Collection of Serum

from blood
Objective

To understand the various techniques applied to process

a blood sample and obtain serum.
Principle
Blood serum can be defined in a number of ways. Clear, watery fluid of the blood
that separates when blood clots or blood plasma from which the protein fibrinogen
or clotting factors, has been removed.

Clotting factors are the proteins which causes the clotting of blood. when the blood is
allowed to clot after its withdrawn from a vein, the clot slowly shrinks and a clear
watery fluid squeezed out from the clot is known as serum.
Why use Serum for Studies?
Serum includes antibodies, antigens, electrolytes, hormones
and any exogenous substances such as drugs and
microorganisms and all proteins expect that used in blood
clotting. Thus it has applications not only in the field of clinical
diagnostics, but also in research
.
A very simple example could be their use in the different types
of ELISA tests. They are therefore used to detect the presence
of a particular antibody, antigen, hormone, exogenous
substances, etc.
Why use Serum for Studies?

Blood serum and plasma are biofluids that are

increasingly important in NMR-based metabolomics
analysis. Metabolite analysis of fluids from the
circulatory system provides a view of the metabolic
state of an organism. Unlike urine analysis, which
measures an organism's waste products, serum or
plasma analysis measures homeostatic levels of
metabolites throughout the organism.
Why use Serum for Studies?

Abnormal levels of hormones and various other

indicators can also be detected from serum, and
are being used in clinical diagnostic labs for
routine tests. The presence of elevated levels of
antibodies indicates that the body is not at
homeostasis.
Why use Serum for Studies?

It is important to study the parameters like

pharmacokinetics (what the body does to the drug)
and pharmacodynamics (what the drug does to the
body) while conducting clinical trials. Also it is
necessary to know; how the drug is metabolized, what
the half-life of the drug is, and after metabolism in
what chemical from it persists in the body.
 Blood sampling
Normally, blood is collected from the
Intravenous route. It is directly transferred
to a sterile container that is stoppered and
also has a label on it.

When serum is desired to be collected, it is

not heparenised, since we want the clot to
form and remove the clotting factors from
the sample!
 Blood sampling
It is critical to take care when an individual is dealing
with blood since blood is a biohazardous material.

The container must be stoppered to prevent spillage,

contamination, etc.
The container must be properly labeled, since there is
high chance of confusion while conducting the tests.

A wrong diagnosis can have devastating effects!!

The clinician looking at the result makes a wrong conclusion, thus making his
diagnosis also wrong; at the end of the day putting the life of the patient 'on
the line'.
Procedure

1. Collect the blood into a glass container and allow it to clot at room
temperature for 1h.
2. Once the clot has formed, loosen it from the walls of the container
to aid retraction.
3. Keep at 4°C and leave it there overnight.
4. Collect the expressed plasma and centrifuge at 150 g for 5 min (to
sediment erythrocytes) and then at 350g for 15 min.
5. Transfer the serum (straw-colored supernatant) to containers
suitable for long-term storage and heat at 56°C for 30 min to destroy
the heat-labile components of complement.
Mouse Anesthesia and
Blood Collection
Objectives

•To familiarise the user to the common anaesthesia

techniques applied in laboratories on rodents.

•To introduce basic restraining techniques and blood

collection techniques while handling mice.
Principle

 In experimental immunology and biomedical research, the

importance of laboratory animals is indisputable.

 Their care and handling is a contributing factor to the

quality of the science resulting from their use.

 Animals are used as a source of biological materials (cells,

antibodies, and serum) for laboratory use or as
experimental subjects.

 Animal research is best served by a coordinated approach

and sound animal research methodology.
Maintenance parameters
The environment where a laboratory animal is housed has a major impact
on the animal's health and response to experimental manipulation.

Variations in the environment and infectious agents can seriously alter

immunological function.

Details like ;
• caging
• feed
• bedding
• water
• sanitation (to minimize the impact of environmental contamination)
• environmental monitoring (monitoring controlled temperature, humidity,
ventilation, and illumination, etc)
 Anaesthesia
 Anesthetic agents are used in laboratory animals to prevent pain or
distress due to an experimental procedure or for restraint to facilitate
a technically difficult procedure.

 Agents may be used alone or in combination to achieve the desired

effect.

 Anaesthesia is achieved through drug selection and proper dosing.

 Investigators using anesthetic agents should familiarize themselves

with their physiologic effects and the risks involved with their use.
Blood Collection

Blood is the most frequently sampled body fluid for evaluation of

serum antibodies or analysis of surface markers on peripheral
blood cells.

Here we describe a few basic blood collection methodologies

commonly when working on rodents.

Blood collection is the most common interventional procedure.

Selection of an appropriate method will depend on the species,
amount of blood needed, frequency of sampling, and whether
the animal's survival is required.
 Blood Collection

With appropriate techniques, small amounts of blood can be

obtained with little ill effect on the animal. Use of anesthetics
is recommended for some of the protocols. Anesthetics may
alter some hematological and biochemical parameters, but
their use is indicated to prevent undue stress. When
necessary, anticoagulants may be used to prevent clotting.
 Procedure
Materials Required
•Isoflurane
•70% Alcohol
•Animal Model : White Mice
•Anesthesia Chamber
•Cotton
•Dropper
•Microhematocrit Tube
•Test tubes
•Restrainer
•Syringe
•Needle
Inhalant Anaesthesia Using Isoflurane for Mouse and Rat

1. Determine the volume of the anaesthesia chamber in litres. Multiply

the volume by 0.15 to 0.2 ml to obtain the volume, in ml of Isoflurane
to be used.

2. Move the glass chamber and isoflurane to a fume hood/laminar flow.

3. Soak a cotton ball with the required amount of the anesthetic solution
and put it in the glass chamber. Above the cotton ball place a raised
wire gauze platform.

4. Close container lid immediately. Allow the isoflurane to evaporate to

vapour phase over 5-10 minutes.
 Inhalant Anaesthesia Using Isoflurane for Mouse and Rat

5.Transfer animal from its cage into anaesthesia chamber by lifting it, by
holding at the tail and placing it into the chamber. Close lid tightly after
this.

6.Monitor animal closely. Within approximately one minute for mice and 2
minutes for rats, the animal will become anesthetized. Initially, respiratory
rate will increase and then decrease.

7.Allow the animal to become recumbent and reach slow and regular
breathing.

8.Allow the animal to remain at a deep anesthetic plane for ~10 more
seconds before proceeding into the desired experimental procedures.
Blood Collection from Tail Vein of Mouse and Rat Using Microhematocrit
Tube

1. Restrain the animal.

2. Warm the tail with a heat lamp or immerse in warm water to dilate the
vessels.
3. Visualize a sampling site of the lateral tail vein at approximately
midpoint on the length of the tail.
4. Extend the tail with one hand, and with the other hand insert needle 3 to
4 mm into the lateral tail vein.
5. Collect blood from the hub of the needle with the microhematocrit tube.
6. Remove needle from the tail vein and apply a gauze sponge with gentle
pressure on the bleeding site to ensure hemostasis.
Blood Collection from Tail Vein of Mouse and Rat Using Centrifuge Tube

1. Restrain the animal.

2. Warm tail with the heat lamp or immerse in warm water to dilate the
blood vessels.
3. Visualize a sampling site of the lateral tail vein in the distal one-third
of the tail.
4. While extending the tail, swiftly incise across the lateral tail vein with
a new scalpel blade.
5. Collect blood drops into 12- to 50-ml centrifuge tube.
6. Apply gauze sponge with gentle pressure to the wound to ensure
hemostasis.
Blood Collection from Orbital Sinus or Plexus of Mouse and Rat

1.Manually restrain and anesthetize the animal.

2.Introduce the end of the microhematocrit tube at the medial canthus of

the orbit.

3.Slowly, and with axial rotation, advance the tip of the microhematocrit
tube gently towards the rear of the socket until blood flows into the tube.

4.Remove the microhematocrit tube from orbit and dab excess blood from
the site with a gauze sponge or swab moistened in saline or PBS.

5.Observe the animal for recovery from anaesthesia.

Parenteral Injections
Objectives
1.To understand the basics of handling and manually
restraining experimental animals (rodents).

2.To understand how to perform Parenteral injections in

experimental animals.
Principle
This section describes the use of rodents - how to handle them,
manually restrain them, how to inject the experimental animals.

For the safety of the handler and the animal, proper methods for
handling and restraining laboratory animals should be followed.

Many animal facilities provide training in handling techniques to

enable the inexperienced handler to gain confidence and skill.
 Principle

Improper handling can result in increased stress and injury to the

animal. In addition, the handler risks injury from bite wounds or
scratches inflicted when the animal becomes fearful or anxious.

By using sure, direct movements with a determined attitude, the animal

can be easily handled and restrained. It should be remembered that
mice will always try to bite the operator, proper restraint of a mouse is
important for further manipulation.
 Principle
To properly restrain a mouse, pick it up by the base of the tail with your
right hand and place it on the top of a wire cage lid, pull gently back on
the tail, which will ensure the mouse to grasp the bars of the cage lid
with all four feet.

Then using the thumb and index finger of the left hand, quickly grasp
the mouse by the scruff near the base of the head. When the scruff (the
back of the neck) is firmly held, lift the mouse and press the tail base
with third or fourth finger against the palm. Always remember to wear
disposable gloves when you get involved in these lab activities.
Types of Parenteral Injections

a) Intramuscular Injection

Regardless of the method used for intramuscular

injections, it must be noted that the sciatic nerve
runs along the length of the femur.

It is very important to avoid injuring this nerve.

This is best accomplished by pointing the needle,

caudally rather than cranially, into the caudal thigh
muscles
Types of Parenteral Injections

a) Intramuscular Injection

It is imperative that the mouse be

properly restrained.

If the mouse is allowed to kick or

struggle, this could cause injury to
the muscles or the nerve
 b) Subcutaneous Injection
Restrain the mouse by the scruff method.
Use your thumb and forefinger to make a
tent of skin over the scruff.

Prep the area with 70% ethanol.

Insert the needle, bevel up, at the base of

the tent.

The needle should be inserted parallel to

the skin and should be directed toward the
posterior of the animal.

Aspirate to ensure proper placement and

inject the material.
 c) Intraperitoneal Injections
Restrain the mouse by the scruff method.
Expose the ventral side of the animal, tilting
the head down at a slight angle.

Prep the site with 70% ethanol.

The sterile needle should be placed, bevel

up, in the lower right or left quadrant of the
animal’s abdomen.

Insert the needle at a 30° angle.

Aspirate to ensure proper placement and

inject the material.
 d) Intradermal Injections
In order to perform intradermal injections, the mouse should be
anesthetized.

Shave or pluck an injection site on the back of the animal to remove

the hair.

Swab the site with 70% ethanol.

Insert the needle into the skin, bevel up, holding the needle nearly
parallel to the plane of the skin.

Do not aspirate.

Inject the material.

The volume of the injection should be limited to 50 ìl per site to

avoid tissue trauma.
e) Intravenous Injection

The IV injection has been widely used for drug,

etc.

Tail veins of the mouse are most commonly used

for intravenous injection.

They are readily visible especially after alcohol

cleans the skin. Warming a mouse by putting it
under heating light or warming its tail directly by
soaking it in hot water (40-45oC) will dilate the
veins to facilitate the injection (increases blood
circulation).
e) Intravenous Injection

To perform the procedure, place the mouse in a mouse restrainer,

disinfect the tail with 70% ethanol, hold the tail firmly, insert a
small-gauge (25-gauge or smaller, attached to a syringe) needle
with its bevel up through the skin and into the lumen of the vein,
advance the tip in the vein about half centimetre to ensure it not to
slide out of the vein, push the plunger of the syringe slowly and
smoothly (You should be able to see the vein blanch if the needle
is properly positioned).

If the insertion of the needle into the vein is successful, there

should be no resistance felt as the material is given, and the blood
in the vein can be seen to be washed away.
e) Intravenous Injection

If any swelling at the injection site or resistance to injection occurs,

remove the needle and reinsert it slightly above the initial injection
site.

Retract the needle and apply pressure to the injection site to

ensure hemostasis.

Intravenous injection of the mouse is a difficult procedure which

requires practice and patience.

The inexperienced investigator should take the trouble to gain this

skill in several practice sessions with phosphate-buffered saline
(PBS) as the injectate.
f) Footpad Injection

Many protocols for the production of T cell lines and clones in mice
and rats require footpad injection, which is usually done in only one
or two footpads to minimize discomfort.

This procedure is also chosen at times when allergic responses of

an agent are desired to be studied.

Disinfect the skin over a footpad with 70% ethanol. Insert Gauge
needle subcutaneously into the footpad area from the ventral side.

Inject up to 50 μl of the material into subcutis in the footpad area.

Retract the needle.

Place a cotton ball over the injection site for about 1 min to prevent
bleeding and spillage of material.
g) Intrathymic Injection

The thymus is one of the lymphoid organs very

commonly used in studies regarding Lymphocytes,
etc.

Like the organs like spleen, lymph nodes, etc;

thymus is also used to isolate lymphocytes from an
organism; thus having an important role in studies
related to understanding the basic immunologic
responses occurring in an organism under the test
condition.
Thus they are of application at the Cellular, as well
as Molecular level of research.