Blood Collection in Lizards

Video

 

Video 1. This video was produced by Katie Lennox-Phillibeck and narrated by Dr. Angela Lennox. An embedded clip on jugular venipuncture was contributed by Dr. Marco Di Giuseppe.

The video script was written by Dr. Christal Pollock and reviewed by Drs. Byron de la Navarre, DVM, Alyssa Scagnelli, DVM, and Anneliese Strunk. More detailed information is available in the text below, as well as a brief video illustrating jugular transillumination and venipuncture in a chameleon shared by Dr. Charly Pignon.

 

Introduction

Hematology and biochemistry results are an important part of the minimum database for all veterinary patients, including lizards. Proper venipuncture technique is critical for accurate interpretation of laboratory results.

 

 

Equipment needed 

To collect the sample, you will need:^1,8,19,22

• Antiseptic (Fig 1)
• Glass slides and hematocrit tubes
• A 22-27-gauge needle attached to a 1 or 3-ml syringe (or insulin syringe). The length of the needle will vary with the size of the animal, and a long needle is often required to collect samples from large lizards.
• Anticoagulant tubes:  Many clinical practices routinely use lithium heparin for reptile hematology. However, research has identified species in which sodium ethylenediamine tetraacetic acid (EDTA) works or is even superior to heparin, such as the green iguana (Iguana iguana) and Chinese water dragon (Physignathus cocinus).^12,16
• A butterfly catheter can be useful to obtain samples from areas prone to movement, like the tail.
• Disposable gloves to minimize the risk of zoonotic disease

Visit “General Principles of Reptile Venipuncture” for advice on sample handling, use of anticoagulants, and recommended sample volume.

 

 

 

Handling and restraint

Two people are typically required for venipuncture in lizards. One person restrains the animal while the phlebotomist collects the sample. Sedation may be required for large or intractable patients.²¹ Ketamine, alfaxalone, or alpha-2 agonists, like dexmedetomidine, can be used to chemically restrain the lizard.²⁰

 

 

Venipuncture sites

Blood samples can be collected from a variety of sites in the lizard (Fig 2), including the axillary plexus, brachial vein, heart, cranial vena cava, jugular vein, orbital sinus, ventral abdominal vein, and ventral coccygeal vein (Fig 3, Fig 4) (Table 1).^{1–10,13,15,18,19,21} Samples are most frequently collected from the ventral coccygeal vein and the jugular vein.^1,13,18,19

 

 

 

Table 1. Venipuncture sites described in the lizard
Vessel	Landmarks	Advantages	Limitations	Comments	References
Axillary plexus	Near the shoulder joint at the caudal aspect of the humerus		Lymph dilution can occur1,15	Described as the brachial plexus by Brown (2007)1	1,8,15,21
Brachial vein			Often lymph diluted	Blind approach	2
Cardiocentesis	Cardiac position is highly variable in lizards and a Doppler flow monitor can be useful in small lizards	The sample is generally free of lymph	Generally considered an unacceptable route due to the potential for injury and hemorrhage since the lizard heart cannot be stabilized as it is in snakes1,6,8	Utilized in crested geckos* in a laboratory setting. The animals were monitored for over 1 year afterwards and “showed no ill effects”17	1,5,6,8,17,21
Cephalic vein					19
Cranial vena cava				This blind technique was used in leopard geckos** under isoflurane anesthesia 11 and in crested geckos* using manual restraint17	4,11,17
Jugular vein	Between the point of the shoulder and the tympanum or ear scale	Lymph contamination is often less likely A large volume of blood can be collected Collection time can be relatively fast	Often performed as a blind technique, which increases the risk of soft tissue injury	Transillumination has been used in chameleons10	1-3,6,7,10,1819
Orbital sinus					8,21
Ventral abdominal vein	Located on ventral midline within a suspensory ligament just beneath the linea alba		Risk of vessel and visceral lacerations with subsequent uncontrolled hemorrhage or intracoelomic contamination	Often a blind technique although a transilluminator can be useful in some small species Due to the risk of injury, the patient should be anesthetized	1,5,18,19,21,23
Ventral coccygeal vein	Ventral or lateral midline of the tail, approximately one-fourth the length of the tail caudal to the vent	Accessible in most lizards	Avoid or use caution in species with tail autotomy More difficult in smaller lizards Lymph contamination can occur Soft tissue injury, transient skin darkening can occur10 Risk of puncturing the hemipenes in male lizards	The most common venipuncture site in the lizard	1,3,4,12,14,15,16,19
*Crested gecko (Rhacodactylus ciliates) **Leopard gecko (Eublepharis macularius)

Ventral coccygeal vein

The ventral coccygeal vein or “tail vein” is the most commonly used vessel in lizards.^5,8,21 The tail vein extends along the ventral surface of the coccygeal vertebral bodies and is partially protected by the ventral spinous processes.¹

 

Potential complications

Care must be exercised in species with tail autotomy ^2,5, and some reptile veterinarians avoid this site all together in species, such as iguanids and geckos.⁶ If coccygeal venipuncture is utilized in a species with the ability to shed the tail, risk may be minimized by monitoring the tail closely and aborting the procedure if tail twitching is observed.⁴ If venipuncture is attempted too close to the vent there is also a risk of puncturing the hemipenes in male lizards.⁵ Lymphatic contamination may also occur.⁸

 

 

Venipuncture of the tail vein is a blind technique and the rate of failure to obtain a useful sample can be significant.^3,10,14 In a study comparing venipuncture sites in the common chameleon (Chamaeleo chamaeleon), only 67% (34/51) of venipuncture attempts from the ventral tail were successful.¹⁰ Even when a sample is obtained, difficulty during sample collection can lead to soft tissue damage and transient darkening of the skin.^3,10,14

 

Location

The recommended venipuncture site is approximately one-fourth the length of the tail caudal to the vent. Of course the best venipuncture site will vary by species and the ideal site can range between 20% to 80% down the tail.^5,8 The tail vein can be accessed either ventrally or laterally.²²

 

Equipment

Select a needle that is long enough to be able to reach the ventral vertebral processes.¹ When accessing the “tail vein” by the ventral approach, a 13 mm (0.5-in) needle will often suffice in smaller patients but a needle measuring at least 25-50 mm (1-1.5 in) will be required for most medium-sized to large lizards.¹ The lateral approach requires a shorter needle than the ventral approach.¹

 

Ventral approach

To perform the ventral approach:^{1,2,4,5,8,10,19,22}

• Restrain the lizard in dorsal recumbency.
• The phlebotomist uses their non-dominant hand to pin the hind legs beside the tail. The dominant hand is used to collect the blood (Fig 7, Fig 8).
• Aseptically prepare the venipuncture site.
• Insert the needle directly on ventral midline at a 45 to 60-degree angle between two ventral scales (Fig 9).
• Once the needle is through the scales, direct the needle at a 90-degree angle towards the tail OR in a craniodorsal direction.
• Maintain gentle negative pressure as the needle is advanced.
• Once blood appears in the needle hub, hold the needle steady while continuing to apply gentle negative pressure (Fig 10).
• If the needle reaches a ventral vertebral process without getting a flash of blood, then very slowly withdraw a few millimeters while maintaining negative pressure.
• It may be necessary to withdraw the needle and redirect slightly. If a flash is noted but stops, gently rotate the needle and syringe while applying very gentle negative pressure and moving the needle and syringe in tiny increments towards and away from the vertebrae until a better flow of blood is observed.

Some large lizards possess extremely tough, semi-calcified scales along the ventral tail surface that prevent easy insertion of a needle. Consider the lateral approach in these patients.¹

 

 

 

 

Lateral approach

In the lateral approach, the needle is inserted on the lateral midline and directed just beneath the lateral processes of the coccygeal vertebrae.^{1,2,4,5,8,10,22}  The lateral approach can be particularly useful for larger lizard species.⁸ Some individual patients also better tolerate this approach since they are allowed to remain in sternal recumbency, however, lymph dilution may be more likely (Fig 11).⁸

 

Step-by-step technique^1,8,22

• Restrain the lizard in sternal recumbency.
• The lateral midline can be identified by a longitudinal groove where the dorsal and ventral coccygeal muscle groups meet. This landmark can be difficult to identify in overweight lizards.
• Aseptically prepare the venipuncture site.
• Insert the needle at a 90-degree angle to the tail OR advance the needle at a 45 to 60-degree angle in a craniomedial direction (Fig 12, Fig 13).
• Maintain gentle negative pressure as the needle is advanced.
• If the needle contacts bone, slowly shift the needle ventrally in very small increments while maintaining negative pressure.

 

Jugular vein

Experienced reptile veterinarians also sometimes utilize the jugular vein.^{1–3,6,7,10,19} The vessel is sizable and relatively large volumes of blood can be collected with little lymphatic fluid dilution.⁶ In a survey of 51 samples collected from common chameleons, none of the samples appeared to be hemodiluted.¹⁰

Jugular venipuncture has historically been recommended in larger lizards, such as monitors and iguanas.^1,2,8 More recently, the technique has been employed in small lizards, such as agamids, chameleons, lacertids, and geckos, weighing less than 100 grams.^3,6,7

 

Anatomical landmarks

The jugular veins lie lateral and deep and are rarely visible even when caudally occluded by digital pressure.^1,2,8,19 To locate the jugular vein, draw an imaginary line between the point of the shoulder and the tympanum or ear scale.^1,8,18,19 The jugular vein lies caudal to the ramus of the mandible or the tympanum and courses caudoventrally toward the point of the shoulder (Fig 14-Fig 16). In chameleons, an extension of the jugular vein, called the casque vein, can also be accessed behind the casque as it courses vertically along the inside of this bony structure.⁹

 

Potential complications

Jugular venipuncture can pose technical challenges and when using the blind technique there is a risk of causing pain and soft tissue injury to major cervical anatomical structures, such as the carotid artery and vagus nerve.¹⁰ Hemodilution is less common with jugular venipuncture but it is also possible.

 

Blind technique

Jugular venipuncture is often a blind technique.^1–3,6,7,10

• Restrain the lizard in lateral recumbency with the head and neck slightly extended (Fig 17).
• Di Guiseppe (2017) recommends placing the index and middle fingers behind the head, in contact with mandibular branch.⁶
• The right jugular vein is usually larger than the left. Slightly rotate the head to the left to better expose the jugular vein.
• To locate the jugular vein, draw an imaginary line between the point of the shoulder and tympanum.
• Aseptically prepare the venipuncture site.
• Insert the needle behind the tympanum at an angle less than 45-degrees while applying gentle negative pressure .
• Direct the needle in a caudal direction along the imaginary line towards the point of the shoulder (Fig 18).

 

Transillumination

The transillumination method relies upon visualization of the jugular vein in a restrained chameleon by backlighting the cervical region (Video 2, Fig. 19).¹⁰ Any bright, concentrated light source can be used, such as a penlight, flashlight, or transilluminator.¹⁰ Directly viewing the vein using transillumination allows the angle of needle insertion to be adjusted as needed (Fig 20, Fig 21).¹ Since the jugular vein lies relatively close to lymphatics in the common chameleon (Chamaeleo chamaeleon), Eshar (2018) also recommends creating an approximate 45-degree bend in the needle to reduce the risk of hemodilution.¹⁰

Video 2. Jugular transillumination and venipuncture in a chameleon. Video shared by Dr. Charly Pignon.

 

 

 

 

The transillumination technique has been reported to provide more accurate venipuncture, increased sampling success, and reduced handling time, while minimizing soft tissue damage.¹⁰ In a survey of 40 chameleons, the transilluminated jugular venipuncture technique was successful in all animals.¹⁰ The smallest chameleon to be sampled from the jugular vein using this technique weighed only 13 grams.¹⁰

 

Ventral abdominal vein

Lizards possess a large ventral abdominal vein that lies within a suspensory ligament just beneath the linea alba.^1,5,8,9 The ventral abdominal vein is formed by the joining of the pelvic veins, hypogastric veins, as well as a single ventral pubic vein.^1,8,9 At the level of the umbilicus, the ventral abdominal vein begins to dive dorsally and joins with the hepatic vein.¹

 

Anatomical landmarks

The ventral abdominal vein is located on ventral midline between the umbilical scar and pelvic inlet.^5,18

 

Potential complications

The ventral abdominal vein is an extremely fragile vessel and there is the potential for intracoelomic hemorrhage if the vein is lacerated.^1,5,23 This hemorrhage can be uncontrolled since it is difficult to apply pressure to the vessel.^1,8 There is also the risk of visceral lacerations and contamination of the coelom if the gastrointestinal tract or bladder is punctured.^1,8,23

 

Technique

Venipuncture of the ventral abdominal vein is generally a blind technique, although a transilluminator can assist visualization of the vessel in some smaller species (Fig 23).¹ Due to the potential risks, the patient should be anesthetized.¹

• Aseptically prepare the venipuncture site.
• Approach the vessel in the midcoelomic region, at or just proximal to the umbilicus (Fig 22).^1,8
• Insert the needle at a 15 to 45-degree angle.¹
• Apply gentle negative pressure once the needle is inserted and advance the needle in a craniodorsal direction.^8,18

 

 

 

Summary

The venipuncture 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. Blood samples are most frequently collected from the ventral coccygeal vein and jugular vein in lizards.

 

Acknowledgements:  Thank you to Drs. Charles Innis and Stacey Leonatti Wilkinson for helpful feedback.

 

 

References and further reading