University Committee On Animal Care and Supply (UCACS) Standard Operating Procedure (SOP)

Guidelines SOP
C201: Blood Collection – Multiple Species
University Committee on Animal Care and Supply (UCACS)

Standard Operating Procedure (SOP)
SOP # Title
C201 Blood Collection by Different Routes in Different Species
1. Purpose
The purpose of this SOP is to describe the general method for blood collection by different
acceptable routes in various species and recommended blood collection volume and
frequency.
2. General Information/Responsibility
This SOP describes the various blood collection procedures approved by the AREB. Principal
investigators may reference this SOP when blood collection is performed as indicated in the
SOP. Any deviation from the SOP must be identified in the Animal Use Protocol and the
principal investigator must provide justification for the deviation from standard procedure.
Total blood volume of the species determines the acceptable quantity and frequency of
blood sampling. Volume of blood sampled must also consider the size and health status of
the animal, type of sample needed (serum, whole blood), frequency of sampling.
Responsibility: Individuals performing blood collection must be adequately trained.

Individuals collecting blood samples must be aware of the maximum allowable blood
volume that can be collected at any one time or over a period of time to ensure that the
animal is not physiologically compromised. This SOP provides only general guidance on
blood volume collection.
Table of Contents for Blood Collection Techniques by Species

Mouse (Submandibular vein, Sapheneous vein, Tail vein, Cardiac puncture)....................7
Rat (Submandibular vein, Jugular vein, Saphenous vein, Cardiac puncture)....................11
Rabbit (Central ear artery, Marginal ear vein, Carotid artery)..........................................16
Guinea Pig (Cardiac puncture, Vena Cava, Jugular vein)...................................................18
Dog (Cephalic vein, Lateral saphenous vein, Jugular vein)................................................21
Cat (Cephalic vein, Medial saphenous vein, Jugular vein).................................................24
Cattle (Tail vein, Jugular vein)............................................................................................26
Sheep (Jugular vein)...........................................................................................................28
Horse (Jugular vein, Facial sinus).......................................................................................29
Pig (Cranial vena cava, Marginal ear vein..........................................................................31
Chicken/Bird (Cardiac puncture, Jugular vein, Brachial vein)............................................32
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Guidelines SOP
3. Equipment/Materials (items utilized will depend upon the species and the collection
method)
 Vacutainer tubes or Eppendorf  Acepromazine
tubes and holders  Hydrogen peroxide
 Capillary or Hematocrit tube (plain  Isoflurane and anaesthetic machine
or heparinized) – table top with face mask and
 Sterile disposable Vacutainer or induction chamber
hypodermic needles (one inch)  Eye lubricant
o Gauge 18, 20, 22, 23, 25, 27  Circulating warm water blanket
 Needle holders  Halstead mosquito forceps, curved
 Syringes (e.g. 1 cc, 3 cc, 6 cc, 12 cc,  Kelly forceps – curved
60 cc)  Allis tissue forceps
 Gauze or Cotton tipped applicators  Tissue forceps – toothed
 Vaseline  Microsurgery scissors, sharp points,
 Oil of Wintergreen Vein introducer
 Cordless hair trimmer; razor blade;  Fluids for replacement (Lactated
electric clippers Ringer Solution, 0.9% Saline, 5%
 Restraint tube (50 mL tube with Dextrose)
open bottom) or appropriate  Macrodrip administration set
restraint device (e.g. towel, cat bag)  IV Pressure bag
 Hemostatic forceps with wide elastic  2-0 silk ties
band attached  3 French or 4 French x 70 cm
 Butterfly catheter with plastic tubing urethral Catheter, open-end (Cook
cut off Urological)
 2% xylocaine jelly  Sharps container
 Skin disinfectant  Gloves
 Alcohol  Paper clip
 Atropine  White tape, shoelaces
 Ketamine
4. Procedure
A. Type of Blood Collection Tube and Size of Needle
1. Blood Collection Tube: Check the relevant laboratory assay procedure for the type and
method of sampling required (serum, whole blood, plasma). Obtain the correct type and
size of Vacutainer/Eppendorf tube required for assays.
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Guidelines SOP
Table. Type of Vacutainer tubes.

Sample Anticoagulant Color Code
Serum None (Silicone glass + SST) Red/Grey
Serum None (Silicone glass) Red
Plasma Heparin Green
Plasma Na2EDTA Lavender
Plasma Sodium Citrate Blue
Blood Trace Elements None Royal Blue
Note: The specific type of anticoagulant may be critical for the study. A wide variety of
specialized blood collection tubes are available to suit specific needs.
2. Size of needle: The length and bore size (gauge) are important considerations. Use the
largest bore size possible to allow rapid blood withdrawal without collapsing the vein
and without causing a hematoma.
B. Volume of Blood
1. Circulating Blood Volume

a. The maximum allowable blood collection depends on knowledge of the circulating
blood volumes. On average the circulating blood volume is approximately 55-70
mL/kg body weight or 5-15% of body weight.
2. Blood Collection Volume

a. Animal well-being and maintenance of a normal physiological response are the
primary considerations regarding the limits of blood sampling volume and repeated
blood sampling.
Table. Blood volume limits and recovery periods

Single sampling Multiple sampling
% Circulatory
blood volume % Circulatory
removed in a Approximate blood volume Approximate
single sampling recovery period removed in 24 h recovery period
7.5% 1 week 7.5% 1 week
10% 2 weeks 10-15% 2 weeks
15%* 4 weeks 20% 4 weeks
*For single sampling it is not recommended to remove blood volume >15% due
to risk of hypovolaemic shock.
b. Single blood draw – a maximum of 1% body weight can be removed as a single blood
draw without need to provide supplemental replacement fluids. Withdraw the
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Guidelines SOP
minimum volume of blood necessary for the experimental needs. If >10% blood
volume is required, replace collected volume by 3-4 –fold with isotonic fluids (saline,
dextrose, lactated ringers).
Table. Recommended maximum blood volumes for different species*

Species Blood volume (mL) 7.5% (mL) 10% (mL) 15% (mL) 20% (mL)
(weight) [mL/kg BW] [mL/kg BW] [mL/kg BW] [mL/kg BW] [mL/kg BW]
Mouse 1.8 0.1 0.2 0.3 0.4
(25 g) [72] [5.4] [7.2] [10.8] [14.4]
Rat 16 1.2 1.6 2.4 3.2
(250 g) [64] [4.8] [6.4] [9.6] [12.8]
Rabbit 224 17 22 34 45
(4 Kg) [56] [4.2] [5.6] [8.4] [11.2]
Hamster 7.8 0.6 0.8 1.2 1.6
(100 g) [78] [5.8] [7.8] [11.7] [15.6]
Cat 168 12.8 17 25.5 34
(3 Kg) [56] [4.2] [5.6] [8.4] [11.2]
Dog 850 64 85 127 170
(10 Kg) [80] [6.4] [8.5] [12.8] [17.0]
Guinea pig 14.6 1.2 1.5 2.3 3.0
(200 g) [73] [5.5] [7.3] [11.7] [15.6]
Minipig 975 73 98 146 195
(15 Kg) [65] [4.8] [6.5] [9.7] [13]
Pig 1950 146 196 292 390
(30 Kg) [65] [4.8] [6.5] [9.7] [13]
Goat 4650 350 467 700 933
(50 Kg)
Sheep 4550 350 450 600 900
(75 Kg) [60] [4.5] [6] [9] [12]
Deer 5100 385 510 765 1020
(85 Kg) [60] [4.5] [6.0] [9.0] [12]
Poultry 60 4.5 6 9 12
(1 Kg) [60] [4.5] [6] [9] [12]
Cattle 15000 1000 1500 2000 3000
(250 Kg) [60] [4.5] [6] [9] [12]
Bison 21000 1575 2100 3150 4200
(350 Kg) [60] [4.5] [6.0] [9.0] [12]
*Blood volumes that can be removed that do not cause significant alterations in the
animal’s normal physiology is as follows
c. Multiple blood collection – 0.07% of body weight is the maximum volume of blood
that can be taken daily without requiring supplemental fluids.
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Guidelines SOP
d. For exsanguination, expect blood volumes equaling 4-5% body weight when blood is
collected while the heart is beating under a surgical plane of anaesthesia.
e. Monitor the animal during and immediately following the blood sampling for possible
signs of hypovolemic shock. Institute emergency measures if noted (provision of
oxygen, fluid replacement). Signs of hypovolemic shock include: a) fast and weak
pulse; b) pale mucous membranes; c) cold skin and extremities; d) restlessness; e)
hyperventilation.
Table. Normal haematological values in different species

PCV RBC Hb Reticulocyte WBC
Species (%) (1012/L) (g/dL) (%RBC) (109/L)
Mouse 35-45 7.7-12.5 10-20 3.3-13.3 8.0
Rat 35-45 7.2-9.6 12-18 1.7-21.1 14.0
Hamster 39-59 4.0-10.0 2-30 -- 7.6
Guineapig 35-42 4.5-7.0 11-17 1.8-6.1 10
Rabbit 30-50 4.5-7.0 8-15 2.9-8.0 9.0
Cat 30-50 6.0-10.0 8-14 0-1.0 5.5-19.5
Dog 38-53 4.5-8.0 11-18 0-1.5 6.0-17.0
Pig 30-50 5.0-9.0 10-16 -- 7.0-20.0
Chicken 23-55 1.25-4.5 7.0-18.6 -- 9-31
Cattle 24-46 5-10 8-15 -- 4-12
Sheep 29-38 8.0-14.0 10-12 ---- 4.0-12.0
Goat 29-38 13.0-18.0 8-14 5.0-14.0
C. Collection of Blood Samples (General)
1. Manual restraint or sedation/general anaesthesia is required depending upon blood

volume collection and species. General guidelines are as follows:
a. Rodents: manual restraint for small volumes; general anaesthesia for large volumes
b. Rabbits: sedation with or without topical for other than small volumes
c. Dog/Cat/Pig: manual restraint for small volumes, general anaesthesia for large
volumes
d. Ruminants and horses: manual restraint for small volumes, general anaesthesia for
large volumes
e. Fish: general anaesthesia
Table. Advantages/disadvantages of the various blood sampling routes and methods.

Vein General Tissue Repeat Volume Species
Anaesthesia Trauma sampling
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Guidelines SOP
Jugular No Low Yes +++ Rat, dog,

rabbit, cat,
hamster,
large animal
Cephalic No Low Yes +++ Dog, sheep,
cat
Saphenous/latera No Low Yes ++(+) Mouse, rat,
l tarsal dog, cat,
hamster
Marginal ear No (local) Low Yes ++ Rabbit, pig
Submandibular No Low Yes ++ Mouse, rat
Lateral tail No Low Yes ++(+) Mouse, rat
Femoral No Low Yes ++(+) Rabbit
Cranial vena cava No Low Yes +++ Pig
Tail tip Yes Moderate Limited + Mouse, rat
amputation
Cardiac Yes Moderate No +++ Mouse, rat,
rabbit
Coccygeal No Low Yes +++ Bovine
Brachial wing vein No Low Yes ++ Bird
Caudal vein Yes Fish
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Guidelines SOP
D. Blood Sample Collection by Species and Route
MOUSE
I. Submandibular Vein
Orbital vein
Submandibular
vein

Jaw
line

Figure 1 Grey
dot
a. At the back of the jaw the veins draining the eye (orbital vein) and facial region
(submandibular vein) meet to form the beginning of the jugular (Figure 1).
b. Assemble blood collection equipment. Open the protective covering containing the
needle but do not remove the needle cover.
c. Remove the mouse from the cage by picking it up at the base of the tail.
d. Transfer mouse from cage to stainless steel cage top.
e. While holding onto the tail, use the other hand to manually restrain the mouse. Scruff
the mouse by grasping loose skin over the shoulders by starting low on the forelegs of
the mouse and moving upwards behind the ears. Hold mouse in the air to establish a
relaxed position for the mouse. (The proper restraint will result in the forelegs pulled up
and backwards away from the head and the eyes will bulge out. NOTE: this restraint
may cause some respiratory distress to the mouse – try to restrain the mouse for the
shortest period of time possible.
f. Position a needle, bevel up at 90° to the skin at a location using the following landmarks:
i. White mouse will have a grey dot located on the jawline directly below the lateral
canthus of the eye (Figure 1).
ii. Colored mice do not have a landmark; the needle will be inserted on the jaw line in
a line drawn straight down from the lateral canthus of the eye.
g. To determine the appropriate needle gauge use the following criteria:
i. 18 gauge is used to collect a rapid free flow sample into a blood collection tube
when a maximum volume of sample is required.
ii. 20 gauge is used to collect a moderately flowing sample either into a collection tube
or a capillary tube when 100-150 microliters of blood are required.
iii. 22 gauge is used to collect a controlled blood flow into a capillary tube when 50-100
microliters of blood are required.
h. When the needle is removed, blood will flow.
i. Collect blood into the desired blood collection container (e.g. Eppendorf).
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j. After collection, release the mouse from the restraint to stop the flow of blood.
k. If the facial artery is struck instead of vein, blood will be bright in color and after sample
collection the artery will have to be held off to stop the bleeding using a gauze.
l. Return the mouse to its cage.
m. Discard the needle into the sharps container.
Figure. Mouse restraint, facial vein blood collection
Mouse restraint Facial Vein Blood Collection
II. Saphenous Vein Venipuncture

a. Saphenous vein venipuncture is a convenient, rather noninvasive method for collection
of blood samples for purposes of clinical chemistry, hematology, biochemical assays,
and pharmacokinetic studies. This procedure can be performed on both anaesthetized
and unanaesthetized mice.
b. Warming the mouse immediately prior to blood collection by use of a heat lamp over
the cage for five minutes will improve blood collection.
c. Place mouse head first into the restraint tube.
d. Extend the back leg and fix by holding the fold of skin between the tail and the thigh of
the mouse.
e. Shave hair from the hind leg over the saphenous vein using a cordless trimmer.
f. Apply vaseline on the skin to form a thin layer as a barrier between the skin and the
blood. Vaseline will allow the blood to form a droplet for easy collection.
g. Apply digital pressure at the groin to raise the femoral and saphenous veins. The vein
crosses the leg at the ankle and wraps around below the knee joint.
h. Using a 22 gauge needle (one inch) make a puncture by quickly inserting the needle
perpendicular to the vessel on the inner surface of the hind leg. The blood should
immediately flow and form a drop on the skin. Dispose the needle into the sharps
container.
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i. Fill hematocrit tubes from the blood that pools on the surface of the skin.
j. Alternatively, blood can be collected into an Eppendorf tube by collecting each droplet
of blood as it forms.
k. Release leg and apply pressure with a clean gauze to the puncture site to achieve
hemostasis.
l. Release mouse from restrainer and return the animal to the cage after checking that
bleeding has stopped.
m. Serial samples can be obtained over a short period of time by gently removing the scab.
III. Tail Vein Microsampling (5-20 μL Blood Volumes)

a. Tail vein microsampling is used for the collection of small (5-20 μL) blood samples as a
single sample or serial sampling from the same animal.
b. Remove the mouse from the cage by picking it up at the base of the tail.
c. Transfer mouse from cage to the workstation.
d. Gently restrain the mouse at the base of the tail.
e. Using a 25 gauge needle, prick the tip of the tail and transfer the mouse to the top of a
wire cage.
f. Gently stroke the tail from the base of the tail to near the tip to encourage a drop of
blood to form at the site of the prick.
g. Apply a hematocrit or capillary tube to the blood drop to collect the blood.
h. Repeat the tail stroke until a sufficient volume of blood is collected from the mouse.
i. Apply finger pressure on the tail at the blood sampling site for 10-20 seconds to stop
blood flow.
j. Return mouse to its cage.
k. It might be necessary to warm the mouse to dilate the blood vessel prior to sampling
when 20 μL blood volume is required.
IV. Lateral Tail Vein

a. Lateral tail vein sampling is used when larger volumes of blood (50 μL - 0.2 mL) are
required. Ideally, a maximum of two blood samples in a 24 h period is recommended to
avoid damage to the tail vein.
b. Place mouse head first into the restraint tube.
c. Extend the tail from the opening and wash the tail with diluted Hibitaine (1%), if
necessary.
d. Apply Oil of Wintergreen to the sampling site and allow 30 minutes before blood
sampling to assure complete local anaesthesia.
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e. Using a 25 gauge needle, prick the lateral tail vein one-third the distance up the tail from
the tail tip. This will allow additional samples to be taken from the tail vein by using sites
closer to the base of the tail.
f. Apply a capillary tube to the blood drop and collect blood into the tube.
g. Stop blood flow by applying finger pressure at the blood sampling site for 30 seconds.
h. Return the mouse to the cage.
i. It might be necessary to warm the mouse to dilate the blood vessel prior to sampling
when a larger blood volume is required.
V. Cardiac Puncture
a. Cardiac puncture is used when large volumes of blood (0.1 mL – 1 mL) are required.
This procedure is normally done under general anaesthesia and is terminal (if not
terminal consult the Animal Welfare Veterinarian).
b. Remove mouse from cage and place in induction chamber.
c. Turn oxygen to 1 L/min and isoflurane to 5%.
d. Once rodent has fallen asleep and is breathing regularly, remove from chamber and
place rodent in dorsal recumbency on circulating warm water blanket with nose in nose
cone to maintain anesthesia.
e. Align animal so it is straight from nose to tail tip.
f. Prepare 23 gauge needle and 1 mL syringe.
g. With index finger, locate the notch between the Xiphoid process (at the end of the
sternum) and the last rib on the left side of rodent.
h. Aiming toward the midline, insert the needle into the notch, at a 10 – 15 degree angle
above the body of the rodent.
i. As the needle is inserted into the heart, pulsating blood will be seen in the hub of the
needle.
 If no blood is seen, pull the needle straight out the path it went in. Relocate the
landmarks and insert the needle again.
 (alternative needle placement below)
j. When blood appears in the hub, hold the needle and syringe still and withdraw sample.
k. If the blood flow stops, use MINOR manipulations of the needle (rotating left or right,
inserting or withdrawing a millimeter or two) to try to restart the flow.
l. When completed, withdraw the needle out of the heart and chest cavity in a straight
path.
m. Place blood into collection tube
n. Transfer mouse to euthanasia chamber.
o. Turn off anesthetic machine.
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p. Ensure euthanasia has been accomplished by opening the chest cavity before properly
disposing of mouse
q. Alternative needle placement
a) Grasp the rodent’s right front foot and bend the elbow to form a 90° angle.
b) At the point the of the 90° angle, insert the needle though the chest between the
ribs.
 The needle should be inserted closer to the sternum of the rodent as opposed
to its spine.
c) Insert the needle until blood is visible in the hub of the needle.
 If the needle is inserted and no blood appears, pull the needle straight back
out the path it went in.
 Recheck the landmarks for proper position and re-insert the needle to locate
the heart.
d) Continue procedure as above.
RAT
I. Facial (Submandibular) Vein
a. Assemble blood collection equipment. Open the protective covering containing the
needle but do not remove the needle cover.
b. Remove the rat from the cage by picking it up under its armpits.
c. Restrain rat with a scruff hold so that it is lying on the palm of your hand to expose the
jaw line and masseter muscle.
d. Use the following landmarks to determine where to insert the needle:
i. In a white rat located along the jaw line directly below the lateral canthus of the eye
a grey dot is observed (use this as a landmark).
ii. Insert the needle (18 gauge) at a 30° angle 0.5 cm behind the grey dot.
iii. For pigmented rat, insert the needle at a point straight down from the lateral
canthus of the eye to the jaw line.
e. Invert the animal to collect the sample directly into the collection tube.
f. After the sample is collected, apply gauze and pressure at the site of the venipuncture to
stop bleeding.
g. Release restraint and return rat to the cage.
h. Dispose of needle into sharps container.
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II. Jugular Vein (anesthetized)

a. Put a paper towel at the bottom of the anaesthetic induction chamber and place rat in
the chamber.
b. Turn the oxygen flow to 1-2 L/ min and the isoflurane to 5% on the anaesthetic machine.
c. Once the rat is relaxed and has lost its righting reflex dial down isoflurane to 2-3%.
d. Remove rat from chamber and put on the face mask (remember to open the valve to
the face mask and close the valve to the induction chamber) and place rat on an
incontinent pad.
e. Put eye lube in each eye and then place the rat in dorsal recumbency.
f. Using the pedal reflex ensure that the rat is maintained at a light surgical plane (usually
1.5-2 % isoflurane is sufficient).
g. Place a 3 cc syringe underneath the neck of the rat cranial to the shoulder blades.
h. Secure the rat to the pad with masking tape (extend each limb and secure to pad). It is
important that the rat is positioned with its vertebra as straight as possible and the front
limbs at 90o angles to the spine.
i. Hold the skin taut in the neck region and shave the hair from midline and laterally to the
right side of the rat using the small clippers (only shave a 1 inch by 1 inch area). Remove
hair from the area.
j. Sterilize the area with an alcohol swab.
k. Secure a needle to a syringe and unlock the plunger of the syringe. If a plasma sample is
required, first draw up the anticoagulant to allow for coating of the syringe barrel and
discharge to the syringe contents to leave a small volume of anticoagulant in the hub of
your needle.
l. Find the middle point between the sternum and the top of the shoulder at a point just
right to the midline.
m. Advance a 23 gauge one inch needle (bevel side up) through the skin at a 30 o angle in a
direction towards the heart. Create a vacuum within the syringe barrel and then
advance the needle slowly a few millimeters at a time.
n. Once the needle is in the trunk of the vein, a very small drop of blood will appear in the
needle hub. This will indicate that the needle is properly seated into the vein.
o. Once seated, steady the syringe and slowly milk the plunger to fill the barrel. Avoid
continuous pressure as this will collapse the vein.
p. Once the sample is collected, apply digital pressure with gauze to the site before
withdrawing the needle.
q. Remove the needle from the syringe and dispense blood into the collection tube.
Dispose needle into sharps container.
r. Remove the masking tape and syringe from under the animal’s neck.
s. Turn off the isoflurane and deliver 100% oxygen for a few minutes.
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t. Place the animal in a cage on a paper towel until fully conscious. If recovery is slow, the
cage can be placed on a heated circulating water blanket until complete recovery.
u. This can be conducted in an awake rat according to the following steps:
i. This technique requires two people – a restrainer and a phlebotomist.
ii. Remove rat from the cage and calm the rat by gently stroking it.
iii. Restrain the rat by grasping the skin over the nape of the neck and shoulders
with thumb and index fingers.
iv. Turn the rat over such that it is lying in your palm and the body is straight. With
the other hand restrain the head by placing the muzzle between the second and
third fingers and the thumb at the thoracic inlet to hold back the forelegs. Pull
back on the head until it is even with the spine.
v. The other person shaves and prepares the neck region as above.
vi. Apply slight pressure in the thoracic region to raise the jugular vein.
vii. With a 23 guage one inch needle insert the needle (bevel side up) through the
skin at a 30o angle at a midpoint between the sternum and shoulder in a
direction towards the heart. Create a vacuum within the syringe barrel and then
advance the needle slowly a few millimeters at a time.
viii. Once the needle is in the trunk of the vein, a very small drop of blood will appear
in the needle hub. This will indicate that the needle is properly seated into the
vein.
ix. Once seated, steady the syringe and slowly milk the plunger to fill the barrel.
Avoid continuous pressure as this will collapse the vein.
x. Once the sample is collected, apply digital pressure with gauze to the site before
xi. Remove the needle from the syringe and dispense blood into the collection tube.
xii. Release animal from restrainer; give positive reinforcement.
xiii. Dispose of needle into sharps container.
III. Saphenous Vein

a. Use this route for collection of single or serial blood samples without anaesthesia.
b. Assemble all equipment.
c. Remove rat from cage and wrap the head, front legs and body in a towel leaving the
rear legs and tail free.
d. Using the same hand that is holding the restrained rat, gently but firmly squeeze the leg
by placing the thumb on the upper thigh/hip area and the lower leg between the second
and third fingers.
e. Shave hair using clippers over the lateral saphenous vein area and swab shaved area
with 70% alcohol.
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f. The pressure applied by the thumb will result in saphenous vein engorgement. Locate
the vein and apply a thin film of Vaseline® to allow for drop formation.
g. Using a 25 gauge needle puncture the vessel at a 90° angle at the most proximal (closest
to the body) visible site.
h. Collect the blood drops into the appropriate collection tube.
i. Apply dry gauze and pressure to the puncture site and release pressure on the upper
thigh until bleeding ceases.
j. Remove the rat from the towel and return to its cage.
k. Monitor the rat for an addition 5-10 minutes to ensure that bleeding has stopped.
l. Repeated blood samples may simply require removal of the scab with a dry piece of
gauze or a new puncture site can be made distal to the previous site (towards the foot).
IV. Tail Vein

a. Assemble all equipment.
b. Remove rat from cage 20 – 30 minutes before procedure – apply 2% Xylocaine Jelly to
rodent tail & return to cage.
c. Restrain animal in a decapicone (see figure below) using forceps to tighten elastic band
around cone and tail.
d. Clean tail with gauze, disinfectant, and warm water; apply alcohol and let dry.
e. With bevel up, insert butterfly needle (21-23 gauge depending on size of rat) into the
lateral tail vein – if no blood flow, redirect needle.
f. Position tail over blood collection tube to catch drops.
g. After sample collection, remove needle and apply pressure with wet gauze.
h. Release animal from restrainer; give positive reinforcement.
i. Dispose of needle into sharps container.
Figure. Restraint, blood collection, and positive reinforcement with hemostasis
Rat restraint in decapicone and tail preparation
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Butterfly catheter inserted and collection Praising rat and holding off vein following
into blood tube. procedure.
V. Cardiac Puncture (Terminal)

a. Cardiac puncture is only used for terminal blood sampling because of the risk of
damage to the myocardium.
b. Put a paper towel at the bottom of the anaesthetic induction chamber and then place
the rat in the chamber.
c. Turn the oxygen flow to 1-2 L/ min and the isoflurane to 5% on the anaesthetic machine.
d. Once the rat is relaxed and has lost its righting reflex, dial down the isoflurane to 3%.
e. Remove the rat from the chamber, put on the face mask (remember to open the valve
to the face mask and close the valve to the induction chamber) and place rat on a
incontinence pad.
f. Place the rat in dorsal recumbency. Align the rat such that it is straight from nose to tail
tip.
g. Using the pedal reflex, ensure that the rat is maintained at a surgical plane of
anaesthesia (usually 1.5-2% isoflurane).
h. Secure an 18 or 20 gauge needle to the syringe (10 or 20 cc) and unlock the plunger of
the syringe.
 If a plasma sample is required, first draw up the anticoagulant to allow for coating
of the syringe barrel and discharge to the syringe contents to leave a small volume
of anticoagulant in the hub of your needle.
i. Locate the notch between the xiphoid process (at the end of the sternum) and the last
rib on the left side of the rat. Just below or to the side of the xyphoid cartilage advance
the needle (bevel side up) through the skin at a 15-30o angle and toward the midline.
 Create a vacuum within the syringe barrel and then advance the needle slowly a
few millimeters at a time.
j. Once the needle is in the heart, a very small drop of blood will appear in the needle hub.
If no blood is seen, pull the needle out a short distance in the same path as it went in,
redirect the needle and advance it slowly again.
Page 15 of 35
Guidelines SOP
k. Once seated, steady the syringe and slowly withdraw the plunger to fill the syringe
barrel. Avoid continuous pressure as this will collapse the chamber of the heart.
l. When completed, withdraw the needle from the heart and chest cavity in a straight
path.
m. Remove the needle from the syringe and dispense blood into the collection tube.
n. Further exsanguination, cervical dislocation, decapitation, opening the chest cavity, or
removal of a vital organ should be performed to ensure death.
RABBIT
I. Central Ear Artery

a. Place rabbit in a cat bag or wrap in a towel.
b. Grasp ear and remove hair over the artery with a straight razor blade.
c. To dilate the artery apply Oil of Wintergreen with the cotton-tipped applicator to the
skin over the artery.
d. Remove caps from blood collection tubes.
e. Insert a 22 gauge catheter into the artery.
f. Have the rabbit restrainer lightly pinch the artery as the catheter stylette is removed.
g. Place the blood collection tube under the catheter end.
h. Restrainer releases pinch over the artery to allow blood to drip into the blood collection
tube.
i. When filling multiple blood collection tubes, have restrainer pinch artery every time the
blood collection tube is changed.
j. If blood flow stops or slows before all blood is collected, gently rub artery with finger or
with the wintergreen cotton-tipped applicator.
k. Remove catheter and gently apply pressure with gauze until the blood flow stops from
the puncture site.
l. Wash ear with wet gauze and then apply Hibitane ointment.
m. Return rabbit to cage or pen.
II. Marginal Ear Vein

a. Place rabbit in a cat bag or wrap snuggly in a towel.
b. Grasp ear and remove hair over the vein with a straight razor blade.
c. Clean the shaved area with an alcohol swab.
d. Anaesthetize the venipuncture site by application of a lidocaine containing cream (i.e.
EMLA) (this is recommended and if performed the skin requires 45 minutes for the full
skin numbing effect).
Page 16 of 35
Guidelines SOP
e. Remove caps from blood collection tubes.

f. Massage the ear to dilate the vein.
g. Insert a 23 gauge needle or catheter into the vein.
h. Have the rabbit restrainer lightly pinch the artery as the catheter stylette is removed.
i. Place the blood collection tube under the catheter end.
j. Remove catheter and gently apply pressure with gauze until the blood flow stops from
the puncture site.
k. Wash ear with wet gauze and then apply Hibitane ointment.
l. Return rabbit to cage or pen.
III. Carotid Artery

a. Wrap the rabbit snuggly in a towel or cat bag, and give an I.M. injection of Ketamine
25mg/kg and acepromazine 1 mg/kg.
b. Once the sedation has taken effect, place the rabbit on the table and place the face
mask over the rabbit’s nose.
c. Start delivering the isoflurane in small increments every minute from 1- 5% (to prevent
breath holding).
d. Once the rabbit is relaxed, dial down the isoflurane to 2-3%.
e. Tie mask, using gauze, around the head behind the ears.
f. Place the rabbit in lateral recumbency and clip the ear over the marginal ear vein, and
then clean over the vein with chlorhexidine and alcohol.
g. Place a paper clip over the proximal end of the vein and insert the catheter in the raised
vein.
h. Remove the paper clip, remove the stylet of the catheter and attached the pre-flushed
extension set to the catheter.
i. Give a small bolus of heparinized saline to clear the catheter.
j. Using a 8-12” piece of 1/4’ white tape, make a tape butterfly over the hub of the
catheter; place a gauze roll inside the ear; wrap the long end of tape around the ear to
secure the catheter in place.
k. Attach the I.V. fluids of 0.9% NaCl to the extension set and set fluid drip rate to the
maintenance rate of 3 mL/kg/hr.
l. Turn the rabbit into dorsal recumbency and clip the hair on the neck from the start of
the mandible to the thoracic inlet using the electric clippers with #40 blade.
m. Using the knife handle and scalpel blade, make an incision just lateral to the trachea and
about 1 1/2”-2” long.
n. Using blunt dissection with a kelly forcep, separate the connective tissue between the
sternohyoideus and sternocepalicus muscles.
Page 17 of 35
Guidelines SOP
o. Once the carotid artery is exposed, use the mosquito forcep to gently separate the
carotid artery from the vagus nerve.
p. Loop two silk ligature (2-0) lengths (3-4 inches) under the carotid artery.
q. Tie the silk ligature that is most cranial so that it occludes blood flow.
r. Place one throw on the ligature proximally to the heart, so that it is ready to tie down.
s. Have an assistant pull up on both ligatures, so that you have good exposure of the
carotid artery.
t. Make a small cut in the artery about 0.5 cm from the cranial silk tie using vascular
scissors.
u. Place the tip of the vein introducer through the hole and into the artery.
v. Slide the catheter under the introducer and into the artery, past the ligature.
w. Insert the catheter approximately 8 cm into the carotid and then tie the proximal
ligature snuggly over the catheter.
x. Tie the distal ligature over the catheter to anchor it in place.
y. Pull the catheter so that it is taut (but not sliding out of the artery), and tape it to the
face mask with masking tape or white tape.
z. Inflate the pressure bag over the I.V. fluids to 150 mm Hg, and open the line fully so that
it is flowing in.
aa. Start removing the blood from the catheter with a 60 cc syringe.
bb. Put collected blood into 50 ml centrifuge tubes. Label vials 1, 2, 3….. to identify the
order of collection.
*This technique allows for the collection of a much greater percentage of the blood
volume of a rabbit compared to that using cardiac puncture.*
GUINEA PIG
I. Cardiac Puncture
a. NB: This technique is only used when attempts at vena cava collection are
unsuccessful.
b. Day before blood sampling – fast the guinea pig overnight.
c. Weigh the guinea pig and record the weight in the file.
d. Administer 0.1 mg/kg atropine subcutaneously.
e. Fifteen minutes after the atropine administration place the guinea pig into the paper
towel lined induction chamber.
f. Turn the oxygen flow to 1-2 L/ min and the isoflurane to 5% on the anaesthetic machine.
g. Once the guinea pig is relaxed and has lost its righting reflex, dial down the isoflurane to
2-3%.
Page 18 of 35
Guidelines SOP
h. Remove the guinea pig from the chamber onto the incontinence pad and put on the
face mask (remember to open the valve to the face mask and close the valve to the
induction chamber).
i. Put eye lubricant in each eye and then place the guinea pig in dorsal recumbency.
j. Locate the xyphoid process of the sternum.
a. Prepare the area with an alcohol swab.
k. Insert a needle (23 gauge with 6cc syringe on an extension with a T) just lateral to the
xyphoid process at a 30 degree angle directed midline below the sternum.
i. Maintain a slight amount of negative pressure on the syringe while slowly
withdrawing the needle until blood readily flows.
ii. Collect the desired sample volume (5-6 mL).
l. Pour the blood into the appropriate collection tube.
m. Put gentle pressure over the collection site for 30-60 seconds to stop the bleeding;
clean the site with hydrogen peroxide if necessary.
n. Place the animal in sternal recumbency.
o. Turn the anesthetic gas off and deliver 100% oxygen.
p. Give the guinea pig 10 mL of 0.9% NaCl over the dorsal back between the shoulder
blades as a subcutaneous injection.
q. Maintain guinea pig on 100% oxygen for 2-3 minutes.
r. Monitor the guinea pig until mobile and then return to the cage.
s. Monitor the animal every two hours until the end of the work day.
t. Animals that have prolonged recovery from anesthesia (>20 minutes), laboured or
raspy respiration, cyanosis or pale mucous membranes, and decreased heart rate (less
than 150 bpm) will be euthanized with intracardiac Euthanyl under anesthesia.
II. Vena Cava

a. Fast the guinea pig to be bled for 24 hours prior to the procedure.
b. Weigh the guinea pig and record in the file.
c. Administer 0.1 mg/kg atropine subcutaneously.
d. Fifteen minutes after the atropine injection place the guinea pig into the paper towel
lined induction chamber.
e. Turn the oxygen flow to 1-2 L/ min and the isoflurane to 5% on the anaesthetic machine.
f. Once the guinea pig is relaxed and has lost its righting reflex, dial down the isoflurane to
2-3%.
g. Remove the guinea pig from the chamber and put on the face mask (remember to open
the valve to the face mask and close the valve to the induction chamber).
h. Put eye lubricant in each eye and then place the guinea pig in dorsal recumbency.
i. Locate the cranial portion of the sternum, the manubrium.
Page 19 of 35
Guidelines SOP
i. Prepare the area with an alcohol swab.

j. Insert a needle (23 gauge with 6cc syringe on an extension with a T) just lateral to the
manubrium at a 30 degree angle directed at the opposite thigh – mid-lateral abdominal
region.
i. The needle should not be inserted more than ½ inch.
ii. Maintain a slight amount of negative pressure on the syringe while slowly
withdrawing the needle until blood readily flows.
iii. Collect the desired sample volume (5-6 mL).
k. Pour the blood into the appropriate collection tube.
l. Put gentle pressure over the collection site for 30-60 seconds to stop the bleeding;
clean the site with hydrogen peroxide if necessary.
m. Place the animal in sternal recumbency.
n. Turn the anesthetic gas off and deliver 100% oxygen for 5 minutes.
o. Give the guinea pig 10 mL of 0.9% NaCl over the dorsal back between the shoulder
blades as a subcutaneous injection.
p. Monitor the guinea pig until mobile and then return to the cage
q. Animals that have prolonged recovery from anesthesia (>20 minutes), laboured or
raspy respiration, cyanosis or pale mucous membranes, and decreased heart rate (less
than 150 bpm) will be euthanized with intracardiac Euthanyl under anesthesia.
III. Jugular Vein

a. Fast guinea pig overnight (take feed away from the whole tub).
b. Weigh the guinea pig and record the weight in the log.
c. Give 0.1 mg/kg Atropine by subcutaneous injection 15 minutes before inducing
anesthesia.
d. Put a paper towel at the bottom of the induction chamber, and then place the guinea
pig in the induction chamber.
e. Turn the oxygen flow to 1-2 L/ min and the isoflurane to 5% on the anaesthetic machine.
f. Once the guinea pig is relaxed and has lost its righting reflex, remove the guinea pig
from the chamber and put on the face mask (remember to open the valve to the face
mask and close the valve to the induction chamber).
g. Dial down the isoflurane to 2-3%.
h. Put eye lubricant in each eye.
i. Give the guinea pig 10 mL of 0.9% NaCl over the dorsal back between the shoulder
blades.
j. Place in dorsal recumbency on an incontinence pad resting on a stainless steel rodent
cage lid.
k. Position the head into the food trough so that it is at a 30% angle to the body.
Page 20 of 35
Guidelines SOP
l. With the body straight, tie the front limbs at 90o angles to the spine using shoelaces.
m. Hold the skin taut in the neck region and shave the hair using small clippers. Remove
hair from the area.
n. Prepare the area with an alcohol swab.
o. Secure a needle (22 gauge) to the syringe and unlock the plunger of the syringe. Draw
up ~0.5 – 1 cc of anticoagulant to allow for coating of the syringe barrel and leaving a
small amount in the syringe.
p. The landmark for needle insertion is a depression within the jugular furrow cranial to
the clavicle and slightly off the midline.
q. Advance the needle (bevel side up) through the skin. Create a vacuum within the
syringe barrel and then advance the needle slowly a few millimeters at a time.
r. Once the needle is in the trunk of the vein, a very small drop of blood will appear in the
needle hub. This will indicate that the needle is properly seated into the vein.
s. Once seated, steady the syringe and slowly withdraw the plunger to fill the barrel.
Avoid continuous pressure as this will collapse the vein.
t. Once the sample is collected, apply digital pressure with a gauze to the site before
u. Remove the needle from the syringe and dispense blood into the collection tube and
mix gently.
v. After putting gentle pressure over the collection site for 30-60 seconds, clean the site
with hydrogen peroxide if necessary.
w. Remove the leg restraints, massage the feet to promote circulation, and then place the
animal in sternal recumbency.
x. Turn the anesthetic gas off and deliver 100% oxygen.
y. Give 100% oxygen for another 2-3 minutes.
z. Monitor the guinea pig until in sternal recumbency and then return to the cage.
DOG
I. Cephalic Vein
a. Two people are needed for this procedure – one for restraint and raising the vein and
the other for taking the blood sample.
b. Assemble all of the equipment. Attach appropriate sized syringe to a 21 gauge needle.
Manually restrain the dog in sternal recumbency or in a standing position. Extend the
foreleg. For the right cephalic vein the restrainer is positioned on the dog’s left side. The
dog is restrained close to the body of the restrainer and the restrainer’s left hand or arm
is placed under the muzzle to pull the head toward the restrainer’s body. The restrainer
Page 21 of 35
Guidelines SOP
extends the foreleg with the right hand positioned under the dog’s elbow while
occluding the vein with the thumb positioned near the elbow of the dog.
c. If needed, clip hair around the sampling site with small clippers. Sterilize the area (skin
or hair) by using a swab soaked in 70% alcohol.
d. Raise the cephalic vein with one thumb by holding pressure across the proximal end of
the foreleg. This prevents blood flow and the cephalic vein will enlarge and become
turgid.
e. Remove cap from needle. Grab the leg and place a thumb adjacent to the vein to pull
the skin and stabilize the vein. Position the needle with bevel facing upwards parallel to
the skin overlying the cephalic vein.
f. Insert the needle through the skin and into the vein at a 25° angle up to 0.5-0.75 cm and
when blood appears in the syringe hub retract the syringe plunger to let blood fill the
syringe. Do not apply too much suction as this may collapse the vein and disrupt blood
flow into the syringe.
g. If blood is not flowing into the syringe, the needle bevel may be lodged against the
vessel wall. Rotate the needle slightly and reposition. Retract the syringe plunger again
to let blood collect into the syringe.
h. When blood collection complete, release pressure on the vein and remove the needle.
i. Apply pressure with fingers immediately following removal of the needle until the blood
flow stops from the puncture site (about 30 seconds).
j. Detach needle from syringe and add blood to the blood collection tube.
k. Provide dog with a reward (e.g. food treat) and return dog to kennel.
l. Subsequent venipuncture attempts can occur as necessary more proximal to the original
puncture site.
II. Lateral Sapheneous Vein

Manually restrain the dog in lateral recumbency. For the right lateral saphenous vein the
restrainer holds off the vein with the right hand positioned behind the knee.
d. Raise the vein with one thumb by holding pressure across the proximal end of the
saphenous vein. This prevents blood flow and the cephalic vein will enlarge and become
turgid.
e. Remove cap from needle. Grab the leg and place a thumb adjacent to the vein to pull
Page 22 of 35
Guidelines SOP
k. Provide dog with a reward (e.g. food treat) and return dog to kennel.
l. Subsequent venipuncture attempts can occur as necessary more proximal to the original
puncture site.
III. Jugular Vein

b. Assemble all of the equipment. Attach appropriate sized syringe to a 21 gauge one inch
needle. Manually restrain the dog.
d. Raise the jugular vein with one thumb by holding pressure across the jugular groove
near the thoracic inlet, below the venipuncture site. This prevents blood flow and the
jugular vein will enlarge and become turgid.
e. Remove cap from needle and position the needle with bevel facing upwards parallel to
the skin overlying the jugular vein.
f. Insert the needle through the skin and into the vein at a 25° angle and retract the
syringe plunger to let blood fill the syringe. Do not apply too much suction as this may
collapse the vein and disrupt blood flow into the syringe.
j. Provide dog with a reward (e.g. food treat) and return dog to kennel.
Page 23 of 35
Guidelines SOP
CAT
I. Cephalic Vein
Manually restrain the cat in sternal recumbency. For the right cephalic vein the
restrainer is position on the cat’s left side. The restrainer’s left hand or arm is placed
under the jaw to pull the head toward the restrainer’s body and the restrainer extends
the foreleg with the right hand under the elbow while occluding the vein with the right
thumb positioned near the elbow of the cat.
d. Raise the cephalic vein with one thumb by holding pressure across the proximal end of
the foreleg. This prevents blood flow and the cephalic vein will enlarge and become
turgid.
e. Remove cap from needle and grab the leg and place a thumb lateral to the vein to pull
k. Return the cat to the cage.
II. Medial Saphenous or Femoral Vein

a. Use this route to obtain small volumes of blood.
b. Two people are needed for this procedure – one for restraint and raising the vein and
Page 24 of 35
Guidelines SOP
c. Assemble all of the equipment. Attach appropriate sized syringe to a 22 gauge one inch
needle. Manually restrain the cat in right lateral recumbency with the left rear leg
abducted.
d. If needed, clip hair around the sampling site with small clippers. Sterilize the area (skin
e. Grasp the trarsus and extend the right leg. Occlude the vein with pressure applied by the
restrainer’s left hand in the right inguinal region. This prevents blood flow and the vein
will enlarge and become turgid. To visualize the vein it may be necessary to wipe the
area with alcohol and separate the hair over the vessel.
f. Remove cap from needle and position the needle with bevel facing upwards parallel to
g. Insert the needle through the skin and into the vein at a 25° angle and retract the
h. If blood is not flowing into the syringe, the needle bevel may be lodged against the
i. When blood collection complete, release pressure on the vein and remove the needle.
j. Apply pressure with fingers immediately following removal of the needle until the blood
flow stops from the puncture site (at least 60 seconds).
III. Jugular Vein

the other for taking the blood sample. A cat bag can also be used for restraint.
b. Assemble all of the equipment. Attach appropriate sized syringe to a 21 gauge one inch
needle. Manually restrain the dog.
d. Raise the jugular vein with one thumb by holding pressure across the jugular groove
near the thoracic inlet, below the venipuncture site. This prevents blood flow and the
jugular vein will enlarge and become turgid.
e. Remove cap from needle and position the needle with bevel facing upwards parallel to
f. Insert the needle through the skin and into the vein at a 25° angle and retract the
Page 25 of 35
Guidelines SOP
CATTLE AND BISON
I. Tail Vein
a. Use appropriate restraint to ensure the procedure will cause minimal distress and can
be conducted quickly and safely. Stand where you are protected from the hind limbs of
the bison. Do not stand directly behind a bison or you will be kicked.
b. Wear gloves and disinfect gloves between animals. Use a new needle for each
venipuncture. The vacutainer holder can be reused but should be cleaned between
animals.
c. Assemble all of the equipment. Screw the vacutainer needle onto the vacutainer.
d. Insert the collection tube into the vacutainer, using care not to puncture the stopper.
Hold the vacutainer and inserted collection tube in one hand.
e. Restrain the animal.
f. Remove the needle cap and store safely.
g. Raise the tail vertically with one hand until it is horizontal with the ground.
h. Locate the groove lying in the ventral midline of the tail approximately 15 cm from the
base of the tail.
i. Clean the skin using an antiseptic cleanser prior to the venipuncture.
j. Midway along the body of a tail vertebra insert a 20 gauge needle attached to a syringe
or vacutainer holder perpendicularly to the surface of the skin to a depth of a few
millimeters. Once the needle is through the skin, push the collection tube onto the
needle within the vacutainer. Remember that vacuum will be lost if the needle is
removed from the skin, therefore use care if re-directing the needle. If vacuum is lost, a
new collection tube must be used.
k. Advance the needle slowly until blood starts flowing into the collection tube. Hold the
needle in this position until the tube is filled. If more than one tube is required, remove
the tube from the collection needle and insert a new tube without pulling the needle
from the skin.
l. Withdraw needle and apply pressure to the venipuncture site to stop bleeding.
m. Discard the needle into a sharps container, using care to avid needle-stick injury.
n. Label and process tubes as appropriate.
II. Jugular Vein
Page 26 of 35
Guidelines SOP
a. Move animal into squeeze chute, and restrain properly. For bison, in a chute do not raise
the ‘crash gate’ until you are certain that the head has be caught by the head gate.
b. When bleeding in front of the head gate, apply a halter and tie the head to one side
exposing the lower neck region.
c. Identify the jugular vein by raising the vein and inspection and/or palpation. Clip the hair
on the animal’s neck at the area (2 inch x 2 inch) for needle insertion.
d. Clean the clipped area using 70% alcohol and swab.
e. Screw the vacutainer needle onto the vacutainer.
f. Insert the collection tube into the vacutainer, using care not to puncture the stopper.
g. Remove the needle cap and store safely.
h. Raise the jugular vein with one thumb by holding pressure across the base of the neck
within the jugular groove. This prevents blood flow and the jugular vein will enlarge and
become turgid.
i. Insert the needle (20 gauge) into the jugular vein in the upper third of the neck and at
approximately a 45 angle. It is helpful to rest your wrist against the animal’s neck.
j. Once the needle is through the skin, push the collection tube onto the needle within the
vacutainer. Remember that vacuum will be lost if the needle is removed from the skin,
therefore use care if re-directing the needle. If vacuum is lost, a new collection tube must
be used.
k. Advance the needle slowly until blood starts flowing into the collection tube. Hold the
needle in this position until the tube is filled. If more than one tube is required, remove
the tube from the collection needle and insert a new tube without pulling the needle
from the skin.
l. When collection is completed, remove the tube from the collection needle and then
remove the needle from the skin. Lightly press a new swab with 70% alcohol on the site.
m. Discard the needle into a sharps container, using care to avoid needle-stick injury.
n. Remove halter and release animal from chute.
o. Label and process tubes as appropriate.
SHEEP AND DEER
I. Jugular Vein
be conducted quickly and safely.
Page 27 of 35
Guidelines SOP
animals.
c. Assemble all the equipment.
d. Restrain the sheep by straddling the animal, placing knees behind the shoulders of the
animal and backing the animal into a wall to control the hindquarters. Use electric
shears to shave a patch over the jugular groove large enough to visual the vein (e.g. 3
inches by 5 inches).
e. White-tailed deer must be either chemically immobilized (asleep) or secured in a
suitable deer chute. White-tailed deer cannot be safely held otherwise.
f. Have an assistant turn the head at a 30° angle to the side by holding the animal under
the jaw to allow for easy access to the vein.
g. Identify the jugular vein by raising the vein and inspection and/or palpation. The vein
lies along an imaginary line from the middle of the animal’s eye down the side of the
neck. Apply alcohol to clean the skin and facilitate visualization of the vein.
h. Screw the vacutainer needle (20 gauge by one inch) onto the vacutainer holder.
i. Insert the collection tube into the vacutainer holder, using care not to puncture the
stopper. Hold the vacutainer and inserted collection tube in one hand.
j. Remove the needle cap and store safely. Do not remove the needle cap with your teeth.
k. Raise the jugular vein with one hand by applying pressure in the jugular furrow near the
thoracic inlet while using the other hand to stroke the jugular vein until it becomes large
and distended.
l. Insert the needle with a quick thrust into the jugular vein in the upper third of the neck
and at approximately a 45° angle. It is helpful to rest your wrist against the horse’s neck.
m. Once the needle is through the skin, push the collection tube onto the needle within the
therefore use care if re-directing the needle. If vacuum is lost, a new collection tube
must be used.
n. Hold the needle in this position until the tube is filled. If more than one tube is required,
remove the tube from the collection needle and insert a new tube without pulling the
needle from the skin.
o. When collection is completed, remove the tube from the collection needle and then
remove the needle from the skin. Press gently on the site of needle insertion until
bleeding has ceased.
p. Discard the needle into a sharps container, using care to avoid needle stick injury. Do
not recap the needle.
q. Discard disposable materials in the trash.
r. Wash hands, clean area.
Page 28 of 35
Guidelines SOP
HORSE
I. Jugular Vein
be conducted quickly and safely.
animals.
d. Restrain the horse, by having an assistant hold the horse by its halter. Do not tie the
horse. Do not perform this procedure without an assistant holding the horse. Do not
proceed with the venipuncture unless the horse is adequately restrained.
e. Identify the jugular vein by raising the vein and inspection and/or palpation. Apply
alcohol to clean the skin and facilitate visualization of the vein.
f. Screw the vacutainer needle (20 gauge) onto the vacutainer.
g. Insert the collection tube into the vacutainer, using care not to puncture the stopper.
h. Remove the needle cap and store safely. Do not remove the needle cap with your teeth.
i. Raise the jugular vein with one hand by applying pressure in the jugular furrow near the
thoracic inlet while using the other hand to stroke the jugular vein until it becomes large
and distended.
j. Insert the needle with a quick thrust into the jugular vein in the upper third of the neck
and at approximately a 45° angle. It is helpful to rest your wrist against the horse’s neck.
k. Once the needle is through the skin, push the collection tube onto the needle within the
therefore use care if re-directing the needle. If vacuum is lost, a new collection tube
must be used.
l. Hold the needle in this position until the tube is filled. If more than one tube is required,
remove the tube from the collection needle and insert a new tube without pulling the
needle from the skin.
m. When collection is completed, remove the tube from the collection needle and then
remove the needle from the skin.
n. Discard the needle into a sharps container, using care to avoid needle stick injury. Do
not recap the needle.
o. Discard disposable materials in the trash.
p. Wash hands, clean area.
Page 29 of 35
Guidelines SOP
II. Facial Sinus

a. This procedure is used if small volumes of blood are required, e.g. for a PCV/TP
determination. Typically, only a few drops of blood will be obtained.
b. This is a “blind stick”, i.e. you cannot visualize or palpate the venous sinus. Review
anatomy before performing this procedure.
d. Restrain the horse, typically by having an assistant hold the horse by its halter. Do not
tie the horse. Do not perform this procedure without an assistant holding the horse. Do
not proceed with venepuncture unless the horse is adequately restrained.
e. Locate the appropriate site, approximately 0.5-1.cm below the facial crest at
approximately 1/3 of the way between the medial and lateral canthus of the eye.
f. Clean the skin using a moist (not wet) alcohol swab. Take care to avoid any alcohol
entering the horse’s eye. Do not apply alcohol directly from the bottle.
g. Remove the needle cap from a 20 gauge, 1.5 inch needle (or 22 gauge, 1.5 inch) and
store safely. Do not remove the needle cap with your teeth.
h. Rest the hand holding the needle firmly against the horse’s head. Failure to do so can
lead to eye injury of the horse if the horse moves during the procedure.
i. Use your free hand to help restrain the horse by its halter.
j. Rapidly insert the needle through the skin at the appropriate site, using a 90° angle to
the skin (perpendicular orientation). Most horses will flinch at this, therefore let go of
the needle as soon as it is inserted. Failure to do so can make you pull out the needle
inadvertently and you have to repeat the procedure.
k. Slowly advance the needle until blood is obtained. It is helpful to advance the needle in
short, rapid thrusts and to rotate the needle during advancement. Let go of the needle
each time after advancing it.
l. Once blood is obtained, collect blood into hematocrit tubes or blood collection tubes, or
just remove the needle.
m. Remove the needle and discard in a sharps container using care to avoid needle stick
injury. Do not recap the needle.
n. Discard any disposable materials in the trash. Wash hands, clean area.
PIG
I. Cranial vena cava/External jugular vein
Page 30 of 35
Guidelines SOP
a. Move pig into blood collection area. Restrain the pig. For small pigs less than 25 kg one
person can restrain the pig by turning the pig onto its back, holding the front legs and
restraining the body with the person’s arms or by straddling the pig and using their legs.
Larger animals require restraint using a snout snare and will remain standing. Allow the
pig to bite the snare and then close it quickly and apply pressure. The pig will remain
quiet when pressure is maintained. Ensure that the neck is stretched upwards by making
sure the pig leans back against the pull of the rope.
b. A second person will take the blood sample from the jugular vein by using one hand to
restrain the head of the pig by holding the snout and the other hand will take the blood
sample.
c. Insert an appropriate sized needle attached to a vacuum test tube holder and tube or a
syringe into the ventral side of the neck, on the right side just off the midline in the
depression under the neck between the legs. The needle puncture site should be at the
deepest point of the jugular groove formed between the medial sternocephalic and
lateral brachiocephalic muscles. Use of the left side is not recommended due to risk of
causing trauma to a vulnerable nerve. Suggested needle sizes: newborn: 21 gauge x 1
inch; <20 kg: 20 gauge x 1 inch; >20kg: 20 gauge x 1.5 inch.
d. Advance the needle caudo-dorsally towards the opposite scapula. Pull gently using a
syringe or apply vacuum with the vacutainer snapped into place. If more than one
vacutainer fill is required change the vacutainers without removing the needle. For large
pigs the 1.5 inch needle usually must be inserted its full length.
e. Remove the vacuum or release pressure on the syringe prior to removing the needle.
Remove the needle and briefly apply pressure to stop blood flow.
f. The size and type of the vacuum tube depends on the type and amount of sample
required.
II. Marginal Ear Vein

a. Move pig into blood collection area. Restrain the pig. For small pigs less than 25 kg one
person can restrain the pig by turning the pig onto its back, holding the front legs and
restraining the body with the person’s arms or by straddling the pig and using their legs.
Larger animals require restraint using a snout snare and will remain standing. Allow the
pig to bite the snare and then close it quickly and apply pressure. The pig will remain
quiet when pressure is maintained.
b. Warm the ear to dilate the vessel – either gently stroke the ear or apply a swab soaked
in warm water.
c. Occlude the vein at the base of the lateral surface of the ear.
d. Insert an appropriate sized needle (21-23 gauge) attached to a vacuum test tube holder
and tube towards the base of the ear.
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Guidelines SOP
e. Remove the vacuum or release pressure on the syringe prior to removing the needle.
Remove the needle and briefly apply pressure to stop blood flow (about 2 minutes).
f. The size and type of the vacuum tube depends on the type and amount of sample
required.
POULTRY/BIRDS
I. Cardiac Puncture
a. Animal should be fasted overnight.
b. Prepare vacutainers or collection vials with labels.
i. Ensure that the proper anti-coagulant is being used (if necessary).
c. Have an assistant hold the rooster (hen) by the feet and legs with the left hand and the
wings over the back with the right hand.
d. Pluck a few feathers from the breast area near the middle of the body. You will notice a
V-formation of thickened skin (where the larger feathers were anchored). You may use
this as a guide for point of entry of the needle.
e. Wipe the area with alcohol. Try to locate a strong heart beat with the left thumb.
i. Keeping the left thumb over the point of the strongest heart beat as another guide,
hold the vacutainer assembly in the right hand, insert the needle at the tip of the
"V" of skin and follow through at an angle of 35-40. You may have to insert the
total length of the needle before the blood will appear at the tip of the needle
inside the vacutainer holder.
f. If blood is not seen, withdraw the needle and try again (avoid unnecessary jabbing).
NOTE: As there is only a small space allowance on the side of the breast bone where the
needle can pass through to reach the heart, any deviation of puncture site of skin or
angle of needle insertion may bring the needle against the breast bone and dull the
needle tip. If this happens, start again using a fresh needle to minimize the damage to
the heart.
II. Jugular Vein

a. The right jugular vein is the preferred vein due to its larger size relative to the left
jugular.
b. Prepare equipment. Attach appropriate sized syringe to a 22-28 gauge needle
(depending upon size of bird). If an anticoagulant is necessary first rinse the syringe with
the anticoagulant and discharge contents such that a small volume of the anticoagulant
remains in the hub of the needle.
c. Remove the bird from the cage or holding bag.
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Guidelines SOP
d. Hold the bird in one hand with the bird’s right side up (left lateral recumbency) and neck
and head held between two fingers (index and middle fingers) to gently extend the
bird’s neck. If the bird is too large then a towel can be used to restrain the body while
the free hand is used to restrain the head and neck.
e. Gently blow on the neck to expose the unfeathered area on the lateral side of the neck.
Apply a small volume of 70% alcohol to the area to wet the feathers, sterilize the area,
and expose the jugular vein.
f. Apply a small amount of pressure at the base of the neck just cranial to the thoracic inlet
to occlude the jugular vein and allow it to distend and become visible.
g. With the bevel down, pierce the skin with the needle directly beside the jugular at a 30°
angle to the skin. Provide gentle suction on the needle by retracting the plunger of the
syringe and then decisively pierce the jugular vein. (Placement of the needle with the
bevel facing down reduces the potential for lacerating the opposite wall of the vein and
the risk of causing vessel collapse and vacuum-associated cessation of blood flow).
h. Blood will flow into the syringe if the needle is in the jugular vein. Take care to avoid
pushing the needle through the vein.
i. Slowly retract the plunger to allow blood to enter the syringe until the desired volume is
collected.
j. Gently remove the needle and apply gentle pressure to the puncture site for 30 seconds
to 2 minutes. Look for any excess bleeding through the skin and if note apply additional
pressure.
k. Return the bird to the cage or holding bag.
III. Brachial vein

a. Prepare equipment. Attach appropriate sized syringe to a 22-28 gauge needle
(depending upon size of bird). If an anticoagulant is necessary first rinse the syringe with
the anticoagulant and discharge contents such that a small volume of the anticoagulant
remains in the hub of the needle.
b. Remove the bird from the crate, pen or holding bag.
c. For a two person technique, one person gently places the bird on its side on the table
and with one hand holds the head and with the other hand holds the legs. The other
person lifts up the wing.
d. Gently separate the feathers apart to visualize the brachial vein. Apply a small volume of
70% alcohol to the area to wet the feathers, sterilize the area, and expose the brachial
vein.
e. With the bevel down, pierce the skin with the needle at a 30° angle to the skin. Provide
gentle suction on the needle by retracting the plunger of the syringe and then decisively
pierce the brachial vein. (Placement of the needle with the bevel facing down reduces
Page 33 of 35
Guidelines SOP
the potential for lacerating the opposite wall of the vein and the risk of causing vessel
collapse and vacuum-associated cessation of blood flow).
f. Blood will flow into the syringe if the needle is in the brachial vein. Take care to avoid
pushing the needle through the vein.
g. Slowly retract the plunger to allow blood to enter the syringe until the desired volume is
collected.
h. Gently remove the needle and place the bird wing back down so it is tight to the body to
stop the flow of blood. Look for any excess bleeding through the skin and if note apply
additional pressure.
i. Return the bird to the crate, pen or holding bag.
5. Safety
Personal protective equipment (gloves, lab coat) must be worn at all times. Care must be
taken to restrain animals properly and prevent injury, particularly needle sticks. Consult the
MSDS for isoflurane to ensure the proper safety procedures are followed. Any injuries must
be reported to the principle investigator and WSEP.
6. Potential Complications and Troubleshooting
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Guidelines SOP
Problem Response
Animal moves excessively Re-evaluate restraint and/or get help. Operator safety must
during the procedure be the utmost concern when working with large animals.
Accidental intra-arterial injection can result in violent
reactions and poses a serious danger to both the horse
7. References and people around. Do not proceed unless the horse is
appropriately restrained.
Failure to obtain blood Re-evaluate needle position and repeat. Get help from
trained personnel if you are unsuccessful after 2 attempts.
Blood may have clotted in the needle. Use a new needle
and repeat the procedure.
Arterial blood is obtained Re-position the needle. Do not proceed with injection
(bright red, squirting from unless the needle is properly seated in the jugular vein.
needle)(IV injection) Use a new needle if necessary
Vacuum is lost from the Use a new collection tube and repeat the procedure
collection tube (jugular
collection)
Blood flows into the tube Re-evaluate needle position, especially if the animal has
at first but stops (jugular moved. If the needle has been removed from the skin
collection) accidentally and vacuum is lost, use a new collection tube
Blood is seen in the hub of Re-evaluated needle position, especially if the animal has
the needle at first but moved. This is best done by removing the syringe and
cannot be aspirated once repositioning the needle. Do not proceed with injection
the syringe is attached (IV unless the needle is properly seated in the jugular vein.
injection)
and repeat the procdure.
Hematoma formation Apply pressure for 2-3 minutes. Do not allow the horse to
(swelling) after needle lower its head. Inform the principal investigator or UCACS
removal veterinarian.
Needle stick injury Wash your hands with copious amounts of water and soap.
Inform the principal investigator and WSEP.
Diehl KH et al (2001). A good practice guide to the administration of substances and
removal of blood, including routes and volumes. J Appl Toxicol 21:15-23.
Golde WT, Bollobin P, Rodriquez LL (2005). A rapid, simple, and humane method for
submandibular bleeding of mice using a lancet. Lab Animal 34(9):39-43.
McGuill MW, Rowan AN (1989). Biological effects of blood loss: Implications for sampling
volumes and techniques. ILAR News 4:5(Fall).
First Report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement (1993).

Removal of blood from laboratory mammals and birds. Laboratory Animals 27:1-22.
Page 35 of 35
Guidelines SOP
Guidelines for survival bleeding of mice and rats; NIH:

http://oacu.od.nih.gov/ARAC/Bleeding.pdf
8. Revision History
Date Created: February 26, 2013 Written by: Nelles/ Moroz/
Lohmann/Woodbury/Alcorn
SOP Review and Revision History
Revision Number Review/Revision Date Reviewer
List the changes made during the most recent revision, as well as the reasons for the
changes.
Page 36 of 35

University Committee On Animal Care and Supply (UCACS) Standard Operating Procedure (SOP)

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University Committee On Animal Care and Supply (UCACS) Standard Operating Procedure (SOP)

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Guidelines SOP

C201: Blood Collection – Multiple Species

University Committee on Animal Care and Supply (UCACS)

Responsibility: Individuals performing blood collection must be adequately trained.

Table of Contents for Blood Collection Techniques by Species

A. Type of Blood Collection Tube and Size of Needle

Table. Type of Vacutainer tubes.

1. Circulating Blood Volume

2. Blood Collection Volume

Table. Blood volume limits and recovery periods

Table. Recommended maximum blood volumes for different species*

Table. Normal haematological values in different species

C. Collection of Blood Samples (General)

1. Manual restraint or sedation/general anaesthesia is required depending upon blood

Table. Advantages/disadvantages of the various blood sampling routes and methods.

Jugular No Low Yes +++ Rat, dog,

D. Blood Sample Collection by Species and Route

Figure. Mouse restraint, facial vein blood collection

Mouse restraint Facial Vein Blood Collection

II. Saphenous Vein Venipuncture

III. Tail Vein Microsampling (5-20 μL Blood Volumes)

IV. Lateral Tail Vein

II. Jugular Vein (anesthetized)

III. Saphenous Vein

IV. Tail Vein

Figure. Restraint, blood collection, and positive reinforcement with hemostasis

Rat restraint in decapicone and tail preparation

V. Cardiac Puncture (Terminal)

I. Central Ear Artery

II. Marginal Ear Vein

e. Remove caps from blood collection tubes.

III. Carotid Artery

II. Vena Cava

i. Prepare the area with an alcohol swab.

III. Jugular Vein

II. Lateral Sapheneous Vein

III. Jugular Vein

II. Medial Saphenous or Femoral Vein

III. Jugular Vein

CATTLE AND BISON

II. Jugular Vein

SHEEP AND DEER

II. Facial Sinus

I. Cranial vena cava/External jugular vein

II. Marginal Ear Vein

II. Jugular Vein

III. Brachial vein

6. Potential Complications and Troubleshooting

First Report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement (1993).

Guidelines for survival bleeding of mice and rats; NIH:

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