White blood cells (WBCs), or leukocytes, are 1 of the body’s major defenses in preventing and combating infection. The most common types of leukocytes are granulocytes, lymphocytes, and monocytes. This chapter will deal primarily with granulocytes, which include neutrophils, basophils, and eosinophils. The most common of the granulocytes is the neutrophil. Neutropenia, which is a deficiency of neutrophils, is 1 of the most common hematologic abnormalities during childhood and, when severe, can result in life-threatening infection.
The absolute neutrophil count (ANC) and the relative proportions of neutrophils to lymphocytes vary with age. At birth, neutrophils are the majority and decrease during the first few days of life. During infancy, neutrophils compose approximately 20% to 30% of the circulating WBCs. By 5 years of age, neutrophil and lymphocyte counts are equal, and by puberty, approximately 70% of WBCs are neutrophils.
Neutropenia is defined by a decrease in the absolute number of circulating mature and band forms of neutrophils, which can be determined by calculating the ANC. The ANC is calculated by multiplying the number of total WBCs obtained from the complete blood count (CBC) by the combined percentage of segmented neutrophils and bands. The normal resting ANC in the general population ranges between 1500 and 8000 cells/mm3 for white children over 6 years of age, while 30% of African American children have an ANC as low as 800 cells/mm3. Based on the ANC, neutropenia can be classified as mild (ANC of 1000–1500 cells/mm3), moderate (ANC of 500–1000 cells/mm3), or severe (ANC < 500 cells/mm3). Neutropenia is associated with an increased chance of developing an infection; however, only patients with severe neutropenia are likely to develop life-threatening infections.
Patients with neutropenia are most frequently infected with endogenous flora, Gram-negative organisms, Staphylococcus aureus, Staphylococcus epidermidis, streptococci, and enterococci. Susceptibility to bacterial infections varies even in the presence of severe neutropenia. Some patients with chronic neutropenia syndromes with an ANC less than 200 cells do not develop life-threatening infections, while neutropenia induced by immunosuppressive drugs, particularly in conjunction with a malignancy, is associated with higher rates of severe infections, probably due to the additional loss of cellular immunity. Severe neutropenia is associated with skin and soft tissue infections, gingivitis, stomatitis, pneumonia, and septicemia. However, isolated neutropenia is not associated with an increased risk for parasitic, viral, or fungal infections. Acute neutropenia arises when neutrophils are being employed and production is limited. Chronic neutropenia lasting months to years often evolves from impaired production or excessive splenic sequestration. Neutropenia may commonly occur from factors extrinsic to marrow myeloid cells or less frequently as an acquired disorder of myeloid and stem cells. A neutropenia classification schema is shown in Figure 437-1.
A neutropenia classification schema.
CLASSIFICATION OF PRIMARY NEUTROPENIAS
The inherited disorders of bone marrow production, characterized by selective loss of neutrophil production without accompanying congenital anomalies, are diagnosed by clinical and laboratory features and by genetic testing. Many of the inherited forms of congenital neutropenia fall into a category of chronic disorders with a heterogeneous genetic basis. Neutropenia associated with multifaceted syndromes can now be classified molecularly, which provides a basis for understanding the pathophysiology of the neutropenia.
Disorders of Granulopoiesis
Reticular dysgenesis (RD) is a very rare form of severe, combined immunodeficiency characterized by agammaglobulinemia, lymphopenia, and neutropenia. Patients with RD usually die shortly after birth from overwhelming sepsis. Bone marrow transplantation is the only available treatment. Examination of the bone marrow from patients with RD reveals absent myeloid precursors with normal erythroid and platelet differentiation. RD is thought to arise from a defect in a common stem cell that affects maturation of myeloid and lymphoid cell lines. However, the defect may be more specific for granulocyte and T lymphocyte development, since some patients with RD have residual functional B lymphocytes and monocytes of host origin following bone marrow transplantation.
Cyclic neutropenia is an autosomal dominant disorder resulting from a mutation in the ELA2 gene, which encodes for neutrophil elastase. Peripheral blood neutrophil numbers oscillate with a periodicity of 21 ± 3 days, with the nadirs often falling below 200 cells/mm3. During the periods of neutropenia, patients often have episodes of fever, malaise, aphthous stomatitis, cervical lymphadenopathy, and gastroenteritis. While episodes may be asymptomatic, life-threatening infections with Clostridium perfringens and Gram-negative organisms are a risk.
The diagnosis of cyclic neutropenia can be made by monitoring peripheral neutrophil counts 3 times a week over a 6- to 8-week span. The diagnosis can be confirmed by sequencing the ELA2 gene. While management is predominantly symptomatic, aggressive use of antibiotics, including coverage for both Gram-negative and anaerobic organisms, during infections may be required. Improvement in the neutrophil count occurs with daily granulocyte colony-stimulating factor (G-CSF). Unlike patients with severe congenital neutropenia (SCN), patients with cyclic neutropenia are not at an increased risk for hematologic malignancies.
Chronic Benign Neutropenia
Chronic benign neutropenia represents a group of disorders that are characterized by ANCs typically between 200 and 500 cells/mm3. Patients with this disorder are not at an increased risk of developing severe infections but may present with mild to moderate infections of the skin and mucous membranes. The management consists of good skin and dental care and expeditious use of antibiotics for infections. If a patient continues to have infections, G-CSF can be used.
Severe Congenital Neutropenia and Kostmann Disease
SCN, including Kostmann disease, is a group of genetic disorders characterized by severe neutropenia, with ANCs persistently below 500 cells/mm3, and associated monocytosis and eosinophilia. Affected patients suffer from severe, life-threatening bacterial infections beginning in the first few months of life. Examination of the bone marrow reveals an arrest in myeloid differentiation at the promyelocyte or myelocyte stage of development. Approximately 60% of cases of SCN arise from a mutation (autosomal dominant or sporadic) in the ELA2 gene, resulting in an increase in apoptosis of neutrophil precursors in the bone marrow. Additionally, rare cases of autosomal dominant SCN arise from mutations in GFI1, PRDM5, and PFAAP5, which repress transcription of myeloid genes, including ELA2. Kostmann disease was the first described (as an autosomal recessive SCN) and results from mutations in multiple genes, including HAX1, ELA2, GCSFR, GFI1, AP3B1, TAZ1, and MAPBPIP. The HAX1 gene specifically encodes for a mitochondrial protein that protects myeloid precursors from apoptosis.
A majority of patients, up to 90%, respond to G-CSF, resulting in an increase in peripheral neutrophil counts and reduced rates and severity of infections. While their prognosis is greatly improved, they are still at risk for severe bacterial infection, probably due to functional defects in neutrophil function. In addition, they have up to a 40% risk of developing myelodysplasia (MDS) or acute myelogenous leukemia (AML), frequently characterized by monosomy 7 and alterations in expression of the G-CSF receptor.
MULTIFACETED SYNDROMES ACCOMPANIED BY NEUTROPENIA
Neutropenia arising from complex phenotypes may be classified into disorders of ribosomal dysfunction, metabolism, vesicular transport, and immune function.
Disorders of Ribosomal Dysfunction
Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by neutropenia, recurrent infections, exocrine pancreatic insufficiency, short stature, and metaphyseal dysplasia. Metaphyseal chondrodysplasia, associated with dwarfism, occurs in approximately 50% of patients, but other skeletal anomalies include rib cage deformities, osteopenia, clinodactyly, kyphosis, supernumerary metatarsals, coxa vara deformity, and syndactyly. SDS usually presents with steatorrhea, weight loss, and failure to thrive in early infancy due to pancreatic insufficiency. While anemia and thrombocytopenia can occur, neutropenia is the most common hematologic manifestation of SDS. Approximately 90% of patients with SDS have an inactivating mutation in the SBDS gene (located on chromosome 7q11), which codes for a protein involved in rRNA maturation. These patients also have a propensity for monosomy 7–related hematologic disorders, including MDS or AML.
Management of SDS must include the replacement of pancreatic enzymes. G-CSF will raise the ANC into the normal range but should be reserved for persistent neutropenia associated with recurrent bacterial infection. Both MDS and AML are generally preceded by cytogenetic changes in the bone marrow. Bone marrow transplantation may be curative, but its precise role has not been completely defined.
Dyskeratosis congenita (DC) is a rare congenital syndrome in which progressive bone marrow failure is associated with the triad of reticulated skin hyperpigmentation, nail dystrophy, and oral leukoplakia. Common abnormalities include developmental delay, short stature, pulmonary fibrosis, esophageal web, dental caries, hyperkeratosis of the palms and soles, mandibular hypoplasia, and osteoporosis. Inheritance can be X-linked recessive, autosomal dominant, or autosomal recessive. The X-linked recessive form of DC is the most common and most severe and is caused by a mutation in the DKC1 gene coding for dyskerin, a ribosomal RNA processing and telomere maintenance protein. The autosomal dominant form arises from mutations in the telomerase components TERC or TERT.
The hallmark of DC is pancytopenia, with 10 years of age as the mean age of onset. However, 90% of patients have at least a single diminished cell line by 3 years of age, and 50% of patients will develop aplastic anemia. There is an increased incidence of malignancy (9%) in patients with DC. The role of hematopoietic stem cell transplantation is not well defined because of the high prevalence of fatal pulmonary complications, probably due to underlying pulmonary disease.
Barth syndrome is a rare X-linked recessive neuromuscular and metabolic disorder characterized by cardiomyopathy, skeletal muscle weakness, neutropenia, and growth retardation. The cardiomyopathy, which is comprised of either biventricular dilatation or left ventricular dysfunction, is the result of mutations in the TAZ (Tafazzin) gene (located at Xq28), which encodes an enzyme required for cardiolipin synthesis. The pathophysiology of the neutropenia is unknown, but ANCs range between 500 and 1500 cells/mm3. There is no specific treatment for Barth syndrome. Management is directed by symptoms, including the judicious use of antibiotics and G-CSF during infection. Often, heart disease and short stature resolve entirely with puberty.
Pearson syndrome is a rare disorder caused by a large deletion in mitochondrial DNA. Clinical features of Pearson syndrome include failure to thrive; macrocytic sideroblastic anemia; dysfunction of the exocrine pancreas, resulting in malabsorption and chronic diarrhea; lactic acidemia; hepatic dysfunction, which may lead to liver failure; renal disease; and endocrinopathies, such as growth hormone deficiency, hypothyroidism, and hypoparathyroidism. The bone marrow reveals normal cellular numbers, but both erythroid and myeloid precursors demonstrate vacuolization and accelerated apoptosis.
Glycogen Storage Disease Type 1b
Glycogen storage disease type 1b is an autosomal recessive inborn error in glycogenolysis and gluconeogenesis caused by a defect in the glucose-6-phosphatase complex (Chapter 149). Neutrophils in these patients have a flawed transport system that causes defective mobility and subsequent neutropenia (ANC < 500 cells/mm3). Their bone marrow may be either hyper- or hypocellular. Treatment with G-CSF improves the ANC and diminishes the risk of infections.
Disorders of Vesicular Transport
The constellation of autosomal recessive disorders that combine neutropenia with partial albinism are derived from abnormalities in formation or trafficking of lysosome-related organelles.
Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by partial oculocutaneous albinism in conjunction with neutrophil dysfunction. It is frequently associated with recurrent infections affecting the skin, respiratory tract, and mucous membranes. These infections are caused by both Gram-positive bacteria, Gram-negative bacteria, and fungi; S aureus is the most common organism. Enterocolitis; peripheral neuropathy; easy bruising with prolonged bleeding times; and hypopigmentation of the skin, hair, and eyes are also observed.
CHS is caused by a mutation in the lysosome trafficking regulatory gene (LYST), localized to chromosome 1 (q42-43), resulting in a defect in intracellular trafficking that affects vesicle formation and fusion. Leukocytes, fibroblasts, platelets, and melanocytes contain large, irregular storage and secretory granules. Decreased survival of myeloid precursors in the bone marrow results in moderate neutropenia, with total WBC counts of about 2500 cells/mm3. Neutrophils contain giant specific and azurophilic granules exhibiting diminished bactericidal activity. Ingestion and the respiratory burst remain normal; however, chemotactic activity is decreased, and intracellular killing is diminished due to a slow, inconsistent delivery of dilute amounts of hydrolytic enzymes from the giant granules into the phagosomes. Monocyte and natural killer cell function also is impaired. Additionally, a platelet storage pool abnormality is present, although platelet counts are normal.
Most patients exhibit a lymphoma-like accelerated phase caused by a lymphohistiocytic proliferation in the reticuloendothelial systems, which intensifies the already existing neutropenia and leads to pancytopenia. The accelerated phase, which can occur at any age, is associated with recurrent bacterial and viral infections and ultimately results in death. The onset of the accelerated phase may be related to the inability to contain and control the Epstein-Barr virus and leads to features that simulate viral-mediated hemophagocytic lymphohistiocytosis (HLH) syndrome.
Oculocutaneous albinism, secondary to melanocyte dysfunction, is a prominent feature of CHS. Patients with this condition have skin that is usually fair, light blond or silvery gray hair, pale retinas, and translucent irises. Neurological dysfunction, which frequently appears during the accelerated phase, is commonly associated with gait disturbances, seizures, mental retardation, or dementia later in life; parkinsonism; and peripheral neuropathy. Spinocerebellar degeneration, movement disorders such as parkinsonism, or dementia may occur in patients who survive to adulthood, but may also be the presenting symptoms of CHS.
CHS is often fatal before age 10, usually secondary to overwhelming infection. Those who survive longer are at increased risk of developing lymphomas. Early bone marrow transplantation is the preferred therapy, especially if the patient is in the accelerated phase. While a transplant will correct the immune problem and the accelerated phase, it has little effect on the development of neurological sequelae, which typically worsen with age.
Griscelli syndrome (GS) is a rare autosomal recessive disorder characterized by pigmentary dilution of the skin and hair in association with either hematophagocytic syndrome and immunodeficiency or neurological symptoms, depending on the genetic defect. Patients with GS have silver hair and may have neurological manifestations, including obstructive hydrocephalus, bilateral basal ganglia involvement, hypotonia, encephalopathy, hemiparesis, peripheral facial palsy, spasticity, seizures, or psychomotor retardation.
GS is caused by 1 of 2 gene mutations located at band 15q21, RAB27A, which encodes for guanosine-5′-triphosphate–binding protein, or MYO5A, which codes for myosin Va. Both proteins are involved in pigment granule (melanosome) transport. The 2 clinical presentations of GS are due to differential expression of RAB27A and MYO5A. The MYO5A gene is expressed in the brain while the RAB27A gene is not, accounting for the absence of neurological involvement with RAB27A mutations. All patients with RAB27A mutations have uncontrolled activation of T cells and macrophages and develop HLH, which is not seen with MYO5A mutations. Patients with RAB27A mutations can be successfully treated with bone marrow transplantation.
Hermansky-Pudlak Syndrome Type 2
Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disease characterized by neutropenia, oculocutaneous albinism, lysosomal ceroid storage, and hemorrhagic diathesis, which is a result of platelet dysfunction. Pulmonary fibrosis and inflammatory colitis may also occur. HPS2 is caused by mutations in the AP3B1 or HPS2 gene, which encodes the beta subunit of the adaptor protein 3 complex, which is involved in intracellular vesicle formation and trafficking. Mutations in 8 genes, HPS1 through HPS8, have been associated with variations of HPS. In HPS2, melanocytes, platelets, cytotoxic T-lymphocytes, and natural killer cells are preferentially affected; the neutropenia is associated with diminished levels of neutrophil elastase.
P14 deficiency is an autosomal recessive disorder associated with congenital neutropenia, partial albinism, short stature, and B-cell and cytotoxic T-cell deficiencies. The p14 protein is required for the proper biogenesis of endosomes and subcellular relocation of mitogen-activated protein kinase signaling to late endosomes.
Cohen syndrome is an autosomal recessive disorder characterized by intermittent isolated neutropenia; nonprogressive mild to severe psychomotor retardation; facial dysmorphism, including high-arched or wave-shaped eyelids, a short philtrum, thick hair, and low hairline; pigmentary retinopathy; joint laxity; and microcephaly. The gene for Cohen syndrome (COH1) has been mapped to chromosome 8q22, which codes for a transmembrane protein thought to be involved in intracellular vesicle transport.
Disorders of Immune Function
Cartilage-hair hypoplasia (CHH), an autosomal recessive disorder, is characterized by short-limb dwarfism arising from metaphyseal dysplasia, fine hair, immunodeficiency, and an increased incidence of cancer. Patients are noted to have moderate neutropenia of 100 to 2000/mm3 and an increased risk of life-threatening infections, especially varicella zoster virus. Physical manifestations of CHH include fine or sparse hair, including eyebrows; gastrointestinal complications, including Hirschsprung disease; hypermobility and hyperflexibility of the joints; and mild flattening of the vertebral body and lumbar lordosis of the spine. It arises from mutations in the RMRP gene on chromosome 9p13-12, which encodes the RNA component of a ribonuclear protein ribonuclease. Abnormalities of both T-cell function and the humoral immune system have been found. Some patients have benefited from G-CSF treatment.
Hyper-IgM syndrome is an immunodeficiency disorder with elevated immunoglobulin M (IgM), caused by at least 3 different genetic defects. The most common form of the disease, X-linked hyper-IgM syndrome (Xq26-27.2), arises from mutations in the gene for CD40 ligand and in 50% of patients is accompanied by neutropenia. The mechanism of neutropenia is not well understood. Myeloid precursors in the bone marrow express CD40 ligand, and bone marrow examination in these patients reveals maturation arrest at the promyelocyte-myelocyte stage. The CD40 ligand protein is normally expressed on activated T lymphocytes, macrophages, and dendritic cells. It is necessary for an immunoglobulin class switch. Thus, patients with X-linked hyper-IgM syndrome have normal or elevated IgM with extremely low or absent IgG, IgA, and IgE. These patients also have altered cell-mediated immunity with an increased susceptibility to opportunistic infection and are at increased risk of developing autoimmune disorders and malignancies, probably due to altered macrophage and dendritic cell function. Treatment for the immune deficiency by IgG replacement is necessary. The neutropenia responds to G-CSF. Pneumocystis jirovecii prophylaxis with trimethoprim/sulfamethoxazole is indicated as soon as the diagnosis is established.
X-linked agammaglobulinemia (XLA) is an immunodeficiency with a B-cell differentiation defect. It is caused by a mutation on the Bruton’s tyrosine kinase (BTK) gene, which leads to a defect in the toll-like receptor with resultant failure in the regulation of B-cell survival, activation, proliferation, and differentiation. Most patients are diagnosed in early childhood with low serum immunoglobulins and severe bacterial infections after 6 months of age. Infections are commonly caused by S aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Neutropenia is noted in 20% of XLA patients.
X-linked neutropenia is a genetic disorder with variable expression and severe neutropenia that is responsive to G-CSF therapy. Patients have cellular and humoral immunodeficiency, normal platelet numbers, and recurrent infections. The Wiskott-Aldrich syndrome (WAS) gene, located on Xp11.22-23, codes for a protein involved in signal transduction between G-protein coupled receptors and the initiation of actin bundling. WAS mutations affect cellular functions such as growth, endocytosis, exocytosis, and cytokinesis. The bone marrow shows trilineage dysplasia with markedly reduced myelopoiesis.
Myelokathexis is a rare autosomal dominant disorder characterized by moderate to severe neutropenia accompanied by neutrophil hyperplasia in the bone marrow. There are noticeable morphological changes in the neutrophils, including cytoplasmic vacuoles, prominent granules, and nuclear hypersegmentation with very thin filaments connecting pyknotic-appearing nuclear lobes. Recurrent warts and hypogammaglobulinemia often occur with myelokathexis, leading to the acronym WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis). This syndrome is now attributable to a defect in the chemokine receptor CXCR-4. The ligand stromal derived factor 1 (SDF-1) is believed to play a role in regulating neutrophil migration as well as the trafficking of lymphocytes in hematopoietic progenitor cells. Myeloid cells are not mobilized from the bone marrow, where they undergo apoptosis. The neutropenia can often be partially corrected by the use of G-CSF and granulocyte macrophage colony-stimulating factor (GM-CSF).
ACQUIRED NEUTROPENIAS OF MYELOID AND STEM CELLS
Neutropenia can develop as a result of a variety of conditions. Bone marrow failure with neutropenia, associated with anemia and thrombocytopenia, can occur in aplastic anemia (Chapter 428). Vitamin B12, folate, and copper deficiency, as well as starvation can lead to decreased marrow production of neutrophils. Antibodies directed against neutrophils as well as many drugs, including chemotherapeutic agents, can induce neutropenia. Overwhelming bacterial sepsis, some viruses, fungi, and protozoal infections may also cause neutropenia. This condition can arise during hemodialysis and leukapheresis. Many of the acquired neutropenias will respond to removal of the offending agent or treatment with recombinant G-CSF.
Alloimmune neonatal neutropenia is a self-limited condition in which maternal antineutrophil antibodies (IgG) cross the placenta, resulting in the destruction of the newborn’s neutrophils. Maternal antibodies may be the result of an underlying maternal autoimmune condition or may be secondary to direct sensitization to paternally derived fetal neutrophil antigens, in a similar manner to Rh hemolytic disease. The duration and extent of the neutropenia depends on maternal antibody production, but recovery is usually complete within 6 to 12 weeks. During the neutropenic period, the infant should be watched closely for infection and managed aggressively with appropriate antibiotics for clinical indications.
Autoimmune neutropenia of infancy is a relatively benign condition in which children transiently develop moderate to severe neutropenia secondary to the production of antineutrophil antibodies. The age of onset is usually between 5 and 15 months, and the mean duration of neutropenia is 17 months. While the ANCs often fall below 500 cells/mm3, life-threatening infections rarely occur. Patients usually present with otitis media, gastroenteritis, tonsillitis, and skin and soft tissue infections. Approximately one-third of the patients show circulating antibodies against neutrophil antigens HNA-1 or HNA-2. G-CSF is the most effective agent for normalizing peripheral neutrophil counts in patients with recurrent fever or infectious complications.
Autoimmune neutropenia arising from the production of antineutrophil antibodies can occur in patients with no underlying autoimmune disorder or can be associated with a variety of autoimmune conditions such as systemic lupus erythematosus, autoimmune hemolytic anemia, immune thrombocytopenia, or thyroiditis. Autoimmune neutropenia in children frequently is associated with immune deficiencies. Successful management of the underlying autoimmune disease will often result in normalization of peripheral neutrophil counts. In other instances, G-CSF may be required.
Drug-related neutropenia is common and can occur at any age, although more than 90% of cases occur in adults. Drug-induced neutropenia is not always predictable, but when it arises, it may occur by several different mechanisms, including immune-mediated toxic, idiosyncratic, and hypersensitivity reactions. In contrast, cytotoxic chemotherapy predictably affects proliferating myeloid cells by impairing neutrophil production. Therapy of the former causes of neutropenia requires cessation of the responsible drug. Treatment with G-CSF should be employed, but it may not always be effective. Neutropenia accompanying cytotoxic chemotherapy typically occurs 7 to 14 days after administering therapy. It is often accompanied by depressed cellular immunity, thereby predisposing patients to a much greater risk of infection than those disorders associated with the acute onset of isolated neutropenia.
Reticuloendothelial sequestration frequently results in mild to moderate neutropenia often accompanied by platelet and red cell trapping in the spleen, leading to thrombocytopenia and anemia. Splenectomy should be avoided if possible because of the increased risk of infection due to encapsulated bacterial organisms.
Virus-related neutropenia occurs concurrently or shortly following a viral illness and is secondary to viral antibodies cross-reacting to neutrophil antigens. The neutropenia is usually mild, transient, and of minimal clinical significance.
Conditions that result in complement activation, inflammatory disorders, hemodialysis, and extracorporeal membrane oxygenation (ECMO) can also cause neutropenia.
FUNCTIONAL NEUTROPENIA WITH DISORDERS OF PHAGOCYTOSIS
Neutrophils play a key role in protecting the skin and mucous membranes of the oral cavity, the respiratory tract, and the gastrointestinal tract; they are the first line of defense against bacterial invasion. Neutrophils arrive at sites of inflammation during the critical period after microbial tissue invasion. It is at this juncture that infection must be contained. If not, the infection can spread and potentially disseminate. Patients with the following disorders of neutrophil function have difficulty in containing bacterial infection.
Specific Granule Deficiency
This is a rare autosomal recessive disorder resulting in an increased susceptibility to severe bacterial infection, characterized by severely diminished production of neutrophil constituents, defective neutrophil chemotaxis, and diminished bactericidal activity. On peripheral blood smear, the nuclei of the neutrophils have a characteristic bilobed morphology. Eosinophil-specific granules exhibit diminished eosinophil-cationic protein, eosinophil-derived neurotoxin, and major basic protein. The disorder arises secondarily to the functional loss of the transcription factors CCAAT/enhancer binding protein ε or GFI1. Patients are predisposed to infections caused by S aureus and Pseudomonas species. Treatment is supportive.
Leukocyte Adhesion Deficiency
Leukocyte adhesion deficiency (LAD) disorders are a group of autosomal recessive disorders of neutrophil function arising from a deficiency of the β2 antigen subunit of the leukocyte adhesion molecule. In general, these disorders are characterized by recurrent bacterial infections and by impaired pus formation and wound healing as a result of diminished adhesion functions of neutrophils, monocytes, and natural killer (NK) cells.
LAD type 1 is characterized by marked leukocytosis, ranging from 12,000 to 60,000/mm3. Even in the absence of a specific infection, patients exhibit a diminished expression of abnormal function of the β2 integrin family surface glycoproteins, designated the CD11/CD18 complex. A markedly diminished or absent CD11a/CD18 complex accounts for the failure of patients’ neutrophils to adhere to endothelial cells and subsequently migrate to specific sites of infection. Each of the molecules comprising the CD11/CD18 complex contains an α and a β subunit that is noncovalently associated in an α-β structure. Each of the α chains associates with the same β subunit and is distinguished by its unique α subunit protein structure. Many patients have point mutations that result in a single amino acid substitution in the β2 subunit. The resulting mutations lead to the failure of the expression of the β2 subunit and a failure of the assembly of the α-β subunits on leukocyte membranes.
The leukocytes in patients with the most severe clinical form of LAD express less than 0.3% of the normal amount of β2 integrins, while those patients with the moderate phenotype may express 2.5% to 31% of normal amounts of β2integrin molecules. The severe forms of LAD1 suffer from recurrent and chronic or even gangrenous soft tissue infections generated by bacterial and fungal microorganisms such as S aureus, Pseudomonas species, other Gram-negative microorganisms, or Candida species. Patients with a moderate phenotype have fewer and less severe infections but can be chronically afflicted with severe periodontitis. Infectious susceptibility and impaired wound healing are related to diminished or delayed infiltration of neutrophils and monocytes into extravascular inflammatory sites. All patients surviving infancy have profound periodontitis. Delayed separation of the umbilical cord is noted following birth.
LAD2, or congenital disorder of glycosylation type IIc, is an autosomal recessive disorder that is associated with growth and mental retardation. Patients have distinctive dysmorphic features, including microcephaly and a depressed nasal bridge along with seizures and hypotonia, which are related to a defect in fucose metabolism. Laboratory studies reveal decreased neutrophil motility but normal phagocytic activity. Patients lack the red blood cell H antigen, a fucosylated protein, and manifest the Bombay (hh) phenotype. Functionally, the neutrophils from these patients are unable to adhere to E-selectin or cytokine-activated endothelial cells and exhibit an inability to roll on postcapillary venules in vivo. The LAD2 neutrophils express normal levels of CD18 integrins but are deficient in the fucose structure sialyl-Lewis X, which renders the cell unable to roll on activated endothelial cells. The LAD2 defect is secondary to decreased protein fucosylation, which arises from impaired transport of GDP-fucose from cytoplasm to the Golgi lumen.
LAD3 has been described in patients with recurrent infections, leukocytosis, absent pus formation, and a bleeding disorder. It is characterized by a general failure to activate a number of integrins on leukocytes and platelets.
Most cases can be diagnosed by clinical symptoms and marked leukocytosis; however, flow cytometry identifying the expression of CD18 can confirm the diagnosis of LAD1.
Patients with the severe form of LAD1 should be considered for allogeneic bone marrow transplantation. Since only 25% of LAD patients will have a matched sibling donor, other donor sources have been tried, but the toxicity associated with the myeloablative transplant regimens has restricted the more extensive use of alternate donor sources. Patients with moderate LAD can be maintained on prophylactic trimethoprim/sulfamethoxazole. Broad-spectrum antibiotics are indicated for empirical therapy when infection occurs. Since most children with LAD lack a matched sibling donor, gene replacement therapy, which is a new technology used for other immune deficiencies, may be investigated for LAD treatment in the future. LAD1 is an ideal candidate for this approach because some improvement in neutrophil function attenuates the severity of the disease.
Some patients with LAD2 respond to oral fucose, which restores the expression of fucosylated sialyl-Lewis X neutrophils and leads to a disappearance of leukocytosis and an attenuation of recurrent bacterial infection.
The severity of repeated infectious complications correlates with the extent of β2 integrin deficiency. Patients with severe deficiency usually die in infancy, whereas patients with moderate deficiency have infrequent life-threatening infections and relatively long survival.
Hyperimmunoglobulin E Syndrome
Hyperimmunoglobulin E (hyper-IgE) syndrome is a rare autosomal dominant disorder with incomplete penetrance characterized by markedly elevated levels of serum IgE, chronic eczema, recurrent bacterial infections, retention of primary dentition, hyperextensible joints, a predisposition to bone fractures, and a distinctive coarse facial appearance.
There have been reports of more than 200 cases worldwide in patients from diverse ethnic backgrounds. Males and females have been affected with variable expression in succeeding generations, suggesting that the disorder is autosomal dominant with incomplete penetrance. The neutrophils and monocytes from patients with hyper-IgE syndrome exhibit a variable chemotactic defect that appears extrinsic to the neutrophil. Several mutations affecting the signaling molecule STAT3 have been identified in both familial and sporadic cases of hyper-IgE syndrome. The STAT3 molecule plays a pivotal role in embryogenesis and immunomodulation/regulation, including the induction of antigen-specific T-cell tolerance and interleukin 12 (IL-12) regulation, which may explain the diverse clinical features of the syndrome.
Clinical manifestations of hyper-IgE syndrome may begin in the first 2 months of life. Initially, patients may have a chronic eczematoid rash involving the face and extensor surfaces of the arms and legs. Skin lesions are usually sharply demarcated and lack surrounding erythema. By school age, there is a history of recurrent skin abscesses; recurrent pneumonia often leading to pneumatoceles; as well as chronic otitis media. S aureus is the most common infectious agent, but other organisms, including Haemophilus influenzae and E coli, have been documented. Other associated features include coarse facial features, a broad nasal bridge, and facial asymmetry. There is often retention of primary teeth and generalized osteopenia, which can predispose to bone fractures.
Hyper-IgE syndrome needs to be differentiated from atopic dermatitis. In the former disorder, S aureus infections are deep seeded and serious, and respiratory allergy is rare. Atopic dermatitis in characterized by superficial S aureus infections; furthermore, the patients with atopic dermatitis do not have the distinctive anatomic features found in hyper-IgE syndrome.
Serum IgE levels typically exceed 2500 IU/mL. Abnormally low antibody responses and depressed cell-mediated responses to neoantigens are frequently present. The treatment of hyper-IgE syndrome is predominantly supportive and includes the judicious use of antistaphylococcal antibiotic therapy and immunoglobulin replacement therapy. Prophylaxis with trimethoprim/sulfamethoxazole can reduce the frequency of recurrent infections. Incision and drainage are required for managing abscesses, including infected pneumatoceles. At times, the neutrophils of patients manifest impaired chemotactic responses. If hyper-IgE is recognized early in life and if the patient is placed on proper therapy, prognosis is good.
Chronic Granulomatous Disease
Chronic granulomatous disease (CGD) is a genetic disorder affecting the function of both neutrophils and monocytes. The phagocytic cells are able to ingest but not kill catalase-positive microorganisms because of an inability to generate antimicrobial oxygen metabolites. CGD arises from mutations involving 1 of 4 genes that encode some of the components of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
The incidence of CGD in the United States is 1 in 200,000 births with a predominance of males (> 85% of patients). Approximately 70% of children affected with CGD have the X-linked recessive form of the disease resulting from a mutation in the CYBB gene, located on the X chromosome, which encodes for gp91phox. The remainder of patients inherit CGD in an autosomal recessive fashion: Mutations in the NCF1 gene, encoding for p47phox, account for 20% of the remaining cases, while mutations in the genes NCF2 (encoding for p67phox) and CYBA (encoding for p22phox) each account for an additional 5% of cases.
Mutations in the genes comprising components of NADPH mentioned above impair the phagocyte’s ability to generate O2− and H2O2 from molecular O2 following phagocytosis. Mutations in genes giving rise to cytochrome b588 affect electron transport from NADPH to flavin and heme prosthetic groups, whereas mutant genes encoding for p47phox and p67phox affect activation of cytochrome b588 by preventing changes in its tertiary structure.
The failure to activate the NADPH oxidase in CGD predisposes the host to infection with catalase-positive microorganisms. Normal neutrophils generate H2O2 in the phagosome and utilize myeloperoxidase (MPO), which is delivered into the phagosome from primary granules fusing with the phagosome’s membrane to generate hypochlorous acid from chloride and kill microbes. The H2O2 produced by normal neutrophils can exceed the capacity of catalase, a hydrogen peroxide–catabolizing enzyme of many aerobic microorganisms, including S aureus, most Gram-negative bacteria, and Candida albicans and Aspergillus species, to kill the offending microorganisms. In contrast, the CGD neutrophils, which cannot produce H2O2, are unable to kill the catalase-positive microorganisms but are able to kill catalase-negative microorganisms, because the microorganisms produce sufficient amounts of H2O2 to result in a microbicidal effect. Catalase-positive microorganisms can survive for long periods in the CGD phagosome. Eventually, the bacteria are killed but are not digested properly in macrophages and thereby contribute to the formation of granulomata.
Although the clinical presentations can be quite variable, features that suggest a diagnosis of CGD include recurrent lymphadenitis; bacterial abscesses affecting the lung, perianal area, liver, and spleen; or osteomyelitis manifesting at multiple sites or in the small bones of the hands and feet. Patients with a family history of recurrent infections or unusual catalase-positive microbial infections should be evaluated for this disorder. The onset of clinical signs and symptoms may occur from early infancy to young adulthood with two-thirds of patients’ symptoms appearing during the first year of life, although some may present later in life.
S aureus is the microorganism most commonly affecting CGD patients, although infection with Serratia marcescens, Burkholderia cepacia, Salmonella species, Aspergillus species, and Nocardia species are pathognomic for CGD. C albicans is also commonly seen. Patients may develop numerous sequelae, including anemia of chronic disease, lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, chronic purulent dermatitis, restrictive lung disease, gingivitis, hydronephrosis, or pyloric outlet obstruction. Chronic inflammation may lead to granuloma formation, and some patients may develop a Crohn disease–like inflammatory bowel process.
The diagnosis of CGD historically was made using the nitroblue tetrazolium dye (NBT) test, which assesses the ability of activated neutrophils to reduce the yellow water-soluble tetrazolium dye to an insoluble blue formazan pigment using the NADPH oxidase system. This rarely used test is not quantitative and may miss some patients with p47phox deficiency who may have low but insufficient levels of NADPH oxidase activity. The standard diagnostic test is now the dihydrorhodamine (DHR) test, which employs flow cytometry to detect the conversion of DHR to rhodamine upon activation of the neutrophils. Autosomal recessive p22phox, p67phox, p47phox, and gp91phox expression can be determined by western blot analysis, and individual genetic analysis can be employed to identify the specific genetic mutation to determine the inheritance pattern. When suspected, the prenatal diagnosis can be determined either genetically (DNA analysis following chorionic villous biopsy) or functionally by evaluating neutrophil activation on fetal blood via DHR. Similarly, the female carrier state of X-linked CGD can be determined genetically or functionally by activating neutrophils and establishing whether 2 populations of neutrophils exist (ie, 1 that oxidizes DHR and 1 that does not).
Patients with severe forms of glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolysis are unable to generate NADPH and therefore have an impaired respiratory burst and increased susceptibility to infections similar to patients with CGD. A normal G6PD activity level with an impaired respiratory burst is most consistent with CGD.
Treatment involves the aggressive use of bactericidal antibiotics, often for prolonged periods of time, and appropriate drainage of abscesses. Cultures must be obtained as soon as infections are suspected. If fever occurs, it is advisable to obtain studies that aid in managing septic episodes, which may include chest and skeletal x-rays or computed tomography (CT) scans to ascertain whether pneumonia, osteomyelitis, or liver abscesses are present. Aspergillus infections require treatment with antifungal agents and, in refractory cases, granulocyte transfusions. Glucocorticoids may be useful in treating patients with severe inflammation and granuloma formation, such as with antral and urethral obstruction.
Long-term prophylaxis includes therapy with trimethoprim/sulfamethoxazole (5 mg/kg per day of trimethoprim) and itraconazole to reduce the development of bacterial and fungal infections, respectively. Interferon-γ can reduce the number of serious bacterial and fungal infections, although its mechanism of action is unclear.
Hematopoietic stem cell transplantation can be curative for patients with CGD. Transplantation at an early age likely has the greatest chance of success, but because the clinical severity of the disorder is so variable, transplantation is often performed later in life. Gene therapy utilizing the normal gene for a missing component has been successfully employed in 2 adult CGD patients and warrants further evaluation.
The prognosis for patients with CGD is highly variable, with median survival estimated to be between 20 and 25 years. X-linked CGD tends to be more severe than the autosomal recessive forms likely due to the decrease in O2− production in X-linked compared to most autosomal recessive forms.
MPO deficiency, the most common inherited disorder of neutrophil function, is inherited as an autosomal recessive trait with a prevalence of 1 in 2000. MPO, found in neutrophil azurophilic granules, catalyzes the conversion of H2O2 and chloride ions into a potent antibacterial agent, hypochlorous acid.
Although the rate of initial bacterial killing is diminished in MPO deficiency, normal microbicidal activity is observed 1 hour following cellular microorganism ingestion. Thus, the MPO-deficient neutrophil employs an MPO-independent system for killing bacteria that is slower than the MPO–hydrogen peroxide–halide system, but eventually it is effective in eliminating bacteria. MPO neutrophils produce higher H2O2 concentrations than normal, which improves the bactericidal activity of the affected neutrophils. On the other hand, candidacidal activity in MPO-deficient neutrophils is markedly reduced. The genetic mutations in the MPO gene causing this defect have been defined. The primary translation product of the gene is a single-gene peptide that undergoes cotranslational glycosylation in the promyelocyte stage of development. Most patients with MPO deficiency have a missense mutation in the gene that prevents glycosylation and results in the enzyme’s inability to incorporate heme, leading to an inactive enzyme.
MPO deficiency is not usually clinically apparent. Patients rarely may have disseminated candidiasis, usually in conjunction with diabetes mellitus. Acquired partial MPO deficiency has been found in patients with acute myelogenous leukemia or myelodysplastic syndrome. Deficiency of neutrophil MPO can be identified by histochemical analysis. There is no specific therapy. Prognosis is usually excellent.
Leukocytosis is an elevation in the total leukocyte count that is 2 standard deviations above the mean value for age. The various etiologies of leukocytosis are categorized by the class of WBC that is elevated.
Neutrophilia refers to an alteration in the total number of blood neutrophils that is in excess of about 7500/mm3 in older children and adults. During the first few days of life, the upper limit of normal ranges from 7000 to 13,000/mm3. When the child is 1 year of age, the range is 1500 to 8500/ mm3.
Neutrophils increase in the circulation following disruption to the normal equilibrium in their production in the bone marrow and/or their migration into the circulation. Neutrophilia may arise as a result of (1) enhanced mobilization into the circulation from either the bone marrow or peripheral blood marginating pool, (2) reduced neutrophil migration into tissues, or (3) expanded progenitor proliferation and terminal differentiation through the myeloid series. Neutrophilia is usually acquired. Both acute and chronic bacterial infections, trauma, and surgery are among the most common causes. Neutrophilia may accompany sickle cell disease, thermal injury, diabetic ketoacidosis, and chronic hemolytic anemias. Several drugs commonly associated with neutrophilia include epinephrine, corticosteroids, recombinant human G-CSF, and recombinant human GM-CSF. Epinephrine releases neutrophils from the marginating pool to the circulating pool. Corticosteroids enhance the release of neutrophils from the marrow and impair their migration from the circulation into tissues. Acute neutrophilia can also accompany inflammation or infection associated with the generation of endotoxin, tumor necrosis factor, and IL-1.
Leukemoid reaction is a nonmalignant leukocytosis that typically includes a WBC count over 50,000/mm3 alone or with leukocyte precursors (eg, myeloblasts or myelocytes). It arises from inflammatory syndromes, including infection.
Monocytosis may occur in many clinical disorders, including bacterial, fungal, or rickettsial infections. It may also occur in patients with conditions associated with chronic inflammation (eg, systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis) preleukemic states; with malignancies (eg, chronic myelogenous leukemia, juvenile myelomonocytic leukemia, lymphomas, and Hodgkin disease); in patients recovering from myelosuppressive chemotherapy; following drug reactions; as a manifestation of sarcoidosis; with severe chronic neutropenia; and postsplenectomy. Monocytopenia is a feature of the immune deficiency syndrome involving the transcription factor important for hematopoiesis, GATA2. This syndrome is characterized by mycobacteria susceptibility, monocytopenia, and lymphopenia with poor NK cell and dendritic cell function.
Viral illness is the most common etiology of lymphocytosis. It is observed in infectious mononucleosis, cytomegalovirus infection, viral hepatitis, many common viral illnesses, toxoplasmosis, syphilis, tuberculosis, and brucellosis. Severe chronic neutropenia often is accompanied by a relative lymphocytosis. Lymphocytosis may also be present in patients with thyrotoxicosis or Addison disease.
Eosinophils are similar to neutrophils with respect to size and morphology but can be distinguished by their characteristic reddish brown membrane-bound granules when stained with eosin. Their constituent products include major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil peroxidase. Eosinophils differentiate in the bone marrow from myeloid precursors in response to IL-3, IL-5, and GM-CSF, ultimately comprising 2% to 3% of the circulating WBC population. Eosinophils remain in the circulation transiently, for 8 to 12 hours, then migrate to and function in tissue sites where they can persist up to 8 to 12 days in the absence of stimulation. The tissue-to-circulation eosinophil ratio is about 100:1, with the colon being the most densely populated site.
Eosinophils are associated with many specific disease processes. In response to allergens and tissue parasites, eosinophils are recruited to the site of inflammation by chemokines such as eotaxin-1 and -2 and leukotriene B4. At this site, they release reactive oxygen species, granular proteins such as leukotrienes (eg, LTC4, LTE4,), growth factors, and cytokines. Eosinophilia, more than 500 cells/mm3, is associated with infestation by helminths or protozoa, collagen vascular disease, malignancies such as Hodgkin disease, brain tumors, acute myelogenous leukemia, severe eczema, exfoliative dermatitis, and Addison disease, and can occur in response to certain drugs such as penicillins. Eosinophilia can be a response to peritoneal dialysis or a manifestation of several gastrointestinal disorders, including eosinophilic gastroenteritis, inflammatory bowel disease, and chronic hepatitis. It is often observed in primary immunodeficiency syndromes such as hyper-IgE syndrome and Wiskott-Aldrich syndrome.
Hypereosinophilic syndrome is an acquired condition associated with chronic eosinophilia and tissue infiltration without a definable cause. Three criteria for this disorder include (1) eosinophils greater than 1500/mm3, (2) signs and symptoms of organ involvement, and (3) diagnoses not explained by other causes. On the other hand, eosinophilic leukemia can be distinguished by signs that indicate a clonal myeloid disease, such as anemia, thrombocytopenia, splenomegaly, and the presence of cytogenetic abnormalities in the marrow. In both the hypereosinophilic syndrome and eosinophilic leukemia, life-threatening complications can arise from cardiac damage secondary to eosinophil infiltration and release of granule constituents.
Basophils are associated with asthma and other allergic diseases. In allergic late-phase reactions, they are recruited, along with eosinophils and T helper lymphocytes, to the site of inflammation by chemokines such as eotaxin, RANTES (regulated on activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) and MCP-3, where they release histamine and LTC4. When observed under the microscope, basophils contain large blue or purple granules. Basophilia occurs when basophil counts exceed 100 to 150/mm3. Basophil counts may rise in chronic myelogenous leukemia, Hodgkin disease, hemolytic anemias, ulcerative colitis, varicella, juvenile rheumatoid arthritis, and hypothyroidism. Estrogens and antithyroid medications also may increase the basophil count. An increased number of basophils is commonly associated with hypersensitivity disorders of the IgE-associated “immediate” type.
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