Blood withdrawal techniques in lab animals

For repeated blood samples at short interval- 0.6ml/kg/day or 1% of animal blood volume can be

withdrawn. The blood volume in an adult animal is 55 - 70ml/kg.

1.Methods not requiring anaesthesia-

a. Blood collection from saphenous vein (rat, mice and guinea pig)

A slight pressure is applied above the knee joint. The vein is punctured using a needle (20G) and

the blood is collected using a capillary tube or a syringe with a needle. Local anaesthetic cream

may be applied on the collection site. The punctured site is compressed to stop the bleeding. Less

than four blood samples may be collected per day. Continuous sampling should be avoided.

b.Dorsal pedal vein (Rat or mice)

The foot is cleaned with absolute alcohol and medial dorsal pedal vein is punctured with 23G/27G needle. The appeared drops of blood are collected by a capillary tube.

2. Blood withdrawal techniques requiring anaesthesia (local/general anaesthesia).

a.Tail vein blood collection in mouse (rat, mice)- This technique is useful when the blood

requirement is about 2ml per withdrawal. The tail may be dipped in warm water (40oC). Local

anaesthetic cream may be applied 30 min before the blood withdrawal.

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 1

A 23G needle is inserted into the blood vessel and blood is collected using a capillary tube or asyringe with a needle. After the blood collection little pressure and silver nitrate ointment isapplied to stop the blood flow.

b) Tail snip blood collection in mouse.

Local anaesthetic cream is applied on the tail before the blood collection. Using a scalpel blade acut is made 1mm from the tip of the tail. The blood flow is stopped by pressing the tail tip.

c) Orbital sinus blood sample collection (Mice and rats)

Requirements- Animals- (rats or mice), anaesthetic agent, cotton, capillary tube and capillarytubes, eppendorff tubes.

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

canthus. This venous sinus is located just below the conjunctival membrane. Less than 10% of

the blood volume can be removed at one sampling. The blood volume of a mouse is about 8% of

the body weight. For example if the mouse weight is 30g then it has about 2.4ml. So less then

0.24ml blood can be withdrawn at one sampling. Two weeks should be allowed between two

samplings. It is not recommended for repeated blood sampling.

Procedure- Animals must be fully anesthetized. Topical ophthalmic anaesthetic agent (one drop

of tetracaine HCl) is applied to the eye to minimize the discomfort before bleeding. Do not touch

the tip of the applicator to any surface. This will cause the contamination of anaesthetic agent.

The animal is scruffed with thumb and forefinger and the skin around the eye is pulled taut. A

capillary tube is inserted into the medial canthus under the nictitating membrane of the eye.

Slight thumb pressure with rotation motion is applied to puncture the tissue and to enter the

capillary tube tip into the plexus/sinus. As soon as the sinus is punctured, blood enters the tubing

by capillary action. When the desired amount of blood is collected, the tube is withdrawn and a

clean gauze pad is applied on the eye to ensure hemostasis. Do not scratch the cornea with gauze

pad. It is recommended that a small amount of triple antibiotic ophthalmic ointment be applied to

the eye after the technique.

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 2

d)Marginal ear vein/artery blood collection in rabbits

Requirements-Anaesthetic agent, cotton, 26G needle, 95% alcohol, o-xylene and surgical blade.

Blood collection by central artery

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 3

Blood collection by marginal vein

The rabbit should be kept in a restrainer. Ten min before the sampling the eye is cleaned with

alcohol and anaesthetic cream is applied. If possible o-xylene and topical vasodilator is applied

on the eye surface. Size- 11 surgical blade is used to cut the marginal ear vein and the blood is

collected. A 26 G sized needle may also be used to collect the blood from marginal vein.

Sterilized cotton is placed on the injured place with pressure to stop the blood flow.

3.Terminal blood collection procedure (rat, mice, rabbit, guinea pig)

Requirements- Mouse 23-25G needle and 1-3ml syringe. Rat-20-23G needle and 10-20ml

syringe. Anaesthesia.

Anesthetize the animal. Place animal on back. Palpate heart. Insert needle slightly left of and

under sternum, directed toward animal's head. Needle and syringe should be held 20-30 degrees

off horizontal. Insert into heart. Gently apply negative pressure on syringe pressure. Withdraw

needle after blood has been collected. Approximately 1ml (in mouse) and 8ml (in rat) blood can

be collected.

Blood sample processing

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 4

1.Blood serum preparation- Blood serum is blood plasma without the clotting factors.

As serum has fewer proteins it is clear than plasma.

Procedure- The collected blood is transferred the collected blood in to the eppendorf (centrifuge)

tube. The transferred blood should be approximately 2.5 times the volume needed for use. (For

eg 5ml blood is required for 2ml serum). The resultant tube is to be kept for incubation in an

upright position for a period of 30-45min to allow clotting. Then it is centrifuged using a cold

centrifuge for a period of 15min at 1000-2000RPM. Using a clean pipette aspirate the supernatant

serum and pour into another eppendorf tube. Inspect for the turbidity. If turbidity is seen, then it

is centrifuged again. The serum samples should be stored in cold freezer (-80oC).

2.Blood plasma preparation- Blood plasma is the liquid component of blood, in which the

blood cells are suspended. Plasma is the supernatant fluid obtained when anti-coagulated blood

has been centrifuged.

Procedure- Transfer the collected blood into eppendorf tube containing an anticoagulant

(1.8mgK2EDTA/ml of blood). The tube is inverted 10times to mix the blood and anticoagulant.

The resultant mixed blood is centrifuged for 10min at 1000-2000RPM at room temperature. The

supernatant clear plasma is aspirated into another eppendorf tube. Inspect for the turbidity. If

turbidity is seen, then it is centrifuged again. The plasma samples should be stored in cold freezer

(-80oC).

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 5

Eppendorf tube Micropipette Mini-cenrifuge

------------------------------------------------------------------------------------------------------------------------------

APT (P) I MPharm 2013-14 Dr.KPS Gowda PESCPPage 6