Agglutination Testing in Laboratory Medicine
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Agglutination test: Qualitative/ Quantitative(Ab titer)
agglutination test. It is used to determine Ag or Ab
presence and amount(
titer)
•
Carrier particles
: Latex, RBC’s, charcoal, protein A of
Staph. Aureus.
•
It is an indirect test.
•
It is used to detect Ag or Ab in the patient sample.
•
Latex particles are inert polystyrene substance(0.79-
0.81mm).Ag or Ab attach spontaneously to it.
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(
R
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)
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Is traditionally considered a chronic, inflammatory
autoimmune disorder that causes the immune system
to attack the joints.
•
It is a disabling and painful inflammatory condition,
which can lead to substantial loss of mobility due to
pain and joint destruction.
•
RA is a systemic disease, often affecting extra- articular
tissues throughout the body including the skin, blood
vessels, heart, lungs, and muscles.
•
About 60% of RA patients are unable to work 10 years
after the onset of their disease.
•
The Rheumatoid arthritis immune response appears to
be directed against multiple antigenic determinants on
the gamma globulin molecule.( Anti-IgG)
•
In the test kit, latex particles are coated with human
gamma globulin IgG molecule.
•
In the screening test, however, positive results may
also be obtained due to others disorders such as:
•
Systemic Lupus Erythematosis (SLE)
•
Polyarteritis nodosa
•
Dermatomyositis or Scleroderma.
Some diseases such as Cirrhosis, Hepatitis,, Syphilis, Sub
acute bacterial endocarditis and Lymphomas may also
give rise to agglutination reactions. Titers in these
conditions are usually less than 1:20
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Sample collection
:
•
Collect 5 – 10 ml venous blood into sterile tube
without anticoagulant
•
Allow to clot at room temp.
•
Formation of clot must be complete
•
Separate serum and store at 2-8 °C
•
It is preferable to test samples within 2-3 days
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Screening test
•
Bring all reagents to room temp.
•
Prepare 1:20 dilution of test serum by adding 50
μ
l of
serum to 1 ml of glycine buffer
•
Place 50
μ
l of diluted serum on to the test slide
•
Add 1 drop of
well shaken
latex reagent
•
Mix the two drops together with a clean stirrer and
spread out to the edge of the test area
•
Rock the slide gently and observe for macro agglutination
•
Read at
2 minutes
under a direct light source
•
A definite clumping is reported as
reactive
(R).
•
No clumping is reported as
non-reactive
(N).
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C-reactive protein (CRP) is a non-specific test.
•
It is used by a doctor to detect inflammation if
there is a high suspicion of tissue injury or
infection somewhere in the body, but the test
cannot tell where the inflammation is or what
condition is causing it.
•
CRP is not diagnostic of any condition, but it can
be used together with signs and symptoms and
other tests to evaluate an individual for an acute
or chronic inflammatory condition
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The same principle of Latex kits .
•
CRP is a protein present normally in human
serum.
•
Anti-CRP is coated on latex to detect CRP in
patient sample to detect CRP in patient sample.
•
CRP is used mainly as a marker of inflammation
•
It may also be ordered on a regular basis to
monitor conditions such as rheumatoid arthritis
and Lupus and is often repeated at intervals to
determine whether treatment is effective
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(
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R
)
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Syphilis is a sexually transmitted disease caused
by the spirochete
Treponema pallidum
.
•
The
RPR
(Rapid Plasma Reagin) Card test is a
presumptive serologic screening test
for syphilis.
•
The serum of a person with syphilis contains a
non-specific anti-lipid antibody
(termed
Reagin
),
which is not found in normal serum.
•
Syphilis infection starts the breakdown of the
patient's own tissue cells.
•
Fatty substances which are released, combine with
protein from
Treponema pallidum
to form an antigen
which stimulates the body to produce
antibodies
against both the body's tissue lipids (non-specific or
non-treponemal) as well as the
T. pallidum
protein
(specific or treponemal
)
.
•
The RPR Card test detects the nonspecific antilipid
antibody and is referred to as a
non- treponemal test
for syphilis
.
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The RPR test is a non-treponemal slide
agglutination test for the qualitative and semi-
quantitative detection of plasma reagins in
human serum.
•
Carbon particles coated with a lipid complex
are agglutinated when mixed with samples
containing reagins.
•
The
known
RPR
antigen
consists of
Cardiolipin, lecithin, and
cholesterol
bound to
charcoal particles
•
Charcoal makes the reaction visible.
•
If the patient has syphilis, the antilipid
antibodies (Reagin) in his/her serum will
cross-react with the known RPR lipid antigens
giving a visible clumping of the charcoal
particles.
Procedure:
•
Place 50 µl of sample and one drop of each +ve and –ve controls
into separate circles on the test slide.
•
Swirl the RPR-carbon reagent gently before using.
•
Place the micropipette in a vertical position and perpendicular to
the slide, and add one drop (20 µl) of this reagent next to the
samples to be tested.
•
Mix the drops with a stirrer, spreading them over the entire
surface of the circle. Use different stirrers for each samples.
•
Place the slide on a mechanical rotor (shaker) at 80-100 r.p.m. for
8 minutes.
•
Read the test results immediately.
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:
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A definite clumping of the charcoal particles is reported as
reactive (R)
•
No clumping is reported as
non-reactive (N)
.
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Protein A which is a cell wall component of
Staphylococcus aureus
is able to bind to Fc
portion of most IgG Ab leaving Fab portion
free to interact with Ag present in specimens.
•
This test is used to
detect Ag
in specimens or
isolated bacteria from cultures.
•
Example
: streptococcal serotyping, Bacterial
Ag detection kit for meningitis.
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It is an agglutination where Ab is coated on
protein A of Staph. aureus .
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Agglutination testing is a common method used in laboratory medicine to detect the presence and amount of antigens or antibodies in patient samples. It involves the use of carrier particles such as latex, RBCs, charcoal, and protein A of Staph aureus. The testing process includes qualitative and quantitative assessments, with applications in conditions like rheumatoid arthritis. The immune response in RA targets gamma globulin molecules, specifically anti-IgG antibodies. Proper sample collection and handling are crucial for accurate test results. Screening tests involve diluting serum samples, adding latex reagents, and observing for clumping reactions indicative of reactivity. Various autoimmune and infectious diseases can lead to positive agglutination reactions.
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Presentation Transcript
Carrier particle Agglutination Latex agglutination flocculation
Carrier particle agglutination test Agglutination test: Qualitative/ Quantitative(Ab titer) agglutination test. It is used to determine Ag or Ab presence and amount(titer) Carrier particles: Latex, RBC s, charcoal, protein A of Staph. Aureus. It is an indirect test. It is used to detect Ag or Ab in the patient sample. Latex particles are inert polystyrene substance(0.79- 0.81mm).Ag or Ab attach spontaneously to it.
Latex Agglutination Test Rheumatoid arthritis (RA) Is traditionally considered a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and joint destruction. RA is a systemic disease, often affecting extra- articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles. About 60% of RA patients are unable to work 10 years after the onset of their disease.
The Rheumatoid arthritis immune response appears to be directed against multiple antigenic determinants on the gamma globulin molecule.( Anti-IgG) In the test kit, latex particles are coated with human gamma globulin IgG molecule. In the screening test, however, positive results may also be obtained due to others disorders such as: Systemic Lupus Erythematosis (SLE) Polyarteritis nodosa Dermatomyositis or Scleroderma. Some diseases such as Cirrhosis, Hepatitis,, Syphilis, Sub acute bacterial endocarditis and Lymphomas may also give rise to agglutination reactions. Titers in these conditions are usually less than 1:20
Method: Sample collection: Collect 5 10 ml venous blood into sterile tube without anticoagulant Allow to clot at room temp. Formation of clot must be complete Separate serum and store at 2-8 C It is preferable to test samples within 2-3 days
Method: Screening test Bring all reagents to room temp. Prepare 1:20 dilution of test serum by adding 50 l of serum to 1 ml of glycine buffer Place 50 l of diluted serum on to the test slide Add 1 drop of well shaken latex reagent Mix the two drops together with a clean stirrer and spread out to the edge of the test area Rock the slide gently and observe for macro agglutination Read at 2 minutes under a direct light source A definite clumping is reported as reactive (R). No clumping is reported as non-reactive (N).
CRP Latex Kit C-reactive protein (CRP) is a non-specific test. It is used by a doctor to detect inflammation if there is a high suspicion of tissue injury or infection somewhere in the body, but the test cannot tell where the inflammation is or what condition is causing it. CRP is not diagnostic of any condition, but it can be used together with signs and symptoms and other tests to evaluate an individual for an acute or chronic inflammatory condition
CRP Latex Agglutination test The same principle of Latex kits . CRP is a protein present normally in human serum. Anti-CRP is coated on latex to detect CRP in patient sample to detect CRP in patient sample. CRP is used mainly as a marker of inflammation It may also be ordered on a regular basis to monitor conditions such as rheumatoid arthritis and Lupus and is often repeated at intervals to determine whether treatment is effective
CRP Test Elisa Kit Latex kit Latex Kit
Flocculation Test Rapid Plasma Reagin (RPR) Syphilis is a sexually transmitted disease caused by the spirochete Treponema pallidum. The RPR (Rapid Plasma Reagin) Card test is a presumptive serologic screening test for syphilis. The serum of a person with syphilis contains a non-specific anti-lipid antibody (termed Reagin), which is not found in normal serum.
Syphilis infection starts the breakdown of the patient's own tissue cells. Fatty substances which are released, combine with protein from Treponema pallidum to form an antigen which stimulates the body to produce antibodies against both the body's tissue lipids (non-specific or non-treponemal) as well as the T. pallidum protein (specific or treponemal). The RPR Card test detects the nonspecific antilipid antibody and is referred to as a non- treponemal test for syphilis.
Principle of the Method The RPR test is a non-treponemal slide agglutination test for the qualitative and semi- quantitative detection of plasma reagins in human serum. Carbon particles coated with a lipid complex are agglutinated when mixed with samples containing reagins.
The known RPR antigen consists of Cardiolipin, lecithin, and cholesterol bound to charcoal particles Charcoal makes the reaction visible. If the patient has syphilis, the antilipid antibodies (Reagin) in his/her serum will cross-react with the known RPR lipid antigens giving a visible clumping of the charcoal particles.
Procedure: Place 50 l of sample and one drop of each +ve and ve controls into separate circles on the test slide. Swirl the RPR-carbon reagent gently before using. Place the micropipette in a vertical position and perpendicular to the slide, and add one drop (20 l) of this reagent next to the samples to be tested. Mix the drops with a stirrer, spreading them over the entire surface of the circle. Use different stirrers for each samples. Place the slide on a mechanical rotor (shaker) at 80-100 r.p.m. for 8 minutes. Read the test results immediately.
Read the results as follows: A definite clumping of the charcoal particles is reported as reactive (R) No clumping is reported as non-reactive (N).
Co-agglutination Protein A which is a cell wall component of Staphylococcus aureus is able to bind to Fc portion of most IgG Ab leaving Fab portion free to interact with Ag present in specimens. This test is used to detect Ag in specimens or isolated bacteria from cultures. Example: streptococcal serotyping, Bacterial Ag detection kit for meningitis.
Co-agglutination It is an agglutination where Ab is coated on protein A of Staph. aureus . Co-agglutination Y Y Y + Y Y Y Y Y Y Y Coated Ab on Staph protein A TEST Ag
