Disorders of white blood cells

Normal neutrophil development and function

Neutrophils have six distinct phases of proliferation and maturation that occur over 12–14 days. The first three phases, myeloblast, promyelocyte, and myelocytes, constitute the proliferative pool, while the latter three phases, metamyelocytes, bands, and polymorphonuclear neutrophils, are postmitotic and account for the majority of the neutrophils in the marrow space. Neutrophil maturation is characterized by the progressive development of granules within the cytoplasm, transitioning from a rich blue cytoplasm to a predominantly pink cytoplasm on hematoxylin and eosin (H&E) stain upon reaching maturity and increasing nuclear condensation with segmentation until the typical 3–5 lobed nucleus of a polymorphonuclear neutrophil is achieved. Cytoplasmic granules are central to neutrophil function as they contain key degradative enzymes and other components needed for effective microbial killing.

• Primary granules, the azurophilic granules most notable in promyelocytes, contain elastase and myeloperoxidase.

• Secondary granules first seen in myelocytes contain plentiful lactoferrin as well as other chemotactic, opsonic, and adhesion protein receptors, for example, CD11b/CD18, and the gp91 ^phox /p22 ^phox component of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

• Tertiary granules first evident in bands and secretory granules also contain adhesion protein receptors and components of the NADPH oxidase. Notably, secretory granules also contain alkaline phosphatase.

Once fully matured and released from the marrow, neutrophils in the vasculature or peripheral tissues survive for only a few hours up to 5 days. Neutrophil cell death is via a combination of apoptosis and macrophage engulfment.

Mature neutrophils have several distinct functions, including the ability to egress from the marrow space into the peripheral vasculature mediated by granulocyte colony-stimulating factor (G-CSF) and the interaction between CXCL12/SDF-1 and CXCR4, adhesion to and subsequent migration through the vasculature wall through the utilization of integrins (CD11a, CD11b, CD11c, and CD18) and selectins, chemotaxis, phagocytosis, and microbial killing largely reliant on functional granule fusion and NADPH oxidase activity. The most recently appreciated neutrophil function is NETosis, a form of neutrophil cell death characterized by the release of intracellular contents into the extracellular space that serves to physically ensnare microbes and enhance the overall localized immune response.

Disruption in the normal neutrophil maturation leading to either neutrophilia or neutropenia, and/or neutrophil dysfunction results in clinically apparent disease as detailed next.

Neutrophilia

By definition, neutrophilia is an increase in the ANC that is more than two standard deviations above the mean for age.

Bands are included in the ANC calculation as they are fully functional phagocytes. In adults the normal WBC range is 4400–11,000 cells/µL, with the typical neutrophil predominance of 60–70% of the total WBC count attained during puberty. Hence, neutrophilia in adults is generally an ANC>7700 cells/µL. The normal ANC ranges for children vary based on age with the highest ANCs seen in the first 24 hours after birth (range 5000–28,000 cells/µL). Neutrophilia may occur either through:

• enhanced granulopoiesis,

• accelerated mobilization of neutrophils from the bone marrow to the blood,

• release of neutrophils from the marginated pool, or

• impaired neutrophil egress into tissues.

Neutropenia

Similarly, neutropenia is a decrease in the absolute number of peripherally circulating segmented neutrophils and bands. Mild neutropenia is defined as an ANC between 1001 and 1500 cells/µL, moderate between 501 and 1000 cells/µL, and severe ≤500 cells/µL. An ANC<200 cells/µL is also referred to as agranulocytosis. The ANC must be considered in the context of both age and ethnicity.

Neonatal ANC values are affected by the surge of neutrophils released from the marrow secondary to the stress of delivery and the subsequent decrease in the marrow proliferative pool postbirth. The lower limit of normal for an ANC during the first 24 hours after birth is 5000 cells/µL, then 1500 cells/µL during the first 2 weeks, followed by 1000 cells/µL between 2 weeks and 1 year. After 1 year the lower limit of normal for the ANC is 1500 cells/µL.

In addition to age, many healthy individuals of African and Middle Eastern descent have a baseline lower ANC as compared to their peers with Western European or Asian heritage. The difference in the ANC is linked to a polymorphism in the Duffy antigen receptor chemokine ( DARC ) gene that encodes the Duffy antigen (FyA/B), a chemokine receptor expressed on the surface of RBCs used by Plasmodium vivax to infect RBCs. Patients with homozygous DARC null polymorphisms are often regarded as having benign ethnic neutropenia (BEN), although Duffy-null status is not required for the diagnosis of BEN. Patients with BEN are not at risk for infection or malignancy. Heterozygous patients have normal mean neutrophil counts.

Neutrophil dysfunction

Neutrophil dysfunction is defined as the partial or complete failure of the neutrophil to execute one or more of the key neutrophil functions. The International Union of Immunological Societies (IUIS) divides disorders of neutrophil and other phagocyte function into three broad categories:

• 1.

  defects of motility

• 2.

  defects of respiratory burst

• 3.

  other nonlymphoid defects

Additionally, several other primary immunodeficiencies and immune dysregulation disorders have neutrophil dysfunction as a key element of their phenotype despite being classified under alternate IUIS categories.

Support for neutrophil dysfunction can be gleaned from careful review of the blood smear, for example, identification of giant azurophilic granulocyte granules in Chediak–Higashi syndrome (CHS) or the absence of granules and bilobed neutrophils in neutrophil-specific granule deficiency. More specific testing through flow cytometry for members of the beta-2 (β2) integrin family crucial to leukocyte adhesion to and migration through vasculature walls, namely, CD11b and CD18, is readily accessible. Similarly, assessment of the respiratory burst via flow cytometry with dihydrorhodamine (DHR), which has largely replaced the older, qualitative nitroblue tetrazolium (NBT) test, is also widely available. DHR relies on the oxidation of the test substrate DHR by hydrogen peroxide to produce rhodamine 123, a florescent compound detectable by flow cytometry, and the NBT relies on reduction by the NADPH oxidase complex to produce a colorimetric change that can be identified by microscopy. Other assays of neutrophil dysfunction, for example, chemotaxis and bactericidal activity, are performed in highly specialized laboratories, but interpretation of these tests is often hindered by the technical difficulty in performing the assay as well as methods of blood drawing, age of the specimen, influences of shipping/travel conditions, and need for normal controls. With the limitations of functional testing of neutrophils, genetic testing for pathogenic variants is being increasingly utilized.

Approach to suspected neutrophil disorders

Signs of neutrophil disorders include:

• 1.

  recurrent fevers,

• 2.

  oral ulcers,

• 3.

  chronic gingival or dental problems,

• 4.

  poor wound healing,

• 5.

  recurrent or unusual infections,

• 6.

  lack of pus, and

• 7.

  abnormal blood counts.

The increasing recognition of neutropenia as a feature of primary immunodeficiency disorders and autoimmune/immune dysregulatory disorders, such as ADA2 deficiency, reinforces the need for a broad differential in the evaluation of patients with quantitative and qualitative neutrophil defects.

Approach to neutrophilia

By definition, 2.5% of the normal population will have baseline mild–moderate neutrophilia. Neutrophilia is often a physiologically normal response of the hematopoietic system to:

• stress from acute or chronic infections,

• underlying inflammatory disease states,

• medication exposures, and

• nonhematologic malignancies.

A careful medical history to ascertain symptoms of infection (e.g., fever, cough, diarrhea, and rash), inflammation (e.g., periodic fevers, photosensitive rashes, joint pain, and chronic diarrhea), malignancy (e.g., fevers, weight loss, fatigue, and night sweats), drug exposures (including cigarette smoking), and physiologic stress (e.g., obesity, exercise, and pregnancy) can often determine the underlying etiology of the neutrophilia. Screening questions for sickle cell anemia, chronic hemolytic anemias, trisomy 21, and leukocyte adhesion deficiency are also appropriate, particularly in younger patients. Physical examination for localized infection (e.g., otitis media), joint swelling, rashes, or lymphoproliferation should be undertaken. Both the complete blood count (CBC) with differential and review of the blood smear can offer helpful insights to support infection/inflammation (e.g., toxic granulation, Döhle bodies), hemolysis, or raise concern for dysplastic processes. Additional labs to test for specific microbes, screen for autoimmunity, assess inflammation, and/or tumor lysis may be appropriate but should be guided by the patient-specific presentation. For patients with significant or persistent neutrophilia in the absence of overt infection, inflammation or medication exposure, or with coincident anemia and/or thrombocytopenia, strong consideration should be given to bone marrow aspirate and biopsy with flow cytometry, cytogenetics, and molecular testing to assess for myeloproliferative neoplasms (MPN), hematologic malignancy, or other infiltrative marrow processes. Additionally, flow cytometry for CD11b/CD18 to screen for leukocyte adhesion deficiency can be considered in patients with a concerning personal or family history.

Approach to neutropenia

The majority of patients presenting with neutropenia will have transient neutropenia secondary to acute infections or medication exposures. Nonetheless, a complete history should be ascertained, focusing on the presence or absence of:

• recurrent fevers,

• oral ulcers or gingivitis,

• frequent or unusual skin or deep-seated infections,

• failure to thrive or malabsorption,

• dietary/nutritional deficiencies,

• prior or family history of malignancy,

• liver or lung fibrosis,

• early graying,

• significant viral infections [e.g., warts, Epstein–Barr virus (EBV)],

• lymphedema, and

• evidence of autoimmune disease.

On physical examination the oral cavity should be thoroughly assessed for oral ulcers, gingival inflammation, as well as dental abnormalities. Additional focus should include skeletal anomalies, rashes, warts, and lymphoproliferation. Patient height should also be obtained as many of the bone marrow failure syndromes and primary immunodeficiencies that manifest with neutropenia also have short stature. The blood smear should be reviewed for signs of marrow stress and a CBC with differential should be obtained to assess for other cell lineage involvement as multilineage involvement may prompt further evaluation for autoimmune disease (i.e., Evans syndrome), malignancy, or bone marrow failure. Depending on the patient history, additional testing with pancreatic enzymes (serum isoamylase, fecal elastase) and pancreatic ultrasound for Shwachman–Diamond syndrome (SDS), chromosomal breakage for Fanconi anemia, telomere length for dyskeratosis congenita, or lymphocyte subsets and quantitative immunoglobulins to screen for immunodeficiency may be appropriate. If a satisfactory etiology of the neutropenia is not identified, a bone marrow aspirate and biopsy is warranted. Given the overlap of neutropenia with primary immunodeficiency disorders, genetic sequencing that includes both inborn errors of myelopoiesis and immunity is increasingly being utilized to secure a molecular diagnosis to support biologically rationale therapy selection, provide an informed framework for supportive care and prognosis as well as influence decisions regarding definitive therapy, for example, hematopoietic stem cell transplant (HSCT).

Approach to neutrophil dysfunction

For patients with a normal ANC but with symptoms and signs consistent with neutrophil dysfunction, a thorough history should be ascertained with a focus on:

• infection history with unusual organisms (e.g., catalase-positive organisms, nontuberculous mycobacteria, salmonella, severe viral infections);

• oral health (e.g., ulcers, gingivitis, periodontitis);

• rashes [e.g., pyoderma gangrenosum–like lesions that may raise concern for leukocyte adhesion deficiency type 1 (LAD-1), extensive warts concerning for GATA binding protein 2 (GATA2) haploinsufficiency or eczema consistent with Wiskott–Aldrich syndrome (WAS) or STAT3 LOF];

• delayed umbilical cord detachment;

• history of omphalitis; and

• lack of neutrophilic tissue infiltration and pus formation in the setting of infection.

Similar to neutropenia, the physical examination should focus on the oral cavity for evidence of oral ulcers and chronic gingival inflammation and skeletal anomalies as well as skin findings, lymphedema, and lymphoproliferation. Review of the blood smear along with functional testing with flow cytometry for CD11b/CD18 or DHR testing can be pursued, although the majority of patients will rely on identification of pathogenic genetic variants for diagnosis.

Primary neutrophilia disorders

Primary neutropenia disorders

Primary disorders of neutrophil dysfunction