College of American Pathologists
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Laboratory Report: Preparing a Peripheral Blood Smear

Posted January 1, 2005

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.

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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 © 2006 by the College of American Pathologists.
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