None declared.

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
<ol>
	<li>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,...

<ol>
	<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology, Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Korfmacher, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+capillary+microsampling+for+rat+pharmacokinetic+studies:+Comparison+of+tail-vein+bleed+to+jugular+vein+cannula+sampling.">Utility of capillary microsampling for rat pharmacokinetic studies: Comparison of tail-vein bleed to jugular vein cannula sampling.</a> Journal of Pharmacological and Toxicological Methods. 76, 7-14 (2015).</li><li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheterization+of+the+carotid+artery+and+jugular+vein+to+perform+hemodynamic+measures,+infusions+and+blood+sampling+in+a+conscious+rat+model.">Catheterization of the carotid artery and jugular vein to perform hemodynamic measures, infusions and blood sampling in a conscious rat model.</a> Journal of Visualized Experiments. (95), (2015).</li><li>Wickremsinhe, E. R., Renninger, M., Paulman, A., Pritt, M., Schultze, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Repeated+Tail+Clip+and+Saphenous+Vein+Phlebotomy+on+Rats+Used+in+Toxicology+Studies.">Impact of Repeated Tail Clip and Saphenous Vein Phlebotomy on Rats Used in Toxicology Studies.</a> Toxicologic Pathology. 44 (7), 1013-1020 (2016).</li><li>Takahashi, M., Makino, S., Kikkawa, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+suitable+for+mammalian+whole+embryo+culture.">Preparation of rat serum suitable for mammalian whole embryo culture.</a> Journal of Visualized Experiments. (90), e51969 (2014).</li><li>Cochran, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+Glucose+Metabolism+Kinetics+Using+18F-FDG+Micro-PET/CT.">Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT.</a> Journal of Visualized Experiments. (123), (2017).</li><li>Beeton, C., Garcia, A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drawing+blood+from+rats+through+the+saphenous+vein+and+by+cardiac+puncture.">Drawing blood from rats through the saphenous vein and by cardiac puncture.</a> Journal of Visualized Experiments. (7), 266 (2007).</li><li>Zou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Blood+Collection+from+Tail+Vein+of+Non-Anesthetized+Rats+with+a+Vacuum+Blood+Collection+System.">Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Kilkenny, C., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+ARRIVE-ing+at+a+solution.">Improving bioscience research reporting: ARRIVE-ing at a solution.</a> Lab Animal. 44 (4), 377-378 (2010).</li><li>National Institute of Health. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Antonelli, D., Feldman, A., Freedberg, N. A., Turgeman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axillary+vein+puncture+without+contrast+venography+for+pacemaker+and+defibrillator+leads+implantation.">Axillary vein puncture without contrast venography for pacemaker and defibrillator leads implantation.</a> Pacing and Clinical Electrophysiology. 36 (9), 1107-1110 (2013).</li><li>Luis, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+magnetic+resonance+imaging+of+the+thoracic+venous+system+in+rats+with+congenital+diaphragmatic+hernia.">Microscopic magnetic resonance imaging of the thoracic venous system in rats with congenital diaphragmatic hernia.</a> Pediatric Surgery International. 27 (2), 175-180 (2011).</li><li>Tsukamoto, A., Serizawa, K., Sato, R., Yamazaki, J., Inomata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+signs+monitoring+during+injectable+and+inhalant+anesthesia+in+mice.">Vital signs monitoring during injectable and inhalant anesthesia in mice.</a> Experimental Animals. 64 (1), 57-64 (2015).</li></ol>

This work was supported by the grant from the National Natural Science Foundation of China No. 81670269, No. 81500355 and No. 81500226.

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<sup clas...

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 artery1,2,3,4. Sometimes, blood could be obtained from the abdominal aorta, vena cava or even the heart5,6,7. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>rats</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan Province, China)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stastical software</td>
			<td>SPSS Statistics 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>epilating agent</td>
			<td>France Yi Sha Cosmetics Co.,Ltd(Guangzhou, Guangdong Province ,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringe</td>
			<td>Shandong Weigao Group Medical Polymer Co.,Ltd(Weihai,Shandong Province ,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hair shaver</td>
			<td>Shanghai FLYCO Electric Co., Ltd(Shanghai,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>adhesive tape</td>
			<td>3M Deutschland GmbH(EdisonstraBe 6,59157 Kamen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Department of Pharmacy,The Second Xiangya Hospital of Central South Univesity</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>blood collection tube</td>
			<td>Hubei Jinxing Technology&#38;Development Co.,Ltd (Wuhan Hubei Province,China) (2ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electronic scale</td>
			<td>Dongguan Shengheng Electronics Co.,Ltd (Dongguan,Guangdng Province,China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>canvas gloves</td>
			<td>for anethesia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hepain</td>
			<td>Nanjing Xinbai Pharmaceutical&#160; Co.,Limited (Nanjing, Jiangsu Province, China) (2mL, 12500 IU)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>physical saline</td>
			<td>Hunan Kelun Pharmaceutical Co., Ltd(Yueyang ,Hunan Province,China) (100ml)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

subclavian vein puncture as an alternative method of blood sample collection in rats

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.

A total of 20 SD rats (male n=10, female n=10) were used here.&#160;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...

Watch this Scientific Journal Video about Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats at JoVE.com

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

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

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

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.

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13.7K Görüntüleme.  Central South University. Sıçanlarda Alternatif Kan Örneği Toplama Yöntemi Olarak Subklavian Ven Delinme. Genel anesteziden sonra sıçanları supine pozisyona yerleştirin ve %75 etanol ile infraklaviküler alanı sterilize edin. Delinmeden önce üstün sternal fossa tanımlayın.Aynı tarafta köprücük dış üçte bir bölgeye kaudal, delinme alanı bulun. İğnenin yönü üstün sternal fossa doğru konumlandırılmış ve neredeyse köprücük kemiği paralel olmalıdır, sadece posterior. Kan top...