Comments & suggestions  should be directed to jroese@lssu.edu Visit my website Blood Collection A safe maximum for a single sample is 1.25 ml per 100 g body weight taken every 2 weeks. Animals on chronic studies requiring multiple blood samples may need to have hematocrit and serum protein levels monitored.  The total blood volume of the rat ranges from 20 - 40 ml depending on the weight of the animal.  Several techniques may be employed to collect blood samples from the rat. Retroorbital bleeding is one of the most commonly used for routine blood collection.  The rat must be anesthetized for this procedure.  Pressure is placed on the top and bottom lids of one eye to keep the eye open and the globe pushed forward slightly.  A glass microcapillary tube is placed in the medial canthus of the eye at a 30° - 45° angle toward the back of the eye.  Use firm, steady forward pressure and rotate the tube between the thumb and forefinger to cut through the conjunctiva at the back of the eye and enter the retroorbital sinus at which time blood should flow into the tube.  If no blood is obtained gently back off on the position of the tube.  After collecting the sample, close the eyelids and apply pressure with a piece of gauze until hemostasis has been achieved.  A small amount of ophthalmic ointment containing an antibiotic may be placed on the eye after bleeding has stopped to act as a bandage and help prevent infection.  If the technique of the sampler is good the eye should not be damaged.  If frequent samples must be collected, alternate the eye used for sampling. The dorsal metatarsal vein is a convenient location for collection of small blood samples.  The foot is lightly furred, eliminating the need to clip the fur.  A small amount of petroleum jelly, applied over the foot, will prevent blood from flowing into the fur of the leg.  Using a 22 - 25 gauge needle, insert the tip into the vein and immediately remove it.  As blood flows, collect it in a microcapillary tube. The tail vein is another site commonly used to collect small blood samples from rats.  The tail must be warmed with water or a heat lamp for dilation of the vessel, and it is helpful to have an assistant who can hold the tail steady and apply pressure to the base of the tail to further encourage dilation.  A 21 gauge or smaller needle or a butterfly needle can be used to enter the vein.  Start as close to the tip of the tail as possible.  After blood has been collected, apply pressure with a piece of gauze until bleeding has stopped. The saphenous vein is rapidly becoming the site of choice for blood collection for many investigators.  This procedure does not require that the rat be anesthetized to collect a blood sample, and is less invasive than retroorbital bleeding.  The rat is restrained by an assistant while the hind leg is extended by applying gentle downward pressure just above the knee.  The hair over the tarsal area is shaved with clippers or depillatory cream, and the vein is pricked with a needle (25 gauge is usually adequate).   Blood can be collected in a microcapillary tube.  Smearing a small amount of silicone grease over the area to be punctured helps to prevent the blood from corning into contact with the skin and minimizes blood clotting.  When the blood has been collected, gentle pressure applied with a piece of gauze should be used to effect hemostasis. Terminal bleeding procedures are commonly used to collect larger volume samples from anesthetized rats at the time of euthanasia, and 8 - 12 ml may be obtained from exsanguination.  In one technique, the brachial vessels are exposed after removing the skin over the axilla and the vessels are then cut.  It is helpful to pin the skin away from the axilla before cutting the vessels. Pooling blood is collected using a pasteur piptte.  Blood collection must be done quickly because clotting is initiated by contact of the blood with the tissues. Terminal bleeding can also be accomplished by collecting blood directly from the heart.  Heart puncture may be done “blindly” by directing a needle into the thoracic cavity under the xiphoid cartilage while raising the cartilage slightly with the index finger.  The needle should be directed at approximately a 30° - 35° angle slightly to the left of the midline.  A 21 gauge needle may be used, and the plunger should be pulled back on the syringe to create a vacuum.  When the heart has been entered blood will then flow into the syringe.  The syringe may also be held lightly and moved in a horizontal position until the heart beat can be felt through the syringe, at which time the syringe is raised to a 30° angle and the heart is penetrated.  Alternatively, the chest cavity may be opened so that the heart can be visualized, at which time the needle can be introduced into the right ventricle to collect blood.  Blood may also be collected terminally from the posterior vena cava or the dorsal aorta.  To collect blood from the posterior vena cava or the dorsal aorta, the entire gut must first be deflected to the right of the worker (left of the rat).  The liver must be pushed forward to expose the vena cava.  Using a 19 or 21 gauge needle slowly withdraw blood until the vessel wall collapses, then wait for it to refill, and withdraw again.  This procedure is repeated until no more blood can be obtained.  The dorsal aorta may be entered just anterior to its bifurcation into the iliac arteries.