Neutrophil Function Tests

Neutrophil function tests assess whether circulating neutrophils can adhere, migrate, ingest and kill microbes. The tests in routine use are the dihydrorhodamine 123 (DHR) flow cytometry assay and the nitroblue tetrazolium (NBT) test for the respiratory burst, and flow cytometry for adhesion molecules (CD18, CD11b, CD15s). Chemotaxis, phagocytosis and degranulation assays are confined to specialist centres. They answer one question: does a patient with recurrent, severe or unusual infection and a normal neutrophil count have a primary phagocyte disorder?

Chronic granulomatous disease (CGD) is the most common defect found, with an incidence of about 1 in 200 000 to 250 000 live births. About two thirds of cases are X-linked (CYBB, gp91phox) and the rest are autosomal recessive (NCF1, CYBA, NCF2, NCF4). Leucocyte adhesion deficiency, Chediak-Higashi syndrome and specific granule deficiency are rarer.

Indications. Recurrent or deep seated bacterial or fungal infection, hepatic or other visceral abscess, invasive aspergillosis in a child or previously healthy adult, infection with Staphylococcus aureus, Burkholderia cepacia complex, Serratia marcescens, Nocardia or Salmonella, granulomatous inflammation or early onset inflammatory bowel disease, delayed umbilical cord separation (over 3 weeks) with marked neutrophilia, and family history of an X-linked phagocyte disorder.

Specimen. Fresh heparinised whole blood kept at room temperature, never refrigerated, and tested within 24 hours. A healthy control is collected and processed on the same day.

Dihydrorhodamine 123 assay. Neutrophils loaded with non-fluorescent dye are stimulated with phorbol myristate acetate. NADPH oxidase activity produces hydrogen peroxide, which oxidises the dye to fluorescent rhodamine 123, read by flow cytometry. Results are reported as the stimulation index (stimulated over unstimulated fluorescence) and as the histogram pattern compared with the control.

NBT test. Normal neutrophils reduce yellow NBT to blue black formazan after stimulation. In CGD, cells remain unstained. It is slide based and subjective, and has been largely replaced by the DHR assay.

Adhesion molecule expression. Flow cytometry for CD18, CD11b and CD15s (sialyl Lewis X) on neutrophils.

Other assays. Chemotaxis (Boyden chamber), phagocytosis and killing assays, NADPH oxidase subunit immunoblot, and gene sequencing.

X-linked CGD. No fluorescence shift on DHR stimulation, and no NBT reduction.

Autosomal recessive CGD. Reduced but detectable shift, usually a single dim population. Residual activity can be difficult to separate from a mild variant.

X-linked carriers. Two populations on DHR (normal and non-functioning), because of random X inactivation. The normal fraction varies from under 5% to over 95%. Carriers with under about 20% functioning cells have an increased risk of infection, and carriers can develop discoid lupus like lesions and aphthous ulceration.

Myeloperoxidase deficiency. DHR is reduced or absent, since the dye conversion needs myeloperoxidase, while the NBT test is normal. It affects about 1 in 2000 to 4000 people and is usually asymptomatic, with risk of Candida infection in diabetes.

Leucocyte adhesion deficiency type I (ITGB2). CD18 and CD11b absent or reduced (under 2 to 5% of normal in severe disease, 2 to 30% in moderate disease), with marked neutrophilia, absence of pus, delayed cord separation and periodontitis.

Leucocyte adhesion deficiency type II (SLC35C1). Absent CD15s with the Bombay blood group phenotype and developmental delay.

Chediak-Higashi syndrome. Giant cytoplasmic granules on the film, partial albinism, neuropathy, and a risk of accelerated haemophagocytic lymphohistiocytosis.

Reduced burst without a primary defect. Severe G6PD deficiency (class I), glutathione synthetase deficiency, severe sepsis, burns, drugs, and aged samples.

Critical findings needing immediate action. Absent or severely reduced oxidative burst in a febrile patient, or one with pneumonia or deep abscess, needs urgent specialist assessment, since infection progresses rapidly and often needs tissue sampling.

Pitfalls.

• Delayed or chilled samples give falsely abnormal results, and every batch needs a same day control.
• The DHR assay is myeloperoxidase dependent, so a low signal can reflect myeloperoxidase deficiency.
• The NBT test can miss carriers with mosaic populations, so the DHR assay is preferred.
• Mild variants with residual activity may give a reduced but not absent signal.
• A normal result does not exclude other phagocyte defects, such as chemotaxis or granule defects.
• Neutropenia limits interpretation.

Special groups. Neonates and preterm infants have reduced chemotaxis and phagocytosis, so abnormal results in the first weeks need repeating. Mothers and sisters of boys with X-linked CGD need carrier testing.

Follow up tests. Full blood count and film, immunoglobulins with IgE, lymphocyte subsets, complement studies, HIV testing, CYBB, NCF1, CYBA, NCF2 and NCF4 sequencing, NADPH oxidase subunit immunoblot, CD18 and CD15s flow cytometry, imaging and culture of infected sites, tissue biopsy for granulomas, serum galactomannan and beta-D-glucan.