The present report describes the single tail vein blood sampling method in non-anesthetized rats using vacuum extraction tube system. This is accomplished by first restricting rat, cleaning the tail, and wiping it with 70%ethanol. Then, a butterfly needle tip is inserted in one of the dilated lateral tail veins and the blood is collected in the vacuum collection tube in the rubber end of the butterfly needle.
Finally the needle is removed, and the blood puncture is stopped. The highlight of this sampling technique is the usage of vacuum extraction tube system. It can reduce the risk of direct exposure to blood and make it easier to take multiple samples from a single venipuncture.
The procedure will be demonstrated by two members of our laboratory, Yanyang Cheng and Zhong Peng. To start, prepare plastic holder, vacuum tubes with rubber stopper, and a double pointing needle. Place this material on a sterile surface.
Next, place the rat in the plastic restraining holder, taking care of that fore and hind paws are in a comfortable position, and breathing is unrestricted. Then, put the holder on a solid work surface. Let the tail hanging off the edge of the counter extended.
To obtain the blood sample, wash the tail with 20 to 30 degrees Celsius warm water to remove any visible dirt. Wipe it with 70%ethanol to dilate the tail blood vessels. Then, locate the right or left tail veins, lateral to the artery, for blood sampling.
Taking a section of the vein in the lower portion of the tail. Grasp the tail gently and firmly near the tip to keep the tail straight during the sample collection. Now, insert the 22g butterfly needle tip into one of the lateral tail veins around five millimeters at the position approximately two to three centimeters away from the tip of the tail, at angle of approximate 20 degrees.
When is the vein is penetrated, blood will flow into needle. Now, quickly insert the rubber end of the butterfly needle into the vacuum blood collection tube. Blood will flow into the vacuum collection tube slowly.
The planned blood volume is one point two milliliters each time. When adequate sample volume has been collected withdraw the needle from the vein and briefly apply pressure to the insertion site to stop the bleeding. To collect blood plasma, use tubes that contain EDTA as an anticoagulant.
Gently invert the tube several times to mix and decoagulate blood. And place the samples on ice vertically. Spin whole blood sample collection tubes in refrigerated centrifuge for ten minutes to separate plasma and serum.
Elute the plasma, taking care not to disturb the red and white blood cell layers. To collect blood serum, use tubes without anticoagulant. Both types of samples can be used immediately or stored at under 80 degrees Celsius for up to one year.
In order to test and verify the feasibility of this method, two medical students were trained for two hours before performing this blood collection protocol as beginner group. And two experienced fellows served as experimental group. The results showed that the success rate was 95%in the experimental group and 90%in the beginner group.
The success rate and puncture times were similar between the two groups. The sampling duration was significantly shorter in the experimental group compared to the beginner group. This sampling technique is simple and efficient for repeated blood collection in rats, and could obtain sufficient blood volume each time.
Moreover, this method is easy to learn, and it could be performed in non-anesthetized rats and cause the minimal stress to the animals. When adopting a vacuum blood collection method because the vacuum and pressure will obtain continuous blood drawing without the risk of blood contact, which also reduces the chance of contamination and hemolysis.
