SOP No injection plunger

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					   Griffith University
 Animal Ethics Manual- Laboratory
 STANDARD OPERATING PROCEDURES

SOP No:             L-6

SUBJECT:            Cardiac Puncture to Collect Blood from Rodents (Non
                    Recovery)

POLICY:             The Code (1) specifies;

    3.3.13 Animals must be handled only be personnel instructed and competent in
         methods that avoid pain or distress.

    3.3.32 For non-recovery surgery, the animal must remain unconscious
         throughout the procedure.

PRECAUTIONS:       Personnel conducting this procedure must have been
                   vaccinated against Tetanus and Hepatitis B and should wear
                   Personal Protective Equipment (Lab coat/gown, gloves, eye
                   protection and masks).

EQUIPMENT:         26 gauge ½ inch needle and 1 cc syringe. Ketamine
                   (100mg/ml), Xylazine (20mg/ml) and 1.5ml Eppendorf tube.

                   Micro-centrifuge.

PROCEDURE:
               1. Procedure is to take place in a quiet, clean environment,
                  separate from other animals.
               2. Draw required dosages of anesthetic solutions into the syringe.
                  Mouse (50mgs/kg Ketamine and 10mgs/kg Xylazine).
                  Rat (50mgs/kg Ketamine and 10mgs/kg Xylazine).
               3. Injecting dosage intra-peritoneally.
                  a. Restrain animal securely at the scruff with its head lower
                        than body to avoid injury to organs during injection.
                  b. Swab lower right quadrant of abdomen with alcohol.
                  c.    Introduce needle slowly through skin, subcutaneous tissue
                        and abdominal wall.
               4. Withdrawal plunger marginally to check needle is not in bladder
                  or intestines. If no fluid is withdrawn begin to slowly inject
                  dosage.
               5. Place animal into cage free from structural obstacles and
                   cover with towel to create a quiet, dim environment.
               6. Monitor animal closely until signs of 2nd stage anesthesia
                  occurs. Loss of consciousness may involve involuntary
                  movement and breath holding.
                7. Remove animal from cage monitor until 3rd stage anesthesia
                   occurs. Confirm anesthetic depth, overall body muscle
                   relaxation and absence of; Pedal or Withdrawal reflex,
                   Palpebral reflex and Jaw tone reflex.
                   8. Place animal in a dorsal recumbency position on a clean and
                      sterile surface and swab injection site with alcohol.




                        Collecting blood from a terminally anesthetized rat.
                   9. Using a sterile syringe insert needle just behind the xiphoid
                      cartilage and slightly left of the midline (as above). Advance
                      needle at a 10-30 degree angle from the horizontal axis of the
                      sternum, aspirating lightly while advancing.
                   10. Slowly withdraw blood until required sample is obtained;
                          Mouse: ≥1ml, Rat: ≥10ml.
                   11. Humanely euthanize animal via cervical dislocation
                       immediately after blood collection. Death must be established
                       by lack of responses (pedal/withdrawal reflex, palpebral reflex,
                       body relaxation) and loss of pulse before disposal of the
                       carcass.
                   12. Transfer whole blood sample to a 1.5ml Eppendorf tube.
                   13. If serum sample is required allow blood to clot at ambient
                       temperature for a minimum of 30 minutes, refrigerate at 4C for
                       1 hour to allow clot to contract. Centrifuge on low speed for 5-
                       10 minutes and then extract serum with a sterile pipette and
                       transfer to sterile 1.5ml Eppendorf tube.

RECOMMENDATIONS:

The procedure must be conducted by technicians previously certified competent in
small animal care, restraint, IP injections and species specific anaesthesia /
euthanasia techniques or, under the direct supervision of such a competent person.


DATE ISSUED:           May 2008
REVISED:               2011
                                                                 Dr G Harrison
                                                              CHAIR OF GUAEC

                                       REFERENCES
    1. NHMRC (2004) Australian code of practice for the care and use of animals for
    scientific purposes (7th edition). Australian Government.
    2. http://www.animalethics.org.au/reader/research-procedures/arrp-blood-collection.htm
    3. http://www.lal.org.uk/pdffiles/BLOOD.PDF
    4. http://www.jhu.edu/animalcare/Mouse.HTM#blood
    5. http://www.jhu.edu/animalcare/rat.htm#blood

				
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