Neutrophils: Overview, Quantity, Morphology

Overview

Origin

Neutrophils are produced in the bone marrow, released into blood, circulate briefly, and migrate into tissue spaces or on to epithelial surfaces such as those in the respiratory, digestive, or urogenital tracts.

Production is continuous in order to provide for the continual demand for neutrophils in the tissues and maintain the circulating pool in the blood.

Transit time for generation of neutrophils in marrow is approximately 4-6 days and the marrow maintains a five day supply of mature neutrophils in storage.

Injury or bacterial invasion of tissue results in the production and release of colony-stimulating factors (CSFs) that govern the proliferation and maturation of immature neutrophils in the marrow.

Granulopoiesis is a continuum of cell division, differentiation, and maturation (Fig. 5-1).

> Stem cells differentiate into myeloblasts which are the earliest recognizable cell in the granulocytic series.

> Division and differentiation of myeloblasts result in sequential generations of progranulocytes and myelocytes.

> Subsequent generations consist of metamyelocytes, bands, and segmented neutrophils which are postmitotic cells that undergo nuclear and cytoplasmic changes to become capable of phagocytosis and microbicidal activity.

Figure 5-1. Normal granulopoiesis in canine bone marrow. The majority of cells are developing granulocytes. Segmented neutrophils and bands predominate with fewer numbers of metamyelocytes, myelocytes, progranulocytes (60x).

Neutrophils circulate for about 10 hours in blood and are compartmentalized into a circulating neutrophil pool and a marginal neutrophil pool.

> Neutrophils in the circulating neutrophil pool circulate with other blood cells and are measured in the CBC.

> The marginal neutrophil pool consists of neutrophils that adhere intermittently to endothelium especially in small veins and capillaries. These cells are not counted in the CBC.

> In dogs the ratio of neutrophils in the circulating to marginal pools is 1:1. For cats, the ratio is 1:3.

Migration of neutrophils into tissues occurs randomly and is unidirectional.

> Neutrophils survive for 1-4 days in tissues and undergo programmed cell death or apoptosis.

> Neutrophils are also destroyed in spleen, liver, and bone marrow by resident macrophages.

Factors that influence the numbers of circulating neutrophils include the relative rates of:

• Bone marrow production and release.
• Exchange between circulating neutrophil pool and marginal neutrophil pool.
• Migration into tissue.

Function

Neutrophils serve as the primary defense against invasion of tissues by microorganisms. Neutrophils kill bacteria and can also damage or participate in the destruction of mycotic agents, algae, and viruses.

Neutrophils accumulate at sites of inflammation or bacterial infection by a process of directional migration or chemotaxis.

> Cellular and molecular mediators of inflammation generate chemotactic substances, stimulate marrow release, and promote margination and adhesion of neutrophils to vascular endothelium at sites of inflammation.

> Neutrophils leave the bloodstream and enter the tissues by transmigration between endothelial cells.

At the site of inflammation, neutrophils are capable of phagocytosis and microbicidal activity. Fusion of lysosomal granules with the phagocytic vesicle releases lytic enzymes and chemicals capable of killing bacteria.

Quantity

Changes in the rates of marrow production and release, the exchange between marginal neutrophil pool and circulating neutrophil pool, and/or the rate of tissue migration directly influence the number of neutrophils measured in a CBC.

WBCs and neutrophils counted electronically via impedance can be falsely increased by large platelets, platelet clumps, and Heinz bodies. Leukocyte clumping causes a false decrease in WBC count.

Neutropenia

A decrease in the absolute number of neutrophils.

In dogs and cats, neutropenia occurs when the absolute count is less than 3000 - 4000/μl.

Neutropenia is the most frequent cause of leukopenia.

Mechanisms of neutropenia include (Fig. 5-4):

• Acute demand or consumption in tissues
• Decreased marrow production
• Ineffective granulopoiesis (dysgranulopoiesis)
• Increased margination from the circulating neutrophil pool to the marginal neutrophil pool

Neutropenia due to Acute Tissue Demand

> Neutrophils can rapidly sequester in a well-vascularized tissue that becomes acutely inflamed or septic (Fig. 5-2).

> Neutropenia results when the rate of migration into tissue exceeds the capacity of the marrow storage pool of neutrophils.

• The inflammatory process is so severe and acute that there is insufficient time for granulopoiesis to replenish the supply of mature neutrophils.
• Bands and some metamyelocytes are released from the marrow causing a severe left shift.

> Toxic change often will be evident in neutrophils in blood and in precursors in marrow.

> Neutropenia with a severe left shift and toxic neutrophils is seen in conditions such as acute peritonitis, ruptured GI viscus, acute metritis, gangrenous mastitis, and acute cellulitis.

> A poor or guarded prognosis is indicated because of the extent and severity of inflammation necessary to produce this neutrophil response (See Case 11 and Case 13).

Figure 5-2. Neutrophilic exudate from a dog with bacterial peritonitis. The majority of cells are degenerate neutrophils. Rod-shaped bacteria (arrow) have been phagocytosed by a neutrophil (60x).

Neutropenia due to Decreased Marrow Production

> Severe toxic insults to marrow can result in decreased marrow production of neutrophils.

> The bone marrow in these animals is usually hypocellular with a severe reduction in granulocytic, erythroid and megakaryocytic precursors (Fig. 5-3). On occasion, the marrow is very cellular due to extensive replacement by neoplastic cells (myelophthisis).

> Potential causes include: adverse drug reactions, exposure to toxic chemicals and plants, infectious agents, myelophthisis, and suspected immune-mediated marrow destruction.

> Drugs that have been incriminated include: estrogen, phenylbutazone, trimethoprim-sulfadiazine, chloramphenicol, griseofulvin, and several chemotherapeutic agents.

> Infectious agents include: parvovirus, panleukopenia virus, feline leukemia virus, Ehrlichia.

> Production of RBCs and platelets can also be affected resulting in concurrent nonregenerative anemia and thrombocytopenia (See Case 10).

Figure 5-3. Severe marrow hypoplasia in a dog resulting in neutropenia, anemia, and thrombocytopenia. The hematopoietic cells are markedly reduced and have been replaced by fat tissue (40x).

Neutropenia Due to Ineffective Granulopoiesis (dysgranulopoiesis)

> Neutropenia can occur because of arrested development or a reduction in marrow release in spite of adequate numbers of granulocytic progenitor cells in marrow.

> The bone marrow in these animals is cellular with adequate or increased numbers of granulocytic precursors.

> Diseases associated with this neutrophil response include: myelodysplasia, acute myeloid leukemia, and infections caused by feline leukemia or feline immunodeficiency virus.

Neutropenia due to Increased Margination

> A sudden shift in neutrophils from the circulating neutrophil pool to the marginal neutrophil pool can cause a transient acute neutropenia.

> Causes include:

• Anaphylaxis
• Endotoxemia

> Neutropenia is an early, transient event and may disappear before the animal is presented for medical treatment (Fig. 5-4).

Figure 5-4. Mechanisms of neutropenia. Changes in the production, distribution, release, and circulation are indicated in bone marrow and blood. Relative size of arrows indicates changes in rate of release from blood and bone marrow and the rate of migration into tissue. Modified from Schalm’s Veterinary Hematology.

Neutrophilia

Neutrophilia is defined as an increase in the absolute numbers of circulating neutrophils. In adult dogs and cats, neutrophil counts exceed 12,000 - 13,000/μl. Neutrophilia is the most frequent cause of leukocytosis.

Causes of neutrophilia include (Fig. 5-5):

• Physiologic or epinephrine-induced
• Corticosteroid- or stress-induced
• Acute inflammation
• Chronic inflammation with marked neutrophilia
• Chronic inflammation with new steady state
• Hemorrhage or hemolysis
• Granulocytic leukemia
• Inherited granulocyte defects

Physiologic Neutrophilia

> Epinephrine release causes a transient (1 hour) mature neutrophilia by shifting neutrophils from the marginal neutrophil pool to the circulating neutrophil pool.

> Epinephrine release is caused by fear, excitement, vigorous exercise, and seizure activity.

> In cats, a marked lymphocytosis (6000 to 20,000/μl) can occur concurrently or be the prevalent finding (See Case 1).

Corticosteroid or Stress-Induced Neutrophilia

> Increased circulating levels of glucocorticoids cause increased release of mature neutrophils into the circulating neutrophils and decreased migration of neutrophils into tissue.

> The response can occur after endogenous secretion or exogenous administration of corticosteroids. Common causes of endogenous release include pain, traumatic injury, boarding, transport, or other painful conditions.

> Following exogenous administration, leukocytosis (17,000 - 35,000/μl) and neutrophilia occur within 4 - 8 hours and return to normal 1 - 3 days after treatment (See Case 2).

Neutrophilia of Acute Inflammation

> Inflammation, sepsis, necrosis, or immune-mediated disease cause increased tissue demand and increased marrow release of segmented and band neutrophils.

> Leukocytosis (15,000 - 30,000/μl) characterized by neutrophilia with left shift and variable monocytosis is the usual response. Toxic neutrophils may be observed.

> Lymphopenia and eosinopenia, reflections of stress and elevated circulating glucocorticoids, are also common.

> Surgical removal or drainage of septic focus may transiently increase the magnitude of the neutrophilia (See Case 11).

Neutrophilia of Chronic Inflammation

> Some chronic suppurative lesions (eg, pyometra, abscesses, pyothorax, pyoderma) and some neoplasms can cause marrow granulocytic hyperplasia that results in severe leukocytosis (50,000 - 120,000).

> Laboratory features include neutrophilia with a left shift, variable numbers of toxic neutrophils, monocytosis, and often hyperglobulinemia.

> The anemia of inflammation (mild to moderate nonregenerative anemia) is usually present.

> The term "leukemoid response" is used to describe such inflammatory neutrophilias with WBC counts >100,000/μl (See Case 9).

Chronic Inflammation with New Steady State

> A second form of chronic inflammation is seen where a new steady state has been reached between marrow production and release of granulocytes and tissue demand.

> Total white cell counts are normal or only slightly elevated.

> Neutrophil counts are high normal or only slightly elevated and there is minimal to no left shift.

> Lymphocyte numbers tend to be in the reference range.

> The most consistent leukogram abnormality is monocytosis.

> The anemia of inflammatory disease and hyperglobulinemia are often present (See Case 6).

Figure 5-5. Mechanisms of Neutrophilia. Changes of production, distribution, release and circulation are indicated in bone marrow and blood. Relative size of arrows indicates change in rate of release from blood and bone marrow and the rate of migration into tissue.

Hemolytic or Hemorrhagic Anemias

> Neutrophilias with left shift frequently occur in animals with immune-mediated hemolytic anemia. Leukocytosis can be marked (>50,000/μl).

> Mature neutrophilia occurs 3 hours following acute hemorrhage (See Case 8).

Chronic Granulocytic Leukemia

> Usually presents with marked neutrophilic leukocytosis (>80,000/μl).

> Left shift is present and may be disordered with evidence of a maturation arrest. Very young neutrophil precursors (promyelocytes and myeloblasts) may be seen.

> Thrombocytopenia and/or nonregenerative anemia are observed in varying degrees.

> Hepatomegaly and/or splenomegaly may be present due to neoplastic infiltration.

> This condition must be differentiated from the neutrophilia of chronic inflammation (See Case 18).

Inherited Neutrophil Disorders

> Consider only when other causes can be eliminated.

> B2 integrin deficiency has been recognized in Irish Setters and results in decreased neutrophil adhesion to endothelium, diminished chemotaxis, and decreased bactericidal activity. These dogs have persistent neutrophilia and recurrent infections.

> Cyclic hematopoiesis or grey colic syndrome is characterized by cyclic fluctuations in neutrophils, monocytes, eosinophils, platelets, and reticulocytes at 11 - 13 day intervals. Neutrophil changes are most pronounced with neutrophilia following 2 to 4 day neutropenic episodes.

Morphology

Normal

Normal circulating neutrophils have the following features (Fig. 5-6 and Fig. 5-7):

• Size - 12 - 15 μ in diameter or 2 - 2.5 times the diameter of a RBC.
• Nucleus - lobulated or partially segmented with dense, dark purple chromatin
• Cytoplasm - pale pink or light blue, finely granular, smooth.

Figure 5-6. Normal canine neutrophils. Both neutrophils have a lobulated nucleus in a light pink finely granulated cytoplasm (100x).

Figure 5-7. Immature neutrophils. A band (arrow) and metameclocyte are located in the center. A normal segmented neutrophil is located in the lower left (100x).

Most of the circulating neutrophils (95 - 100%) in normal animals are segmented forms. Very few are band neutrophils.

Variation in normal morphology (Fig. 5-8 and Fig. 5-9).

> Prolonged exposure to EDTA prior to preparing the blood film can produce discrete, clear, cytoplasmic vacuoles in the cytoplasm.

> Normal neutrophils have two to four nuclear lobes.

> Five or more lobes indicate hypersegmentation, an aging change, which occurs with prolonged exposure to EDTA, glucocorticoid therapy, hyperadrenocorticism, or neutrophilias associated with chronic infections.

Figure 5-8. Hypersegmented neutrophil. Dog and cat neutrophils may have up to five nuclear lobes. This neutrophil has 8 nuclear lobes and is evidence of prolonged lifespan (100x).

Figure 5-9. Canine neutrophil with Barr body. The small tennis racket shaped appendage on the neutrophil nucleus is a Barr body, or sex lobe, indicating that the dog is a female. This can be a useful morphologic feature in dogs and cats if there is a question of gender or patient identity (100x).

In Disease

Toxic neutrophils (Fig. 5-10A, Fig. 5-10B, and Fig. 5-10C).

> Morphologic changes are apparent in neutrophils of dogs and cats with severe inflammatory disease or toxemia.

> The severity of the morphologic changes is proportional to the intensity of the inflammatory or toxemic disease.

> Morphologic features of toxic neutrophils:

• Diffuse cytoplasmic basophilia - color of the cytoplasm becomes blue-grey.
• Foamy vacuolation of the cytoplasm - irregular clearing in the cytoplasm produces vacuolation.
• Dohle bodies - one or more, irregular, basophilic cytoplasmic inclusions.
• Abnormal nuclear shapes - irregular lobulation or ring shaped nuclei.

> Toxic change is scored as mild, moderate, or severe on a scale of 1+ to 3+.

Figure 5-10a. Toxic neutrophils. With severe inflammation or toxemia, neutrophils develop morphologic changes that include cytoplasmic basophilia, cytoplasmic vacuolation, Dohle bodies, cytoplasmic granulation, and bizarre nuclear configuration. All three pictures demonstrate cytoplasmic basophilia. Neutrophils in A and B have Dohle bodies (arrows).

Figure 5-10b. Toxic neutrophils. With severe inflammation or toxemia, neutrophils develop morphologic changes that include cytoplasmic basophilia, cytoplasmic vacuolation, Dohle bodies, cytoplasmic granulation, and bizarre nuclear configuration. All three pictures demonstrate cytoplasmic basophilia. Neutrophils in A and B have Dohle bodies (arrows).

Figure 5-10c. Toxic neutrophils. With severe inflammation or toxemia, neutrophils develop morphologic changes that include cytoplasmic basophilia, cytoplasmic vacuolation, Dohle bodies, cytoplasmic granulation, and bizarre nuclear configuration. All three pictures demonstrate cytoplasmic basophilia. Neutrophils in A and B have Dohle bodies (arrows). C shows feline blood with the neutrophils having a basophilic granular cytoplasm and a bizarre circular nucleus.

Infectious agents (Fig. 5-11 and Fig. 5-12)

> Organisms that can be found in neutrophil cytoplasms include Ehrlichia, Hepatozoon, and Histoplasma.

> Canine distemper inclusions are seen occasionally in neutrophil and lymphocyte cytoplasms.

Neutrophil changes with inherited disorders

> Lysosomal Storage Disease - Cats and dogs with mucopolysacharidoses, gangliosidosis have fine, purple cytoplasmic granules in neutrophils (Fig. 5-13).

> Chediak-Higashi Syndrome - small, round pink granules are present in neutrophil cytoplasm.

> Pelger Huet Anomaly (Fig. 5-14)

• Occasionally seen in dogs.
• Neutrophil and eosinophil nuclei are hyposegmented but have dark, condensed mature chromatin patterns.
• Neutrophils and eosinophils are functionally normal so the condition is not clinically significant.
• Must be differentiated from a true left shift.
• Also occurs in rabbits where the anomaly occurs in association with other fatal heritable abnormalities.

Figure 5-11. Canine Ehrlichia: Round, granular, basophilic inclusion in neutrophil cytoplasm (arrow) is an Ehrlichia morula (100x).

Figure 5-12. Canine distemper inclusion bodies. Pale light pink, round cytoplasmic droplets in the neutrophil are canine distemper viral inclusions (100x).

Figure 5-13. Neutrophil from a dog with lysosomal storage disease. Numerous small basophilic granules are noted in the cytoplasm of a segmented neutrophil of a dog with gangliosidosis. Neutrophils from cats with mucopolysaccaridosis have a similar appearance (100x).

Figure 5-14. Canine blood, Pelger Huet anomaly. This inherited disorder causes hyposegmentation of neutrophil and eosinophil nuclei, giving the appearance of a persistent left shift. However, the neutrophils do not have toxic change and the nuclear chromatin is very dark and condensed indicating maturity (100x).