REPTILIAN HEMATOLOGY

51 REPTILIAN HEMATOLOGY



1 How are reptilian blood cells different from the blood cells of mammals?


Reptilian blood cells are similar to mammalian blood cells in some aspects but differ significantly in others, particularly morphology. As in mammals, reptilian blood cells include erythrocytes, leukocytes, and thrombocytes (platelets), all of which are easily evaluated by examination of air-dried, Romanowsky-stained blood smears.



2 What are some important principles to understand about the identification of reptilian leukocytes by blood smear evaluation?



a. Proper identification of leukocytes depends on familiarity with the morphology of leukocytes and the species of reptile, prompt processing of the blood sample, quality of the blood smear, and staining techniques used to visualize the leukocytes.

b. Fresh blood (no anticoagulant) and heparin anticoagulated blood samples are both satisfactory for use in the identification of leukocytes, but fresh blood smears are preferred. EDTA causes lysis of erythrocytes in multiple reptile species so its use as an anticoagulant is not recommended. When cell enumeration is required, it is best to keep samples at a cool temperature when shipping. Long transit times may result in inaccurate leukocyte counts, abnormal morphology, or hemolysis.

c. Gaining familiarity with leukocyte morphology requires frequent examination of blood samples from normal and sick patients of various species. Morphology of cells and staining affinity of cytoplasmic granules vary between species of reptiles.

d. Use of consistent staining techniques and methods are important to avoid excessive variation in cell staining and to ensure consistent and accurate leukocyte identification.

e. Formalin fumes, even if the source is a few feet away, will alter cellular proteins in such a way that the staining intensity and affinity of leukocytes will be altered. Blood smears should be kept away from formalin sources and stained promptly.

f. If a blood smear must be stored for longer periods, for later examination or for archiving, immersion oil should be removed with appropriate solvents, and the slide permanently coverslipped.


3 What are some important considerations in evaluating leukocyte responses in reptiles?



a. Leukocyte parameters vary greatly between species of reptiles.

b. Within a species, significant differences may result from temperature, photoperiod, season of the year, gender, and age. The degree of variation may be marked in reptiles and is greater than in mammals and birds.

c. Few controlled clinical studies specifically address leukocyte reference intervals and leukocyte patterns associated with specific disease conditions. Information often originates from individual case reports and anecdotal accounts.

d. Absolute numbers of leukocytes are more diagnostically accurate than relative percentages of leukocytes. Whenever possible, a total leukocyte count should be performed.

e. Because of all these factors, available reference intervals should be used as a guide and not as a rigid standard.

f. Pathophysiologic mechanisms and causes that lead to changes in the reptilian leukogram are generally believed to be similar to those in mammals and birds.

g. Practitioners should develop in-house reference intervals for the species of reptiles most frequently seen in their practice. This may help interpret differences in reference intervals caused by the uniqueness of the local pet reptile population, environment, temperature, and other factors.


4 Describe the various reptilian leukocytes.


The heterophil is the predominant leukocyte in health in some reptiles and predominates in many disease conditions, as discussed in the following questions. In most species of reptiles (chelonians, crocodilians, snakes, some lizards) the nucleus is round to oval, although the nucleus is lobulated in a few lizards (e.g., Green iguana). The heterophil of most reptile species has a cytoplasm that is densely packed with elongate, rod- to spindle-shaped, orange to brick-red granules that may partially obscure the nucleus (Figure 51-1). In other species the granules may not be as numerous.


image

Figure 51-1 Mature heterophils from a Green iguana (A) and a ball python (B). In contrast to the lobulated nucleus of iguana heterophils, the mature heterophil from snakes and other reptiles is characterized by a round to oval nucleus. (Wright’s stain; original magnification 1000×.)


The eosinophil has prominent round, small cytoplasmic granules that vary in color from bright red to pink depending on the species (Figure 51-2). In a few species (e.g., Green iguana) the granules may appear lavender blue in color using Romanowsky-type stains. One must keep in mind that the granules of the eosinophil will stain differently than those of the heterophil within the same blood smear.


image

Figure 51-2 Eosinophils (E) from a Green iguana (A) and a snake (B). In general, reptilian eosinophils have a round nucleus and round to oval granules. The color of the granules varies between species. In the iguana the granules of the eosinophil stain lavender blue with Romanowsky-type stains. In other reptiles the granules may be eosinophilic/pink. A mature heterophil (H) is adjacent to the eosinophil from the snake. (Wright’s stain; original magnification 1000×.)


The basophil has prominent, small, deep-magenta to purple, round cytoplasmic granules and, unlike the mammalian basophil, has a round to oval, nonlobed nucleus (Figure 51-3). The contents of basophil granules can be dissolved by Romanowsky-type stains, giving the basophil a foamy, vacuolated appearance with few granules, if any. Careful examination of these cells in a blood smear may reveal a few characteristic granules, particularly over the nucleus, helping to identify them as basophils. Some species of reptiles (e.g., turtles) may have naturally high numbers of circulating basophils.


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Figure 51-3 Basophils from a snake. Basophils are characterized by a cytoplasm that is packed full of deep purple granules that partially obscure the nucleus (A). Basophil granules sometimes dissolve during staining, giving the basophil a foamy, vacuolated appearance (B). A thrombocyte (T) is adjacent to the basophil in A. (Wright’s stain; original magnification 1000×.)


The monocyte is similar to its mammalian counterpart. It is a large mononuclear cell with abundant cytoplasm and a round to oval to indented nucleus (Figure 51-4). Cytoplasmic vacuolation may be seen. The azurophil is morphologically similar to the monocyte and is unique to some species of reptiles (snakes, iguanas). Some species of snakes in particular have greater numbers of azurophils than other reptiles. The azurophil is generally regarded as a cell of monocytic origin that contains numerous fine, red intracytoplasmic granules. The granules may be numerous enough to impart a red color to the cytoplasm.


image

Figure 51-4 Azurophils from a snake (A) and a monocyte from a Green iguana (B). Azurophils, generally regarded to be of monocytic origin, are round, mononuclear cells with round to oval nuclei and numerous dustlike, fine, pink cytoplasmic granules. The pink granules are not evident in this monochrome image. The reptilian monocyte (B) is similar to the mammalian monocyte. (Wright’s stain; original magnification 1000×.)


The lymphocyte is also morphologically similar to that of mammals. It is the predominant leukocyte in many species of reptiles (up to 80% of circulating lymphocytes). The lymphocyte is a mononuclear cell, with low to scant amounts of blue cytoplasm, high nuclear/cytoplasmic ratio, and a round nucleus (Figure 51-5). Reptiles normally have small and larger lymphocytes in circulation. Care must be taken not to confuse the small lymphocytes with thrombocytes. Thrombocytes may be smaller, more oval, with a darker, more condensed nucleus and indistinct, pale to nonstaining cytoplasm, and in contrast to lymphocytes, may readily clump in blood smears.


image

Figure 51-5 Thrombocyte (T) and lymphocyte (L) from a snake. Notice the colorless cytoplasm of the thrombocyte compared with the darker cytoplasm of the lymphocyte. (Wright’s stain; original magnification 1000×.)



5 What are common methods of enumerating total numbers of leukocytes?


Leukocyte enumeration methods include indirect methods (blood smear estimation, eosinophil Unopette 5877 system) and direct methods (Natt and Herrick’s). Natt and Herrick’s method is more labor intensive because it involves manual enumeration of cells using a hemocytometer. Some authors do not recommend the use of the eosinophil Unopette 5877 system in reptiles because of discrepancies with the Natt and Herrick’s method. The discrepancies may be greater in species with normally low heterophil counts.



a. Estimation of the leukocyte count by blood smear evaluation is not as accurate as other methods but may be the only available option in a veterinary practice.

b. Natt and Herrick’s method allows the direct measurement of total leukocytes and erythrocytes. In the hands of an experienced veterinarian or veterinary technician, this method produces accurate results and can be readily performed in a clinical practice setting.

For faster, automated, direct quantification, there are automated cell counters based on flow cytometric technology. Unfortunately, this methodology has not been adequately validated in reptiles. Known drawbacks of this technology include the potential of including nucleated erythrocytes and thrombocytes in the leukocyte count and generally inconsistent automated differential cell counts. Manual examination of a blood smear is therefore essential when performing differential cell counts in conjunction with an automated cell counter. Flow cytometric technology is expensive and available primarily to commercial laboratories with the time, finances, and dedicated personnel to utilize and service the technology appropriately. As with any automated technology, daily quality control and quality assurance measures must be in place to produce reproducible, accurate results.


For detailed discussion on leukocyte count techniques, the reader is directed to the list of references.



6 What are the most common leukocyte alterations?


The most common alterations include leukocytosis and leukopenia. Leukocytosis refers to an absolute increase in the total number of white blood cells (WBCs) in the peripheral blood. Leukopenia refers to an absolute decrease in the total number of leukocytes in the peripheral blood. These two alterations are the result of increases (-cytosis or -philia) or decreases (-penia) in the number of individual leukocyte cell types.



7 What causes heterophilia in reptiles?


Heterophilia in reptiles may be caused by the following two conditions, acting singly or in combination (Box 51-1):


a. Corticosteroid release or administration is characterized by heterophilia accompanied by lymphopenia in most animals. It may be associated with trauma, pain, disease, stressful conditions, and exogenous glucocorticoid administration. A common term used for the characteristic leukogram changes associated with corticosteroids is stress leukogram.

b. Inflammation is an important cause of heterophilia in pet reptiles and results from infectious and noninfectious causes.


Box 51-1 Causes of Heterophilia in Reptiles



Inflammation



Infectious


Atypical mycobacteriosis


Mycoplasma (alligators)


Miscellaneous bacterial infections/abscesses


Fungal infections



Noninfectious


Tissue trauma/necrosis



• Traumatic injuries

• Ischemia

• Renal disease

• Surgery

Foreign bodies


Neoplasms



Corticosteroids



Exogenous


Glucocorticoid administration



Endogenous (“stress”)


Excessive handling, physical restraint, unfamiliar environment


Transportation, high density


Food restriction, disease


The reptile’s response to these conditions may be modified by other factors, such as temperature and season of the year. Increased numbers of dysplastic or immature heterophils may be seen in cases of granulocytic leukemia, but mature heterophilia caused by chronic myelogenous leukemia is not documented in reptiles. Physiologic leukocytosis is another cause of heterophilia/neutrophilia in animals, but whether it occurs in reptiles is not known.



8 How do corticosteroids and inflammation result in heterophilia?


The mechanisms leading to heterophilia include heterophil redistribution, decreased egress from the circulation to tissues, and/or increased hematopoietic production.



9 Describe the mechanism of heterophil redistribution.


The marginal pool (MP) is not sampled during routine blood collection and consists of heterophils within blood vessels but found rolling along endothelial surfaces or having temporarily ceased moving. Under the influence of corticosteroids, sudden exercise, and epinephrine, mammalian neutrophils and perhaps reptile heterophils redistribute from the MP to the circulating pool (CP).



10 What causes decreased egress of heterophils from the peripheral blood in reptiles?


Decreased egress for the peripheral blood to the tissues is believed to result primarily from the effects of corticosteroids and may be mediated by down-regulation of adhesion molecules on the surfaces of leukocytes or endothelial cells.



11 When does hematopoietic tissue increase production of heterophil precursors in reptiles?


Increased hematopoietic production of heterophil precursors occurs when there is increased demand for heterophils (e.g., inflammation) in the peripheral tissues. Infection (pneumonia, enteritis, dermatitis), trauma, infarction, and neoplasms may result in increased demand for heterophils. Chemical mediators released from inflammatory cells, infectious agents, and damaged tissue stimulate the bone marrow to increase production. In healthy reptiles, hematopoietic production occurs primarily in the bone marrow. During conditions of increased demand, however, granulopoiesis may occur in extramedullary sites, such as the spleen, liver, and kidney.



12 What are the expected complete blood count findings in reptiles with heterophilia caused by corticosteroid effects?


The complete blood count (CBC) changes typical of corticosteroid-induced leukograms in other species have not been extensively studied in reptiles. However, a few studies indicate that heterophilia may occur with “stress” and exogenous glucocorticoids. Changes may be confounded by the presence of inflammation or environmental effects (e.g., low temperature).



13 What are the expected CBC findings in reptiles with heterophilia of inflammatory disease?


Mild heterophilia may be the only CBC abnormality in mild inflammatory disease. With increasing severity, left shift (increased presence of immature heterophils, e.g., band forms, metamyelocytes, myelocytes, promyelocytes; Figure 51-6), heterophil toxicity (cytoplasmic basophilia, abnormal vacuolation, abnormally shaped cytoplasmic granules, degranulation, large heterophils), and greater numbers of heterophils may be present and reflect a significant demand for heterophils in tissues. Monocytosis or azurophilia may also be observed. Overwhelming inflammation may result in heteropenia with left shift and heterophil toxicity (see Question 15). The number of lymphocytes may be decreased, normal, or increased with inflammatory disease. Environmental factors (e.g., temperature) may also influence the magnitude of the changes.


image

Figure 51-6 Blood smear from a Green iguana with a heterophil left shift. Three heterophils are shown. Two immature heterophils with round nuclei (arrows) and one mature heterophil with a lobulated nucleus (arrowhead) are shown. Notice also the rounding of granules and cytoplasmic vacuolation.



14 What are causes of heteropenia in reptiles?


Heteropenia may be caused primarily by overwhelming inflammation resulting in excessive tissue demand for heterophils (Box 51-2).



Box 51-2 Causes of Heteropenia in Reptiles



Inflammation


Overwhelming bacterial infection


Viral infection


Fungal infection



Other Possible Causes


Myelophthisic syndromes (leukemia, lymphosarcoma)


Myelosuppressive therapies (radiation, cancer chemotherapies)


Heterophil redistribution caused by endotoxemia or gram-negative bacterial infections

Related posts:

  1. ERYTHROCYTE DISORDERS
  2. OVERVIEW: HEMOSTATIC COMPONENTS AND DISORDERS
  3. INTERPRETATION OF THE LEUKOGRAM
  4. AVIAN BIOCHEMICAL ANALYSIS

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Aug 26, 2016 | Posted by in INTERNAL MEDICINE | Comments Off on REPTILIAN HEMATOLOGY

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