RF Test: Introduction, Principle, Testing Procedure, Result -Interpretation, Normal Range, Clinical Significance, and Keynotes

Introduction of RF Test

RF test uses to detect autoantibody of rheumatoid factor which stands for rheumatoid factor. 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 suppurative proliferative and inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosing of joints. Rheumatoid factor (RF) is common in about 1% population. It is higher in females than males. Rheumatoid factor (RF) is the autoantibody that was 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  (an antibody that precipitates on the 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 individuals with RA have antibodies to the Fc portion of autologous IgG (i.e. RF).

Detection Methods of RF

Detection of RF by following methods

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

RF Test by Latex Particle Agglutination Test

Test Principle of RF Test

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 particles 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 for RF Test

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

 Test Procedure of RF Test

Qualitative Method

1. Allow test reagents and patient serum to reach room temperature.
2. Transfer 50μl of the 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 patient’s 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 of RF Test

Qualitative Method 

1. A positive result is indicated by the obvious agglutination pattern of the red cell reagent, in a clear solution.
2. A negative result is indicated by no change in the red cell suspension on the test slide.
3. The latex particle agglutination test has a detection limit of 20 IU/ml. Positive results will be obtained at an RF serum concentration of 20 IU/ml or more and negative results will be obtained at an 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 titer appears at 1/8 the approximate serum RF concentration is 20 x 8 = 160 IU/ml.

Clinical Significance of RF Test

• Diseases Associated with RF

1. Rheumatic disease: RA, Systemic Lupus Erythematosus (SLE), scleroderma, mixed connective tissue disease
2. Viral disease: AIDS, Infectious Mononucleosis (IM), hepatitis, influenza, after vaccination
3. Chronic bacterial disease: Tuberculosis(TB), Leprosy, Yaws, Syphilis, Brucellosis
4. Parasitic disease: Trypanosomiasis, Kala-azar, Malaria, and Filariasis.
5. Antibodies to a citrullinated peptide (anti-CCP antibodies) have been recently shown to be present in many people with RA and rarely in people with other inflammatory diseases or healthy individuals.

Further Reading 

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