Avian and reptilian hematology: Scratching the surface (Parts 1 and 2) (Proceedings)

Introduction

The title of this presentation is apropos, considering the fact that there are over 9,000 species of birds and nearly 8,000 species of reptiles. Knowing how different the hematology is between dogs and cats exemplifies how different eagle hematology may be from a parrot, or an iguana from a snake. As such, we truly are scratching the surface when talking about hematology of these species.

Every discussion involving hematology should begin with sample collection and handling. EDTA retains premium staining quality and morphologic preservation and is preferred in most cases. However, it will hemolyze or otherwise detrimentally alter samples collected from crows, jays and many reptiles, especially chelonians (turtles). For these species, heparin is a reasonable alternative. Ostrich blood fairs best in citrate however one should be aware of the 9:1 dilution effects. An added benefit to using heparin is that the same sample can be used for most biochemistry analyses. As with all species, regardless of the anticoagulant used, fill the tube at least half full (full for citrate), mix well, and prepare a blood film as soon as possible. Some recommend preparing a film directly from the needle so as to avoid anticoagulant-associated artifacts altogether. Films can be made using the wedge technique and clean beveled edge slides. Some prefer to use coverslips to minimize rupturing cells. Mixing one drop of 22% bovine albumin with 4-5 drops blood prior to film preparation will also minimize ruptured cells, however this will alter the sample for additional testing. The cells most commonly ruptured in our laboratory are erythrocytes, and while they may contribute to the complexity of evaluating a film, they shouldn't alter the accuracy of the differential.

A good rule of thumb for blood collection volume is 1% of the animal's weight if collecting no more frequently than every other week. This works out to .8 ml for an 80 g animal. If the animal is very ill, or if sampling will be more frequent, taking half or less that volume is necessary. There are many sites that can be used for blood collection and ideal sites vary between species. The most common for birds and turtles is probably the right jugular, as it tends to be the largest and most readily accessible vein. It is also less likely to be diluted with lymph in reptiles. Another common site for reptiles is the tail vein. Toe-nail clipping is not recommended.

The first examination of a blood film from a bird or reptile can be overwhelming since all the cells are nucleated. It is recommended that when first examining a film, pause a minute and review all the cells present for staining characteristics and other hints as to the cell types present before initiating the differential count.

There are some similarities between avian and reptilian hematology and common mammalian species including the basic cell groups of granulocytes, lymphocytes and monocytes. However, within granulocytes, birds and reptiles have heterophils rather than neutrophils. These are typically the most common cells observed in bird samples. Mature heterophils have oval to rod-shaped, reddish granules that often obscure a purple-staining lobed nucleus and colorless cytoplasm. Immature heterophils may appear with inflammation. Band forms are difficult to identify because the nuclear shape is obscured by the granules, therefore, myelocytes and metamyelocytes are more frequently observed and are identified by more deeply basophilic cytoplasm, fewer granules, immature granulation (blue-purple rounded granules), and round to indented nuclei. Toxic changes may also be observed in animals with severe systemic disease. Mild to more severe toxic changes, in order, include increased cytoplasmic basophilia, vacuolization, degranulation, large fused granules, and smudging of nuclear detail.

Heterophils may be mistaken for another granulocyte, eosinophils. Eosinophils can usually be identified by the numerous, intensely pink, usually round, cytoplasmic granules, although the shape and color of granules may vary somewhat between species. Eosinophil granules of psittacines and iguanas typically stain blue to lavender and staining artifacts may create swollen, rounded granules in various specimens. Eosinophils have a more deeply basophilic cytoplasm and also have a lobed nucleus that may be obscured by granules.

Avian and reptilian basophils are more commonly observed than in mammals and contain purple or metachromatic granules and have an oval to round rather than lobed nucleus. These appear very similar to mammalian mast cells. Quick stains, Romanowsky stains, and heparin may alter the staining and/or retention of granules however there are usually at least a few granules visible to help identify these cells.

Lymphocytes appear very similar to mammalian lymphocytes. They have a round to slightly indented nucleus containing clumpy chromatin. The cells are often oval to round with scant, pale to moderately basophilic cytoplasm that lacks granules and vacuoles. Some forms have moderate amounts of cytoplasm with irregular borders that fold around adjacent cells. Reactive lymphocytes have deeply basophilic cytoplasm and plasma cells (B lymphocytes that produce antibody in response to antigenic stimulation) can be identified by an eccentric nucleus and more abundant cytoplasm containing a golgi body that appears as a perinuclear clear zone. Immature lymphocytes may be larger, have a higher N/C ratio, fine chromatin and may have a prominent nucleolus. High numbers of these are suspicious for neoplasia. Mature lymphocytes are easily confused with thrombocytes, the cells responsible for clotting. Thrombocytes tend to have very clear, colorless cytoplasm that may contain a few vacuoles, and often contain one to two azurophilic granules at one pole. Small clumps of thrombocytes may be scattered throughout the film or concentrated at the feathered edge. It can be helpful to observe their morphology to better distinguish them from lymphocytes before proceeding with the differential.

Monocytes also resemble mammalian monocytes and tend to be the largest leukocytes observed. They have an amoeboid nucleus containing more open, lacey chromatin. The cell borders may be round, irregularly round or amoeboid. The cytoplasm is typically moderately abundant, blue-grey and may contain fine granulation with an overall ground glass texture. Monocytes migrate into tissues to become macrophages but activated forms with vacuolated cytoplasm and/or cells containing phagocytized debris are occasionally observed in the blood. The older term "azurophil" has been used to classify certain reptilian monocytes with similar morphology but a relatively high concentration of the fine pink granulation. It is recommended that these be included as monocytes in the differential.

Erythrocytes of birds and reptiles are flat, oval cells with orange-pink cytoplasm containing a single oval nucleus with condensed chromatin. Reptilian nuclei may have irregular borders. Erythrocytes are significantly larger than mammalian erythrocytes with mean cell volumes (MCV) ranging from (for example) 120 fl to over 600 fl. Interestingly, the physiologic standard observed in the mean corpuscular hemoglobin concentration (MCHC) of mammals (33-36 g/dl) is roughly maintained in the lower vertebrates with MCHC values ranging from (for example) 29-34 g/dl. Since hematocrit values tend to approximate mammalian values, the number of erythrocytes is inversely proportional to the MCV. A desert tortoise may have a PCV of 26%, but the erythrocytes are huge (MCV of 638 fl) and few in number (only .53 x10^9/ml). A broad normal PCV interval for birds is 35-55% while that of reptiles is lower, ~20-40%. Low values indicate anemia and high values indicate dehydration or polycythemia. The plasma protein can help differentiate the latter with high or low/normal protein values, respectively.

Erythrocytes should be evaluated for the nucleus (location, shape, presence), size (microcytosis, macrocytosis, anisocytosis), shape (round, teardrop), color (hypochromic, polychromatic), cytoplasmic inclusions (parasites, Heinz bodies, basophilic stippling) and agglutination (immune-mediated disorder. Microcytosis and hypochromasia (patchy pale cytoplasm) in an anemic animal suggest iron deficiency. Erythroplastids are oval erythrocytes lacking a nucleus. These can be observed in low numbers in normal animals, but higher numbers may suggest abnormal maturation. As in mammals, polychromatophilic erythrocytes are equivalent to reticulocytes and contain immature hemoglobin. Normal reptiles often have a higher proportion of reticulocytes than birds. Reticulocyte stain may be used to quantitate reticulocytes and cells with aggregated reticulum surrounding the nucleus are counted as reticulocytes. Increased numbers, with or without more immature erythrocyte forms indicate a regenerative response. Very immature erythrocytes (basophilic rubricytes and prorubricytes) and even occasional mitotic figures may be observed with marked regeneration. Young erythrocytes tend to be smaller, rounder and more basophilic. Abnormalities should be semi-quantitated; a grading scheme for birds has been proposed [Campbell, 2004].

Familiarity with hemoparasites that may be encountered can be daunting. Extracellular parasites including Trypanosoma and microfilaria of nematode parasites along with malarial organisms (Plasmodium) and Hemoproteus may be seen in either birds or reptiles. The protozoon parasites Hemoproteus and Leukocytozoon are commonly observed in certain birds, while Hemogregarina may be observed in reptiles. Less common parasites include pyroplasmids (Aegyptianella of birds and Sauroplasma of reptiles) and Atoxoplasma (canaries). Reptilian erythrocytes may contain dark spherical inclusions. These are presumed to be remnant organelles and resemble mammalian Howell-Jolly bodies but may be confused with parasites. Many parasites are incidental, or when present in high numbers indicate an immunocompromised animal (Hemoproteus); the numbers of organisms decline as the animal's health improves. Plasmodium, however, is often associated with hemolytic anemia (malaria) and other organisms are associated with disease in select species as leukocytozoon in waterfowl and turkeys, Hemoproteus in pigeons, quail, and nestlings, Borrelia in galliformes, waterfowl, Atoxoplasma in passarines especially canaries, Aeyptionella in tropical birds, and hemogregarines in unnatural hosts.

The quantitative analysis of blood can be performed by manual and automated methods. Manual methods include the basic PCV and plasma protein with one of several means for quantifying leukocytes, erythrocytes and thrombocytes. Estimation of leukocytes and thrombocytes is an imprecise method but may be the only means available or provide a means for confirming a count obtained by other means. A crude estimate may be obtained by multiplying the average number of leukocytes in five 1000x monolayer fields by 3,500, assuming an average of 200 erythrocytes per 1000x oil field. Note that microscope field sizes may vary and abnormally low or high PCVs will affect the estimate.

Leukocytes may be quantified indirectly by diluting the sample 1:32 with phloxine B stain (ENG Scientific, Inc. marketed by Fisher Scientific) or use commercially prepared premeasured vials (Exotic Animal Solutions, LLC). Plate the mixture on a Neubauer-ruled hemacytometer. Since the phloxine B only stains acidophils (heterophils and eosinophils), the total white count per mm3 is calculated from the number of acidophils observed in 18 large squares (both sides of the chamber) and the % of these cells obtained from performing a differential. The formula is:

*Where 136 is the total acidophils from both sides of the chamber.

The Natt & Herrick method is probably a more precise method since it is direct, is generally necessary for snakes and turtles since they have low numbers of heterophils, and with practice, can be used to obtain leukocyte, thrombocyte and erythrocyte counts if desired. A 1:100 or 1:200 dilution is made with either a standard red blood cell-diluting pipette or by adding 20 l to 4 ml of Natt-Herrick* solution (ENG Scientific, Inc. marketed by Fisher Scientific) or use commercially prepared premeasured vials (Exotic Animal Solutions, LLC) and follow the package insert instructions. The hemacytometer is loaded and counted and the cell count is calculated taking in to account the number of squares counted and the dilution. Leukocytes stain more intensely than the intact erythrocytes with the erythrocyte nuclei being the only portion to take up stain. Leukocytes and thrombocytes may be difficult to distinguish. Referring to the blood film prior to counting may be helpful.

The phloxine stain can be made as follows:

Add 1 Gram Phloxine B and 500 ml propylene glycol to a 1000 ml flask and add dH20 to the final 1000ml volume. A few aliquots of 775ul stain can be prepared to which 25ul of sample is added. Protect stock and aliquots from light, dehydration and moisture.

References

Campbell TW Hematology of Common Nondomestic Animals in Thrall MA, ed. Veterinary Hematology and Clinical Chemistry, Lippincott, Williams & Williams, 2004:225-276. (New edition in press).

Stockman, C Personal Communication re Phloxine B Stain, Tufts University, Feb 2010.