Laboratory evaluation of neutrophil disorders

INTRODUCTION — The polymorphonuclear neutrophil (PMN) is the primary defense against bacterial and fungal invasion. Defects of neutrophil number and function commonly present with recurrent and severe bacterial and fungal infections, often involving the skin and respiratory tract as well as deep tissue sites.

This topic reviews the laboratory evaluation of neutropenia and neutrophil dysfunction. The overall approach to the patient with neutropenia, including a discussion of indications for bone marrow evaluation and the individual neutrophil function defects, are discussed separately:

●Neutropenia (children) – (See "Overview of neutropenia in children and adolescents".)

●Neutropenia (adults) – (See "Approach to the adult with unexplained neutropenia".)

●Neutrophil dysfunction – (See "Primary disorders of phagocyte number and/or function: An overview".)

TESTING FOR NEUTROPENIA — Initial testing for neutropenia is done with the complete blood count and differential, along with a review of the peripheral blood smear. Recognition of neutropenia is straightforward (ie, absolute neutrophil count [ANC] <1500/microL), although there are some age and racial differences in the normal values (eg, higher ANC during the first few days of life, lower ANC in some Black individuals and Yemenites). (See "Overview of neutropenia in children and adolescents", section on 'Definitions and normal values'.)

The ANC is calculated as the product of the white blood cell (WBC) count and the fraction of polymorphonuclear neutrophils (PMNs) and band forms in the differential analysis (calculator 1).

The peripheral blood smear may show additional helpful findings, including the presence of malignant cells, specific neutrophil inclusions or missing components, or other morphologic defects (eg, bilobed nuclei). (See "Evaluation of the peripheral blood smear", section on 'White blood cells'.)

Testing for polymorphisms associated with mild neutropenia (eg, Duffy-null associated neutrophil count [DANC]) that is most often seen in individuals of African, Sephardic, West Indian, Yemenite, and Arab Jordanian descent (previously called benign ethnic neutropenia) is described separately. (See "Approach to the adult with unexplained neutropenia", section on 'Normal variants <1500/microL'.)

Overview of causes (neutropenia) — Neutropenia can be caused by decreased production of granulocytes in the bone marrow, shift of circulating granulocytes to marginated or tissue pools, neutrophil destruction, or a combination of these causes. Information about the pathogenesis, clinical features, and diagnosis of these syndromes is discussed in separate topic reviews:

●Decreased production:

•Congenital neutropenia, including severe congenital neutropenia (SCN), Shwachman-Diamond syndrome (SDS), Chediak Higashi syndrome, and monoMAC syndromes – (See "Congenital neutropenia" and "Shwachman-Diamond syndrome" and "Chediak-Higashi syndrome".)

•Cyclic neutropenia – (See "Cyclic neutropenia".)

•Infections – (See "Infectious causes of neutropenia".)

•Bone marrow failure syndromes – (See "Treatment of acquired aplastic anemia in children and adolescents" and "Aplastic anemia: Pathogenesis, clinical manifestations, and diagnosis" and "Clinical manifestations and diagnosis of Fanconi anemia".)

•Paroxysmal nocturnal hemoglobinuria – (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria".)

●Peripheral destruction or shift out of the circulation:

•Immune neutropenia – (See "Immune neutropenia".)

•Hypersplenism – (See "Splenomegaly and other splenic disorders in adults", section on 'Hypersplenism'.)

Importantly, the cause of the neutropenia has implications for the ability of the patient to handle infections. Depending on the underlying cause, fever and neutropenia may either be a medical emergency with a risk of life-threatening sepsis, or the risks may be similar to the general population. (See "Management of the adult with non-chemotherapy-induced neutropenia" and "Overview of neutropenia in children and adolescents" and "Management of children with non-chemotherapy-induced neutropenia and fever", section on 'Risk-based management approach'.)

Bone marrow evaluation — Bone marrow evaluation, including an aspirate and a biopsy, often plays an important role in evaluating neutropenia and distinguishing between decreased neutrophil production and peripheral neutrophil destruction/sequestration.

Specific findings that may be helpful include the bone marrow cellularity, presence of an arrest at a specific stage of neutrophil maturation (see "Regulation of myelopoiesis", section on 'Neutrophil production and maturation'), and/or abnormalities of myeloid cells (eg, nuclear findings, malignant populations of cells).

An early myeloid arrest is associated with a higher risk of infection due to inability to deliver mature neutrophils to the tissues, whereas even severe neutropenia in the face of a late myeloid arrest is rarely associated with increased propensity for infection due to the neutropenia (figure 1). (See "Overview of neutropenia in children and adolescents", section on 'Propensity to infection and significance of neutropenia' and "Approach to the adult with unexplained neutropenia", section on 'Risk of infection'.)

While the bone marrow evaluation allows classification of neutropenia as due to decreased neutrophil production or peripheral neutrophil destruction/sequestration, it rarely provides a specific diagnosis. Thus, once a defect in neutrophil production is documented, a search for related disorders such as lupus, or genetic testing for specific neutrophil defects, is often the next step. In fact, due to the common availability of exome panels, we often go directly to genetic testing even before bone marrow aspiration as marrow morphology often does not provide a specific diagnosis. (See 'Genetic testing' below.)

Notable exceptions where marrow morphology can be diagnostic include the following:

●Arrest at the promyelocyte stage in an infant, which suggests severe congenital neutropenia. (See "Congenital neutropenia", section on 'Severe congenital neutropenia'.),

●Arrest at the promyelocyte stage adult can be seen after administration of rituximab. (See "Drug-induced neutropenia and agranulocytosis", section on 'Rituximab'.)

●The presence of the large myelocytic granules that are typical of Chediak-Higashi syndrome are often more easily seen in the bone marrow if missed on peripheral smear. (See "Chediak-Higashi syndrome", section on 'Laboratory and imaging findings'.)

●Tetraploid nuclei in myeloid cells suggests myelokathexis, which may indicate an inherited syndrome. (See "Congenital neutropenia", section on 'GATA2 deficiency/MonoMAC syndrome'.)

Tests for immune neutropenia — We do not feel that testing for anti-neutrophil antibodies is clinically useful, though a positive result may provide some reassurance regarding the mechanism. Details about this testing and its use in research are presented separately. Anti-neutrophil antibodies can be present in the absence of neutropenia, and neutropenia can be present in the absence of detectable antibodies, even if the setting suggests autoimmune disease. (See "Immune neutropenia", section on 'Detection of antineutrophil antibodies'.)

TESTING FOR NEUTROPHIL DYSFUNCTION — Testing for neutrophil function defects is appropriate in a patient whose clinical presentation is consistent with defective phagocytic function (eg, chronic bacterial or fungal infections) and who has a normal absolute neutrophil count (ANC) (calculator 1). Although there are some subtle findings that may lead one to suspect either neutropenia or neutrophil dysfunction as a cause of recurrent infection, there is a great deal of overlap in the clinical presentations with those of other immune deficiency states. Additional discussion of typical presenting findings for immunodeficiency and patterns seen in specific syndromes are presented separately. (See "Approach to the child with recurrent infections", section on 'Clinical features suggestive of a primary immunodeficiency' and "Approach to the child with recurrent infections", section on 'Clinical patterns suggestive of immunodeficiency'.)

Although a large number of neutrophil function assays have been described, the extreme rarity of primary disorders and lack of special expertise needed to properly interpret the results makes them inappropriate for routine clinical laboratories. Most of the functional assays are appropriate only in a research setting. The general aspects of a few selected tests that can be performed outside the realm of a neutrophil research laboratory will be described here.

Overview of causes (neutrophil dysfunction) — Neutrophil dysfunction disorders can be understood as defects in one or more steps in normal neutrophil processes, including adhesion to the vascular wall, chemotaxis into the tissues, phagocytosis of pathogens, and bacterial killing; these steps are illustrated in the figure (figure 2). These normal neutrophil functions are described in more detail in review articles and in a separate topic review [1]. (See "An overview of the innate immune system", section on 'Neutrophils'.)

All of the genetic neutrophil dysfunction disorders that result from defects along this pathway are very rare disorders and, with the exception of those listed in this section and in the table (table 1), cannot be diagnosed by non-molecular approaches without the help of a specialized research laboratory and an immunologist with special expertise in neutrophil disorders. Currently, an early, if not the first step in diagnosis of neutrophil dysfunction disorders is to obtain a focused exome panel.

●Chronic granulomatous disease – Chronic granulomatous disease (CGD) is due to defects in the neutrophil NADPH oxidase and related pathways that result in an inability of neutrophils to make superoxide. Superoxide production is the major mechanism of intracellular killing of bacteria (the final step in the figure (figure 2)). Neutrophils in patients with CGD have markedly impaired ability to kill bacteria. (See "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis", section on 'Pathogenesis'.)

Approximately 70 percent of patients with CGD are males who have X-linked disease; the remaining cases are autosomal recessive. Female carriers of X-linked disease have no increased risk of infection. Rarely, in cases of extreme lyonization (skewed X-inactivation), female carriers who have <5 to 10 percent of neutrophils able to make superoxide may have clinical symptoms of mild CGD. (See "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis".)

The diagnosis of CGD is established by showing absence of the ability to make superoxide in response to stimulation with phorbol myristate acetate (PMA) (table 1). Superoxide production is indirectly assayed as an inability to reduce dihydrorhodamine (DHR) to its fluorescent form (which can be assayed using flow cytometry) or to reduce nitroblue tetrazolium (NBT), which is assayed using the NBT slide test (figure 3) [2]. The DHR flow cytometry testing is generally preferred, though in experienced hands, the NBT test is excellent for detection of the disease state.

•Individuals with X-linked or recessive disease are unable to reduce DHR and have no blue staining on the NBT slide test.

•Individuals with recessive CGD, particularly defects in p47phox component of the oxidase, and some very rare patients with X-linked disease may make a very small amount of superoxide that is detectible by DHR reduction. This small amount of superoxide production is generally not detectable by the NBT slide test. The patients who make a small amount of superoxide have fewer infections and improved survival [2].

•In female carriers of X-linked disease, approximately 50 percent of the neutrophils have no superoxide production, and 50 percent have completely normal superoxide production. Thus, there is usually a clear bimodal distribution of DHR fluorescence by flow cytometry and a bimodal distribution of NBT reduction on the NBT slide test (figure 3).

Of note, the reaction with DHR depends on myeloperoxidase; thus, patients with concomitant complete myeloperoxidase deficiency can have a false positive DHR-based test (ie, no fluorescence detected) and a CGD-like phenotype. Some female patients with a high degree of lyonization may have low percentages of DHR-positive or NBT-positive cells.

Carriers of X-linked disease with less than 10 to 15 percent activity may have symptomatic infections and may require treatment for mild disease. Some patients, particularly the patients with defects in p47phox, may make a very small amount of superoxide and have a somewhat milder clinical course [2].

Genetic testing is recommended for the proband, to classify the defect and permit prenatal diagnosis if required. NBT or DHR on fetal blood samples has been performed but is very difficult and unreliable. Essentially, all of the patients have a detectable genetic defect.

●LAD-I – Leukocyte adhesion deficiency I (LAD-I) is caused by reduction or absence of the transmembrane protein CD18, the common chain of the beta 2 integrin family, which in turn reduces the amount of the linked alpha subunit receptors CD11a, CD11b, and CD11c used for neutrophil adhesion to the vessel wall (the second step in the figure (figure 2)). These integrins mediate firm adhesion at sites of endothelium where intercellular adhesion molecules (ICAMs) are upregulated on vascular endothelium due to local infection and cytokines. Subsequently, transmigration and chemotaxis are triggered. This receptor also mediates complement-mediated phagocytosis. Motility is affected because the cells cannot grip the endothelial substrate. Patients with LAD-I have high white blood cell (WBC) counts and severe infections from birth, due to motility and phagocytosis defects. The diagnosis is established by flow cytometry with fluorescently labeled antibodies against CD11b (figure 4) or CD18 (figure 5) that show absent (severe form) or markedly reduced (mild phenotype) surface labeling (table 1 and figure 5). A more extensive discussion is presented separately. (See "Leukocyte-adhesion deficiency", section on 'LAD I'.)

●LAD-II – LAD-II is an extremely rare disorder caused by decreased sialyl-Lewis-X on the neutrophil surface; this is the ligand that mediates selectin-mediated weak rolling adhesion of neutrophils to the vascular endothelium, chemotaxis, and phagocytosis (the first step in the figure (figure 2)). These form the "marginated pool" of neutrophils that move slowly on the vessel wall and are prepared to engage the strong adhesion medicated by CD18/CD11b and ICAMs. Patients have intellectual disability, short stature, and distinctive facial appearance with a depressed nasal bridge, and mild neutrophilia. Diagnosis is based on flow cytometry that shows absence of CD15a (also called SLeX) (figure 5 and table 1). A more extensive discussion of LAD-II is presented separately. (See "Leukocyte-adhesion deficiency", section on 'LAD II'.)

●Chediak-Higashi syndrome – Chediak-Higashi syndrome (CHS) is primarily a granule fusion defect that affects leukocytes, nerve endings, and melanocytes, resulting in partial albinism, nystagmus, and defects in chemotaxis and bacterial killing (the second and final steps in the figure). The diagnosis is usually easy from presence of huge granules visible in the peripheral blood smear (picture 1 and picture 2 and table 1), although the granules may occasionally be very subtle in the peripheral blood and obvious only in the bone marrow. Patients may also have mild to moderate neutropenia (eg, ANC 500 to 2000/microL). (See "Chediak-Higashi syndrome".)

●Hyperimmunoglobulin E syndrome – Hyperimmunoglobulin E syndrome (also called hyperimmunoglobulin E recurrent infection syndrome (HIES), Job syndrome) is a disorder of cytokine function associated with abnormal T cell function, increased IgE production, and intermittent defects in neutrophil chemotaxis (the second step in the figure (figure 2)) due to mutations in STAT3. The inheritance is dominant, recessive, or sporadic. The diagnosis is based on marked elevated IgE levels, T helper cell type 17 (Th17) count, and genetic testing. A more extensive discussion is presented separately. (See "Autosomal dominant hyperimmunoglobulin E syndrome".)

An additional overview of phagocytic disorders is also presented separately. (See "Primary disorders of phagocyte number and/or function: An overview".)

Presentation and indications for evaluation — Patients with neutrophil dysfunction present with chronic recurrent bacterial infections. Severe chemotactic defects in particular have been associated with chronic skin and mucosal infections. Patients with severe chemotactic defects may also present with omphalitis (infection/inflammation of the umbilical cord stump) at birth. These are significant lesions and are characterized by the absence of exudate in the setting of significant leukocytosis [2]. The skin infections associated with severe chemotactic defects tend to be severe enough to lead to permanent scarring. Attempts to drain these lesions are often unsuccessful because neutrophils cannot migrate to the site of infection to form abscesses.

Unlike patients with severe neutropenia, patients with neutrophil dysfunction rarely present with overt sepsis but rather with more indolent infections in the lymph nodes, lung, or liver. The fevers are usually low-grade, and it is often difficult to culture organisms. Certain organisms such as Serratia, Nocardia, Aspergillus, and Burkholderia cepacia (formerly known as Pseudomonas cepacia) are unusual in normal hosts and immediately raise the possibility of chronic granulomatous disease (CGD). Staphylococcus is also a common organism in CGD.

Choice of assay — Primary neutrophil dysfunction significant enough to cause clinical disease accounts for less than 10 percent of all primary immune deficiency states. Thus, unless there is a family history suggestive of a neutrophil dysfunction syndrome (eg, known familial syndrome, history of frequent skin and soft tissue infections without evidence of cellular immunodeficiency), it is appropriate to eliminate other disorders first, including those of immunoglobulins, complement, or T cells, before a primary neutrophil dysfunction syndrome is considered. Details of the diagnostic evaluation for humoral and T cell disorders are presented separately. (See "Laboratory evaluation of the immune system".)

One possible exception to this rule is an evaluation for CGD, because of the ease of performing the screening NBT slide test or DHR florescence assay. (See "Primary disorders of phagocyte number and/or function: An overview", section on 'Chronic granulomatous disease'.).

Once other immune defects have been eliminated, the choice of initial assay for neutrophil dysfunction depends on a variety of factors, including the presence of a known familial defect, characteristic physical or laboratory findings (eg, depressed nasal bridge and intellectual disability in LAD-II, giant azurophilic granulocyte granules in Chediak-Higashi syndrome (picture 2 and picture 1), absence of granules and bilobed neutrophils in neutrophil-specific granule deficiency (picture 3)) and the urgency of diagnosis.

●Patients who have characteristic clinical or laboratory findings, or a known family history of a specific disorder, may proceed directly to genetic testing. (See 'Genetic testing' below.)

●Patients without findings characteristic for a specific disorder should be evaluated for CGD using flow cytometry with dihydrorhodamine (DHR). This test is quantitative and is useful in the identification of affected individuals and heterozygotes (ie, carriers). This procedure is widely available in reference laboratories and has generally replaced the nitroblue tetrazolium test (NBT test). However the NBT test can be used for screening purposes. (See "Flow cytometry for the diagnosis of inborn errors of immunity" and "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis".)

●Patients suspected of having LAD-I can be evaluated with flow cytometry for deficiency of CD11b/CD18 (characteristic of LAD-I). (See "Leukocyte-adhesion deficiency", section on 'Evaluation and diagnosis'.)

●Patients suspected of having hyperimmunoglobulin E syndrome (Job syndrome) can have IgE levels measured. (See "Autosomal dominant hyperimmunoglobulin E syndrome", section on 'Laboratory findings'.)

●Individuals who remain without a diagnosis despite this testing may require referral to a clinician with more specialized expertise in immunodeficiency or hematologic disorders. Ultimately, definitive diagnosis may require specialized flow cytometric or molecular methods available in reference laboratories or specialized centers [3-5].

Challenges in more specialized neutrophil function testing — A number of assays have been developed to assess neutrophil adhesion, transmigration, shape change, chemotaxis, phagocytosis, bactericidal killing, and respiratory burst. These have been employed in clinical settings. However, these assays measure cellular behavior; they have tremendous inherent variability and require special laboratory expertise and constant practice to make them work, along with careful determination of normal age-related controls. Thus, while these assays have been useful in the research laboratory setting to clarify the cellular physiology of various specific disease states, they have essentially no place in routine clinical practice, with the exception of measurement of the respiratory burst for the diagnosis of CGD.

In addition to the challenges of performing these biological assays, another issue is the effect of clinical factors other than primary neutrophil dysfunction on the results. As examples, neutrophil functions are significantly affected by general inflammation, methods of blood drawing, age of the specimen, and myriad other issues, making the interpretation very difficult. Thus, with the exception of genetic disorders that impair neutrophil production, it is very difficult, if not impossible, to tell if the measured change in neutrophil function is the cause of infection or if the infection and attendant inflammation is causing the decrease in neutrophil function.

Genetic testing — Genetic testing is appropriate for patients with a suspected genetic cause of neutropenia or neutrophil dysfunction based on findings from the patient and family history, physical examination, complete blood count, peripheral blood smear, bone marrow, and/or neutrophil function assay. Specific gene defects associated with each of the disorders are presented in the table (table 1). The choice of gene test, and decision of whether to test for all genetic defects or to test a panel of genes, is individualized depending on specific clinical factors, available testing, and input from a clinician with expertise in immunodeficiency and hematologic disorders. Resources for genetic testing include the Cincinnati Children’s hospital and the Genetic Testing Registry.

SUMMARY

●Role of neutrophils – Neutrophils are the primary defense against bacteria and fungi. Decreased numbers or functional abnormalities impair immunity and cause recurrent or severe infections.

●Initial evaluation – History, physical examination, complete blood count (CBC), and review of the blood smear are performed initially. (See 'Testing for neutropenia' above.)

●Neutropenia

•Definition – Neutropenia is an absolute neutrophil count (ANC) <1500/microL, calculated as follows (calculator 1):

     ANC  =  WBC (cells/microL)  x  percent (PMNs  +  bands)  ÷  100

•Causes of neutropenia – Isolated neutropenia can be caused by an acquired condition (usually immune destruction) or an inherited condition. (See 'Overview of causes (neutropenia)' above.)

Individuals with the Duffy null [Fy(a-b-)] red blood cell phenotype have isolated mild neutropenia that is not associated with defective immunity; the condition was formerly called benign ethnic neutropenia. (See "Approach to the adult with unexplained neutropenia", section on 'Normal variants <1500/microL'.)

•Evaluation of neutropenia – Evaluation of isolated neutropenia is described separately. (See "Overview of neutropenia in children and adolescents" and "Approach to the adult with unexplained neutropenia".)

Neutropenia together with anemia and/or thrombocytopenia is considered pancytopenia. Evaluation of pancytopenia is described separately. (See "Approach to the adult with pancytopenia".)

•Diagnostic testing

-Bone marrow examination – Bone marrow aspirate and biopsy are important for evaluating neutropenia and distinguishing decreased production from peripheral destruction/sequestration. (See 'Bone marrow evaluation' above.)

-Genetic testing – Individuals with a family history of neutropenia and/or characteristic somatic abnormalities (eg, abnormal skeletal findings, skin disorders, others) should be considered for genetic testing for an inherited condition. (See 'Genetic testing' above.)

Physical findings that suggest an inherited condition (eg, Fanconi anemia, dyskeratosis congenita) are described separately. (See "Clinical manifestations and diagnosis of Fanconi anemia" and "Dyskeratosis congenita and other telomere biology disorders".)

●Neutrophil dysfunction

•Presentation – Clinical manifestations of functional neutrophil disorders include recurrent, severe, or chronic bacterial or fungal infections. The manifestations overlap considerably with those of neutropenia and other immune deficiencies. (See 'Presentation and indications for evaluation' above.)

•Causes of neutrophil dysfunction – Inherited neutrophil disorders (table 1) are rare and account for <10 percent of primary immune deficiency states. They result from a defect in one or more steps in normal neutrophil processes (figure 2), including adhesion to the vascular wall, chemotaxis into the tissues, phagocytosis of pathogens, and bacterial killing.

•Initial evaluation – We test for neutropenia and other immunodeficiencies before testing for a functional neutrophil disorder, as described separately. (See 'Choice of assay' above.)

Details of our approach to testing are described separately. (See "Approach to the child with recurrent infections".)

•Diagnostic testing – Testing for a functional neutrophil disorder is appropriate with a suggestive family history or an individual with an adequate ANC in whom other immunodeficiency disorders have been excluded. (See 'Challenges in more specialized neutrophil function testing' above and 'Genetic testing' above.)

ACKNOWLEDGMENT — We acknowledge the contributions of the late Laurence A Boxer, MD, who served as a section editor for this topic.