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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. Hamish McMath D:\769860297.doc Page 1 6/05/2017 Standard Operating Procedure Animal Facilities 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: 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 220l) amounts of blood. The procedure must be performed competently by a trained technician to ensure injuries cannot occur to the mice. Guidelines: 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 Hamish McMath D:\769860297.doc Page 2 6/05/2017 Standard Operating Procedure Animal Facilities 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: 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: 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 Hamish McMath D:\769860297.doc Page 3 6/05/2017 Standard Operating Procedure Animal Facilities 3. 4. 5. 6. 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 Hamish McMath D:\769860297.doc Page 4 6/05/2017 Standard Operating Procedure Animal Facilities 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: 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: 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: Level 1: Trainee – Practice on culled or culling mice in a training Hamish McMath D:\769860297.doc Page 5 6/05/2017 Standard Operating Procedure Animal Facilities 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 Hamish McMath D:\769860297.doc Page 6 6/05/2017 Standard Operating Procedure Animal Facilities bleeds; 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; . To achieve level 4 competency the following is required: 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 D:\769860297.doc Approval Date: 24/08/2009 Page 7 Review Date: 24/08/2010 6/05/2017