Introduction

Interpretation of the avian and reptile hemogram can be complex and confusing. While subtle differences exist in the non-diseased patient, the response to disease is quite varied. Extrinsic and intrinsic factors will affect the hemogram numbers, distribution of the leukocytes, and the patient’s response to disease. Understanding the normal variation between the avian and reptile hemogram, along with preservation of the blood sample to ensure reliable results, will be addressed.

Blood Collection and Preparation

Although it is not the scope of this paper to address sites of blood collection and sample preparation, a few important points need to be explained. To preserve cell morphology and decrease sample artifacts, hematologic testing should be performed as soon as possible following blood collection. Prolonged exposure to any anticoagulant (EDTA or lithium heparin) will distort cell morphology and increase sample artifacts such as smudge cells.

EDTA is not recommended for use in certain avian species (crows, jays, ravens, magpies, ducks, cranes, and hornbills) and reptiles (chelonians). Incomplete anticoagulation or partial hemolysis may occur in these species. Although EDTA has preserving power for 6–12 hours and may preserve cellular elements better than heparin, blood smears should be made within 15 minutes. Excess EDTA will cause shrinkage of the erythrocytes resulting in erroneously low packed cell volume (PCV), and degeneration of the white blood cells. EDTA blood samples cannot be used for chemistry evaluation.

In general, lithium heparin is recommended and has the advantage of being used as an anticoagulant for hematology and plasma for blood chemistry evaluation. However, prolonged exposure to heparin may result in inadequate staining of the white blood cells (affecting cell morphology) or result in clumping of the white blood cells and thrombocytes. On average, heparin will not prevent clotting longer than 8 hours.

Regardless of the anticoagulant of choice, blood smears should be made immediately without exposure to anticoagulants. This preserves cell morphology and helps to prevent clumping of the white blood cells and sample artifact. Also important is to air dry the blood smears with a rapid fanning of the slides. Smears allowed to dry slowly may result in perinuclear ring artifacts. These artifacts have been mistaken for Hemoproteus sp. or Plasmodium sp. in the avian or reptile patient. In reptiles, lymphatic vessels often accompany blood vessels, which may result in a collection of a mixture of blood and lymph. Lymphatic dilution of the sample will affect the hemogram by decreasing the packed cell volume (PCV), hemoglobin concentration, and total red and white blood cell counts.

The total blood volume of reptiles and birds will vary between species. The reported reptile range is estimated at 5–8% of the body weight in grams, while in birds it is estimated at 6–12% of the body weight. In the healthy reptile or bird, the amount of blood that can safely be drawn is 0.5–0.8% and 0.6–0.12% of the body weight in grams, respectively. It should also be noted that the reported packed cell volume (PCV) in reptiles ranges from 20–40% and in birds from 35–55%. In general, due to the lower PCV in reptiles, the yield of a plasma sample is often greater versus the bird.

Gross hemolysis of the sample tends to occur when the concentration of hemoglobin exceeds 0.02 g/dl. This is when it can be detected in the plasma or serum. Here are some hemolysis facts:

• Icteric serum may hide the appearance of a hemolyzed sample.
• Serum lipase values may be lowered.
• Urea nitrogen may be falsely elevated.
• Increase in inorganic phosphate, potassium, and alkaline phosphatase.

A lipemic blood sample elevates the optical density with the colorimetric method and falsely elevates the total protein, hemoglobin, and amylase. Lipemia also interferes with the staining capabilities of the blood smear and will make cell morphology evaluation difficult.

Erythrocytes

Reptile erythrocytes are generally larger than avian erythrocytes but smaller than amphibian erythrocytes. Depending on the species, the average lifespan of the reptile erythrocyte in the peripheral blood is 600–800 days and only 28–45 days in the bird. The slow metabolic rate of reptiles and age may play a role in the red blood cell lifespan. In reptiles, erythrocytic indices are at their highest prior to hibernation and lowest immediately following hibernation. The packed cell volume (PCV) and the total erythrocyte count increases with age in both the avian and reptile species.

The color of the adult erythrocyte is orange and contains a nucleus. A nuclear remnant (blue dot in the cytoplasm) may be seen in the reptile red blood cell and can be considered normal in turtles and tortoises. Circulating erythrocytes that have a polychromatic color to the cytoplasm are termed polychromatophilic erythrocytes. They are similar in size and shape to the mature erythrocytes. These polychromatic erythrocytes reflect the final stage of maturation and are often termed reticulocytes. However, a new methylene blue stain is required to verify the aggregate form of the reticulum encircling the erythrocyte nucleus to properly classify. In the avian species, up to 5% percent of the circulating erythrocytes are polychromatic cells, which reflect the short lifespan of the avian erythrocyte. In contrast, the slower turnover rate of reptilian erythrocytes and the long erythrocyte lifespan result in less than 1% percent of the circulating erythrocytes as polychromatic cells. For the avian patient, polychromasia is graded on a scale of 1+ to 4+, with 1+ considered normal in the non-anemic patient. In the reptile, polychromasia is usually not graded since polychromasia is less than 1% of the total erythrocyte population. In either species, the presence of increased polychromatic erythrocytes and reticulocytes is evidence of active erythropoiesis.

In most instances, the classification of erythrocyte maturation is not detailed as it is in mammals. The different stages of the immature erythrocytes are just referred to as immature erythrocytes. The presence of immature erythrocytes indicates a marked erythropoietic response to anemia. Immature erythrocytes are occasionally seen in the peripheral blood of very young birds and reptiles or reptiles going through ecdysis. The response to anemia is slower in the reptile compared to birds, requiring up to 30 days to be visible in the hemogram and up to 8 weeks before the maximal response is noted. This may be due to prolonged development time of the rubriblasts in the bone marrow and other hematopoietic sites.

Hypochromasia increases in either species may be a result of nutritional deficiencies leading to iron deficiencies, lead toxicity, or chronic inflammatory disease. Occasional anisocytosis and poikilocytosis are considered normal in the peripheral blood of both species. Increases in anisocytosis with an increase in polychromasia suggest a regenerative anemia. Increases of poikilocytosis may suggest erythrocyte dysgenesis. In addition, anisocytosis and poikilocytosis may also be found in the post hibernation reptile or those with severe inflammatory disease. Basophilic stippling may be associated with erythrocyte regeneration. It is rarely associated with lead toxicity; however, in reptiles it may suggest iron deficiency.

White Blood Cells

Lymphocytes: The function of the lymphocyte is like mammals and reflects immune stimulation. In many reptile species, the lymphocyte can be the dominant cell with ranges up to 80% of the leukocyte numbers.

Heterophils are functionally equivalent to the mammalian neutrophil. They are highly phagocytic with bactericidal activity. Heterophils also participate in viral and parasitic infections. An interesting observation is that the heterophil of snakes and tortoises has a round nucleus, while lizards have a lobed nucleus.

Monocytes have phagocytic capabilities and are active with chronic inflammatory conditions; chronic antigenic stimulation; and bacterial, fungal, or parasitic diseases. Unique to reptiles, circulating monocytes and macrophages that contain melanin pigment (melanomacrophages), nucleoproteinaceous debris, or lipid vacuoles and erythrophagocytic macrophages can be observed in the peripheral blood.

The reptile monocytic cell has been referred to as a monocyte, monocytoid azurophil, azurophilic monocyte, or azurophil.

Reptile azurophils: An azurophilic leukocyte, the azurophil causes confusion in classification and identification. The granulopoietic origin of the azurophil has not been documented or confirmed. Many researchers believe this cell should be considered a monocyte and not a separate cell type. They may represent an immature form of the monocyte. The azurophil is slightly smaller than the monocyte, has an eccentrically placed round to oval to bilobed nucleus, basophilic cytoplasm, and contains small numbers of cytoplasmic azurophilic granules. Like the monocyte, this cell has phagocytic capabilities. Increased numbers are often seen with acute bacterial and inflammatory conditions of the snake and chronic inflammatory conditions of the other types of reptiles.

Plasma cells may occasionally be seen in the peripheral blood smear. With immune stimulation, the cell will become distended with round clear structures (Russell bodies) and is now referred to as a Mott cell. May be seen with severe infection or inflammatory disease.

Eosinophils are similar in size to heterophils. In both species, the granules are primarily round. However, in the avian species, the nucleus is lobed, while in reptiles the nucleus is round to oval and eccentrically placed (lobed in some lizards). Although their function is not entirely known, these cells may play a role in parasitic infections or hypersensitivity reactions. Eosinophils may compose 7–20% of the leukocytes in the healthy reptile, with lower percentages in the lizard and higher percentages in the turtles. This contrasts with birds, where eosinophils are not a common occurrence in the peripheral blood.

Basophils are round cells often small versus the eosinophil. The cytoplasmic granules contain histamine, and their function is similar to the mammalian basophil (processing surface immunoglobulins and releasing histamine). Basophils may participate in acute inflammation and type 1 hypersensitivity reactions in the avian and reptile species. In addition, reptile basophils have been associated with parasitic and viral infections. Whereas basophils may be found in small numbers in avian peripheral blood, the normal basophil percent in reptiles may range from 0–40% (desert tortoise) depending on the species and up to 65% in healthy freshwater turtles.

Thrombocytes: A small, nucleated cell, the thrombocyte is the second most numerous cell in the peripheral blood. Thrombocytes are derived from a mononuclear precursor in the bone marrow. The primary function of the thrombocyte is hemostasis. Thrombocytes are also considered to be nonspecific scavengers, capable of removing foreign material including bacteria from the blood.

References

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2.  Giordano N, Stefania L, Mattia B. Clinical hematology in reptilian species. Vet Clin Exot Anim. 2013;16:1–30.

3.  Nicole S, Rick A, Katherine AS. Diagnostic hematology of reptiles. J Clin Lab Med. 2011;31:87–108.

4.  Raffaella C, Lorenzo C. Overview of psittacine blood analysis and comparative retrospective study of clinical diagnosis, hematology and blood chemistry in selected psittacine species. Vet Clin Exot Anim. 2013;16:71–120.