8.3 Blood Smear Preparation and CBC Concepts

Blood Smear Preparation and CBC Concepts

A Complete Blood Count (CBC) is a standard diagnostic test that evaluates the cellular components of blood: red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). The veterinary assistant plays a key role in sample collection, select preparation tasks, and assisting with basic measurements like Packed Cell Volume (PCV) and Total Protein (TP). Performing these steps carefully ensures that cell morphology is preserved and numerical results are accurate.

Blood Collection Tubes and Anticoagulants

When collecting blood for laboratory analysis, the choice of collection tube and anticoagulant is critical to preserving sample integrity. The tube must be filled to the correct volume and gently inverted 8 to 10 times immediately after collection to mix the anticoagulant with the blood. Never shake blood tubes, as this causes mechanical hemolysis (rupturing of red blood cells).

1. Lavender Top Tube (LTT):
   • Anticoagulant: Ethylenediaminetetraacetic acid (EDTA).
   • Mechanism: Prevents clotting by binding (chelating) calcium ions, which are essential components of the coagulation cascade.
   • Primary Use: Complete Blood Count (CBC) and blood smears. EDTA is the anticoagulant of choice for hematology because it preserves cell size, shape, and staining characteristics (morphology) better than any other anticoagulant.
2. Red Top Tube (RTT) and Serum Separator Tubes (SST):
   • RTT (Plain Red Top): Contains no anticoagulant or additives. Blood is allowed to clot naturally (typically taking 20 to 30 minutes). After clotting, the sample is centrifuged to separate the liquid portion, which is serum. Used for blood chemistry panels, therapeutic drug monitoring, and serological testing.
   • Tiger Top / Gold Top (SST): Contains a clot activator to speed up clotting and a silicone gel separator. During centrifugation, the gel moves to form a physical barrier between the red blood cell clot at the bottom and the serum at the top. This prevents the cells from metabolizing glucose and altering other chemistry parameters.
3. Green Top Tube (GTT):
   • Anticoagulant: Lithium Heparin (or Sodium Heparin).
   • Mechanism: Prevents clotting by activating antithrombin, which inhibits thrombin and other clotting factors.
   • Primary Use: Plasma chemistry panels and blood gas analysis. It is also the anticoagulant of choice for avian and reptile hematology because EDTA can cause lysis of red blood cells in certain exotic species.
4. Blue Top Tube (BTT):
   • Anticoagulant: Sodium Citrate.
   • Mechanism: Reversibly binds calcium.
   • Primary Use: Coagulation assays (such as PT and aPTT).
   • Rule: The tube must be filled to the exact fill line to maintain a precise 9:1 ratio of blood to anticoagulant. An incorrect ratio will invalidate coagulation test results.

Wedge Blood Smear Preparation

A blood smear is a thin film of blood spread across a glass slide, used for manual white blood cell differentials, cell morphology evaluation, and checking for platelet clumps or blood parasites. The 'wedge' smear is the most common preparation technique.

Step-by-Step Wedge Smear Technique:

1. Place a clean glass slide flat on a clean table.
2. Place a small drop of fresh, well-mixed EDTA blood (using a microcapillary tube) approximately 1 cm from the frosted end of the slide.
3. Hold the specimen slide flat with one hand. With the other hand, hold a second clean slide (the 'spreader slide') at a 30- to 45-degree angle in front of the blood drop.
4. Draw the spreader slide backward until its edge makes contact with the blood drop. Allow the blood to spread along the contact line of the spreader slide via capillary action.
5. Push the spreader slide forward in a single, smooth, rapid, and continuous motion. Maintain the angle and do not lift the slide until the smear is complete.
6. Air dry the slide immediately by waving it gently or using a fan on a cool setting. Never apply heat, as heat will lyse cells and distort their structures.

Characteristics of a High-Quality Smear:

• The smear should cover 1/2 to 2/3 of the glass slide.
• It must have a smooth, even transition without ridges, lines, or holes.
• The smear should terminate in a bullet-shaped feathered edge.
• The Monolayer: Located just behind the feathered edge, the monolayer is the area where cells are distributed in a single, non-overlapping layer. This is the only zone of the slide suitable for evaluating cell morphology and performing manual counts. (Note: Platelet clumps and microfilariae often accumulate at the very tip of the feathered edge).

Diff-Quik Staining Protocol

Diff-Quik is a rapid Romanowsky-type stain widely used in veterinary clinics to stain blood smears and cytology slides. It consists of three solutions:

1. Fixative (Light Blue/Clear): Contains absolute methanol. It attaches (fixes) the blood cells to the glass slide so they do not wash off during subsequent rinsing. Dip the slide 5 to 10 times for 1 second each.
2. Solution I (Red/Orange): Contains an acidic eosinophilic dye (Eosin Y). This stains basic cell components, such as cytoplasm, hemoglobin, and eosinophilic granules. Dip the slide 5 to 10 times for 1 second each.
3. Solution II (Dark Blue/Purple): Contains a basic basophilic dye (Thiazine dye). This stains acidic cell components, such as nuclear chromatin (DNA) and ribosomes (RNA). Dip the slide 5 to 10 times for 1 second each.
4. Rinsing: Gently rinse the back of the slide with a slow stream of distilled water (do not spray water directly on the smear, as it can wash the cells off). Stand the slide vertically on a paper towel to air dry completely.

Packed Cell Volume (PCV) and CBC Concepts

Packed Cell Volume (PCV), also known as microhematocrit, is the percentage of whole blood that is made up of red blood cells. It is a rapid and highly accurate test used to screen for anemia and monitor hydration status.

PCV Procedure:

1. Fill a microcapillary tube (plain for EDTA blood, heparin-treated for non-anticoagulated blood) 2/3 to 3/4 full with well-mixed blood.
2. Wipe the outside of the tube clean with a tissue.
3. Seal one end of the tube with clay sealant (making sure to push it in straight to form a flat plug).
4. Place the tube in the microhematocrit centrifuge with the sealed clay end facing outward (against the outer rubber gasket).
5. Balance the centrifuge by placing a second tube of equal volume directly opposite.
6. Secure the inner safety lid and outer centrifuge lid. Spin at high speed (10,000 to 15,000 RPM) for 3 to 5 minutes.
7. Align the tube on a microhematocrit reader card. The interface between the clay and the RBCs is placed at the 0% line, and the top of the liquid plasma is placed at the 100% line. The line that intersects the top of the red blood cell column (excluding the buffy coat) represents the PCV percentage.

Centrifuged Tube Layers:

A centrifuged microcapillary tube contains three distinct layers:

1. Red Blood Cells (RBCs): Packed at the bottom of the tube (heaviest layer).
2. Buffy Coat: A thin, whitish-gray layer sitting directly on top of the RBCs. It contains white blood cells and platelets. An abnormally thick buffy coat suggests a high white blood cell count (leukocytosis).
3. Plasma: The liquid portion of the blood at the top of the tube.

Plasma Color and Turbidity:

Evaluating the physical appearance of the plasma provides important clinical clues:

• Normal: Clear, straw-colored (or light yellow).
• Hemolyzed (Pink to Red): Indicates ruptured red blood cells. Hemolysis can occur in vivo (hemolytic anemia) or in vitro due to improper sample handling (e.g., forcing blood through a needle, excessive shaking, or thermal trauma).
• Icteric (Bright Yellow to Orange): Indicates hyperbilirubinemia (excess bilirubin). Common in animals with liver disease, gallbladder/bile duct obstruction, or intravascular hemolysis.
• Lipemic (Cloudy, Milky White): Indicates high concentrations of lipids (fats). Often seen in animals that were not fasted prior to blood collection (postprandial sample) or those with metabolic disorders like diabetes mellitus, pancreatitis, or hypothyroidism.

Total Protein (TP) Measurement:

The veterinary assistant can measure Total Protein (TP) using the plasma from the centrifuged capillary tube.

• Procedure: Break the tube carefully just above the buffy coat (avoiding glass shards). Tap or blow the plasma onto the prism of a refractometer. Close the cover plate. Look through the eyepiece toward a light source and read the value at the boundary line of the total protein scale, which is measured in grams per deciliter (g/dL).