Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

In pharmacokinetics and toxicokinetics studies, we need to collect a certain amount of blood at specific time points in rats to detect drug exposure. This study describes a simple and efficient method for collecting blood from the subclavian vein in rats in this protocol. This technique enables repeat, timely, and easily identifiable sampling without anesthesia, and obtains high quality blood through repetitive sample collection.

You should know the anatomy of the subclavian vein, capable rat restraint and the needle point. The method is risky, and it calls for a qualified professional. To begin, find the lowest point of the subclavian triangular fossa between the neck and the forelimb in a rat.

Move approximately two to three millimeters towards the head and reach the needle point at the blood collection site. Remove the hair using an electric shaver. To keep the rat's head, neck, and breast upright, grasp the skin on the back of the neck with one hand and expose the injection site.

Straighten the forelimb on the side of the injection site to maintain its level. Keep the syringe parallel to the rat's head with the other hand and tilt the syringe outward five to 10 degrees, so that the tip is tilted toward the ventral direction. Insert the needle fully into the anterior cavity.

Draw back the syringe to maintain negative pressure with the tube. Slowly move the needle from deep to shallow and back up the same path. When blood has entered the syringe needle, fix the position of the needle.

Depending on the weight and health of the rats, monitor the maximum blood quantity to be drawn in accordance with the standards set by the institution's animal care and use committee. In the absence of other requirements, do not remove more than 20%of the animal's total blood volume within 24 hours. When sufficient blood sample is collected, immediately withdraw the syringe, and prepare for blood treatment.

Pressurize the rat's injection site for one to two minutes to stop the bleeding. Since the collection site is in the lower part of the neck, pinch the skin of the subclavian vein to stop the bleeding by pressing on it. Remove the needle from the syringe, and discard it in the sharp tool container.

Slowly transfer the blood from the syringe to a 1.5-milliliter tube. Press the syringe against the wall to avoid bubble formations. Close the lid of the microcentrifuge tube and gently flick it.

Mix the blood with the anticoagulant thoroughly by turning it upside down at least five times. Within 120 minutes after collecting the plasma, centrifuge the whole blood sample at 2, 000 RCF for 10 to 15 minutes at room temperature. Use a pipette gun to transfer the upper plasma into a fresh microcentrifuge tube.

Do not touch the pipette head with the lower whole blood. Dispose of, or re-centrifuge the plasma contaminated with the red blood cells. Plasma samples accurately collected from the subclavian vein were translucent pale yellow.

Improper blood collection, or manipulation resulted in hemolysis. During the first sampling, the body weight of the male animal was between 268 to 297 grams, and around 214 to 239 grams for the females. During the last sampling, the body weight of the male animal was between 376 to 462 grams, and around 254 to 300 grams for the females.

There was a gap of 28 days between the toxicokinetic blood collection. The animal's body weight increased steadily every week, and the clinical observation was normal. The precise needle insertion method and angle can ensure smooth blood collection and avoid risks.

This operation requires repeated practice.