Blood Smear Review: How to Identify and Interpret Neutrophil Abnormalities

Blood smear evaluation remains a critical part of a hematologic evaluation of veterinary patients, complementing the results of a CBC by enabling clinicians to identify morphologic abnormalities that may not be detected by an in-house benchtop analyzer.^1-3 This article focuses on the identification of abnormalities of peripheral blood neutrophils that may facilitate the early detection of inflammation, monitor response to treatment, and inform long-term prognosis (BOXES 1 AND 2). In some instances, neutrophils may contain certain infectious agents or other abnormal inclusions, and the ability to recognize these abnormalities may lead the clinician to an immediate diagnosis.

Neutrophil Maturation and Normal Morphology

Neutrophils are the first immune cells to respond to inflammation and infection and are derived from myeloid (granulocytic) precursors in the bone marrow. Granulocytic precursors exhibit pyramidal maturation progressing from the earliest precursors, myeloblasts, to progranulocytes; myelocytes; metamyelocytes; band cells; and, lastly, mature, segmented neutrophils. The stages can be distinguished by their nuclear and cytoplasmic features.

• Myeloblast (FIGURE 1A): Myeloblasts have high nuclear-to-cytoplasmic ratios with round to oval, eccentric nuclei containing finely stippled chromatin with 1 to multiple prominent nucleoli. They have small amounts of medium basophilic cytoplasm that often display prominent paranuclear clearing (Golgi zone).
  

  Figure 1A. Myeloblast in bone marrow aspirate from a dog. Myeloblasts are uncommonly encountered in peripheral blood, and their presence raises concern for myeloid leukemia; however, myeloblasts may also be seen with severe inflammation as a part of left shift. Wright-Giemsa stain, 100× objective.

• Progranulocyte (or promyelocyte) (FIGURE 1B): Progranulocytes have round to oval, eccentric nuclei containing coarsely stippled chromatin and lightly basophilic cytoplasm that contains few fine, magenta-colored primary granules. Primary granules are the first granules seen as myeloblasts transition to progranulocytes. They contain many antimicrobial compounds and are myeloperoxidase positive.
  

  Figure 1B. Progranulocyte in canine blood. Wright-Giemsa stain, 100× objective.

• Myelocyte (FIGURE 1C): Myelocytes have round to oval, often eccentric nuclei containing coarse or ropy chromatin and have paler blue cytoplasm than progranulocytes.
  

  Figure 1C. Neutrophilic myelocyte in canine blood. Wright-Giemsa stain, 100× objective.

• Metamyelocyte (FIGURE 1D): Metamyelocytes have reniform nuclei with coarse chromatin and clear to variably basophilic cytoplasm that, depending on the degree of immaturity, contains neutral-staining secondary granules. Secondary granules are smaller and less dense than primary granules and are specific to the type of granulocyte present (e.g., neutrophils have neutral-staining granules, eosinophils have pink granules). These granules contain compounds involved with the formation of reactive oxygen species and other proteins that aid in neutrophil migration.
  

  Figure 1D. Neutrophilic metamyelocyte in canine blood. Wright-Giemsa stain, 100× objective.

• Band cell (FIGURE 1E): Bands have nuclei with no lobulations and virtually parallel sides of which no area has a diameter less than two-thirds the diameter of any other area, often resembling a horseshoe with more coarse, paler-staining chromatin.⁸ The cytoplasm resembles that of a mature neutrophil. Bands are rarely seen in the blood of healthy animals.
  

  Figure 1E. Band cell in canine blood. Wright-Giemsa stain, 100× objective.

• Segmented (mature) neutrophil (FIGURE 2A): Mature neutrophils have a segmented nucleus containing 3 to 5 separate lobes; condensed, dark purple chromatin; and colorless to pale blue cytoplasm that contains neutral (i.e., nonstaining) secondary granules and measure approximately 10 to 14 µm in diameter (twice the size of an erythrocyte). Female animals may have a few neutrophils containing a Barr body (FIGURE 2B), which appears as a small, teardrop-shaped protrusion of the nucleus and represents the inactivated X chromosome.⁶ In cats, low numbers of neutrophils may contain a few Döhle bodies that appear as small, round to ovoid, blue cytoplasmic inclusions and represent aggregates of rough endoplasmic reticulum (FIGURE 2C). Certain cat breeds (e.g., Birman, Siamese, Himalayan) may have neutrophils containing fine, red to pink cytoplasmic granules that are often discovered incidentally on blood smear examination and are not an indication of illness.^8,9

Figure 2A. Normal neutrophil in canine blood. Wright-Giemsa stain, 100× objective.

Figure 2B. Normal neutrophil in canine blood. Note the small teardrop-shaped Barr body (arrow) that can be present in females. Wright-Giemsa stain, 100× objective.

Figure 2C. Normal neutrophil in feline blood. Note the 2 small Döhle body inclusions (arrows). Wright-Giemsa stain, 100× objective.

Abnormal Neutrophil Morphology

Nuclear Abnormalities

Left Shifting

The presence of increased numbers of band neutrophils and earlier precursors is called a left shift, which is a response to inflammation and increased peripheral demand. Left shift should be orderly and pyramidal with higher numbers of more mature precursors than less mature precursors. When the number of immature neutrophils is greater than the number of mature segmented neutrophils, a degenerative left shift is present, which may be seen with severe inflammation and may confer a guarded clinical prognosis.^5,10

Pelger–Huët Anomaly

Pelger–Huët anomaly (PHA) is a congenital morphologic abnormality that has been documented in dogs, cats, rabbits, and horses.^11-14 PHA is suspected to result from a mutation in the gene that encodes the lamin B receptor (a protein involved with nuclear envelope structure and function).^11-14 Neutrophils of animals with PHA contain condensed chromatin and cytoplasmic features similar to a typical mature neutrophil; however, their nuclei appear hypolobulated (round, oval, or reniform-shaped) (FIGURE 3). The nuclei of affected granulocytes and monocytes fail to undergo segmentation but retain normal function. PHA is often incidentally discovered during blood smear evaluation, typically after suspected abnormalities are flagged by an analyzer (e.g., suspected bands) because affected cells can resemble bands. PHA can often be distinguished from left shift because many cells will be affected and their cytoplasm will lack typical features of immature cells (e.g., cytoplasmic basophilia, primary granules).^2,4,8 An acquired form, called pseudo-PHA, has been documented in association with myeloid leukemias, severe infections, and administration of chemotherapy.^8,15 In contrast to congenital PHA, pseudo-PHA is transient and generally only a subset of neutrophils appear hyposegmented.⁴

Figure 3. Neutrophils in the blood of a dog with Pelger–Huët anomaly. Wright-Giemsa stain, 100× objective.

Hypersegmentation

Hypersegmentation occurs as a part of cell aging after neutrophils remain in peripheral blood for an extended period. In most domestic species, a neutrophil is considered hypersegmented if the nucleus consists of 5 or more distinct segments (FIGURE 4A). Hypersegmentation may be seen in patients with chronic inflammation, excess endogenous or exogenous glucocorticoids, myeloid leukemias, and certain congenital conditions.^4,8,16 Botryoid neutrophils are neutrophils with hypersegmented nuclei that are radially arranged and exhibit a distinct “pinwheel” appearance; they are often associated with canine heatstroke (FIGURE 4B).¹⁷

Figure 4A. Hypersegmented neutrophil in the blood of a dog with a history of long-term prednisone administration. Wright-Giemsa stain, 100× objective.

Figure 4B. Botryoid neutrophil in the blood of a dog with heatstroke. Wright-Giemsa stain, 100× objective.

Cytoplasmic Abnormalities

Toxic Change

Toxic change is one of the most common morphologic abnormalities of neutrophils in the peripheral blood and suggests accelerated granulopoiesis resulting from systemic inflammation.^2,4 Neutrophils are classified as toxic if they exhibit certain cytoplasmic features (e.g., increased cytoplasmic basophilia, vacuolated cytoplasm, Döhle bodies) (FIGURE 5). Toxic change grading is often subjectively based on the features and severity, which can vary among laboratories. Toxic changes are often most severe in animals with sepsis, endotoxemia, and/or tissue necrosis.² The morphology of neutrophils with severe toxic changes may be similar to that of monocytes, making differentiation difficult and limiting the accuracy of a leukocyte differential. Pseudotoxic change represents an in vitro change secondary to prolonged storage (Döhle body–like inclusions have been noted as soon as 4 hours after blood collection).¹⁸

Figure 5A. Toxic neutrophil (asterisk) adjacent to normal neutrophil in canine blood. Wright-Giemsa stain, 100× objective.

Figure 5B. Toxic neutrophils in feline blood. Wright-Giemsa stain, 100× objective.

Sideroleukocytes

Sideroleukocytes are neutrophils that contain hemosiderin inclusions within their cytoplasm that appear as coarse, dark blue–black granules (FIGURE 6).⁴ The inclusions may be seen in animals with hemolytic anemia and can be confirmed only through special cytochemical staining for iron (e.g., Prussian blue stain).⁴

Figure 6. Sideroleukocyte containing coarse blue-green hemosiderin inclusions (arrow) in canine blood. Wright-Giemsa stain, 100× objective.

Lipofuscin-like Inclusions

Lipofuscin-like inclusions, also referred to as critical green inclusions, are fine to coarse, dark blue–green granules within the cytoplasm of neutrophils and monocytes (FIGURE 7).¹⁹ In dogs and cats, the inclusions are suspected to represent lipofuscin pigment and are often associated with severe hepatocellular injury and higher risk for death.²⁰

Figure 7. Neutrophil containing coarse green-brown lipofuscin-like inclusions (arrow) in the blood of a dog with acute hepatocellular necrosis. Wright-Giemsa stain, 100× objective.

Infectious Organisms

Certain species of Rickettsia (e.g., Ehrlichia ewingii, Anaplasma phagocytophilum) may be seen within the cytoplasm of neutrophils of dogs during the acute phase of infection. The morulae of these species appear similar and cannot be distinguished via cytology alone, necessitating the use of PCR or serology for further differentiation (FIGURE 8). Affected patients often have characteristic hematologic abnormalities (e.g., thrombocytopenia, anemia, leukopenia), which can contribute to the clinical suspicion of tick-borne diseases.²

Figure 8. Neutrophil containing a single morula (arrow) in equine blood and round to irregular dark blue coccobacilli ~0.5 µm in diameter. PCR confirmed the diagnosis of anaplasmosis (Anaplasma phagocytophilum). Wright-Giemsa stain, 100× objective.

Gamonts of Hepatozoon americanum protozoa may occasionally be seen within the cytoplasm of neutrophils and monocytes of dogs (FIGURE 9).^2,8 Because these intracellular parasites are often found in low numbers in the peripheral blood, a buffy coat preparation may be needed for visualization.² Dogs with hepatozoonosis may also have an extreme neutrophilia, often with a left shift.^2,4 Infrequently, phagocytized intracellular bacteria may be seen in the peripheral blood, which indicates bacteremia.

Figure 9. Toxic neutrophil containing a nonstaining Hepatozoon americanum gamont (arrow) in canine buffy coat that appears as negatively staining and light blue cigar-shaped inclusions that contain a small purple nucleus. Wright-Giemsa stain, 100× objective.

Abnormal Neutrophil Concentrations

Abnormal neutrophil concentrations are best determined by evaluating the absolute neutrophil counts reported by a hematology analyzer and confirming the automated findings via manual blood smear evaluation.² Differentials for abnormalities, their general mechanisms, and defining characteristics are summarized in BOX 2. Detailed descriptions of the mechanisms of these abnormalities and less common differentials can be found elsewhere.^2,4,8,9

Summary

Neutrophil abnormalities are common among veterinary patients. In addition to an automated CBC, blood smear evaluation is encouraged to recognize atypical morphology and confirm an analyzer’s findings. Ultimately, the ability to interpret neutrophil abnormalities is a useful diagnostic skill that can provide further valuable insight into patient health, response to treatment, and prognosis.