Rheumatoid factor (RF)
- Rheumatoid factor (RF) is an autoantibody (usually of the IgM class, less often IgG or IgA) specific for 'self' immunoglobulin (IgG).
- The target immunoglobulin is considered to be bound in turn to an antigen, and this interaction of antigen and antibody creates a structural (conformational) change in the IgG that permits binding of the RF. The identity of the target antigen(s) is presently unknown.
- Studies on rheumatoid factor are relatively limited in horses. While it has been shown to be present in diseased joints, there is currently no evidence to support a primary causative role in equine joint disease or other autoimmune diseases.
- RF is a non-specific autoantibody that may be induced in a wide range of inflammatory, infectious or neoplastic diseases.
- Clinically normal animals may have serum RF. However, in these situations RF is generally of low titer.
- Higher titers of RF occur in autoimmune disease in some species, although there is limited data available in horses.
- Classically, RF is present in patients with rheumatoid arthritis, but may be found in patients with other autoimmune diseases particularly those involving connective tissue.
- Detection of significantly titered serum or synovial RF is a useful test for determining whether polyarthritis may be immune-mediated Joint: immune-mediated polysynovitis in some species.
- Classically, a patient with rheumatoid arthritis will have positive serum RF and be negative for serum antinuclear antibody (ANA).
- Conversely, a patient with polyarthritis due to systemic lupus erythematosus (SLE) Systemic lupus erythematosus-like syndrome will have serum ANA but be negative for RF.
- In practice, such distinctions are not always possible.
Source of test material
- RF may be detected in serum or synovial fluid.
- Anecdotally, the chance of a positive test is increased when synovial fluid is used as the sample.
Quantity of test material
- Relatively small volumes are required for the RF test.
- A 1 ml volume of serum is adequate, and a lesser of volume of synovial fluid will suffice.
Timing of test
- The serum or synovial fluid samples should be submitted to the laboratory as soon as possible after collection.
- These antibodies are robust and are unlikely to be affected by routine postage of samples.
- Samples that are to be assayed within one week may be kept refrigerated (4°C/39.2°F).
- Serum should be harvested from clotted blood or the gel tube.
- For longer term storage, freezing the sample (at -20°C/-4°F or -70°C/-94°F) will preserve the antibodies for many years.
- Routine postal submission to the laboratory is acceptable Transportation of diagnostic specimens .
- Two tests are recommended for detection of serum or synovial fluid rheumatoid factor in veterinary species.
- The most commonly used test is theRose Waller Assay:
- In this test, a washed suspension of sheep or canine erythrocytes are preincubated with an antiserum specific for sheep or dog erythrocytes.
- The antiserum must be used at a sub-agglutinating dose, so that the red cells are coated with antibody but do not agglutinate.
- This preparation of antibody-coated red blood cells (RBC) is then incubated with serum or synovial fluid from the patient.
- The test samples are serially diluted, usually in a microtiter system and a standard volume of antibody-coated RBC suspension is added to each dilution.
- The antibody-RBC complex mimics the antibody-antigen complex to which RF binds in vivo.
- If RF is present in the serum or synovial fluid sample it will bind to the anti-RBC antibody and cause cross-linking of these molecules, and thus agglutination of the red cells.
- This agglutination is scored visually, and the last well in the microtiter plate in which agglutination has occurred is recorded in order to calculate the titer of the reaction.
- A duplicate titration of the sample is incubated with a suspension of RBC that has not been pre-incubated with anti-RBC antibody as a negative control.
- The second method used for detection of RF isELISA Enzyme linked immunosorbent assay (ELISA) :
- In this assay, a purified immunoglobulin is coated to the plastic wells of the ELISA microplate.
- Serum or synovial fluid from the patient is titrated into replicate wells, and if RF is present it will bind to the immunoglobulin.
- Presumptively, the process of immunoglobulin adhering to the plastic surface of the ELISA plate is sufficient to alter the conformation of the molecule to permit RF binding.
- Any bound RF is subsequently detected using an antiserum, eg specific for canine IgM, that is conjugated to an enzyme (typically alkaline phosphatase Blood: biochemistry - alkaline phosphatase (SAP, ALP) ).
- The appropriate substrate is added and the ensuing color change is recorded spectrophotometrically.
- In addition to the internal control for RF binding (non-coated RBC), the RF assay will include a negative and positive control serum of known RF content.
- The positive control serum will produce an agglutination reaction with antibody-coated RBC, but not uncoated RBC.
- In the ELISA method, the positive control serum may be used to generate a standard curve, the slope of which can be compared to those obtained with test sera for calculation of the result.
- The RF assay will usually only be available from specialist diagnostic or research laboratories.
- The ELISA is a more sensitive test than the Rose Waller Assay, however the ELISA is generally used as a research tool rather than a routine diagnostic test.
- RF is a non-specific autoantibody.
- It may be present in clinically normal animals, or in animals with non-autoimmune disease.
- In most cases these will be relatively low-titered RFs.
Normal (reference) values
- The testing laboratory should provide some interpretation of the significance of the titer obtained.
- In general, a RF titer of </=32 is not considered clinically significant.
Errors and artifacts
- Autoantibodies may wax and wane within the circulation, so a patient that is seronegative on one occasion and still strongly suspected of having autoimmune disease should be retested some weeks later.
- Recent references from PubMed and VetMedResource.
- Carter S D, Osborne A C, May S A & Bennett D (1995) Rheumatoid factor, anti-heat shock protein (65 kDa) antibodies and anti-nuclear antibodies in equine joint diseases. Equine Vet J 27 (4), 288-295 PubMed.
- Osborne A C, Carter S D, May S A & Bennett D (1995) Anti-collagen antibodies and immune complexes in equine joint diseases. Vet Immunol Immunopathol 45 (1-2), 19-30 PubMed.
- Day M J & Penhale W J (1986) Immunodiagnosis of autoimmune skin disease in the dog, cat and horse. Aust Vet J 63 (3), 65-68 PubMed.
Other sources of information
- Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, UK.