INTRODUCTION

Immune System

• The immune system is a system of biological structures and

processes within an organism that protects against disease.

• To function properly, an immune system must detect a wide

variety of agents, from viruses to parasitic worms, and

distinguish them from the organism's own healthy tissue.

Antigen - Antibody

Types of Ag : RBCs .

WBCs

Bacteria .

Latex particles .

Ag/Ab reaction applications : Agglutination

Direct agglutination

Indirect agglutination

Precipitation

Eliza ( Enzyme linked Immunosorbent

Assay )

RIA ( Radio Immuno Assay )

FPIA

MEIA

Factors affecting the Ag/Ab reaction: 1- Buffer 2- Concentration of Ag 3- Concentration of Ab4- Temperature 5- Electrolytes

Agglutination• Agglutination: The reaction between an antigen and particulate antibody

appear as visible clumping.

• The antibody is called agglutinins.

• The principle of Agglutination reactions depend on the cross linking of polyvalent antigens. The Ab react with Ag, present in the surface of particles.

• When the antigen is an erythrocyte it is called hemagglutination.

• Theoretically all antibodies can agglutinate antigens but IgM, due to its high specificity is a particularly good agglutinin.

• Prozone effect is defined as the invisibility of agglutination at high concentrations of antibodies. It is due to the reason that excess antibody forms very minute complexes that do not clump to form visible agglutination.

• The Ag/Ab precipitation differs from agglutination , that the Ag in precipitation is soluble.

Type of Reactions:• Qualitative agglutination test• Agglutination tests can be used in a qualitative manner to assay for the

presence of an antigen or an antibody. The antibody is mixed with the antigen and a positive test is indicated by the agglutination of the antigen.

• Quantitative agglutination test • To measure the level of antibodies to antigens, agglutination test can be

widely used. For this test, serial dilutions of the sample can be made and it is tested for antibody. Then a fixed amount of antigen or bacteria or red blood cells can be added to it. Determine the maximum dilution which forms agglutination and this maximum dilution which gives observable agglutination is known as the titer. The results is shown as the reciprocal of the maximum dilution that forms visible agglutination.

Advantages of agglutination test 1. a low individual test cost2. ability to obtain semiquantitative results3. short time to obtain result4. don't need expensive instrument

Rheumatoid Factor • RA is a chronic inflammatory disorder, affecting primary the joints of

particular tissue.

• The most common area affecting:

1. Tendon

2. Subcutaneous tissues

3. Heart blood vessels

4. Lungs

• RA is an autoimmune disease, a disorder in which the body’s immune

system attempts to destroy its own tissues.

• RA begins with inflammation of the synovial membranes, the

membrane thickness of the joints swell as synovial fluid accumulates .

Pathological changes during the disease :-

1. Proliferation of synovial membrane with the formation of granulated

tissue that extends as vascular layer from the margin toward the center

of the effected joints.

2. Hypochromic anemia.

3. Toxemia.

4. Elevated ESR.

5. Characteristic changes in the serum protein.

6. Generalized lymphadenopathy.

Principle of (RF) rheumatoid factor :-• RA and related disease causes a production of globulin known as RF (IgG and IgM

antibodies) .

• IgM is a macroglobulin produced by lymphocyte react with other antibodies IgG or IgM to produce immune complex which activated of tissue destruction.

Interfering factors :-1. activated complements2. high lipid or cryoglobulin give false positive result .3. high IgG level in patient serum give false positive result .

Clinical conditions which give positive RF :-1. rheumatoid arthritis2. SLE3. systemic sclerosis4. mixed connective tissue disease

RF is detected in serum by their ability to agglutinate with latex particle .

Latex agglutination and Rose Waller agglutination

Sensitized RBCs is more sensitive than Latex particle, that the

sensitized RBCs react with the low antibody; so the Latex maybe

negative and the sensitized RBCs may be positive.

Latex Rose-waller Antigen is suspended on polystyrene reagent

Antigen is suspended on sensitized RBC

Less sensitive More sensitive

(Antistreptolysin O Titer) ASOT• Normal value: less than 200 IU/ml • Alternative names: • ASO• ASLO

• Anti- streptolysin O (ASO or ASLO) is the antibody produced against an antigen produced by Lancefield group A streptococci. • The enzyme is called streptolysin O, the O stands for oxygen-

labile; the other antigen being oxygen stable streptolysin-S. Both the enzymes are involved in producing hemolysis, i.e., digestion of blood, in particular, beta-hemolysis.

Significance• ASO testing is a procedure that demonstrates the presence of

antibodies generated by the body against infections by group A Streptococcus (Streptococcus pyogen), • Group A Streptococci produce the enzyme streptolysin O,

which can cause destruction of red blood cell walls. It is also antigenic, that is, the body produces antibodies against it. • The antibody may be detected in the blood for weeks or

months after the primary source of the infection has been eradicated.

Result Interpretation

• Antibodies begin to appear 7 – 10 days after infection.

• Incidence of positive result is during the :-

• 3rd weak → 90 – 95 %

• 2nd month → 70 – 75 %

• 6th month → 35 %

• 12th month → 20 %

• If the titer does not decrease, a recurrent or chronic infection

may exist.

• Why the test is performed?This test can be used to confirm a recent or ongoing infection with Group A streptococcus

• Elevated levels may indicate:• active streptococcal infection• bacterial endocarditis• post-streptococcal glomerulonephritis• rheumatic fever • scarlet fever • Strep throat

• The ASO antibody may be found in the blood weeks or months after the strep infection has gone away.

ASOT test:latex agglutination test for the qualitative screening and semi-quantitative determination of antistreptolysin O antibodies (ASO) in serum.

• Principle of the test:ASO is a stabilized buffered suspension of polystyrene latex particles that have been coated with streptolysin O. when the latex reagent is mixed with a serum containing antibodies to streptolysin O, agglutination occurs.

• Specimen:The test should be performed on serum.

• False positive ( rise in titer may occur in ) :-• Plasma: may cause nonspecific agglutination of the latex

particles, due to fibrinogen.• high lipid in serum.• contamination with bacteria.• gross hemolysis,• reagent may oxidized • The test reaction must be read immediately following the

two minutes rotating . Delayed readings may result in false positive results.

• Qualitative results:• Positive result: An agglutination of the latex particle suspension will occur

within two minutes, showing ASO level of 200 lU/ml or more, to determine the exact titer do serial dilution.

• If No agglutination observed so ASOT is less than200 lU/ml, don't write negative result.

• Quantitative results:• The serum ASO concentration can then be calculated approximately by

multiplying the dilution factor (i.e. 2, 4, etc.) by the detection limit (200 lU/ml).

• e.g. if the agglutination titer appears at 1:4 the approximate serum ASO level is 4 x 200 = 800 lU/ml.

C reactive protein (CRP):-

• C-reactive protein was originally discovered as a substance in the serum of patients with acute inflammation that reacted with the C polysaccharide of pneumococcus.• This protein which is cause the reaction called CRP.

Characteristics of CRP :• C-Reactive Protein (CRP) is alpha – globulin produced in the liver.• CRP appears in sera of individuals in response to inflammatory

conditions (rheumatoid arthritis, lupus), and with some forms of cancer.• CRP can also be elevated when there is inflammation in the arteries of

the heart and is a "marker" for coronary artery disease, called high sensitive CRP (hs-CRP).

• CRP is not specific. • A high result serves as a general indication of acute inflammation. • a CRP test cannot show where the inflammation is located or • what is causing it. • Other tests are needed to find the cause and location of the inflammation.

• CRP combines with T- lymphocyte to inhibit their functions.• CRP inhibits the aggregation of platelets by aggregated human gamma

globulin and thrombin.• Thermo labile destroyed by heating at 700 c for 30 minutes.• Don 't cross human placenta.

Why the test is done?• Identify and keep track of infections and diseases that cause

inflammation.• Check for infection after surgery. CRP levels normally rise

within 2 to 6 hours of surgery and then go down by the third day after surgery. If CRP levels stay elevated 3 days after surgery, an infection may be present.• Check to see how well treatment is working, such as treatment

for cancer or for an infection. CRP levels go up quickly and then become normal quickly if you are responding to treatment measures.

• Both CRP (C reactive protein) and ESR (erythrocyte sedimentation rate) give similar information about non-specific inflammation. • CRP appears and disappears more quickly than changes in ESR.

Therefore, your CRP level may drop to normal following successful treatment, whereas ESR may remain elevated for a longer period.• CRP present at all times when the ESR increase but CRP remains

normal , this condition happen in case of :-• Pregnancy due to increase fibrinogen.• Nephrosis in which globulin and albumin increase.• Anemia.• Hypoalbuminemia.• Drugs.

• Normal value: 0.6 mg/dl or 6 mg/L

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