1. INTRODUCTION

2. Immune System The immune system is a system of biological structures and processes within an organism that protects against disease. processesorganismdisease 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.virusesparasitic wormstissue

3. 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

4. Factors affecting the Ag/Ab reaction: 1- Buffer 2-Concentration of Ag 3-Concentration of Ab 4-Temperature 5-Electrolytes

5. 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.

6. 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.

7. Advantages of agglutination test 1. a low individual test cost 2. ability to obtain semiquantitative results 3. short time to obtain result 4. don't need expensive instrument

8. 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.

10. 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.

11.  Interfering factors :- 1. activated complements 2. 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 arthritis 2. SLE 3. systemic sclerosis 4. mixed connective tissue disease  RF is detected in serum by their ability to agglutinate with latex particle.

12. 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.

13. (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.

14. 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.

15. Result Interpretation Antibodies begin to appear 7 – 10 days after infection. Incidence of positive result is during the :- 3 rd weak → 90 – 95 % 2 nd month → 70 – 75 % 6 th month → 35 % 12 th month → 20 % If the titer does not decrease, a recurrent or chronic infection may exist.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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 70 0 c for 30 minutes. Don 't cross human placenta.

22. 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.

23. 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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