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Blood Smear Preparation

When performing in-house hematology, the evaluation of a peripheral blood smear is an essential task to ensure a thorough examination of a patient’s hemogram. The first step in this process is to create a high-quality blood film. A diagnostic smear permits the RVT to accurately assess the populations and morphologies of the erythrocytes, leukocytes and thrombocytes, and complements the automated hematology results generated from an in-house analyzer.

Before collection, potential pre-analytical variables should be taken into consideration. Patient preparation and blood collection techniques can influence both the blood values, and the microscopic appearance of the blood cells. Low-stress handling protocols for blood collection should be followed, to minimize the excitement response in the patient. Failure to do so can result in a leukocytosis, caused by a mature neutrophilia. A fresh, well-mixed EDTA blood sample is used when preparing blood films of mammals, and it is important to fill the blood tubes according to the manufacturer’s recommendations. Failure to do so can result in altered cell morphology, which could be confused with pathological changes. Underfilling the blood tube results in excess amounts of anticoagulant present for the blood volume, which will cause cell crenation. Overfilling the blood tube will result in not enough anticoagulant being present to prevent platelet clumping. Blood smears should be made within two hours of collection, to minimize cellular changes such as hypersegmentation of the neutrophils or platelet clumping.

Slide Preparation - Procedure Review

To begin, clean glass microscope slides should be used, free of chipped edges and any debris. One slide will serve as the sample slide and the other as the spreader slide. Slides should be handled in a manner that will avoid any fingerprint deposits on the surface.

To transfer the blood sample onto the sample slide, the use of a microhematocrit tube will allow for a small, controlled drop size. A well-formed drop, three millimeters in diameter, should be deposited towards the frosted end of the sample slide.

To create the smear, a spreader slide is held at a 30-degree angle in front of the blood drop. The slide is backed into the drop, and a short pause is taken to allow the blood drop to spread laterally. Before the blood has reached the sides of the slide, the spreader slide is pushed forward rapidly while maintaining consistent contact with the sample slide. While spreading, the 30-degree angle should be maintained until the smear is complete.

When performing the blood smear technique, several factors can be manipulated to change the shape and appearance of the smear: the size of the blood drop; the angle of the spreader slide; the speed of the spreader slide; and the length of the pause at the drop itself. These adjustments can be made as needed, to correct for errors, and would also be required to accommodate varying blood samples. For example, a blood sample from an anemic patient is thinner, and has a “watery” consistency to it. This variable would cause the blood to spread faster and thinner. Increasing the angle of the spreader slide and/or increasing the speed will address this change. A sample from a dehydrated or hemoconcentrated patient is thick, and would be slow to spread, requiring a decreased angle and slower speed when spreading the drop of blood. (See Table 1)

An acceptable smear should possess a monolayer of at least two to three mm width, and would have a rainbow appearance when held to the light. There should be a gradual transition from a thick area in the body of the smear, to the monolayer, and then to the feathered edge. The smear should be absent of any waves or ridges. The feathered edge should also be free of any tails or streaks. The size of the overall smear is also important. It should cover at least half of the slide length, to allow for an area of adequate size for cell counts and evaluations. (See Figure 2)

Figure 2: The rainbow appearance of the monolayer of an unstained slide (left). An example of a well-prepared, stained blood smear (right).

The smear should be allowed to dry fully before staining to prevent distortion of the cells or partial washing off of the smear during the staining process. Typically, a Romanowsky-type stain is used to give a differential staining result. Follow the manufacturer’s recommendations, to produce predictable staining of the cells for identification. To reduce artifact caused by the stain, such as debris or precipitate, the stain should be changed regularly. Also, blood smears should be processed with “clean stains,” which are those reserved for samples such as blood smears and clean cytology. Other types of samples, such as urine smears and dirty cytology (ex. ear swabs), should be stained using a separate set of “dirty stains.” This avoids potential cross-contamination of debris, which could be mistaken for pathology.

Step-By-Step Method

1. Begin with two clean, high-quality microscope slides that are free of debris and scratches, or nicked edges.

2. Deposit a three mm drop of well-mixed EDTA blood towards the frosted end of the sample (base) slide.

3. Holding the spreader slide at a 30-degree angle, back into the blood drop and pause to allow the drop to spread laterally.

4. In a smooth, quick motion, advance the spreader slide forward while maintaining the 30-degree angle.

5. Allow the slide to dry fully before staining.

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