Refining Bld Cllectin Techniques t Imprve Animal Welfare and Sample Quality Amy Allaire RLATG 1, Jennifer Jhnsn 2, Kimberly Maratea DVM PhD 2, Steven Bulé CMAR RLATG 1, Sara Savage DVM DACLAM 1 1 Dispsitin, Safety and Animal Research, Sanfi, Framingham, MA 01701 2 Sanfi-Genzyme R&D Center, Sanfi Framingham, MA 01701
Abstract An effrt t refine rdent bld cllectin techniques resulted in an examinatin f alternative methdlgies t imprve animal welfare and sample quality. Disease mdel r repeated sample cllectin resulted in inadequate sample vlume, ccasinal hemlysis, and cmplicatins including bruising, scarring, and necrsis f sample cllectin sites. A study was designed t determine whether the use f a vibrating device in cmparisn t standard straight stick methds wuld decrease health cmplicatins as well as imprve sample quality and quantity. C57BL/6 mice were used as a cntrl strain fr bth tail and saphenus venipuncture. NOD mice were used fr the diseased tail stick grups. Athersclersis induced ApB mice were used as a disease mdel fr saphenus venipuncture. Tail sticks were perfrmed twice daily fr tw mnths and saphenus bleeds were perfrmed bi-weekly fr tw mnths, t mimic study schedules. Sample vlume increased with the use f the vibrating device and bld cllectin was achieved withut the use f heat, unlike the standard direct venipuncture methd. Cmparisn f the tw bleeding methds in the ApB strain shwed that we were able t cllect cmplete vlume in 73% f samples via the standard methd versus 84% with the use f the vibrating needle. Full sample vlumes were cllected with single sticks in 38% f attempts f the standard methd versus 70% f attempts with vibrating needle in ApB mice. An incidence f bruising was nted at 36% with standard methd versus 12% with the use f vibrating needle. Tail circumference at the site f standard venipuncture f C57Bl/6 mice averaged 13.2mm; this was a 3mm increase frm bth the cntrl and vibrating needle grups which averaged 10mm tail circumference. This wrk demnstrates that, as cmpared t standard bld cllectin methds, the vibrating needle allws fr increased sampling cmpliance while decreasing assciated trauma.
Backgrund As an alternative t retr rbital eye bleeds, saphenus venipuncture is used with increased frequency in an attempt t decrease cular cmplicatins, including blindness, crneal and retrbulbar trauma r lss f the eye. Saphenus venipuncture des nt require the use f anesthesia, which culd cause cmplicatins related t disease mdel r strain as well as alter bld chemistry. Cmplicatins frm saphenus venipuncture can result in incnsistent bld sample vlumes and lcalized pst-bleed trauma - factrs related t strain, disease mdel, and size f the animal. Daily tail stick sample cllectin fr bld glucse mnitring als causes multiple risks. SID and BID venipuncture f a nn-bese diabetic (NOD) muse tail increases incidence f bruising, swelling, and necrsis and may adversely affect animal welfare. Required mnitring can als disrupt the strain s disease prgressin, as NOD mice can reverse t nn-diabetic if infectin is intrduced. Prmising phlebtmy alternatives include the use f the GentleSharp device. The GentleSharp attaches t a needle hub and uses an scillatry mtin that reprtedly requires less effrt t pass thrugh tissue. The reductin in frce required t mve the needle thrugh tissue is believed t cause less trauma, stress and pain t the animals while allwing fr adequate bld sample vlume. A study was designed t cmpare standard versus GentleSharp saphenus and tail venipuncture bld cllectin methds in bth nrmal and diseased mice.
Grup Strain Lcatin f Cllectin Study Design and Prtcl NOD and C57BL/6 mice were allcated int Grups 2-5 (tail venipuncture). 27 gauge needles were used and changed between each animal. Venipuncture sites were indicated n the animals tail with permanent marker. Samples cllected BID t mimic current diabetic study prtcl. ApB and C57BL/6 mice were allcated int Grups 2, 3, 6, and 7 (saphenus venipuncture). 25 gauge needles were used and changed between each animal. Samples cllected bi-weekly frm the left saphenus vein t mimic athersclersis study prtcl. Upn cmpletin f the study, histlgy samples were cllected frm animals in grups 1-5. Cllectin Technique Animals/Grup In-Life Perid Histlgy 1 C57 (female) Nne Nne 10 8 weeks in-life Leg and Tail 2 C57 (female) Saphenus and Tail Standard 10 5 mice - 4 weeks inlife Leg and Tail 5 mice 8 weeks inlife 3 C57 (female) Saphenus and Tail GentleSharp 10 5 mice 4 weeks inlife Leg and Tail 5 mice 8 weeks inlife 4 NOD (female) Tail Standard 5 8 weeks in-life Tail 5 NOD (female) Tail GentleSharp 5 8 weeks in-life Tail 6 ApB (male) Saphenus Standard 32 2 weeks in-life NA 7 ApB (male) Saphenus GentleSharp 23 2 weeks in-life NA
Cmparisn f Saphenus Venipuncture in Diseased ApB Mice Percent f Single-Stick Attempts 100 80 60 40 20 0 Day 1 Day 14 GentleSharp Methd Standard Methd Percent Incidence f Bruising 45 40 35 30 25 20 15 10 5 0 0 0.5 1 1.5 2 2.5 GentleSharp Methd Standard Methd ApB mice are used t mimic human cardivascular diseases. Thrughut disease prgressin it becmes increasingly difficult t btain full sample vlume thrugh saphenus venipuncture due t cmplicatins frm altered metablism, including athersclersis. Repeated venipuncture attempts ften result in insufficient sample size and quality, bruising and scarring making future cllectins increasingly difficult. The graph abve indicates use f the GentleSharp ver a tw week perid in ApB mice imprved cmplete sample cllectins in a single attempt as well as decreasing the amunt f pst-bleed bruising.
Saphenus Cllectin in Diseased ApB Mice Percent f Mice per Stick 100 80 60 40 20 6 Sticks 5 Sticks 4 Sticks 3 Sticks 2 Sticks 1 Stick Day 1 Bleed 28% f mice that had standard cllectin required the use f heat lamp Average sample cllectin size fr standard cllectin was 181ul Average sample cllectin size fr GentleSharp was 198ul 0 GentleSharp Standard 100 Day 14 Bleed Percent f Mice per Stick 80 60 40 20 3 Sticks 2 Sticks 1 Stick 42% f mice that had standard cllectin required the use f heat lamp Average sample cllectin size fr standard cllectin was 168ul Average sample cllectin size fr GentleSharp was 191ul 0 GentleSharp Standard
Saphenus Cllectin in C57BL/6 Mice 100 Percent f Mice per Stick 80 60 40 20 0 GentleSharp Standard Methd 3 Sticks 2 Sticks 1 Stick A. Attempts Average Number f Attempts Needed t Acquire Cmplete Sample Vlume 1.8 1.7 1.6 1.5 1.4 GentleSharp 1.3 1.2 1.1 1 0.9 Day 1 Day 14 Day 28 Day 42 Day 56 Standard Methd A. Bruising nted ne day pst saphenus bleed with the use f the standard methd. Animals in the GentleSharp grups had n evidence f bruising. Heat lamp nt used fr nn-diseased mice Cmplete sample vlume btained fr all cllectin methd time pints
Cmparisn f Tail Circumference A. 14 B. 14 13 13 Millimeters 12 11 10 C57BL/6 GentleSharp C57BL/6 Standard Methd C57BL/6 Cntrl Millimeters 12 11 10 NOD GentleSharp NOD Standard Methd NOD Cntrl 9 9 8 Day 1 Day 56 8 Day 1 Day 56 Tail measurements were taken Day 1 and Day 56 using a Medline Surgical Ruler. Figure A. indicates the use f the GentleSharp in nn-diseased C57BL/6 mice increases tail circumference by 6% cmpared t the use f the standard methd which increased tail circumference by 38%. Figure B. indicates the use f the GentleSharp in NOD mice increases tail circumference by 17% cmpared t the use f the standard methd which increased tail circumference by 47%
Cmparisn f Tail Venipuncture Methds in NOD Mice A. B. 100 90 Incidence f Bruising Percentage f Mice 80 70 60 50 40 30 20 GentleSharp Methd Standard Methd A. NOD muse pst GentleSharp venipuncture. B. NOD muse pst standard methd venipuncture. 10 0 Day 1 Day 28 Day 56 Alng with increased tail circumference, the standard methd increased the incidence f bruising in NOD mice.
Tail Venipuncture Histlgy Findings Standard GentleSharp NOD C57BL/6 N differences were bserved between the standard cllectin and GentleSharp grups r between NOD and C57BL/6 mice. On day 56, micrscpic lesins were characterized by fcally extensive acanthsis (epidermal thickening), minimal t mild dermal inflammatin and hemrrhage, and fibrsis in the dermis and subcutis, with extensin int muscle, tendns, and nerves. (H&E stain, 100x magnificatin) Day 56
Saphenus Venipuncture Histlgy Findings Standard GentleSharp N differences were bserved between the standard cllectin and gentle sharp grups in C57BL/6 mice. Micrscpic lesins were nt bserved arund the saphenus venipuncture site f either grup n day 35. On day 56, lesins in bth grups cnsisted f minimal t mild perivascular hemrrhage and neutrphilic inflammatin. (H&E stain, 100x magnificatin)
Cnclusins Study prtcls were effectively mimicked by using strains specific t disease mdels. The use f the GentleSharp device fr BID tail sticks effectively maintained tail circumference in C57Bl/6 mice while the use f the standard methd increased circumference by 38%. The use f the GentleSharp device fr BID tail sticks in NOD mice increased tail circumference by 17% while the use f the standard methd increased circumference by 47%. The use f the GentleSharp device fr bi-weekly saphenus venipuncture yielded a 72% success rate f single stick attempts versus a 40% success rate f single stick attempts while using the standard methd in ApB mice. Histlgical data indicates there was n significant difference in tail and saphenus vein puncture site trauma between the GentleSharp grups cmpared t the standard methd grups. Future Directins Determine the efficacy f the GentleSharp device fr muse mandibular bld cllectin Evaluate tissue pathlgy with the use f the GentleSharp n animals n lng term diabetic studies t determine if it can decrease incidence f bruising in symptmatic NODs. Acknwledgements Fr yur dedicatin and supprt, thank yu t Cmparative Medicine.