Kendal E. Harr, DVM, MS, DACVP
Sample Collection
Collection of blood volume equivalent to 0.5% of a reptile's body weight is usually not associated with any adverse effects. All samples for biochemical analyses should be placed in a lithium heparin vacutainer appropriate for volume to avoid variable clotting time and gelling of serum samples. In the United States, many exotics practitioners perform venipuncture using a needle that is heparinized in house with injectable sodium heparin. If most of the heparin is expelled, it will minimally affect the sample. However, this can vary between practitioners and samples. Any droplets remaining may cause dilutional effects as well as interfere with some analytical tests, such as sodium and albumin. Additionally, many practitioners use tuberculin or insulin syringes that do not have detachable needles. These needles can easily be cut from the syringe using a pair of large veterinary nail clippers.
Reptilian plasma samples, especially those from herbivores, may be yellow due to carotenoid pigments, not bilirubin. Pink or red plasma is indicative of hemolysis. Hemolysis may cause increases in LDH, AST, potassium, and bile acids (colorimetric). Extended transport time or increased temperature during transport can cause variable potassium, calcium, and phosphorous values, decreased glucose concentration, and increased LDH, AST, and potentially protein concentration due to diffusion/transport across the RBC membrane. Biochemical samples should always be separated within 30 minutes to prevent artifact that may cause misdiagnosis. Lipemia is rarely observed in reptilian samples.
Many veterinarians are not adequately equipped to manage exotic species and receive minimal training in appropriate handling of blood. Small needles and syringes such as insulin syringes may be required. Any syringe that does not have a removable needle requires removal with nail clippers or similar equipment prior to the expulsion of blood. There is quite a range of equipment needed to address the amazing variation in reptiles. For example, in the larger lizard species such as alligators, 3 inch needles and 12 cc syringes may be required for cervical sinus blood collection.
Temperature
Reptiles are ectotherms and can survive a range of body temperatures that would kill domestic mammals. Active reptiles will behaviorally thermoregulate to maintain their core body temperature within 15 to 20°F (8–11°C), for instance - between 70–85°F (21–32°C). However, they can survive physiological core body temperature ranges of 30 to 40°F (17–22°C) or more in states of torpor or hibernation. Changes in blood temperature of this magnitude will cause marked changes in pH as well as the concentrations of other ionized electrolytes. The preprogrammed computerized calculations based on Sigaard-Anderson nomograms made by the I-STAT and other pH, oxygen, and electrolyte monitors are not accurate if the body temperature is significantly different from 98.6°F or 37°C. This should be considered when clinically assessing an animal or analyzing data from research or clinical studies.
Function of enzymes changes dramatically at these different temperatures. Laboratory instruments measure enzyme kinetic activity (not concentration) at 37°C which may not reflect the activity that occurs in the animal in question. Actual enzymes that function in reptiles have not been investigated in the same detailed manner as in mammals and so function and origin are questionable.
White cell count may double or more with change in temperature from the low end of the thermoneutral zone to the high end. Additionally, if this is done rapidly, more immature cells may be observed in the blood stream which can mimic a left shift in species with segmented nuclei such as iguanas.
Hibernation and Postprandial Artifact
During hibernation and dry periods reptiles may not drink for weeks to months at a time. This results in a dehydrated state and marked changes due to hemoconcentration that should not be overly interpreted. Carnivores and piscivores may have marked uratemia postprandially that mimics renal disease. Preprandial samples from rehydrated animals are therefore recommended for evaluation.
Lymph Dilution
Artifactual changes should be ruled out prior to interpretation of anemia in reptiles. Lymphatic vessels are present in close proximity to blood vessels in the tail, forelimb, and other regions of the body.11,13 Dilution of the blood sample with lymph results in decreased PCV, hemoglobin concentration, etc.8 If the sample has a decreased PCV with no evidence of regeneration and increased numbers of small lymphocytes, submission of a new sample should be requested to verify results. Clinical chemistry values will vary dependent on which organ or disease process the lymph is draining, however reports in reptiles reveal a decreased protein as well as decreased potassium in lymph fluid.
Anticoagulants
Blood is a tissue and will autolyze like any other tissue removed from the body. Blood autolyzes quickly and, for some reason, blood from reptile and especially avian species autolyzes faster than mammalian blood. In bird species, half of cells on a blood smear may be autolyzed at 12 hours while the window of transport approaches 24 hours for reptilian blood.6 Shorter transport time, including in clinic and in lab time, will decrease artifactual changes in the blood and error in the quantitation of the cells. Ca EDTA (purple tops) generally minimize morphologic artifacts, however ratites, corvids, and chelonian, are susceptible to lysis using EDTA. This is the anticoagulant recommended in Campbell's Avian and Exotic Animal Hematology and Cytology (3rd ed).2 EDTA is a calcium chelator and cannot be used if chemistries are to be performed on the sample.
Heparin, an antithrombin III agonist, is preferred if blood is to be used for both hematology and chemistry. Heparinized blood samples have an altered cell morphology which makes evaluating toxicity difficult. Additionally, heparin causes cell clumping which makes quantitation less accurate. Make sure that the green top tube is labeled lithium heparin and not sodium heparin which can affect electrolyte values. Citrated blood (blue tops) typically are not used and have resulted in increased lysis when examined in controlled studies.6 Always use a tube that is appropriate for blood volume to reduce the likelihood of cell lysis and artifact. Microtainers should have at least 0.5 cc while low volume (2 ml tubes) should have at least 1 cc of blood volume.
Erythrocyte Morphology and Function
Unlike mammalian erythrocytes, reptilian, avian, amphibian and piscine red blood cells (RBC) have nuclei. Nucleated RBCs are elliptical and larger than non-nucleated RBCs with amphibian RBCs being the largest. In general, mature reptilian erythrocytes have nuclei that are irregularly round to oval, with dense pyknotic chromatin and homogenous eosinophilic (red) cytoplasm. Reticulocytes, immature erythrocytes, have a similar shape but are slightly rounder with light blue cytoplasm upon Romanowsky staining. Reticulocyte nuclei contain clumped chromatin with obvious, pale euchromatin indicative of the active hemoglobin production occurring in these cells. Rubricytes, immature reticulocytes, have round, slightly irregular nuclei with clumped chromatin and round, dark blue cytoplasm. This cell type should not be confused with small lymphocytes. This stage of erythrocyte may be present in the blood stream and is capable of replication. Therefore, mitotic activity may be seen in the erythrocyte line in blood smears, and especially in samples with active regeneration. Mitotic activity in reptilian peripheral blood is not indicative of a neoplastic process.
Erythrocyte size, number and hemoglobin content have been compared between 441 species of mammals, birds, and reptiles.7 Reptiles have lower total number RBC, hemoglobin concentration, and PCV than either mammals or birds. These findings indicate that the oxygen-carrying capacity of the blood is highly conserved in birds and mammals but is lower in exothermic animals such as reptiles.
Erythrocyte function is similar to that of mammals though adaptations exist across this diverse class of animals. Hemoglobin tetramers appear to be relatively well conserved across the species of the class Reptilia.3 However, small changes in molecular structure result in significant variation in oxygen affinity.15 In general, lizards tend to have a significantly higher oxygen affinity while Chelonia have decreased oxygen affinity.20 Two functionally different hemoglobin tetramers have been separated from the blood of adult red-eared freshwater turtles (Trachemys scripta) which exhibit marked differences in oxygen affinity and in concentration of ATP associated with the hemoglobin.4
Reptilian red cells have an increased life span in comparison to mammalian red cells. For instance, turtle erythrocytes may live for up to 11 months.10 Nucleated red blood cells undergo programmed cell death and offer an excellent model for the study of apoptosis. Characterization of disease processes associated with abnormal erythrocyte morphology has been limited in reptiles.12 Polychromasia (multiple colors) is the presence of bluish or immature RBCs on stained blood smears. It is observed with some frequency in moderately to severely anemic reptiles. This represents a regenerative response and an attempt by the animal to return to homeostasis. Low numbers (0–1 rubricyte/100 WBC) of rubricytes may be present in normal blood smears. Reptilian erythroid regenerative response appears to be slower than that observed in mammals. When anemia was induced in turtles (Pseudemys elegans) with phenylhydrazine hydrochloride, 30 days elapsed prior to any regenerative response and the authors report up to eight weeks prior to maximal regenerative response. Rabbits showed a regenerative response in 5 days in the same study.16 Decreased mean corpuscular hemoglobin concentration (MCHC) and decreased mean cell volume (MCV) have been documented to be associated with reticulocytosis and polychromasia in reptiles.16 Both mammalian and reptilian reticulocytes contain decreased quantities of hemoglobin which is actively produced in these immature cells, resulting in decreased MCHC. In mammals, MCV generally increases during a regenerative response due to the slightly larger size of mammalian reticulocytes. However, reptilian reticulocytes are generally smaller in size than mature reptilian RBCs, resulting in decreased MCV.
Upon confirmation that the sample is representative of the patient, anemia should be characterized based on polychromasia and rubricyte count as either regenerative or nonregenerative. Anemia may be caused by increased RBC destruction, decreased RBC production (nonregenerative), or blood loss. It should be noted that reptiles have increased time to regenerative response so one must consider the chronicity of the anemia. In general, if the anemia has persisted for more than one month with no significant response it may be classified as nonregenerative which may be caused by chronic inflammation, metabolic disease, neoplasms, toxins, and starvation.
Intraerythrocytic Inclusions
Normal intracytoplasmic inclusions may be seen in several chelonians. These single, small, blue, punctate inclusions may be present in a few erythrocytes or a majority of erythrocytes in a blood smear with no known clinical significance. Ultrastructural investigation reveal that these inclusions are consistent with degenerate organelles.1
Similar intracytoplasmic inclusions in American alligators (Alligator mississipiensis) are only seen in animals with significant infection.14 Generally, these red cell inclusions are concurrently observed with toxic change in the heterophil cell line. Upon ultrastructural examination, some of the vacuoles contained degenerate organelles and ferritin aggregates, leading to the hypothesis that they may be autophagosomal and formed during the maturation process of the erythrocyte.
Square to rectangular to occasionally hexagonal, pale, crystalline-like cytoplasmic inclusions consistent with hemoglobin crystals were initially investigated in Rhinoceros iguanas (C. cornuta and C. figgensi) using transmission electron microscopy.17,18 In the author's experience, similar crystals are observed with some frequency in various species of lizards, snakes, and tortoises. These have been documented in the literature in the green iguana (Iguana iguana), and crystals may also be observed in the nucleus in this species.5
Viral inclusions have been observed in erythrocyte cytoplasm. A fer de lance (Bothrops moojeni) snake, that was being evaluated for renal carcinoma, was found to have two types of inclusions present concomitantly in the same RBC. One type of inclusion contained viral particles and the other inclusion was crystalline and contained an unknown protein.9 The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis revealed an iridovirus consistent with snake erythrocyte virus and the crystalline structures that were different than typical hemoglobin crystals. The viral inclusions are similar to acidophilic (blue) inclusions in east African Chameleons (Chamaeleo dilepis) documented on electron microscopy to contain viral particles consistent with the family Iridoviridae.19 Erythroparasites, such as Hepatozoan sp. and Hemogregarina sp. are also present in RBC cytoplasm and may be associated with anemia and other pathologic disease states.
Inclusion body disease of boas and pythons has classic, pathognomonic inclusions in lymphocytes creating a half moon appearance of the nucleus. Upon ultrastructural evaluation, only protein has been found in lymphocytes. Sensitivity and specificity of inclusion identification is currently unknown. Previous publications identified a potentially associated retrovirus but it was later found that the ophidian renal culture was contaminated with retrovirus. Current investigations are underway to identify alternate etiologies.
References
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2. Campbell TW, Ellis CK. Avian and Exotic Animal Hematology and Cytology, 3rd ed. Blackwell Publishing Limited/John Wiley and Sons, Inc., Hoboken, NJ. 320 pg. 2007.
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