Three-minute peripheral blood film evaluation: Preparing the film


Three-minute peripheral blood film evaluation: Preparing the film

No single hematology procedure produces more valuable information yet requires so little time and expense than a peripheral blood film evaluation. Proper preparation and staining of the film are critical.

A peripheral blood film evaluation should be part of all complete blood counts (CBCs), regardless of whether hematology is performed in-house or at a reference laboratory. Proper sample collection, slide preparation, and staining are essential to accurately evaluate a blood film, as is the correct use of a high-quality microscope. This article describes the steps in preparing a blood film and the equipment you'll need.


Important components of a CBC include evaluating the erythron (hematocrit, total red blood cell [RBC] count, hemoglobin concentration, absolute reticulocyte count, and RBC indices), the leukon (total white blood cell [WBC] count, five-part differential count including immature neutrophils), the thrombon (platelet count and platelet indices), and the total protein concentration. As mentioned earlier, always include a peripheral blood film evaluation in the CBC.

A CBC should be included in evaluations of every sick patient, every patient with vague signs of disease, and every patient receiving long-term medications. In addition, a CBC should be performed as part of every preanesthetic workup, for adult wellness and geriatric profiles, and as a recheck test for patients in which RBC, WBC, or platelet abnormalities were previously diagnosed.


Artifacts must be avoided for proper hematologic interpretation. Causes of artifacts include poor blood collection techniques, inadequate sample volumes, prolonged sample storage, and delayed sample analysis.

Proper blood collection is vital to prevent erroneous results from sample clotting and cellular lysis. Obtain hematology samples from the largest blood vessel possible to minimize cellular trauma and to prevent the activation of clotting mechanisms. For accurate results, discard clotted samples and collect fresh samples. Common venipuncture sites in dogs and cats include the jugular, cephalic, and lateral and medial saphenous veins. Anticoagulants include EDTA, heparin, and citrate. EDTA is the preferred anticoagulant for blood film preparation because it preserves cellular detail better than other anticoagulants do and does not interfere with Romanowsky staining of WBCs.

Inadequate sample volume is a common cause of inaccurate hematologic results. Properly fill anticoagulated blood collection tubes to avoid falsely decreased hematocrits and cell counts and to prevent RBC shrinkage.

Hematologic samples must be analyzed as soon as possible to prevent artifacts created by exposure to anticoagulants and cell deterioration due to storage and shipment. Analyze samples within three hours or refrigerate them at 39.2 F (4 C) to avoid an artificially increased hematocrit, increased mean corpuscular volume, and decreased mean corpuscular hemoglobin concentration.1 Prepare blood films within one hour of collection to avoid morphologic artifacts. RBC crenation, neutrophil hypersegmentation, lymphocytic nuclear distortion, and general WBC degeneration including vacuolization in neutrophils may occur in aged samples. In addition, monocyte vacuolization, monocyte cytoplasmic pseudopod formation, and platelet agglutination can be encountered in stored samples.2 If you use a reference laboratory for primary hematologic analyses, we recommend submitting freshly prepared blood films along with the anticoagulated blood.


1. The steps in preparing a blood film. A small drop of blood is placed near the end of a slide (top). A spreader slide is drawn back into the blood drop at a 30-degree angle (middle). Then the spreader slide is pushed away from the blood drop, creating a uniform film across the slide (bottom).
Producing high-quality blood films begins by using clean, new slides. Used slides frequently have imperfections such as scratches and other physical defects. Slides must be free of fingerprints, dust, alcohol, detergents, and debris. Using a microhematocrit tube, place a small drop (2 to 3 mm in diameter) of well-mixed EDTA blood about 1 to 1.5 cm from the end of the slide. Next, draw a spreader slide back into the blood drop at about a 30-degree angle until the spreader slide edge contacts the sample drop and capillary action disperses the sample along the edge. Then, using a smooth steady motion, push the spreader slide away from the blood drop, creating a uniform film that covers nearly the entire length of the slide (Figure 1). Changing the angle of the spreader slide will change the length and thickness of the blood film. For blood samples with low hematocrits (severe anemia), you may need to decrease the angle of the spreader slide to make a good-quality slide. In contrast, for samples with high hematocrits (severe dehydration and polycythemia due to a variety of conditions), you may need to increase the angle of the spreader slide.