OUR LADY OF FATIMA UNIVERSITY

COLLEGE OF MEDICAL LABORATORY SCIENCE

Dela Paz Norte, San Fernando, Pampanga

LABORATORY MODULE FORMAT

COURSE TITLE: Immunology and Serology

LABORATORY ACTIVITY: Rheumatoid Factor Latex Agglutination Test

Intended Learning Outcomes:

At the end of this unit, the student is able to:

 Correctly perform and interpret RF test

 Explain the clinical significance of RF test

LABORATORY GUIDE:

Week Teaching-Learning Activities Assessment Task Resources

o Correctly perform and interpret RF test Perform Laboratory activities

IMS Laboratory
o Explain the clinical significance of RF test
7 Manual
Post Lab Quiz
.

COURSE CONTENT
PRE-ANALYTICAL PHASE:
The work area of the experiment should be sterilized. The Students should wear proper personal
protective equipment before the experiment (laboratory gown, eye protector, laboratory mask, gloves and hair
net). Standard precaution is applied to every phase of experiment.

MATERIALS:
Rf latex suspension Positive and Negative Controls
Black test cards Disposable stirrers
Mechanical rotator

LABORATORY DISCUSSION GUIDE:

I. Principle of the test
The RF reagent is a suspension of polystyrene latex particles sensitized with specially prepared human
IgG. The reagent is based on an immunological reaction between human IgG bound to biologically inert
latex particles and rheumatoid factors in the test specimen. When serum containing rheumatoid factors
is mixed with the latex reagent, visible agglutination occurs. The RF latex reagent sensitivity has been
adjusted to detect a minimum of 8 IU/mL of rheumatoid factors according with the WHO International
Standard without previous sample dilution.

Sensitivity: 8 IUml or above.

I. Reagents of the test

 OUR LADY OF FATIMA UNIVERSITY
COLLEGE OF MEDICAL LABORATORY SCIENCE
Dela Paz Norte, San Fernando, Pampanga

1. RF Latex Reagent: A suspension of uniform polystyrene particles coated with IgG (human) in
glycine buffer, pH 8.2; reagent sensitivity is standardized with the World Health Organization RF
Standard. MIX WELL BEFORE USING.
2. RF Positive Control Serum: A stabilized, prediluted human serum containing at least 8 IU/mL of RF.
3. RF Negative Control Serum: A stabilized, prediluted human serum containing less than 8 IU/mL of
RF.
4. Glycine-Saline Buffer (20x): pH 8.2 ± 0.1M glycine and 0.15M NaCl
5. Reaction Slide.
6. PipetteStir Sticks

II. Procedure
QUALITATIVE METHOD
1. Allow each components of the test kit (reagents, controls) to reach room temperature.
2. Gently shake the latex reagent to disperse the particles.
3. Place a drop of undiluted serum into a circle of a test slide.
4. Add one drop of Rf Latex reagent next to the drop of serum.
5. Spread the reagent and serum sample over the entire area of the test circle using a separate stirrer
for each sample.
6. Gently tilt the test slide backwards and forwards for two minutes.
7. Observe and interpret the results.

NOTE: Positive and Negative Controls should be run at regular intervals

SEMI-QUANTITATIVE DETERMINATION

The semi-quantitative test can be performed in the same way as the qualitative test using dilutions of the
serum in saline, phosphate buffer saline or glycine saline as follows:

Dilutions ½ ¼ 1/8 1/16 1/32

Sample 100 ul
serum

Saline 100ul 100ul 100ul 100ul 100ul

100ul 100ul 100ul 100ul

Volume of 50ul 50ul 50ul 50ul 50ul

sample

8 x Dilution 8 x 2 8x4 8x8 8 x16 8 x32

factor

IU/ml 16 32 64 128 256

Normal levels (adults): less than 8 IU/ml

CALCULATION: In the semi-quantitative test, the titer is expressed as the reciprocal of the highest
dilution showing macroscopic agglutination. For example, if agglutination occurs in dilution 3, the titer is
64.
 OUR LADY OF FATIMA UNIVERSITY
COLLEGE OF MEDICAL LABORATORY SCIENCE
Dela Paz Norte, San Fernando, Pampanga

III. Results

Qualitative Test:
Negative Result: A negative reaction is indicated by a uniform milky suspension with no agglutination
observed with the RF Negative Control.
Positive Result: A positive reaction is indicated by any observable agglutination in the reaction mixture.
The specimen reaction should be compared to the RF Negative and Positive Controls

Semi-quantitative Test:
The titer of the serum is the reciprocal of the highest dilution, which exhibits a positive reaction. An estimate
of the RF concentration in the specimen can be expressed in IUml by using the following equation:
IUml of specimen = IUml control  specimen titer
The approximately RF level (IU/ml) present in the sample may be obtained by the multiplying the titer by
the analytical sensitivity of the Rf Latex reagent (8 IU/ml)

IV. Clinical Significance

RA is another example of a systemic autoimmune disorder. It affects about 0.5% to 1.0% of the adult
population, but prevalence varies with ethnicity and geographic location. Women are three times as likely
to be affected as men; in addition, the prevalence of the disease is highest in women who are more than
65 years of age. RA can be characterized as a chronic, symmetric, and erosive arthritis of the peripheral
joints that can also affect multiple organs such as the heart and the lungs. Associations of RA with more
than 30 genetic regions have been discovered. The strongest associations have been between a subset
of patients with RA and specific HLA-DRB1 alleles or PTPN22 gene polymorphisms. These patients are
positive for rheumatoid factor (RF) or antibodies to CCP (see the section on Laboratory Diagnosis). The
strongest environmental risk factor for RA is believed to be cigarette smoking, which doubles the risk of
developing the disease.

The pathology of RA is caused by an inflammatory process that results in the destruction of bone and
cartilage. The lesions in rheumatoid joints show an increase in cells lining the synovial membrane and
formation of a pannus, a sheet of inflammatory granulation tissue that grows into the joint space and
invades the cartilage. Infiltration of the inflamed synovium with T and B lymphocytes, plasma cells,
dendritic cells, mast cells, and granulocytes is evidence of immunologic activity within the joint.

It is not known what role autoantibodies play in the initiation of the inflammatory response. Two key
antibodies found in the disease are RF and anti-CCP. RF is an antibody that is most often of the IgM
class and is directed against the FC portion of IgG. It has been postulated that RFs may play a role in the
pathogenesis of RA by increasing macrophage activity and enhancing antigen presentation to T cells by
APCs. In RA, autoantibodies such as RF and anti-CCP are thought to combine with their specified
antigen, and the resulting immune complexes become deposited in the joints, resulting in a type III (or
immune complex) hypersensitivity reaction. The complement protein C1 binds to the immune complexes,
activating the classical complement cascade. During this process, C3a and C5a are generated, which
act as chemotactic factors for neutrophils and macrophages. The continual presence of these cells and
their associated cytokines leads to chronic inflammation, which damages the synovium itself.

Diagnosis of RA is based on a combination of clinical manifestations, radiographic findings, and

laboratory testing. RF is the antibody that is most often tested to aid in making the initial diagnosis. The
importance of testing for the presence of RF is also reflected in the fact that it is one of the classification
criteria for RA.

Recall that RF is an autoantibody, usually of the IgM class, that reacts with the Fc portion of IgG.
Approximately 70% to 90% of patients with RA test positive for RF. Thus, a negative result does not rule
out the presence of RA. Conversely, a positive test result is not specific for RA because RF is also present
in about 5% of healthy individuals and in 10% to 25% of those over the age of 65.
 OUR LADY OF FATIMA UNIVERSITY
COLLEGE OF MEDICAL LABORATORY SCIENCE
Dela Paz Norte, San Fernando, Pampanga

In addition, RF can be found in patients with other connective tissue diseases such as SLE, Sjögren’s
syndrome, scleroderma, and mixed connective tissue disease, as well as in people with some chronic
infections. Manual agglutination tests using charcoal or latex particles coated with IgG have been used
for many years to detect RF. These tests, however, only detect the IgM isotype, found in about 75% of
patients, and have been largely replaced by ELISA, chemiluminescence immunoassay, and
nephelometric methods, which are automated, have greater precision and sensitivity, and can also detect
other RF isotypes.

Once a diagnosis of RA is made, the most helpful tests for following the progress of the disease are
general indicators of inflammation, such as measurement of ESR, CRP, and complement components.
Typically, CRP and ESR are elevated and the levels of serum complement components are normal or
increased because of increased acute-phase reactivity. CRP levels correlate well with disease activity
because levels reflect the intensity of the inflammatory response.

And other important information

1. Results should be read two (2) minutes after the mixing or the reagent on the slide.
2. Existence of prozone at high titers has not been encountered.
3. Increased levels of RF may be round in some diseases other than rheumatoid arthritis such as
infectious mononucleosis, sarcodosis, lupus erythematosus, Sjogren's syndrome. 6,7
4. Certain patients with rheumatoid arthritis will not have the RF present in their serum.

Expected values:
1. The diagnosis of rheumatoid arthritis is based largely on clinical examination, but laboratory tests are
useful to support the clinical diagnosis and to evaluate the severity and course or the disease in the
individual patient. One of the most useful clinical markers for rheumatoid arthritis is rheumatoid factor in
serum. Rheumatoid factor is a term used to describe a variety of antibodies or immune complexes or
both, that occur with rheumatoid arthritis as well as in a variety of other diseases. 8
2. Different studies have shown positive serological reactions for rheumatoid factor in as high as 90 of
patients with rheumatoid arthritis compared with less than 5 in control groups.

STORAGE
1. Reagents are stable until stated expiration date on bottle label when stored refrigerated (2 - 8°C).
2. DO NOT FREEZE.
3. The RF latex reagent, once shaken must be uniform without visible clumping. When stored refrigerated,
a slight sedimentation may occur and should be considered normal.
4. Do not use the latex reagent or controls if they become contaminated