Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Summary

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Abstract

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Introduction

Blood collection is essential in animal research. The target veins for blood collection include the retro-orbital plexus, the jugular vein, the saphenous vein, tail blood vessels, and the carotid artery^1,2,3,4. Sometimes, blood could be obtained from the abdominal aorta, vena cava or even the heart^5,6,7. On such occasions, the animals must be sacrificed and cannot be used for subsequent observation; thus, these methods are used less in daily experimental work. Blood collection from the tail vein of rats is popular and less stressful compared to the above mentioned methods⁸.

However, blood collection from the tail vein is sometimes limited by an unsatisfactory success rate. Occasionally, it is also difficult to obtain enough blood volume by this method. As the subclavian vein is large enough and is located just under the clavicle bone, subclavian vein puncture can be an alternative method for blood sampling if routine blood collection methods are unsuccessful. Here, we introduce a method of blood collection by means of subclavian vein puncture in rats.

Protocol

This study was approved by the Central South University Ethics Committee for Animal Research from the Second Xiangya Hospital (Changsha, China). The manuscript was prepared according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines⁹.

1. Material and Animal Preparation

1. Prepare required materials: adhesive tape, epilating agent, 75% ethanol, blood collection tube, 2.5 mL syringe connected with needle (24 G, 0.6 mm x 25 mm), hair shaver, electronic scale, and gauge (see Table of Materials).
2. Use 20 Sprague-Dawley (SD) rats, aged 8-10 weeks and weighing 153-200 g (see Table of Materials). Maintain rats in accordance with the Guide for the Care and Use of Laboratory Animals¹⁰.
3. House rats under standard conditions with free access to food and drinking water. Keep them in 530 cm² cages with wood-shaving bedding (2 rats per cage). Maintain animals in a temperature-controlled room temperature at around 25 °C.
4. Induce general anesthesia (sodium pentobarbital 40 mg/kg) in all animals through intraperitoneal injection before the subclavian vein puncture.
5. Place the rat in a supine position. Fix the hind limbs in a comfortable position and fix the upper limb parallel to the body axis just beside the trunk.
   NOTE: No mechanical ventilation is needed for this procedure (Figure 1).
6. Clean both sides of the infraclavicular space to remove fur (with hair shaver and epilating agent, see Table of Materials) and any visible dirt. Wipe the neck and thoracic skin with 75% ethanol. Keep the area for puncture clean and dry with gauze.

2. Blood Collection Procedure

NOTE: Both sides of the subclavian vein are suitable for puncture, and here we choose the right side for puncture. The location and direction of the right clavicle are identified by the operator's left thumb (Figure 2). The suprasternal fossae are gently touched with the index finger as the suprasternal fossae is in the direction of the needle (Figure 2).

1. Rinse a sterile 2.5 mL syringe with heparin solution (10 IU/mL).
2. Locate the puncture site 0.5 mm caudal to the outer 1/3 region of the right clavicle (Figure 3). Move the syringe gently into the skin of the puncture site. Once the needle enters the infraclavicular skin, apply negative pressure with the operator's right hand (Figure 4).
   NOTE: The average and maximal blood volume are 1.0 mL and 1.4 mL, respectively. The direction of the needle is positioned toward the suprasternal fossae and should be almost parallel to the clavicle just posterior to it. Usually, the subclavian vein can be reached by inserting the needle about 2 mm in this direction. Once the needle enters the vein, blood will enter the syringe under negative pressure.
   1. Maintain negative pressure until enough volume of blood is drawn (Figure 5).
3. If the subclavian vein blood collection is not successful, withdraw the needle to the subcutaneous area and adjust the direction of the needle slightly interior to the sternum.
   1. If three attempts fail, avoid the subclavian vein from the same side and use the contralateral side for blood sampling.
4. After blood collection, remove the puncture needle, and apply pressure to the puncture site for 1-2 minutes to stop bleeding.
   NOTE: The pressure must be very gentle to prevent choking.

3. Blood Sample Processing

1. Transfer the blood sample into an anticoagulant-coated vacuum tube (with EDTA) and centrifuge for 10 min at 1600 x g to collect plasma.
2. Aspirate the supernatant to a clean tube and store at -80 °C (Figure 6).

Results

A total of 20 SD rats (male n=10, female n=10) were used here. Blood collectionwas performed by skilled operators, who have performed more than 20 blood samplings from the subclavian vein in rats, and by the beginner operators, who have no subclavian vein puncture experience in rats or other animals. Before operations, beginners watched at least 3 procedures done by skilled operators.

Blood sampling succeeded in 17 rats thr...

Discussion

Here, we introduce an alternative method for blood extraction from the subclavian vein and we show that this method is feasible, safe, and applicable for blood sampling in rats. This method is derived from the subclavian vein puncture in pacemaker lead implantation for patients¹¹. The subclavian vein is located just posterior and caudal to the clavicle and is a continuation of the axillary vein. It runs from the outer border of the first rib to the medial border of anterior scalene muscle

Materials

Name	Company	Catalog Number	Comments
rats	Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan Province, China)
stastical software	SPSS Statistics 17
epilating agent	France Yi Sha Cosmetics Co.,Ltd(Guangzhou, Guangdong Province ,China)
2.5 mL syringe	Shandong Weigao Group Medical Polymer Co.,Ltd(Weihai,Shandong Province ,China)
hair shaver	Shanghai FLYCO Electric Co., Ltd(Shanghai,China)
adhesive tape	3M Deutschland GmbH(EdisonstraBe 6,59157 Kamen, Germany)
Pentobarbital sodium	Merck
75% ethanol	Department of Pharmacy,The Second Xiangya Hospital of Central South Univesity
blood collection tube	Hubei Jinxing Technology&Development Co.,Ltd (Wuhan Hubei Province,China) (2ml)
electronic scale	Dongguan Shengheng Electronics Co.,Ltd (Dongguan,Guangdng Province,China)
canvas gloves	for anethesia
hepain	Nanjing Xinbai Pharmaceutical  Co.,Limited (Nanjing, Jiangsu Province, China) (2mL, 12500 IU)
physical saline	Hunan Kelun Pharmaceutical Co., Ltd(Yueyang ,Hunan Province,China) (100ml)