Week 8: Rheumatoid Factor Latex Agglutination Test Flashcards
Materials used
Rf latex suspension/ reagent
Black test card (reusable): to visualize agglutination
• 3 circles: positive control, negative control, rf latex reagent
Mechanical rotator
Positive and negative controls Disposable stirrer
Principle of the test
Passive or indirect agglutination reaction
A suspension of polystyrene latex particles sensitized with specially prepared human igG
Rheumatoid factor reagent
Uses carrier particles
Latex particle
Other carrier particles that can be used in passive agglutination
Erythrocytes
Gelatin
Charcoal
When polystyrene latex particle meets with the human igG in the serum it forms?
Agglutination
It is based on an immunological reaction between human igG bound to biologically inert latex particles and rheumatoid factors in the test specimen
Reagent
The rf latex reagent sensitivity has been adjusted to derect a minimum of __ rheumatoid factors
8 IU/ml
Sensitivity
8 IU/ml or above
Reagents
Rf latex reagent
Rf positive control serum
Rf negative control serum
Glycine saline buffer (20x)
It is a suspension of uniform polystyrene particles coated with igG in glycine buffer ph is 8.2
Rf latex reagent
Stabilized, pre-diluted human serum containing less than 8 IU/ml of rf (should not be reactive to rf)
Rf negative control serum
Stabilize pre-diluted human serum containing at least eight IU/ml of rf
Rf positive control serum
Only use in semiquantitative method
pH 8.2
+/-0.1M glycine
0.15M sodium chloride
Glycine saline buffer (20x)
If there is a positive result in the qualitative method the next step would be
Detecting the titer by the semi quantitative method
Qualitative method procedure or screening
7 steps
- Allow each component of the test gates reagents control to reach room temperature
- Gently shake the latex reagent using your palms to disperse the particles
- Place a drop of undiluted serum into the circles of the test slides
- Add one drop of rf latex reagent to the next drop of serum sample
- Spread the reagent and serum sample over the entire area of the test circle using a separate stirrer for each sample
- Gently tilt the test slide backwards and forwards or place in a mechanical rotator for 2 minutes 7. Observe and interpret the results
1st well
Positive control
2nd well
Negative control
3rd well
For serum sample
Positive and negative controls should be run at_____ to compare the results
Regular interval
Negative result shows or indicate
Uniform milky suspension with no agglutination observed in the rf negative control
Positive result a reaction is indicated by
Any observable agglutination in the reaction mixture
Semi quantitative method procedure or 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
Dilutions of tube 1 2 3 4 and 5
1/2
1/4
1/8
1/6
1/32
Sample serum
100 ul
What tube does have sample serum only
Tube 1
How many ul of saline is used in tubes 1 to 5
100 ul
What is the sample volume of tube 1 to 5
50 ul
Titer of tube 1 to 5
16, 32, 64, 128, 256
8×Df= IU/ml
Reciprocal of the highest dilution showing macroscopic of glutination
Titer
If agglutination occurs in dilution 3 the titer is
64
Dilution factor is eight because this is the sensitivity of
Rf latex agglutination test
IU/ml of specimen =
IU/ml control specimen titer
The approximately rf level present in the sample may be obtained by
Multiplying the titer by the analytical sensitivity of rf latest reagent which is 8 IU/ml
Rheumatoid arthritis is another example of a
Systemic autoimmune disorder
How many percent does rheumatoid arthritis affects the adult population
0.5% to 1.0%
Prevalence of rheumatoid arthritis varies with
Ethnicity and geographic location
Women are ___ as likely to be affected as men
3 times
The prevalence of the disease is highest in women who are
More than 65 years of age
Rheumatoid arthritis can be characterized as
Chronic, symmetric, and erosive arthritis of the peripheral joints
It can also affect multiple organs such as heart and the lungs
Association of RA with more than ____ genetic regions have been discovered
30
The strongest associations have been between a subset of patients with ra and specific ____ and ____.
HLA-DRB1 alleles or PTPN22 gene polymorphisms
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 ______ in rheumatoid joints show an increase in cell lining the synovial membrane and formation of a panus
Lesions
Organized mass cell/ a sheet of inflammatory granulation tissue that grows into the joint space and invades the cartilage
Pannus
Evidence of immunologic activity within the joint is the infiltration of the inflamed synovium with?
(Cells)
T and B lymphocytes
Plasma cells
Dendritic cells
Mast cells
Granulocytes
What is the role of autoantibodies in the initiation of the inflammatory response
Not known
Two key antibodies found in the disease are:
Rheumatoid factor
Anti–cyclic citrullinated peptide (anti-CCP)
Rheumatoid factor is an antibody that is most often of the igM class and is directed against which portion of igG
FC Portion
It has been postulated that rheumatoid factors may play a role in the pathogenesis of rheumatoid arthritis by increasing ___ and ___.
Macrophage activity and enhancing antigen presentation to T cells by apcs (antigenpresenting cells)
In rheumatoid arthritis, autoantibodies such as rf and anti ccp are thought to combine with their specific antigen and resulting immune complexes become deposited in the joints resulting in what type of hypersensitivity reaction
Type 3 hypersensitivity reaction or immune complex
What complement protein binds to the immune complexes, activating the classical component cascade?
C1
During this process ___and ___ are generated, which act as chemotactic factors for neutrophils and macrophages
C3a and c5a
It acts as a chemotactic factor for neutrophils and macrophages
C3a and c5a
The continual presence of this cells (neutrophils and macrophages) and their associated cytokines leads to
Chronic inflammation which damages the synovium itself
Diagnosis of rheumatoid arthritis is based on a combination of
Clinical manifestations
Radiographic findings and Laboratory testing
It is the antibody that is most often tested to aid in making the initial diagnosis
Rf or rheumatoid factor
The importance of testing for the presence of rf is also reflected in the fact that it is one of the classification criteria for
Rheumatoid arthritis
It is an auto antibody that is usually in the igm class that reacts with the fc portion of igg
Rheumatoid factor
Approximately _____of patients with rheumatoid arthritis test positive for rf
70% to 90% of patients
A negative test or result does not rule out the presence of ra
True
A positive test is not specific for RA because RF is also present in about _____ of healthy individuals and in _____ of those over the age of 65
5% of healthy individuals
10% to 25% of those over the age of 65
Rf is present in about ___ in healthy individuals
5%
Rf can be found in patients with other connective tissue diseases such as
Systemic lupus erythematosus Sjogren’s syndrome
Scleroderma
Mixed connective tissue disease
People with some chronic infections
Manual agglutination test using __ or ___ have been used for many years to detect rf
Charcoal or latex particles coated with igG
These test, however only detect the igM isotype found in about 75% of the patients
Manual agglutination tests
Manual agglutination test only detect the
IgM isotype
Igm isotype is found in about how many percent of patients
75%
Manual agglutination tests have been largely replaced by automated methods. These have also a greater precision and sensitivity, can also detect other isotypes
These are the following:
Enzyme link immunosorbent assay chemiluminescence immunoassay Nephelometric methods
Once a diagnosis of rheumatoid arthritis is made the most helpful test for following the progress of the disease are general indicators of inflammation, such as:
Measurement of erythrocyte sedimentation rate
C-reactive protein
Complement components
What is the typical result of esr and crp
Elevated
Resutls of the levels of serum complement components are
Normal or increased because of increased acute phase reactivity
Levels of serum component complements are normal or increase because of increase
Acute-phase reactivity
The result should be read in____ mixing the reagent on the slide
2 minutes after
Existence of __ at high titers has not been encountered
Prozone
Increased levels of rheumatoid factor may be around in some diseases other than rheumatoid arthritis such as
Mononucleosis or sarcoidosis
Lupus Erythematosus
Sjogren’s syndrome
Certain patients with rheumatoid arthritis will not have the rf present in their serum
True
Reagents are stable until stated expiration date on bottle label and stored refrigerated at what degree celsius
2 to 8 degrees celsius
If the reagents came from the refrigerated temperature we must
Cool down first
The rf latest reagents one shaken must be uniform without
Visible clumping
The Rf latex reagent when stored refrigerated there is a presence of______. And it should be considered normal
Slight sedimentation
Do not use the latex reagent or control if they become
Contaminated
Using contaminated latex reagent results in
Invalid results
We should interpret the result strictly in 2 minutes because it is sensitive to
False positive results
Rf is not specific
True
Specific for RA
Anti-CCP
It is simply an autoimmune disease compared with _____ which is a consequence of streptococcus infection
Rheumatoid arthritis
Acute RHEUMATIC FEVER
Passive agglutination reaction has a carrier particle which is
Latex particle
Crp and aso carries antigens
RF carries___
Antibodies (IgG)
Rf detects ___
Antibody
