A blood smear involves spreading a thin layer of blood onto a glass slide, creating a film for microscopic examination of blood components. This fundamental technique provides a visual representation of blood cells, enabling assessment of their morphology, including size, shape, and structure of red blood cells, white blood cells, and platelets. The quality of this preparation directly impacts the accuracy of subsequent analysis, making proper technique essential for obtaining reliable insights into an individual’s blood composition.
Essential Materials
Creating a high-quality blood smear begins with assembling the correct materials. You will need clean, grease-free microscope slides, preferably with a frosted end for labeling, and a spreader slide, which is another clean glass slide used to spread the blood. Blood collection devices, such as a sterile lancet for finger pricks or an EDTA-anticoagulated blood tube for venous samples, are necessary to obtain the specimen. Additionally, personal protective equipment like gloves, alcohol wipes, cotton balls, and a sharps container are required. Staining reagents, typically a Romanowsky stain like Wright’s or Giemsa, are also required to visualize cellular details.
Sample Collection and Slide Preparation
The initial steps for a blood smear involve careful sample collection and meticulous slide preparation. If obtaining blood via a finger prick, select a finger, typically the middle or ring finger, and clean the area thoroughly with an alcohol wipe, allowing it to air dry completely. After pricking the finger with a sterile lancet, gently wipe away the first drop of blood with a clean gauze to remove tissue fluid contamination. For samples from an EDTA-anticoagulated tube, gently invert the tube several times to ensure the blood is well-mixed before use.
Immediately after obtaining a fresh drop of blood, or from a well-mixed EDTA tube, place a small, round drop onto one end of a clean microscope slide. This drop should be approximately 1 to 2 millimeters in diameter, or about 2 microliters, positioned about 0.5 to 1 centimeter from the frosted end of the slide. The precise size and placement of this blood drop are important, as they influence the length and thickness of the final smear. Prompt action is necessary to prevent the blood from clotting or drying prematurely.
Performing the Smear Technique
Once the blood drop is correctly positioned, the next step involves the precise technique of spreading the blood to create an optimal film. Take a second clean glass slide, known as the spreader slide, and place its edge on the surface of the first slide, just in front of the blood drop. Hold the spreader slide at an angle, typically between 30 to 45 degrees, relative to the horizontal plane of the bottom slide. The specific angle can be adjusted; a steeper angle (e.g., 40-45 degrees) creates a shorter, thicker smear, while a shallower angle (e.g., 30 degrees) results in a longer, thinner smear.
Carefully draw the spreader slide backward until its edge makes contact with the blood drop, allowing the blood to spread evenly along the entire width of the spreader by capillary action. It is important to wait for the blood to fully traverse the edge of the spreader slide before moving forward. Maintain a consistent angle and apply steady, even pressure as you smoothly and rapidly push the spreader slide forward across the length of the bottom slide. This motion should be continuous and free of jerky movements to ensure an even distribution of cells. The goal is to produce a thin, uniform film that covers about two-thirds to three-fourths of the slide’s length, ending with a feathered edge where the blood gradually thins out.
Drying and Staining
After creating the blood film, the next steps involve drying and staining the smear to prepare it for microscopic examination. Immediately following the spreading technique, the blood smear must be air-dried completely. This can be achieved by waving the slide in the air or allowing it to dry on a flat surface, protected from dust. Rapid drying prevents cellular distortion and the formation of artifacts that can compromise the quality of the sample.
Once thoroughly air-dried, the smear is ready for staining, a process that enhances the visibility of cellular components under a microscope. Common Romanowsky stains, such as Wright’s or Giemsa, contain a mixture of dyes like eosin and methylene blue.
The staining procedure typically begins with fixation, often by immersing the dried slide in absolute methanol for about 30 seconds. This step adheres the cells to the slide and preserves their morphology. Following fixation, the stain is applied, allowed to react for a specified time, and then rinsed with buffered water or distilled water to remove excess stain. The slide is then allowed to air dry completely again before it is ready for microscopic analysis.