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

1. 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)

2. 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).

3. Exsanguination

a. The animal must be anesthetized or euthanized prior to terminal blood collection.

4. 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a. When blood is withdrawn above the maximum amount (10% blood volume), fluid replacement must be implemented.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.

5. 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

6. 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: August 2008, December 2012, March 2016, July 2019