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Laboratory Report: Preparing a peripheral blood smear

The Laboratory Report series is written for those who collect patient specimens. By
providing this educational background on medical tests, it is intended that fewer errors
in specimen collection will be made.

Examination of the blood smear is an important part of the hematologic evaluation. The
reliability of the information obtained depends on well-made and well-stained films that
are systematically examined. Peripheral blood or potassium EDTA anticoagulated blood
(1-2 mg EDTA/1 mL blood) may be used. Smears of peripheral blood must be made
immediately. Smears made from EDTA-anticoagulated blood should be made within 2
hours of collection. There are 2 main manual methods described; the 2-slide or wedge
method and the coverglass method.

The wedge method: First, place a 2-3 mm drop of blood about 1 cm from the frosted end
of a clean slide that is on a flat surface. Second, with the thumb and forefinger of the right
hand, hold the end of a second slide or coverglass (“spreader slide”) against the surface of
the first slide at an angle of 30-45 degrees. Third, draw it back to contact the drop of
blood. Allow the blood to spread and fill the angle between the 2 slides. Finally, push the
“spreader slide” at a moderate speed forward until all of the blood has been spread into a
moderately thin film.

The coverglass method: First, place a 2-3 mm drop of blood on 1 coverglass and then
place another coverglass crosswise over the drop so that the corners appear as an 8-
pointed star. Just as it stops spreading, pull the coverglasses quickly but firmly apart on a
plane parallel to their surfaces. The coverglasses should then be dried film side up.

Using either method, the films should be fixed in absolute methanol for 1 to 2 minutes.
The slide is next exposed to undiluted Wright stain solution for 2 minutes. Then, without
removing the stain from the horizontal slide, an equal amount of buffer is added and
mixed by blowing gently. The stain is flushed from the slide using water for about 30
seconds. The slide is allowed to air dry in a tilted position. Slides can be cover-slipped or
coverglasses can be mounted (2 on each slide) with standard mounting media.

The film should not cover the entire surface of the slide. In a good film, there is a thick
portion and a thin portion and a gradual transition from one to the other. The film should
have a smooth, even appearance and be free from ridges, waves or holes. The end of the
smear (the “feathered edge”) should be smooth and even. A well-stained smear will have
pink-colored red cells and the nuclei of the leukocytes will be purple, with well-
differentiated chromatin and parachromatin.
NewsPath® Editor: Megan J. DiFurio, MD, FCAP
This newsletter is produced in cooperation with the College of American Pathologists’ Public Affairs Committee and may
be reproduced in whole or in part as a service to the medical community. Copyright © 2005