Safe Work Method Statement. Mouse Blood Collection

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Flinders University Safe Wrk Methd Statement Muse Bld Cllectin Versin 6 Cllege f Medicine and Public Health Animal Facility SWMS Number RA Number RA Scre Cntents The SOP Muse

2 MEDIUM Cntact Persn SWMS prepared by AWC Apprval Date Review Date Rxanne Cllingwd Rxanne Cllingwd & Lewis Vaughan 14 th June 2016 June 2021 Legislatin University Plicy Lcal

1 Flinders University Safe Wrk Methd Statement Muse Bld Cllectin Versin 6 Cllege f Medicine and Public Health Animal Facility SWMS Number RA Number RA Scre Cntents The SOP Muse Bld Cllectin cntains the fllwing sectins: SWMS- 1.2 RA- 1.2 MEDIUM Cntact Persn SWMS prepared by AWC Apprval Date Review Date Rxanne Cllingwd Rxanne Cllingwd & Lewis Vaughan 14 th June 2016 June 2021 Legislatin University Plicy Lcal Plicy Safe Wrk Methd Statement Persnal Prtective Equipment Required Hazards and Cntrls Befre Wrk Cmmences General Infrmatin NHMRC Bld Cllectin Guidelines Saphenus Vein Puncture Tail Vein Puncture Methd Facial Vein (Cheek Bleed) Cardiac Puncture - Terminal SWMS 1.2 Muse - Bld Cllectin Page 1 19/12/2017

2 Legislatin Australian Cde fr the Care and Use f Animals fr Scientific Purpses 8 th Ed. Animal Welfare Act 1985 Animal Welfare Regulatins 2012 Gene Technlgy Act 2000 (the Act) Gene Technlgy Regulatins 2001 Wrk Health and Safety Regulatins 2012 University Plicy Wrk Health and Safety Plicy 2013 Respnsible Cnduct f Research Plicy 2016 NHMRC Guidelines Versin 6 Lcal Plicy Use f the Cllege f Medicine and Public Health Animal Facilities by all staff and researchers f the Cllege f Medicine and Public Health, Flinders University, is subject t awareness f, and adherence t the fllwing: Research Invlving Animals: The University hlds a licence fr the use f animals fr teaching and research purpses. T satisfy the requirements f the licence, anyne wishing t undertake teaching and research using animals must submit a prpsal t the Animal Welfare Cmmittee (via the Animal Ethics Review Sub- Cmmittee. N wrk with animals may cmmence until written apprval has been received frm the Animal Welfare Cmmittee. Standardised applicatin frms fr Research and Teaching can be fund n the Flinders University website listed belw. It is yur respnsibility t regularly check this site fr updates t guidelines, frms etc All staff and students invlved in animal research must cmplete Animal Ethics Online Training (AEOT) and must als regularly attend Animal Researcher Infrmatin Sessins (ARIS). All persnnel wrking with Genetically Mdified Animals (GMO) r wrking with in a PC1 r PC2 facility must attended a Bisafety Training Day every 3 years Safe Wrk Methd Statement Refer t Risk assessments, Safe Wrk methd Statements fr chemicals, prcesses and plant equipment where apprpriate. All prjects must have an accmpanying Risk Assessment signed by the Animal Facility Manager SWMS 1.0 Muse- Sexing, Handling, Restraint and Ear Ntching RA 1.0 Muse- Sexing, Handling, Restraint and Ear Ntching SWMS 1.1 Muse- Injectin techniques RA 1.1 Muse- Injectin techniques SWMS 1.7 Muse Transprtatin RA 1.7 Muse Transprtatin SWMS Emergency Cntingency RA Emergency Cntingency Refer t Risk assessments, Safe Wrk Methd Statement fr chemicals, prcesses and plant equipment where apprpriate. All prjects must have an accmpanying Risk Assessment signed by the Animal Facility Manager SWMS 1.2 Muse - Bld Cllectin Page 2 19/12/2017

3 Versin 6 Glves Gwn Mask Hair Net She Cvers Persnal Prtective Equipment Required Hazards and Cntrls Animal bites- training, demnstrate cmpetency, adhere t SWMS. Animal Scratches- training, demnstrate cmpetency, adhere t SWMS. Needle Stick- DO NOT recap needles, dispse immediately int sharps cntainers, adhere t SWMS. Chemical expsure- wear PPE and gggles. Befre Wrk Cmmences Ensure that yu are aware f the lcatins f the fllwing: Spill Kit Fire Extinguisher Eye Wash Exits Risk Assessment and SDS (Safety Data Sheet) - Ensure that yu have read and understd fr all the substances being used. Equipment Check fr safety and electrical cmpliance Ensure that yu have read and understd the Risk Assessment and Safe Wrk Methd Statement Obtain training befre using any equipment General Infrmatin All prcedures are t be perfrmed by trained cmpetent staff. Training is available frm senir animal huse staff r Animal Welfare Officer. Evidence f training is available in the Staff Training Needs Analysis. If sedatin r anaesthesia is required fr the bld cllectin prcedure SWMS 1.3 Muse Anaesthesia and Analgesia shuld be fllwed. Suitable sedatives may include acetylprmazine with r withut butrphanl/buprenrphine, r xylazine, r medetmidine fr all bld cllectin in this SWMS, except fr cardiac bleeding. General anaesthesia is required fr cardiac bleeding. Isflurane r ketamine/xylazine, ketamine/medetmidine, Zletil/xylazine r Zletil/medetmidine, r pentbarbitne may be used fr this purpse. SWMS 1.2 Muse - Bld Cllectin Page 3 19/12/2017

4 Versin 6 NHMRC Bld Cllectin Guidelines The apprximate bld vlume can be calculated using the assumptin that the animals ttal bld vlume is 7% f animal s bdyweight. Bld vlume limits can be expressed as a percentage f bld vlume, assuming that an animal s ttal bld vlume can be estimated at 70mL/kg. Up t 10% f the circulating bld vlume can be taken n a single ccasin frm nrmal healthy animals with minimal adverse effect. This vlume may be repeated after 2 weeks. Fr repeat bleeds at shrter intervals, a maximum f 1.0% f an animal s circulating bld vlume can be remved every 24 hurs. Maximum vlumes and recvery perids fr bld cllectin Perid f cllectin % f bld vlume cllected Apprximate recvery perid in weeks Single bleed Up t 7% (minr bleed) 1 10% (mderate bleed) 2 15% (severe bleed) 3 Recmmended site and vlume f bld cllectin using calculated bld vlume Species Ttal bld vlume (using vl. f 7% bdy weight[ml]) Recmmended site fr bld cllectin <7% Minr bleed (ml) 10% Mderate bleed (ml) 15% Majr bleed (ml) Muse (26gm) 1.82 (0.026g x 70mL/kg) Saphenus vein, facial vein, tail vein < Saphenus Vein Puncture Anaesthesia r sedatin is recmmended fr saphenus vein puncture, althugh it is pssible t perfrm the bleed with simple restraint. 1. Restrain the muse in an pen falcn tube, with the hind leg extended. The lateral saphenus vein is in the hind leg, which runs drsally and then laterally ver the tarsal jint. 2. Immbilise the leg in the extended psitin by gently applying dwnward pressure abve the knee jint. This technique stretches the skin ver the ankle, making it easier t shave, and immbilising the saphenus vein. 3. Carefully shave the lateral area prximal t the ankle. The vein shuld be clearly visible under the skin. 4. Apply a small amunt f Vaseline ver the site befre puncturing the vein (this aids the bld t run freely frm the skin, and prvides mre efficient bld cllectin). SWMS 1.2 Muse - Bld Cllectin Page 4 19/12/2017

5 5. Using a 23 t 25g needle, puncture the vein. Use a micr haematcrit tube r micrvette tube t cllect the bld as it flws frm the puncture site. Versin 6 6. Once an adequate bld sample has been cllected, gentle apply pressure ver the wund site t stp further bleeding. 7. Serial sampling may be perfrmed frm the same puncture site by gently rubbing ff the scab that frms. Saphenus Vein Puncture Tail Vein Puncture Methd 1. The tail vein dilatin can be prmted by ne f several ways: - Occluding the vessel with the thumb and frefinger, r turniquet. - Heat the tail by either immersing in warm water (37 C) fr 1-2 minutes. - Place the tail f the muse under a 40 watt incandescent light fr up t 5 minutes. - Place the muse in a therma-cage (incubatr) set at 35 C fr up t 10 minutes. - Administering a subcutaneus r intraperitneal injectin f sterile Nrmal Saline fr injectin at a dse vlume f up t 5% f bdyweight. - Administering a subcutaneus r intraperitneal injectin f acetylprmazine at a dse rate f up t 2.5 mg/kg 15 minutes prir t the bld cllectin. 2. Smear tpical lcal anaesthetic cream (EMLA Cream) ver the surface f the tail 10 minutes prir t the bleeding prcedure. 3. General anesthesia is nt required, place the muse int an apprpriate size restrainer. 4. The lateral (side) veins are immediately belw the skin, and shuld be entered at a shallw angle t the vein. SWMS 1.2 Muse - Bld Cllectin Page 5 19/12/2017

5. If using: Versin 6 A scalpel blade, sizes 10, 11, 14, 15, 16, 20, 21 r 22, r a Glden Rd

sufficient t penetrate the skin and ne side f the wall f the vein. See https://www.nc3rs.rg.

Tail Vein Puncture Available: https://www.nc3rs.rg.

6 5. If using: Versin 6 A scalpel blade, sizes 10, 11, 14, 15, 16, 20, 21 r 22, r a Glden Rd Lancet, and make a light transverse incisin with the base f the blade ver the tail vein, just sufficient t penetrate the skin and ne side f the wall f the vein. See fr a vide that depicts this technique. Tail Vein Puncture Available: accessed: June 29, 2016 Available: accessed: June 29, 2016 SWMS 1.2 Muse - Bld Cllectin Page 6 19/12/2017

7 Versin 6 A 23 t 25 gauge needle, insert the needle int the vein with the bevel upwards. Remve the needle and a small bld drp shuld frm. 6. The bld is cllected int a capillary tube r micrvette tube, r allwed t drip int a suitable cntainer. 7. Once an adequate bld sample has been cllected, gently apply pressure ver the wund site t stp further bleeding, r use a chemical cautery pen r a cttn bud impregnated with ferric chlride crystals, sliver nitrate, r ptassium permanganate. Anesthesia is nt required. Facial Vein (Cheek Bleed) Nte the maximum bld vlumes that may be safely cllected frm a 26 gram muse: Minr bleeds up t 0.12 mls maximum f nce weekly. Mderate bleeds up t 0.18 mls maximum f nce frtnightly. Majr bleed up t 0.27 mls maximum f nce every 3 weeks. If a majr bleed is cllected, the animal shuld be given 1.0 t 1.5 mls f bdytemperature sterile Nrmal Saline fr Injectin 15 minutes prir t the prcedure by subcutaneus r intraperitneal injectin. Recvery cages shuld be placed n warm mats, and supplementary 100% xygen must be available if signs f shck ccur. 1. Select an Eppendrf tube, and fill this with the target vlume that has been identified. This will assist yu t determine if yur cllectin vlumes are t great and whether yu need t prvide fluid therapy, warmth, and supplementary xygen. 2. Scruff the muse (this will cause the eyes t bulge and reduce the flw frm the face). Nte: Scruff mre skin if the muse is able t mve during the prcedure. If the muse s nse and lips start t turn blue, release the muse and re-scruff using less skin and pressure. 3. Glden rd lancets (see belw fr sizes) shuld be used. Lancet sizes - 4 mm pint fr mice f between 2-8 weeks ld. - 5 mm pint fr mice f between 2-6 mnths f age mm pint fr mice lder than 6 mnths f age. 4. Insert the needle, r the lancet, ne eye length caudal, and ne eye width drsal, t the freckle n the face, and puncture the skin angling the lancet/needle up twards the muse s ear. The sub-mandibular r facial vein lies just under the skin. 5. Remve the lancet and cllect the required vlume f bld. If a larger bld sample is required, use yur index finger t mve the muse s head gently up and dwn. 6. Apply gentle pressure until bleeding ceases. SWMS 1.2 Muse - Bld Cllectin Page 7 19/12/2017

8 Versin 6 Submandibular Vein Freckle Bld Cllectin int tube Apply gentle pressure until bleeding stps SWMS 1.2 Muse - Bld Cllectin Page 8 19/12/2017

Versin 6 Cardiac Puncture - Terminal 1. Animals must be surgically anaesthetised when using this technique. See Muse Anaesthesia Safe Wrk Methd Statement. 2.

9 Versin 6 Cardiac Puncture - Terminal 1. Animals must be surgically anaesthetised when using this technique. See Muse Anaesthesia Safe Wrk Methd Statement. 2. Psitin the muse n its back, and feel fr the heart beat by palpating the mid ventral thrax with yur index finger and thumb. 3. Using a 23 t 25G needle, carefully insert the needle either between the ribs r beneath the sternum, int the heart. If inserting the needle beneath the sternum, the needle must be inserted slightly left f the midline. Alternative appraches fr cardiac bld cllectin, which may be used are sternal and diaphragmatic. 4. The required vlume f bld can then be withdrawn. The animal must be euthanized at the end f the prcedure. 5. Death after exsanguinatin shuld be cnfirmed by cervical fracture whilst still under anaesthesia. SWMS Review This SWMS currently applies t the animals hused in the Cllege f Medicine and Public Health Animal Facility. This SWMS will be reviewed 5 yearly, but als updated mre frequently as plicies, techniques and animal care requirements change. Psitin Name Cntact Details Manager Animal Facility Rxanne Cllingwd rxanne.cllingwd@flinders.edu.au Animal Welfare Officer Lewis Vaughan aw@flinders.edu.au Useful References SWMS 1.2 Muse - Bld Cllectin Page 9 19/12/2017

10 Versin Any questins regarding the abve guidelines and any technical advice/ assistance required can be directed t Animal Facility Manager. SWMS 1.2 Muse - Bld Cllectin Page 10 19/12/2017

Safe Work Method Statement. Mouse Anaesthesia and Analgesia

Safe Work Method Statement. Mouse Anaesthesia and Analgesia Flinders University Safe Wrk Methd Statement Muse Anaesthesia and Analgesia Cllege f Medicine and Public Health Animal Facility SWMS Number RA Number RA Scre SWMS- 1.3 RA- 1.3 MEDIUM Cntact Persn SWMS