Obtaining Blood in Small Laboratory Animals by Cardiac Puncture

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Obtaining Blood in Small Laboratory Animals by Cardiac Puncture

http://dir.niehs.nih.gov/dirlep/Webpages/techniq.html#anchor1248635

Principle

A large amount of blood can be obtained from small laboratory animals by cardiac or posterior vena cava puncture. This procedure should be used only when exsanguinating the animal. If a study protocol requires repeated blood collections from the same animal, then retro-orbital bleeding is the method of choice. The method of sample collection should remain consistent throughout a study; e.g., if blood was collected by retro-orbital bleeding at the beginning of the study, it must always be collected by that method, even at the terminal sacrifice.

Materials and Equipment

A. Anesthesia

1. Acrylic Anesthesia Chamber (70% CO2/30%O2) OR

2. Bell Jar (Dry ice placed in the bottom of a bell jar is sufficient for random animals. The animal should be separated from the dry ice by a platform [desiccator dish] and paper towels.)

B. Collection tubes, plain or with anticoagulant

C. Test tube rack

D. 3-5 mL syringes with:

1. 20 to 22 gauge needles for rats

2. 22 gauge needles for mice

E. Gauze (2" x 2")

F. Anticoagulant to coat the inside of the syringe (when hematology studies are requested)

G. Micro dissecting forceps, 4", serrated, slightly curved or tissue forceps, 1 X 2 teeth, 5"

H. Operating scissors, 5 1/2" or 6 1/2", straight, sharp/blunt

I. Foerster sponge holding forceps, 9 1/2", straight, serrated jaws

J. Necropsy board or plastic-backed sheet of absorbent material

Procedure

When drawing blood, it is always desirable to have an assistant because:

1. If there are any anticoagulated tubes, they must be mixed immediately upon collection. While the assistant is mixing the anticoagulated tubes, the phlebotomist can continue to fill the remaining tubes.

2. The assistant can prepare the next animal to be drawn (begin the anesthetization) and remove the bled animal from the work area.

A. Label the collection tubes with the proper identification numbers. Arrange the tubes in the test tube rack so that the anticoagulated tubes, if any, are drawn first.

B. Anesthetize the animal by placing it in the chamber or bell jar. Place the cover on the apparatus. Remove the animal immediately after it achieves sternal or lateral recumbency (usually less than 30 seconds). Use the pedal or palpebral reflex to assure the animal is sufficiently anesthetized and will not awaken during the procedure.

C. Place the animal on its back on a necropsy board or plastic-backed sheet of absorbent material.

D. Using the dissecting or tissue forceps, pick up the loose skin over the xiphoid process; moving anteriorly with the scissors, cut away the skin over the thorax.

E. Grasp the xiphoid through the muscle with the forceps and cut through the muscle into the abdominal cavity directly caudal to the tip of the process.

F. Lifting up on the xiphoid, carefully cut through the diaphragm and through each side of the rib cage to the axillary regions. Be careful not to damage the heart or lungs.

G. Snip the mediastinal membranes attached to the base of the heart.

H. Fold the xiphoid/rib cage anteriorly with the holding forceps.

I. Insert the needle into the right ventricle and slowly pull back on the plunger, allowing time for the cardiac chamber to refill with blood. The blood may not flow freely if the bevel of the needle is up against the ventricle wall.

J. When the desired amount of blood has been obtained, withdraw the needle from the heart. Remove the needle from the syringe and slowly deliver the blood into the collection tube(s).

K. Immediately cap the collection tube(s) containing anticoagulant and invert the tube(s) approximately 10 times mixing gently.

L. Ensure that the animal has been thoroughly exsanguinated by cutting the anterior vena cava with the scissors.

Comments

Blood may also be collected from the POSTERIOR VENA CAVA by making a mid-line incision along the length of the abdomen. The posterior vena cava can be exposed by sliding the small intestines to one side. The needle is inserted into the vein and the blood is drawn in the same manner as from the heart.