Sampling Blood from the Lateral Tail Vein of the Rat

The overall goal of this procedure is to minimize the stress of collecting blood from rats by sampling via the lateral tail vein. This is accomplished by first restraining the rat and warming the tail. In the second step, a butterfly catheter is inserted into one of the dilated lateral tail veins, and then the blood is withdrawn in the final step.

The catheter is removed and the blood flow from the puncture site is stopped. Ultimately, sampling blood from the lateral tail vein causes minimal stress to the rat during blood collection, reducing the influence of the acute stress response on the composition of the blood. The main advantages of this technique over existing methods like tail tip amputation about sampling from the lateral tail vein is far less distressing to the animal and that it allows repeated within subject sampling.

The procedure will be demonstrated by two members of my laboratory, Graham Lee, a postdoc and Elia Harmatz, a technician To start, attach a butterfly catheter to the syringe, keeping the syringe shield over the needle to protect the tip. Then withdraw a volume of heparin slightly greater than the volume of blood that will be collected. Detach the syringe and fill the syringe with air.

Reattach the syringe to the catheter and expel all but trace amounts of the heparin solution from the tubing, needle, and syringe. Then place the catheter and syringe on a sterile surface. Now, quickly secure the rat in a clean cloth, taking care that the four and hind paws are in a comfortable position and that the breathing is unrestricted.

Secure the wrap with hook and loop tape. Then on a solid work surface, have an assistant gently and firmly restrain the rat by the abdomen and the base of the tail with the tail hanging off the edge of the counter to obtain the blood sample. Next, dilate the tail blood vessels in 42 degrees Celsius water for 40 to 50 seconds.

Then dry the tail with a paper towel and rotate the whole body with the tail to locate either the right or left tail vein, lateral to the artery for bleeding. Target a section of the vein in the lower portion of the tail and wipe the area with a 2%chlorhexidine antiseptic solution. Next, withdraw the plunger of the syringe from zero to approximately 50 microliters to create negative pressure in the syringe and grasp the tail gently and firmly near the tip to keep the tail straight during the sample collection.

Now, slowly insert the catheter into the vein at a shallow angle, approximately five centimeters from the tip of the tail. When the vein is penetrated, blood will flow into the catheter, slowly withdraw the plunger of the syringe to collect the desired volume at a steady rate of about 20 microliters per second. To facilitate the blood flow, run a finger along the length of the vein from the base towards the tip of the tail while remaining more than two centimeters from the tip of the inserted needle to milk the blood.

If blood cannot be successfully collected from the initial site of catheter penetration, reinsert the needle farther up the vein. When an adequate sample volume has been collected, disconnect and reconnect the catheter to release the pressure in the syringe. Then withdraw the needle from the vein and briefly apply pressure to the insertion site to stop the bleeding when the bleeding has stopped.

Wipe the area with antiseptic solution and return the animal to its home cage. To process the blood sample, aspirate the air to ensure that no blood remains inside the catheter needle, and use scissors to cut the catheter tubing just above the needle. Remove the needle, then collect the blood plasma and expel the blood into micro centrifuge tubes containing EDTA.

As an anticoagulant. Gently flick the tube to mix the anticoagulant in the blood and then place the samples on ice. Within 10 minutes of collection, spin down the whole blood and elute the plasma, taking care not to disturb the red and white blood cell layers to collect the blood serum.

Place the blood in micro centrifuge tubes without an anticoagulant at room temperature for up to 30 minutes. Then after the blood has clotted, spin the collection tubes in a refrigerated centrifuge and elute the serum. Both types of samples can then be used immediately or stored at minus 80 degrees Celsius for up to one year.

Blood plasma collected from the lateral tail vein as demonstrated, gives a plasma sample that is translucent and pale yellow in appearance. If hemolysis has occurred, a red tint will be imparted to the plasma. An acute stress response can rapidly alter the composition of a blood sample.

For example, the circulating corticosterone concentration markedly increases within 10 minutes of exposure to a stressor as in this representative experiment. Importantly, however, the low basal level of corticosterone obtained by this method prior to stressor exposure reveals that the sampling procedure itself is not a significant source of stress. We suggest that you practice this procedure on anesthetized animals until you've mastered the needle insertion and blood collection.

Then once mastered, the technique can be completed in five minutes if it's performed properly. While performing this procedure, it's important to remember to minimize any potential sources of stress, such as ambient noise, and to complete the procedure as rapidly as possible.