Rheumatoid Factor(RF): Introduction, Associated Diseases, Detection methods, Test procedure, Result Interpretation

Introduction of Rheumatoid factor test

Rheumatoid arthritis ( RA) is a chronic systemic inflammatory disorder that may affect many tissues and organs like  skin, blood vessels,heart,lungs, and muscles but particularly attacks joints, producing a supppurative proliferative and inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosing of joints. Rheumatoid factor is common in about 1% population.

Female>male

40-70 years age

• Rheumatoid factor(RF) is the auto antibody that  first found in rheumatoid arthritis.
• It defines as an antibody against the Fc portion of IgG (an antibody against an antibody).
• RF is present among IgM, IgA and IgE classes
• RF and IgG join to form immune complexes that contribute to the disease process.
• Rheumatoid factor can also be a cryoglobulin  (antibody that precipitates on cooling of a blood sample); it can be either type 2 (monoclonal IgM to polyclonal IgG) or type 3 (polyclonal IgM to polyclonal IgG) cryoglobulin.
• About 80% of individual with RA have antibodies to the Fc portion of autologous IgG (i.e. RF)

Diseases associated with RF

Rheumatic disease: RA, Systemic Lupus Erythematosus (SLE) , scleroderma, mixed connective tissue disease

Viral disease: AIDS, Infectious Mononucleosis (IM), hepatitis,influenza, after vaccination

Chronic bacterial disease: Tuberculosis(TB), Leprosy, Yaws, Syphilis, Brucellosis

Parasitic disease: Trypanosomiasis, Kala-azar, Malaria, Filariasis, Visceral leishmaniasis,etc.

Antibodies  to citrullinated peptide (anti-CCP antibodies) have been recently shown to be present in many people with RA and rarely in people with other inflammatory disease or healthy individuls.

Detection of RF

• Latex particle agglutination
• Rose-Waller test
• ELISA
• RIA
• CFT
• Precipitation, etc

 Latex particle agglutination test

Test principle

Latex particle agglutination test is an agglutination test for the detection of Rheumatoid Factor (RF) in human serum. This  test reagent is a suspension of stabilized latex particle  with rabbit anti-sheep IgG. When RF is present in the sample, clear agglutination is seen.

Latex particle agglutination test has a detection limit of 20 IU/ml of RF in the patient serum and the reagent is calibrated against the World Health Organization (WHO) International Reference Preparation.

 Requirements

• Test slide
• Latex particle
• Blood sample
• Centrifuge
• Micro pipette and tips/ droppers
• Mixer (plastic sticks provided in test kits)
• Negative control
• Positive control- provided in test kits

 Test Procedure

Qualitative Method

1. Allow test reagents and patient serum to reach room temperature.
2. Transfer 50μl of patient’s serum to a test circle on the slide.
3. Shake the Reagent, then add one drop of suspension to the test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the mixture.
5. Place the test slide on the bench and leave for 2 minutes.
6. Gently and evenly, rock and rotate the test slide once and place the test slide on the bench once more.
7. One minute later, read the test slide for the presence of agglutination.

Semi Quantitative Method

1. Using isotonic saline prepare serial dilutions of the patients serum (1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and so on)
2. Transfer 50μl of each serum dilution to a test circle on the slide.
3. Shake the reagent, then add one drop of suspension to the test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the mixture.
5.  Place the test slide on the bench and leave for 2 minutes.
6.  Gently and evenly, rock and rotate the test slide once and place the test slide on the bench once more.
7. One minute later, read the test slide for the presence of agglutination.

Result Interpretation

Qualitative Method 

A positive result is indicated by the obvious agglutination pattern of the red cell reagent, in a clear solution.

A negative result is indicated by no change in the red cell suspension on the test slide.

Latex particle agglutination test has a detection limit of 20 IU/ml. Positive results will be obtained at a RF serum concentration of 20 IU/ml or more and negative results will be obtained at a RF concentration below 20 IU/ml.

Semi-Quantitative Method 

The serum RF concentration can then be calculated approximately by multiplying the dilution factor (i.e. 2, 4, 8 or 16) by the detection limit, i.e. 20, to give the number of IU/ml concentration e.g. if the agglutination titre appears at 1/8 the approximate serum RF concentration is 20 x 8 = 160 IU/ml.

Further Reading 

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845430/
2. https://www.hindawi.com/journals/dm/2013/726598/
3. https://medlineplus.gov/lab-tests/rheumatoid-factor-rf-test/
4. https://www.uptodate.com/contents/diagnosis-and-differential-diagnosis-of-rheumatoid-arthritis/print
5. https://www.mayoclinic.org/tests-procedures/rheumatoid-factor/about/pac-20384800
6. https://onlinelibrary.wiley.com/doi/pdf/10.1002/1529-0131(200001)43:1%3C155::AID-ANR20%3E3.0.CO;2-3
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832720/