Sea Turtle Venipucture and Leukocyte Morphology

Jeanne M. Harding, DVM; Fernando Torres-Velez, DVM; Kenneth S. Latimer, DVM, PhD; Heather L, Tarpley, DVM; Bruce E. LeRoy, DVM, PhD

Class 2005 (Harding) and Department of Pathology (Torres-Velez, Latimer, Tarpley, LeRoy), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Introduction

Hematological evaluation of leukocytes or white blood cells is important in evaluating the health of an individual. Information such as the presence of leukocytosis or leukopenia can be further qualified based on the percentage of the different leukocytes present. This information helps the clinician in determining the potential cause of the abnormality and appropriate treatment options. Data from overtly healthy individuals is compiled to establish "normal" reference intervals for a particular species. Reference intervals are continually updated and modified, especially for species where the data is scant. In wild animals, reference intervals can vary depending on the season and natural lifecycles (i.e., hibernation).

Data collected from an individual is compared to the reference interval to determine if disease is present. Automated cell counters that count nucleated cells are convenient and accurate (inherent error of approxiamately 5%) ¹ and can be used for mammalian species. Since avian and repitilian erythrocytes are nucleated, manual counts must be performed using a Unopette® system, a hemocytometer, and a microscope (inherent error 20%).¹ A blood smear using blood that has not been in contact with an anticoagulant, should be made immediately after collection to preserve cell morphology. Once the smear is made, the remaining blood should be placed in a tube containing lithium heparin as the anticoagulant, since the use of EDTA can lead to hemolysis.^2,6

Venipuncture Techniques

Before performing venipuncture, the skin above the collection site should be aseptically prepared to prevent introduction of contaminants to the turtle or the blood sample.

A common site for blood collection in chelonians is the dorsal cervical sinus. The sinuses are found bilaterally just lateral to the cervical vertebrae and cranial to the carapace. The turtle should be restrained and the neck outstretched at a lower plane from the carapace to allow the sinuses to fill with blood.^3,7 With the neck outstretched, the needle should be inserted perpendicular to the skin and on the outside edge of the tendons that run parallel to the vertebral column.^3,7 (Fig. 1)

Figure 1. Venipuncture technique from the dorsal cervical sinus of Green Sea Turtle

For larger species, such as the Leatherback, the hind flipper can be used while the female is laying eggs. During oviposition, the female enters a trance and remains relatively still for approximately 10 minutes. The collection site is located by palpation of the popliteal vein. It is found approximately 5 cm from the edge of the carapace and 1 cm medial to the tibia.³ (Fig. 2).

Figure 2. The hind flipper is a practical venipuncture site in large sea turtles, such as this Leatherback.

Leukocyte Morphology

Direct examination of a stained blood smear is required to examine the white blood cells from an individual. A dried blood smear should be stained with a Romanowsky stain, such as Wright’s stain. To perform a differential, 100 white blood cells are counted and the type of cell is recorded to determine the relative percentage of each cell.

Granulocytes

Granulocytes include the leukocytes that contain specific granules that identifies the cell lineage. These cells include heterophils, eosinophils, and basophils. In mammals, the nuclear lobulation is distinct, but this is not a prominent feature of turtle granulocytes

Heterophils - The heterophil is the most abundant leukocyte found in health. It is the equivalent of a mammalian neutrophil. Heterophils (10 to 23 µm) contain large, eosinophilic, ovoid, cytoplasmic granules (Fig. 3).^2,6,7 The cytoplasm, which can be difficult to visualize, is light blue or clear.^2,7 The nucleus is frequently displaced toward the edge of the cell. It is basophilic with dense chromatin.² The reference interval can vary between 47 – 70%, depending on the species.^2,4,5,6,7

Figure 3. Heterophils containing numerous, red granules (green sea turtle (left) and hawksbill turtle (right); Wright stain).

Eosinophils - The eosinophil (9 to 20 µm) is distinguished by its round, eosinophilic cytoplasmic granules which do not fill the colorless cytoplasm (Fig. 4).^2,7 The nucleus contains coarse, clumped chromatin and stains red.^2,7 It is round to oval, single or bi-lobed, and eccentrically placed within the cytoplasm. The reference range for eosinophils is approximately 6-20%, depending on the species and age. ^2,4,5,6,7

Figure 4. Eosinophil from a hawksbill turtle containing an oval nucleus and large, round, red, cytoplasmic granules (Wright stain).

Basophils - The basophil (7 to 20µm) has pale blue cytoplasm with scattered dark metachromatic (purple) to dark gray granules(Fig. 5).^2,6,7 The nuclear chromatin is densely arranged and stains eosinophilic.² Basophils are the least encountered leukocyte in sea turtles.⁶ The reference interval is 0 – 0.5%.⁶

Figure 5. Basophil from a hawksbill turtle with dark purple-gray cytoplasmic granules (Wright stain).

Mononuclear Cells

The mononuclear cells include lymphocytes and monocytes. These cells generally lack significant lobulation of the nucleus and do not contain an abundance of cytoplasmic granules.

Lymphocytes - The lymphocytes ( 5.5 to 14.5 µm) are usually the second – most prolific and often surpass the percentage heterophils, especially in the summer.^6,7 They have a relatively large, round, eosinophilic, coarsely granular nucleus with a very small amount of blue cytoplasm (Fig. 6).^2,6,7 The reference interval varies between 20 to > 50%.^2,4,5,6,7

Figure 6. Lymphocyte from a green sea turtle. The nucleus is round and slightly indented. A thin rim of light blue cytoplasm is apparent (Wright stain).

Monocytes - The monocyte (8 to 20 µm) contains a large amount of light blue-gray, finely granular or vacuolated cytoplasm, and a oval or indented nucleus with a dense chromatin pattern that stains eosinophilic (Fig. 7).^2,6,7 The reference interval is 0 – 13%.^2,4,5,6,7

Figure 7. Monocytes from a hawksbill turtle. The nucleus is round to oval with moderately granular chromatin. The cytoplasm may range from blue to gray (Wright stain).

References

1. Latimer KS, Mahaffey EA, Prasse KW (eds): Duncan and Prasse’s Veterinary Laboratory Medicine, Clinical Pathology, 4th ed. Iowa State Press / Blackwell Publishing Co., Ames, 2003, pp 57-58.

2. Bradley TA, Norton TM, Latimer KS: Hemogram values, morphological characteristics of blood cells and morphometric study of loggerhead sea turtles, Caretta Caretta, in the first year of life. Proc ARAV 8(3): 8-12, 1998.

3. Dutton P. Methods for collection and preservation of samples for sea turtle genetic studies. Proc Internatl Symp Sea Turtle Conservation Genetics, Miami, FL. Pp. 17-22, 1995.

4. Cray C, et al. Altered in vitro immune responses in green turtles (Chelonia Mydas) with fibropapillomatosis. J Zoo Wildlife Med. 32(4):436-440, 2000.

5. Christopher MM, et al. Reference intervals and physiologic alterations in hematologic and biochemical values of free-ranging desert tortoises in the Mojave Desert. J Wildlife Diseases. 35(2):212-238, 1999.

6. Fudge A (ed): Laboratory Medicine: Avian and Exotic Pets. W B Saunders, Philadelphia, 2000.

7. Personal observations, Summer 2003, Fajardo and Culebra, Puerto Rico

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