General Principles of Reptile Venipuncture

  • Video

    Dr. Sean Perry is the co-creator of this presentation and all images in this recording were provided by Dr. Perry unless otherwise indicated. Dr. Charly Pignon generously shared the video clips incorporated into this video recording. This video script was created by Dr. Christal Pollock, and reviewed by Drs. Byron de la Navarre and Annelise Strunk.

    View the video recording OR the slideshow with text and still images.

  • Introduction

    iguana

    Photo credit:  Dr. Sean Perry

    Hematology and biochemistry results serve as an important part of the minimum database for all veterinary patients.

  • Collection of blood samples can be challenging

    Turtles and chameleon

    Photo credit:  Dr. Sean Perry

    Although collection of blood samples can be a clinical challenge in reptiles, the method of patient handling, blood collection and sampling techniques are all critical for proper interpretation of laboratory results.

  • Check Marks

    This slideshow reviews the basic principles of reptile venipuncture that should be considered before, during, and after the procedure.

  • Before venipuncture

     

    Green Check Mark

    • Gather all equipment that will be needed
    • To pre-heparinize or not pre-heparinize?
    • Address hypothermia
    • Calculate blood sample volume

     

  • Gather all equipment that will be needed

    equipment

    To collect the blood sample you will need: 4,9,25

    • Glass slides
    • Hematocrit tubes
    • 1 or 3-ml syringe, or an insulin syringe with a needle that can be removed with hemostats
    • 22-27-gauge needles
    • Standard size needles generally suffice, however larger reptiles may require longer (38-50 mm or 1.5-2 in) needles
    • Disposable gloves are advisable due to the risk of zoonoses.
    • Butterfly catheters (optional) can be used to collect samples from areas prone to movement, such as the tail or neck, or in fractious animals.
  • Select the largest needle size appropriate for your patient

    erythrocytes

    Vertebrate red blood cell types with measurements

    Photo credit:  John Alan Elson via Wikimedia Commons

    Reptile erythrocytes are relatively large and fragile, and the use of very small needles can lead to hemolysis. Therefore always select the largest needle size appropriate for your individual patient.23

    The low blood pressure and slow heart rate of the reptile supports the selection of a smaller syringe than one would typically choose for a bird or mammal of similar size. This smaller syringe paired with a relatively large bore needle will encourage blood flow while maintaining gentle negative pressure that will not damage the large, delicate reptile erythrocyte.9

  • Scrub the venipuncture site

    scrub venipuncture site

    Photo credit:  Dr. Charly Pignon

    You will need

    • Antiseptic
    • Small synthetic brush (optional)

    Scrub the venipuncture site thoroughly before sampling. A small synthetic brush, such as a toothbrush, nail brush or scrub brush, may even be required to adequately clean a heavily scaled species.4,9

  • Anticoagulant tubes

    Microtainers

    Photo credit:  Dr. Sean Perry

    Sodium ethylenediamine tetraacetic acid (EDTA) can potentially cause hemolysis in reptiles, particularly some chelonian species.12,19,24 However, research has also identified reptile species in which EDTA works or is even superior to heparin 2,7,15,16, and some reptile veterinarians recommend EDTA for most snakes and lizards.4,18

    Nevertheless, lithium heparin is the anticoagulant of choice for plasma biochemistry analysis, and some clinical practices only use lithium heparin for all reptile blood samples to minimize confusion.9,25

    Always check first with your laboratory before collecting blood.17

    Shown above:  Microtainers allow fast and easy mixing of small blood volumes (0.25-0.50 ml). The tubes contain a dry form of anticoagulant that prevents dilution artifact. BD Vacutainer Systems; Beckton, Dickinson and Co., Franklin Lakes, NJ USA

  • To pre-heparinize or not pre-heparinize the syringe?

    Heparin

    Collection of a viable sample can be challenging in small reptile patients and the practice of pre-heparinizing the syringe has been commonly practiced in zoological medicine, however this technique is often unnecessary and can confound laboratory results by diluting the blood sample.4,911 Sample dilution can increase sodium or potassium, and artificially lower ionized calcium levels.13,27,28 Heparin can also create color artifact and abnormal white cell morphology on peripheral blood smears.2,9

     

  • To pre-heparinize…

    Syringe

    Photo credit:  Dr. Sean Perry

    If you do elect to pre-heparinize the syringe, draw heparin into the syringe with a needle and push the heparin back into the bottle. Remove the needle from the bottle and then forcibly expel all visible heparin. Always replace the needle used to pre-heparinize the syringe with a new, sharp needle to perform venipuncture.

     

     

  • Body temperature & peripheral perfusion

    Terrarium

    Photo credit:  Dr. Sean Perry

    Many reptiles are hypothermic by the time they arrive at the hospital. Keeping the reptile warm will help to maintain peripheral perfusion. A gauze pad soaked in warm water can also be applied over the venipuncture site to promote vasodilation prior to venipuncture. For brachial plexus venipuncture in chelonians, the forelimb can be gently held, squeezing the limb to improve circulation.1

  • Sample volume from a healthy reptile

    Tiny turtles

    Photo credit:  Dr. Mark Mitchell

    When compared to a mammal of similar size, the reptile has lower total blood volume approximating 4-8% of body weight (BW) in kilograms.4,17,26

    Ten percent of estimated blood volume, or up to 4-8 ml/kg or 0.4-0.8 ml per 100 grams BW, can be safely collected from a healthy reptile.4,17,18,23,25,26

  • Sample volume for debilitated or small patients

    venipuncture tiny turtle Mitchell

    Photo credit:  Dr. Mark Mitchell

    Collect a smaller volume, approximately 5% of blood volume (or 0.5% of body weight in kilograms), from critically ill patients or small patients with a history of dehydration or blood loss.17,18

    When calculating a safe volume to collect, also be sure to consider the possibility of blood loss through hemorrhage, such as hematoma formation.

     

    Example #1 Healthy 1 kg (1000-gram) reptile

    1) Shortcut calculation:  Theoretically up to 8 ml/kg or 8 ml of blood can be collected.

    2) Long form calculation:

    Estimated blood volume 4-8% total BW (kg)
    Up to 10% of estimated blood volume can be collected Or 1% of BW (kg)
    The possibility of hemorrhage or hematoma formation must be taken into account, so clinically a smaller volume is collected:  ~0.8%  BW
    0.8      ÷ 100   = 0.008
    0.008 x 1000 =
    8 ml

    Collect no more than half this volume or 4 ml from a debilitated patient.

  • During venipuncture

    Green Check Mark

    Select a venipuncture site

    Be consistent

    Practice optimal patient positioning

    Monitor the sample for hemodilution

  • Each venipuncture site has advantages & limitations

    Chameleon and gecko vemnipuncture

    Photo credit:  Dr. Sean Perry

    Depending on your species of interest, blood can be collected from a variety of venipuncture sites and the location selected can depend on a variety of factors, including the preferences and experience of the phlebotomist, the volume of blood needed, patient size and temperament, and of course the species involved.25,26 Each venipuncture site has advantages and limitations

    View the LafeberVet videos (or text with still images):  Blood Collection in Lizards, Blood Collection in Snakes, and Blood Collection in Turtles and Tortoises for details.

  • Be consistent

    jug stick Perry

    Photo credit:  Dr. Sean Perry

    Once a venipuncture site has been selected, be consistent in that individual patient, whenever possible. Studies in select species have shown there can be significant differences in test results in samples obtained from different venipuncture sites.3,6,14,24

    If performing blood work as part of a routine health check, also make every effort to be consistent with respect to the time of year. Season is another major source of variability affecting hematologic and biochemical values.22,24

  • Optimal patient positioning is critical

    Venipuncture in turtle and lizrd

    Photo credit:  Dr. Sean Perry

    Venipuncture is often a blind technique in reptiles and optimal patient positioning is important. For information on reptile restraint, visit the LafeberVet series: Chelonian Handling and Restraint, Lizard Handling and Restraint, Snake Handling and Restraint.

    Some animals may require sedation in order to exteriorize the legs, tail or head for blood sampling.23 Ketamine (2-5 mg/kg IM), alfaxalone (5-10 mg/kg IM), or alpha-2 agonists, like dexmedetomidine (0.025-0.05 mg/kg IM), can be used to chemically restrain the reptile.21

  • Lymph contamination is a common complication in reptiles

    Cardiocentesis Snake

    Photo credit:  Dr. Sean Perry

    Reptiles possess well-developed lymphatics that often run very close to blood vessels, making lymph contamination and hemodilution a frequent complication.3,17,25

  • Hemodilution is particularly common in chelonians

    hemolymph

    Photo credit:  Dr. Charly Pignon

    A blood sample that has been contaminated with lymph (arrow) will appear pink, pale yellow, whitish, or even clear.17 Hemodilution can occur in all reptiles, but is particularly common in chelonians.

  • The jugular vein is the best source of high-quality samples

    Turtle jugular

    In all reptiles, the jugular vein (arrow) or heart* are the most likely sites to yield a high-quality, representative blood sample.3,18,25  Hemodilution is still possible with the jugular vein, but it is fortunately less likely than with other venipuncture sites.

    *Cardiocentesis is only routinely performed in the snake.

  • If hemodilution is recognized…

    syringe Perry

    Photo credit:  Dr. Sean Perry

    If hemodilution is recognized, the procedure should ideally be aborted, the sample discarded, and a new sample collected, preferably from a different anatomic location.18

  • Take lymph contamination into account when interpreting results

    HCT tubes Perry

    Photo credit:  Dr. Sean Perry

    In reality, it may be inadvisable or even impossible to repeat blood collection in a tiny or debilitated patient. Instead it may be necessary to take lymph contamination into account when interpreting results, particularly packed cell volume, potassium, and protein levels.3,17

  • After venipuncture 

    Green Check Mark

    Prevent clotting

    Prevent hemolysis

    Prepare blood smears

    Process samples as soon as possible

    Wash your hands!

        

  • Prevent clotting

    Prevent clotting

    Photo credit:  Dr. Sean Perry

    Immediately transfer blood samples into tubes containing anticoagulant to prevent clotting.

  • Prevent hemolysis

    Blood through needles

    Photo credit:  Dr. Sean Perry

    Large reptile erythrocytes are relatively fragile.26 To reduce the risk of hemolysis, remove the needle hub from the syringe when transferring the blood sample to a collection tube or preparing a blood smear.26

  • Bovine albumin

    Bovine Albumen

    Photo credit:  Dr. Ankë Stohr

    To reduce the likelihood of white blood cell damage during the blood smear process, some reptile veterinarians also premix the blood sample with bovine albumin (Gamma Biologicals, Inc., Houston, TX USA), which is reported to stabilize cell membranes.18 The blood sample is mixed with 22% albumin, adding 1 drop of albumin per 5 drops of whole blood.

    One small study in pancake tortoises (n=11) suggested that the use of bovine serum albumin minimized cell lysis 20, however the addition of albumin did not prevent cell breakage in a small number of Burmese pythons (Python bivittatus) (n=10).8

  • Prepare peripheral blood smears

    blood smear Perry

    Photo credit:  Dr. Sean Perry

    Peripheral blood films should ideally be prepared directly from the syringe using a fresh blood sample free of anticoagulant and air dried.9,18,19,23,25

     

  • Centrifuge biochemistry samples immediately

    Centrifuges

    Photo credit:  Dr. Sean Perry

    Be prepared to centrifuge plasma biochemistry samples immediately to separate cells from plasma. If blood cells are not separated swiftly, then phosphorus and potassium levels will be falsely elevated while blood glucose levels will be lower than normal. Protein, enzymes, and electrolytes are also very likely to be affected by storage.10

    Few studies have focused on the best way to store reptile blood samples and the effect of delay is poorly understood.5,8,9,25 Therefore analysis should be performed as soon as possible and at the very least within 12 to 24 hours.9,25

  • Wash your hands

    wash your hands

    Photo credit:  Brenda Kochevar/Flickr Creative Common

    Once your reptile patient has been returned to its enclosure and the sample has been processed, wash your hands!

  • Obtaining the next blood sample

    Sick beardie

    If a relatively large volume of blood was collected from a debilitated or small reptile patient, it is recommended to wait at least 5-10 days before repeating venipuncture.21b

     

  • Summary

    reptile venipuncture images

    When preparing to collect blood from a reptile patient, gather all equipment that will be needed, including anticoagulant tubes. Although lithium heparin is often used in reptiles, EDTA may be superior to heparin in select species, particularly some snakes and lizards. Also be sure to address hypothermia, and maintain the reptile within its preferred optimum temperature zone. Additional measures can also be taken to promote peripheral perfusion while a safe sample volume is calculated. Up to 4-8 ml/kg BW can be collected in healthy animals. Halve this volume in critically ill patients or small patients with a history of dehydration or blood loss.

    Once a venipuncture site is selected, position your patient carefully. Blood collection in reptile patients is often a blind technique and optimal patient positioning is important. While the sample is being aspirated, use gentle negative pressure and monitor the sample for evidence of hemodilution. Immediately afterwards, transfer blood samples into anticoagulant tubes. Remove the needle hub from the syringe when transferring blood to the collection tube or preparing a blood film to minimize the risk of hemolysis. Also be prepared to centrifuge plasma biochemistry samples promptly.

References and further reading

References


1. Berry KH, Demmon A, Bailey T. Protocols for drawing blood from the brachial plexus of desert tortoises. USGS Website. July 2005. Available at https://www.werc.usgs.gov/fileHandler.ashx?File=/…/Protocolbloodnasalrev0705.pdf. Accessed September 27, 2016.

2. Bogan JE, Antonio F, Zachariah TT, Stacy NI. Comparison of the effects of three anticoagulants on hematological analytes in the Eastern indigo snake (Drymarchon couperi). J Herp Med Surg. 2020;30(2):96–100. doi: 10.5818/17-07-118.2.

3. Bonnet X, El Hassani MS, Lecq S, et al. Blood mixtures: impact of puncture site on blood parameters. J Comp Physiol B. 2016;186(6):787-800. doi: 10.1007/s00360-016-0993-1. Epub 2016 May 4. PMID: 27146147.

4. Divers SJ. Diagnostic techniques and sample collection. In: Divers SJ, Stahl SJ (eds). Mader’s Reptile and Amphibian Medicine and Surgery, 3rd ed. St. Louis, MO: Elsevier. 2019:1085-1091.

5. Eisenhawer E, Courtney CH, Raskin RE, Jacobson E. Relationship between separation time of plasma from heparinized whole blood on plasma biochemical analytes of loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med. 2008;39(2):208-215. doi: 10.1638/2007-0166R.1. PMID: 18634211.

6. Gottdenker NL, Jacobson ER. Effect of venipuncture sites on hematologic and clinical biochemical values in desert tortoises (Gopherus agassizii). Am J Vet Res. 1995;56(1):19-21. PMID: 7695143.

7. Hanley CS, Hernandez-Divers SJ, Bush S, Latimer KS. Comparison of the effect of dipotassium ethylenediaminetetraacetic acid and lithium heparin on hematologic values in the green iguana (Iguana iguana). J Zoo Wildl Med. 2004;35(3):328-32. doi: 10.1638/03-057. PMID: 15526887.

8. Harr KE, Raskin RE, Heard DJ. Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons. Vet Clin Pathol. 2005;34(4):383-388. doi: 10.1111/j.1939-165x.2005.tb00065.x. PMID: 16270264.

9. Heatley JJ, Russell KE. Hematolgy. In: Divers SJ, Stahl SJ (eds). Mader’s Reptile and Amphibian Medicine and Surgery, 3rd ed. St. Louis, MO: Elsevier; 2019:857-865.

10. Heatley JJ, Russell KE. Clinical chemistry. In: Divers SJ, Stahl SJ (eds). Mader’s Reptile and Amphibian Medicine and Surgery, 3rd ed. St. Louis, MO: Elsevier; 2019:899-909.

11. Johnson JG, Nevarez JG, Beaufrere H. Effect of manually preheparinized syringes on packed cell volume and total solids in blood samples collected from American alligators (Alligator mississippiensis). J Exot Pet Med. 2014;23(2):142-146. doi:  10.1053/j.jepm.2014.02.008.

12. Klein K, Gartlan B, Doden G, et al. Comparing the effects of lithium heparin and dipotassium ethylenediaminetetraacetic acid on hematologic values in Eastern box turtles (Terrapene carolina carolina). J Zoo Wildl Med. 2021;51(4):999-1006. doi: 10.1638/2020-0109. PMID: 33480581.

13. Landt M, Hortin GL, Smith CH, et al. Interference in ionized calcium measurements by heparin salts. Clin Chem. 1994;40(4):565-570.

14. López-Olvera JR, Montané J, Marco I, et al. Effect of venipuncture site on hematologic and serum biochemical parameters in marginated tortoise (Testudo marginata). J Wildl Dis. 2003;39(4):830-836. doi: 10.7589/0090-3558-39.4.830. PMID: 14733278.

15. Martinez-Jiménez D, Hernandez-Divers SJ, Floyd TM, et al. Comparison of the effects of dipotassium ethylenediaminetetraacetic acid and lithium heparin on hematologic values in yellow-blotched map turtles (Graptemys flavimaculata). J Herp Med Surg. 2007;17(2):36–41. doi:  10.5818/1529-9651.17.2.36.

16. Mayer J, Knoll J, Innis C, Mitchell MA. Characterizing the hematologic and plasma chemistry profiles of captive Chinese water dragons, Physignathus cocincinus. J Herp Med Surg. 2005; 15(3):45–52. doi: https://doi.org/10.5818/1529-9651.15.3.45.

17. Mitchell MA. Reptile biochemistries. Proc International Conference on Avian, Herpetological and Exotic Mammal Medicine. 2013:22-23.

18. Mitchell MA. Managing the reptile patient in the veterinary hospital:  Establishing a standard of care model for nontraditional species. J Exotic Pet Med. 2010;19(1):56-72. doi:  10.1053/j.jepm.2010.01.015.

19. Muro J, Cuenca R, Pastor J, et al. Effects of lithium heparin and tripotassium EDTA on hematologic values of Hermann’s tortoises (Testudo hermanni). J Zoo Wildl Med. 1998;29(1):40-44.

20. Myers DA, Mitchell MA, Fleming G, et al. Determining the value of bovine albumin as a blood cell stabilizer for pancake tortoise, Malacochersus tornieri, blood smears. J Herp Med Surg. 2008;18(3–4):95–99. doi: 10.5818/1529-9651.18.3-4.95.

21. Nevarez J. Spotlight on anesthesia and analgesia in reptiles. LafeberVet website. Available at https://lafeber.com/vet/spotlight-anesthesia-analgesia-reptiles/. Accessed April 23, 2022.

21b. Nevarez J. Lizards. In: Mitchell MA, Tully TN (eds). Manual of Exotic Pet Practice. Saunders Elsevier; St. Louis; 2009:179-180.

22. Pagés T, Peinado VI, Viscor G. Seasonal changes in hematology and blood chemistry of the freshwater turtle Mauremys caspica leprosa. Comp Biochem Physiol A. 1992;103(2):275–278.

23. Pendl H. Avian and reptilian haematology Proc International Conference on Avian, Herpetological and Exotic Mammal Medicine. 2015:177-178.

24. Perpiñán D, Armstrong DL, Dórea F. Effect of anticoagulant and venipuncture site on hematology and serum chemistries of the spiny softshell turtle (Apalone spinifera). J Herpetological Med Surg. 2010;20(2-3):74-78. doi: 10.5818/1529-9651-20.2.74.

25. Proença LM. Blood sampling and intravenous access in exotic species. Proc International Conference on Avian, Herpetological and Exotic Mammal Medicine. 2015:122-126.

26. Saggese MD. Clinical approach to the anemic reptile. J Exotic Pet Med 2009;18(2):98-111. doi:  10.1053/j.jepm.2009.04.003.

27. Tappin S, Rizzo F, Dodkin S, et al. Measurement of ionized calcium in canine blood samples collected in prefilled and self-filled heparinized syringes using the i-STAT point-of-care analyzer. Vet Clin Pathol. 2008;37(1):66-72. doi: 10.1111/j.1939-165X.2008.00001.x. PMID: 18366547.

28. van Berkel M, Scharnhorst V. Electrolyte-balanced heparin in blood gas syringes can introduce a significant bias in the measurement of positively charged electrolytes. Clin Chem Lab Med. 2011;49(2):249-52. doi: 10.1515/CCLM.2011.047. Epub 2010 Dec 14. PMID: 21143021.
Further reading

de la Navarre BJS. Current diagnostic techniques and therapeutic techniques in reptiles and amphibians. Proc International Conference on Avian, Herpetological and Exotic Mammal Medicine 2015:73-74

de la Navarre BJ. Common procedures in reptiles and amphibians. Vet Clin North Am Exot Anim Pract. 2006;9(2):237-267, vi. doi: 10.1016/j.cvex.2006.04.002. PMID: 16759946.

Heatley JJ. Box turtle (Terrapene spp.) hematology. J Exotic Pet Med 2010;19(2):160-164. doi:  10.1053/j.jepm.2010.06.002.

Glassman AR, Gamblin KM, Zachariah TT. Comparison of biochemistry values from plasma and lymph in Krefft’s river turtles (Emydura macquarii krefftii). J Herp Med Surg 2022;32(1):63-72.  doi:  10.5818/JHMS-D-20-00017.

Lloyd M, Morris PJ. Phlebotomy techniques in crocodilians. Bulletin of the Association of Reptilian and Amphibian Veterinarians. 1999;9(3):12–14. doi: 10.5818/1076-3139.9.3.12.

Mader DR. Clinical pathology in reptiles: What do these results mean? Proc International Conference on Avian, Herpetological and Exotic Mammal Medicine. 2015:59-60.

Sykes JM 4th, Klaphake E. Reptile hematology. Vet Clin North Am Exot Anim Pract. 2015;18(1):63-82. doi: 10.1016/j.cvex.2014.09.011. PMID: 25421027.

To cite this page: Pollock CG, Perry S. General principles of reptile venipuncture. April 25, 2022. LafeberVet web site. Available at  https://lafeber.com/vet/gen-prin-video-or-slideshow/