Basic Biomethodology
For Laboratory Mice

It is important to select the proper method of blood collection that corresponds to the volume required for your research purposes.

Some methods are intended for survival and others are not.

Consult your veterinarian for more information.

Retro-orbital Sinus Blood Collection

The retro-orbital sinus is the site located behind the eye at the medial or lateral canthus.

This venous sinus is located just underneath the conjunctival membrane.

This method is intended for survival blood collection.

No more than 10% of the blood volume should be removed at one sampling.

The blood volume of a mouse is approximately 8% of the body weight.

For example, a 25 gram mouse has a blood volume of approximately 2 ml, so no more than 200 μl of blood can be removed at a single bleeding without scientific justification and approval of the Animal Care and Use Committee.

Mice should not be bled more frequently than every 3 weeks unless smaller volumes are collected.

Restrain the mouse by the scruff method. It is imperative that the mouse be properly restrained.

If the mouse is allowed to move its head, severe injury to the eye or surrounding tissues could occur.

A topical ophthalmic anesthetic must be used prior to performing this procedure.

Apply one drop of an anesthetic such as proparacaine or tetracaine hydrochloride to the eye.

Be careful not to touch the tip of the applicator to any part of the mouse.

This will cause contamination of the anesthetic.

Wait 5 - 10 seconds after the anesthetic is applied before attempting this procedure.

Gently blot away excess anesthetic with a clean gauze pad, being careful not to scratch the cornea.

An alternative to topical anesthesia for this procedure is general anesthesia.

With a gentle rotating motion, insert the tube through the sinus membrane.

Continue rotating the tube at the back of the orbit until blood flows.

Collect the appropriate volume of blood.

Upon completion, ensure good hemostasis with a clean gauze pad before returning the animal to its cage.

Be careful not to scratch the cornea with the gauze pad.

To become proficient at this technique, additional training outside the scope of this text is required.

Please contact your veterinarian for appropriate training.

The NHGRI Guideline 00.1 Procedures for Retro-Orbital Bleeding in Mice can also be used as a reference.

Blood Collection Via the Lateral Tail Veins

Tail nicking is a survival procedure that can be used to collect up to 200 μl of blood from the lateral tail veins.

This method must be used with caution, as when improperly performed, permanent tail injury or amputation may occur.

Revised 4/6/04 11

Warm the animal under a heat source, being careful NOT to overheat.

The temperature at the level of the animal should not exceed 85 - 90° Fahrenheit.

Place the mouse in a restrainer.

Prep the tail with 70% ethanol.

Stabilize the tail with the thumb and forefinger of the hand that will not be used to nick the tail.

Using a #11 scalpel blade, gently nick the lateral tail vein in the general area around the midline of the tail.

Start at least half way down the tail so that if there is a problem, you can nick the tail above the initial site and still obtain your blood sample.

Allow the blood to flow into an appropriate receptacle.

Do not squeeze the tail or attempt to milk blood from the tail.

This may cause tissue damage and contamination of the blood sample with tissue fluids.

When an appropriate volume has been collected, ensure good hemostasis with a dry, sterile gauze pad, surgical glue or silver nitrate.

Intracardiac Puncture



Intracardiac puncture must be performed under deep anesthesia and is considered a nonsurvival procedure.

Once the mouse is deeply anesthetized, prep the ventral chest area with 70% ethanol.

Insert the needle at the base of the sternum, bevel up, into the thoracic cavity at a 15 - 20° angle directed just to the left of the midline.

Aspirate slowly.

If blood starts to flow into the syringe, continue to aspirate with steady, even pressure.

If no blood is seen, reposition the needle and attempt aspiration.

Once the required blood volume is collected, the mouse is euthanized while still deeply anesthetized.

Up to one milliliter or more of blood may be collected from an adult mouse using this method.

Alternatives to the methods described here include collecting blood from the saphenous vein.

A description of this method can be found in the reference section.






Remember

• Select the appropriate restraint technique and method of collection that corresponds to the volume of blood required for your research purposes.
• No more than 10% of the blood volume should be removed at one sampling.
• Always use ophthalmic anesthetic prior to retro-orbital blood collection.
• When warming animals, DO NOT OVERHEAT.
• Ensure good hemostasis.
• The method of blood collection must be described in your Animal Study Proposal.
• Consult your veterinarian for further information on blood collection.