Download TITLE: Blood collection from the retro-orbital sinus

Standard Operating Procedure
Animal Facilities
TITLE: Blood
collection from the retro-orbital sinus
This technique requires special approval from the Animal Ethics
Committee and may only be used with good justification e.g.
(Alternative routes for blood sampling such as submandibular bleeds
have proved unsuccessful).
Retro-orbital bleeding can only be performed by approved
technicians who have been signed off as being competent by an
approved trainer.
Introduction
The mouse has a large peri-orbital venous sinus that fills the bony orbit of
the eye. Blood can be collected from either medial or lateral canthus
approach.
Lateral Canthus: Pick-up the mouse and restrain it in one hand. Inset
the tube into the lateral canthus. The tube should be at about a 30 degree
angle to the side of the head.
Medial Canthus: Place the mouse on a table or cage lid in lateral
recumbancy. The body of the mouse is restrained against the table with
the palm of the hand. The thumb and forefingers of the same hand
restrain the hand and gently open the eyelids to expose the eye. Insert
the tube into medial canthus and hold it at a 30 degree angle to the nose.
Both Procedures: A microhematocrit tube or pipette is inserted into the
orbital sinus by quickly rotating the tube. The eye is not damaged
because the tube passes under the eye.
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Standard Operating Procedure
Animal Facilities
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Mice can only be bled from 6 weeks of age, depending on their body
weight which should ideally be 20gms or above.
Animals must be given a suitable veterinary eye medication (Conoptal)
after blood sample collection.
Before and after performance of the procedure; any abnormalities
regarding the health of the mouse must be recorded. A record must be
kept on the health status of the animal.
The interval between bleeds must be a minimum of 2 weeks.
Each mouse can be bled no more than six (6) times
Only 10% of the blood volume of the mouse can be collected (e.g. If
the mouse is between 22 and 25 grams then 220 µl is the maximum
amount of blood to be collected)
If a mouse has one damaged eye, then it is preferable to use this eye
for bleeding to minimize the risk of damaging the healthy eye.
If a mouse cannot be bled via the retro orbital sinus (both blind/
cataract/small/infected eyes), then alternative routes of collection of
blood sample must be performed (***unless the decision not to collect
is ethical – heavily pregnant, with small litter, sick, runted, or injured
requiring the animal to be humanely culled)
Possible Complications to avoid:
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Retro-orbital haemorrhage resulting in haematoma and excessive
pressure on the eye
Corneal ulceration, keratitis, pannus formation, rupture of the globe and
micro-ophthalmia
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
This is why it is very important that staff are competently trained before
they attempt this procedure on live mice
Purpose:
Retro-orbital bleeding in mice is used to collect moderate (up to 220l)
amounts of blood. The procedure must be performed competently by a
trained technician to ensure injuries cannot occur to the mice.
Guidelines:
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The use of retro-orbital bleeding must only be done on mice in a
protocol approved by the Griffith University AEC.
All technicians that are to independently perform this procedure must
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Standard Operating Procedure
Animal Facilities
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be competently trained by their supervisor and to have completed:
Basic Handling training
Intermediate Training
Alternative Blood collection Procedures
o Lateral tail blood collection
o Submandibular bleeds
o Saphenous blood collection
Technical Requirements:
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Competent in restraint
Competent in cervical dislocation
Competent in Mouse Identification
Competent in ear punch collection
Able to use the blood / punch program
Competent in Health Observation
Able to competently bleed in both eyes
EQUIPMENT:
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75 µl haematocrit tubes (heparinised or non-heparinised – depending
on purpose
o Heparinised – do not want the blood to clot, blood is used for
assay work ( FAC’s)
o Non – heparinised – want the blood to clot to be able to collect
the sera (ELISA’s, health screening for antibody responses in
the white blood cells)
Cluster tubes
Cluster tube rack
Paper towel
Sharps bin
Gloves
Gown
Face mask
An appropriate disinfectant e.g. virkon
Ethanol
Bleed request numbers printout
Sample collection grid (as backup)
PROCEDURE:
1. Collect the plate and bleeding template so that you are aware of which
mice to bleed
2. Enter animal holding room
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3.
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Locate mice to be bled on the racks
Identify mice for blood sampling.
Check that all mice are healthy.
Identify the mice to be bled within the cage and select mice as per the
cage card
7. Use grid to identify animals and sample tubes, ensuring that only the
mice that are to be bled are entered.
8. Place mouse on wire and restrain in a secure grip ensuring that the
middle or index finer are restricting movement of the head. (Refer to
“Restraint Procedure”). The eyes should slightly bulge.
9. Pick up the haematocrit tube, which is placed in the cluster tube with
your spare hand.
10. Place the haematocrit tube parallel (outer side of the eye away from
the nose) to the bulging eye, applying gentle pressure. The eye should
now bulge further.
11. Gently tilt the tube at an angle until it slides into the outer edge of the
bulging eye. The tube should not go any deeper than the coloured
marking.
12. Slowly rotate the haematocrit tube until the orbital blood vessel is
nicked and blood freely flows down the haematocrit tube into the
cluster tube.
13. Allow it to flow until the required amount of blood is collected.
14. Remove haematocrit tube from eye and immediately release restraint.
15. Tap cluster tube gently onto a level surface before replacing in rack.
This mixes the blood with the anticoagulant in the tube to prevent
clotting.
16. Discard used haematocrit tube into designated sharps container.
17. Place mice that have already been bled into a clean cage or the lid of
their cage to make identification of remaining mice simpler.
18. Cross off mouse number on mouse request document. If cannot be
bled add reason to blood request form and comment section on blood
program.
19. Select the next mouse, if there is one and repeat steps 8 to 17.
20. When finished with the cage, use grid ensuring that only the mice that
are to be bled are entered.
21. Return cage to it’s rack and position
22. When finished bleeding wipe down laminar flow hood or procedure
bench thoroughly, with an appropriate disinfectant e.g. virkon and then
80% ethanol, ensuring that no blood is left on any surface.
23. Tape lid onto plate and attached blood request form. Place plate inside
sealed bag before leaving mouse-holding / procedure room.
24. Leave the completed plate on the collection bench.
25. Contact researcher to let them know the blood is ready for collection.
The mouse will stop bleeding when it is released (no longer restrained,
lower stress, lower blood pressure). The mouse is able to be placed
back into cage with other mice, normal behavior is that the mouse or its
cage mates will clean its eye and the bleed will stop within seconds
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COMPLICATIONS:
****Should you notice any of these complications please record it immediately
and seek veterinary attention if required
Injuries to the eye caused by retro-orbital bleeding can be seen in the
following ways
 The mouse squinting when the procedure has finished
 The eye looks bulged and swollen
 The area around the eye is swollen
 Opacification of the eye, (clouding over), usually happens the
following day
 Orbital discharge after the procedure
 Clean fluid entering the capillary tube during the procedure may
mean that you have nicked the eye ball and ocular fluid is leaking. If
this occurs the animal must be humanely culled immediately.
HANDY HINTS:
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High blood pressure occurs when mice are restrained; therefore
always ear punch mice before bleeding to avoid hemorrhaging.
Sometimes the mouse’s nictitating membrane (third eyelid) comes
out, the mouse will rectify this problem itself when grooming.
Check blood plate program at regular intervals to insure that
samples are aligned in sequence on the physical plate and the plate
program.
If plate program is unavailable keep copy of written template to
rescan at the earliest possible chance. It is the responsibility of the
bleeder to ensure all plates are entered on the plate program.
Competency Levels and Assessment:
Training:
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Retro-orbital Bleeding Supervised training
o 2 small bleeds of <30mice supervised (assessment checking)
o 2 medium bleeds of <60 mice supervised (assessment
checking)
o Independently bleed 4 small to medium bleeds with assessment
checking
There are 4 Levels of Competency:
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Level 1: Trainee – Practice on culled or culling mice in a training
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environment, No bleeding unsupervised
Level 2: Trainee – Able to bleed groups of up to 30 culling mice
supervised in a training environment
Level 3: Supervised Bleeding Trainee– Able to participate in the
bleeding schedule for plates of less than 30 mice, must be
supervised
Level 4: Competent Bleeder - able to bleed any number of mice
unsupervised
Level 5: Trainer – Able to bleed any number of mice unsupervised
and train others in the technique
Level 1 – Trainee
Undertaking training from a level 4 person with all bleeding done under
supervision
During level 1 training the trainee should practice the techniques in a training
environment.
1. Restraint and ear punching can be practiced on mice that are to be
culled
2. Orbital bleeding can be practiced on already culled mice.
(*** 3 training sessions)
Level 2 - Trainee
Undertaking training from a level 4 person with all bleeding done under
supervision
During level 2 training the trainee should practice the techniques in a training
environment.
1. Restraint and ear punching can be practiced on mice that are to be
culled
2. Orbital bleeding can be practiced on mice that are to be culled.
3. Should be able to competently bleed 30 live mice in a row, without
causing injuries or stress (2 weeks)
4. Should be able to competently bleed 45 live mice in a row, without
causing injuries or stress (2 weeks)
5. Should be able to competently bleed 60 live mice in a row, without
causing injuries or stress (2 weeks)
(*** 6 training sessions)
Level 3 – Supervised Bleeding Trainee
To achieve Level 3 competency the trainee is required to participate in the
bleeding schedule by undertaking bleeds that have <30mice. The trainee is
closely supervised and is able to perform the following in 2 consecutive
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bleeds;
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Be competent with orbital bleeding restrains, within 2 attempts
Be able to ear punch and orbitally bleed in one restraint
Be able to bleed from the left and right eye in one restraint
Have haematocrit tube in correct position whilst bleeding
Be able to restrain a mouse without it struggling or going into
respiratory distress, with a limited time of handling of 20-25 seconds
Be able to collect a consistent 200ul of blood without contamination,
with the ability to control the amount of blood
Level 4 –Competent Bleeder
To achieve Level 4 competency the Competent bleeder is required to
participate in the bleeding schedule by undertaking bleeds that have >30mice.
The Bleeder is closely supervised for the first bleed and is able to perform the
following in all consecutive bleeds;
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To achieve level 4 competency the following is required:
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Must be able to bleed a full plate in one sitting with a consistent
200ul of blood in all tubes on two consecutive bleeding sessions
Must be able to achieve level 4 competency steps on a minimum of
95% of animals in any one bleeding session on two consecutive
occasions
Level 5 – Trainer
Experienced invivo personnel able to demonstrate and apply training of
relevant techniques and assess trainee at all levels of competency.
Author:
Approved by:
Signature
Signature
Hamish McMath
Type Name
Hard Copy Location:
Hamish McMath
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Approval Date:
24/08/2009
Page 7
Review Date:
24/08/2010
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