Nemours Biomedical Research
The Clinical Immunology Lab at Nemours/Alfred I. duPont Hospital for Children provides a wide range of pediatric land clinical lab services. We conduct diagnostic tests related to autoimmune disorders and research focused on the study of infectious and inflammatory diseases that affect pediatric patient populations.
Paul T. Fawcett, PhD: Dr. Fawcett has been the head of the research and clinical immunology laboratories since 1986. In addition, he serves as Chair of the IACUC and is a member of the IRB.
Our CAP-certified pediatric lab specializes in the serologic detection of Lyme disease and autoimmune disorders. Diagnostic testing services may be requested by physicians and are provided at Nemours/Alfred I. duPont Hospital for Children in Wilmington, Delaware.
The immunofluorescence ANA assay (Antibodies, Inc) utilizes the method considered the gold standard for ANA testing by the American College of Rheumatology; circulating autoantibodies reactive with nuclear antigens of HEp-2 cells are detected. Unlike multiplex or ELISA assays, the HEp-2 cell substrate provides 100-150 possible autoantigens to detect ANAs with greater sensitivity than the solid phase assays.1, 2
The pattern and semi-quantitative titer provide useful information for both the diagnosis and the monitoring of therapy for patients with systemic lupus erythematosus (SLE) and other connective tissue diseases such as rheumatoid arthritis (RA), scleroderma, and Sjogren’s Disease.
The ANCA assay (INOVA) detects the presence of IgG antibodies that bind to human neutrophil antigens using direct immunofluorescence.¹,² Screening all samples with ethanol fixed slides allows ANCA reactivity to be separated into two diagnostically useful categories. The pattern and semiquantitative titer are reported. Current research indicates that two patterns may be clinically useful:
A granular cytoplasmic pattern (cANCA) is a serologic marker in up to 96% of Wegener’s granulomatosis patients. This severe systemic vascular disease that causes irreversible injury to the kidneys and lungs presents with initial symptoms and biopsy findings that are frequently non-specific. Early diagnosis and treatment can greatly improve renal outcome. The degree of reactivity of cANCA has been found to follow the disease course so repeat testing can be important in disease management. The antigenic specificity of this pattern is primarily serine protease 3 (PR3).
A classic perinuclear pattern (pANCA) is primarily due to antibodies to myeloperoxidase (MPO) but may also be caused by others including elastase and lactoferrin. This pattern has been associated with more organ-limited vasculitis, in particular rapidly progressive glomerulonephritis. All pANCA reactions on ETOH-fixed slides are confirmed by testing on formalin slides.
ANCA antibodies have been associated with ulcerative colitis² and primary sclerosing cholangitis but the specific antigen is unknown at this time.
Reactivity to other cytoplasmic and nuclear antigens may be observed. Concurrent antinuclear antibody (ANA) screening with Hep-2 cells may aid in determining the specificity of the antibodies.
The Cardiolipin antibody (IgG) test (GenBio) is an ELISA for the quantitative detection of autoimmune antibodies to cardiolipin (aCL), a subset of antiphospholipid antibodies. This assay includes standardized cofactor beta 2 glycoprotein (b2-GP1) and phosphotydyl serine to assure measurement of both anti-cardiolipin and anti-b2-GP1 activity.
Elevated levels of anti-cardiolipin have been associated with thrombosis in patients with "anti-phospholipid syndrome" and systemic lupus erythematosus (SLE).
Results are reported as GPL units.
The double-stranded DNA (dsDNA) antibody test (INOVA) is an ELISA for the semi-quantitative detection of auto-antibodies to double-stranded DNA. Presence of these antibodies may be helpful in the diagnosis and monitoring of therapy for patients with systemic lupus erythematosus (SLE).
This assay has been calibrated against the Wo/80 dsDNA standard from the World Health Organization.
Results are reported in International Units/milliliter (IU/ml).
The presence of antibodies to Helicobacter pylori is assayed by means of a qualitative ELISA (INOVA). This assay serves as an alternative to more invasive procedures and is intended to aid in the diagnosis of H. pylori infections in patients with clinical signs and symptoms of gastrointestinal disease.
Antibodies to Helicobacter pylori, a bacterium that colonizes the gastric mucosa and is associated with gastritis and peptic ulcers, are demonstrated qualitatively with this Western blot assay. This method provides a characterization of the immune response by detecting specific reactivity to discrete bacterial antigens. If the samples contain antibodies to H. pylori in sufficient quantities then specific bands will be detected. Additional studies will hopefully elucidate the diagnostic implications of the proteins.¹ Reactivity is determined by comparison to controls.
Antibodies to Borrelia burgdorferi are detected with a semi-quantitative enzyme immunoassay (ELISA) technique. Our validated in-house assay has been in use for over 20 years1 and adheres to strict quality control criteria for lot-to-lot reactivity. Patient serum is diluted in an adsorbent solution that reduces the incidence of false positives.2, 3 Serum giving a positive reaction is assigned a titer based on the standard curve and assayed by Lyme Western Blot for confirmation.
The Lyme Western Blot assay qualitatively detects antibodies to Borrelia burgdorferi, the etiologic agent of Lyme disease. This method provides a detailed characterization of the immune response by detecting specific reactivity to discrete borrelial antigens. Presence of IgG and IgM antibodies are detected.
If the samples contain B. burgdorferi antibodies in sufficient quantities, then specific bands will be present. Reactivity is determined by comparison to controls.4, 5, 6 and 7.
Rheumatoid factor is an immunoglobulin with antigenic specificity for the Fc fragment of human IgG. Detection and quantitation of RF is clinically useful due to their presence in many rheumatic and chronic inflammatory diseases.
Rheumatoid arthritis usually produces the highest titers but systemic lupus erythematosus, polyarthritis, and other chronic inflammatory diseases also produce significant RF. The majority of patients with juvenile rheumatoid arthritis (JRA) are RF negative. However, a positive RF may provide prognostic information in a JRA patient with polyarticular disease, as these children are more likely to develop severe chronic arthritis.
Our lab uses latex agglutination, one of the most widely used methods to detect RF. Serum containing RF will cause agglutination of latex particles that have been coated with human gamma globulins.
Titration of positives provides semi-quantitative results. Positive samples are reported as the highest titer exhibiting RF activity.
Research efforts of the Immunology Research Lab focus on the study of infectious and inflammatory diseases affecting Nemours patient populations. The lab's approach to these investigations emphasizes the role of the immune system as both an effector/modulator and as an indicator of disease in these disorders.
Current investigations include studies of rheumatic (in collaboration with Rheumatology and Orthopedics) and gastrointestinal inflammatory diseases (in collaboration with Gastroenterology) of idiopathic and infectious etiologies. These investigations utilize a multidisciplinary approach, which enables the lab to combine the medical and clinical diagnostic skills of physician collaborators with their own research and lab-based diagnostic capabilities.
Two infectious agents are under study as part of these investigations. They are Borrelia burgdorferi, the bacteria that causes Lyme disease, and Helicobactor pylori, the leading cause of ulcers. H. pylori is also associated with other inflammatory gastric disorders. The lab is also studying the role of the immune system in initiation and perpetuation of inflammatory joint and soft-tissue disease associated with both rheumatic and gastrointestinal disorders. The latter studies are focused on detecting various antibodies and cytokines using immunoassays and a cell culture system; the former is focused on identifying relevant antigenic determinants of the infectious agents and elucidating immune markers that may serve as diagnostic and prognostic indices of disease.
The lab is dedicated to investigations that are based upon current patient-related needs of clinical divisions. Current areas of study (gastroenterology and rheumatology) were identified in response to the need for improved ability to diagnose and formulate a prognosis for related diseases. Several gastrointestinal and rheumatic diseases are associated with profound degradation in the quality of life for affected patients. Furthermore, the mechanisms of disease induction and perpetuation for these infectious and inflammatory disorders have not yet been determined. It is in this latter area that the lab’s research expertise can provide significant improvement in patient care.
The unique union of the clinical and research laboratories in Immunology ensures that research efforts are directly relevant to patient care for the Nemours patient population. The lab currently provides immunodiagnostic testing services for several medical divisions. Available tests include assays for autoantibodies and for infectious diseases. The lab is currently working to develop the capability to test for cell markers that could be useful diagnostically and in formulating a prognosis for several diseases.
The primary focus of our research during the preceding year involved investigations on the cellular mechanisms responsible for inflammatory joint diseases. Studies are being conducted in collaboration with the research department’s Transplant Lab and physicians at the Wilmington campus. We have developed a series of in-vitro culture techniques and serologic assays that enable us to examine cells and blood from patients with inflammatory joint diseases in an effort to determine the cause of this class of disease. Samples from patients with both autoimmune (idiopathic) and infectious (Lyme disease) causes of inflammatory joint disease are being studied.
Our research efforts are focused on development of new or improved lab-based assays to aid in the diagnosis and treatment of pediatric disorders.
Phone: (302) 651-6776
Fax: (302) 651-6881
Nemours/Alfred I. duPont Hospital for Children
1600 Rockland Road
Wilmington, DE 19803