49 AVIAN HEMATOLOGY
Avian blood cells are similar to mammalian blood cells in some aspects but differ significantly in others, particularly morphology. As in mammals, avian 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 avian leukocytes by blood smear evaluation?
Figure 49-2 Blood smear from a Bald eagle. A heterophil (H) and an eosinophil (E) are shown. The heterophil has a lobulated nucleus with characteristic spindle-shaped cytoplasmic granules, and the eosinophil has a round nucleus and round granules. (Wright’s stain; original magnification 1000×.)
The most common leukocyte alterations include leukocytosis and leukopenia. Leukocytosis refers to an absolute increase in the total number of white blood cells (WBCs) in circulation. Leukopenia refers to an absolute decrease in the total number of WBCs in circulation. These two alterations are the result of increases (-cytosis or -philia) or decreases (-penia) in the number of individual leukocyte cell types.
These methods include indirect (blood smear estimation, eosinophil Unopette 5877) and direct (Natt and Herrick’s) methods. The Unopette system and Natt and Herrick’s are labor intensive because they involve manual enumeration of cells using a hemocytometer.
For faster, automated, direct quantification, automated cell counters are available based on flow cytometric technology. These automated counters are capable of quantifying the total number of leukocytes in avian blood. However, known drawbacks of this technology include the potential for 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/quality assurance measures must be in place to produce reproducible, accurate results. (For detailed discussion on leukocyte count techniques, see the Bibliography following Chapter 53.)
Box 49-1 Causes of Heterophilia in Birds
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, or epinephrine, mammalian neutrophils and probably bird heterophils redistribute from the MP to the circulating pool (CP).
Decreased egress for the peripheral blood to the tissues is believed to be primarily caused by the effects of corticosteroids and may be mediated by down-regulation of adhesion molecules on the surfaces of leukocytes or endothelial cells.
Increased hematopoietic production of heterophil precursors occurs when there is increased demand for heterophils in the peripheral tissues. Infection (e.g., pneumonia, enteritis, dermatitis), trauma, immune-mediated injury, 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 birds, hematopoietic production occurs primarily in the bone marrow; however, during conditions of increased demand, granulopoiesis may occur in extramedullary sites (e.g., spleen, liver, kidney).
11 What are the expected complete blood count (CBC) findings in birds with heterophilia of physiologic leukocytosis?
Typically, in other domestic animals, absolute increases in neutrophils are mild to moderate in degree; with few exceptions, however, the magnitude of the heterophilia in multiple avian species with physiologic leukocytosis has not been well documented or reported in the literature. In general, leukocyte counts may be higher than 10,000 cells per microliter (μl). For example, nestling psittacines may have leukocyte counts of 20,000 to 40,000 cells/μl, with most of the leukocytes being mature heterophils. Other helpful CBC clues are that physiologic leukocytosis is associated with normal or increased numbers of lymphocytes and that no heterophil left shift or heterophil toxicity occurs.
As with physiologic leukocytosis, the magnitude of heterophilia and leukocytosis in corticosteroid-induced leukograms may be mild to moderate in degree, with most leukocytes being mature heterophils (no left shift, no toxic changes). A defining characteristic is the presence of lymphopenia, which helps distinguish corticosteroid-induced changes from physiologic leukocytosis. CBC changes typical of corticosteroid-induced leukograms may be confounded by the presence of inflammation or myeloproliferative disease.
Mild heterophilia may be the only abnormality in mild inflammatory disease. With increasing severity, left shift (increased presence of immature heterophils, e.g., band forms, metamyelocytes, myelocytes, promyelocytes), 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 may also be observed. In severe inflammatory disease (e.g., chlamydophilosis, mycobacteriosis), total leukocyte counts may reach or exceed 100,000 cells/μl. Keep in mind that overwhelming inflammation may result in heteropenia with left shift and heterophil toxicity (see following questions). The numbers of lymphocytes may be decreased, normal, or increased with inflammatory diseases.
Box 49-2 Causes of Heteropenia in Birds
Overwhelming inflammation is primarily caused by an overwhelming insult (infectious or noninfectious) that elicits massive inflammatory cytokine release and marked, sudden tissue demand for heterophils. The differential list should include, but should not be limited to, disseminated bacterial infections, coelomitis, severe enteritis, and massive tumor necrosis. Any inflammatory condition where the rate at which heterophils exit the circulation exceeds the rate of production by the bone marrow will produce heteropenia. Ultimately, if the demand for heterophils is decreased or bone marrow production is sufficiently augmented, heterophil numbers may increase to within reference intervals or may result in heterophilia.
Heterophil production abnormalities may be the result of destruction of heterophil precursors, hormonal/chemical suppression of granulopoiesis, or myelophthisic processes. Destruction of heterophil precursors in birds may be caused by viral infection or adverse drug reactions (e.g., fenbendazole in painted storks, piperacillin and/or doxycycline in one budgerigar). Suppression of granulopoiesis may occur with hormonal treatment (progesterone) and other myelosuppressive therapy (cyclophosphamide, cancer chemotherapies, radiation). Myelophthisic processes, such as neoplastic disease (leukemia, lymphosarcoma), may infiltrate the bone marrow and displace hematopoietic precursors.
In domestic animals, during endotoxemia and gram-negative bacterial infections, neutrophils redistribute from the CP to the MP. Redistribution of heterophils from the CP to the MP caused by endotoxemia may also occur in birds.
Although heteropenia may be the only hematologic abnormality, birds with inflammatory diseases may have inflammatory leukograms (left shift, heterophil toxicity). Birds with leukemia, myelosuppressive therapy, or idiosyncratic drug reactions may have pancytopenias. Atypical cells in circulation may indicate leukemia.
20 How do physiologic leukocytosis, antigenic stipulation, and lymphoproliferative disease result in lymphocytosis?
The lymphocytosis is transient and frequently accompanied by mild to moderate heterophilia with no left shift or toxic change in heterophils. The magnitude of the lymphocytosis is mild to moderate. Lymphocytosis may be greater than the heterophilia, particularly in species with predominant lymphocyte numbers in health.
The lymphocytosis may be mild to moderate. Lymphocytosis in one reported case in a crane was approximately 45,000 lymphocytes/μl. The magnitude of the lymphocytosis may overlap with that of physiologic leukocytosis or of early lymphoproliferative disease. An important difference is that the lymphocytosis of chronic inflammatory disease is not transient as in physiologic leukocytosis. Heterophilia with left shift or toxic change or monocytosis may accompany lymphocytosis of chronic inflammatory disease. Reactive lymphocytes may be seen in circulation. Vaccination (duck plague, pasteurellosis) in some species of birds produces lymphocytosis secondary to antigenic stimulation.
Lymphocytosis may be greater than that seen with lymphocytosis of physiologic leukocytosis or of chronic inflammatory disease, but these may overlap. The lymphocyte counts in a cockatoo and an Amazon with lymphoid leukemia were approximately 38,000 to 49,000 cells/μl. Lymphoid leukemia in other birds species (emus, ducks) may have lymphocyte counts as high as 200,000 cells/μl. Atypical-appearing lymphocytes may be present in circulation and serve as a clue to the presence of neoplastic disease; however, the lymphocytes may also appear morphologically normal. Serial CBCs may help in differentiating mild neoplastic lymphocytosis from that of physiologic leukocytosis or chronic inflammatory disease. Anemia or pancytopenia may also be present and suggest bone marrow infiltration.
Box 49-4 Causes of Lymphopenia in Birds
25 How do corticosteroids, inhibition of lymphocyte production, and acute infection result in lymphopenia?
26 Describe the mechanisms of lymphocyte redistribution and lymphocyte trapping in lymphoid tissues.
Corticosteroids (endogenous or exogenous) promote redistribution of lymphocytes from the CP to the MP and other locations (bone marrow, lymphoid tissues). Under the influence of inflammatory cytokines released during acute inflammation, and sometimes in combination with endogenous corticosteroid release, lymphocytes may be redistributed from the CP to the MP or other locations (bone marrow, site of inflammation, lymphoid tissue). Inflammatory cytokines may also promote the temporary sequestration of lymphocytes in lymphoid tissues as part of the antigen presentation process.
Lymphoid tissue, as with bone marrow, is an actively proliferating tissue susceptible to interference by substances that inhibit proliferation. Cancer chemotherapies, toxins, radiation, cyclophosphamide, and extended corticosteroid exposure may directly inhibit or destroy lymphocyte production. Viruses that infect lymphocytes may also be cytolytic. Congenital defects in lymphocyte production are extremely rare in domestic animals and are not documented in birds.
Lymphopenia secondary to acute infections is believed to result from a combination of corticosteroid-induced effects, lymphocyte redistribution, and perhaps interference with lymphocyte production. This has not been adequately investigated in birds.