When automated differentials do not meet specified criteria programmed into the automated hematology instrument, the technologist/technician must perform a manual differential count from a prepared smear. There are two types of blood smears: the wedge smear and the spun smear. The wedge smear will be discussed in this procedure. Smears are prepared by placing a drop of blood on a clean glass slide and spreading the drop using another glass slide at an angle. The slide is then stained and observed microscopically. A well-stained peripheral smear will show the red cell background as red orange. White cells will appear with blue purple nuclei with red purple granules throughout the cytoplasm. A well made, well distributed peripheral smear will have a counting area at the thin portion of the wedge smear which is approximately 200 red cells not touching. A good counting area is an essential ingredient in a peripheral smear for evaluating the numbers of and types of white cells present and evaluating red cell and platelet morphology.
Reagents and Equipment
1. Glass slides (frosted)
2. Wooden applicator sticks
3. DIFF-SAFE (an apparatus designed to avoid removing the tube top)
Specimen Collection and Storage
1. EDTA specimen or EDTA Microtainer
2. Smears are made from EDTA
a. Microtainers within 1 hour of collection b. EDTA blood within 2 to 3 hours c. Check all Microtainers for clots with applicator sticks
A random slide is picked after it has been stained and a technologist/technician checks the quality of the stain for the WBCs and RBCs, platelets, and the distribution of cells (see Principle)
1. Insert the DIFF-SAFE dispenser through the stopper of the tube held in an upright position.
2. Turn the tube upside down and apply pressure at the frosted end of the slide. When the drop of blood appears, discontinue pressure.
3. Using a second slide (spreader slide), place the edge of the second slide against the surface of the slide at an angle between 30 and 45 degrees (Fig. 20.6).
4. Bring the spreader slide back into the blood drop until contact is made with the drop of blood.
5. Move the spreader slide forward on the slide, so a smear is made approximately 3 to 4 cm in length. The smear should be half the size of the slide, with no ridges, and a "feather edge" should be toward the end of the smear.
6. Label the frosted end of the slide with the patient's last name and first initial, specimen number, and the date.
8. Proceed with staining. Manual Wright staining is not found often in the clinical laboratory setting. Most clinical laboratories have an automated staining instrument attached to their automated CBC analyzer. If there is no automated stainer attached to the analyzer, there still is a separate staining instrument.
1. The angle between the slides is dependent upon the size of the blood drop and viscosity of the blood. The optimal angle is 45 degrees.
2. The larger the drop of blood and lower the hematocrit, the higher the angle needs to be so the blood smear is not too long.
3. Blood with a higher hematocrit needs to have a lower angle so the smear is not too short and thick.
4. Glass slides must be clean; otherwise, this results in imperfect distribution of cells and improper staining.
5. Once the drop of blood has contact on the slide, the smear needs to be made immediately. Otherwise, the blood will clump and dry, again resulting in uneven distribution of WBC and platelets.
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