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The complete blood cell count: a powerful diagnostic tool
Vet Clin Small Anim 33 (2003) 1207–1222
The complete blood cell count: a powerful diagnostic tool Anne M. Barger, DVM, MS Department of Pathology, University of Illinois, College of Veterinary Medicine, 1008 Hazelwood Drive, Urbana, IL 61801, USA
The complete blood cell count (CBC) is an important and powerful diagnostic tool as well as a component of a minimum database. It can be used to monitor response to therapy, to gage the severity of an illness, or as a starting point for formulating a list of differential diagnoses. Interpretation of the CBC can be broken down into three sections: evaluation of the erythrocytes, leukocytes, and platelets. Each of these parameters can be interpreted individually; however, integration of the data is important for the highest diagnostic yield.
Erythrocytes To evaluate erythrocytes appropriately, results of the red blood cell count (RBC), packed cell volume (PCV), hemoglobin, mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH) must be scrutinized. The peripheral blood smear can provide additional information through examination of the red blood cell morphology. The PCV is measured as a percentage of packed cells in whole blood spun in a microhematocrit tube. The hematocrit, however, is a calculation using MCV and RBC values from an automated hematology analyzer. For the purpose of this article, PCV is used throughout. The evaluation of erythrocytes should begin by interpreting the results of the PCV and total protein. The PCV is a reflection of the circulating red cell mass. If the PCV is decreased, the animal is anemic, whereas an elevated PCV indicates polycythemia. Concurrent measurement of the total protein can further assist in interpretation of the PCV. When the total protein is elevated, dehydration or inflammation should be considered. It is important to
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remember that the presence of dehydration may ‘‘mask’’ an anemia. Thus, the PCV may be normal or even elevated, but once the patient has been rehydrated, an anemia may be evident. Dehydration may be the underling cause for polycythemia; fluid losses from the vascular compartment may result in a relative increase in the red cell mass. A decreased total protein in an anemic patient could indicate blood loss as the cause of anemia. Anemia Anemia is characterized by a decreased PCV, hemoglobin, and RBC in a normally hydrated animal. Further evaluation of the CBC is important to classify the anemia as regenerative or nonregenerative. This is likely to assist in determining the underlying cause of the anemia. The reticulocyte count is considered the gold standard in evaluating the animal’s response to the anemia. Reticulocytes are immature red blood cells with increased RNA and organelles, such as mitochondria and ribosomes. Methodology for reticulocyte counts includes vital stains, such as New Methylene Blue, and flow cytometry [1]. Additionally, peripheral blood smears stained with Romanowsky stain can be evaluated for polychromasia, which is a reflection of the reticulocyte response. All polychromatophilic red blood cells are reticulocytes; however, all reticulocytes are not polychromatophilic [2]. Thus, evaluation of the blood smear for polychromasia should only serve as a subjective estimate for the presence or absence of a regenerative response and may, in fact, underestimate the actual reticulocyte response. Calculation of the absolute reticulocyte count is the preferred method of reticulocyte enumeration [3]. Multiplying the percentage of aggregate reticulocytes by the RBC gives the absolute number of circulating reticulocytes. A normal dog can have up to 1% or about 60,000 reticulocytes/lL of blood. Healthy cats generally have less than 0.4% or up to 40,000 reticulocytes/lL of blood. Thus, an absolute value of 60,000 reticulocytes/lL in the dog and an absolute value of 40,000 reticulocytes/lL in the cat are the minimum values for indicating a regenerative response. An additional distinction must be made in cats between aggregate and punctate reticulocytes (Fig. 1). The aggregate reticulocytes are similar to those observed in dogs and are a reflection of current bone marrow activity. Aggregate reticulocytes in the cat mature into punctate reticulocytes, however. Punctate reticulocytes increase with erythropoiesis, but the increase is delayed and may persist for 3 to 4 weeks after the bone marrow response [4]. A healthy cat may have up to 17% punctate reticulocytes in circulation. The absolute reticulocyte count should only include aggregate reticulocytes, because these cells are used to evaluate the regenerative capability of the bone marrow. Many factors influence the reticulocyte response, including duration and severity of anemia, species difference, and age and health status of the animal. All these factors must be considered in determining the adequacy of response. Ultimately, the anemia must cause hypoxia at the level of the kidney to
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Fig. 1. New methylene blue–stained blood smear. Aggregate reticulocytes from a cat with regenerative anemia.
stimulate erythropoietin release, leading to a bone marrow response. It may take 3 to 5 days before the presence of a marrow response is reflected in the peripheral blood. Before this, the anemia may appear nonregenerative. In early blood loss or destruction, erythroid hyperplasia in the marrow precedes an elevated reticulocyte count. Therefore, bone marrow aspiration can be beneficial for immediate assessment of the regenerative response. The red blood cell indices MCV, MCH, and MCHC are used to evaluate overall red cell size and hemoglobin concentration. These indices can be beneficial in assessment of anemic patients. The terms macrocytic, normocytic, and microcytic are used to reflect the MCV and overall cell size. The terms hypochromic and normochromic refer to the MCH and MCHC and the overall hemoglobin concentration. Rarely is the term hyperchromic used. An elevated MCHC or MCH is usually the result of in vitro or in vivo hemolysis. In addition, the presence of interfering substances, such as lipemia or Heinz bodies, may significantly elevate these two indices. Most anemias are normocytic and normochromic. In markedly regenerative anemias, indices may indicate a macrocytic and hypochromic anemia (ie, increased MCV, decreased MCHC), reflecting the increased size and decreased hemoglobin of the reticulocytes. This morphologic classification is consistent with a regenerative anemia. Indices are insensitive indicators of regeneration, however, and may be normal despite the presence of a regenerative response. A slightly more sensitive indicator is the combination of an elevated MCV and red cell distribution width (RDW) [5]. Nevertheless, the presence of an elevated absolute reticulocyte count is the best measure of regeneration. As outlined in Box 1, an elevated MCHC or
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Box 1. Interpreting instrument-derived red blood cell indices Mean corpuscular volume Macrocytosis (increased mean cell volume) Reticulocytosis (regenerative anemia) Acute blood loss (>3 days) Acute hemolytic anemia (>3 days) Early iron deficiency in young animals Hereditary microcytosis of toy and miniature poodles (no anemia) Stomatocytosis Hereditary (Alaskan Malamute, Miniature Schnauzer, Drentse Patrijshond) Acquired Red blood cell agglutination (immune-mediated hemolytic anemia) Macrocytosis of feline leukemia virus Vitamin B12 or folic acid deficiency (unlikely in animals) Artifact (old blood sample, hypertonic red blood cells in isotonic or hypotonic fluid) Numerous large platelets or white blood cells measured as red blood cells; most frequently observed in severely anemic cats Microcytosis (decreased mean cell volume) Absolute iron deficiency (decreased bone marrow iron stores, serum ferritin, and serum iron) Dietary deficiency in puppies and kittens Chronic blood loss in young and adult animals Ineffective iron use (increased bone marrow iron and serum ferritin and decreased serum iron) Anemia of chronic disease (generally normochromic) Portal systemic shunts may or may not be associated with anemia Hereditary microcytosis (Akita, Shiba Inu) Familial dyserythropoiesis of English Springer Spaniels Red blood cell crenation Overanticoagulated sample Acute reaction of red blood cells to hypertonic fluid (hyperglycemia, hypernatremia, azotemia) Red blood cell fragments or platelets measured as red blood cells Mean corpuscular hemoglobin concentration Hyperchromasia (increased mean corpuscular hemoglobin concentration)
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Hemolysis in vitro or in vivo Spectrophotometric interference (turbidity/color) Lipemia Heinz bodies White blood cell count greater than 50,000 cells/lL Icterus (can interfere with spectrophotometric reading of hemoglobin) Spherocytes formed by membrane loss Transfusion with hemoglobin-based oxygen-carrying solution Hypochromasia (decreased mean corpuscular hemoglobin concentration) Reticulocytosis Acute blood loss for longer than 3 days Acute hemolytic anemia for longer than 3 days Early iron deficiency in young animals Absolute iron deficiency (decreased bone marrow iron stores, decreased serum ferritin) Dietary deficiency in puppies and kittens Chronic blood loss in young and adult animals Ineffective iron use (increased bone marrow iron and serum ferritin) Anemia of chronic disease (usually normochromic) Copper deficiency Vitamin B6 deficiency From Barger A, Grindem C. Analyzing the results of a complete blood cell count. Vet Med 2000;534–46; with permission.
MCH may indicate intravascular hemolysis if in vitro hemolysis and the presence of interfering substances are ruled out. Red blood cell destruction (hemolysis) and loss are causes of regenerative anemia. Differentiation of these ‘‘categories’’ requires evaluation of other parameters, including total protein, bilirubin, plasma color, and red blood cell morphology. A decreased PCV and total protein are often observed in blood loss anemia with normal bilirubin and plasma color. In contrast, the total bilirubin may be increased in certain hemolytic anemias, and, particularly with intravascular hemolysis, hemoglobin-tinted (reddish) plasma may be observed. The presence of abnormal red cell morphology, including spherocytes and Heinz bodies, may provide clues as to the cause of the anemia. Finally, the blood smear must be evaluated for red cell parasites, such as Babesia sp, Mycoplasma sp (formerly Haemobartonella sp), and Cytauxzoon sp, which may cause red cell destruction. Nonregenerative anemias are more common and are the result of decreased red blood cell production. The morphologic classification of these
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anemias is usually normocytic and normochromic or microcytic and hypochromic, having a count of less than 60,000 reticulocytes/lL and less than 40,000 reticulocytes/lL in the dog and cat, respectively. Many diseases can result in a nonregenerative anemia, with the most common cause being anemia of chronic disease (ACD). This anemia is associated with inflammatory processes, chronic infections, and disseminated neoplasia. A combination of mechanisms is implicated in ACD, including decreases in iron availability, erythrocyte survival, and response to erythropoietin [6]. ACD is typically normocytic and normochromic but may progress to a microcytic and hypochromic anemia. The PCV usually does not fall below 20% in ACD. Additional causes of normocytic and normochromic nonregenerative anemia include diseases that infiltrate or replace the normal architecture of the marrow (eg, myelofibrosis, myelophthisis), decreased erythropoietin concentrations associated with chronic renal disease, and infectious diseases that affect red cell maturation (eg, feline leukemia virus [FeLV], feline immunodeficiency virus [FIV], and Ehrlichia canis). FeLV subtype C has been also reported to cause pure red cell aplasia [7]. Many drug toxicities, including estrogen compounds, doxorubicin, and vincristine (in dogs), result in a nonregenerative anemia [8]. When immunemediated destruction is directed at erythroid precursors rather than at mature red cells or early in the course of hemolytic and blood loss anemias, a regenerative response is absent. In these instances, doing a bone marrow aspiration and running serial CBCs to evaluate the PCV are important for an appropriate interpretation. It must also be emphasized that the regenerative response that is typical of a hemolytic or destructive anemia may be dampened or absent in patients having a concurrent ACD. The patient’s history and physical examination findings along with additional testing, such as blood chemistry, urinalysis, bone marrow aspiration, and, if warranted, specific testing for infectious causes, must also be evaluated. Microcytic anemias are associated with either iron deficiency or inadequate iron use. Specific diseases in which the MCV may be decreased include chronic blood loss, portal systemic shunts, and iron deficiency in young animals or associated with chronic blood loss. Although microvascular dysplasias do not generally have a decreased MCV, if combined with a portal systemic shunt, they can result in a microcytosis [9]. The MCV and MCHC are insensitive indicators of changes in red cell size and hemoglobin content and thus may not detect mild or early changes [10]. Polycythemia Polycythemia is characterized as an elevated PCV, RBC, and hemoglobin and may be further classified as relative or absolute. A PCV greater than 60%, except in sighthound breeds, should arouse suspicion of polycythemia. Relative polycythemia is most commonly encountered in dogs and cats; their total red cell mass is normal but appears increased as a result of
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reduction in plasma volume or redistribution of the red cell mass. Physiologic and pathologic causes of relative polycythemia may occur. Physiologic polycythemia results from the injection of a mass of concentrated erythrocytes into the circulating blood from the spleen. This response is caused by epinephrine and is usually transient. A pathologic process that often leads to a relative polycythemia is dehydration. In this case, the PCV is increased because of an overall decrease in plasma fluid volume. The patient’s clinical signs as well as other abnormal test values, which often include increases in albumin, total protein, blood urea nitrogen, and creatinine, may help to support this diagnosis. In absolute polycythemia, there is a true increase in the circulating red cell mass, which may be primary or secondary. The PCV, RBC, and hemoglobin are increased without an elevation in the total protein. Secondary polycythemia is caused by an underlying disease that results in overproduction of erythropoietin; it is relatively common in dogs and cats. The elevated concentration of erythropoietin is caused by either a compensatory physiologic response of the kidney to tissue hypoxia or an ancillary source of the hormone, such as a renal carcinoma. Differentials for secondary polycythemia should include pulmonary disease, cardiac disease, high altitude or other causes of overall tissue hypoxia, and renal neoplasia. In these cases, blood gas results should show that the arterial oxygen (PO2) is low. Primary polycythemia is an uncommon myeloproliferative disease known as polycythemia vera (PV). These patients have a low normal to decreased erythropoietin concentration with normal arterial oxygen levels. Further, the absolute numbers of reticulocytes are not increased in these patients, their red cell fragility is normal, and Na/K pump seems to be functional [11] Human patients with PV often have elevated platelets and white blood cell count (WBC); however, this is an inconsistent finding in dogs [12]. Causes of secondary polycythemia need to be eliminated first before a diagnosis of PV can be made (Fig. 2). Bone marrow evaluation is not beneficial in these patients, because erythroid hyperplasia is commonly observed for both primary and secondary polycythemia. Ultimately, a detailed history through a physical examination, chest radiographs, cardiac evaluation, and laboratory tests may be needed to distinguish primary and secondary polycythemia. Red blood cell morphology Further information can be gained from evaluation of red blood cell morphology on the peripheral blood smear. Morphologic changes that may be associated with a regenerative response to anemia include an increased amount of polychromasia, which is often associated with the presence of Howell-Jolly bodies and nucleated red blood cells (nRBCs). When present in the absence of polychromasia, nRBCs may indicate pathologic processes like myeloproliferative diseases, bone marrow toxicity like lead poisoning,
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Fig. 2. Diagnostic approach to polycythemia. Elevated packed cell volume patient is dehydrated.
and splenic disease or lack of a spleen. Anisocytosis indicates an overall variation in red cell size; it is most commonly caused by macrocytes or microcytes in combination with red cells that are normal in size. Macrocytes are often indicative of reticulocytes, because reticulocytes are larger immature cells. The presence of increased numbers of microcytes is commonly associated with iron deficiency. Other differentials for microcytes and macrocytes are outlined in the list in this article. Variation in cell shape (poikilocytosis) is also a useful morphologic change that may assist the clinician with formulating a diagnosis. For example, certain shape changes are highly suggestive of a particular pathologic process. The presence of red blood cell fragments or schistocytes resulting from mechanical trauma to circulating erythrocytes are often associated with disseminated intravascular coagulation. Spherocytes are densely stained red blood cells that have lost their central pallor (Fig. 3). These cells are commonly observed in immune-mediated hemolytic anemia (IMHA); if present in high numbers, they are almost pathognomic for this disease. Acanthocytes are red blood cells with multiple irregular surface projections. Their presence has been associated with underlying liver disease and with splenic hemangiosarcoma in dogs. The presence of Heinz bodies,
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Fig. 3. Canine peripheral blood smear from a dog with immune-mediated hemolytic anemia. Numerous spherocytes are observed as indicated by the arrows.
keratocytes, or eccentrocytes indicates oxidative damage to the red cell. Cats can have significant numbers of Heinz bodies with no clinical significance; however, increased numbers with anemia may indicate a pathologic process. Disorders in cats that are commonly associated with an increased incidence of Heinz body formation include hyperthyroidism, lymphoma, and diabetes mellitus [13]. Oxidant drugs or other compounds that cause a Heinz body anemia in cats include acetaminophen, propylene glycol, fish-based diets, propofol, and onions [14]. Although the hemoglobin of the dog is less vulnerable to oxidant damage, onions, zinc, naphthalene, and, rarely, vitamin K can induce Heinz body formation. Other specific red cell shape changes include echinocytes, commonly known as crenated red blood cells, which have numerous, short, evenly spaced surface projections. These cells are usually an artifact but have been reported in some dogs with lymphoma, glomerulonephritis, and snake envenomation [15]. Dacryocytes are teardrop-shaped cells and can be observed with myeloproliferative diseases, myelofibrosis, and hypersplenism. Leptocytes, or target cells, can be observed with iron deficiency anemia. These cells can result in pseudomacrocytosis; they are thin and appear to be increased in size, but the cell volume is not increased [2]. Evaluation of the peripheral blood smear is also beneficial for identification of red blood cell parasites. In the cat, Mycoplasma haemofelis, Mycoplasma haemominutum, and Cytauxzoon felis may be observed. The hemotrophic mycoplasmas (M haemofelis and M haemominutum) can occur
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as individualized cocci, chains, or ring structures on the surface of the red blood cell. If there are many parasites on each of the red blood cells and the cat is severely anemic, M haemofelis infection is suspected. The parasitemia is often low and undetected by microscopic evaluations, however. It is recommended that a polymerase chain reaction (PCR), which is specific for these parasites, be run to determine if these cats are infected. C felis is a protozoan parasite that causes an often fatal disease in domestic cats, whereas the disease is usually asymptomatic in wild cats. Parasitized red blood cells usually contain a single ring-shaped structure or piroplasma-like organism (Fig. 4). In the dog, red cell parasites that have been reported include Babesia canis (Fig. 5), Babesia gibsoni, Mycoplasma hemocanis, and M haemominutum. The latter parasite has only been detected using PCR and not by microscopic methods. Leukocytes Evaluation of the leukocytes involves interpretation of the white blood cell parameters, including the WBC, differential count, and white blood cell morphology. An elevated WBC is called a leukocytosis, whereas a decreased WBC is a leukopenia. A markedly elevated leukocytosis, greater than 70,000 lL in the cat and greater than 65,000 lL in the dog is a poor prognostic indicator [16,17]. Further evaluation of the leukocytosis or leukopenia involves examination of the differential. A manual differential count is performed by counting 100 to 200 cells on the peripheral blood smear, giving
Fig. 4. Peripheral blood smear from a cat infested with Cytauxzoon felis. Piroplasmic intracellular organisms are indicated by the arrow.
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Fig. 5. Canine blood smear. Intracellular Babesia canis organisms are indicated by the arrow. These organisms are often observed as large paired piroplasms.
a percentage of each cell type. This percentage must then be multiplied by the WBC to give an absolute value before attempting to interpret the results. It is the absolute numbers rather than the percentages that should be used to classify the differential as consistent with inflammation, stress, excitement, hypersensitivity, or neoplasia. Inflammation The most common changes associated with inflammation include a leukocytosis with mature neutrophilia, often with increased numbers of bands present, which is described as a left shift. If the number of bands is less than the segmented neutrophils, this is described as a regenerative left shift. Conversely, if the number of bands is greater than the segmented neutrophils, this is called a degenerative left shift and is a poor prognostic indicator. The total WBC is usually elevated in a regenerative left shift. The neutrophils should also be evaluated for toxic changes. These changes may include cytoplasmic basophilia, Dohle bodies, azurophilic granules, and foamy cytoplasm. In severe inflammatory responses, leukopenia can occur rather than leukocytosis. It is not uncommon to observe a degenerative left shift in these situations. The identification of toxic changes within leukocytes can be a valuable ‘‘clue’’ for the clinician when struggling to distinguish a leukopenia of inflammation from that of decreased production. The finding of toxic changes strongly supports an inflammatory response, whereas their absence
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favors a production problem. A careful evaluation of the patient’s history as well as measurement of fibrinogen or other acute-phase proteins may be beneficial in distinguishing a chronic inflammatory leukon from a stress response. Chronic inflammation often is associated with a mature neutrophilia, having no or few bands. These leukocyte changes are also seen in conjunction with a stress response. The presence of an elevated fibrinogen concentration in conjunction with toxic changes strongly supports a chronic inflammatory response. The degree of leukocyte elevation is also important, because these numbers should not exceed 30,000 leukocytes/lL in stress. Immune stimulation/lymphocytosis Persistent antigenic stimulation caused by infectious agents, such as E canis and certain protozoal organisms, or in response to a vaccination may result in a significant lymphocytosis. This reactive lymphocytosis can be difficult to differentiate from chronic lymphocytic leukemia (CLL) or lymphoma with spillover into the blood. A reactive lymphocytosis is generally accompanied by the presence of immunocytes or reactive lymphocytes having deeply basophilic cytoplasm. Further, the lymphocytosis usually disappears with recovery from disease. These features help to distinguish a reactive lymphocytosis from CLL. The presence of a lymphocytosis with immature lymphocytes in the blood, favors a diagnosis of lymphoma. A complete history, physical examination, and additional testing (ie, Ehrlichia serology) are also essential components necessary to define a lymphocytosis. Stress leukogram The stress leukogram is mediated by endogenous or exogenous glucocorticoid release rather than by the epinephrine release associated with excitement. Characteristic changes include a mature neutrophilia, occasional hypersegmented neutrophils, lymphopenia, monocytosis (in the dog), and eosinopenia. The expression of L-selectin is downregulated by glucocorticoids in certain species, which may play an important role in the development of the mature neutrophilia [18]. The effects of glucocorticoids usually last for about 24 hours. With continuous endogenous release or long-term steroid use, however, the changes may be more sustained, especially the lymphopenia and eosinopenia. There is often a component of stress that can be recognized on the leukogram of any patient with an inflammatory lesion. A characteristic stress leukogram is a common finding in hyperadrenocorticism. Excitement The response to epinephrine is immediate but short-lived. It causes a transient neutrophilia by shifting cells from the marginal pool into the
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circulating pool. Because the total WBC is a reflection of the numbers of cells in the circulating pool, a leukocytosis is observed. The leukocytosis is caused by increases in lymphocytes (lymphocytosis) and mature neutrophils (neutrophilia). The absence of toxic changes in the neutrophils and presence of a lymphocytosis help to distinguish this leukocytosis from an inflammatory leukocytosis. Another change on the CBC that may support an epinephrine response is the presence of an elevated PCV (relative polycythemia, physiologic). The epinephrine response is more common in young animals, cats, and horses. If a second sample could be taken under more ‘‘relaxed’’ conditions, these changes would likely resolve. Hypersensitivity Eosinophils mediate the hypersensitivity response. Allergic, parasitic, and paraneoplastic syndromes should be considered as possible causes of eosinophilia. Depending on the level of the eosinophilia, eosinophilic leukemia and hypereosinophilic syndrome (HES, persistent eosinophilia of undefined cause) should also be considered. The absolute numbers of eosinophils may be quite high ([5000 eosinophils/lL) in these conditions. All possible causes of eosinophilia should be ruled out before a diagnosis of eosinophilic leukemia or HES can be made. Neoplasms that have been associated with eosinophilia as a paraneoplastic response include T-cell lymphoma and mast cell neoplasia. Hematopoietic neoplasia A leukocytosis or leukopenia may be observed with hematopoietic neoplasias. It is more common for patients with leukemia to present with a marked leukocytosis. If the patient has an acute leukemia, many immature cells, or blasts, are observed on the blood smear. There are two broad categories of acute leukemias: acute lymphoblastic leukemia (ALL) and acute myeloid leukemias (AML). Circulating blasts, usually in low numbers, may be also observed with stage V lymphoma. Chronic leukemias can be more difficult to diagnose but usually have gradually increasing numbers of differentiated hemopoietic cell in the blood, resulting in a marked leukocytosis (chronic myelogenous leukemia [CML] and chronic lymphocytic leukemia [CLL]), erythrocytosis (PV), or thrombocytosis (essential thrombocythemia [ET]). CML and CLL can only be diagnosed when no evidence of underlying inflammation or antigenic stimulation is identified in the presence of a marked leukocytosis. The absence of reactive lymphocytes or toxic changes in the neutrophils may also support a diagnosis of chronic leukemia, because these changes are more commonly associated with immune stimulation or inflammation, respectively. Hematopoietic neoplasia can result in a leukopenia, especially if myelophthisis or myelofibrosis has occurred or if the patient is receiving chemotherapy. Neutropenia can be a limiting factor for treatment.
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Additionally, aleukemic leukemia has been reported [19]. These patients commonly have a cytopenia of one or more cell lineages, which prompts a marrow evaluation. The presence of greater than 30% blasts in the bone marrow aspiration is diagnostic for an acute leukemia, despite the absence of blasts in the peripheral blood.
Platelets Platelets are small, round to elongate, cytoplasmic fragments of megakaryocytic origin. They have fine reddish-purple granules scattered throughout their cytoplasm and primarily function in hemostasis. The platelet number, size, and morphology are evaluated as part of the CBC. Platelet counts can be performed on an automated cell counter, performed manually via a hemacytometer, or estimated from a peripheral blood smear. The blood smear should also be evaluated for platelet clumps because they can falsely decrease the platelet count by all three methods. If platelet clumps are absent, an estimate of 8 to 20 platelets per 100 oil immersion field indicates that the numbers are adequate in the dog and cat (1 platelet 20,000/lL or 8–20 platelets per 100 oil immersion field ¼ 160,000– 400,000/lL). Platelet size can also affect the platelet count via automated methods, because the larger platelets may be counted with the red blood cells; if they are too small, the platelets are not counted at all. A platelet estimate from the blood smear may be beneficial in identifying any discrepancies between methods. Despite a low platelet count, the presence of large densely stained platelets or macroplatelets suggests active thrombopoiesis, whereas smaller platelet may suggest a production problem. Additionally, the mean platelet volume (MPV) can be determined on automated counters, which is a more accurate determination of their overall size. Thrombocytopenia Thrombocytopenia refers to a true decrease in platelet numbers, which is the most common platelet abnormality encountered. Thrombocytopenia, like anemia, may be caused by decreased production, increased destruction, or sequestration or loss. The presence of giant platelets suggests a regenerative response from the bone marrow; therefore, if giant platelets are observed, decreased production is a less likely cause. Additionally, an elevated MPV can be a good indicator of bone marrow response [20]. A normal MPV may indicate acute thrombocytopenia or nonregenerative disorders. Microplatelets with a decreased MPV have been reported in dogs suspected of having immune-mediated thrombocytopenia. Destruction of platelets is usually immune mediated; however, infectious etiologies like Ehrlichia platys and canine distemper virus can cause thrombocytopenia via destruction [21]. Other infectious causes may lead to platelet
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consumption by triggering disseminated intravascular coagulation; these include leptospirosis, infectious canine hepatitis, and salmonellosis [22]. Those causes associated with decreased production of platelets are most often accompanied by other cytopenias. These include bone marrow toxicity, myelophthisis, myelofibrosis, and primary bone marrow neoplasia. Certain infectious causes, including FeLV, FIV, panleukopenia, and parvoviral infections, result in decreased platelet production and other cytopenia. Similarly, drugs associated with bone marrow toxicity that may lead to the development of multiple cytopenias include estrogen, trimethoprim sulfa, busulfan, 5-fluorouracil, 6-thioguanine, doxorubicin, daunomycin, cisplatin, and carboplatin [8]. Thrombocytosis Thrombocytosis is an increased platelet count and may be reactive or primary. Reactive thrombocytosis has been reported in various conditions, including acute or chronic inflammation, iron deficiency, and hyperadrenocorticism. In dogs and cats, the most common diseases categories associated with thrombocytosis are neoplasia, gastrointestinal disorders, and endocrine disorders. Physiologic thrombocytosis may occur as a result of increased mobilization of platelets from splenic and nonsplenic stores; pulmonary pools of platelets can be mobilized during mild exercise, whereas splenic pools are mobilized as part of an epinephrine response. These responses are transient, and only mild to moderate increases are usually seen. Autonomous thrombocytosis is a myeloproliferative disorder that occurs as a primary disorder, ET, or in association with other hematopoietic neoplasias, including PV and chronic granulocytic leukemia (it also occurs inconsistently with acute megakaryocytic leukemia). It is not uncommon for platelet counts to be greater than 1 million/lL of blood in these proliferative disorders. Bone marrow aspiration is beneficial to differentiate these disorders. Summary In conclusion, the CBC can be a powerful diagnostic tool. Appropriate evaluation of all aspects of the CBC can lead to a specific diagnosis or assist in ruling out many diseases. To gain the full benefit of the CBC, it must be used in conjunction with a good history and physical examination as well as with additional components of the minimum database, which include a chemistry panel and urinalysis. References [1] Perkins P, Grindem C, Cullins L. Flow cytometric analysis of punctate and aggregate reticulocytes in phlebotomized cats. Am J Vet Res 1995;56(12):1564–9. [2] Pierre R. Red cell morphology and the peripheral blood film. Clin Lab Med 2002;22(1): 25–60.
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[3] Tvedten H, Weiss D. Classification and laboratory evaluation of anemia. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th edition. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 143–50. [4] Barger A, Grindem C. Analyzing the results of a complete blood cell count. Vet Med 2000;534–46. [5] Neiger R, Hadley J, Pfeiffer D. Differentiation of dogs with regenerative and nonregenerative anemia on the basis of their red cell distribution width and mean corpuscular volume. Vet Rec 2002;150(14):431–4. [6] Waner T, Harrus S. Anemia of inflammatory disease. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th edition. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 205–9. [7] Abkowitz J. Retrovirus-induced feline pure red cell aplasia: pathogenesis and response to suramin. Blood 1991;77:1442–51. [8] Barger A, Grindem C. Hematologic abnormalities associated with cancer therapy. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th edition. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 676–81. [9] Allen L, Stobie D, Mauldin N, Baer K. Clinicopathologic features of dogs with hepatic microvascular dysplasia with and without portosystemic shunts: 42 cases (1991–1996). J Am Vet Med Assoc 1999;214(2):218–20. [10] Tvedten H. Morphologic classification of anemia. Vet Clin Pathol 1999;28(3):80–2. [11] Comazzi S, Sacchet A, Milani F, Paltrinieri S, Agnes F. Some aspects of erythrocyte metabolism in a dog with polycythemia vera. Vet Rec 2000;147(12):331–4. [12] McGrath C, Krawiec D, Johnston S. Canine polycythemia vera: a review of diagnostic features. Vet Med Small Anim Clin 1982;4:611–3. [13] Christopher M. Relation of endogenous Heinz bodies to disease and anemia in cats: 120 cases (1978–1987). J Am Vet Med Assoc 1989;194:1089–95. [14] Desnoyers M. Anemias associated with Heinz bodies. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th edition. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 178–84. [15] Walton R, Brown D, Hamar D, Meador V, et al. Mechanisms of echinocytosis induced by Crotalus atrox venom. Vet Pathol 1997;34:442–9. [16] Lucroy M, Madewell B. Clinical outcome and associated diseases in dogs with leukocytosis and neutrophilia: 118 cases (1996–1998). J Am Vet Med Assoc 1999;214(6):805–7. [17] Lucroy M, Madewell B. Clinical outcome and diseases associated with extreme neutrophilic leukocytosis in cats: 104 cases (1991–1999). J Am Vet Med Assoc 2001; 218(5):736–9. [18] Nakagawa M, Bondy G, Waisman D, et al. The effect of glucocorticoids on the expression of L-selectin on polymorphonuclear leukocytes. Blood 1999;93(8):2730–7. [19] Helfand S. Low-dose cytosine arabinoside-induced remission of lymphoblastic leukemia in a cat. J Am Vet Med Assoc 1987;191(6):707–10. [20] Sullivan P, Manning K, McDonald T. Association of MPV and bone marrow megakaryocytopoiesis in thrombocytopenic dogs: 60 cases (1984–1993). J Am Vet Med Assoc 1995;206(3):332–4. [21] Russell K, Grindem C. Secondary thrombocytopenia. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th edition. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 487–95. [22] Grindem C. Infectious and immune-mediated thrombocytopenia. In: Bonagura J, editor. Kirk’s current veterinary therapy XIII small animal practice. Philadelphia: WB Saunders; 2000. p. 438–42.
The complete blood cell count: a powerful diagnostic tool Anne M. Barger, DVM, MS Department of Pathology, University of Illinois, College of Veterinary Medicine, 1008 Hazelwood Drive, Urbana, IL 61801, USA
The complete blood cell count (CBC) is an important and powerful diagnostic tool as well as a component of a minimum database. It can be used to monitor response to therapy, to gage the severity of an illness, or as a starting point for formulating a list of differential diagnoses. Interpretation of the CBC can be broken down into three sections: evaluation of the erythrocytes, leukocytes, and platelets. Each of these parameters can be interpreted individually; however, integration of the data is important for the highest diagnostic yield.
Erythrocytes To evaluate erythrocytes appropriately, results of the red blood cell count (RBC), packed cell volume (PCV), hemoglobin, mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH) must be scrutinized. The peripheral blood smear can provide additional information through examination of the red blood cell morphology. The PCV is measured as a percentage of packed cells in whole blood spun in a microhematocrit tube. The hematocrit, however, is a calculation using MCV and RBC values from an automated hematology analyzer. For the purpose of this article, PCV is used throughout. The evaluation of erythrocytes should begin by interpreting the results of the PCV and total protein. The PCV is a reflection of the circulating red cell mass. If the PCV is decreased, the animal is anemic, whereas an elevated PCV indicates polycythemia. Concurrent measurement of the total protein can further assist in interpretation of the PCV. When the total protein is elevated, dehydration or inflammation should be considered. It is important to
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remember that the presence of dehydration may ‘‘mask’’ an anemia. Thus, the PCV may be normal or even elevated, but once the patient has been rehydrated, an anemia may be evident. Dehydration may be the underling cause for polycythemia; fluid losses from the vascular compartment may result in a relative increase in the red cell mass. A decreased total protein in an anemic patient could indicate blood loss as the cause of anemia. Anemia Anemia is characterized by a decreased PCV, hemoglobin, and RBC in a normally hydrated animal. Further evaluation of the CBC is important to classify the anemia as regenerative or nonregenerative. This is likely to assist in determining the underlying cause of the anemia. The reticulocyte count is considered the gold standard in evaluating the animal’s response to the anemia. Reticulocytes are immature red blood cells with increased RNA and organelles, such as mitochondria and ribosomes. Methodology for reticulocyte counts includes vital stains, such as New Methylene Blue, and flow cytometry [1]. Additionally, peripheral blood smears stained with Romanowsky stain can be evaluated for polychromasia, which is a reflection of the reticulocyte response. All polychromatophilic red blood cells are reticulocytes; however, all reticulocytes are not polychromatophilic [2]. Thus, evaluation of the blood smear for polychromasia should only serve as a subjective estimate for the presence or absence of a regenerative response and may, in fact, underestimate the actual reticulocyte response. Calculation of the absolute reticulocyte count is the preferred method of reticulocyte enumeration [3]. Multiplying the percentage of aggregate reticulocytes by the RBC gives the absolute number of circulating reticulocytes. A normal dog can have up to 1% or about 60,000 reticulocytes/lL of blood. Healthy cats generally have less than 0.4% or up to 40,000 reticulocytes/lL of blood. Thus, an absolute value of 60,000 reticulocytes/lL in the dog and an absolute value of 40,000 reticulocytes/lL in the cat are the minimum values for indicating a regenerative response. An additional distinction must be made in cats between aggregate and punctate reticulocytes (Fig. 1). The aggregate reticulocytes are similar to those observed in dogs and are a reflection of current bone marrow activity. Aggregate reticulocytes in the cat mature into punctate reticulocytes, however. Punctate reticulocytes increase with erythropoiesis, but the increase is delayed and may persist for 3 to 4 weeks after the bone marrow response [4]. A healthy cat may have up to 17% punctate reticulocytes in circulation. The absolute reticulocyte count should only include aggregate reticulocytes, because these cells are used to evaluate the regenerative capability of the bone marrow. Many factors influence the reticulocyte response, including duration and severity of anemia, species difference, and age and health status of the animal. All these factors must be considered in determining the adequacy of response. Ultimately, the anemia must cause hypoxia at the level of the kidney to
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Fig. 1. New methylene blue–stained blood smear. Aggregate reticulocytes from a cat with regenerative anemia.
stimulate erythropoietin release, leading to a bone marrow response. It may take 3 to 5 days before the presence of a marrow response is reflected in the peripheral blood. Before this, the anemia may appear nonregenerative. In early blood loss or destruction, erythroid hyperplasia in the marrow precedes an elevated reticulocyte count. Therefore, bone marrow aspiration can be beneficial for immediate assessment of the regenerative response. The red blood cell indices MCV, MCH, and MCHC are used to evaluate overall red cell size and hemoglobin concentration. These indices can be beneficial in assessment of anemic patients. The terms macrocytic, normocytic, and microcytic are used to reflect the MCV and overall cell size. The terms hypochromic and normochromic refer to the MCH and MCHC and the overall hemoglobin concentration. Rarely is the term hyperchromic used. An elevated MCHC or MCH is usually the result of in vitro or in vivo hemolysis. In addition, the presence of interfering substances, such as lipemia or Heinz bodies, may significantly elevate these two indices. Most anemias are normocytic and normochromic. In markedly regenerative anemias, indices may indicate a macrocytic and hypochromic anemia (ie, increased MCV, decreased MCHC), reflecting the increased size and decreased hemoglobin of the reticulocytes. This morphologic classification is consistent with a regenerative anemia. Indices are insensitive indicators of regeneration, however, and may be normal despite the presence of a regenerative response. A slightly more sensitive indicator is the combination of an elevated MCV and red cell distribution width (RDW) [5]. Nevertheless, the presence of an elevated absolute reticulocyte count is the best measure of regeneration. As outlined in Box 1, an elevated MCHC or
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Box 1. Interpreting instrument-derived red blood cell indices Mean corpuscular volume Macrocytosis (increased mean cell volume) Reticulocytosis (regenerative anemia) Acute blood loss (>3 days) Acute hemolytic anemia (>3 days) Early iron deficiency in young animals Hereditary microcytosis of toy and miniature poodles (no anemia) Stomatocytosis Hereditary (Alaskan Malamute, Miniature Schnauzer, Drentse Patrijshond) Acquired Red blood cell agglutination (immune-mediated hemolytic anemia) Macrocytosis of feline leukemia virus Vitamin B12 or folic acid deficiency (unlikely in animals) Artifact (old blood sample, hypertonic red blood cells in isotonic or hypotonic fluid) Numerous large platelets or white blood cells measured as red blood cells; most frequently observed in severely anemic cats Microcytosis (decreased mean cell volume) Absolute iron deficiency (decreased bone marrow iron stores, serum ferritin, and serum iron) Dietary deficiency in puppies and kittens Chronic blood loss in young and adult animals Ineffective iron use (increased bone marrow iron and serum ferritin and decreased serum iron) Anemia of chronic disease (generally normochromic) Portal systemic shunts may or may not be associated with anemia Hereditary microcytosis (Akita, Shiba Inu) Familial dyserythropoiesis of English Springer Spaniels Red blood cell crenation Overanticoagulated sample Acute reaction of red blood cells to hypertonic fluid (hyperglycemia, hypernatremia, azotemia) Red blood cell fragments or platelets measured as red blood cells Mean corpuscular hemoglobin concentration Hyperchromasia (increased mean corpuscular hemoglobin concentration)
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Hemolysis in vitro or in vivo Spectrophotometric interference (turbidity/color) Lipemia Heinz bodies White blood cell count greater than 50,000 cells/lL Icterus (can interfere with spectrophotometric reading of hemoglobin) Spherocytes formed by membrane loss Transfusion with hemoglobin-based oxygen-carrying solution Hypochromasia (decreased mean corpuscular hemoglobin concentration) Reticulocytosis Acute blood loss for longer than 3 days Acute hemolytic anemia for longer than 3 days Early iron deficiency in young animals Absolute iron deficiency (decreased bone marrow iron stores, decreased serum ferritin) Dietary deficiency in puppies and kittens Chronic blood loss in young and adult animals Ineffective iron use (increased bone marrow iron and serum ferritin) Anemia of chronic disease (usually normochromic) Copper deficiency Vitamin B6 deficiency From Barger A, Grindem C. Analyzing the results of a complete blood cell count. Vet Med 2000;534–46; with permission.
MCH may indicate intravascular hemolysis if in vitro hemolysis and the presence of interfering substances are ruled out. Red blood cell destruction (hemolysis) and loss are causes of regenerative anemia. Differentiation of these ‘‘categories’’ requires evaluation of other parameters, including total protein, bilirubin, plasma color, and red blood cell morphology. A decreased PCV and total protein are often observed in blood loss anemia with normal bilirubin and plasma color. In contrast, the total bilirubin may be increased in certain hemolytic anemias, and, particularly with intravascular hemolysis, hemoglobin-tinted (reddish) plasma may be observed. The presence of abnormal red cell morphology, including spherocytes and Heinz bodies, may provide clues as to the cause of the anemia. Finally, the blood smear must be evaluated for red cell parasites, such as Babesia sp, Mycoplasma sp (formerly Haemobartonella sp), and Cytauxzoon sp, which may cause red cell destruction. Nonregenerative anemias are more common and are the result of decreased red blood cell production. The morphologic classification of these
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anemias is usually normocytic and normochromic or microcytic and hypochromic, having a count of less than 60,000 reticulocytes/lL and less than 40,000 reticulocytes/lL in the dog and cat, respectively. Many diseases can result in a nonregenerative anemia, with the most common cause being anemia of chronic disease (ACD). This anemia is associated with inflammatory processes, chronic infections, and disseminated neoplasia. A combination of mechanisms is implicated in ACD, including decreases in iron availability, erythrocyte survival, and response to erythropoietin [6]. ACD is typically normocytic and normochromic but may progress to a microcytic and hypochromic anemia. The PCV usually does not fall below 20% in ACD. Additional causes of normocytic and normochromic nonregenerative anemia include diseases that infiltrate or replace the normal architecture of the marrow (eg, myelofibrosis, myelophthisis), decreased erythropoietin concentrations associated with chronic renal disease, and infectious diseases that affect red cell maturation (eg, feline leukemia virus [FeLV], feline immunodeficiency virus [FIV], and Ehrlichia canis). FeLV subtype C has been also reported to cause pure red cell aplasia [7]. Many drug toxicities, including estrogen compounds, doxorubicin, and vincristine (in dogs), result in a nonregenerative anemia [8]. When immunemediated destruction is directed at erythroid precursors rather than at mature red cells or early in the course of hemolytic and blood loss anemias, a regenerative response is absent. In these instances, doing a bone marrow aspiration and running serial CBCs to evaluate the PCV are important for an appropriate interpretation. It must also be emphasized that the regenerative response that is typical of a hemolytic or destructive anemia may be dampened or absent in patients having a concurrent ACD. The patient’s history and physical examination findings along with additional testing, such as blood chemistry, urinalysis, bone marrow aspiration, and, if warranted, specific testing for infectious causes, must also be evaluated. Microcytic anemias are associated with either iron deficiency or inadequate iron use. Specific diseases in which the MCV may be decreased include chronic blood loss, portal systemic shunts, and iron deficiency in young animals or associated with chronic blood loss. Although microvascular dysplasias do not generally have a decreased MCV, if combined with a portal systemic shunt, they can result in a microcytosis [9]. The MCV and MCHC are insensitive indicators of changes in red cell size and hemoglobin content and thus may not detect mild or early changes [10]. Polycythemia Polycythemia is characterized as an elevated PCV, RBC, and hemoglobin and may be further classified as relative or absolute. A PCV greater than 60%, except in sighthound breeds, should arouse suspicion of polycythemia. Relative polycythemia is most commonly encountered in dogs and cats; their total red cell mass is normal but appears increased as a result of
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reduction in plasma volume or redistribution of the red cell mass. Physiologic and pathologic causes of relative polycythemia may occur. Physiologic polycythemia results from the injection of a mass of concentrated erythrocytes into the circulating blood from the spleen. This response is caused by epinephrine and is usually transient. A pathologic process that often leads to a relative polycythemia is dehydration. In this case, the PCV is increased because of an overall decrease in plasma fluid volume. The patient’s clinical signs as well as other abnormal test values, which often include increases in albumin, total protein, blood urea nitrogen, and creatinine, may help to support this diagnosis. In absolute polycythemia, there is a true increase in the circulating red cell mass, which may be primary or secondary. The PCV, RBC, and hemoglobin are increased without an elevation in the total protein. Secondary polycythemia is caused by an underlying disease that results in overproduction of erythropoietin; it is relatively common in dogs and cats. The elevated concentration of erythropoietin is caused by either a compensatory physiologic response of the kidney to tissue hypoxia or an ancillary source of the hormone, such as a renal carcinoma. Differentials for secondary polycythemia should include pulmonary disease, cardiac disease, high altitude or other causes of overall tissue hypoxia, and renal neoplasia. In these cases, blood gas results should show that the arterial oxygen (PO2) is low. Primary polycythemia is an uncommon myeloproliferative disease known as polycythemia vera (PV). These patients have a low normal to decreased erythropoietin concentration with normal arterial oxygen levels. Further, the absolute numbers of reticulocytes are not increased in these patients, their red cell fragility is normal, and Na/K pump seems to be functional [11] Human patients with PV often have elevated platelets and white blood cell count (WBC); however, this is an inconsistent finding in dogs [12]. Causes of secondary polycythemia need to be eliminated first before a diagnosis of PV can be made (Fig. 2). Bone marrow evaluation is not beneficial in these patients, because erythroid hyperplasia is commonly observed for both primary and secondary polycythemia. Ultimately, a detailed history through a physical examination, chest radiographs, cardiac evaluation, and laboratory tests may be needed to distinguish primary and secondary polycythemia. Red blood cell morphology Further information can be gained from evaluation of red blood cell morphology on the peripheral blood smear. Morphologic changes that may be associated with a regenerative response to anemia include an increased amount of polychromasia, which is often associated with the presence of Howell-Jolly bodies and nucleated red blood cells (nRBCs). When present in the absence of polychromasia, nRBCs may indicate pathologic processes like myeloproliferative diseases, bone marrow toxicity like lead poisoning,
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Fig. 2. Diagnostic approach to polycythemia. Elevated packed cell volume patient is dehydrated.
and splenic disease or lack of a spleen. Anisocytosis indicates an overall variation in red cell size; it is most commonly caused by macrocytes or microcytes in combination with red cells that are normal in size. Macrocytes are often indicative of reticulocytes, because reticulocytes are larger immature cells. The presence of increased numbers of microcytes is commonly associated with iron deficiency. Other differentials for microcytes and macrocytes are outlined in the list in this article. Variation in cell shape (poikilocytosis) is also a useful morphologic change that may assist the clinician with formulating a diagnosis. For example, certain shape changes are highly suggestive of a particular pathologic process. The presence of red blood cell fragments or schistocytes resulting from mechanical trauma to circulating erythrocytes are often associated with disseminated intravascular coagulation. Spherocytes are densely stained red blood cells that have lost their central pallor (Fig. 3). These cells are commonly observed in immune-mediated hemolytic anemia (IMHA); if present in high numbers, they are almost pathognomic for this disease. Acanthocytes are red blood cells with multiple irregular surface projections. Their presence has been associated with underlying liver disease and with splenic hemangiosarcoma in dogs. The presence of Heinz bodies,
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Fig. 3. Canine peripheral blood smear from a dog with immune-mediated hemolytic anemia. Numerous spherocytes are observed as indicated by the arrows.
keratocytes, or eccentrocytes indicates oxidative damage to the red cell. Cats can have significant numbers of Heinz bodies with no clinical significance; however, increased numbers with anemia may indicate a pathologic process. Disorders in cats that are commonly associated with an increased incidence of Heinz body formation include hyperthyroidism, lymphoma, and diabetes mellitus [13]. Oxidant drugs or other compounds that cause a Heinz body anemia in cats include acetaminophen, propylene glycol, fish-based diets, propofol, and onions [14]. Although the hemoglobin of the dog is less vulnerable to oxidant damage, onions, zinc, naphthalene, and, rarely, vitamin K can induce Heinz body formation. Other specific red cell shape changes include echinocytes, commonly known as crenated red blood cells, which have numerous, short, evenly spaced surface projections. These cells are usually an artifact but have been reported in some dogs with lymphoma, glomerulonephritis, and snake envenomation [15]. Dacryocytes are teardrop-shaped cells and can be observed with myeloproliferative diseases, myelofibrosis, and hypersplenism. Leptocytes, or target cells, can be observed with iron deficiency anemia. These cells can result in pseudomacrocytosis; they are thin and appear to be increased in size, but the cell volume is not increased [2]. Evaluation of the peripheral blood smear is also beneficial for identification of red blood cell parasites. In the cat, Mycoplasma haemofelis, Mycoplasma haemominutum, and Cytauxzoon felis may be observed. The hemotrophic mycoplasmas (M haemofelis and M haemominutum) can occur
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as individualized cocci, chains, or ring structures on the surface of the red blood cell. If there are many parasites on each of the red blood cells and the cat is severely anemic, M haemofelis infection is suspected. The parasitemia is often low and undetected by microscopic evaluations, however. It is recommended that a polymerase chain reaction (PCR), which is specific for these parasites, be run to determine if these cats are infected. C felis is a protozoan parasite that causes an often fatal disease in domestic cats, whereas the disease is usually asymptomatic in wild cats. Parasitized red blood cells usually contain a single ring-shaped structure or piroplasma-like organism (Fig. 4). In the dog, red cell parasites that have been reported include Babesia canis (Fig. 5), Babesia gibsoni, Mycoplasma hemocanis, and M haemominutum. The latter parasite has only been detected using PCR and not by microscopic methods. Leukocytes Evaluation of the leukocytes involves interpretation of the white blood cell parameters, including the WBC, differential count, and white blood cell morphology. An elevated WBC is called a leukocytosis, whereas a decreased WBC is a leukopenia. A markedly elevated leukocytosis, greater than 70,000 lL in the cat and greater than 65,000 lL in the dog is a poor prognostic indicator [16,17]. Further evaluation of the leukocytosis or leukopenia involves examination of the differential. A manual differential count is performed by counting 100 to 200 cells on the peripheral blood smear, giving
Fig. 4. Peripheral blood smear from a cat infested with Cytauxzoon felis. Piroplasmic intracellular organisms are indicated by the arrow.
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Fig. 5. Canine blood smear. Intracellular Babesia canis organisms are indicated by the arrow. These organisms are often observed as large paired piroplasms.
a percentage of each cell type. This percentage must then be multiplied by the WBC to give an absolute value before attempting to interpret the results. It is the absolute numbers rather than the percentages that should be used to classify the differential as consistent with inflammation, stress, excitement, hypersensitivity, or neoplasia. Inflammation The most common changes associated with inflammation include a leukocytosis with mature neutrophilia, often with increased numbers of bands present, which is described as a left shift. If the number of bands is less than the segmented neutrophils, this is described as a regenerative left shift. Conversely, if the number of bands is greater than the segmented neutrophils, this is called a degenerative left shift and is a poor prognostic indicator. The total WBC is usually elevated in a regenerative left shift. The neutrophils should also be evaluated for toxic changes. These changes may include cytoplasmic basophilia, Dohle bodies, azurophilic granules, and foamy cytoplasm. In severe inflammatory responses, leukopenia can occur rather than leukocytosis. It is not uncommon to observe a degenerative left shift in these situations. The identification of toxic changes within leukocytes can be a valuable ‘‘clue’’ for the clinician when struggling to distinguish a leukopenia of inflammation from that of decreased production. The finding of toxic changes strongly supports an inflammatory response, whereas their absence
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favors a production problem. A careful evaluation of the patient’s history as well as measurement of fibrinogen or other acute-phase proteins may be beneficial in distinguishing a chronic inflammatory leukon from a stress response. Chronic inflammation often is associated with a mature neutrophilia, having no or few bands. These leukocyte changes are also seen in conjunction with a stress response. The presence of an elevated fibrinogen concentration in conjunction with toxic changes strongly supports a chronic inflammatory response. The degree of leukocyte elevation is also important, because these numbers should not exceed 30,000 leukocytes/lL in stress. Immune stimulation/lymphocytosis Persistent antigenic stimulation caused by infectious agents, such as E canis and certain protozoal organisms, or in response to a vaccination may result in a significant lymphocytosis. This reactive lymphocytosis can be difficult to differentiate from chronic lymphocytic leukemia (CLL) or lymphoma with spillover into the blood. A reactive lymphocytosis is generally accompanied by the presence of immunocytes or reactive lymphocytes having deeply basophilic cytoplasm. Further, the lymphocytosis usually disappears with recovery from disease. These features help to distinguish a reactive lymphocytosis from CLL. The presence of a lymphocytosis with immature lymphocytes in the blood, favors a diagnosis of lymphoma. A complete history, physical examination, and additional testing (ie, Ehrlichia serology) are also essential components necessary to define a lymphocytosis. Stress leukogram The stress leukogram is mediated by endogenous or exogenous glucocorticoid release rather than by the epinephrine release associated with excitement. Characteristic changes include a mature neutrophilia, occasional hypersegmented neutrophils, lymphopenia, monocytosis (in the dog), and eosinopenia. The expression of L-selectin is downregulated by glucocorticoids in certain species, which may play an important role in the development of the mature neutrophilia [18]. The effects of glucocorticoids usually last for about 24 hours. With continuous endogenous release or long-term steroid use, however, the changes may be more sustained, especially the lymphopenia and eosinopenia. There is often a component of stress that can be recognized on the leukogram of any patient with an inflammatory lesion. A characteristic stress leukogram is a common finding in hyperadrenocorticism. Excitement The response to epinephrine is immediate but short-lived. It causes a transient neutrophilia by shifting cells from the marginal pool into the
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circulating pool. Because the total WBC is a reflection of the numbers of cells in the circulating pool, a leukocytosis is observed. The leukocytosis is caused by increases in lymphocytes (lymphocytosis) and mature neutrophils (neutrophilia). The absence of toxic changes in the neutrophils and presence of a lymphocytosis help to distinguish this leukocytosis from an inflammatory leukocytosis. Another change on the CBC that may support an epinephrine response is the presence of an elevated PCV (relative polycythemia, physiologic). The epinephrine response is more common in young animals, cats, and horses. If a second sample could be taken under more ‘‘relaxed’’ conditions, these changes would likely resolve. Hypersensitivity Eosinophils mediate the hypersensitivity response. Allergic, parasitic, and paraneoplastic syndromes should be considered as possible causes of eosinophilia. Depending on the level of the eosinophilia, eosinophilic leukemia and hypereosinophilic syndrome (HES, persistent eosinophilia of undefined cause) should also be considered. The absolute numbers of eosinophils may be quite high ([5000 eosinophils/lL) in these conditions. All possible causes of eosinophilia should be ruled out before a diagnosis of eosinophilic leukemia or HES can be made. Neoplasms that have been associated with eosinophilia as a paraneoplastic response include T-cell lymphoma and mast cell neoplasia. Hematopoietic neoplasia A leukocytosis or leukopenia may be observed with hematopoietic neoplasias. It is more common for patients with leukemia to present with a marked leukocytosis. If the patient has an acute leukemia, many immature cells, or blasts, are observed on the blood smear. There are two broad categories of acute leukemias: acute lymphoblastic leukemia (ALL) and acute myeloid leukemias (AML). Circulating blasts, usually in low numbers, may be also observed with stage V lymphoma. Chronic leukemias can be more difficult to diagnose but usually have gradually increasing numbers of differentiated hemopoietic cell in the blood, resulting in a marked leukocytosis (chronic myelogenous leukemia [CML] and chronic lymphocytic leukemia [CLL]), erythrocytosis (PV), or thrombocytosis (essential thrombocythemia [ET]). CML and CLL can only be diagnosed when no evidence of underlying inflammation or antigenic stimulation is identified in the presence of a marked leukocytosis. The absence of reactive lymphocytes or toxic changes in the neutrophils may also support a diagnosis of chronic leukemia, because these changes are more commonly associated with immune stimulation or inflammation, respectively. Hematopoietic neoplasia can result in a leukopenia, especially if myelophthisis or myelofibrosis has occurred or if the patient is receiving chemotherapy. Neutropenia can be a limiting factor for treatment.
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Additionally, aleukemic leukemia has been reported [19]. These patients commonly have a cytopenia of one or more cell lineages, which prompts a marrow evaluation. The presence of greater than 30% blasts in the bone marrow aspiration is diagnostic for an acute leukemia, despite the absence of blasts in the peripheral blood.
Platelets Platelets are small, round to elongate, cytoplasmic fragments of megakaryocytic origin. They have fine reddish-purple granules scattered throughout their cytoplasm and primarily function in hemostasis. The platelet number, size, and morphology are evaluated as part of the CBC. Platelet counts can be performed on an automated cell counter, performed manually via a hemacytometer, or estimated from a peripheral blood smear. The blood smear should also be evaluated for platelet clumps because they can falsely decrease the platelet count by all three methods. If platelet clumps are absent, an estimate of 8 to 20 platelets per 100 oil immersion field indicates that the numbers are adequate in the dog and cat (1 platelet 20,000/lL or 8–20 platelets per 100 oil immersion field ¼ 160,000– 400,000/lL). Platelet size can also affect the platelet count via automated methods, because the larger platelets may be counted with the red blood cells; if they are too small, the platelets are not counted at all. A platelet estimate from the blood smear may be beneficial in identifying any discrepancies between methods. Despite a low platelet count, the presence of large densely stained platelets or macroplatelets suggests active thrombopoiesis, whereas smaller platelet may suggest a production problem. Additionally, the mean platelet volume (MPV) can be determined on automated counters, which is a more accurate determination of their overall size. Thrombocytopenia Thrombocytopenia refers to a true decrease in platelet numbers, which is the most common platelet abnormality encountered. Thrombocytopenia, like anemia, may be caused by decreased production, increased destruction, or sequestration or loss. The presence of giant platelets suggests a regenerative response from the bone marrow; therefore, if giant platelets are observed, decreased production is a less likely cause. Additionally, an elevated MPV can be a good indicator of bone marrow response [20]. A normal MPV may indicate acute thrombocytopenia or nonregenerative disorders. Microplatelets with a decreased MPV have been reported in dogs suspected of having immune-mediated thrombocytopenia. Destruction of platelets is usually immune mediated; however, infectious etiologies like Ehrlichia platys and canine distemper virus can cause thrombocytopenia via destruction [21]. Other infectious causes may lead to platelet
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consumption by triggering disseminated intravascular coagulation; these include leptospirosis, infectious canine hepatitis, and salmonellosis [22]. Those causes associated with decreased production of platelets are most often accompanied by other cytopenias. These include bone marrow toxicity, myelophthisis, myelofibrosis, and primary bone marrow neoplasia. Certain infectious causes, including FeLV, FIV, panleukopenia, and parvoviral infections, result in decreased platelet production and other cytopenia. Similarly, drugs associated with bone marrow toxicity that may lead to the development of multiple cytopenias include estrogen, trimethoprim sulfa, busulfan, 5-fluorouracil, 6-thioguanine, doxorubicin, daunomycin, cisplatin, and carboplatin [8]. Thrombocytosis Thrombocytosis is an increased platelet count and may be reactive or primary. Reactive thrombocytosis has been reported in various conditions, including acute or chronic inflammation, iron deficiency, and hyperadrenocorticism. In dogs and cats, the most common diseases categories associated with thrombocytosis are neoplasia, gastrointestinal disorders, and endocrine disorders. Physiologic thrombocytosis may occur as a result of increased mobilization of platelets from splenic and nonsplenic stores; pulmonary pools of platelets can be mobilized during mild exercise, whereas splenic pools are mobilized as part of an epinephrine response. These responses are transient, and only mild to moderate increases are usually seen. Autonomous thrombocytosis is a myeloproliferative disorder that occurs as a primary disorder, ET, or in association with other hematopoietic neoplasias, including PV and chronic granulocytic leukemia (it also occurs inconsistently with acute megakaryocytic leukemia). It is not uncommon for platelet counts to be greater than 1 million/lL of blood in these proliferative disorders. Bone marrow aspiration is beneficial to differentiate these disorders. Summary In conclusion, the CBC can be a powerful diagnostic tool. Appropriate evaluation of all aspects of the CBC can lead to a specific diagnosis or assist in ruling out many diseases. To gain the full benefit of the CBC, it must be used in conjunction with a good history and physical examination as well as with additional components of the minimum database, which include a chemistry panel and urinalysis. References [1] Perkins P, Grindem C, Cullins L. Flow cytometric analysis of punctate and aggregate reticulocytes in phlebotomized cats. Am J Vet Res 1995;56(12):1564–9. [2] Pierre R. Red cell morphology and the peripheral blood film. Clin Lab Med 2002;22(1): 25–60.
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