Vet Clin Small Anim 33 (2003) 13351357

Clinical assessment of leukocytosis: distinguishing leukocytoses caused by inammatory, glucocorticoid, physiologic, and leukemic disorders or conditions
Steven L. Stockham, DVM, MS*, Kerry S. Keeton, DVM, PhD, Balazs Szladovits, DVM
Department of Diagnostic Medicine/Pathobiology, 1800 Denison Avenue, Kansas State University, Manhattan, KS 665065705, USA

Leukocytosis is an increased leukocyte concentration in a sample collected from the circulating blood pool. Leukocytosis results from changes in production, distribution, or use of neutrophils, lymphocytes, monocytes, eosinophils, basophils, or mast cells. Because the concentration of each type of leukocyte is determined by dierent factors, it is important to recognize that a leukocytosis is created by increased concentrations of individual cell types. The purpose of this article is to describe criteria that are used to distinguish the major disorders or conditions that produce a leukocytosis:  Inammation: depending on the type and duration of the inammatory or immune response, inammation may result in a neutrophilia, lymphocytosis, monocytosis, eosinophilia, mastocytemia, or, probably, basophilia. The leukocytosis involves altered production, distribution, and use of leukocytes.  Glucocorticoid response: glucocorticoid hormones (eg, cortisol) or drugs (eg, prednisolone) can alter the distribution and use of leukocytes to create a neutrophilia and perhaps a monocytosis.  Catecholamine response: catecholamines (ie, epinephrine, norepinephrine) can alter the distribution of leukocytes to create a neutrophilia, lymphocytosis, and possibly monocytosis or eosinophilia. The catecholamine response is associated with excitement, fright, and exercise.

* Corresponding author. E-mail address: Stockham@vet.k-state.edu (S.L. Stockham). 0195-5616/03/$ - see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0195-5616(03)00098-6

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 Neoplasia: the neoplastic transformation of a leukocyte precursor may result in uncontrolled leukocyte production; lymphoid neoplasia is the most common form in dogs and cats. If nonhemic neoplastic cells release granulocyte colony-stimulating factor (G-CSF) or similar substances, the animal may have a paraneoplastic neutrophilia or eosinophilia. Detection and characterization of a leukocytosis The complete blood cell count (CBC) includes a group of tests that characterize the cells in the peripheral blood, including the total leukocyte concentration (total white blood cell [WBC] count), individual leukocyte concentrations, and the microscopic appearance of leukocytes. Total leukocyte concentration It is important to know how the leukocyte concentration was determined so that the increased concentration can be correctly interpreted. Basically, there are four WBC count methods that can be a part of a CBC:  Manual WBC count: typically, this is completed with the Unopette (Becton Dickinson and Company, Franklin Lakes, NJ) system, in which the number of nucleated cells is counted in a dened volume of diluted blood in a hemocytometer. Thus, the result is the total nucleated cell concentration (TNCC), which represents the total WBC concentration if nucleated erythrocytes (eg, metarubricytes, rubricytes) are not present. If nucleated erythrocytes are present, their contribution to the TNCC must be removed to obtain a corrected WBC count [1].  Impedance WBC count: in the classic impedance count using the Coulter principle, the white cell impedance count (WIC) determines the number of nucleated cells in a dened volume of diluted blood. Thus, the WIC is a TNCC.  Optical WBC count: in the white cell optical count (WOC) of ow cytometers, the instrument directs a laser beam at cells as they pass in a diluted blood sample. The system distinguishes the cells based on the way they scatter light. Because nucleated erythrocytes scatter light differently than leukocytes, the WOC determines a total leukocyte concentration.  QBC method: in the quantitative buy coat (QBC) method, the total leukocyte concentration is estimated based on the percentage of blood volume occupied by leukocytes after centrifugation of blood in special tubes. This percentage (a leukocrit) is converted to a leukocyte concentration by a factor derived from the analysis of blood of healthy animals. Because of dierent buoyant properties, cells are separated into four major layers (erythrocytes, granulocytes, agranulocytes, and platelets) and leukocyte layers are distinguished by uorescence. Nucleated erythrocytes are located between the mature erythrocyte and granulocyte

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layers. Assuming that there is appropriate separation of cells, appropriate uorescent staining and detection, and the leukocytes have typical cell volumes, the total leukocyte concentration represents the sum of the estimated concentrations of granulocytes and agranulocytes. Determining concentrations of each type of leukocyte A major method of distinguishing the leukocytoses is by recognizing the patterns formed by the concentrations of each leukocyte type (eg, neutrophil concentration, lymphocyte concentration). To interpret those concentrations, it is important to know how the concentrations were obtained. Basically there are three methods:  Calculated from manual leukocyte dierential count. Leukocytes are dierentiated on a stained blood lm by a microscopic examination, and dierential percentages are obtained (eg, 70% of the leukocytes are neutrophils). The neutrophil concentration is the product of the total leukocyte concentration times the neutrophil percentage. Because there are many factors that inuence the analytic accuracy and precision of the dierential leukocyte count, the calculated concentration of leukocytes is at best a reasonable estimate and should be interpreted accordingly [2]. Factors that inuence the precision include quality of the sample (eg, fresh blood free of clots), distribution of cells on the slide (wellprepared lm), skills of microscopist (ability to identify cells accurately), and number of cells included in the dierential count. If a blood sample contains 10.0103 leukocytes/lL and 10 lL of blood is used to make the blood lm, the blood lm contains 100,000 leukocytes. If only 100 cells are included in the dierential count, only 0.1% of the leukocytes are identied. This low percentage can lead to sampling errors and poor reproducibility of the dierential leukocyte count. These potential variations are illustrated in Table 1. Table 1 was constructed using the 95% condence intervals of a 100-cell leukocyte dierential count [3] and assuming that the total WBC concentration was 20.0103/lL . For example, if the actual neutrophil percentage in a blood sample was 50% (last row in neutrophil group) and 100-cell dierential counts were completed 100 times, 95% of the determined neutrophil percentages would be between 39% and 61%. Accordingly, 95% of the calculated neutrophil concentrations would fall between 7.8103/lL and 12.2 103/lL, whereas, the true concentration would be 10.0103/lL. Thus, some results indicate a neutrophilia (>11.5103/lL), but others do not. Examination of Table 1 data shows that the imprecision of a manual leukocyte dierential count can result in variation in the calculated cell concentrations and thus, potentially, variations in interpretations. Precision of the dierential count can be improved by including more cells (eg, 200-cell count or 500-cell count), but then cost/benet ratios need to be considered.

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Table 1 Alterations in calculated cell concentrations because of analytic variation in 100-cell leukocyte dierential cell counts Total WBCs Lowa % Neutrophils Neutrophils Neutrophils Neutrophils Lymphocytes Lymphocytes Lymphocytes Lymphocytes Eosinophils Eosinophils Eosinophils 70 60 49 39 30 21 12 4 4 1 0 103/lL 14.0 12.0 9.8 7.8 6.0 4.2 2.4 0.8 0.8 0.2 0.0 % 80 70 60 50 40 30 20 10 10 5 2 20.0103/lL Actualb 103/lL 15.0 14.0 13.0 10.0 8.0 6.0 4.0 2.0 2.0 1.0 0.4 % 88 79 70 61 51 40 30 18 18 12 8 Highc 103/lL 17.6 15.8 14.0 12.2 10.2 8.0 6.0 3.6 3.6 2.4 1.6 Reference interval 103/lL 3.011.5 3.011.5 3.011.5 3.011.5 1.04.8 1.04.8 1.04.8 1.04.8 0.10.8 0.10.8 0.10.8

Concentrations outside the reference intervals are in bold. Abbreviation: WBC, white blood cell. a Low: lower limit of a 95% condence interval as reported by Rumke [3]. b Actual: actual percentage or concentrations of leukocytes in the blood. c High: upper limit of a 95% condence interval as reported by Rumke [3].

 Calculated from an electronic leukocyte dierential count. Some optical and impedance counters are designed to dierentiate leukocytes based on dened properties. Typically, the properties are dened to distinguish leukocytes from healthy animals in a specied species. Therefore, the settings need to be changed for each species and may not be able to identify abnormal leukocytes accurately. The electronic cell count is more reproducible, because the instrument examines thousands of leukocytes and thus removes the errors created by examining a small sample of a population [2].  QBC (Becton Dickinson and Company, Franklin Lakes, NJ) method. As described previously, this method separates leukocytes into two groups: granulocytes (neutrophils, eosinophils, and basophils) and agranulocytes (lymphocytes and monocytes); an eosinophil layer can be identied in canine blood. A microscopic leukocyte dierential count is needed to calculate the concentrations of individual leukocyte populations. Absolute versus relative changes Even though calculated leukocyte concentrations are prone to errors, interpreting the calculated concentrations is highly recommended compared with interpreting the percentages of a leukocyte dierential count. A percentage is always an expression of relative values; thus, it is dicult to identify

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true changes consistently. For example, 80% lymphocytes could be found in a sample with lymphocytosis (lymphocytes = 16.0103/lL of a total WBC count of 20.0103/lL) or with a lymphopenia (lymphocytes = 0.8103/lL of a total WBC count of 1.0103/lL). Presampling information Historical and physical examination ndings Interpretation of all laboratory data should made be with knowledge of appropriate historical and therapeutic information and physical ndings that characterize the animals illness. Findings suggesting an inammatory state might include fever, presence of exudate, swollen lymph nodes, or many other physical ndings of inamed tissues or organs. Because of the eects of glucocorticoid hormones or drugs on leukocyte movements in the body, stressful states and knowing if the animal has received therapeutic amounts of glucocorticoid drugs, such as prednisone, prednisolone, or dexamethasone, should be considered. Also, because of the responses to catecholamines (epinephrine and norepinephrine), interpretations of leukocytoses can be inuenced by knowing if the animal was excited or frightened while the blood sample was being collected. Criteria of leukocytes used to distinguish leukocytoses Concentration of each type of leukocyte The rst step in distinguishing the causes of a leukocytosis is determining the leukocyte population that has accumulated in the circulating blood (ie, determining if the leukocytosis is caused by a neutrophilia, lymphocytosis, monocytosis, eosinophilia, basophilia, mastocytemia, or combination of these ndings). As mentioned previously, the accuracy of the leukocyte concentrations should be considered while evaluating the concentrations. Once the abnormalities are dened, one or more of the lists found in this article (Boxes 17) can be examined to determine diseases and conditions that should be considered for each abnormality. Also, the concentrations of other leukocytes may dene a pattern of one or more of the common leukocytosis disorders or conditions (Table 2). Magnitude of leukocytosis The magnitude of altered leukocyte concentrations should be considered when attempting to dierentiate a leukocytosis for two major reasons: the more pronounced the abnormality, the more likely it is that the abnormality represents a biologic change and not just an analytic change, and inammatory and neoplastic states have the potential to create higher leukocyte concentrations than the physiologic or glucocorticoid leukocytoses.

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Box 1. Diseases and conditions that cause neutrophilia Inammatory Infections: bacterial, fungal, viral, protozoan Immune hemolytic anemia Necrosis: hemolysis, hemorrhage, infarcts, burns, neoplasia, sterile inammation Sterile foreign body Glucocorticoid-associated Stress (physical or neurogenic) Hyperadrenocorticism Glucocorticoid therapy Adrenocorticotropic hormone (ACTH; corticotropin) administration Physiologic (shift) Fight or ight response: excitement, fright, pain, exercise, anxiety Catecholamine injections: epinephrine or norepinephrine Neoplastic Granulocytic (myelogenous) leukemia Paraneoplastic neutrophilia Others or unknown mechanisms Neutrophilia of leukocyte adhesion deciency Granulocyte colony-stimulating factor administration Estrogen toxicosis (early)
Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

Neutrophilia The magnitude of the inammatory and neoplastic neutrophilias can vary from mild (\20.0103/lL) to extreme ([100.0103/lL). Dierentiating the extreme neutrophilias typically involves the microscopic evaluation of cells. In an inammatory neutrophilia, there is an orderly maturation sequence in the blood and bone marrow characterized by more segmented neutrophils than band neutrophils and more band neutrophils than metamyelocytes, for example. In most neoplastic neutrophilias, the neutrophilic cells are poorly dierentiated; thus, it may be dicult to establish the cell lineage. The major exception to these concepts is chronic myeloid leukemia, in which the blood and marrow are dominated by segmented and band neutrophils [4]. The magnitudes of the neutrophilias associated with glucocorticoid hormones/drugs and catecholamines are limited. In response to catecholamines,

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Box 2. Diseases and conditions that cause lymphocytosis Chronic inammation Bacterial infections, especially rickettsial Fungal infections, primarily systemic Viral infections Protozoan infections, especially babesial Physiologic (shift) Fight or ight response: excitement, fright, pain, exercise, anxiety Catecholamine injections: epinephrine or norepinephrine Neoplasia Lymphoma (feline leukemia virus, idiopathic), leukemic phase Lymphoid leukemia Hypoadrenocorticism
Lists of specic disorders or conditions are not complete but are provided to give examples. Puppies and kittens have higher lymphocyte concentrations than mature animals of the respective species. Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

neutrophils shift from the marginated neutrophil pool to the circulating neutrophil pool. Because the cell concentrations in the two pools are nearly equal in the dog, the canine physiologic neutrophilia is not expected to exceed twice the upper reference limit (URL) of the reference interval (2URL). The marginal neutrophil pool in the cat is nearly three times larger than the circulating pool [5]; thus, the feline physiologic neutrophilia can (in theory) approach 4URL. The neutrophilia created by the eects of glucocorticoids also involves shifting of neutrophils from marginal to circulating pools. This neutrophilia may be enhanced by the release of neutrophils from the marrow storage pool and the decreased emigration of neutrophils to tissues. As a guideline, a glucocorticoid-associated neutrophilia is typically less than 2URL and is not expected to exceed 3URL. Lymphocytosis The inammatory lymphocytosis is typically a mild lymphocytosis (\2URL) but occasionally exceeds 30.0103/lL in dogs and cats in response to chronic stimuli to the lymphoid system. Frequently, this lymphocytosis is accompanied by the presence of reactive lymphocytes (plasmacytoid lymphocytes, immunocytes, and virocytes), and these cells may be dicult to distinguish from neoplastic cells via light microscopy.

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Box 3. Diseases and conditions that cause lymphopenia Acute inammation Acute bacterial infections Acute viral infections Endotoxemia Glucocorticoid-associated See list for steroid neutrophilia (Box 1) Depletion Lymphoid effusion: chylothorax, feline cardiomyopathy Loss of afferent lymph: alimentary lymphoma, enteric neoplasms, granulomatous enteritis, protein-losing enteropathy, lymphangiectasia, ulcerative enteritis Lymphoid hypoplasia or aplasia Immunosuppressive drugs or whole body irradiation Destruction of lymphoid tissues: multicentric lymphoma, generalized lymphadenitis Combined immunodeciency of dogs (Basset Hound, Cardigan Welsh Corgi, Jack Russell Terrier)
Lists of specic disorders or conditions are not complete but are provided to give examples. Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

A neoplastic lymphocytosis can range from mild to extreme ([100.0 103/lL). Typically, this lymphocytosis is accompanied by the presence of atypical neoplastic lymphocytes. The magnitude of a physiologic lymphocytosis, like a physiologic neutrophilia, is determined by the shift of cells from the marginal pool to the circulating pool. The lymphocytosis is not expected to exceed 2URL in dogs but may be slightly higher in cats. The lymphocytosis of hypoadrenocorticism is typically mild (\2URL). The key diagnostic clue for recognizing this lymphocytosis is that it occurs in a stressed animal in which lymphopenia is expected. Monocytosis An inammatory monocytosis is typically mild (\2URL) but may occasionally exceed 10.0103/lL. It is usually accompanied by an inammatory neutrophilia; the concurrent lymphocyte concentrations can vary from decreased to increased. A glucocorticoid-associated monocytosis is typically mild (\2URL) and is usually accompanied by a mature neutrophilia and lymphopenia.

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Box 4. Diseases and conditions that cause monocytosis Inammation Infections: bacterial (including rickettsial), fungal, protozoan Necrosis: hemolysis, hemorrhage, neoplasia, infarction, trauma Glucocorticoid-associated Stress (physical or neurogenic) Hyperadrenocorticism Glucocorticoid therapy ACTH administration Neoplasia: monocytic or myelomonocytic leukemia Secondary to immune neutropenia Cyclic hematopoiesis Granulocyte colony-stimulating factor administration
Lists of specic disorders or conditions are not complete but are provided to give examples. Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

A neoplastic monocytosis (ie, monocytic leukemia) is usually characterized by a moderate to extreme monocytosis ([50.0103/lL) with many atypical forms of monocytes in blood and marrow. Eosinophilia A slightly increased eosinophil concentration should be interpreted carefully because of analytic variations. An eosinophilia can be used as a clue to explore a variety of pathologic states (see Box 5), most of which cause a mild to moderate eosinophilia (\10.0103/lL). When eosinophilias of greater magnitude (especially[20.0103/lL) are found, hypereosinophilic syndrome and eosinophilic leukemia should be considered. These may be dicult to dierentiate if the neoplastic state produces well-dierentiated eosinophils. Basophilia The same basic concepts mentioned for eosinophilias apply to basophilias (see Box 6). The most dramatic basophilias are seen with the rare basophilic leukemias. Mastocytemia Mast cells are not expected to be found in peripheral blood of healthy dogs and cats; thus, nding mast cells in a blood lm represents a mastocytemia

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Box 5. Diseases and conditions that cause eosinophilia Hypersensitivity (allergic) disorders Flea bite dermatitis Hypersensitivity to staphylococcal or streptococcal proteins Asthma and eosinophilic respiratory disorders Parasitism Ectoparasites Heartworms Tissue nematodes, trematodes, and protozoa Dogs: Dirolaria, Dipetalonema, Spirocerca, Strongyloides, Trichuris, Paragonimus, Habronema; larval migration of hookworms and roundworms Cats: Paragonimus, Aelurostrongylus Idiopathic eosinophilic conditions Dog: eosinophilic myositis, eosinophilic gastroenteritis, eosinophilic panosteitis, eosinophilic pneumonitis, eosinophilic granuloma complex in Siberian Huskies Cat: eosinophilic granuloma complex, eosinophilic enteritis, hypereosinophilic syndrome Mast cell degranulation caused by inammation: cutaneous, respiratory, intestinal, genital, urinary Mast cell neoplasia Hypoadrenocorticism Neoplastic eosinophilia (eosinophilic leukemia, paraneoplastic eosinophilia)
Lists of specic disorders or conditions are not complete but are provided to give examples. Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

(the term mastocytemia is preferred over mastocytosis, because mastocytosis can also refer to an accumulation of mast cells in tissues other than blood). Because mast cells are not typically detected during the dierential leukocyte count, a mast cell concentration is rarely calculated. When only a few mast cells are found in the canine blood lm, the mastocytemia can be caused by an inammatory disease or mast cell neoplasia. When mast cells are relatively common (eg, [1% of the leukocytes), the likelihood of a neoplastic mastocytemia increases. Well-granulated and poorly-granulated canine mast cells can be seen in inammatory and neoplastic states. Mastocytemia in cats is associated with splenic and systemic mastocytosis.

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Box 6. Diseases and conditions that cause basophilia Allergic reactions (immediate or delayed) Drugs, foods, inhalants, insect stings/bites Parasitism Fleas Gastrointestinal parasites, such as nematodes Vascular parasites, such as Dirolaria immitis and Dipetalonema reconditum Neoplasia Basophilic leukemia Mast cell neoplasia Feline myeloproliferative diseases Lymphomatoid granulomatosis Essential thrombocythemia Polycythemia vera
Lists of specic disorders or conditions are not complete but are provided to give examples. Adapted from Stockham SL, Scott MA: Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

Presence of a left shift An increased concentration of immature neutrophils in the blood indicates a left shift. Classically, the left shift is the hallmark of an inammatory neutrophilia, but mild left shifts (typically band neutrophil concentration \1.0103/lL) may occur in glucocorticoid-associated neutrophilias. Left shifts may vary in severity from mild increases in band neutrophils to more severe responses indicated by the presence of metamyelocytes, myelocytes, and, on rare occasions, promyelocytes or myeloblasts. The degree to which the neutrophil series is shifted to the left is suggestive of the severity of the underlying inammatory disease. Left shifts are often classied as regenerative, degenerative, or leukemoid. Regenerative left shifts are characterized by a leukocytosis caused by neutrophilia with the presence of immature neutrophils [6]. Mature neutrophils outnumber the immature neutrophils, with the immature cells being distributed in an orderly manner (eg, segmented [ bands [ metamyelocytes [ myelocytes). A regenerative left shift is considered to be an appropriate response to an inammatory process because it implies that neutrophils are being introduced into the blood at a rate exceeding the rate of use and that the marrow is able to mature most of the cells before release.

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Box 7. Disorders reported to be associated with mastocytemia Neoplastic disorders Cutaneous mast cell neoplasms Visceral mast cell neoplasia Nonneoplastic disorders in dogs Inammatory Enteritis, especially parvovirus Fibrinous pericarditis and pleuritis Bacterial peritonitis Aspiration pneumonia Acute pancreatic necrosis Immune hemolytic anemias Renal failure associated with acute inammation Inammatory skin diseases: ea-bite hypersensitivity, atopy, sarcoptic mange, food allergy; some with secondary pyoderma Hemorrhage secondary to hemophilia in dogs Gastric torsion in dogs
Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames (IA): Iowa State Press; 2002. p. 4983.

Degenerative left shifts are recognized when immature neutrophils outnumber the segmented neutrophils and are often associated with a leukopenia and neutropenia [6]. A degenerative left shift is considered an inappropriate inammatory response because the marrows ability to supply neutrophils to the blood is exceeded by the rate of neutrophil egress into
Table 2 Major leukocytosis patterns based on leukocyte concentrations Leukogram pattern Acute inammatory Chronic inammatory Glucocorticoid Physiologic leukocytosis Hemic neoplasia Total Segmented Nonsegmented WBCs neutrophil neutrophil Lymphocyte Monocyte Eosinophil " " " " """a " " " " ? " WRI-" WRI-slight" WRI ? # WRI-" # " ? WRI-" WRI-" " WRI-" ? #-WRI WRI # WRI ?

Abbreviation: WBC, white blood cell. a The concentration of the neoplastic cell line is expected to be increased; concentrations of other cell lines typically are within reference intervals (WRI) or decreased. Adapted from Stockham SL, Scott MA. Leukocytes. In: Fundamentals of veterinary clinical pathology. Ames, IA: Iowa State Press; 2002. p. 4983.

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inamed tissues. As a result of the high marrow release rate, the mature storage pool is rapidly depleted, resulting in release of predominantly immature cells. If mostly nonsegmented neutrophils are observed in a blood lm, Pelger-Huet anomaly should also be considered [79]. The term leukemoid response or leukemoid blood picture is applied when a marked or extreme leukocytosis is present, most commonly associated with a neutrophilia and severe left shift. In this case, the term leukemoid denotes a blood picture resembling that of chronic myelogenous leukemia; however, the inciting cause is increased tissue demand for neutrophils and not a myeloproliferative disorder. Leukemoid reactions may also involve a significant lymphocytosis or eosinophilia. Again, the inciting cause is not a primary hematopoietic disorder. More commonly, lymphoma and mast cell tumor are the underlying cause for a massive eosinophilia, whereas an extreme lymphocytosis has been reported with persistent antigenic stimulation as the result of an infectious etiology and, rarely, in response to vaccination. Absence of a left shift A left shift is not expected when a neutrophilia is caused by a physiologic (catecholamine) response and usually is not present with a glucocorticoid neutrophilia. A left shift also may be absent in an inammatory neutrophilia when one of two states is present. First, a left shift would not be present if there is a mild inammatory stimulus and only segmented neutrophils are released from marrow. Second, the magnitude of a left shift diminishes and perhaps disappears in a chronic inammatory process when granulocytic hyperplasia results in the replenishment of the neutrophil storage pool so that the rate of band neutrophil release from marrow is not increased. Presence of toxic neutrophils Toxic neutrophils are neutrophils with any or all of the following characteristics, which are called toxic changes: foamy cytoplasm, diuse cytoplasmic basophilia, Dohles inclusion bodies (focal cytoplasmic basophilia, Dohle bodies), asynchronous nuclear maturation, and toxic granules. The toxic changes represent defective or incomplete maturation of neutrophils, which occurs during the rapid neutropoiesis of a marked to severe inammatory state. Toxic neutrophils are not seen with the other leukocytoses, but atypical cells with similar features can be seen in neoplastic leukocytoses. Presence of hypersegmented neutrophils Hypersegmented neutrophils contain ve or more nuclear lobes separated by laments, and they are occasionally seen in blood samples with either a chronic inammatory or glucocorticoid-associated neutrophilia. Increased

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concentration of hypersegmented neutrophils is sometimes described as a right shift. Hypersegmented neutrophils are usually considered as old cells resulting from an increased circulating lifespan. A common cause of longer blood transit times and a right shift is increased endogenous or exogenous glucocorticoid compounds, which decrease the rate of migration of neutrophils from blood to tissues. Hypersegmented neutrophils may also be encountered in resolving chronic suppurative inammation. As chronic inammation takes a course toward resolution, the dynamics of increased neutrophil production combined with a decrease in tissue demand lead to longer blood transit times for neutrophils. This allows for increased nuclear segmentation as part of the normal cell aging process. In vitro development of hypersegmentation can also occur when processing of blood samples is delayed for several hours. Hypersegmented neutrophils in dogs and cats may also be associated with myelodysplastic diseases, myeloid leukemias, Poodle marrow dyscrasia, and cobalamin deciencies [1,10,11]. Presence of giant neutrophils Giant neutrophils are neutrophils with an increased cell diameter ([13 lm) on a stained blood lm and are usually segmented or band neutrophils. Giant neutrophils are typically associated with inammatory neutrophilias but may also be seen as a feature of a myelodysplastic syndrome or myeloproliferative disease (especially if associated with feline leukemia virus). In inammatory responses, increased neutropoiesis may result in the formation of giant neutrophils, which are generally thought to be the product of a skipped division. Giant neutrophils are more commonly seen in cats than in dogs. When giant neutrophils are associated with increased neutropoiesis, they are considered a toxic change. Presence of a lymphopenia The concurrent ndings of lymphopenia and leukocytosis typically reect responses to an acute inammatory disease or to glucocorticoid hormones or drugs. In inammatory disease, some of the same cytokines that promote altered neutrophil movements also promote migration of lymphocytes to the inamed tissue and homing of lymphocytes to lymphoid tissues [12]. In response to glucocorticoids, there is a redistribution of lymphocytes from circulating blood to other sites, probably marrow or lymph nodes [1315]. Because well-dierentiated lymphocytes of dogs and cats are relatively steroid-resistant, only long-term steroid treatments have lympholytic eects [16]. A lymphopenia may also occur when there has been a loss of lymph from the body or when there is hypoplasia of lymphoid tissues. There may or may not be a leukocytosis associated with these types of disorders.

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Presence of atypical cells or immature cells A reactive lymphocyte (plasmacytoid lymphocyte, immunocyte, or virocyte) is a lymphocyte that has microscopic features that represent reactive changes: increased amount of cytoplasm; enhanced cytoplasmic basophilia; perinuclear halo; prominent focal Golgi zone; and eccentric, enlarged, cleaved, convoluted, lobulated, or bilobed nucleus. The most consistent changes are an increased amount of cytoplasm and increased cytoplasmic basophilia. Rare reactive lymphocytes can be found in most blood samples, but an increased number indicates an inammatory disease or an immune response. Individual reactive lymphocytes can be impossible to distinguish from neoplastic lymphoid cells. The more pleomorphic the cells are, the more likely it is that they represent neoplasia and not hyperplasia. Neoplastic leukocytes may have microscopic features of well-dierentiated leukocytes (eg, chronic granulocytic leukemia, chronic lymphocytic leukemia) but more commonly have features of poorly dierentiated or undierentiated hemic cells [4,10]. Poorly dierentiated cells have nuclei with nely granular to agranular chromatin and varying degrees of cytoplasmic basophilia. There may be cytoplasmic or nuclear features, which suggest dierentiation toward neutrophils, eosinophils, basophils, lymphocytes, or monocytes. It may be dicult to establish cell lineage from microscopic features on a Wright-stained blood lm. High concentrations of poorly dierentiated cells indicate the presence of either a myeloproliferative or lymphoproliferative disease, however.

Transient versus persistent changes Because of the circulation time of blood leukocytes and the factors that alter the concentration of leukocytes in the circulating blood, leukocyte concentrations in the circulating blood can change quickly. Conversely, persistent changes in leukocyte concentrations typically represent persistence of the factors that alter the concentration. In the physiologic leukocytosis of excitement or fright, the leukocytosis is a response to catecholamines, which induce the shifting of leukocytes from the marginal to circulating blood pool. These changes persist as long as there is an increased catecholamine concentration, but the changes disappear soon after the concentration returns to baseline values. The same general concepts apply to altered leukocyte concentrations created by glucocorticoid hormones or drugs. Because these compounds can circulate in blood for hours or days, however, their eects may persist for hours or days. If altered leukocyte concentrations are not caused by physiologic or pharmacologic factors, duration of the altered leukocyte concentrations typically reects the duration or variations of the pathologic state. Persistence of an inammatory neutrophilia typically indicates continued stimulus from

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cytokines for neutrophil production and release from marrow or other hematopoietic tissues. Persistence of a lymphocytosis also represents an ongoing pathologic state whether it is caused by inammation, neoplasia, or hypoadrenocorticism. Changes in the rate of leukocyte migration to tissues can also aect leukocyte concentrations. For example, the magnitude of a neutrophilia may increase after the removal of a pyometra uterus, because tissue demand suddenly decreased but neutrophil production and release remained increased. In another example, an inammatory neutrophilia may suddenly change to an inammatory neutropenia if the tissue demand suddenly becomes overwhelming. Other laboratory criteria used to distinguish leukocytoses Anemia Anemia can be a concurrent nding in inammatory or neoplastic leukocytosis. The anemia of inammation is typically a mild to moderate, normocytic, normochromic, nonregenerative anemia; it develops more rapidly in the cat than in the dog. The anemia occurs because of decreased erythrocyte production and decreased erythrocyte lifespan. Hemolytic anemias, especially immune-mediated or spherocytic, are frequently accompanied by an inammatory leukocytosis. The anemia associated with a neoplastic leukocytosis may be similar to the anemia of inammation but also can progress to a severe anemia when there has been extensive replacement of marrow by neoplastic cells. Cats with myeloproliferative diseases may have a macrocytic, normochromic, nonregenerative anemia because of erythroid dysplasia. Erythrocytosis Erythrocytosis may be found in animals with a physiologic leukocytosis, because catecholamines stimulate splenic contraction, which results in the injection of erythrocyte-rich blood into the systemic circulation. This response is not expected in cats because they lack the muscular spleen and sinusoid splenic blood. Erythrocytosis may also be found concurrently with inammatory or neoplastic leukocytoses if the animal is dehydrated. Rouleaux Rouleaux are common in blood lms of animals with hyperproteinemia, hyperglobulinemia, or hyperbrinogenemia. Thrombocytosis Inammation can produce a reactive thrombocytosis in which cytokines stimulate increased thrombopoiesis. Although rarely recognized, the clonal

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proliferations in polycythemia vera may result in leukocytosis, erythrocytosis, and thrombocytosis. Splenic contraction stimulated by catecholamines can cause a thrombocytosis by adding platelet-rich blood to the peripheral blood. Thrombocytopenia Many infectious and noninfectious inammatory disorders can produce a thrombocytopenia by suppressing platelet production, altering platelet distribution, increasing platelet consumption, or increasing platelet destruction. A clonal proliferation of leukocytes can damage the marrow and result in megakaryocytic hypoplasia and decreased thrombopoiesis. Marrow biopsy results A variety of changes may occur in marrow and its cell populations in response to an inammatory disease. The major ndings typically would include granulocytic hyperplasia and an increased amount of iron pigment; other ndings could include lymphoid hyperplasia, plasmacytosis, megakaryocytic hypoplasia, and erythroid hypoplasia. More dramatic changes may occur if the inammatory disease directly involves the marrow. When there are neoplastic leukocytes in blood, the same type of leukocytes typically are found in marrow samples. Lymph node biopsy results A variety of changes may occur in lymph node tissue and its cell populations in response to an inammatory disease [17]. The major ndings typically would include lymphoid hyperplasia and plasmacytosis. More dramatic changes may occur if the inammatory disease directly involves the lymph node. Because lymphoid leukemias in dogs and cats frequently represent the leukemic manifestations of lymphoma, lymph node samples may contain the neoplastic lymphocytes. Although rarely recognized, the neoplastic cells in a lymph node can be cells of a myeloproliferative disease (eg, myelogenous leukemia, myelomonocytic leukemia). Hyperproteinemia Inammatory cytokines trigger increased production of several globulins (positive acute-phase proteins) by hepatocytes. Also, stimulation of Blymphocytes may increase the production of immunoglobulins. A clonal proliferation of B-lymphocytes or plasma cells may result in increased production of one type of immunoglobulin to generate a monoclonal gammopathy. Hyperglobulinemia Occasionally, there is an inammatory hyperglobulinemia without a hyperproteinemia because of a concurrent hypoalbuminemia or the broad

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range of total protein reference intervals in dogs and cats. The monoclonal gammopathy of lymphoid neoplasia may produce a hyperglobulinemia, and there may or may not be a concurrent hyperproteinemia. Hypoalbuminemia Inammatory cytokines trigger the decreased production of albumin (a negative acute-phase protein) by hepatocytes. Hypoalbuminemia may be found in animals with hemic cell neoplasia because of a variety of mechanisms, including decreased albumin production by hepatocytes and increased albumin loss from plasma. Hyperalbuminemia Glucocorticoid drugs or hormones may increase albumin synthesis to cause a hyperalbuminemia [18]. Hyperalbuminemia is more commonly associated with hemoconcentration as a result of dehydration, which can be found in animals with either inammatory or neoplastic leukocytoses. Hyperbrinogenemia Fibrinogen is one of the positive acute-phase proteins. Hypoferremia Altered iron distribution during inammatory states results in a shift of iron from plasma (bound to transferrin) to macrophages. Decreased total iron-binding capacity The total iron-binding capacity (TIBC) reects the serum concentration of transferrin; transferrin is one of the negative acute-phase proteins. Hyperferritinemia Ferritin is one of the positive acute-phase proteins. Hyperglycemia Hyperglycemia is an early response to an endotoxemia that may be associated with an inammatory leukocytosis [19]. Hyperglycemia also may be associated with the hyperglycemia of acute pancreatitis. Catecholamines promote hyperglycemia by stimulating hepatic glycogenolysis and promoting growth hormone (GH) release. Increased GH activity promotes hyperglycemia by interfering with glucose uptake by myocytes and adipocytes. Glucocorticoid drugs or hormones may produce a hyperglycemia by promoting gluconeogenesis and reducing cellular uptake of glucose by reducing the number or eciency of glucose membrane transporters.

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Hypoglycemia Hypoglycemia can be associated with the leukocytosis of sepsis. The hypoglycemia may be caused by increased use of glucose by tissues and decreased glucose production. A pseudohypoglycemia may occur with an extreme inammatory or neoplastic leukocytosis because of in vitro consumption of glucose before the harvesting of serum or plasma. Azotemia A renal azotemia may be present because of inammation or neoplasia involving kidneys, but it also may be present because of a hypercalcemic nephropathy (see section on hypercalcemia). The azotemia may also be present because of prerenal mechanisms (especially dehydration). Decreased serum urea concentration Cortisol and glucocorticoid drugs can reduce the secretion of antidiuretic hormone (ADH) [20], which may result in a lowered serum urea concentration, because less urea is resorbed by the renal tubules in the absence of ADH. Pseudohyperkalemia A pseudohyperkalemia may be linked with an inammatory leukocytosis by two methods. Potassium is released from leukocytes during blood clotting, and there may be enough potassium released to produce a pseudohyperkalemia if there is an extreme inammatory leukocytosis. The same erroneous values may occur with extreme neoplastic leukocytosis. Second, potassium is released from platelets during clotting; thus, a reactive thrombocytosis may contribute to a pseudohyperkalemia. Hypercholesterolemia Glucocorticoid drugs and hormones alter lipoprotein metabolism by increasing hepatocyte production of very low-density lipoproteins (VLDLs) and decreasing intravascular processing of lipoproteins. Both mechanisms may result in increased concentrations of cholesterol-containing lipoproteins and thus hypercholesterolemia. Hypertriglyceridemia The glucocorticoid eects on lipoprotein metabolism may also cause a hypertriglyceridemia, but it is not recognized as frequently as hypercholesterolemia.

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Hypercalcemia Hypercalcemia is a paraneoplastic state when it is caused by the actions of parathyroid hormonerelated protein (PTH-rp) or other hypercalcemic agents of neoplastic lymphocytes. Dogs with inammatory diseases (especially fungal) may have a hypercalcemia because of the formation of 1,25-dihydroxycholecalciferol (calcitriol) by macrophages. Hypophosphatemia Hypophosphatemia is also a paraneoplastic state when it is caused by production of PTH-rp by neoplastic cells. A more common reason for a hypophosphatemia would be anorexia associated with an inammatory or neoplastic state. Increased serum enzyme activity Increases in common serum enzymes (eg, alanine transaminase, creatine kinase) typically do not directly help to determine the cause of a leukocytosis. Perhaps the one exception to this concept is alkaline phosphatase (ALP). In the presence of glucocorticoid compounds, serum ALP activity frequently increases because of the increased production of liver-ALP isoform and a unique corticosteroid-induced ALP isoform. Serum ALP activity could also be increased because of inammatory or neoplastic diseases that cause cholestasis or osteoblast proliferation; thus, the total serum ALP activity may not help to distinguish the causes of leukocytosis. Dilute urine Glucocorticoid drugs and hormones inhibit the secretion of ADH [20], and ADH is needed for the optimal renal concentrating ability. Depending on several factors, the glucocorticoid eect may result in the kidneys producing dilute urine (osmolality \100 mOsm/kg of water, specic gravity \1.003). There may be impaired renal concentrating ability as a result of the destruction of nephrons by neoplasia or inammation, a hypercalcemic nephropathy, or the direct eects of free calcium ions on ADH activity. Pyuria Inammation of the kidneys (eg, pyelonephritis) may result in an inammatory leukocytosis, but it is unusual for a lower urinary inammation (ie, cystitis, urethritis) to cause a leukocytosis. Pyuria may also be the result of prostatitis. Proteinuria Proteinuria may be a manifestation of urinary tract inammation that has resulted in a leukocytosis; alternately, some inammatory disease may

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lead to the development of a protein-losing nephropathy. Proteinuria can be directly linked to a clonal proliferation of lymphocytes if those lymphocytes are secreting the light chains of immunoglobulins.

New techniques for identifying or classifying cells Some leukocytosis samples pose diagnostic challenges when the microscopic examination of stained leukocytes does not dierentiate leukemia cells (eg, lymphoid versus myeloid) or does not dierentiate neoplastic from hyperplastic lymphocytes. New techniques are emerging from research laboratories that promise better identication of cell lineage in leukemias and better dierentiation of some neoplastic and inammatory leukocytoses. Immunophenotyping As mentioned in the earlier section on total leukocyte concentrations (optical WBC count), ow cytometric methods can be used to dierentiate cells by the way they scatter laser light. Adding immunouorescent markers (or probes) to the cells greatly expands the potential uses of a ow cytometer. Immunophenotyping can be accomplished by using monoclonal antibodies against leukocyte antigens, which have been classied as cluster of dierentiation (CD) antigens [21]. The immunophenotyping may indicate if the leukocytosis is caused by one type of cell (eg, T lymphocyte) and can help to determine the lineage of poorly dierentiated neoplastic leukocytes. Similar antibodies can be used to generate an immunophenotypic classication of cells in tissue sections via immunouorescence or immunochemistry. Ploidy analysis Determining DNA content (ploidy) via ow cytometry can be valuable in dierentiating neoplastic from nonneoplastic cells. Most normal cells contain two sets of chromosomes (diploid, 2N or 2C), except in the DNA synthesis stage (S-phase) or mitosis stage (M-phase), in which there is chromosome duplication (tetraploid, 4N or 4C). Malignant cells can have abnormal chromosome numbers (aneuploid). S-phase determinations can indicate how quickly the neoplastic cells are proliferating based on the numbers of nuclei that are in S-phase [22]. Polymerase chain reaction assays for clonality Because neoplastic lymphocytosis typically represents an uncontrolled clonal proliferation, assays have been developed to attempt to determine if a lymphocyte population represents monoclonal (neoplastic) or polyclonal (hyperplastic) cells [23]. Detection of clonality strongly suggests neoplasia,

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even though it is important to realize that it is not exclusive. Lymphomas, lymphocytic leukemias, and myelomas represent clonal expansions of neoplastic lymphoid cells. The current polymerase chain reaction (PCR) assays for clonality are directed against the genes encoding the heavy chains hypervariable region (CDR3 [complementarity-determining region 3]) of immunoglobulin (for B cells) and T-cell receptor gamma (TCRc) (for T cells). These regions in each lymphocyte clone are formed through DNA recombination to provide diversity against a variety of antigens and thus are highly variable between lymphocyte clones. If inammation or another immune stimulus is the cause of lymphocyte proliferation, a variety of PCR products (mixtures of dierent CDR3 or TCRc sequences) would be detected and thus indicate a polyclonal proliferation. If lymphoid neoplasia is the cause of the lymphocyte proliferation (clonal proliferation), the PCR product would contain the one unique CDR3 or TCRc sequence if it results from a monoclonal proliferation or two products if it results from a biclonal proliferation. In a recent study, a mono-, bi-, or oligoclonal pattern was found in 91% of the lymphoid malignancies [24]. If the neoplastic cells had deleted receptor genes or had a natural killer (NK) cell lineage, the PCR method of the study would not help to characterize the cells. The authors reported that an unstained cytologic preparation (eg, ne-needle biopsy sample) has enough material that can be washed o and used for the PCR analysis of clonality.

Summary The four major types of leukocytoses are inammatory, glucocorticoidassociated, catecholamine-associated, and neoplastic. These leukocytoses are distinguished by leukocyte concentrations, microscopic features of leukocytes, and associations with other laboratory data. All laboratory ndings need to be interpreted within the context of the case information, including signalment, history, and physical examination ndings. Newer assays are being used to dierentiate the dierent forms of leukocyte neoplasia and to distinguish between hyperplastic and neoplastic proliferations.

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