The majority of video clips incorporated into this recording were created and shared by Dr. Charly Pignon of the École Nationale Vétérinaire d’Alfort. The video illustrating brachial venipuncture was created by videographer, Katie Lennox-Phillibeck. View the recording or go to the text with still images below for more detailed information.
Successful venipuncture can be a challenge in turtles and tortoises (Fig 1), however hematology and biochemistry results serve as an important part of the minimum database in chelonians just as they do for all veterinary patients.
- Antiseptic – The venipuncture site should always be scrubbed thoroughly before sampling.
- Glass slides
- Hematocrit tubes
- Blood collection tubes with lithium heparin
- A 22-25-gauge needle attached to a 1 or 3-ml syringe (or insulin syringe) – Always select the largest gauge needle appropriate for your patient.
- A butterfly catheter (optional) can be useful to collect samples from areas prone to movement or in fractious animals.
- Wear disposable gloves to minimize the risk of zoonotic disease.
Although sodium ethylenediamine tetraacetic acid (EDTA) has been shown to be superior to lithium heparin for hematology in some reptiles 13, EDTA causes hemolysis in other species (Table 1).4,11,16,17,18,19,21 Therefore many practices routinely use heparin for hematology samples in all reptiles (Fig 2).7,11,18,19
|Table 1. Anticoagulant of choice in select chelonian species based on studies evaluating clotting and cellular morphology|
|Species||Sample size||Anticoagulant of choice||Comments||
|Eastern box turtle|
(Terrapene carolina carolina)
|Lithium heparin||EDTA caused frequent and significant hemolysis|
|Hermann’s tortoise |
Lithium heparin provided more optimal blood smears; thrombocyte and leukocyte aggregation was not observed.
EDTA did not appear to interfere with hemoglobin determinations
|Spiny softshell turtles|
|Lithium heparin||EDTA caused severe hemolysis|
|Yellow-blotched map turtles, (Graptemys flavimaculata)|
|EDTA: ethylenediamine tetraacetic acid|
Restraint and handling
Optimal patient positioning is important for successful venipuncture 22, and some chelonians may require sedation in order to exteriorize the legs, tail or head for blood sampling.19 Ketamine (2-5 mg/kg IM), alfaxalone (5-10 mg/kg IM), alpha-2 agonists, like dexmedetomidine (0.025-0.05 mg/kg IM), can be used to chemically restrain the patient.16
A high quality blood sample is needed for precise examination and diagnosis.1 Poor quality blood data can be useless or even misleading.22 The presence of blood clots, hemolysis, excessive anticoagulant, and hemodilution of the sample with lymph or extravascular fluid can all alter the diagnostic value of test results.1,5,6,12,17,19,21
The blood of some chelonians can clot very quickly, so obtain all blood samples as quickly as possible. Chelonian erythrocytes are also one of the largest of all vertebrates and the use of very small needles can lead to hemolysis (Fig 3).19 Select the largest needle size appropriate for your individual patient and always apply gentle negative pressure as too much suction increases the risk of damaging the large chelonian erythrocyte.19 To reduce the risk of lysing large, fragile chelonian erythrocytes, also be sure to remove the needle hub from the syringe before transferring blood to the blood collection tube or preparing the blood film (Fig 4).7,17,22
Visit “General Principles of Reptile Venipuncture” for additional advice on preparation, sample handling, and recommended sample volume.
Common venipuncture sites
Chelonian blood samples can be collected from a variety of sites 7, however blood samples are routinely collected from the jugular vein, subcarapacial vessel, dorsal coccygeal sinus, or brachial vein.16,17 The site 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.7,22 All venipuncture sites have advantages and limitations (Table 2). When comparing test results in an individual patient over time, it is prudent to consistently use the same venipuncture site. Comparing results taken from different venipuncture sites may offer little correlation or agreement.21
|Table 2. Advantages and disadvantages of common venipuncture sites in chelonians7,16,17,21,22|
|Brachial venous sinus||
|Dorsal venous sinus||
Well-developed lymphatics often run very close to blood vessels in turtles and tortoises, making lymph contamination and hemodilution a common complication.1,5,16,22 The jugular vein, and to a lesser degree the brachial vein, usually produce the least amount of lymphatic contamination resulting in a better quality sample that will presumably yield more accurate results.1,22 A blood sample that has been contaminated with lymph can appear pink, pale yellow, whitish, or even clear (Fig 5).16 When mixed with whole blood, lymph dramatically decreases the white cell count, hematocrit, and total solids.19 Lymph can also dilute plasma biochemistries, making it difficult, if not impossible, to accurately interpret test results.1,3,16 If a diluted blood sample or clear fluid is obtained, the procedure is generally aborted, the sample discarded, and a new sample collected, ideally from a different anatomic location.5,17
However, a recent study in Krefft’s river turtles (Emydura macquarii krefftii) (n=11) comparing plasma samples collected from the jugular vein and lymph samples collected from the dorsal lymphatic ring near the subcarapacial plexus has added further complexity to chelonian venipuncture.5 No statistically significant differences were found between lymph and plasma for chloride, glucose, alanine aminotransferase, aspartate aminotransferase, creatine kinase, urea nitrogen, and total bilirubin.5 Statistically significant differences were found between lymph and plasma for gamma-glutamyltransferase (GGT), total protein, globulin, and uric acid.5 Researchers concluded that severe lymph dilution (1:1 with plasma) can cause significant decreases in potassium, total protein, globulin, and LDH, and increases of GGT and uric acid in both sexes, as well as clinically significant decreases of calcium and triglycerides in female river turtles.5
The jugular vein is located on the lateral aspect of the neck. It courses superficially from the angle of the mandible in line with the dorsal edge of the tympanum or ear scale (Fig 6a, Fig 6b).7,15,16,20,21 In many individuals, the right jugular vein is larger.2,7 In some species, dorsal and ventral jugular veins appear to be present.2,15,20 The veins run directly along the length of the neck from the dorsal and ventral borders of the tympanum.2 The dorsal jugular vein is associated with the carotid artery, which lies deeper and parallel to the jugular vein.2,15,20 There can also be species variation with respect to vessel size. Some aquatic turtles (Emydidae, Geoemydidae) have small jugular veins that are difﬁcult to sample 20, while this vessel may be grossly visible in some tortoises before taking any measures to raise the vein (Fig 7).2
The jugular vein is considered the vessel of choice in chelonians as a relatively large volume of blood can be collected. The jugular veins also have few collateral lymphatic vessels and lymph dilution is rare.7,17,22 However this technique can be technically challenging in many chelonians, particularly when the patient is relatively large or strong. In many patients the vessel is also not easily observed. In thin chelonians, relatively thick, wrinkled and/or folded skin lies over the vessel and in overweight animals excess fat or muscle obscures the vessel.7 The skin in this area may also be highly pigmented, which can further hinder the phelebotomist’s ability to observe the vein (Fig 8).7
To collect the sample, an assistant restrains the chelonian in lateral recumbency while applying pressure to the thoracic or carapacial inlet or the lateral cervical neck area.7 It may also help if the assistant holds both of the forelimbs back.15,20 The phlebotomist extends the head and neck by placing a thumb and finger behind the back of the head (Fig 9).15,17 If the vessel cannot be visualized, it sometimes helps to position the head down and pull the skin of the neck taut to allow better visualization.22 Nevertheless, jugular venipuncture is often a blind technique in chelonians.7,22 Aseptically prepare the venipuncture site, then insert a needle very superficially into the vessel in a caudal-pointing direction that is parallel to the neck (Fig 10).15,17,20
Afterwards, apply firm, but gentle pressure (Fig 11). Adequate hemostasis is important. Hematoma formation is more likely if excessive compression or raising of the vessel was performed or if the underlying carotid artery was inadvertently entered.2,15
Brachial venous sinus
The brachial (ulnar) vein or brachial venous sinus is located behind the elbow joint, beneath the biceps brachii tendon of insertion (Fig 12).3,16,22 Lymph dilution is less likely to occur with brachial venipuncture when compared to the dorsal coccygeal sinus 12,24, but when compared to the jugular vein, only a relatively small volume of blood can be collected.7 For instance, approximately 0.10-0.25 ml of blood can typically be collected from the brachial venous sinus of a box turtle (Terrapene spp.).7 Brachial venipuncture is sometimes reserved for larger chelonians when it is not possible to access the jugular vein without chemical restraint.17,24 In a chelonian the size of a gopher tortoise (Gopherus polyphemus) (~4.5 kg) or desert tortoise (Gopherus agassizii) (~4 to 7 kg) enough blood can typically be collected to perform a complete blood count and chemistry panel (J. Nevarez, written communication).
Brachial venipuncture is a ”blind stick’’ but fortunately the technique is relatively easy to learn. To collect blood from the brachial vein, extend the front leg and palpate for the prominent biceps brachii tendon. Aseptically prepare the venipuncture site, then insert the needle at a 45 to 90-degree angle to the body caudoventral to the tendon (Fig 13).2,16 Apply gentle negative pressure immediately after penetrating the skin and slowly advance the needle towards the elbow joint.2,16
Subcarapacial vessel or venous sinus
The subcarapacial venous sinus or vessel, also known as the subvertebral sinus or dorsal subcarapacial cervical plexus, lies caudal to the central carapacial scute or nuchal scute and dorsal to the eighth cervical vertebra (Fig 14, Fig 15).1,2,7,8,16 The vessel is found at an anastomosis of the common intercostal veins and the caudal cervical branch of the external jugular veins.2,7,8
The subcarapacial vessel is another popular site for venipuncture in the chelonian. The technique is relatively easy for beginners to learn, requires minimal handling, and can be performed with the head and neck extended or retracted.17,22 In some patients, this vessel may be one of the few sites that is easily accessible since it is a great approach for shy species that tend to pull back into the shell.4,7,22
Unfortunately, large lymphatics sit cranial to the subcarapacial vessel and blood samples collected from this site may be heavily diluted by hemolymph.7,22 Additionally, the proximity of the vessel to the vertebral column means that there is the potential for spinal cord trauma and contamination of the sample with cerebrospinal fluid.4,9,10 Therefore the phlebotomist should exercise caution when utilizing this technique, particularly in fractious animals (Fig 16). Fortunately ensuring the head is retracted helps to minimize the risk of spinal injury as this allows flexion of vertebrae away from the needle insertion site (Fig 17). Forceps wrapped in bandage material can be offered to aggressive aquatic turtles. When the patient bites the forceps, the head can then be gently pushed back into the shell.2
To collect blood from the subcarapacial sinus or vessel, clean the skin thoroughly, then insert the needle over the cervical vertebrae, directly on midline where the skin of the neck meets the upper shell or carapace (Fig 18).17,22 A hypodermic needle can be used for most chelonians, however a spinal needle may be needed to reach the vessel in large animals, particularly Geochelone tortoises.2,22 The exact angle of insertion will vary with the shape of the carapace, but the needle is advanced along the ventral aspect of the carapace using mild negative pressure (Fig 19).15,16,22 Some clinicians find it helps to bend the needle up to 60 degrees.2,4,20
If the needle encounters a vertebra or the underside of the carapace, withdraw the needle slightly while maintaining gentle negative pressure and redirect cranially or caudally.15,17,22 Alternatively, the phlebotomist can use a shorter needle or abort the procedure.22
Dorsal coccygeal sinus
The coccygeal sinus or vein is typically found on the dorsal midline of the tail, although its location can vary by species to be dorsal, lateral, or even ventral.15,16,22 The dorsal coccygeal sinus is a popular site for venipuncture because it is accessible and the technique is relatively easy for beginners to learn.15,21 Nevertheless, like the subcarapacial vessel, this venipuncture site also carries the rare potential for spinal trauma because of its proximity to the spinal cord.10,20,22 Therefore the phlebotomist should exercise caution, particularly in fractious animals. The dorsal coccygeal sinus is also closely associated with lymphatic vessels and a poor quality, hemodiluted sample can be collected.12,16
To collect blood from the dorsal coccygeal sinus, either extend the tail or flex it ventrally (Fig 20).16,22 In large, strong tortoises or in female chelonians with very short tails, the vein can be sampled with the tail tucked inside the shell, although this is more challenging.2,20 The sinus is best approached at the tail base or at least as far cranial as possible.3 Aseptically prepare the venipuncture site, then slowly insert the needle on dorsal midline in a cranioventral direction at an angle between 45 to 90 degrees while applying gentle negative pressure (Fig 21).15,16,20,22
Other venipuncture sites
There are a variety of other venipuncture sites in chelonians, including the interdigital vessels, femoral vein, and occipital venous sinus.2,4,13,14,17,20,23 The occipital venous sinus arises from the right jugular vein and is located between the occipital protuberance and the cranial border of the carapace beneath the nuchal ligament.2,4,14
Although successful venipuncture can indeed be a challenge in turtles and tortoises, there are a variety of sites from which to choose. The venipuncture site selected can depend on a variety of factors, including the experience of the phlebotomist, the volume of blood needed, patient size and temperament, and of course the species involved. The novice chelonian phlebotomist may find the dorsal venous sinus or subcarapacial vessel relative easy to access, however hemodilution can be a significant concern with these locations. Jugular venipuncture is the most likely site to yield a high-quality sample, but it may also be the most challenging technique to perform depending on the size and strength of your individual patient. Comparison of test results made from samples taken from different venipuncture sites can unfortunately be unreliable. Therefore once the decision has been made to use a specific venipuncture site, make every effort to consistently use the same vessel in the future.
Acknowledgements: Thank you to Drs. Paul Gibbons, Charles Innis, and Stacey Leonatti Wilkinson for helpful feedback.
References and further reading
1. 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. PMID: 27146147.
2. Chitty J, Raftery A. Venipuncture and sample handling. In: Essentials of Tortoise Medicine and Surgery, 1st John Wiley & Sons; 2013:80-86.
3. 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.
4. Divers SJ. Diagnostic techniques and sample collection. In: Divers SJ, Stahl SJ (eds). Mader’s Reptile and Amphibian Medicine and Surgery, 3rd St. Louis, MO: Elsevier. 2019:1088-1090.
4b. Frye FL. Hematology as applied to clinical reptile medicine. In: Frye FL (ed). Reptile Care, Vol. 1. Neptune City: TFH Publishing; 1992:209–279
5. 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: 5818/JHMS-D-20-00017.
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. Heatley JJ. Box turtle (Terrapene) hematology. J Exotic Pet Med 2010;19(2):160-164. doi: 10.1053/j.jepm.2010.06.002.
8. Hernandez-Divers SM, Hernandez-Divers SJ, Wyneken J. Angiographic, anatomic, and clinical technique descriptions of a subcarapacial venipuncture site for chelonians. J Herp Med Surg 2002;12(2):32-37. doi: 5818/1529-9618.104.22.168.
9. Innis C, DeVoe R, Mylniczenko N, et al. A call for additional study of the safety of subcarapacial venipuncture in chelonians. Proc Annu Conf Assoc Reptilian and Amphibian Veterinarians 2010:8-10.
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