Abnormalities in the Canine and Feline Blood Film

Introduction

The evaluation of a blood smear will allow the practitioner to gain rapid, valuable information regarding the health of the patient when the evaluation is performed in a systematic fashion. The important clinical information needed for the hematologic evaluation of an animal can all be obtained by estimating cell numbers and evaluating the morphologic changes in erythrocytes, leukocytes and platelets. The value of these findings, many of which are not recognized by automated cell counters, cannot be overemphasized.

Blood Collection and Slide Preparation

Vacutainer tubes containing EDTA should be filled to the designated amount. Partial filling of vacutainer tubes with blood may cause artifactual changes in cell morphology and numerical values. Blood smears should be prepared as quickly as possible in order to minimize artifactual changes in erythrocytes and leukocytes such as red cell crenation, leukocyte vacuolation and nuclear swelling and pyknosis. In addition, prolonged exposure to EDTA may make it more difficult, or even impossible to identify infectious agents, such as Mycoplasma haemofelis (formerly Haemobartonella felis), in the blood of infected cats. The coverslip technique for making smears is preferred over the glass slide technique. This technique minimizes traumatic injury to cells during slide preparation. This technique produces a more even distribution of cells, allowing more accurate estimation of leukocyte and platelet numbers. Smears should be rapidly dried with a blow drier to eliminate artifacts of air-drying red blood cells. This is particularly important when attempting to identify red cell parasites such as haemoplasmas or evaluation of erythrocyte shape changes.

Scanning the Smear

The first step in the evaluation of a blood smear is to scan the slide using a 10x or 20x objective. With regard to the red blood cells, we should observe red cell density and presence of rouleaux or agglutination. (Rouleaux may be differentiated from agglutination by saline test where 1 drop of blood is mixed with 0.5 to 1.0 ml of physiological saline solution and observed on wet mounts, unstained. Rouleaux will disperse with saline dilution.). With regard to nucleated cells we should confirm that the mature neutrophil is the predominant cell type. The presence of any left-shifted neutrophils or large or atypical leukocytes, as well as the presence of any nucleated erythrocytes should also be recorded. Platelet clumps should also be identified at this magnification because they will affect how we interpret platelet numbers later in the evaluation. This is particularly crucial in the evaluation of the feline blood film because platelet clumping is a common occurrence in this species. Leukocyte numbers may be estimated using the following formula. Formula: # cells/:l = (avg. # of cells per field) x (objective power)2. The objective used to estimate leukocyte numbers should be one where approximately 5–10 leukocytes are seen per field. Example: If an average of 5 cells were counted for each 50x field, the total leukocyte count would be (5) x (2,500) = 12,500 cells/:l.

Erythrocyte Evaluation

Erythrocyte evaluation begins with the search for agglutination or rouleaux formation using a scanning objective. Erythrocyte morphology should be evaluated using the 100x, oil immersion lens in an area of the smear where red cells are evenly spaced, usually slightly behind the feathered edge. Red blood cells are evaluated for changes in size, shape, color and inclusions. Erythrocytes are normally very uniform in size. Typically, there is minimal variation in the size, shape or color of the erythrocytes in the blood smear. In most domestic species, red cells are normally a biconcave disc shape (discocyte). However, the central pallor (a paler staining area in the center of the erythrocyte) that is normally prominent in the blood cells of dogs of often not seen in the cat. This is associated with the smaller erythrocyte size in the cat (5–6 µm) than in the dog (7 µm). Therefore, in the feline species, spherocytosis (small, round, dense cells with no central pallor) cannot reliably be identified by examination of a blood film. Spherocytosis can only reliably be identified in the canine.

Anisocytosis is defined as a variation in cell size. This usually indicates the presence of abnormally large erythrocytes (macrocytes) which are commonly seen in regenerative anemias. Macrocytes and anisocytosis may be seen in nonregenerative anemias in cats with FeLV infection and some preneoplastic (dysplastic) and neoplastic (leukemias) diseases. Animals with regenerative anemias from any cause typically have marked anisocytosis due to the large polychromatophilic erythrocytes present. Dogs with IMHA usually have marked anisocytosis due to the presence of small spherocytes and large polychromatophilic cells.

Poikilocytes are abnormally shaped cells (See attached figure). There are several different types of poikilocytes each with a different specific cell morphology. Different types of poikilocytes suggest the occurrence of specific disease processes (See chart on last page). Most poikilocytes are due to pathologic changes in erythrocytes; however, echinocytes (crenated red cells) may be formed iatrogenically from insufficient blood in EDTA tubes or delay in slide preparation from collected blood. Low numbers of echinocytes are often seen in the peripheral blood of cats. Two of the most commonly observed shape abnormalities are schistocytes and acanthocytes. Schistocytes are small, irregularly shaped red cell fragments (Figure above). They result from mechanical trauma to circulating erythrocytes and, thus, are considered the hallmark of red cell fragmentation. In dogs, they are most frequently seen with DIC, microangiopathic hemolytic anemia, and neoplasms, particularly HSA; the latter can result in both DIC and microangiopathic hemolytic anemia. Schistocytes have been identified in 25% to 50% of dogs with HSA. However, other conditions, such as congestive heart failure, glomerulonephritis, myelofibrosis, chronic doxorubicin toxicosis, and increased red cell fragility associated with severe iron deficiency, may also result in the formation of schistocytes. Acanthocytes, or spur cells, are irregularly shaped erythrocytes containing membrane spicules that are unevenly distributed around the red cell surface. Unlike schistocytes, however, acanthocytes have not been fragmented and are similar in size to normal erythrocytes. These cells result from alterations in the cholesterol and/or phospholipid concentration in the red cell membrane and are seen in dogs with severe iron deficiency anemia, diffuse liver disease, portocaval shunts, high-cholesterol diets, or HSA. The mechanism underlying acanthocyte formation in dogs with HSA is not completely understood. Coexistence of acanthocytes and schistocytes in a dog with anemia is highly suggestive of HSA.

The density of the red color in erythrocytes is dependent on the concentration of hemoglobin. Changes in hemoglobin concentration can be in the form of polychromasia or hypochromasia. Polychromasia is defined by the presence of basophilic appearing erythrocytes. Polychromatophilic erythrocytes are seen in regenerative anemias when immature erythrocytes with decreased hemoglobin and increased amounts of RNA are released from the bone marrow. Numbers of polychromatophilic cells correlate well with numbers of reticulocytes. There are two types of reticulocytes in feline blood, aggregate reticulocytes and punctate reticulocytes (these are discussed in more detail below). Polychromasia or the number of reticulocytes present is the only peripheral blood findings that can be used to determine if an anemia is regenerative or not. Erythrocytes are termed hypochromic if they stain less intensely red than normal. Generally, the hemoglobin in hypochromic patients is concentrated around the periphery of the cells causing a larger than normal central pallor. Normal canine erythrocytes have a central pallor with a diameter that is equal to approximately one-third of the diameter of the cell. In iron deficient dogs, the central pallor of most of the erythrocytes is half the diameter of the cell or greater, allowing the morphologic identification of hypochromasia. Hypochromatic erythrocytes result from decreased hemoglobin, most often associated with iron deficiency. Most iron deficiencies in domestic animals are due to chronic blood loss. Normal feline erythrocytes have a very small or no central pallor. In iron deficient cats, the central pallor is visualized in most, if not all of the erythrocytes and usually occupies 1/3 to 1/2 the cell diameter, allowing the morphologic identification of hypochromasia. Most (>95%) of all iron deficiencies are the result of prolonged or chronic blood loss. This is typically due to either external parasitism or GI neoplasms.

Platelet Evaluation

Platelet evaluation begins with the search for clumps using a scanning objective (Figure upper right). If platelet clumps are observed, quantitative assessment of platelets will be falsely reduced. An estimation of platelet numbers is done using the 100x oil emersion lens. The formulas for estimation of platelet numbers are (Dog): # cells/µl = (# cells per 100x oil field) x 15,000, and for the (Cat): # cells/µl = (# cells per 100x oil field) x 20,000. The technique of platelet evaluation is particularly useful in evaluating platelets in the cat since automated cell counts are often unreliable in assessing platelet numbers for this species. The overlap in platelet and erythrocyte size, along with the propensity for feline platelets to clump or aggregate makes it very difficult to obtain accurate platelet counts using most automated hematology analyzers. Mega platelets (Figure, lower right), platelets as large as or larger than erythrocytes, may indicate platelet regeneration due to a peripheral destruction or consumption of platelets. However, in the cat, platelets are often larger than erythrocytes due to both the larger size of the platelet (than in dogs) and the smaller size or the erythrocyte. Thrombocytopenia may result from a production problem in the marrow, or a loss in the peripheral circulation due to destruction or consumption of platelets. A bone marrow evaluation may be necessary to make this distinction.

Leukocyte Evaluation

Leukocyte evaluation begins using a scanning objective by observing the mature neutrophil as the predominant cell type and identifying the presence of immature neutrophils (bands, metamyelocytes or myelocytes) or reactive changes in monocytes and lymphocytes. Large, immature blast cells should also be identified at this time. These pathological changes and other changes are then evaluated more closely using the 100x, oil immersion lens.

Left shift - the presence of excessive numbers of immature neutrophils (>300 bands/Fl of blood) in the peripheral blood. Most of these cells are band neutrophils with fewer metamyelocytes (Figures on right), myelocytes and progranulocytes (promyelocytes) in decreasing order of frequency. When the most immature forms (metamyelocytes and myelocytes) are relatively few compared to the band neutrophil population, the left shift is said to be pyramidal and complete. This is a favorable response. The extent of the left shift (how immature the cells are) will indicate the severity of the disease. The magnitude of the response (numbers of immature cells) will indicate the ability of the bone marrow to respond to the disease.

Regenerative left shift - a left shift in which there is typically a neutrophilia and there are a higher numbers of mature cells than immature. This is a favorable response where the bone marrow has had sufficient time (3–5 days) to respond to peripheral demands for neutrophils.

Degenerative left shift - a left shift in which there are more immature neutrophils (bands, metamyelocytes and myelocytes) than mature neutrophils (segmented). Total neutrophils counts are typically low or only slightly elevated. This indicates that the reserve of mature neutrophils in the bone marrow has been depleted, has had insufficient time to respond, or cannot meet the overwhelming demand for neutrophils. In most species this is an unfavorable prognostic indicator.

Toxic change - One important change in neutrophils is the presence of cytoplasmic toxicity. It is particularly important to evaluate toxicity of neutrophils in patients with a leukocytosis, leukopenia or a left-shift. Toxicity is a cytoplasmic change which is usually associated with the presence of bacterial infections or toxins. It results from a maturation arrest in cell development, and therefore occurs in bone marrow precursor cells. Toxicity is semi-quantitated in order of increasing severity from +1 to +4. A +1 Toxicity is defined by the presence of Döhle bodies; small, basophilic aggregates of RNA in cytoplasm of cells. This may be normal if seen in low numbers of neutrophils in cats. A +2 Toxicity is defined as Döhle bodies and diffuse cytoplasmic basophilia. A +3 Toxicity would contain all of the above plus foamy cytoplasmic vacuolation, and a +4 Toxicity would have all of the above plus giantism and/or nuclear lysis.

Reactive Change

Lymphocytes do not develop toxicity, but may become reactive as a result of some antigenic stimulation from an infectious agent, neoplasm, or immune-mediated disease. The cytoplasm of reactive lymphocytes becomes more intensely basophilic, almost a royal blue. Reactive monocytes may also be seen if the cytoplasm becomes more intensely basophilic and vacuolated. This usually indicates a chronic inflammatory process or may be seen with hemoplasmas in the cat.

Neoplastic Cells in Circulation

Neoplastic cells, primarily those of hematopoietic origin, can also be identified in the peripheral blood. These cells would alert the clinician to the possibility of leukemia, however, in many cases this diagnosis is confirmed by evaluating the bone marrow. Hematopoietic blast cells may be of erythroid, granulocyte, monocyte, or rarely megakaryocyte origin (myeloproliferative disease), or lymphoid origin (lymphoproliferative disease). In general, blast cells are identified as large cells with nuclei often or more times the size of erythrocytes. They often have abnormal nuclear morphology including high N:C ratio, diffuse, altered chromatin pattern, and prominent nucleoli. The cytoplasm of these cells is often deeply basophilic. The presence of blast cells in the peripheral circulation would alert the clinician to the possibility of an acute leukemia, whereas the presence of unexplained elevations in mature leukocytes (neutrophils, lymphocytes, monocytes, eosinophils, or basophils) would suggest the possibility of a chronic leukemia. In addition to being classified as acute or chronic depending on the maturity of the neoplastic cell, leukemias are also classified as a myeloproliferative (erythroid, myelogenous, or monocytic leukemia) or a lymphoproliferative (lymphoid leukemia) depending on the cell line from which the neoplastic population arises. Since leukemia is defined as a bone marrow neoplasm of the hematopoietic cells, a bone marrow evaluation is often required to make a definitive diagnosis.

A list of the various types of acute and chronic leukemias is provided. In general, chronic leukemias have a better long-term prognosis than acute leukemias and lymphoid leukemias have a better prognosis than myeloproliferative disorders.

• Lymphoid leukemias
• Chronic lymphoid leukemia
• Acute lymphoblastic leukemia (ALL)
• Chronic lymphocytic leukemia (CLL)
• Acute myeloid leukemias
• Chronic myeloid leukemias
• Acute myelogenous leukemia (AML)
• Chronic myelogenous leukemia (CML)
• Erythroid leukemia
• Eosinophilic leukemia
• Acute myelomonocytic leukemia
• Basophilic leukemia
• Acute monocytic leukemia
• Polycythemia vera
• Megakaryoblastic leukemia
• Acute myelogenous leukemia
• Acute lymphocytic leukemia
• Chronic lymphocytic leukemia