NHGRI ANIMAL CARE AND USE COMMITTEE (ACUC) GUIDELINE AND
PROCEDURES FOR RETRO-ORBITAL BLEEDING IN MICE*
The NHGRI ACUC has determined that IN THE HANDS OF A SKILLED
PRACTITIONER, retro-orbital bleeding is a humane method to obtain blood samples from
mice. Investigators should be aware that this procedure may cause transient pain and
distress and appears to members of the lay community and others to be unpleasant to the animal.
Training by the Animal Program Director (APD), or his/her designee, is absolutely
required to achieve proficiency in retro-orbital bleeding.
Authorization for an investigator to
perform retro-orbital bleeding will be granted by the ACUC only after the APD has certified
that an individual has become proficient at the procedure.
Alternative methods* of blood
collection such as saphenous or tail vein puncture are available, and investigators are
encouraged to consider these as alternatives to retro-orbital bleeding.
Refer to the ARAC
Guidelines for Survival Bleeding of Mice and Rats for additional information
Although this guideline recommends rotating
orbits for successive blood collection from the retro-orbital sinus, the NHGRI ACUC does
not make this same recommendation unless the individual demonstrates proficiency in both
Collection of blood from the opposite orbit is sometimes an awkward undertaking
and may increase, rather than decrease, the possibility of injuring the animal.
The NHGRI ACUC believes that it is safer and more humane to collect blood from the orbit you are
- All blood sampling (including frequency of sampling and volume of sample) must be
approved in the Animal Study Proposal.
- Ideally, mice should be at least three weeks of age.
- Manually restrain mouse by grasping near base of tail with one hand and grasping the
nape of the neck with the opposite hand. Place tail between fingers to secure and
- Apply one drop of topical ophthalmic anesthetic (e.g., proparacaine, tetracaine) to eye to
be used. Wait a few seconds. Gently blot away excess with clean gauze, being careful
not to scratch the cornea. (An alternative to topical anesthesia is general anesthesia.)
- Place hematocrit tube or Pasteur pipette at the canthus of eye. Sterile hematocrit tubes
or Pasteur pipettes are recommended for use, but are required for immune compromised
animals. It is imperative that you know the volume of the tube you are using to collect
blood and that you do not exceed the approved volume for the weight of the animal. For
example, the volume of a standard microhematocrit tube is 70 ìl and the volume of a
Pasteur pipette to the tapered section (shoulders) is 250 ìl.
- With a gentle rotating motion, insert tube through membrane.
- Continue rotating tube on back of orbit until blood flows.**
- In most cases, no measures need to be taken to ensure good hemostasis. Should
excessive bleeding occur, apply gentle pressure with a gauze pad, being careful to avoid
scratching the cornea.
*See http://www.uib.no/vivariet/mou_blood/Blood_coll_mice_.html for the use of the
saphenous vein as an alternative to retro-orbital bleeding.
**No more than 10% of the blood volume should be removed at one sampling. Blood
volume of a mouse is ~8% of the body weight. (For example a 25 gm mouse has a blood
volume of ~2 ml – not more than 200 ìl could be removed at a single bleeding without
scientific justification and approval by the NHGRI ACUC.
Mice should not be bled anymore frequently than every 3 weeks unless smaller volumes
Approved by the NHGRI ACUC 1/28/00
Source: US National Institutes of Health