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Veterinarian Technician October 2008 (Vol 29, No 10)

Hematologic Evaluation Is Mainstay of Diagnosis for Reptiles

by Jenni Jenkins-Perez, LVT, CVT, BAS(VT)

    CETEST This course is approved for 0.5 CE credits

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    With the increasing popularity of reptiles as pets, they are becoming routine patients in more veterinary practices. Just as with cats and dogs, basic hematologic evaluation is a mainstay of diagnosis for reptiles, and the technician who can conduct these tests is becoming a vital part of daily operations in many clinics.

    As with any pet, reptiles should undergo annual checkups to safeguard their health and well-being. One of the basic parameters used in these evaluations is the complete blood count (CBC). Veterinary technicians should develop proficiency in reptilian hematology so they can assist the veterinarian in making a diagnosis.

    Blood Collection

    The size of reptiles varies from the tiny green anole to the formidable Nile crocodile and can weigh from 3 or 4 g to 700 kg. To conduct a valid CBC requires about 0.3 ml of blood. The blood volume in reptiles is approximately 5% to 8% of their body weight, and about 10% of the total blood volume from a healthy patient can be collected.1 For example, a snake weighing 100 g has a total blood volume of 5 to 8 ml. Ten percent of that volume can be safely drawn from the patient.4 So, the 100 g healthy snake can safely have approximately 0.5 to 0.8 ml of its blood drawn.

    The collection site should be disinfected with either alcohol or diluted chlorhexidine solution (3 oz chlorhexidine in 1 gallon of water). The choice of needle size and syringe depends on the size of the reptile as well as the blood vessel size. For smaller patients or if a slow draw is anticipated, the syringe can be heparinized with lithium heparin to prevent hemolysis or clotting. This can be accomplished by "rinsing" the hub of the needle and syringe with heparin and then drawing a small amount of anticoagulant and expelling it back into the bottle. Doing so reduces the chance of sample dilution while still allowing enough anticoagulant to prevent clotting. However, if cells are allowed to remain in the anticoagulant for too long, heparin can cause thrombocyte and leukocyte aggregation.2 Ethylene diamine tetraacetic acid (EDTA) anticoagulant should be chosen as a last resort because it causes hemolysis in certain species of reptiles, such as sea turtles and iguanas.3,4

    Snakes

    The collection site depends on the type and size of the snake and the availability of sedatives and anesthetics. For larger, unsedated snakes, the preferred collection site is the tail (ventral coccygeal) vein, with the needle inserted between the scutes (the underside of the scales). Because this vessel narrows as it descends from the cloaca, blood should be collected as close to the cloaca as possible. In male snakes, however, the insertion site must be far enough away from the cloaca to avoid puncturing the hemipenes or musk glands.4Holding smaller snakes in a vertical position with the head up facilitates collection by promoting "pooling" of the blood in the tail area. For larger snakes, only the tail portion can be comfortably held in a vertical position.

    Alternatively, blood can be collected from snakes through cardiac puncture. This works well for smaller snakes or if larger sample volumes are required. Brief sedation with sevoflurane is generally recommended because of the mobility of the heart.4 Some practices might choose isoflurane, but it takes longer for the sedation to take hold in reptiles. The heart is located about one-third of the way down the snake's body. It is best to restrain the snake in dorsal recumbency to visualize the heartbeats on the ventral side. Snakes with darker pigmentation may require the use of Doppler ultrasonography to locate the heart. Once it has been located, the heart can be marked with a piece of tape or a surgical marker. The heart should then be held firmly in position with one hand or held by an assistant and the site swabbed with alcohol or diluted chlorhexidine. The needle should then be inserted with the other hand into the heart ventrally between the scutes. If the needle is not placed into the heart on the first attempt, remove it completely before redirecting to prevent dire consequences.

    Turtles and Tortoises

    For turtles and tortoises, there are five locations for phlebotomy: the occipital sinus, jugular vein, dorsal coccygeal venous sinus, subvertebral venous sinus, and brachial vein. The occipital sinus, the site preferred in aquatic turtles, is located dorsolaterally to the cervical vertebrae on the right side of the neck and caudally to the base of the skull. The head is held firmly, and the needle is inserted at a 30° angle. Pulling the head outward and slightly downward helps to visualize the base of the skull and the spinous process.

    The jugular vein can be used in aquatic turtles for larger sample requirements. Access may be hindered by the strength of the neck so that sedation with a gas anesthetic may be required. The needle and syringe should be placed parallel to the vein, with the needle inserted in the direction of the venous blood flow, which is away from the head.

    Although the dorsal coccygeal sinus is an alternative collection site, this small vessel is not ideal for large-volume sampling (i.e., 1 ml or more). It is easily accessible with a 25-gauge or tuberculin needle and does not require sedation. The turtle can be placed on a surface with a smaller diameter than the ventral shell; then the tail is pulled out and held firmly with one hand while the blood is collected using the other hand.

    The subvertebral sinus (also known as the subcarapacial sinus) is another location commonly used for collection. It is considered a sinus because of the grouping of several blood vessels in this location. It is located at the level of the eighth cervical vertebra, just caudal to the nuchal scute.4 It should be used for collection only as a last resort because of the risk for sample contamination from the lymphatic vessels.5 The advantage of using this site is that blood can be drawn whether the head is extended or retracted, meaning that no sedation is required.

    Lizards

    In larger lizard and crocodilian species, blood can be collected from the ventral tail vein with minimal restraint . The occipital sinus is commonly used for crocodilian species. If necessary, cotton balls can be placed over the eyes and secured with an elastic bandage around the head. This restraint technique calms the patient without the use of chemical restraint. This technique also can be used for other procedures, such as radiology and the physical examination. The vein, which lies just beneath the vertebrae, can be accessed laterally or ventrally. For the ventral approach, the tail can be held horizontally and draped off the end of the examination table. The needle is then inserted perpendicular to the vein between the scales and distal enough from the cloaca in male animals to avoid the hemipenes. Some prefer the lateral approach. The needle is inserted midway between the dorsal and ventral side of the tail, where there is often a natural groove or midline to serve as a landmark. Caution must be observed when collecting from the tail of larger lizards, as they often use the tail as a defense mechanism and can injure the phlebotomist.

    The central abdominal vein can be used for blood collection in smaller species and can be visualized as a dark line along the midline of the ventral abdomen in lizards with lighter pigmentation (e.g., leopard geckos, bearded dragons). This site has disadvantages in that the vein is easily lacerated, and there is a risk for hematoma formation because pressure cannot be easily applied after venipuncture. Using cotton-tipped applicators may help to focus pressure directly on the puncture point.

    Smear Preparation and Staining

    Blood smears should be made immediately after blood collection (before any hemoparasites can leave the blood cells)6, or before placing the blood into anticoagulant. Regardless of the technique used, a thin monolayer is needed for proper evaluation.

    The common 30° angle or "push" smear technique, which works well for mammals, may cause reptilian blood cells to rupture.7 The coverslip method avoids this problem but uses a smaller drop of blood, representing a smaller portion of the circulating blood than the 30° angle method. In addition, the coverslip may be difficult to stain if a Coplin jar is not used for dipping. Finally, the coverslip technique requires permanent mounting solution; some of these solutions are extremely caustic, necessitating adequate ventilation and protective equipment.

    The parallel (slide-to-slide) smearing technique uses a larger drop of blood and works well for all nonmammalian species. This technique requires practice, but once it has been perfected, there is minimal distortion or rupturing of the cells. The blood is placed in the center of a slide with another slide on top, and the slides are gently pulled apart. The cells are evaluated from the center of the smear rather than from the feathered edge as with the 30° angle technique.7

    Two types of stains can be used with little time or labor. Quik-Dip stains work well, but some brands do not adequately stain erythrocyte nuclei. They also do not stain the granules of basophils; the basophils are visible, but the granules may appear "ghost-like" or the cells take on a "spider-web" appearance. These cells are often mistaken for smudged cells.8 By contrast, Wright's-Giemsa stain takes longer than the Quik-Dip methods, but all of the cells stain well. Although stain must be filtered regularly because of the formation of precipitate, it is the stain of choice.

    Methods of Analysis

    Phloxine B Method— Because reptiles have nucleated erythrocytes, accurate results cannot be obtained from commercially available automated cell counters. Therefore, CBCs must be done manually. The Eosinophil Unopette #5877 test used historically and referenced in many publications is no longer available. The solution can be made in-house or can be ordered through Vetlab, which now makes a Unopette substitute called the Leukopet (Vetlab Supply).

    This is an indirect counting method, so a differential analysis must be done to obtain the white blood cell count (WBC). Furthermore, a red blood cell (RBC) count cannot be obtained using this method. Finally, studies have shown that there are discrepancies in WBC results for reptiles compared with other manual direct WBC methods, so this is not the recommended technique for conducting a WBC.9

    Natt-Herrick Method —A better method uses the Natt-Herrick's (NH) solution, which requires only 20 µl of blood (compared with 25 µl of blood for the phloxine B solution). It also accommodates both WBC and RBC counts. Most important, this is a direct counting method2 (Table 1). One disadvantage is that the blood cells tend to aggregate in the hemacytometer chamber. This can be avoided by charging the hemacytometer immediately after mixing the blood and stain.2 It may be difficult to distinguish lymphocytes from thrombocytes when counting; the ability to differentiate these cells comes with experience, but thrombocytes tend to stain lighter purple and may have a "fluffy" appearance, whereas lymphocytes tend to be round and stain darker purple. NH solution is now available commercially from ENG Scientific.

    Estimated WBC Method —Another method of counting is the estimated WBC count4,12-15 (Table 2). This method is only recommended if the sample size is insufficient for testing by any other means. Many factors can render the estimated WBC count inaccurate. Most important, the blood smear must be of the highest quality (i.e., good monolayer, no clots, and few smudged cells). Accurate counts can be obtained only if the sample is not diluted by anticoagulant. This method works primarily for establishing a "ballpark" high or low WBC count. It is not recommended for establishing normal values for a species.4

    Differential Evaluation

    After the blood smears have been stained and calculations completed, the differential evaluation can begin. If evaluating the species for the first time, it is always best to scan the slide and try to identify the cell types before obtaining the differential count. The morphology of reptilian blood cells can be quite challenging, with great variation among species. In addition, different reference texts classify the cell types differently. Table 3 lists normal hematologic values for common reptile patients.4,16

    The erythrocytes are round-to-oval, with a nucleus that stains dark purple. The nuclear chromatin pattern also becomes denser, and the cytoplasm becomes less basophilic as the cell ages.4 Reptiles occasionally have immature polychromatic erythrocytes in the peripheral blood that may be mistaken for azurophils or monocytes. These cells also will be rounder and more basophilic than more mature erythrocytes.

    Thrombocytes typically appear as small cells that are oval to spindle-shaped, with a central, dark purple nucleus. In some snake species, they almost have an "airplane propeller" appearance. Thrombocytes are often confused with lymphocytes in that they are generally the same size. However, thrombocytes may be identified in small clusters on the slide, and their cytoplasm tends to be colorless to pale blue. Activated thrombocytes tend to be clustered, with decreased cytoplasm, vacuoles, and irregular cytoplasmic borders.4

    Reptile lymphocytes are nongranulocytes and may be the predominant WBC in green iguanas. Mature lymphocytes are round and have an eccentric nucleus with a chromatin pattern that is dark, clumped, and dense. The cytoplasm is pale blue but darker than that of the thrombocytes. The lymphocytes also have a larger nuclear-to-cytoplasmic ratio, so that the nucleus takes up most of the cell.7 Both lymphocytes and thrombocytes may have an occasional azurophilic granule.

    Monocytes are another type of nongranulocytic cell found in reptiles. They tend to be the largest WBC in the peripheral blood smear. The nuclear shape is usually lobed but may sometimes appear round. The nuclear chromatin pattern is lighter and more lacy than that of the lymphocytes. The cytoplasm is typically a battleship gray and may or may not have vacuoles or a foamy appearance.4,7

    Azurophils are found only in reptiles, and there is much debate as to their classification; they are counted as separate cells in the differential evaluation. They resemble the reptilian monocyte in many ways but differ in that they are irregularly shaped and often smaller. The nucleus is round-to-oval with a coarse chromatin pattern and may occasionally have two lobes (bilobate).17 The cytoplasm is darker blue than that of the monocytes and contains very fine azurophilic granules. They may or may not have vacuoles.

    Reptiles may occasionally have plasma cells in their blood. The nucleus is round and eccentric and has a very clumped and coarse chromatin pattern. Plasma cells are similar to azurophils in size and may be mistaken for them. Unlike azurophils, however, plasma cells have dark blue staining in the cytoplasm and do not contain granules. They also have a "halo," called the Golgi apparatus, surrounding the nucleus.4

    As with mammalian cell types, nonmammalian WBCs can be divided morphologically into granulocytes and nongranulocytes (i.e., mononuclear cells).4 Reptilian granulocytes are predominantly heterophils. The granules are usually refractile, appearing bright orange to brick red. The granules can be round, rod-shaped, or spindle-shaped, depending on the species.4 Their nuclei are eccentric and vary from round to lobed, with a dense chromatin pattern. The cytoplasm is usually colorless and contains granules.

    The eosinophil, another granulocyte, has a round-to-oval nucleus that is slightly eccentric. Some lizard species have eosinophils with lobed nuclei. The chromatin pattern is usually dense. They can be distinguished from heterophils in that their cytoplasm is light blue, and most of the granules within the cytoplasm tend to be round. The granule color will vary among species17; for example, in green iguanas, the granules are blue, whereas in some tortoises (e.g., the gopher tortoise), the granules are bright orange (similar to heterophils). In sea turtles (e.g., the loggerhead), the granules are brick red and sparse, and the cells may appear to be partially degranulated heterophils.

    Basophils are generally the easiest granulocyte to identify. They have the typical round shape and are smaller than heterophils and eosinophils. The nucleus is generally round, with a dense chromatin pattern. It is often difficult to see the nucleus because of the abundance of granules in the cytoplasm. Some basophils are easily distorted on a smear, clearly revealing the nucleus. The granules stain very dark purple and may appear as smudged cells. Again, basophil granules do not stain with some of the Quik-Dip methods; if this is the only stain available, fixing the slide with methyl alcohol for 1 minute before staining and allowing the slide to dry may help prevent the granules from dissolving.4 By contrast, Wright's-Giemsa stain delineates the granules clearly.

    Conclusion

    The veterinary technician's ability to conduct in-house hematologic diagnostic procedures can be vital to the practice. Most veterinary practices can treat the reptile patient but must send samples to outside laboratories and then wait for the results. The veterinary technician who can conduct in-house blood tests for a critically ill reptile patient obtains results in minutes, benefiting the patient and practice as an indispensable asset to the team.

    Acknowledgment

    The author would like to thank Judy Lethbridge for her assistance with the photography and Neil Sweetman for providing the animal "models."

    1. Jacobson E. Blood collection techniques in reptiles: laboratory investigations. In: Fowler M (ed). Zoo and Wild Animal Medicine, Current Therapy, ed 3. Philadelphia: WB Saunders; 1993:144.

    2. Arnold J. Natt-Herrick cell count method. In: National Aquarium Laboratory Procedure Manual. Baltimore, Md.: National Aquarium; 2002:1-7.

    3. Jenkins J. Basic amphibian hematology: a study of collection, preparation and identification techniques. Proceedings of the Association of Zoo Veterinary Technicians Meeting; 1997:13-23.

    4. Campbell T, Ellis C. Avian and Exotic Animal Hematology and Cytology, ed 3. Ames, Iowa: Blackwell; 2007:51-81.

    5. Barrows M, McArthur S, Wilkinson R. Anatomy and physiology (chapter 3), clinical pathology (chapter 7). In: McArthur R, Wilkinson R, Meyer J (eds). Medicine and Surgery of Tortoises and Turtles. Malden, Ma.: Blackwell; 2004.

    6. Neely E. Avian hematozoa. Proceeding of the Association of Zoo Veterinary Technicians Meeting; 1986:27-44.

    7. Jenkins J. Basic avian hematology: techniques of collection, preparation, and identification. In: Exotic Animals, A Veterinary Handbook: A Collection of Articles From Veterinary Technician. Yardley, Pa.: Veterinary Learning Systems; 1995:126-130, 135.

    8. Shields-Mayer C. Comparisons of stains in avian hematology. Proceedings of the Association of Zoo Veterinary Technicians Meeting; 1991:101-106.

    9. Arnold J, Gargan C, Belovarac J. Method validation study for total white blood cell counts: Natt-Herrick versus eosinophil Unopette methods. Proceedings of the 27th Association of Zoo Veterinary Technicians Meeting; 2007.

    10. Wilmoth K. Utilizing toluidine blue stain for direct avian and reptilian blood counts [reprint]. Association of Zoo Veterinary Technicians Newsletter; March 1991.

    11. Pond J. Lincoln Park Zoo, Chicago, Il. Personal communication.

    12. Atinoff N. Avian laboratory diagnostics. Veterinary Laboratory Association Annual Meeting, 2004. Reprinted on the VLA website November 07, 2005; www.vetlabassoc.com/display.php?id=7

    13. Morrisey J. Avian hematology, Lecture from Minnesota Veterinary Medical Association Proceedings, Avian and Reptilian Medicine and Surgery; 2005; www.mvma.org/proceedings/

    14. Feldman B, Jain N, Schalm Stein C, Zinkl J. Normal hematology of reptiles. In: Weiss D, Wardrop K (eds). Schalm's Veterinary Hematology, ed 5. Ames, Iowa: Blackwell; 2000:1126-1132.

    15. Campbell T. Avian Hematology and Cytology, ed 2. Ames, Iowa: Iowa State University Press; 1995:11.

    16. Johnson-Delaney C, Harrison L (eds). Exotic Companion Medicine Handbook for Veterinarians. Lake Worth, Fla.: Wingers Publishing; 1996:Reptile Section 2-3.

    17. Ballard B, Cheek R. Exotic Animal Medicine for the Veterinary Technician. Ames, Iowa: Iowa State University Press; 2003:307-312.

    References »

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