detection of Rheumatoid detection of Rheumatoid factor by using factor by using

LatexAgglutinationLatexAgglutination

Dr Laila Hammed DamanhouriDr Laila Hammed Damanhouri

Agglutination testAgglutination test• It is one of important laboratory method to It is one of important laboratory method to

detect antigen antibody reaction.detect antigen antibody reaction.• It provides flexible and useful method for It provides flexible and useful method for

semi quantitating of either antigen or semi quantitating of either antigen or antibody concentration.antibody concentration.

• The reaction occurs between The reaction occurs between insoluble insoluble antigenantigen and appropriate antibody. and appropriate antibody.

• The reaction will results in forming The reaction will results in forming aggregate or agglutinate. aggregate or agglutinate.

Stages of agglutination Stages of agglutination reactionreaction

Phase onePhase one• Antibody reacts Antibody reacts

with single with single antigenic antigenic

determinants on or determinants on or close to particle close to particle

surface.surface.• It is a rapid It is a rapid

reaction.reaction.

Secondary phaseSecondary phase• A single antibody A single antibody

molecule binds to molecule binds to antigenic determinants antigenic determinants on adjacent particles.on adjacent particles.

• The visible reaction The visible reaction occur under occur under appropriate conditions appropriate conditions and over time, particles and over time, particles remain connected and remain connected and interconnected by interconnected by antibody bridge. antibody bridge.

Types of agglutination reactionTypes of agglutination reaction1. Direct agglutination.1. Direct agglutination.• To test patient’s To test patient’s

sera (contain sera (contain antibody) against antibody) against large antigen.large antigen.

• Direct Direct agglutination can agglutination can be used to be used to determine antibody determine antibody titer.titer.

Indirect agglutinationIndirect agglutination• serum is mixed with serum is mixed with

latex spheres (inert latex spheres (inert substance) with the substance) with the soluble antigens soluble antigens attachedattached..

• Antibodies will then Antibodies will then cause visible cause visible agglutination of the agglutination of the latex spheres with the latex spheres with the soluble antigens soluble antigens attached. attached.

Indirect hemagglutinationIndirect hemagglutination

• the red blood cell are coated the red blood cell are coated with soluble .with soluble .

• then incubated with patient then incubated with patient serum (contain AB against serum (contain AB against Ag.Ag.

• the interaction between AB the interaction between AB in the patients sera and in the patients sera and antigen on the surface of red antigen on the surface of red blood cell resulting on blood cell resulting on agglutination of the red blood agglutination of the red blood cell cell

• If the reaction not occur the If the reaction not occur the red blood cell will form as a red blood cell will form as a button-shaped deposit at the button-shaped deposit at the bottom of reaction vessel. bottom of reaction vessel.

advantages of agglutination advantages of agglutination methodsmethods

• ease of performance.ease of performance.• speed of performance, usually speed of performance, usually

requiring few minutes.requiring few minutes.• high degree of sensitivity.high degree of sensitivity.

Disadvantages of agglutination Disadvantages of agglutination methodsmethods

• the reaction are only semiquantitative.the reaction are only semiquantitative.• the occurrence of the prozone the occurrence of the prozone

phenomenon, in which agglutination is phenomenon, in which agglutination is inhibited by extreme antibody excess inhibited by extreme antibody excess as a result of poor lattic formation.as a result of poor lattic formation.

prozoneprozone• Absence of agglutination at higher antibody Absence of agglutination at higher antibody

concentration.concentration.• It is due to many factors includingIt is due to many factors including• Presence of blocking antibodies at low titersPresence of blocking antibodies at low titers• Inaccessible antigenic determinantsInaccessible antigenic determinants• Weak avidityWeak avidity• Poor lattice formation.Poor lattice formation.• The problem can be avoided by use of The problem can be avoided by use of

standard serial dilution.standard serial dilution.

Application of agglutination Application of agglutination testtest• several antibodies can be detected several antibodies can be detected

by this method such as Rheumatoid by this method such as Rheumatoid factor.factor.

Rheumatoid Factor (RF)Rheumatoid Factor (RF)• his test is done to his test is done to

diagnosed Rheumatoid diagnosed Rheumatoid arthritis, which is one of arthritis, which is one of important autoimmune important autoimmune disease.disease.

• RF is an antibody ( IgM RF is an antibody ( IgM or IgG classes) bind to or IgG classes) bind to the Fc portion of other the Fc portion of other IgG molecules, and form IgG molecules, and form IgG-anti-IgG complexes IgG-anti-IgG complexes in the circulation or in the circulation or joint fluid.joint fluid.

Rheumatoid factor (RF)Rheumatoid factor (RF)• RFs are detected in serum in up to RFs are detected in serum in up to

80% of adult patients with RA.80% of adult patients with RA.• RFs are not specific for RA and occur RFs are not specific for RA and occur

in other autoimmune disease, in in other autoimmune disease, in chronic infectious diseases, such as chronic infectious diseases, such as infective endocarditis, tuberculosis, infective endocarditis, tuberculosis, and hepatitis B.and hepatitis B.

• usually at low titer, in up to 20% of usually at low titer, in up to 20% of overtly normal elderly individualsovertly normal elderly individuals

General Feature of Rheumatoid General Feature of Rheumatoid arthritisarthritis• RA is a systemic chronic inflammatory RA is a systemic chronic inflammatory

disease of unknown etiology. disease of unknown etiology. • is characterized by polyarthritis which is characterized by polyarthritis which

may be progressive and permanently may be progressive and permanently deforming and by extra-articular deforming and by extra-articular manifestations(rheumatoid nodules, manifestations(rheumatoid nodules, pericarditis, and arteritis).pericarditis, and arteritis).

• Adult RA is commonly associated with Adult RA is commonly associated with rheumatoid factors. rheumatoid factors.

Symptoms of Rheumatoid Symptoms of Rheumatoid ArthritisArthritis• Symptoms first begin in the small joints Symptoms first begin in the small joints

of the fingers, wrists and feet, with of the fingers, wrists and feet, with warm, swollen and tender joints that warm, swollen and tender joints that are painful and difficult to move. are painful and difficult to move.   

• Joints of both sides of the body Joints of both sides of the body (symmetrical) are typically affected. (symmetrical) are typically affected.   

• People with RA often experience People with RA often experience fatigue, loss of appetite and low-grade fatigue, loss of appetite and low-grade fever. fever.   

• There is often stiffness in the morning There is often stiffness in the morning that lasts for several hours or more. that lasts for several hours or more.   

• Nodules may form under the skin, often Nodules may form under the skin, often over the bony areas exposed to over the bony areas exposed to pressure (such as the elbows). pressure (such as the elbows).   

• Over time, damage to the cartilage and Over time, damage to the cartilage and bone of the joints may lead to joint bone of the joints may lead to joint deformities.deformities.

diagnosis of rheumatoid diagnosis of rheumatoid arthritisarthritis• medical history and physical examination, medical history and physical examination,

looking for distribution of joints affected, joint looking for distribution of joints affected, joint swelling, warmth and range of motion, as well swelling, warmth and range of motion, as well as the presence of nodules under the skin. as the presence of nodules under the skin.   

• Imaging studies such as X-rays, sonograms or Imaging studies such as X-rays, sonograms or magnetic resonance imaging may be used to magnetic resonance imaging may be used to detect the degree of joint involvement or joint detect the degree of joint involvement or joint damage. damage.   

• A blood test can indicate the presence of an A blood test can indicate the presence of an rheumatoid factor, which is found in 80 rheumatoid factor, which is found in 80 percent of people with RA.percent of people with RA.

X rayX ray

Methods used to detect RFMethods used to detect RF

• Latex Latex agglutination method.agglutination method.

mixes the blood being tested with (latex) beads that are mixes the blood being tested with (latex) beads that are covered with human antibodies. If rheumatoid factor covered with human antibodies. If rheumatoid factor (RF) is present, the latex beads clump (agglutinate).(RF) is present, the latex beads clump (agglutinate).

• Haemagglutination test.Haemagglutination test.

mixes the blood being tested with a sheep's red blood mixes the blood being tested with a sheep's red blood cells that have been covered with rabbit antibodies. If cells that have been covered with rabbit antibodies. If RF is present, the red blood cells agglutinate.RF is present, the red blood cells agglutinate.

• Nephelometry testNephelometry testUsing an automated machine based on laser light Using an automated machine based on laser light

scattered. scattered.

Latex agglutination testLatex agglutination test

Interpretation of the testInterpretation of the test• Agglutination test is positive. Do titration and Agglutination test is positive. Do titration and

determine the end of titration.determine the end of titration.• Normal range differ from lab to another, but Normal range differ from lab to another, but

in most lab titration of >1:20 consider in most lab titration of >1:20 consider positive.positive.

• Positive test in 80% of Rheumatoid Arthritis.Positive test in 80% of Rheumatoid Arthritis.• It also positive in other autoimmune disease.It also positive in other autoimmune disease.• Positive in viral hepatitis.Positive in viral hepatitis.• Positive in TB. Positive in TB.

Factors interfere with positive Factors interfere with positive resultresult• Hyperlipedemia. Blood that is Hyperlipedemia. Blood that is

very high in fats.very high in fats.• Age. About 5% to 10% of people Age. About 5% to 10% of people

over age 65 have an elevated RF over age 65 have an elevated RF level.level.