Guidelines

Guidelines on Blood Collection

Unit for Laboratory Animal Medicine
Apr 15, 2016 12:00 am

This document is designed to provide general information on blood collection methods for common laboratory animals. All procedures must be approved by the Institutional Animal Care and Use Committee (IACUC). The method of blood collection to be used, the intervals between blood collection procedures, and the volume of blood to be removed, must be listed in the approved protocol specific to each study. 

The ULAM veterinary staff provides the following criteria to determine the maximum safe amount of blood to be withdrawn. It is recommended to take no more blood than is absolutely necessary. Remember to calculate beforehand the minimum amount of blood necessary to perform all tests and assays, and that the serum fraction is about ½ of the total blood volume. When calculating blood volumes based on body weights, remember that body weights in kilograms (kgs) will convert to blood volumes in liters, and weights in grams will convert to volumes in milliliters (mls).

  • Procedures

    1. Approximate Blood Volume

    1. 5-10% of body weight = total blood volume
      1. The circulating blood volume can generally be estimated as 55-70 ml/kg of total body weight. However, care should be taken in these calculations as the % of total blood volume will be lower (-15%) in sick, obese and older animals.
      2. See Table 13 at end of document for some specific blood volumes.

    2. Blood Collection Volumes

    1. 1% of body weight = maximum volume per collection every 14 days, without requiring supplemental fluids. This applies for single blood collections as well as repeated collections. For irregular sampling schedules, calculate the total amount needed over a 14-day span.
    2. 0.07% of body weight = amount that can be taken daily without requiring supplemental fluids
    3. 4-5% of body weight = amount expected at exsanguinations

    3. Single Blood Draw

    1. Maximum of 1% of body weight can be removed as a single blood draw every 14 days, without requiring administration of supplemental replacement fluids. Withdrawing the minimum amount of blood necessary is strongly recommended. Examples:
      1. 0.15 ml from a 15 gm mouse
      2. 3 ml from a 300 gm rat
      3. 50 ml from a 5 kg cat
      4. 100 ml from a 10 kg monkey
      5. 400 ml from a 40 kg dog

    4. Multiple Blood Draws

    1. If the total volume withdrawn over a 14-day period is less than 1% BW, then no additional action needs to be taken.
    2. If the total volume withdrawn over a 14 day period is up to 2% BW (or over), fluid volume replacement must be considered. Withdrawing the minimum amount of blood necessary is strongly recommended. Examples:
      1. Up to 0.15 ml withdrawn from a 15 g mouse over 2 weeks is OK
      2. Up to 0.3 ml withdrawn from a 15 g mouse over 2 weeks, replace volume with 0.3 ml saline SQ
      3. Up to 3 ml from a 300 gm rat over 2 weeks is OK
      4. Up to 6ml from a 300 gm rat over 2 weeks, replace volume with 6 ml saline SQ
      5. Up to 200 ml from a 20 kg dog over 2 weeks is OK
      6. Up to 400 ml from a 20 kg dog over 2 weeks, replace volume with 400 ml saline SQ or IV
    3. As a helpful guideline, daily blood draws under 0.07% BW will keep the total 2-week withdrawal under 1% BW.

    5. Exsanguination

    1. Approximately 50-75% of total blood volume (4-5% of body weight) can be obtained by terminal exsanguination. The animal must be deeply anesthetized, or recently euthanized, prior to exsanguination. Since the amount of blood obtained is substantially increased if the heart is beating during the bleeding procedure, use of a surgical plane of anesthesia is recommended. The procedure for anesthesia and/or euthanasia must be described fully in the approved IACUC protocol. Examples:
      1. 0.60-0.75 ml from a 15 gm mouse
      2. 12-15 ml from a 300 gm rat
      3. 200-250 ml from a 5 kg cat
      4. 400-500 ml from a 10 kg monkey
      5. 1600-2000 ml from a 40 kg dog

    6. Fluid Replacement

    1. If the volume of blood removed from an animal exceeds the maximum recommended blood collection volumes (i.e., > 1% body weight every 14 days), replacement of the removed volume of blood with warm (30-35ºC) isotonic solution (e.g., 0.9% saline, lactated Ringer's solution) constitutes accepted veterinary practice. When this volume of blood is collected, it should be withdrawn at a slow, steady rate, and the volume of solution to be infused should be administered similarly.

    7. Monitoring

    1. If too much blood is withdrawn too rapidly or too frequently without replacement (approximately 2% of the animal's body weight at one time), the animal may go into hypovolemic shock. If signs of shock are observed, contact the ULAM veterinary staff immediately. Signs of shock include:
      1. Fast and thready pulse
      2. Pale dry mucous membranes
      3. Cold skin and extremities
      4. Restlessness
      5. Hyperventilation
      6. Sub-normal body temperature
    2. If 15-20% of total blood volume is removed, cardiac output and blood pressure will be reduced.
    3. If 30-40% of total blood volume is removed, death will result in at least 50% of animals.
    4. If > 40% of total blood volume is removed, death of the animal is expected.
    5. Stressed, sick, or otherwise compromised animals may not tolerate the blood collection criteria noted above, which is for healthy animals.
    6. By monitoring hematocrit (Hct or PCV) and/or hemoglobin (Hb) it is possible to evaluate if the animal has sufficiently recovered from single or multiple blood draws. Remember it may take up to 24 hours for hematocrit or hemoglobin to reflect a sudden or acute blood loss. In general, if the animal is anemic (below the normal PCV range for the species), or if the hemoglobin concentration is less than 10 gm/dL, it is not safe to remove the volumes of blood listed above.

    8. Blood Collection Sites & Methods

    The tables below list the most frequently used blood collection sites for common laboratory animal species. They are listed from most common/desirable to least common/desirable based on ease of collection from the site. For uncommon laboratory animal species, please contact the ULAM veterinarians at ulam-vets@umich.edu. For smaller species, the volume of blood attainable for each site is listed based; however, this is an estimation and will also depend on the size, heath, and hydration status of the animal as well as the experience level of the person collecting the sample. Based on the goals and requirements of the study, certain sites may be preferable). Additionally, publications have indicated that the results from blood analysis (especially cellular indices) may vary based on the site of blood withdrawal; consult the literature for more information. In all cases, cardiac puncture may be used to obtain a single, large volume of blood from heavily anesthetized (terminal procedure only) or euthanized animals. 

    1. Table 1: Mouse

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Lateral Tail Vein   

         No   

         Yes   

         50 - 100 ul  

         Saphenous Vein   

         No   

         Yes   

         100 - 200 ul  

         Facial Vein (submandibular & submental veins)   

         No   

         Yes   

         200 - 500 ul  

         Distal Tail Transection (less than 3mm)   

         Required a   

         Yes - limited   

         < 100 ul  

         Retro-Orbital Sinus   

         Required   

         Yes - limited   

         200 ul  

         Sublingual Vein   

         Required   

         Yes   

         500 ul  

         Jugular Vein   

         Recommended   

         Yes   

         

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         ~ 1 ml  

      a Distal tail transection in gerbils, and adult rats and mice (>21 days) requires the use of general anesthesia and preemptive analgesia (e.g. NSAIDs, opioids) unless scientifically justified and approved in the IACUC protocol.
    1. Table 2: Rat

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Saphenous Vein   

         No   

         Yes   

         300 - 400 ul  

         Lateral Tail Vein   

         No   

         Yes   

         200 - 400 ul  

         Distal Tail Transection (less than 3mm)   

         Required a   

         Yes   

         200 - 400 ul  

         Dorsal Metatarsal   

         No   

         Yes   

         100 - 200 ul  

         Submandibular / Facial Vein

         No   

         Yes   

         200 - 500 ul  

         Jugular Vein   

         Required   

         Yes   

         0.5 - 2.0 ml  

         Sublingual Vein   

         Required   

         Yes   

         0.5 - 1.0 ml  

         Retro-Orbital Plexus   

         Required   

         Yes - limited   

         0.5 - 1.0 ml  

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         ~3 ml  

      a Distal tail transection in gerbils, and adult rats and mice (>21 days) requires the use of general anesthesia and preemptive analgesia (e.g. NSAIDs, opioids) unless scientifically justified and approved in the IACUC protocol.
    1. Table 3: Hamster

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Lateral Tarsal Vein   

         No   

         Yes   

         100 - 200 ul  

         Toenail Clipping   

         No   

         Yes   

         100 - 200 ul  

         Retro-Orbital Sinus   

         Required   

         Yes - limited   

         100 - 200 ul  

         Jugular Vein   

         Required   

         Yes   

         0.5 - 2.0 ml  

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         ~3 ml  

    1. Table 4: Gerbil

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Lateral Tail Vein   

         No   

         Yes   

         200 - 400 ul  

         Toenail Clipping   

         No   

         Yes   

         100 - 200 ul  

         Distal Tail Transection (less than 3mm)   

         Required a   

         Yes (1-2 times only)   

         100 - 200 ul  

         Retro-Orbital Sinus   

         Required   

         Yes - limited   

         100 - 200 ul  

         Jugular Vein   

         Required   

         Yes   

         0.5 - 2.0 ml  

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         ~3 ml  

      a Distal tail transection in gerbils, and adult rats and mice (>21 days) requires the use of general anesthesia and preemptive analgesia (e.g. NSAIDs, opioids) unless scientifically justified and approved in the IACUC protocol.
    1. Table 5: Guinea Pig

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Auricular Vein   

         No   

         Yes   

         50 - 100 ul  

         Cephalic Vein   

         No   

         Yes   

         50 - 100 ul  

         Saphenous Vein   

         No   

         Yes   

         400 - 500 ul  

         Jugular Vein   

         Recommended   

         Yes   

         2 - 3 ml  

         Cranial Vena Cava   

         Recommended   

         Yes   

         2 - 3 ml  

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         

    1. Table 6: Rabbit

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Marginal Ear Vein / Central Ear Artery   

         Local Anesthesia Recommended      

         Yes   

         1 - 3 ml  

         Lateral Saphenous Vein   

         No   

         Yes   

         

         Cephalic Vein   

         No   

         Yes   

         

         Jugular Vein   

         Recommended   

         Yes   

         

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         

    1. Table 7: Ferret
         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Cephalic Vein   

         No   

         Yes   

         

         Jugular Vein   

         Recommended   

         Yes   

         

         Anterior Vena Cava   

         Recommended   

         Yes   

         

         Cardiac Puncture (Terminal Only)   

         Required   

         Terminal   

         

    1. Table 8: Cat

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Medial Saphenous Vein   

         No   

         Yes   

         

         Cephalic Vein   

         No   

         Yes   

         

         Jugular Vein   

         No   

         Yes   

         

    1. Table 9: Dog

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Lateral Saphenous Vein   

         No   

         Yes   

         

         Cephalic Vein   

         No   

         Yes   

         

         Jugular Vein   

         No   

         Yes   

         

    1. Table 10: Sheep

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Jugular Vein   

         No   

         Yes   

         

         Cephalic Vein   

         No   

         Yes   

         

         Saphenous Vein   

         No   

         Yes   

         

    1. Table 11: Pig

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Marginal Ear Vein   

         No   

         Yes   

         

         Cephalic Vein   

         No   

         Yes   

         

         Right Jugular Vein   

         No   

         Yes   

         

         Anterior Vena Cava   

         Recommended   

         Yes   

         

    1. Table 12: Non-Human Primate

         Site   

         Anesthesia   

         Repeat Bleeds   

         Expected Volume   

         Femoral Vein   

         Recommended   

         Yes   

         

         Saphenous Vein   

         Required   

         Yes   

         

         Cephalic Vein   

         Required   

         Yes   

         

         Brachial Vein   

         Required   

         Yes   

         

    1. Table 13: Circulating Blood Volumes in Common Lab Animal Species (adopted from Heinz-Diehl, 2001 and Hawk et al. 2005)

         Species   

         Mean Blood   
         Volume (ml/kg)   

         Range of Mean   
         Blood Volume (ml/kg)   

         Mouse   

         72   

         63 - 80   

         Rat   

         64   

         58 - 70   

         Hamster   

         78   

         

         Gerbil   

         67   

         

         Guinea Pig   

         75       67 - 92   

         Rabbit   

         56       44 - 70   

         Ferret   

         75       

         Cat   

         55       47 - 66   

         Dog (Beagle)   

         85       79 - 90   

         Sheep   

         66       60 - 74   

         Minipig

         65       61 - 68   

         Macaque (Rhesus)

         56       44 - 67   

         Macaque (Cynomolgus)   

         65       55 - 75   

         Marmoset   

         70       58 - 82   
  • References

    1. Clemons DJ, Seeman JL. 2011. The Laboratory Guinea Pig. Boca Raton (FL): CRC Press, p. 89-98.
    2. Ebert RV, Stead EA, Gibson JG. 1941. Response of normal subjects to acute blood loss. Arch Int Med; 68:578-90.
    3. BVA/FRAME/RSPCCA/UFAW Joint Working Group on Refinement. 1993. Removal of blood from laboratory mammals and birds (first report). Laboratory Animals; 27:1-22.
    4. Field KJ, Sibold AL. 1999. The Laboratory Hamster and Gerbil. Boca Raton (FL): CRC Press, p.108-112.
    5. Hawk CT, Leary SL, Morris TH. 2005. Formulary for Laboratory Animals. Ames (IO): Blackwell Publishing. p. 157.
    6. Heimann M, Käsermann HP, Pfister R, Roth DR, and Bürki K. 2009. Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage. Lab Animal; 43: 255-260.
    7. Heinz-Diehl K, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal JM, Vorstenbosch C. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol; 21:15-23.
    8. Hoff J. Methods of blood collection in the mouse. 2000. Lab Animal; 29(10):47-53.
    9. McGuill MW, Rowan AN. 1989. Biological effects of blood loss: Implications for sampling volumes and techniques. ILAR Journal; 31(4):5-20.
    10. Nerenberg ST, Zedler P, Prasad R, Biskup N, Pedersen L. 1978. Hematological response of rabbits to chronic, repetitive, severe bleedings for the production of antisera. J Immunol Meth; 24:19-24.
    11. Otto G, Rosenbald WD, Fox JG. 1993. Practical venipuncture techniques for the ferret. Lab Anim 27:26-29
    12. Scipioni RL, Diters RW, Myers WR, Hart SM. 1997. Clinical and clinicopathologic assessment of serial phlebotomy in the Sprague Dawley rat. Lab Anim Sci; 47(3):293-299.
    13. Scipioni RL, Guidi DA, Stehr JE, Hart SM, Diters RW. 1996. Clinical, hematologic, and clinicochemical assessment of serial blood sample collection in Sprague-Dawley rats. Contemp Top Lab Anim Sci; 35(6):90. [Abstract]
    14. Skavlen PA, Baron SJ, Stevens JO. 1992. Effect of blood collection volumes on the hemograms of rabbits. Contemp Top Lab Anim Sci; 31(4):23. [Abstract]
    15. Villano JS, Sharp PE. 2013. The Laboratory Rat. Boca Raton (FL): CRC press, p. 202-212.
    16. Yale CE, Torhortst JB. 1972. Critical bleeding and plasma volumes of the adult germfree rat. Lab Anim Sci; 22(4):497-502.
Questions?

Questions or concerns about the content of this document should be directed to the Unit for Laboratory Animal Medicine (ULAM) at (734) 764-0277 or ulam-questions@umich.edu.

For training on specific blood collection methods and techniques, contact the ULAM Training Core via email at ulam-trainingcore@umich.edu or call 734-763-8039.