detection of Rheumatoid factor by using LatexAgglutination

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detection of Rheumatoid factor by using
LatexAgglutination

• Dr Laila Hammed Damanhouri

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Agglutination test

• It is one of important laboratory method to
  detect antigen antibody reaction.
• It provides flexible and useful method for semi
  quantitating of either antigen or antibody
  concentration.
• The reaction occurs between insoluble antigen and
  appropriate antibody.
• The reaction will results in forming aggregate or
  agglutinate.

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Stages of agglutination reaction

• Phase one
• Antibody reacts with single antigenic
  determinants on or close to particle surface.
• It is a rapid reaction.

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Secondary phase

• A single antibody molecule binds to antigenic
  determinants on adjacent particles.
• The visible reaction occur under appropriate
  conditions and over time, particles remain
  connected and interconnected by antibody bridge.

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Types of agglutination reaction1. Direct
agglutination.

• To test patients sera (contain antibody)
  against large antigen.
• Direct agglutination can be used to determine
  antibody titer.

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Indirect agglutination

• serum is mixed with latex spheres (inert
  substance) with the soluble antigens attached.
• Antibodies will then cause visible agglutination
  of the latex spheres with the soluble antigens
  attached.

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Indirect hemagglutination

• the red blood cell are coated with soluble .
• then incubated with patient serum (contain AB
  against Ag.
• the interaction between AB in the patients sera
  and antigen on the surface of red blood cell
  resulting on agglutination of the red blood cell
• If the reaction not occur the red blood cell will
  form as a button-shaped deposit at the bottom of
  reaction vessel.

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advantages of agglutination methods

• ease of performance.
• speed of performance, usually requiring few
  minutes.
• high degree of sensitivity.

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Disadvantages of agglutination methods

• the reaction are only semiquantitative.
• the occurrence of the prozone phenomenon, in
  which agglutination is inhibited by extreme
  antibody excess as a result of poor lattic
  formation.

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prozone

• Absence of agglutination at higher antibody
  concentration.
• It is due to many factors including
• Presence of blocking antibodies at low titers
• Inaccessible antigenic determinants
• Weak avidity
• Poor lattice formation.
• The problem can be avoided by use of standard
  serial dilution.

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Application of agglutination test

• several antibodies can be detected by this method
  such as Rheumatoid factor.

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Rheumatoid Factor (RF)

• his test is done to diagnosed Rheumatoid
  arthritis, which is one of important autoimmune
  disease.
• RF is an antibody ( IgM or IgG classes) bind to
  the Fc portion of other IgG molecules, and form
  IgG-anti-IgG complexes in the circulation or
  joint fluid.

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Rheumatoid factor (RF)

• RFs are detected in serum in up to 80 of adult
  patients with RA.
• RFs are not specific for RA and occur in other
  autoimmune disease, in chronic infectious
  diseases, such as infective endocarditis,
  tuberculosis, and hepatitis B.
• usually at low titer, in up to 20 of overtly
  normal elderly individuals

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General Feature of Rheumatoid arthritis

• RA is a systemic chronic inflammatory disease of
  unknown etiology.
• is characterized by polyarthritis which may be
  progressive and permanently deforming and by
  extra-articular manifestations(rheumatoid
  nodules, pericarditis, and arteritis).
• Adult RA is commonly associated with rheumatoid
  factors.

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Symptoms of Rheumatoid Arthritis

• Symptoms first begin in the small joints of the
  fingers, wrists and feet, with warm, swollen and
  tender joints that are painful and difficult to
  move.  
• Joints of both sides of the body (symmetrical)
  are typically affected.  
• People with RA often experience fatigue, loss of
  appetite and low-grade fever.  
• There is often stiffness in the morning that
  lasts for several hours or more.  
• Nodules may form under the skin, often over the
  bony areas exposed to pressure (such as the
  elbows).  
• Over time, damage to the cartilage and bone of
  the joints may lead to joint deformities.

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diagnosis of rheumatoid arthritis

• medical history and physical examination, looking
  for distribution of joints affected, joint
  swelling, warmth and range of motion, as well as
  the presence of nodules under the skin.  
• Imaging studies such as X-rays, sonograms or
  magnetic resonance imaging may be used to detect
  the degree of joint involvement or joint damage.
   
• A blood test can indicate the presence of an
  rheumatoid factor, which is found in 80 percent
  of people with RA.

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X ray
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Methods used to detect RF

• Latex agglutination method.
• mixes the blood being tested with (latex) beads
  that are covered with human antibodies. If
  rheumatoid factor (RF) is present, the latex
  beads clump (agglutinate).
• Haemagglutination test.
• mixes the blood being tested with a sheep's red
  blood cells that have been covered with rabbit
  antibodies. If RF is present, the red blood cells
  agglutinate.
• Nephelometry test
• Using an automated machine based on laser light
  scattered.

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Latex agglutination test
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Interpretation of the test

• Agglutination test is positive. Do titration and
  determine the end of titration.
• Normal range differ from lab to another, but in
  most lab titration of gt120 consider positive.
• Positive test in 80 of Rheumatoid Arthritis.
• It also positive in other autoimmune disease.
• Positive in viral hepatitis.
• Positive in TB.

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Factors interfere with positive result

• Hyperlipedemia. Blood that is very high in fats.
• Age. About 5 to 10 of people over age 65 have
  an elevated RF level.