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Box Turtle (Terrapene spp.) Hematology
Topics in Medicine and Surgery
Box Turtle (Terrapene spp.) Hematology J. Jill Heatley, DVM, MS, Dip. ABVP (Avian), Dip. ACZM, and Karen E. Russell, DVM, PhD, Dip. ACVP
Abstract Box turtles (Terrapene spp.) maintained as pet animals are commonly presented to the veterinary clinic for health care. A complete blood count (CBC) is recommended for box turtle patients as part of the annual physical examination, when they are ill, and before owner-induced brumation. Box turtles from the wild also are commonly presented for injuries and illness, including vehicular trauma, bot fly infestation, and predator attack. The initial health assessment of a wild turtle patient may be enhanced by evaluating the results of a CBC. This article will review venipuncture sites and hematologic techniques for obtaining blood samples to submit for a CBC, as well as give comments on interpreting CBC results in box turtles based on author experience and literature review. Hopefully, this article will promote further exploration and publication relevant to the evaluation of box turtle hematology. Copyright 2010 Elsevier Inc. All rights reserved. Key words: blood; box turtle; hematology; Terrapene spp.; venipuncture
A
lthough successfully collecting blood from most box turtles (Terrapene spp.) is challenging, there are many venipuncture sites to choose from when making an attempt. Patient temperament, size, and body temperature all affect the phlebotomist’s ability to collect blood from box turtles. Anatomical sites for venipuncture in these small shy turtles include (listed from cranial to caudal): postoccipital venous plexus, jugular vein, subcarapacial sinus, brachial vein, ulnar (radiohumeral) venous sinus, and dorsal coccygeal vein.1,2 Although all of the sites listed above may be subject to lymphatic hemodilution, the jugular vein is considered best to reduce contamination of a blood sample by this artifact. The jugular vein, subcarapacial sinus, and brachial vein are considered the best sites for blood collection from a box turtle patient based on author preference; thus, only approaches for these preferred sites are detailed in this article.
Jugular Vein The jugular vein is visible in some box turtle patients; however, in the majority of animals, it may be difficult
160
to identify because of a relatively thick, wrinkled, and/or folded skin in the lateral cervical region. The skin in this area may also be highly pigmented, which might also hinder the clinician’s ability to observe the vein. To access this vessel, which is preferred by many veterinarians based on a reduced chance of lymphatic contamination of the sample, chemical restraint may be necessary for proper patient positioning and blood collection. The right jugular vein may be larger than the left in some species. The application of pressure to From the Department of Small Animal Clinical Sciences, Zoological Medicine and Surgery Service, Texas A&M University, Veterinary Medical Teaching Hospital, College Station, TX USA, and the Department of Pathobiology, Texas A&M University, Veterinary Medical Teaching Hospital, College Station, TX USA. Address correspondence to: J. Jill Heatley, DVM, MS, Dip. ABVP (Avian), Dip. ACZM, Texas A&M University, Veterinary Medical Center, College Station, TX 77843-4474. E-mail: [email protected]. © 2010 Elsevier Inc. All rights reserved. 1557-5063/10/1902-$30.00 doi:10.1053/j.jepm.2010.06.002
Journal of Exotic Pet Medicine, Vol 19, No 2 (April), 2010: pp 160 –164
161
Box Turtle (Terrapene spp.) Hematology
the lateral cervical neck area and/or the carapacial inlet on the side from which the blood will be collected may aid in visualization of this vessel, which courses superficially from the angle of the mandible, in line with the dorsal edge of the tympanum. The general course of the jugular vein in box turtles is approximately the ten and two o’clock positions, relative to the carapacial inlet.
Subcarapacial Sinus The subcarapacial sinus is located caudal to the nuchal scute at the level of the eighth cervical vertebra and is the intersection of the internal jugular vein, the anterior pulmonic vein and artery, vertebral veins and arteries, subclavian veins and arteries, and lymphatic vessels. This site may be subject to marked hemolymphatic dilution. This approach may be used in shy species that tend to retract into the shell. With head and neck retracted, the needle is angled over the head along the midline and dorsally toward the ventral aspect of the carapace. On bone contact, withdraw and slightly aspirate. Because large lymphatic vessels are cranial to the subcarapacial sinus, additional sampling attempts should be made caudal to the prior efforts
Ulnar (Radiohumeral) Venous Sinus In box turtles, the ulnar (radiohumeral) venous sinus will generally yield a small sample volume of blood (0.10-0.25 mL) and is the location most prone to lymphatic contamination. To collect blood from this site, the front leg is extended and the needle is inserted at an angle perpendicular to the body behind the tendon caudal to the radiohumeral joint. Advance the needle toward the radiohumeral joint during syringe aspiration. Choice of the venipuncture site can cause variability in the complete blood count (CBC) results based on lymphatic contamination, extravascular fluid, or serum. The most often encountered incorrect CBC parameter due to artifact effect (hemodilution) is a decreased packed cell volume. Artifact effect to the total and differential white blood cell count (WBC) has not been investigated in box turtle species. Samples from the jugular vein or carotid artery are considered the least likely to be hemodiluted.
Venipuncture and Blood Preparation Techniques The choice of needle and syringe for blood collection in box turtle species is complicated by their
relatively large red blood cell size, small patient size, and relatively low venous refill time. A 25-gauge needle is the authors’ preference, but a 23-gauge needle may be less likely to promote cell lysis when blood is collected. Chelonian blood is relatively slow to clot; therefore, one should avoid excessive aspiration pressure because in most cases there is a slow flow of blood into the syringe. A 1-mL syringe generally gives adequate aspiration and maneuverability to obtain adequate sample volumes from adult box turtles. Heparin is generally regarded as the anticoagulant of choice for most but not all chelonians. Heparin appears to be acceptable for box turtle species based on the authors’ experience. The use of a heparinized syringe to collect blood from a chelonian would be tempered by many drawbacks to sample quality such as sample dilution, increased sodium or potassium in the sample, the addition of color artifact to blood smears, and the effect of heparin on leukocyte morphology. The author prefers using a nonheparinized syringe and placing the sample in a lithium heparin microtainer. These tubes contain a dry form of heparin that avoids dilution artifact. Blood smears should be made without any anticoagulant (from the syringe) and air dried to avoid artifactual effect. Fresh blood smears may be made by the cover-slip-to-cover-slip, coverslip-to-slide, or slide-to-slide techniques; the choice should be based on operator comfort and familiarity to produce the fewest damaged cells and best smears that one can evaluate. Because the WBC is determined from the blood smear slides, preparation of blood smears is the most important aspect to consider for quality control. A poor blood smear may produce results that are not representative of the patient’s true blood parameters at the time the sample was collected. If blood smear slides are being forwarded to a reference laboratory, no further fixation is necessary. However, if slides are being stained “in house,” one should be aware of the technical difficulties associated with “self preparation” of blood smears. For example, Diff-Quik stain may result in the degranulation of basophils, which can result in cell misidentification and an incorrect WBC differential count. Standard staining with modified Wright stains are acceptable to prepare smears for evaluation from box turtle patients.
Complete Blood Count The singular best hematological reference range for box turtles does not mention site of venipuncture, species, sex, or season.3 However, the reasonable
162
Heatley and Russell
ranges reported suggest many box turtle species’ normal hematology results may be comparable (Table 1). An excellent pictorial reference for the granulocytes is also available in the same reference source.
Clinical Hemogram Interpretation There are many potential sources for variability in the box turtle hemogram, including those caused by brumation/hibernation, season (and likely patient temperature), signalment of the patient (species, sex, age), venipuncture site, technique, and laboratory performing the analysis. In torpor, for example, hemoconcentration results in an increased hematocrit, hemoglobin concentration, and erythrocyte size and count. The following is a list of potential factors that may influence box turtle hemogram results and should be considered when interpreting a sample: 1)
Intrinsic: species, sex, age, physiologic status, and
2)
Extrinsic: season, temperature, habitat, diet, disease, stress of captivity, venipuncture site
There are a few published case reports of box turtles in which the hemogram was reported.4-6 A single reference has previously presented images of box turtle blood cells, although only the granulocytes.7 In many of the published clinical case reports it was concluded that the CBCs were not very useful when formulating the disease diagnosis or prognosis; however, the authors suggest that our understanding of box turtle hematology is in its infancy, and, as veterinary medical knowledge increases, the capability of this diagnostic tool will improve. The images found in Appendix A are presented to facilitate the veterinary practitioners’ ability to perform a WBC differential count for box turtle patients in their own clinic. This diagnostic tool may prove increasingly useful when cell types are more clearly defined for these species and the WBC differential count can be performed rapidly when using only a single drop of blood.
References 1.
2. 3.
Table 1. Turtle reference ranges n ⴝ 1043 Parameter
Units
Mean
Range
White blood cell Cells/dL 10,800 8000-13,000 count Packed cell volume % 31.6 27-38 Heterophils % 44.3 21-69 Lymphocytes % 45.7 32-63 Monocytes % 1.06 1-3 Eosinophils % 0.19 0-1 Basophils % 1.92 0-4
4.
5. 6. 7.
Campbell TW, Ellis CK: Reptile hematology, in Campbell TW, Ellis CK (eds): Avian and Exotic Animal Hematology and Cytology (ed 3). Ames, IA, Blackwell, pp 51-82, 2007 Perpinan D: Chelonian hematology: science or myth. Proc Assoc Reptile Amphibian Vet, Milwaukee, WI, pp 8-15, 2009 Fudge AM. Laboratory references ranges for selected species: turtle, box, in Fudge AM (ed): Laboratory Medicine—Avian and Exotic Pets. Philadelphia, PA, W.B. Saunders, pp 376-399, 2000 De Voe R, Geissler K, Elmore S, et al: Ranavirusassociated mortality in a group of captive eastern box turtles (Terrapene carolina carolina). J Zoo Wildl Med 35:534-543, 2004 Joyner PH, Schreve AA, Spahr J, et al: Phaeohyphomyocis in a free-living eastern box turtle (Terrapene carolina carolina). J Wildl Dis 42:883-888, 2006 Evans RH: Chronic bacterial pneumonia in free-ranging eastern box turtles (Terrapene carolina carolina). J Wildl Dis 19:349, 1983 Ryerson DL: Separation of the two acidophilic granulocytes of turtle blood, with suggested phylogenetic relationships. Anat Rec 85:25-46, 1943
163
Box Turtle (Terrapene spp.) Hematology
Appendix A. Blood Cells of Box Turtles (Terrapene carolina): An Atlas
Figure 1. Lymphocyte (Modified Wright’s stain, 1000⫻): A typical small lymphocyte. These can be differentiated from thrombocytes by the blue cytoplasm.
Figure 2. Monocyte (Azurophil) (Modified Wright’s stain, 1000⫻).
Figure 3. Heterophil (Modified Wright’s stain, 1000⫻): The heterophil typically has a round nucleus. The granules of heterophils are rod-shaped, but may be difficult to observe if granules have fused (a common artifact).
Figure 4. Basophil (Modified Wright’s stain, 1000⫻): A typical basophil with dark purple granules and a round to oval nucleus.
164
Figure 5. Eosinophil (Modified Wright’s stain, 1000⫻): The granule shape of the eosinophil can be used to differentiate it from the heterophil. Eosinophils have small, round granules. Moreover, there can be subtle differences in the staining color of the granules. A thromobocyte is also present in this image.
Figure 6. Thrombocytes (Modified Wright’s stain, 1000⫻): This image shows a small thrombocyte aggregate. The thrombocytes often have cytoplasm that is either colorless or very pale blue. There is a lymphocyte at the top that can be used for comparison.
Heatley and Russell
Figure 7. Erythrocytes (Modified Wright’s stain, 1000⫻): There is a mild degree of anisocytosis in the erythrocytes. Many of the erythrocytes have 1 to 2 small, faint blue inclusions in their cytoplasm. These are thought to be artifact and have no known clinical significance.
Box Turtle (Terrapene spp.) Hematology J. Jill Heatley, DVM, MS, Dip. ABVP (Avian), Dip. ACZM, and Karen E. Russell, DVM, PhD, Dip. ACVP
Abstract Box turtles (Terrapene spp.) maintained as pet animals are commonly presented to the veterinary clinic for health care. A complete blood count (CBC) is recommended for box turtle patients as part of the annual physical examination, when they are ill, and before owner-induced brumation. Box turtles from the wild also are commonly presented for injuries and illness, including vehicular trauma, bot fly infestation, and predator attack. The initial health assessment of a wild turtle patient may be enhanced by evaluating the results of a CBC. This article will review venipuncture sites and hematologic techniques for obtaining blood samples to submit for a CBC, as well as give comments on interpreting CBC results in box turtles based on author experience and literature review. Hopefully, this article will promote further exploration and publication relevant to the evaluation of box turtle hematology. Copyright 2010 Elsevier Inc. All rights reserved. Key words: blood; box turtle; hematology; Terrapene spp.; venipuncture
A
lthough successfully collecting blood from most box turtles (Terrapene spp.) is challenging, there are many venipuncture sites to choose from when making an attempt. Patient temperament, size, and body temperature all affect the phlebotomist’s ability to collect blood from box turtles. Anatomical sites for venipuncture in these small shy turtles include (listed from cranial to caudal): postoccipital venous plexus, jugular vein, subcarapacial sinus, brachial vein, ulnar (radiohumeral) venous sinus, and dorsal coccygeal vein.1,2 Although all of the sites listed above may be subject to lymphatic hemodilution, the jugular vein is considered best to reduce contamination of a blood sample by this artifact. The jugular vein, subcarapacial sinus, and brachial vein are considered the best sites for blood collection from a box turtle patient based on author preference; thus, only approaches for these preferred sites are detailed in this article.
Jugular Vein The jugular vein is visible in some box turtle patients; however, in the majority of animals, it may be difficult
160
to identify because of a relatively thick, wrinkled, and/or folded skin in the lateral cervical region. The skin in this area may also be highly pigmented, which might also hinder the clinician’s ability to observe the vein. To access this vessel, which is preferred by many veterinarians based on a reduced chance of lymphatic contamination of the sample, chemical restraint may be necessary for proper patient positioning and blood collection. The right jugular vein may be larger than the left in some species. The application of pressure to From the Department of Small Animal Clinical Sciences, Zoological Medicine and Surgery Service, Texas A&M University, Veterinary Medical Teaching Hospital, College Station, TX USA, and the Department of Pathobiology, Texas A&M University, Veterinary Medical Teaching Hospital, College Station, TX USA. Address correspondence to: J. Jill Heatley, DVM, MS, Dip. ABVP (Avian), Dip. ACZM, Texas A&M University, Veterinary Medical Center, College Station, TX 77843-4474. E-mail: [email protected]. © 2010 Elsevier Inc. All rights reserved. 1557-5063/10/1902-$30.00 doi:10.1053/j.jepm.2010.06.002
Journal of Exotic Pet Medicine, Vol 19, No 2 (April), 2010: pp 160 –164
161
Box Turtle (Terrapene spp.) Hematology
the lateral cervical neck area and/or the carapacial inlet on the side from which the blood will be collected may aid in visualization of this vessel, which courses superficially from the angle of the mandible, in line with the dorsal edge of the tympanum. The general course of the jugular vein in box turtles is approximately the ten and two o’clock positions, relative to the carapacial inlet.
Subcarapacial Sinus The subcarapacial sinus is located caudal to the nuchal scute at the level of the eighth cervical vertebra and is the intersection of the internal jugular vein, the anterior pulmonic vein and artery, vertebral veins and arteries, subclavian veins and arteries, and lymphatic vessels. This site may be subject to marked hemolymphatic dilution. This approach may be used in shy species that tend to retract into the shell. With head and neck retracted, the needle is angled over the head along the midline and dorsally toward the ventral aspect of the carapace. On bone contact, withdraw and slightly aspirate. Because large lymphatic vessels are cranial to the subcarapacial sinus, additional sampling attempts should be made caudal to the prior efforts
Ulnar (Radiohumeral) Venous Sinus In box turtles, the ulnar (radiohumeral) venous sinus will generally yield a small sample volume of blood (0.10-0.25 mL) and is the location most prone to lymphatic contamination. To collect blood from this site, the front leg is extended and the needle is inserted at an angle perpendicular to the body behind the tendon caudal to the radiohumeral joint. Advance the needle toward the radiohumeral joint during syringe aspiration. Choice of the venipuncture site can cause variability in the complete blood count (CBC) results based on lymphatic contamination, extravascular fluid, or serum. The most often encountered incorrect CBC parameter due to artifact effect (hemodilution) is a decreased packed cell volume. Artifact effect to the total and differential white blood cell count (WBC) has not been investigated in box turtle species. Samples from the jugular vein or carotid artery are considered the least likely to be hemodiluted.
Venipuncture and Blood Preparation Techniques The choice of needle and syringe for blood collection in box turtle species is complicated by their
relatively large red blood cell size, small patient size, and relatively low venous refill time. A 25-gauge needle is the authors’ preference, but a 23-gauge needle may be less likely to promote cell lysis when blood is collected. Chelonian blood is relatively slow to clot; therefore, one should avoid excessive aspiration pressure because in most cases there is a slow flow of blood into the syringe. A 1-mL syringe generally gives adequate aspiration and maneuverability to obtain adequate sample volumes from adult box turtles. Heparin is generally regarded as the anticoagulant of choice for most but not all chelonians. Heparin appears to be acceptable for box turtle species based on the authors’ experience. The use of a heparinized syringe to collect blood from a chelonian would be tempered by many drawbacks to sample quality such as sample dilution, increased sodium or potassium in the sample, the addition of color artifact to blood smears, and the effect of heparin on leukocyte morphology. The author prefers using a nonheparinized syringe and placing the sample in a lithium heparin microtainer. These tubes contain a dry form of heparin that avoids dilution artifact. Blood smears should be made without any anticoagulant (from the syringe) and air dried to avoid artifactual effect. Fresh blood smears may be made by the cover-slip-to-cover-slip, coverslip-to-slide, or slide-to-slide techniques; the choice should be based on operator comfort and familiarity to produce the fewest damaged cells and best smears that one can evaluate. Because the WBC is determined from the blood smear slides, preparation of blood smears is the most important aspect to consider for quality control. A poor blood smear may produce results that are not representative of the patient’s true blood parameters at the time the sample was collected. If blood smear slides are being forwarded to a reference laboratory, no further fixation is necessary. However, if slides are being stained “in house,” one should be aware of the technical difficulties associated with “self preparation” of blood smears. For example, Diff-Quik stain may result in the degranulation of basophils, which can result in cell misidentification and an incorrect WBC differential count. Standard staining with modified Wright stains are acceptable to prepare smears for evaluation from box turtle patients.
Complete Blood Count The singular best hematological reference range for box turtles does not mention site of venipuncture, species, sex, or season.3 However, the reasonable
162
Heatley and Russell
ranges reported suggest many box turtle species’ normal hematology results may be comparable (Table 1). An excellent pictorial reference for the granulocytes is also available in the same reference source.
Clinical Hemogram Interpretation There are many potential sources for variability in the box turtle hemogram, including those caused by brumation/hibernation, season (and likely patient temperature), signalment of the patient (species, sex, age), venipuncture site, technique, and laboratory performing the analysis. In torpor, for example, hemoconcentration results in an increased hematocrit, hemoglobin concentration, and erythrocyte size and count. The following is a list of potential factors that may influence box turtle hemogram results and should be considered when interpreting a sample: 1)
Intrinsic: species, sex, age, physiologic status, and
2)
Extrinsic: season, temperature, habitat, diet, disease, stress of captivity, venipuncture site
There are a few published case reports of box turtles in which the hemogram was reported.4-6 A single reference has previously presented images of box turtle blood cells, although only the granulocytes.7 In many of the published clinical case reports it was concluded that the CBCs were not very useful when formulating the disease diagnosis or prognosis; however, the authors suggest that our understanding of box turtle hematology is in its infancy, and, as veterinary medical knowledge increases, the capability of this diagnostic tool will improve. The images found in Appendix A are presented to facilitate the veterinary practitioners’ ability to perform a WBC differential count for box turtle patients in their own clinic. This diagnostic tool may prove increasingly useful when cell types are more clearly defined for these species and the WBC differential count can be performed rapidly when using only a single drop of blood.
References 1.
2. 3.
Table 1. Turtle reference ranges n ⴝ 1043 Parameter
Units
Mean
Range
White blood cell Cells/dL 10,800 8000-13,000 count Packed cell volume % 31.6 27-38 Heterophils % 44.3 21-69 Lymphocytes % 45.7 32-63 Monocytes % 1.06 1-3 Eosinophils % 0.19 0-1 Basophils % 1.92 0-4
4.
5. 6. 7.
Campbell TW, Ellis CK: Reptile hematology, in Campbell TW, Ellis CK (eds): Avian and Exotic Animal Hematology and Cytology (ed 3). Ames, IA, Blackwell, pp 51-82, 2007 Perpinan D: Chelonian hematology: science or myth. Proc Assoc Reptile Amphibian Vet, Milwaukee, WI, pp 8-15, 2009 Fudge AM. Laboratory references ranges for selected species: turtle, box, in Fudge AM (ed): Laboratory Medicine—Avian and Exotic Pets. Philadelphia, PA, W.B. Saunders, pp 376-399, 2000 De Voe R, Geissler K, Elmore S, et al: Ranavirusassociated mortality in a group of captive eastern box turtles (Terrapene carolina carolina). J Zoo Wildl Med 35:534-543, 2004 Joyner PH, Schreve AA, Spahr J, et al: Phaeohyphomyocis in a free-living eastern box turtle (Terrapene carolina carolina). J Wildl Dis 42:883-888, 2006 Evans RH: Chronic bacterial pneumonia in free-ranging eastern box turtles (Terrapene carolina carolina). J Wildl Dis 19:349, 1983 Ryerson DL: Separation of the two acidophilic granulocytes of turtle blood, with suggested phylogenetic relationships. Anat Rec 85:25-46, 1943
163
Box Turtle (Terrapene spp.) Hematology
Appendix A. Blood Cells of Box Turtles (Terrapene carolina): An Atlas
Figure 1. Lymphocyte (Modified Wright’s stain, 1000⫻): A typical small lymphocyte. These can be differentiated from thrombocytes by the blue cytoplasm.
Figure 2. Monocyte (Azurophil) (Modified Wright’s stain, 1000⫻).
Figure 3. Heterophil (Modified Wright’s stain, 1000⫻): The heterophil typically has a round nucleus. The granules of heterophils are rod-shaped, but may be difficult to observe if granules have fused (a common artifact).
Figure 4. Basophil (Modified Wright’s stain, 1000⫻): A typical basophil with dark purple granules and a round to oval nucleus.
164
Figure 5. Eosinophil (Modified Wright’s stain, 1000⫻): The granule shape of the eosinophil can be used to differentiate it from the heterophil. Eosinophils have small, round granules. Moreover, there can be subtle differences in the staining color of the granules. A thromobocyte is also present in this image.
Figure 6. Thrombocytes (Modified Wright’s stain, 1000⫻): This image shows a small thrombocyte aggregate. The thrombocytes often have cytoplasm that is either colorless or very pale blue. There is a lymphocyte at the top that can be used for comparison.
Heatley and Russell
Figure 7. Erythrocytes (Modified Wright’s stain, 1000⫻): There is a mild degree of anisocytosis in the erythrocytes. Many of the erythrocytes have 1 to 2 small, faint blue inclusions in their cytoplasm. These are thought to be artifact and have no known clinical significance.