Lab --2
Rheumatoid Factors (RF(
[Link]. Amjed Hussein
What Is Rheumatoid Factor (RF(?
Rheumatoid Factors (RF) : are autoantibodies that react with individuals
own immunoglobulin. These antibodies are usually directed against the Fc fragment of
the human IgG. RF have been associated with three major immunoglobulin classes:
IgM, IgG, and IgA. Of these IgM and IgG are the most common. The formation of
immune complex in the joint space leads to the activation of complement and
destructive inflammation, causing rheumatoid arthritis (RA(
Rheumatoid Factors (RF(?
• As indicated by its name, RF has particular application to diagnosis and monitoring of
rheumatoid arthritis.
• Rheumatoid arthritis (RA) :is a chronic inflammatory disease affecting primarily the
joints and periarticular tissues. Rheumatoid factor is detected in 60-80% of cases of
diagnosed rheumatoid arthritis. However, it is also detectable sometimes in the serum of
patients with Systemic Lupus Erythematosus (SLE) and in certain non-rheumatic
conditions. Elevated values may also be observed in normal elderly population.
Principle of Rheumatoid
Factor test
• A number of methods are available for testing
of RF. The most commonly used
serological method is based on latex agglutination
test. As RF is an IgM class of antibody directed
against the Fc portion of the IgG molecule, it
is detected by it’s ability to agglutinate the latex
particles coated with IgG molecule.
• Reagent used is a suspension of polystyrene
latex particles in glycine-saline buffer
with pH: 8.6 ± 0.1, coated with human gamma
globulin.
Why did doctor order this test?
❑ Doctors may order a blood test to check for the presence of RF if they suspect you have an
autoimmune condition, such as rheumatoid arthritis or Sjögren's syndrome.
❑ Other health problems that can cause higher-than-normal levels of RF include:
•Chronic infection
• Cirrhosis, which is scarring of the liver ،
• inflammatory lung disease
• mixed connective tissue disease
• lupus
• cancer
Materials provided with the test kit
1. RF Latex Direct Reagent
2. Positive Control Serum
3. Negative Control Serum
4. -6Well Test Slide
5. Disposable Pipettes
Materials required but not provided:
1. Test Tubes (for quantitative method.)
2. Serological Pipettes.
3. Laboratory Timer.
[Link] Rotator (optional.)
.5Isotonic Saline (0.85% sodium chloride, for quantitative method.)
Specimens:
• This test should be performed on fresh serum. The samples maybe stored refrigerated(2°-
8°C) for maximum of 7days.
• If longer storage is required, store at (-20° C.)
• Heavy bacterial contamination may cause positive agglutination.
Qualitative Method:
1. Bring all reagents and specimens to room temperature.
2. Place one drop of the positive control and 40ul of the patient serum into separate circles on
the slide.
3. Gently and add one drop of RF latex reagent on each circle of sample to be tested and
control.
4. Use separate Applicator sticks/stir sticks to spread reaction mixture over entire area of the
particular field.
5. Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates
slowly.
6. Observe for agglutination after 2 minutes under bright artificial light.
Procedure for Rheumatoid Factor test
Procedure of RF Test
Interpretation:
Agglutination of latex particles is considered a
positive reaction, indicating the presence of
rheumatoid factor at a significant and
detectable level.
Positive result: An agglutination of the latex
particles suspension will occur within two
minutes, indicating a RF level of more than 8
IU/ml.
Negative result: No agglutination of the latex
particles suspension within two minutes.
Semi-quantitative Method:
1. Using isotonic saline prepare serial dilutions of the test sample positive in the qualitative
method 1:2, 1:4, 1:8, 1:16,1:32, 1:64, 1:128 and so on as follows:
2. For each specimen to be tested, add 100 µL of 0.9% saline into test tubes numbered 1 to .5
3. Add 100 µL of specimen onto test tube .1
4. Mix the mixture. Avoid formation of bubbles.
5. Transfer 100 µL of mixed sample from tube 1 to .2
6. Repeat this serial dilution procedure in tube 3 to 4, and then 5. Dispose 100 µL from test
tube 5 after mixing
7. Tubes 1 to 5 now represent a dilution series as follows:
Tube
1 2 3 4 5
Number
Dilution 1:2 1:4 1:8 1:16 1:32
Perform the qualitative test procedure using each dilution as test specimen.
Procedure of RF Test
Interpretation for RF Test
Interpretation
The titer is reported as the reciprocal of the highest
dilution, which shows a positive test
result. Read the titer in the last dilution step with visible
agglutination and the approximate concentration of the
rheumatoid factor can be determined as follows:
RF in IU/mL= Sensitivity of latex Gamma
globulin reagent in IU/mL × Titer For example
: 8 *8 =64 IU/ML
Limitations of RF Test
1. RF is not detected in all patients diagnosed with RA.
2. RF may be detected in increased amounts in patients with
infectious mononucleosis, sarcoidosis, systemic lupus
erythematosus, TB or leprosy, and other conditions of acute
or chronic immune response.
3. The significance of a positive result should be
interpreted with caution. Quantitative testing should
be done to confirm diagnosis of RA.
4. Highly hemolyzed and lipemic serum as well as plasma
interfere with the test.