Neutrophil flow cytometry (NFC) is a specialized diagnostic technique that uses laser-based technology to rapidly analyze the characteristics and function of a patient’s neutrophils. Neutrophils are the most abundant type of white blood cell, acting as the body’s first line of defense against infection. This advanced laboratory test provides detailed, single-cell measurements, assessing their ability to perform defensive duties beyond a simple count. By analyzing hundreds of thousands of cells in minutes, NFC offers physicians a powerful tool to identify subtle defects in the immune system and provide a precise diagnosis when neutrophil function is compromised.
The Primary Functions of Neutrophils
Neutrophils are highly mobile, short-lived cells that represent approximately 50 to 70 percent of all circulating white blood cells. They are a primary component of the innate immune system, constantly patrolling the bloodstream for signs of microbial invasion. Once an infection is detected, they are the first cells to migrate out of the blood and into the infected tissue, a process known as chemotaxis.
The main defensive action of neutrophils is phagocytosis, where they engulf and destroy invading pathogens like bacteria and fungi. They utilize a diverse arsenal of antimicrobial mechanisms, including the release of toxic enzymes stored in their granules and the generation of reactive oxygen species (ROS) through the respiratory burst. Additionally, neutrophils can release a meshwork of DNA and proteins, called Neutrophil Extracellular Traps (NETs), to capture and neutralize microbes outside the cell.
How Flow Cytometry Works
Flow cytometry is an analytical technique that measures the physical and chemical characteristics of cells as they pass individually through a beam of light. The technology relies on three interconnected systems: fluidics, optics, and electronics. The fluidics system transports cells in a single-file stream, ensuring that only one cell at a time passes through the laser beam.
When a cell intercepts the laser, the optics system measures how light is scattered. Forward Scatter (FSC) measures the light diffracted along the laser’s path, correlating with the cell’s size or volume. Simultaneously, Side Scatter (SSC) measures light refracted at a 90-degree angle, providing information about the cell’s internal complexity, such as the density of granules. The combination of FSC and SSC allows the instrument to distinguish neutrophils from other cell types, such as lymphocytes and monocytes, based on their distinct size and high granularity.
The electronics system converts the scattered light signals and any fluorescence signals into digital data points that a computer can analyze. This process enables the rapid, quantitative assessment of thousands of cells per second. By using these scatter profiles, researchers can “gate” the data, isolating the neutrophil population to focus the analysis specifically on this cell type.
Analyzing Neutrophil Activity and Surface Markers
Neutrophil flow cytometry measures both the physical presence of molecules and the functional capacity of the cells. Specific surface markers, known as Cluster of Differentiation (CD) antigens, are labeled using fluorescently-tagged antibodies. These antibodies bind only to their specific target molecules on the cell surface, and the resulting fluorescence is detected by the cytometer.
By analyzing the expression of different CD markers, researchers can determine the neutrophil’s maturity and activation state. For instance, the expression levels of markers like CD10 and CD16 can indicate whether a cell is a mature circulating neutrophil or a more immature form released prematurely from the bone marrow. A marker like CD64 is often upregulated on neutrophils during severe systemic inflammation, serving as an indicator of sepsis severity.
Functional capacity is measured through specialized assays, most commonly the respiratory burst assay. This test uses a non-fluorescent dye, such as dihydrorhodamine 123 (DHR 123), which is converted into a fluorescent compound when oxidized by the reactive oxygen species produced during a successful respiratory burst. The increase in fluorescence directly correlates with the cell’s ability to generate these microbicidal agents, providing a quantitative measure of its killing power.
Diagnosing Disease Using Neutrophil Flow Cytometry
Neutrophil flow cytometry is an indispensable tool for diagnosing primary immunodeficiency disorders where a defect in neutrophil function is suspected. One of the most common applications is the definitive diagnosis of Chronic Granulomatous Disease (CGD). CGD is caused by a genetic defect in the NADPH oxidase enzyme complex, which prevents neutrophils from generating the necessary reactive oxygen species to kill internalized pathogens.
In the DHR 123 assay, neutrophils from a patient with CGD will fail to produce fluorescence even when stimulated, indicating a non-functional respiratory burst. This test is highly sensitive and can also identify female carriers of X-linked CGD by showing two distinct populations of neutrophils. Another condition diagnosed using NFC is Leukocyte Adhesion Deficiency (LAD), where a defect in adhesion molecules, like the CD18 component of integrins, prevents neutrophils from migrating out of the bloodstream to the site of infection. Flow cytometry quantifies the reduced or absent expression of these specific surface markers, confirming the diagnosis.
Beyond inherited disorders, NFC helps monitor acquired conditions, including severe infections and hematological malignancies. Measuring the expression of CD64 on neutrophils can provide prognostic information for patients with sepsis, where higher expression is associated with the severity of the inflammatory response. Flow cytometry can detect the presence of immature or functionally altered neutrophils in inflammatory conditions and cancers, which helps guide treatment decisions in complex clinical scenarios.