5. chapter 5 Serologic test for Autoimmune disease.pdf

Serology of CRP and RF 6/29/2024 Menber (BSc, MSc) 1  At the end of this lesson student will able to: Describe serologic diagnosis of CRP, RF Determine the factors that influences serologic tests of CRP, RF 6/29/2024 Menber (BSc, M...

Serology of CRP and RF 6/29/2024 Menber (BSc, MSc) 1  At the end of this lesson student will able to: Describe serologic diagnosis of CRP, RF Determine the factors that influences serologic tests of CRP, RF 6/29/2024 Menber (BSc, MSc) 2  C-reactive protein (CRP) CRP is a protein which is produced by the liver in response to inflammatory stimuli. An early indicator of infections, inflammation or other disease associated with tissue injury. MW 118,000Da, with five homogeneous sub units. Normally trace levels in serum (0.1 mg/dl or less). Acute inflammation indicator; increases within 4-6 hrs of infection, surgery or other trauma (tumors, heart attack) 6/29/2024 Menber (BSc, MSc) 3  100 to 1000 fold increase, reaching a peak value within 48 hours. When stimulus removed levels drop rapidly CRP is synthesized only in the liver, and synthesis is stimulated by IL-6 ,IL-1 and TNF alpha. CRP got its name because it was first identified in the serum of patients with pneumonia because it precipitated with the C-polysaccharide on the pneumococcal cell wall. 6/29/2024 Menber (BSc, MSc) 4  The biological effects of CRP are like those of immunoglobulin, including the ability to precipitate, to function as opsonin through binding to macrophages, and to fix complement.  CRP increases faster than ESR in responding to inflammation, whereas the leukocyte count may remain with in normal limits despite infection.  CRP is the method of choice for screening for inflammatory and malignant organic disease and monitoring therapy in inflammation. 6/29/2024 Menber (BSc, MSc) 5  Elevations of CRP occurs in nearly 70 disease state, including: septicemia and meningitis in neonates, infections in immuno suppressed patients, burns complicated by infection, serious postoperative infections, myocardial infraction, malignant tumors, and rheumatic disease. TB 6/29/2024 Menber (BSc, MSc) 6  The course of the CRP level may be useful for monitoring the effect of treatment and for detection of post operative complications or internal infections. Note: The half life of CRP is 5 to 7 hrs. It falls much more rapidly than other acute phase proteins when patient recovers. 6/29/2024 Menber (BSc, MSc) 7 Laboratory tests for C-reactive protein (CRP)  Rapid latex agglutination test  Complement fixation test  Fluorescent antibody  precipitation 6/29/2024 Menber (BSc, MSc) 8  Rapid latex agglutination test Principle  The agglutination test is based on the reaction between patient serum containing CRP as the antigen and the corresponding antihuman (CRP) antibody coated to the surface participles.  The coated particles enhance the detection of an agglutination reaction when antigen is present in the serum. 6/29/2024 Menber (BSc, MSc) 9 Materials: 1. C -Reactive Protein test kit(s). 2. Patient and control serum specimens. 3. Timer 4. Disposable sampling pipettes 5. Disposable test slides 6/29/2024 Menber (BSc, MSc) 10  Procedure: 1. Be sure reagents and specimens are at room temperature. 2. Add 50 ul or one free-falling drop of serum to the center of one oval on the slide. 3. Re-suspend the latex reagent by gently mixing the vial until the suspension is homogeneous. Place one drop of CRP latex reagent next to each serum specimen. 4. Using separate stirrers, mix each specimen and control until the entire area of each oval is filled. 5. Tilt the slide back and forth, slowly and evenly, for 2 minutes. Place the slide on a flat surface and observe for agglutination using a direct light source. 6/29/2024 Menber (BSc, MSc) 11  Interpretation: Agglutination of latex particles is considered a positive reaction, indicating the presence of C– reactive protein at a significant and detectable level. (> 0.6 mg/dL or higher gives a positive result). Specimens which do not contain human CRP will not cause agglutination. The CRP positive control serum must show distinct agglutination, and the negative control must be nonreactive. If the reagent fails to agglutinate with the positive control, or does agglutinate with the negative control, it should be discarded. 6/29/2024 Menber (BSc, MSc) 12 Limitations Specimens with markedly high CRP may demonstrate post-zone (antigen excess) effect. 6/29/2024 Menber (BSc, MSc) 13 Other test includes  Complement fixation, not for routine clinical laboratory  Fluorescent antibody Used to study binding of CRP to lymphocytes and their subpopulation Used primary as a research tools for localizing CRP in tissue.  precipitation Tube method Gel electrophoresis 6/29/2024 Menber (BSc, MSc) 14  Serology of Rheumatoid factor 6/29/2024 Menber (BSc, MSc) 15  Rheumatoid Factor (RF) Rheumatoid factor : antibodies which specifically recognizes antigenic determinants on the Fc fragment of human or certain animal IgG. Approximately 75 percent of patients with RA have an antibody that has been called the rheumatoid factor (RF). It is most often of the IgM class and is directed against the FC portion of IgG. However, this antibody is not specific for RA, as it is found in 5% of healthy individuals and in 10 to 20% of those over the age of 65. affects all races, F:M ratio is 3:1, peak age is b/n 35 -50 yrs 6/29/2024 Menber (BSc, MSc) 16  As indicated by its name, RF has particular application to diagnosis and monitoring of rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic syndrome in which chronic inflammation of the joints initiated by auto-antibodies. Can also affect multiple organs such as the heart and the lungs Results in decline in functional ability due to joint deformity and disability. 6/29/2024 Menber (BSc, MSc) 17  The formation of immune complexes in the joint spaces leads to activation of complement and destructive inflammation. The most common symptoms include a symmetric arthritis usually involving the small joint of the hands or feet and knees. Morning stiffness around the joints lasting at least 1 hour Swelling of the soft tissue around the joints. Joint involvement progresses to the larger joints in a symmetric fashion, often affecting the knees, hips, elbows and shoulders. 6/29/2024 Menber (BSc, MSc) 18  The earliest lesions in rheumatoid joints show an increase in cells lining the synovium and an infiltration of mononuclear cells, mostly CD4 T lymphocytes. Macrophages and neutrophils are attracted to the area, and this results in the formation of an organized mass of cells called a pannus, which grows into the joint space and invades the cartilage. 6/29/2024 Menber (BSc, MSc) 19  Collagenase and other tissue-degrading enzymes are also released from synoviocytes and chondrocytes that line the joint cavity. The end result is destruction of connective tissue, cartilage and bone. 6/29/2024 Menber (BSc, MSc) 20 Serologic tests Two types of agglutination tests for RF have been developed: using sheep red blood cells coated with IgG using latex particles coated with IgG.(predominantly used) 6/29/2024 Menber (BSc, MSc) 21  a negative result does not rule out the absence of RA. Conversely, a positive test result is not specific for RA, because RF can be found in other diseases such as SLE, chronic active hepatitis, tuberculosis, leprosy, infectious mononucleosis, malaria, and Sjörgren’s syndrome 6/29/2024 Menber (BSc, MSc) 22  RF latex agglutination Procedure 1. Bring reagents and specimens to room temperature before use. Shake the RF- latex reagent gently to obtain uniform suspension. 2. place 40 ul of the undiluted specimens on each field of the reaction slide. 3. Add one drop of RF latex reagent to each test field, Using the stirring sticks, mix and spread reaction mixture over entire test field. 4. Rotate the slide for 2 minutes either by hand or with a rotator (80-100rpm) and read. 5. Interpretation A negative reaction is indicated by a uniform milky suspension with no agglutination. A positive reaction is indicated by any observable agglutination in the reaction mixture. 6/29/2024 Menber (BSc, MSc) 23  Thank you!!! 6/29/2024 Menber (BSc, MSc) 24