Introduction
Autoimmune diseases
Autoantibodies - Introduction
Autoantibodies - Determination
 
Autoantibodies
Rheumatoid Factor
Antinuclear Antibodies (ANA)
Specific Antibodies
Anti-neutrophil Cytoplasmic Antibodies
(ANCA)
Anti-phospholipid Antibodies
Anti-mitochondrial Antibodies (AMA)
Anti-endothelial Cell Antibodies (AECA)
Anti CCP antibodies
Antibodies against DNases
 
Quality Assurance
 
Reference ranges
 
Algorithm
ANA and incidence of diseases
Proposed stepwise diagnosis scheme
Positive Immunoflourescence -
Nucleoplasmic
Positive Immunoflourescence - Nucleolar
Positive Immunoflourescence -
Cytoplasmic
Type of autoimmune diseases
Conditions associated with antinuclear
antibodies (ANA)
 
Slide show
 
References
 
Collaborators
Rheumatoid Factor - Determination


Initial tests for RF were based on the agglutination of IgG-coated red blood cells or latex particles. These methods detected predominantly the ”classical” RF of the IgM class (IgM RF), the presence of RFs of other antibody classes (which are much poorer agglutinins) was often not recognized. IgG, IgA, IgD and lgE RF have therefore not been identified for a long time.
The presence of IgA RF in the serum of RA patients could be first shown by means of immunoelectrophoresis of serum proteins eluted from insoluble IgG (9). There was also indirect evidence for IgA RF in saliva.
The more sensitive techniques of radioimmunoassays (RIA) (10) and ELISA (11, 12) have subsequently confirmed these findings. The diagnostic and prognostic test qualities of the ELISA for IgG, IgA and IgM RF factor for RA vs. controls was 72%, 44%, and 69% respectively; the specificity was 52%, 84%, and 86%. In comparison for the Latex fixation test the sensitivity was 66% and the specificity 91% (13).
IgA RF has also been detected in synovial fluid, nasal and duodenal secretions. Although usually found in association with IgM RF, IgA RF can occur in ”seronegative” patients. Its frequency in RA has been reported to be from 56% up to 88%, with several reports of high incidence in patients with Sjögren’s syndrome. SLE and bacterial endocarditis patients can also have elevated serum levels of IgA RF. In a prospective study of early RA patients, raised IgA RF levels were found to be significantly associated with the later development of erosive disease (14, 15). Consequently, those patients with high levels received more treatment with specific drugs. A similar relationship between IgA RF and erosions in hands was found in a retrospective study which has lead to the suggestion that IgA RF may be related to the development of the erosions (16). In contrast there is no such association with the ”classical” RF levels.
IgA RF-positive patients have more aggressive RA than negative cases. Measuring IgA1 RF and IgA2 RF subclasses did not give more information about clinical status than measuring total IgA RF alone (17). RA patients with a predominantly increase of IgA RF had a high frequency of associated sicca-syndrome (18) and associated with extraarticular manifestations in RA (19). In RA patients with early erosions on magnetic resonance imaging scans the levels of the three RF factor isotypes were significantly higher than in patients without early erosions (20).
Evidence for the existence of the IgG RF first came from ultracentrifugation studies (21), and since then a number of methods for its detection have been developed. These include radioimmunoassay (22), immunofluorescence assay (23) and, more recently, ELISA (24, 25). Animal IgG or the isolated Fc region of human IgG are generally used as antigen. Estimates of the frequency of IgG RF in RA patients have varied from below 50 to over 90%, and although it is also usually found in association with IgM RF, it can - similar to IgA RF - occasionally occur alone in ”seronegative” patients. IgG RF is rare in juvenile RA but can be detected in some patients with other connective tissue diseases and bacterial endocarditis. It also occurs at low frequency and at relatively low level in normal healthy individuals. High levels of IgM RF are associated with a poor prognosis but changes in the level of this class do not correspond to disease activity. However, IgG RF in serum has been shown to correlate with the severity of disease (26). Very high levels of IgG RF in RA patients have also been found to have a strong association with vasculitis and a poor prognosis (27, 28).
Assays for determination of RF subclasses can provide additional important information for the patient management than simple agglutination assays which are only sensitive to IgM class antibodies.

Rheumatoid Factor -
Sample

Use only fresh serum or serum stored at + 2 °C to +8 °C for no longer than 72 hours. For longer storage freeze the serum. Reject any lipemic serum.


Rheumatoid Factor - Methods

- Rheumatoid Factor - Agglutination

Agglutination test have been used for many years for the qualitative and quantitative measurements of antigen and antibodies. Latex agglutination assays are simple to perform and require little equipment. The major interference in the direct agglutination assay test for the presence of antigen is rheumatoid factor. Latex agglutination employs polystyrene latex particles coated with the appropriate test antigens. When the latex particle reagent is mixed with human serum that contains antibodies to the test antigen, a reaction occurs that produces visible agglutination (clumping) in the tube or on the slide or test card surface. The assay is read visually. In the absence of test antigen-specific antibodies, no agglutination occurs and a smooth, milky appearance is observed. Serial two-fold dilutions of the test solution may be done to obtain a relative titer of antibody level. In the direct latex aggluntination assay for the presence of antigen in the sera of patients, the particles are coated with an antibody to the test antigen. In the presence of antigen the particles agglutinate

 

Figure 3 Negative and positive latex test

- Rheumatoid Factor - Radioimmunoasay

Radioimmunoassay (RIA) is based on competition of a known excess amount of radiolabeled antigen (or hapten) and an unknown amount of the same antigen in the The most frequently used procedure is the solid-phase RIA in which the antibody to the substance being assayed is bound to polystyrene tubes. The coupled antibody is still able to interact with the antigen, and after equilibrium is reached, separation of the bound from the free antigen is achieved by washing the tube. The bound antibody remains adsorbed to the tube surface.The use of RIA methods has been hindered by the intrinsic hazard and instability of the isotopes and by the deluge of governmental regulations that control their manufacture and use. The search for nonisotopic labels has produced the enzyme immunoassay (EIA), which shares with RIA the specificity, sensitivity, and rapidity and has the advantage of safety. In many cases assays formerly performed by RIA are now done by EIA techniques.The most common RIA techniques used in serology to detect the presence of antigen use a "sandwich principle", where plastic beads coated with antibody to the  test antigen are incubated with serum or plasma and appropriate test controls. If the test antigen is present, it will bind to the solid phase antibody. After aspiration of the unbound material and washing of the bead, human 125I-antibody is added and allowed to react with the antibody-antigen complex on the bead. This forms the sandwich - "ab-ag-ab". The beads are washed to remove unbound 125I-antibody and the beads are read through the gamma counter. Specimens giving counts per minute (cpm) equal to or greater than the cutoff value are considered reactive for the test antigen.

 

- Rheumatoid Factor – ELISA

ELISA can be performed with nuclear antigens isolated from tissue or cell sources, although most preparationsnow are cloned recombinant products produced in bacteria. In an ELISA, an antigen is adhered to wells of a plastic microtiter plate and exposed sequentially to a serum, enzyme conjugated anti-immunoglobulin reagent, and a substrate, the breakdown product of which can be measured colorimetrically (54, 55).Immunoblot and ELISA are less time consuming than immunodiffusion and show good interassay sensitivity without loss of specificity. The combination of immunoblot and enzyme immunoassay are reported to yield excellent assay sensitivity (100%) and specificity (99%) for detection of autoantibodies.

 

- Rheumatoid Factor – Turbidimetry (nephelometry)

Turbidimetric and Nephelometric Assays are convenient techniques for measuring the rate of formation of immune complexes in solution.Nephelometry is a technique of determining the concentration of solids suspended in a liquid or a gas by use of a nephelometer.Turbidimetry is a measurement of the turbidity (cloudiness) of a solution or suspension in which the amount of transmitted light is quantified with a spectrophotometer or estimated by visual comparison with solutions of known turbidity.Nephelometry measures the amount of light scattered or deflected from its path by macromolecules in solution. Turbidometry measures the reflection of transmitted light The technique utilizes an incidental light beam that passes through a test solution in a given direction. In dilute solution antigens and antibodies form complexes that scatter light. If the amount of antibody is kept constant, the number of complexes varies in direct proportion to the antigen concentration. The amount of scattered light or the reduction of transmitted light of an unknown sample can therefore be compared with the signal from dilutions of a reference serum of knownantigen concentration, and the antigen concentration present in the unknown sample can then be determined.The method can be used to measure most serum proteins including antibodies.In this assay partially purified antigen is mixed with the test serum resulting in the  formation of precipitating antibody-antigen complexes. The complexes are measured by the increased scattering of light as they are formed.


The presence of IgA RF in the serum of RA patients could be first shown by means of immunoelectrophoresis of serum proteins eluted from insoluble IgG (9). There was also indirect evidence for IgA RF in saliva.