HOW TO WITHDRAW

BLOOD SAMPLES
FROM EXPERIMENTAL
ANIMALS

September 09, 2019

Submitted to: Dr. Gul Shehnaz

Submitted by: Group 5
Subject: Pharmaceutics V A
(Biopharmaceutics and
Pharmacokinetics) Lab
Course code: PHM 416

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 GROUP MEMBERS

1. HAFSA SHAHID…………………………… 06331613009

2. SYEDA KOMAL FATIMA……………….06331613011
3. NIDA RASHID………………………………06331613026
4. AYESHA AHMAD………………………….06331613034
 GROUP 5

Contents
1. Introduction, requirements, general principle and considerations of blood sample withdrawal
from experimental animals ................................................................................................................... 3
➢ Introduction ............................................................................................................................... 3
➢ Requirements ............................................................................................................................. 3
➢ General principles of blood collection in animals ..................................................................... 3
➢ General methods for blood collection ........................................................................................ 4
➢ Decision tree ............................................................................................................................... 4
➢ Techniques for taking blood samples from experimental animals ........................................... 5
➢ Use of anti-coagulants ................................................................................................................ 5
➢ Method ....................................................................................................................................... 5
2. Procedure to withdraw blood sample from guinea pig. ................................................................ 6
➢ Requirements ............................................................................................................................. 6
➢ Procedure ................................................................................................................................... 6
1. Blood collection from tarsal vein (preferred route) .............................................................. 6
2. Blood collection from thoracic blood vessel........................................................................... 6
3. Blood collection by cardiac puncture .................................................................................... 6
4. Blood collection from lateral saphenous vein ........................................................................ 7
3. Procedure to withdraw blood sample from rat ......................................................................... 8
➢ Requirements ............................................................................................................................. 8
➢ Procedure ................................................................................................................................... 8
1. Blood collection from tail vein ............................................................................................... 8
2. Blood collection from saphenous vein.................................................................................... 8
3. Blood collection from retro-orbital plexus ............................................................................ 8
4. Blood collection via cardiac puncture .................................................................................... 9
5. Blood collection via jugular vein ............................................................................................ 9
4. To withdraw bood sample from mice. ......................................................................................... 10
➢ Requirements ........................................................................................................................... 10
1. Blood vessel cannulation ...................................................................................................... 10
2. Tail Snip Blood Sample Collection ...................................................................................... 10
3. Tail Vein Blood Sample Collection (most preferred blood collection technique) ............... 10
4. Saphenous Vein blood Sampling (most preferred ruote) .................................................... 11
5. Orbital Sinus Blood Sampling ............................................................................................. 11

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6. Thoracic blood vessel sampling ........................................................................................... 11

7. Cardiac Puncture blood sampling ....................................................................................... 11
5. To withdraw blood sample from rabbit and storage conditions ................................................ 12
➢ Requirements ........................................................................................................................... 12
1. Blood collection from marginal ear vein ............................................................................. 12
2. Blood collection from Lateral saphenous vein (most preferred ruote) ............................... 12
3. Blood collection from central ear artery.............................................................................. 13
4. Blood collection from cardiac puncture (non-survival): ..................................................... 13
➢ Advantages and Disadvantages of sites of injections .............................................................. 14
➢ Storage conditions for blood sampling of laboratory animals ................................................ 14
1. Sample size: Take a large enough sample of blood ............................................................. 14
2. Temperature: Maintain appropriate sample temperature ................................................. 14
3. Length of storage: Make sure conditions are right ............................................................. 14
References ............................................................................................................................................ 15
Learning questions .............................................................................................................................. 16

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 GROUP 5

1. Introduction, requirements, general principle and considerations of blood

sample withdrawal from experimental animals
➢ Introduction
Collection of blood from small laboratory animals is necessary for a wide range of scientific
research and there are a number of efficient methods available for that. It is important that blood
sample collection from experimental animals should be least stressful because stress will affect the
outcome of the study. Various regulatory agencies and guidelines have restricted the use of
animals and the techniques used for blood collection in laboratory animals. Permission of the
Institute Animal Ethics Committee has been obtained for the use of animals for demonstrating
the techniques.
Blood collection is a common requirement for research studies that commonly involves mice and
rats. The method of blood withdrawal in animals is dependent upon

• the volume of blood needed

• the frequency of the sampling
• the health status of the animal to be bled
• the skill level of the technician

➢ Requirements
• Animal
• Anesthetic agent
• Cotton
• Gloves
• 22G needle
• Hair remover
• Anti-coagulant
• Blood sample collection tubes

➢ General principles of blood collection in animals

• The method of blood collection should be described in the protocol approved by the Institute
animal ethics committee.
• It should be least painful and stressful. Blood sample may be collected under anesthesia or
without anesthesia.
• Adequate training is required for blood collection using any method in any species.
• In general, blood sample is withdrawn from venous, arterial blood vessels or heart chambers.
• Frequency of blood collection is important. Once in two weeks is ideal for nonrodents. If the
study needs multiple blood samples, lagomorphs (e.g., hares and rabbit) can be used.
• All nonterminal blood collection without replacement of fluids is limited up to 10% of total
circulating blood volume in healthy, normal, adult animals on a single occasion and collection
may be repeated after 3 to 4 weeks. In case repeated blood samples are required at short
intervals, a maximum of 0.6 ml/kg/day or 1.0% of an animal’s total blood volume can be
removed every 24 hour.
• If the study involves repeated blood sample collection, the samples can be withdrawn through
a temporary cannula. This may reduce pain and stress in the experimental animals.

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➢ General methods for blood collection

On average, mice have around 58.5 ml of blood per kg of bodyweight. A mouse weighing
25 g would therefore have a total blood volume (TBV) of approximately 58.5 ml/kg x 0.025
kg = 1.46 ml.

➢ Decision tree
The two tables below are designed to assist in determining the amount of blood to sample
from the animal, and depending on that volume, the most appropriate techniques to use.
1. Do you require more than one blood sample from the same animal?

YES NO
Maximum <10% TBV (= 0.14 ml) on any single Maximum <10% TBV ( = 0.14 ml)
occasion AND <15% TBV ( = 0.21 ml) in 28 days

For repeat bleeds at short intervals, suggested limit OR terminal sample under general anesthesia
<1% TBV ( = 0.01 ml) in 24 (volume unrestricted)
hours AND consider cannulation

2. How much blood do you require?

Total of <0.20 ml Total of >0.20 ml

General anesthesia required General anesthesia not required General anesthesia required
Non recovery
Saphenous vein Saphenous vein Cardiac puncture

Tail vein Tail vein Abdominal / thoracic blood vessel

Sublingual vein Mandibular vein Retro-orbital

Retro-orbital Blood vessel cannulation Decapitation

Tail snip

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➢ Techniques for taking blood samples from experimental animals

Non surgical Techniques

Surgical techniques

Terminal Techniques
Tail Vessel micro
Blood Vessel Retro Orbital
sampling
Cannulation
Thoracic Blood
Tail Vein
Tail Snip vessel

Cardiac Puncture Sephanous Vein

➢ Use of anti-coagulants
Prior to the bleeding procedure, the type of sample required must be determined. Experimental
procedures could require whole blood, plasma, or serum. For whole blood, an anticoagulant must
be added to the sample. Plasma, which contains fibrinogen and other clotting factors when
separated from the red blood cells, can be extracted from an anticoagulated sample. Serum is
obtained through blood collection without an anticoagulant. The serum will result from
centrifugation of the sample once a clot has formed. As the sample has clotted, the serum will not
contain fibrinogen or other clotting factors. Both plasma and serum are obtained through the use
of a centrifuge run at 2200-2500 RPM for a minimum of 15 minutes.
For a sample that must yield whole blood or plasma, an appropriate anticoagulant must be used.
Commonly used anticoagulants for laboratory animals are

• Heparin
• sodium citrate
• ethylenediamine tetraacetic acid (EDTA), selection of which is based on research needs.

➢ Method
Sequester-a liquid form of EDTA, heparin, and sodium citrate can be loaded directly into the
syringe to coat the surfaces. This allows contact of the anticoagulant directly as the blood is
drawn, aiding in the prevention of clotting. As rat blood clots faster than most mammalian blood,
it is essential that the correct ratio of anticoagulant to blood be used for blood collection.

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2. Procedure to withdraw blood sample from guinea pig.

➢ Requirements
Guinea pig (Cavia porcellus) of weight 700 g, gloves, towel, cotton, sample collection tubes and
21G, 23G, 25G needle, EDTA, anaethesia.

➢ Procedure
1. Blood collection from tarsal vein (preferred route)
a. The guinea pig is held and the foot restrained. No anesthesia is required in this technique.
b. Gentle pressure is applied by massaging above the point
(proximally to the animal) at which the blood sample is
taken to dilate the vessel.
c. The collection site is shaved with an electric clipper and
Lateral
cleaned with 70% alcohol and betadine.
saphenous
d. 0.2ml blood is collected using a 23 gauge needle coated
vein
with EDTA from the tarsal vein of the leg.
e. Blood flow is stopped by applying pressure with sterile
gauze for approximately 2 minutes to achieve hemostasis.
f. No more than six samples, using both hind legs, should be
taken in any 24-hour period.

2. Blood collection from thoracic blood vessel

Number of samples One

Sample volume Up to 15 ml, depending on the size of the guinea pig.
Equipment 19G - 21G needle
Staff resource One person is required to take the blood sample.

3. Blood collection by cardiac puncture

Number of
One
samples
1 - 25 ml, depending on the
Sample volume
size of the guinea pig
Equipment 20 - 21G needle
One person is required to
Staff resource
take the blood sample.

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4. Blood collection from lateral saphenous vein

Number of samples No more than four blood samples should be taken within any 24-hour
period, depending on sample volumes.
Sample volume A single sample equivalent to 0.5% of the animal's bodyweight may be
taken, which can usually be repeated at fortnightly intervals without
disturbances to its haematological status. Alternatively, daily samples
corresponding to 0.05% bodyweight may be taken.
Equipment 23G needle or lance
Staff resource One person is required if a restraint tube is used. Where manual restraint is
used two people may be required: one for handling the guinea pig and one
for taking the blood sample.
Adverse effects

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3. Procedure to withdraw blood sample from rat

➢ Requirements
Brown rat (Rattus norvegicus) of weight 230g, Guaze size : 21G, 23G, towel, collection tubes

➢ Procedure
Blood collection from tail vein and saphenous vein is preferred.

1. Blood collection from tail vein

a. Clean the tail with 70% alcohol.
b. Restrain the rat and collect 2 ml blood using 21-23 gauge
needle from the lateral tail vein.
c. Blood flow is stopped by applying pressure with sterile gauze
to achieve hemostasis.

2. Blood collection from saphenous vein Tail vein

a. The rat is restrained (state method of restraint) and the hind leg is
immobilized in the extended position by applying gentle downward
pressure immediately above the knee joint.
b. The collection site is shaved with an electric clipper and cleaned
with three alternating scrubs of 70% alcohol and betadine.
c. 0.2ml blood is collected using a 23 gauge needle from the lateral
saphenous vein.
d. Blood flow is stopped by applying pressure with sterile gauze to
achieve hemostasis.

3. Blood collection from retro-orbital plexus

Number of samples It is recommended that only one sample be taken.

Sample volume Up to 0.2 ml with recovery; Up to 0.5 ml non-recovery.
Equipment A glass capillary tube or Pasteur pipette.
Staff resource One person is required to take the blood sample.
Careful monitoring for adverse effects is necessary both peri- and post-
Other
operatively.
▪ Retro-orbital haemorrhage resulting in haematoma and excessive pressure
on the eye
▪ Corneal ulceration, keratitis, pannus formation, rupture of the globe and
micro-ophthalmia caused by proptosis of the globe
Adverse effects ▪ Damage to the optic nerve and other intra-orbital structures which can lead
to deficits in vision and blindness
▪ Fracture of the fragile bones of the orbit and neural damage by the micro-
pipette
Penetration of the eye globe itself with a loss of vitreous humour

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Injection in retro-orbital plexus

4. Blood collection via cardiac puncture

Number of samples One

Sample volume Up to 15 ml
Equipment 19G - 21G needle
Staff resource One person is required to take the blood sample.

Cardiac puncture

5. Blood collection via jugular vein

No more than eight blood samples should be taken

Number of samples
in a 24-hour period.
Sample volume 0.1 - 2 ml (normally 0.1 - 0.3 ml)
Equipment 23G ( 1" long) needle
Two people: one to take the blood sample and
Staff resource
another to restrain and monitor the rat.
▪ Bruising
Adverse effects ▪ Infection <1%
Haemorrhage <1%
Rats are restrained in an unnatural position, which
Other can cause stress. A high degree of competence is
required to perform this technique.

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4. To withdraw bood sample from mice.

➢ Requirements
Animal, anesthetic agent, cotton, 25G needle, I.V. cannula, surgical blade, heparin (or
any anticoagulant) and blood sample collection tubes, towel, cotton, 19 to 25G needle
with, surgical blade, tube (internal diameter of 0.1 to 0.3 mm) for thoracotomy, plastic
disposable bag and blood sample collection tubes.
1. Blood vessel cannulation

Number of samples Up to six samples may be taken in a 24-hour period, depending on sample volume.
Sample volume 0.01 - 0.02 ml
Equipment 25G cannula
Staff resource One person is required to take the blood sample. Further staff resource is required for surgery,
post-operative care for up to five days after surgery, and daily animal observations post-
surgery.
Adverse effects ▪
and Infection 5-10%
incidence ▪ Haemorrhage 5-10%
▪ Poor recovery after surgery 5-10%
▪ Blocked cannula 5-10%
▪ Swelling around the jacket 5-10%
▪ Skin sores from the jacket 5-10%
Further considerations Mice should be back at their pre-operative weight before blood sampling starts.

2. Tail Snip Blood Sample Collection

• This method is recommended for blood collection only in mice.

• This method should be avoided as far as possible because it can cause potential permanent
damage on the animal tail. If needed, it should be done under terminal anesthesia only.

3. Tail Vein Blood Sample Collection (most preferred blood collection technique)

Number of samples One or two blood samples can be taken per session and in any 24-hour period, depending on sample
volume.
Sample volume 50 ul to 0.2 ml
Equipment 25G needle or lance
Staff resource One person is required to take the blood sample if a restraint tube is used. For large groups of animals,
more staff members are required.
Adverse effects ▪ Infection <1%
▪ Hemorrhage <1%
Other Mice may be warmed, to dilate the blood vessel. Care should be taken to avoid hyperthermia and
dehydration

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4. Saphenous Vein blood Sampling (most preferred ruote)

Number of samples No more than four blood samples should be taken within any 24-hour period.
Sample volume Up to 0.15 ml for a single sample, which usually can be repeated at 2-weeks intervals without
disturbance in hematological status. Alternatively, multiple smaller samples (e.g. 0.01 ml
daily), taking into account limits on sample volume.
Equipment 27G or 25G needle or lance
Staff resource One person is required to take the blood sample.
Adverse effects ▪ Bruising, Hemorrhage, Infection, Temporary favoring of the opposite limb

5. Orbital Sinus Blood Sampling

Number of It is recommended that only one sample be taken.

samples
Sample volume Up to 0.2 ml with recovery; Up to 0.5 ml non-recovery.
Equipment A glass capillary tube or Pasteur pipette.
Staff resource One person is required to take the blood sample.
Other Careful monitoring for adverse effects is necessary both peri- and post-operatively.
Adverse effects▪ Retro-orbital hemorrhage resulting in hematoma and excessive pressure on the eye
▪ Corneal ulceration, keratitis, pannus formation, rupture of the globe and micro-ophthalmia caused
by proptosis of the globe
▪ Damage to the optic nerve and other intra-orbital structures which can lead to deficits in vision and
blindness
▪ Fracture of the fragile bones of the orbit and neural damage by the micro-pipette
▪ Penetration of the eye globe itself with a loss of vitreous humour

6. Thoracic blood vessel sampling

Number of samples One

Sample volume Up to 1 ml
Equipment 25G needle
Staff resource One person is required to take the sample.

7. Cardiac Puncture blood sampling

Number of samples One

Sample volume Up to 1 ml
Equipment 23G - 25G needle
1 to 5 ml syringe
Staff resource One person is required to take the blood sample.

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5. To withdraw blood sample from rabbit and storage conditions

➢ Requirements
2 kg rabbit, 19-23G needle, blood sample collection tubes, 21-24G needle, 20-22G.

1. Blood collection from marginal ear vein

Number of samples Up to 8 samples may be taken in any 24-hour period, depending on sample volume.
Sample volume Up to 0.5-1oml, depending on the size and strain of the rabbit.

Equipment 19G-23G butterfly needle, depending on the strain and size of the rabbit

Staff resource Two persons; one to restrain the rabbit and other to take the blood sample.

Adverse effects bruising/hemorrhage<1%, infection<1%

2. Blood collection from Lateral saphenous vein (most preferred ruote)

1. The simplest and the most convenient site to collect the
blood is from lateral saphenous vein located at the leg.
2. An attendant restrains the rabbit, exposing the rear leg
to the person drawing the specimen.
3. Hair is clipped from the lateral saphenous vein on the
lower rear limb.
4. The vein is occluded by the attendant and the
phlebotomist cleans and dries the skin over the vein with
alcohol and sterile gauze.
5. Using a scalp vein set primed with sodium heparin, a 21-
24 gauge needle is inserted into the occluded vessel and blood is aspirated into the connected
3ml syringe prefilled with 1ml of air.
6. The syringe may be gently rocked while the blood is collected to avoid clotting.
7. Pressure is placed on the venipuncture site for about 20 seconds after withdrawing the needle.

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3. Blood collection from central ear artery

Sample volume Up to 20ml

Equipment 20-22G needle

4. Blood collection from cardiac puncture (non-survival):

Number of samples One

Sample volume 60-120ml,dependimg on the size of the rabbit
Equipment 19-21G needle
Staff resource One person is required to take the blood sample.

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➢ Advantages and Disadvantages of sites of injections

Collection site Advantages Dis-advantages

Marginal ear vein Vein is easily accessed Secure restraint required
Special equipment recommended
Topical anesthesia recommended
Central ear artery Large samples can be collected Topical anesthesia is strongly advised.
Special equipment recommended
Training required.
Lateral saphenous vein Anesthesia is not required Training required
Special collection set is required
Small to moderate sample sizes.
Cardiac puncture Maximum quantity of blood is required Non-survival procedure only.

➢ Storage conditions for blood sampling of laboratory animals

1. Sample size: Take a large enough sample of blood
➢ Each volume of blood taken should be enough to run two-either separate or duplicate-of the desired
tests.
➢ A good rule of thumb is to collect between 2 to 2.5 times the volumes that the test requires.
➢ Blood should be tested very soon after it’s drawn, usually within about 4 hours.
➢ Serum samples should be separated from whole blood within 2 hours of the blood draw.
➢ Additives such as a clot accelerator, anticoagulant, or heparin might be used, depending in the
testing need.

2. Temperature: Maintain appropriate sample temperature

➢ Depending on the simple use, one of three temperatures will typically be specified for blood sample
storage: room temperature, refrigerated, or frozen.
➢ Room temperature is specified as between 15 and 30°C refrigeration temperature is between 2 and
10 °C; frozen temperature is at or below 20 C.
➢ Samples should be cultured or frozen in liquid nitrogen for future use.

3. Length of storage: Make sure conditions are right

➢ Blood banks consider six weeks to be the ‘shelf life’ of blood, but a study from John Hopkins
University has shown that after three weeks, red blood cells are less effective at delivering oxygen-
rich cells throughout the body.
➢ Blood stored longer than three weeks becomes less flexible and less able to fit in the body ’smallest
capillaries.
➢ Depending on the blood’s future use, longer storage without refrigerated poor frozen temperatures
can jeopardize its visibility.

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References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3043327/
2. https://www.research.psu.edu/arp/training/videos/blood-collection-in-mice.html
3. https://www.medschool.umaryland.edu/media/SOM/Offices-of-the-Dean/OAWA/docs/Blood-
Collection/blood-collection-guidelines-mice.pdf
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540092/
5. https://research.uci.edu/compliance/animalcare-use/research-policies-and-guidance/blood-
collection-rodents.html
6. https://research.uci.edu/compliance/animalcare-use/research-policies-and-guidance/blood-
collection-rodents.html
7. https://www.nhs.uk/conditions/anticoagulants/

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Learning questions

Q1: What are the factors on which the method of blood sampling depends?
A1: The volume of blood needed, the frequency of the sampling, the health status of the animal to
be bled, the skill level of the technician.
Q2: Why is the proper insertion of needle in veins difficult?
A2: It is difficult to locate specific veins and improper insertion may lead to hematoma formation,
nerve damage, bleeding, inflammation of veins.
Q3: Name the mode of injection available in both rats and mice but abssent in guinea pig?
A3: No tail injecction in guinea pig as they are devoid of a tail.
Q4: Name the non-surgical procedures for withdrawing blood samples from the mouse?

A4: Blood collection via tail vein, lateral saphenous vein, orbital sinus, t
Q4: Why is cardiac puncture a terminal technique?

A5: Because this is a surgical procedure and the animal does not survive this type of technique.

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