Blood Smear Preparation

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COHscientist
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Blood Smear Preparation

http://dir.niehs.nih.gov/dirlep/Webpages/techniq.html#anchor1248635

Clinical Significance

A well prepared blood smear is necessary for microscopic examination of blood. Blood smears are used to determine leukocyte differentials, to evaluate erythrocyte, platelet and leukocyte morphology, and, if necessary, to estimate platelet and leukocyte counts. Desirable qualities of a blood smear include:

A. Sufficient reading area

B. Acceptable morphology within the working area

C. Even distribution of leukocytes

Principle

The Clinical Pathology Laboratory uses the wedge technique for preparation of blood smears. This method produces a gradual decrease in thickness of the blood from thick to thin ends with the smear terminating in a feathered edge approximately 2 mm long. The smear is greater than 25 mm long and the feathered edge stops approximately 10 mm from the end of the slide. The blood film occupies the central portion of the slide and has definite margins on all sides that are accessible to examination by oil immersion. The thin end of the film becomes thinner gradually and does not have grainy streaks, troughs, ridges, waves or holes - features that can result in an uneven distribution of leukocytes. In preparations from normal animals, the thin section of the smear occupies approximately 1/3 of the total area and, within that area, erythrocytes are distributed in a monolayer. The thickness of the spread is influenced by the angle of spreader slide (the greater the angle, the thicker and shorter the blood smear), the size of the drop of blood and the speed of spreading. Glass coverslips are mounted on all blood smears to prevent damage to smear during examination, cleaning, handling and storage.

Materials and Equipment

A. Gauze (2" x 2" squares)

B. Plain microhematocrit capillary tubes

C. Clean 3 x 1 inch microscopic slides of good quality, frosted at one end

Sample Collection and Preparation

A. Use whole venous blood collected in tubes containing tripotassium ethylenediaminetetraacetic acid (K3EDTA).

B. Do not make smears from clotted samples. Leukocyte distribution and platelet numbers (clumping) will be affected.

C. Smears may be made immediately from non-anticoagulated whole blood. Smears prepared this way will usually have platelet clumps.

D. Blood smears are prepared within one hour of sample collection. Those made from EDTA-anticoagulated blood specimens that have been at room temperature for less than three hours will have minimal degenerative changes.

Procedure

A. Invert the tube approximately 10 times, mixing gently.

B. Place two microscopic slides flat on the counter top. One will be used as a spreader slide.

C. The smear is made in the middle of the slide. This allows space for a label at the thick end of the smear and adequate area for viewing at the feathered edge.

D. Using a plain microhematocrit tube as a pipette, place a drop of blood, 2-3 mm in diameter, approximately one-third the distance from the frosted end of the slide. Gauze may be used to remove excess blood from the tip of the microhematocrit tube prior to placing the drop on the slide.

E. Grasp a second slide (spreader) between the thumb and forefinger of one hand.

F. Hold the edge of the spreader slide against the surface of the first at an angle of 30 to 45 degrees.

G. Draw the slide backwards until it contacts the drop of blood. Allow the blood to spread by capillary action to within several mm of the edges of the slide. The blood will generally spread very fast.

H. Quickly push forward, using a smooth even motion. The weight of the slide is the only pressure applied.

I. Slides are labeled on the frosted end, using a soft No. 2 pencil. Labels will contain the following information:

1. Study identification number

2. Time Point

3. Animal identification number

4. Date

J. Allow smear to air dry. Use a well ventilated area or a heater block to promote rapid drying.

K. Place the slides on a commercial stainer or process manually by Wintrobe method.

The smear should be saturated with the stain and stand for one minute. After one minute, dilute the stain drop by drop with distilled water. The same number of drops of water is used as was stain. The solution should not run over the edges of the coverglass or slide. A greenish scum will appear and the margins will be a reddish tint. Let stand three to four minutes. Wash with water until the film is yellowish or pink. The slide should remain horizontal to avoid scum formation on smear. While the slide is still horizontal, "float" the stain off of the slide by adding water slowly, then briskly. After the slide is washed thoroughly, stand it on its edge to dry or it may be dried between papers. For coverglasses, once thoroughly dry, mount in one of the synthetic resins designed for this purpose. (Maxwell M. Wintrobe, Clinical Hematology, 6th ed., Lea & Febiger, Philadelphia, 1967, p. 447.)