as possible after preparation. White cells tend to become distorted and disintegrate very rapidly, thus causing considerable difficulty in identification.
  7. After the staining is complete, do not blot the smear but air-dry it. To speed up the drying process, the smear can be placed in the heat of the substage light. It is important that the slide not be heated too intensely or too long since overheating tends to darken the staining reaction.
  8. A good quality smear should macroscopically pinkish-gray in hue. It should not be blue, green, or red. Microscopically, the red blood cells should be pink to orange and the white blood cells bluish if they display their true staining color.
  9. If the RBCs are bluish or green, this indicates that the stain is too alkaline. With an alkaline stain, the WBCs stain heavily and generally display fair distinguishing characteristics. However, the heavy stain masks any abnormalities of the RBCs. Heavy staining can be caused by:
  • Blood smears which are too thick.
  • Over-staining (prolonged buffer action).
  • Evaporation of the methanol in the stain.
  • Stain or diluent which is alkaline.
  • Alkaline fumes.
  1. If the red blood cells are bright red, the stain is too acid. In this condition they stain well but the white blood cells (except eosinophilic granules) stain very poorly if at all. Thus, the stain is of no value for differential studies. ''Tendency toward acid staining is caused by:
  • Incomplete drying before staining.
  • Insufficient staining (insufficient buffer action).
  • Overdilution of the stain with buffer.
  • Prolonged washing of the slide after staining.
  • Stain or buffer which is acid.
  • Acid tunes.
(14) The staining reactions of blood are as given in table 3-1.

Table 3-1. Staining reactions.

Type of blood cell or component Good stain Acid stain Alkaline stain
Erythrocytes
All nuclei
Eosinophilic granules
Pink to orange
Purple-blue
Granules red
Bright red
Pale blue
Brilliant red, distinct
Blue or green
Dark blue
Deep gray or blue
Neutrophilic Granules
Lymphocyte
Cytoplasm
Violet-pink
Blue
Pale
Pale blue
Dark, prominent
  1. A poorly stained smear can sometimes be saved by washing rapidly with 95 percent alcohol, washing quickly in water, then restraining.

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