Blood Collection: The method and volume of blood to be collected will depend on the animal species, frequency of collection, and experimental needs.

• The total blood volume in mammals is about 7% of body weight. In general the volume of blood removed at any one bleeding should not exceed 1.5% of body weight (assuming that 1 ml of blood weighs 1 gram). If blood is collected at repeated intervals, much less volume should be collected at any one time to avoid serious hematological problems. The following guidelines are recommended. If blood is collected at two week intervals, 10% of the animal's total calculated blood volume may be collected at one time. If blood is collected at four week intervals, or less frequently, 15% of the animal's total blood volume may be safely collected at one time. Example: a 4 kg rabbit is calculated to have a total blood volume of 280 ml (0.07 x 4000 gm). Thus, 28 ml may be collected once every two weeks or 42 ml once every four weeks.
   

• Sterile procedures should be used in all blood collection methods. The only exception to sterile collection procedure will be terminal procedures (cardiac exsanguination).
   

• Cardiac puncture: This technique can be used for collection of small serial samples as well as for collection of a one-time single, large-volume sample. However, it is recommended that this technique be limited to terminal collections due to the difficulty of the technique and the danger of lung injury or cardiac tamponade and death when serial samples are taken. Safer alternative techniques are available for large volume serial sampling. Cardiac puncture should be done only under general anesthesia.
   

• Retro-orbital sinus collection: This is a reliable and relatively safe method for serial blood sampling that will give reasonable sample volumes in rodents. The eye and health of the animal are seemingly unaffected when the procedure is properly done by experienced personnel. This technique should only be performed under general anesthesia. The technique involves application of slight pressure (thumb) to the jugular vein to occlude venous return from the orbital sinus followed by penetration of the retro-orbital sinus (medial to the eye) with a small glass capillary tube. Blood flow is terminated by release of pressure to the jugular vein and removal of the capillary tube.  The method will require the P.I. to justify why other less invasive methods are not appropriate for blood collection.  For more information, see Retro-Orbital Bleeding in Mice.
   

• Tail bleeds: This is a reliable and safe method for serial collection of small blood volumes. This involves nicking a tail vein (with a lancet) and collection of a few drops of blood. If tissue analysis requires tail tip amputation, it should be done under general anesthesia for mice older than 21 days of age. Care should be taken to take the smallest piece of tissue possible and stop bleeding by application of gauze pad or silver nitrate, if necessary. Animals should not be placed in a cage with other animals immediately after performing this procedure as the presence of blood on the tail can cause mutilation and further trauma.  Use finger pressure to assure hemostasis.  Finish this procedure with a wipe of peroxide over the blood collection site to eliminate any blood residue remaining.  Dried blood on skin is a highly irritating substance, and will encourage the animal to lick or chew at the dried blood..
   

• Ear vessels: Blood collection from ear vessels is a technique commonly used with rabbits. Ear vessels are very accessible in rabbits and the large white ears of the New Zealand White rabbit have more obvious and accessible vessels than do small black ears, thus explaining the popularity of the New Zealand White for antibody production. Scalpel incision of ear vessels is not a recommended technique. Blood should be collected using a sterile 18_23g needle into the vessel and collection by gravity flow into a tube or aspiration with a syringe or vacuum tube. For large volumes of rabbit blood (over 5 cc), the central ear artery is recommended. Hemostasis should be achieved by direct pressure with a gauze sponge. Arterial blood collection should be done under local or general anesthesia to diminishes vasoconstriction. For general anesthesia of rabbits, intramuscular injection of 25 mg/kg ketamine and 5 mg/kg xylazine is recommended. Alternatively, for local anesthesia, 0.1 cc of 2% lidocaine solution infiltrated into the skin adjacent to the needle entry site may be used.
   

• Dilation of vessels: Use of topical irritants such as xylene is not recommended. Xylene tends to cause leukocytosis and if it comes in contact with blood, will cause hemolysis. Adequate dilation of vessels can generally be achieved by use of a heat lamp, alcohol or gentle massaging of the ear.