Cardiac Blood Collection via Subclavian Puncture in Mice

Summary

Subclavian cardiac puncture is a simple, reliable method for collecting terminal blood in mice. It is easy to operate and ensures high-purity samples.

Abstract

In experimental studies, mouse blood is a common experimental sample. However, there are still some challenges for the collection of large volumes of blood. The common methods of terminal large-volume blood collection include large-vessel blood collection and cardiac puncture collection. Large-vessel blood collection often requires surgery and is difficult to perform. In contrast, cardiac puncture is simple and easy to operate. However, there are also different methods for blood sampling by cardiac puncture. Different methods have different puncture sites, and each has its advantages and disadvantages. In this study, we provided a detailed examination of the subclavian cardiac puncture technique for blood collection, emphasizing its ease of operation and its ability to yield high-quality samples. This study validates that the subclavian cardiac puncture blood collection method can recover approximately 50% of the total blood volume and significantly reduce tissue fluid contamination. The method can be applied to multiple research disciplines, including immunology, toxicology, and metabolic studies.

Introduction

Blood collection from mice is essential in scientific research and various other fields¹. Non-terminal blood collection methods, including tail vein, femoral vein, and submandibular sampling, provide advantages in repeatability, yet they carry potential risks of tissue damage, animal stress, and compromised sample quality². In terminal procedures, cardiac puncture and large-vessel blood collection are commonly used to obtain large blood samples, typically yielding approximately 50% of the total blood volume^3,4.

Large-vessel blood collection requires a surgical incision and carries a high bleeding risk. In contrast, cardiac puncture is minimally invasive, reduces animal stress, and is relatively simple to perform⁵. By directly collecting blood from the heart, this method ensures a swift blood flow, minimizing the risk of coagulation and improving sample quality⁶. As a result, cardiac puncture is the preferred technique for terminal experiments⁷.

In this study, we employed the subclavian approach for cardiac puncture. Compared with traditional puncture methods, this approach offers a more precise needle trajectory. The needle sequentially traverses the skin, subcutaneous tissue, and thoracic muscles, enters the thoracic cavity, and reaches the cardiac chamber for blood collection. Abdominal puncture, for instance, may cause liver injury, which can alter blood components such as serum proteins, coagulation factors, and metabolic enzymes⁸.

Additionally, liver damage can lead to elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, further interfering with experimental results⁹. In practice, the subclavian approach is particularly suitable for experiments that require intact liver tissue, precise assessment of hepatic function, or sensitive measurement of proteins and metabolites. However, it is less suitable for very young or small mice, where anatomical structures are difficult to visualize, and for studies requiring intact cardiac tissue, as puncture may compromise subsequent analyses.

Protocol

All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. The study strictly adhered to ethical regulations for animal experiments and was approved by the Animal Ethics Committee (Approval Number: YD20250310007). A total of 14 healthy C57BL/6 mice (seven males and seven females) were used in this study, with body weights ranging from 21 g to 29 g. The animals were housed at the Laboratory Animal Center of Yanbian University under standard laboratory conditions.

1. Animals

1. Following terminal cardiac blood collection, immediately euthanize mice by overdose of isoflurane in an induction chamber (5%) until respiratory arrest and loss of nociceptive reflexes are observed. Confirm death by absence of spontaneous respiration and cardiac activity.
   NOTE: Handle needles with caution to prevent needlestick injury, and immediately discard all used sharps into designated sharps containers. Dispose of biological waste and animal carcasses in designated biohazard containers.

2. Experimental preparation

1. Get the following materials ready for the experiment: a 2 mL syringe with a 25 G needle, 75% ethanol, 1.5 mL microcentrifuge tubes with a tube rack, heparin, a low-temperature ice box, an electronic scale, a 4 °C centrifuge, a pipette, and pipette tips.
2. For cardiac blood collection in mice, use a 2 mL syringe due to its optimal volume, stable control, enhanced operational flexibility, and reduced risk of blood coagulation.
3. Rinse the syringe with heparin solution (0.3 mL, 1,250 U/mL) to prevent blood clotting.

3. Mouse preparation and positioning

1. Select healthy mice and weigh them.
2. Administer inhalation anesthesia using isoflurane. Place the mouse in an induction chamber and expose it to 5% isoflurane gas until fully anesthetized. Confirm anesthesia when the following criteria are met: loss of the righting reflex, loss of the pain reflex.
3. Monitor respiration, color, and reflexes throughout the procedure to ensure adequate anesthesia.

4. Puncture procedure

1. Place the mouse in a supine position and securely fix it on the operating platform with its forelimbs stabilized (Figure 1). Disinfect the thoracic area with 75% ethanol, ensuring proper identification of the xiphoid process and left clavicle (Figure 2).
   NOTE: When applying ethanol, avoid contact with the mouse's mouth and nose to prevent inhalation of alcohol vapors, which may cause respiratory distress.
2. Hold the syringe with the needle pointing downward.
   NOTE: The primary reason for this orientation is to enhance operational stability when the mouse is fixed in a supine position, reducing needle movement and sampling error during blood collection.
3. Insert the 2 mL syringe from the midline below the left clavicle, directing the needle toward the xiphoid process (Figure 2 and Figure 3).
4. Advance the needle to a depth of 1-1.5 cm while keeping the syringe parallel to the operating table.
5. Confirm the correct positioning of the needle within the heart chamber by observing a pulsatile blood return into the syringe.
   NOTE: The operator should maintain a steady hand to prevent the needle from shifting or penetrating too deeply.

5. Blood collection

1. Once the needle is correctly positioned, slowly draw back the syringe plunger to collect blood. Maintain a steady hand throughout the blood collection process to prevent needle movement, which could disrupt blood flow.
   NOTE: If blood flow is insufficient, slight adjustments to the syringe angle may help improve collection. However, avoid frequent repositioning of the needle to minimize experimental errors and reduce the risk of tissue damage.

6. Sample processing

1. Immediately transfer the blood sample into a pre-heparinized tube to prevent coagulation and preserve sample integrity.
2. Centrifuge the blood sample at 4 °C for 15 min at 2,000 × g to effectively separate plasma from blood cells.
3. Following centrifugation, examine the sample for clear separation of plasma and blood cells (Figure 4A). Record the sample condition to ensure it meets experimental requirements (Figure 4B).
4. Using a pipette, carefully extract the supernatant (plasma) and transfer it to a new tube. Label the tube appropriately for subsequent analysis or storage.

7. Sample testing

1. Analyze the prepared plasma samples for AST and ALT levels using an ELISA assay kit.

Results

In this study, we conducted repeated experiments on 14 C57BL/6 mice (21-29 g), with seven males and seven females. Studies report that the estimated blood volume in an adult mouse is 55 to 70 mL/kg body weight¹⁰. The blood volume collected per session ranged from 700 to 1,200 µL, accounting for approximately 50% of the total circulating blood volume (Figure 5). The operation time per session ranged from 60 to 80 s. No hemolysis or sample conta...

Discussion

Cardiac puncture is a widely used terminal blood collection method in mouse experiments, ideal for obtaining large volumes of blood while minimizing contamination from tissue fluids, thus ensuring sample purity¹². Compared to other blood collection techniques, cardiac puncture is straightforward, requiring only a single procedure, which helps reduce stress in experimental animals. In this study, we employed the subclavian puncture method. This approach features a well-defined needle path, minimizi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Tubes	Labselect	MCT-001-150-S
15 mL Conical Tubes	As One Corp	1-3500-01
2 mL Syringe	Jiangxi Fenglin	0.5x20RWLB
4 °C Centrifuge	Eppendorf	5409CP202285
75% Ethanol	Nanjing Reagent	C069156931
Alanine Aminotransferase (ALT/GPT) Activity Assay Kit	Elabscience®	E-BC-K235-M
Aspartate Aminotransferase (AST/GOT) Activity Assay Kit	Elabscience®	E-BC-K236-M
Electronic Compact Scale	YUHENG	YH-400A
Heparin	solarbio	H8060
Inhalation Anesthesia Machine	Mindray	10-1020
Isoflurane	RWD Life Science	R510-22-16
Pipette	Eppendorf	3111 000 840
Pipette tips	Axygen®	MTX-1250-C