Blood Collection: Maximum Volumes and Fluid Replacement
BACKGROUND
This Standard Operating Procedure (SOP) describes industry standards for blood collection in laboratory animals. If an investigator wishes to deviate from this SOP, all changes must be outlined and justified in the protocol application (approval of the protocol indicates approval of the deviation from the SOP for that protocol only).
Blood collection must be performed in such a manner that the animal's health is not compromised. Collection of excessive blood volume can lead to severe decreases in blood pressure, shock, and death. Hemostasis must be ensured.
IACUC Procedure
- Single blood draw
a. 10% of the animal's total blood volume is the best practice single sample that can be taken once every two weeks. (Table 1)
- Multiple blood draws
a. 15% of the animal's total blood volume can be taken through multiple samplings over a 2-week period.
b. If 20% blood volume is collected over a 2-week period through multiple samplings, double fluid replacement must be administered (see #4 below).
- Exsanguination
a. The animal must be anesthetized or euthanized prior to terminal blood collection.
- Fluid replacement
a. When blood is withdrawn above the maximum amount (10% blood volume), fluid replacement must be implemented.
b. Two times the blood volume removed should be replaced with appropriate isotonic fluids. For example, if 0.27mL of blood is collected from a 25g mouse (representing 15% of blood volume), then 0.5mL fluids must be administered.
- Blood volume calculations
a. Total blood volume (mL) = animal's weight (g) X 0.06
b. 10% maximum allowable sample = total blood volume (mL) = X 0.10
- Hypovolemic shock
a. Single blood collection samples above 15% of total blood volume are not recommended due to potential for hypovolemic shock
b. Clinical signs include:
i. Fast, thready pulse
ii. Pale mucous membranes
iii. Cold skin and extremities
iv. Restlessness
v. Hyperventilation
vi. Subnormal body temperature
Appendix
Table 1. Blood collection % and associated recovery periods
| Single Sample Collection | Multiple Sample Collection |
| % circulatory blood volume removed | Approximate recovery period
| % circulatory blood volume removed
| Approximate recovery period
|
|---|---|---|---|
| 7.5% | 1 week | 7.5% | 1 week |
| 10% | 2 weeks | 10-15% | 2 weeks |
| 15% | 4 weeks | 20% | 4 weeks |
Diehl KH et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl. Toxicol. 21:15-23 (2001).
Table 2. Total blood and sample volumes for species with specific body weight (i.e. volume estimates are based on the example body weight)
| Species | Total blood volume (ml/kg)* | Example Animal Weight | Total blood volume | 7.5% | 10% (max. single sample) | 15% | 20% |
|---|---|---|---|---|---|---|---|
| Mouse | 72 | 25 g | 1.8 mL | 0.14 mL | 0.18 mL | 0.27 mL | 0.36 mL |
| Rat | 64 | 250 g | 16 mL | 1.2 mL | 1.6 mL | 2.4 mL | 3.2 mL |
| Guinea Pig | 60 | 1 kg | 60 mL | 4.5 mL | 6.0 mL | 9 mL | 12 mL |
| Rabbit | 56 | 3 kg | 168 mL | 12.6 mL | 16.8 mL | 25.2 mL | 33.6 mL |
| Cat | 57 | 3 kg | 171 mL | 12.8 mL | 17.1 mL | 25.7 mL | 34.2 mL |
| Dog | 85 | 22 kg | 1.87 L | 140 mL | 187 mL | 281 mL | 374 mL |
| Swine | 65 | 45 kg | 2.93 L | 220 mL | 293 mL | 440 mL | 585 mL |
| Sheep | 60 | 80 kg | 4.8 L | 360 mL | 480 mL | 720 mL | 960 mL |
*For mature, healthy animals with an adequate plane of nutrition.
Table 3. Sites for blood collection by species
| Species | Site of Collection |
|---|---|
| Mouse | Cardiac (terminal only), retro-orbital sinus* (anesthetized only), tail vein, saphenous vein, facial vein, distal tail transection (1-3mm, anesthesia required) |
| Rat | Same as mouse |
| Guinea Pig | Cardiac (terminal only), anterior vena cava/subclavian vein, saphenous vein |
| Rabbit | Cardiac (terminal only), marginal ear vein, jugular vein |
| Dog/Cat | Cephalic, saphenous, and jugular veins |
| Swine | Jugular vein , anterior vena cava (anesthesia recommended), ear veins |
| Ruminants | Jugular vein |
*If retro-orbital collection is necessary, the following guidelines apply:
- General anesthesia is required
- Application of a topical ophthalmic anesthetic during/after collection should be considered to provide post-procedural analgesia.
- Only one eye may be sampled at any time
o If repeated sampling within 8 hours is necessary, the retro-orbital sinus may be re-sampled by disrupting the blood clot without repeated damage to the sinus, provided the 24-hour maximum blood collection limits are not exceeded. Please consult with veterinary staff for demonstration and training of proper technique to reduce risk of trauma.
- Alternate between left and right eyes per session
- No more than 1 collection performed per 7 days; therefore 14 days between collections in the same eye
- A maximum of 3 procedures may be performed per eye (up to 6 collections total)
REFERENCES
- BVA/FRAME/RSPCCA/UFAW Joint Working Group on Refinement. 1993.Removal of blood from laboratory mammals and birds (first report). Laboratory Animals; 27:1-22.
- Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal JM, deVorstenbosch CV. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl. Toxicol. 21:15-23 (2001).
- Lee HB, Blaufox MD. Blood volume in the rat. J Nucl Med. 26(1):72-6 (1985).
- Martin, Brent J. The Laboratory Cat. The Laboratory Animal Pocket Reference Series. CRC Press. 1998.
- Swindle, Michael. Bone marrow access in swine. Sinclair Research Center. 2004. · University of Minnesota Guidelines for Collection of Blood from Experimental Animals
Approved: February 2007
Revisions Approved: 8/2008, 12/2012, 3/2016, 7/2019