Subclavian Vein Puncture as an Alternative Method of Blood Sample Collection in Rats. After general anesthesia, place rats in supine position and sterilize infraclavicular space with 75%ethanol. Identify superior sternal fossa before puncture.
Locate the puncture site, caudal to the outer one-third region of the clavicle in the same side. The direction of the needle was positioned towards the superior sternal fossa and should almost parallel to the clavicle, just posterior to it. After blood collection, press the puncture site for one to two minutes, to stop bleeding.
Blood collection with sufficient volume is always needed in animal experiments. Blood collection from rats'tail vein is popular and less stressful than some aggressive methods such as retro orbital plexus sample collection. However, this method is limited by the success rate.
Here, we introduce our method for blood collection with desired blood volume from the subclavian vein. Our results show that this method is safe and practical, and might be considered in case blood sampling from tail vein is failed. To start, prepare adhesive tape, epilating agent, 75%ethanol, blood collection tube, two point five milliliter syringe, hair shaver, electronic scale, heparin, and physiological saline.
House rats under standard conditions, with free access to food and drinking water, kept in 530 square centimeter cages with wood shaving bedding. Induce general anesthesia in all animals through intraperitoneal injection before puncture. Place the rat in supine position, fix the hind limbs in a comfortable position, and fix the upper limbs parallel to the body, just beside the trunk.
Clean both sides of the infraclavicular space to remove the fur and any visible dirt. Wipe the neck and thoracic skin with 75%ethanol, and keep the area clean and dry with gauze. Both sides of the subclavian vein are suitable for puncture, and here, we choose the right side for puncture.
Identify the location and the direction of the right clavicle with the operator's left thumb, and locate the superior sternal fossa gently with the index finger. Rinse our syringe with 10 unit-per-milliliter heparin solution. Locate the puncture site, point five millimeters caudal to the outer one-third region of the right clavicle.
Move the syringe gently into the skin of the puncture site. Once the needle enters the infraclavicular skin, apply an active pressure through the operator's right hand. The direction of the needle is positioned towards the superior sternal fossa, and should almost parallel to the clavicle, just posterior to it.
Usually, the subclavian vein could be reached by inserting the needle for about two millimeters in this direction. Once the needle enters the vein, blood will enter the syringe under an active pressure. Then active pressure must be maintained until proper volume of blood is drawn.
After blood collection, remove the puncture needle, and press the puncture site for one to two minutes to stop bleeding. Pay attention:the pressure must be very gentle to prevent choke. Transfer the blood sample into an anti-coagulated vacuum tube with EDTA and centrifuge at 1600G for 10 minutes to collect plasma.
Aspirate the supernatent to re-clean tube and store at minus 18 Celsius degrees. To compare the success rate between the skilled operators and the beginners, 20 SD rats were divided into two groups randomly. The results show that through the right subclavian vein, nine rats in the skilled group and eight rats in the beginner group succeeded, respectively.
Then blood collection from the remaining three rats succeeded through the left side. The success rate had no significant difference between these two groups. The time course was slightly different in these two groups.
No severe complications, such as pneumothorax, hemopneumothorax, and hemorrhage occurred during or after the operation. All animals survived after the blood sampling. To demonstrate the difficulties of learning the puncture procedure, we further compared the number of withdrawals between the skilled and the beginner groups.
As shown in Table Two, the average number of withdrawals in skilled group is slightly less than the beginners, but there is no statistical significance. In conclusion, our results show that the subclavian vein puncture strategy for blood collection is safe and feasible in rats. This method is valuable, especially on occasions when large amount blood is needed.
This method might be an optimal alternative if the conventional blood sampling fails.
