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Neutrophil Monitoring Laboratory
Overview
The Neutrophil Monitoring Laboratory performs testing to assess neutrophil dysfunction in patients with various primary immunodeficiencies.
We develop state-of-the-art assays to evaluate a variety of neutrophil functions such as reactive oxygen species production, chemotaxis, cell shape change and spreading, exocytosis, and alterations in surface antigen expression.
The laboratory has developed and performs high complexity, CLIA-certified, assays that are used to diagnose patients with:
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Chronic granulomatous disease, a genetic immunodeficiency characterized by recurrent bacterial and fungal infections caused by a defect in reactive oxygen species production
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Leukocyte adhesion deficiency, a defect in the ability of neutrophils to migrate to sites of infection.
We employ several multiplex analysis platforms to monitor for the levels of inflammatory cytokines and other biomarkers in biological specimens or culture supernatant fluids.
The Neutrophil Monitoring Laboratory provides clinical and basic research in support of the Laboratory of Clinical Immunology and Microbiology at the National Institute of Allergy and Infectious Diseases. We maintain an extensive repository of patient plasma, cryopreserved peripheral blood mononuclear cells, Epstein-Barr virus-transformed B-cell lines, dermal fibroblasts, DNA, and RNA that represent a valuable resource for investigators.
Innovations
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Characterized specimens from more than 450 families with chronic granulomatous disease, totaling 525 individual patients and 333 autosomal and X-linked carriers.
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Developed new approaches (FACS, ddPCR) to further characterize the genetic and protein defects in these patients.
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First characterized a patient with an interleukin-1 receptor associated kinase 4 (IRAK4) defect, a genetic immunodeficiency disrupting one of the body’s innate immune defense signaling systems.
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Using a novel proteomics approach, the laboratory described a novel genetic disease of the neutrophil actin cytoskeleton (WDR1 deficiency) that disrupts actin depolymerization, altering multiple functions including chemotaxis, leading to increased susceptibility to infections.
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Characterized neutrophil chemotactic defects in several other actinopathies – RAC2, HEM1, GNAI1, and ARP5C.
Focus
Diagnosis of chronic granulomatous disease and its subtypes
- Produce reactive oxygen species.
- Perform phagocyte oxidase protein expression by western blotting or fluorescence-activated cell sorting analysis of proteins in permeabilized neutrophils.
- Identify genetic defect using Sanger sequencing or droplet digital polymerase chain reaction.
Neutrophil dysfunction associated with actinopathies
- Perform chemotaxis by modified Boyden chamber (Neuroprobe) or CHEMOTAXIScan instrumentation.
- Use light and electron microscopy for neutrophil morphology alterations and by changing forward angle and right angle light scattering.
- Study increased micropinocytosis and neutrophil vacuolization.
- Study changes in neutrophil surface antigen expression and activation state of neutrophils.
Analysis of inflammatory cytokines and other biomarkers in biological specimens
- Analyze serum and plasma of COVID-19 patients, patients with rare genetic immunodeficiencies, and patients undergoing hematopoietic stem cell transplantation.
- Analyze biomarkers by either ELISA-based or bead based multiplex platforms .
Neutrophil extracellular trap formation and its role in inflammatory disease
- Detect by either image analysis and fluorescence microscopy or fluorescence spectrophotometry monitoring the release of DNA into the extracellular space.
Technology
ddPCR to distinguish p47phox CGD subtypes
- Determines the number of GTGT vs. delta GT at the start of exon 2
- Can be used to identify autosomal carriers of p47phox CGD
Image analysis to characterize neutrophil chemotaxis
- In depth analysis of cell velocity, directionality, randomness, angle of migration, and others.
