A sufficient quantity of blood can be collected from small laboratory animals by puncturing the orbital venous plexus in rats or the orbital venous sinus in mice. Blood can be sampled repeatedly from this site without causing blindness or permanent damage to the eye. Also, collection of blood from this site is much easier and requires less expertise than collection of blood from the tail vein or cardiac puncture.
Materials and Equipment
1. Acrylic anesthesia chamber (70% CO2/30%O2) OR
2. Bell jar (Dry ice placed in the bottom of a bell jar is sufficient for random animals. The animal should be separated from the dry ice by a platform [desiccator dish] and paper towels.)
B. Collection tubes, plain or with appropriate anticoagulant
C. Test tube rack
D. Microhematocrit capillary tubes (heparinized). Score the tube with a triangular file into 25 mm sections (three per tube) and break.
E. Gauze pads (2" x 2" squares)
When drawing blood, it is always desirable to have an assistant because:
1. If there are any anticoagulated tubes, they must be mixed immediately upon collection. While the assistant is mixing the anticoagulated tubes, the phlebotomist can continue to fill the remaining tubes.
2. The assistant can prepare the next animal to be drawn (begin the anesthetization) and remove the bled animal from the work area.
A. Label the collection tubes with the proper identification numbers. Arrange the tubes in the test tube rack so that the anticoagulated tubes, if any, are drawn first.
B. Anesthetize the animal by placing it in the chamber or bell jar. Place the cover on the apparatus. Remove the animal immediately after it becomes recumbent (usually less than 30 seconds).
NOTE: If the animal receives too much anesthesia, the blood will not flow freely and the animal may become asphyxiated.
C. The animal is manually restrained and the loose skin of its head is tightened with the thumb and middle finger of the phlebotomist. The phlebotomist's index finger is used to make the animal's eye protrude slightly by further traction on the skin adjacent to the eye.
D. The tip of the hematocrit tube is placed at the lower or inner corner of the eye and gently, but firmly, slid alongside the eyeball to the ophthalmic venous plexus or sinus which lines the back of the orbit. The venous capillaries rupture on contact with the tip of the hematocrit tube and the resulting hemorrhage fills the orbital cavity. A slight withdrawal of the hematocrit tube frees the tip so that the accumulated blood is immediately drawn into the collection tube by capillary action.
E. Hold the animal over the first collection tube, allowing the blood to drip into it freely. Once filled, immediately cap the tube of blood and mix it by gentle inversion for a few seconds to ensure complete mixture of the blood with the anticoagulant. This step is not necessary if serum is being collected.
F. Continue filling the remaining tubes.
G. When a sufficient quantity of blood is collected, relieve the pressure on the neck and remove the hematocrit tube. Bleeding usually stops immediately upon withdrawal of the tube and re-establishment of normal ocular pressure upon the venous complex. Residual blood around the eye is swabbed clear with a gauze pad to avoid clot formation.
The amount of blood collected must be in correct proportions with the anti-coagulant used, e.g., the collection tube must be sufficiently filled or liquid EDTA will introduce a dilution factor causing incorrect cell counts, shrinking RBCs and decreased MCV. If smaller amounts of blood are desired, smaller collection tubes are available.