Neutrophilia

Neutrophilia refers to a higher than normal number of neutrophils on a CBC with differential. Neutrophilia may result from a shift of cells from the marginal to the circulating pool (shift neutrophilia) without an increase in the total blood granulocyte pool (TBGP) or from a true increase in TBGP size (true neutrophilia).

During established infection, the neutrophil count remains elevated, with equal numbers in the marginal and the circulating pool. During the recovery phase, the flow of cells from the marrow decreases, with a resultant decrease in the number of neutrophils.

The adequate production and distribution of normally functioning neutrophils is vital to host defense. During an infection, chemotactic agents are generated that attract neutrophils to the site of infection, which in turn play a critical role in phagocytosing and killing microorganisms.

Shift neutrophilia

Stimuli such as vigorous exercise or an epinephrine injection can produce shift neutrophilia that is usually transient, lasting 20-30 minutes. ^{[1, 2]} Shift neutrophilia is also seen in cases of seizures and paroxysmal tachycardia. No increase in immature (nonsegmented) neutrophils occurs, because no change occurs in the inflow of neutrophils from the marrow.

True neutrophilia

True neutrophilia occurs in most cases of neutrophilia that are related to infections. The TBGP may be increased 5-6 times normal. During early infection, the neutrophil count may actually decrease briefly because of margination of cells from the blood. This is followed rapidly by egress of cells from the marrow, resulting in an increase in the TBGP and blood neutrophilia. If the demand for cells is high, a shift to the left in the differential count may occur. A left shift is characterized by the appearance of more immature neutrophil forms in the blood.

Acute infections

Neutrophilia can result from acute infections caused by any of the following pathogens:

• Cocci (eg, staphylococci, pneumococci, streptococci, meningococci, gonococci)
• Bacilli (eg, Escherichia coli, Pseudomonas aeruginosa, Actinomyces species)
• Certain fungi (eg, Coccidioides immitis, Candida albicans) ^[3]  
• Spirochetes
• Viruses (eg, rabies, poliomyelitis, herpes zoster, smallpox, varicella),
• Rickettsia
• Parasites (eg, liver fluke)

Neutrophilia is also seen in the following infectious conditions:

• Furuncles
• Abscesses
• Tonsillitis
• Appendicitis
• Otitis media
• Osteomyelitis
• Cholecystitis
• Salpingitis
• Meningitis
• Diphtheria
• Plague
• Peritonitis
• COVID-19 ^[4]

In acute infections, leukocyte counts typically are 15-25 × 10⁹/L. Infections such as typhoid fever, parathyroid fever, mumps, measles, and tuberculosis usually are not associated with leukocytosis.

Noninfectious inflammation

Neutrophilia can occur in noninfectious conditions, such as the following:

• Burns
• Postoperative state
• Acute asthma ^[5]
• Myocardial infarction
• Acute attacks of gout
• Acute glomerulonephritis
• Rheumatic fever
• Collagen-vascular diseases
• Hypersensitivity reactions
• Cigarette smoking

Neutrophilia in severe burns is accompanied by a shift to the left in the differential and the presence of degenerative forms on the peripheral smear, including toxic granulation and Dohle bodies.

Postoperatively, neutrophilia occurs for 12-36 hours as a result of tissue injury–related increases in adrenocortical hormones. Leukocytosis can also occur in intestinal obstruction and strangulated hernia.

Neutrophil activation during cardiopulmonary bypass (CPB) surgery may occur because of the release of complement chemotactic products or the local release of interleukin (IL)-8. The expression of beta-2 integrins on the surface of neutrophils is increased in response to IL-8 and to certain components of complement during CPB. Both IL-8 and the complement system are activated during CPB.

Patients with acute myocardial infarction experience a transient but significant rise in serum IL-8 concentration within 24 hours after the onset of symptoms. An upregulation of messenger RNA (mRNA) for IL-8 occurs in the inflammatory infiltrate near the border between necrotic and viable myocardium. Thus, IL-8 is likely involved in the pathogenesis of myocardial injury following coronary artery bypass graft (CABG) surgery.

Metabolic and toxic causes

Neutrophilia commonly occurs in the following:

• Diabetic ketoacidosis
• Preeclampsia 
• Uremia, especially with uremic pericarditis

Neutrophilia can result from poisoning with lead, mercury, digitalis, camphor, antipyrine, phenacetin, quinidine, pyrogallol, turpentine, arsphenamine, and insect venoms. In patients with lead colic, leukocyte counts as high as 20 × 10⁹/L may be seen.

Hematologic causes

Acute hemorrhage, especially into body spaces such as the peritoneal cavity, pleural cavity, joint cavity, and intracranial cavity (eg, extradural, subdural, or subarachnoid space) is associated with leukocytosis and neutrophilia. This is probably related to the release of adrenal corticosteroids and/or epinephrine secondary to pain. Local inflammation due to pressure necrosis and the generation of chemotactic factors from the lysis of leukocytes also contributes.

During the first 1-3 hours of an acute hemorrhage, neutrophilia occurs because of a shift from the marginal pool to the circulating pool. After 3-6 hours, neutrophils are released from the marrow. Acute hemolysis leukocytosis occurs following a transfusion of mismatched blood or during acute hemolytic disease.

Neutrophilia can occur with the following ^[6] :

• Anemia (eg, in chronic infections)
• Polycythemia (polycythemia vera)
• Increased platelet count (essential thrombocythemia)
• Decreased platelet count (sepsis)
• Nucleated red blood cellsRBCs (myelofibrosis, malignancies)

Neoplasms and blood malignancies

Chronic myelocytic leukemia (CML), polycythemia vera, myelofibrosis, and myeloid metaplasia result in neutrophilia. ^{[7, 8]} Neutrophilia can also occur in association with rapidly growing neoplasms when the tumor outgrows its blood supply. This process is thought to be due to tumor necrosis factor (TNF)-alpha. Some tumor types produce neutrophilic growth factors (eg, granulocyte colony-stimulating factor [G-CSF] production by squamous cell cancers of the head and neck). ^[9]

Physiologic neutrophilia

Strenuous exercise and epinephrine injection can cause transient neutrophilia. Physiologic neutrophilia is also seen in pregnancy, labor, and in newborns.

Genetic causes

Hereditary neutrophilia has been described in one family with an activating germline mutation in colony-stimulating factor 3 receptor (CSF3R).  

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder also associated with mutations in CSF3R that may activate this receptor, leading to the proliferation of neutrophils that are the hallmark of this leukemia. CSF3R is found in about 80% of cases. Also common are mutations in SETBP1 (14%–50%) and various spliceosome proteins. ^[10]  

A novel homozygous IRF8 mutation was reported in the fatality of a neonate with severe neutrophilia. ^[11]   

Other causes

Neutrophilia can occur in association with convulsions and paroxysmal tachycardia. Short- or long-term administration of corticosteroids causes neutrophilia. Neutrophilia is seen in association with Cushing disease.

Neutrophilia may be present without an identifiable cause, in which case it is known as chronic idiopathic neutrophilia. Persistent neutrophilia (PN) has been defined as an absolute neutrophil count of ≥7.8 × 10⁹/L) on at least three CBCs at least 2 months apart without an identifiable etiology. Kushnir and colleagues reported PN with  ≥9.0 × 10⁹/L neutrophils doubled the risk for venous thromboembolism (VTE). ^[12]

Starting, like all blood cells, as multipotent hematopoietic stem cells in bone marrow, neutrophils develop through three initial morphologic stages: myeloblast, promyelocyte, and myelocyte. In  these stages, the cells are capable of replication. In the later stages of neutrophil development, metamyelocyte and polymorphonuclear, only cell differentiation occurs. 

Myeloblast stage

The myeloblast cell has a large nucleus, is round or oval, and has a small amount of cytoplasm. No condensation of chromatin is observed, and 2-5 nucleoli are present. No granules are present in the cytoplasm at this stage.

Promyelocyte stage

The promyelocyte cell is larger than the myeloblast. The nucleus is round or oval, and the nuclear chromatin is diffuse, as in the myeloblast. The nucleoli tend to become less prominent as the cell develops. The azurophilic or primary granules appear at this stage, but the secondary granules are not yet present. The primary granules are budded off the concave surface of the Golgi complex.

Myelocyte stage

In the myelocyte stage, the secondary granules appear. These granules are smaller than the primary granules and stain heavily for glycoprotein. A pinkish ground-glass background, which is the glycoprotein, is observed when the cell is stained. Secondary granules arise from the convex surface of the Golgi complex. The myelocyte nucleus is eccentric and round or oval. The nuclear chromatin is coarse. The nucleoli are smaller and less prominent in the myelocyte stage when compared with the promyelocyte stage.

Primary granule formation is limited to the promyelocyte stage. With each subsequent cell division, the number of primary granules decreases. In mature neutrophils, the ratio of secondary granules to primary granules in humans is approximately 2-3:1.

Metamyelocyte stage  

The next stage, the metamyelocyte stage, is characterized by an indented or horseshoe-shaped nucleus without nucleoli. The nuclear chromatin is dense, with considerable clumping along the nuclear membrane. The cytoplasm is filled with primary, secondary, and tertiary granules. In contrast to its precursors, metamyelocytes are not capable of cell division.

Polymorphonuclear stage

In the last stage, band neutrophils undergo further condensation of the nuclear chromatin. The nucleus has a sausage shape with a uniform diameter throughout its length. The nucleus progressively begins to develop 1 or more constrictions, and, as the cell develops into the polymorphonuclear stage, the nucleus has 2 or more lobes connected with filamentous strands. In the polymorphonuclear stage, the cytoplasm appears faintly pink due to an abundance of specific granules.

The major role of neutrophils is to protect the body against infectious agents. The interaction of bacteria with antibodies and the complement system results in the formation of various chemotactic agents. The initial response of the neutrophil is to migrate directionally toward the source of irritation.

Upon arrival at the site of infection or inflammation, the neutrophils adhere to the vascular endothelium. This adhesive interaction is mediated by adhesion molecules that are present on the neutrophils and the endothelial cells. The major types of adhesion molecules are the selectins, integrins, and immunoglobulin-type molecules. The selectins are the initial mediators of endothelial attachment, followed by the beta-2 integrins. Integrins are proteins on the leukocyte surface that, once activated, anchor the leukocyte to the endothelium.

The next step is migration (diapedesis) through the vascular matrix. Following the increasing gradient of chemotaxins, neutrophils migrate toward the source of tissue irritation. During the migration, the presence of chemotactic agents primes the neutrophil for subsequent activation.

Various chemoattractants, such as N -formyl peptides (eg, FMLP), C5a, ^[13] leukotriene B4, and platelet-activating factor (PAF), are released in response to infection. The chemoattractants bind to specific receptors on neutrophils, initiating cellular signal transduction pathways that set in motion ion fluxes, morphologic changes, and metabolic activation. These processes are governed by G proteins, protein kinases, and phospholipases.

Many chemotactic factor receptors are coupled to G proteins and, when activated, cause phospholipase C activation, which then hydrolyzes phosphatidylinositol bisphosphate (PIP(2)) into 2 messengers. These messengers are inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG).

IP3 binds to specific receptors on intracellular membranes, resulting in the release of intracellular calcium, which is rapidly augmented by an influx of extracellular calcium. This rise in intracellular calcium is thought to be responsible for the release of both specific and azurophil granules. The elevated intracellular calcium is transient and returns to baseline in 1-3 minutes.

Neutrophils move along the gradient of chemotactic agents by projecting a pseudopodium in front of the cell. This involves alterations in the polymerization state of actin, regulated by several proteins including actin-binding protein, gelsolin, and others, and adenosine triphosphate-dependent contraction of the actin network mediated by myosin.

The process of phagocytosis involves the projection of pseudopodia around a foreign particle, which then fuses with the neutrophil through invagination of the cell membrane, forming a phagosome. This process is more efficient if the organism is opsonized by antibodies or complement factors. The contents of the neutrophil storage granules are discharged into this so-called biologic prison. Fusion of azurophil and specific granules with the phagosome follows (phagolysosome formation).

Azurophilic granules contain many antibacterial compounds that are responsible for bacterial cell death. Specific granules contain products that, when released, extracellularly activate the complement cascade. Specific granules also contain collagenase, which helps hydrolyze the extracellular matrix, facilitating locomotion of the neutrophil through the tissues. Tertiary granules contain gelatinase, which plays a similar role in locomotion.

Bacterial cell death in the phagosome results from oxidative and nonoxidative mechanisms. ^[14] Oxidative mechanisms can be mediated by MPO, or they can be independent from MPO. Following activation, a massive increase in the consumption of oxygen by the neutrophil occurs; this is called the respiratory burst. The respiratory burst results in the production of superoxide (O₂[-]), H₂O₂, and glucose oxidation via a hexose monophosphate shunt.

NADPH is nicotinamide adenine dinucleotide phosphate; NADP⁺ is the oxidized form of NADPH, as shown in the following equation:

                     2 O₂ + NADPH (NADPH oxidase) ↔ 2 O₂(-) + NADP⁺ + H⁺

Most O₂(-) is rapidly converted to H₂O₂, either spontaneously or by superoxide dismutase, as follows:

                      2 O₂(-) + 2H⁺ ↔ O₂ + H₂O₂

O₂ and H₂O₂ are not potent microbicides in themselves; rather, they help generate more potent oxidizing agents such as oxidized halogens and oxidizing radicals. MPO in the azurophilic granules is released into the phagosome, which combines with H₂O₂ and a halide (Cl^- or Br^-) to form oxidized halogen, which is a potent antimicrobial, as follows:

                      Cl^- + H₂O₂ (MPO) ↔ H₂O + OCl^-

MPO-independent oxidative mechanisms of bacterial killing involve H₂O₂, superoxide anion (O₂[-]), hydroxyl (OH) radical, and singlet oxygen (1 O₂ *).

Oxygen-independent mechanisms play a role in bacterial killing in anaerobic conditions. These include acid, lysozyme, lactoferrin, defensins, BPI, azurocidin, serine proteinases, elastase, cathepsin G, and proteinase 3. Enzymes and oxidative agents are also released into the extracellular environment to kill invading bacteria. This process may result in tissue destruction.

Hyperglycemia and neutrophil function

Hyperglycemia decreases neutrophil activity, with an increased incidence of infection in patients with diabetes mellitus as the model of this occurrence. Elevated plasma glucose inhibits neutrophil degranulation as well as opsonization. There is evidence that shows hyperglycemia adversely affects neutrophil activity in bacterial and fungal infections. ^{[15, 16, 17, 18]}

In humans, neutrophil production takes place in the bone marrow. ^[19] The life cycle of a neutrophil can be divided into the bone marrow, blood, and tissue phases.

The myeloblast, promyelocyte, and myelocyte are capable of cell division and differentiation. These forms constitute the mitotic compartment.

The more mature neutrophil forms (ie, metamyelocyte, band, and polymorphonuclear cells) are incapable of cell division, but they do undergo cell maturation and differentiation. These cells constitute the maturation compartment and flow into the blood, to be distributed into either the circulating granulocyte pool (CGP) or the marginal granulocyte pool (MGP). The total blood granulocyte pool (TBGP) is the sum of the CGP and the MGP. Cells in these 2 pools are in constant equilibrium. The 2 pools are approximately equal in size.

An estimate of the CGP size can be determined by multiplying the neutrophil count per mm³ of blood by the known circulating blood volume. The MGP consists of cells still within the vascular space, but they are adherent to the walls of small vessels, especially postcapillary venules.

Brief exercise or epinephrine injection can increase the CGP by approximately 50% for a brief period, but the TBGP remains unchanged. This temporary increase is due to the release of cells from the marginal pool. This demargination involves disruption of the bond between the endothelium and leukocyte adhesion receptors, presumably modulated by cytokines.

The response with endotoxin injection is one of initial transient neutropenia followed by a subsequent increase in the TBGP a few hours later. The initial neutropenia is from the shift of the CGP to the MGP. An outpouring of cells from the bone marrow follows, resulting in the increase of TBGP.

Neutrophil kinetics in patients with neutrophilia

To define neutrophilia, one should note to discriminate it from granulocytosis, which could be due to an increased number of neutrophils, basophils, or eosinophils. When the absolute neutrophil count (ANC) is greater than 7700/µL (2 standard deviations above the mean in adults), it is deemed neutrophilia; when a total WBC count greater than 1100/µL accompanies it, that condition is defined as neutrophilic leukocytosis. The ANC can be calculated using the the following formula ^[20] :

                       ANC = WBC (cells/µL) × percent(PMNs + bands) ÷ 100

Leukemoid reaction is leukocytosis with significantly increased WBCs and prevalent neutrophil precursors in blood. ^[21]

Mature neutrophils are terminally differentiated cells that are no longer capable of growth or division. ^[22] Mature neutrophils contain at least 4 types of granules that are specialized lysosomes and serve as microbiocidal mediators designed to destroy microbial invaders. These granules have been classified as (1) primary or azurophil granules, (2) secondary or specific granules, (3) tertiary or gelatinase granules, and (4) secretory vesicles. ^{[23, 24, 25]}

Primary or azurophilic granules

Azurophilic granules fuse with phagocytic vesicles and deliver their contents. Primary or azurophilic granules contain the enzyme myeloperoxidase (MPO) and several other proteins and enzymes. MPO, which constitutes approximately 5% of the dry weight of neutrophils, catalyzes the production of hypochlorite from chloride and hydrogen peroxide (H₂O₂).

Various other components of azurophilic granules include defensins, lysozyme, azurocidin, bacterial permeability–increasing protein (BPI), elastase, cathepsin G, proteinase, and esterase N. Defensins are proteins that defend the body against a variety of bacteria, fungi, and viruses. Lysozyme is an enzyme that degrades bacterial peptidoglycans. Azurocidin demonstrates antibacterial activity and antifungal activity against Candida albicans. BPI has antibacterial activity against some gram-negative bacteria.

Secondary or specific granules

Secondary or specific granules are released into the extracellular space, as opposed to the primary granule content that is released into phagocytic vesicles. Secondary granules contain apolactoferrin, vitamin B-12–binding protein, plasminogen activator, lysozyme, and collagenase. Apolactoferrin binds to iron, thereby depriving bacteria of the iron that is essential for cell growth. Collagenase degrades collagen and thus augments movement of neutrophils through collagen.

Tertiary or gelatinase granules

Tertiary or gelatinase granules contain gelatinase, acetyltransferase, and lysozyme. Tertiary granules are upregulated to the surface with stimulation, as are specific granules.

Secretory vesicles

Secretory vesicles contain alkaline phosphatase, cytochrome b558, and N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) receptors. Secretory vesicles can be upregulated to the surface even in the absence of extracellular calcium, in contrast to specific and gelatinase granules that need extracellular calcium for release.

Neutrophil plasma membrane and cytoplasm

The neutrophil plasma membrane contains several membrane channels, adhesive proteins, receptors for various ligands, ion pumps, and ectoenzymes. Neutrophils contain a complex cytoskeleton, which is responsible for chemotaxis, phagocytosis, and exocytosis. Proteins that make up the cytoskeleton include the following:

• Actin
• Actin-binding protein
• Alpha-actinin
• Gelsolin
• Profilin
• Myosin
• Tubulin
• Tropomyosin

In addition to many components common to all cells, approximately 45% of the neutrophil cytosolic protein is composed of migration inhibitory factor–related proteins (MRPs), such as MRP-8 and MRP-14. Neutrophils contain a large amount of glycogen in the cytoplasm. Glycogen provides neutrophils with a source of energy, especially in areas of low extracellular glucose, such as within abscesses

The approach to a patient with neutrophilia starts with a complete history and physical examination. One should look for signs and symptoms of Cushing syndrome or signs of hyperadrenocorticism or hypercorticism as a primary event, or corticosteroid use as a secondary cause. The physician should ask the patient about medications and previous medical history to rule out the causes of neutrophilia mentioned earlier. If the patient has a fever or an obvious source of infection or inflammation, the next step is assessing it. In every case, a CBC count with differential is necessary, and C-reactive protein and erythrocyte sedimentation rate could be helpful.

In hematologic diseases, it is extremely important to do a peripheral blood smear to assess for abnormally shaped cells. Bone marrow study is of great value to rule out some malignancies. 

The presence of concurrent neutrophilia and basophilia are strong indicators of chronic myeloid leukemia and require BCR-ABL testing by polymerase chain reaction (PCR) for diagnostic confirmation. ^[26]

Diagnosis of chronic neutrophilic leukemia (CNL) requires exclusion other causes of neutrophilia, including infections and inflammatory processes, metastatic cancer, and plasma cell neoplasms with secondary neutrophilia. The presence of a membrane proximal CSF3R mutation in a patient with neutrophilic granulocytosis should be sufficient for the diagnosis of CNL, regardless of the degree of leukocytosis. ^[27]