Anti-streptolysin-O and C-reactive protein Principle, Techniques, and Interpretation. Unit V. DMLT.

Principle, techniques, and Interpretation of Anti streptolysin O

The Anti-Streptolysin O (ASO) test is a serological test. It is used to detect the presence of antibodies against streptolysin O. The streptolysin-O is a toxin produced by Group A Streptococcus bacteria. This test is commonly used to diagnose streptococcal infections, especially those causing diseases like streptococcal pharyngitis (strep throat) and rheumatic fever.

Principle: The principle of the ASO test is based on the immune response of the body to the streptococcal infection. Streptolysin-O is a hemolysin produced by Group A Streptococcus. The human body produces antibodies (anti-streptolysin O antibodies or ASO antibodies) in response to the infection. The ASO test measures the level of these antibodies in the patient's serum.

Techniques

1. Sample Collection: Blood is collected from the patient during the acute phase of the infection and sometimes during the recovery phase for comparison.

2. Serum Separation: The collected blood is allowed to clot, and serum is separated by centrifugation.

3. ASO Test Procedure: The patient's serum is mixed with standardized Streptolysin O antigen. The mixture is incubated. The formation of antigen-antibody complexes occurs during incubation.

4. Detection: After incubation, the reaction is observed for the presence of visible precipitates or turbidity. It indicates the formation of immune complexes.

5. Titration: Similar to the Widal test, the ASO test is often performed in a series of dilutions (titration) to determine the endpoint titer.

Significance of Titers: A rise in ASO titers between acute and convalescent (recovery) samples may indicate a recent streptococcal infection. A high titer suggests a more chronic or recurrent streptococcal infection.

Clinical Correlation: Interpretation of ASO titers should be correlated with clinical symptoms and other diagnostic findings. Elevated ASO titers alone do not confirm a current infection but indicate recent exposure.

It's important to note that the ASO test is a valuable tool in diagnosing streptococcal infections. However, it has limitations. Other streptococcal antibodies, such as anti-DNase B, may also be measured to provide a complete measurement. Additionally, clinical context and other diagnostic tests are crucial for accurate diagnosis and treatment decisions.

Principle, techniques, and interpretation of C-reactive protein

The CRP test is widely used in clinical practice as a valuable tool for assessing inflammation, monitoring disease progress, and guiding treatment decisions. However, it is important to interpret CRP results in the context of the patient's overall clinical condition and other diagnostic information.

Principle:

C-reactive protein (CRP) is an acute-phase reactant produced by the liver in response to inflammation. The CRP test measures the concentration of CRP in the blood, showing the presence and intensity of inflammation in the body. The principle involves the use of an antibody that specifically binds to CRP. This allows the quantification of CRP levels in the patient's serum.

Techniques:

1. Sample Collection: Blood is collected from the patient, usually through venipuncture.

2. Serum Separation: The collected blood is allowed to clot, and serum is separated by centrifugation.

3. CRP Test Procedure: The patient's serum is mixed with a CRP-specific antibody or a substance that binds to CRP

4. Interaction: The interaction between CRP in the serum and the specific antibody or binding substance forms a measurable complex. The level of this complex is then quantified using various techniques, such as immunoturbidimetry or enzyme-linked immunosorbent assay (ELISA)

Interpretation

1. Normal Range: Normal CRP levels in healthy individuals are usually low, typically less than 10 mg/L.

2. Elevated CRP Levels: Elevated CRP levels indicate the presence of inflammation in the body. The degree of elevation may correlate with the severity of the inflammatory process.

3. Clinical Significance: CRP is a nonspecific marker of inflammation. Its elevated levels occur in various conditions, including infections, tissue injury, autoimmune diseases, and cardiovascular diseases.

4. Monitoring Response to Treatment: CRP levels can be monitored over time to assess the effectiveness of treatment in managing inflammatory conditions. A decrease in CRP levels indicates a positive response to therapy.

5. High-Sensitivity CRP (hs-CRP): High-sensitivity CRP assays can detect lower concentrations of CRP and are used in cardiovascular risk assessment. Elevated hs-CRP levels are associated with an increased risk of cardiovascular events.

6. Limitations: CRP is a sensitive marker of inflammation, but it lacks specificity for identifying the cause of inflammation. Elevated CRP can be seen in both infectious and non-infectious inflammatory conditions.

Principle, techniques, and interpretation of VDRL/RPR

The VDRL (Venereal Disease Research Laboratory) and RPR (Rapid Plasma Reagin) tests are blood tests used to screen for syphilis, a sexually transmitted infection caused by the bacterium Treponema pallidum. These tests are non-treponemal, meaning they detect antibodies that are produced in response to syphilis but are not specific to the bacterium itself.

Principle: The VDRL and RPR tests are based on the principle of detecting antibodies, mainly IgM and IgG. IgM and IgG are produced in response to infection with Treponema pallidum. The test uses cardiolipin as an antigen. Cardiolipin is derived from beef heart. The reaction involves the interaction of the patient's serum with cardiolipin. A visible reaction occurs if antibodies against syphilis are present.

Techniques/Procedure

1. Specimen Collection: Collect venous blood using aseptic techniques. Transfer the blood into a sterile tube without anticoagulant. Allow the blood to clot and then centrifuge to separate the serum from the clot.

2. Inactivation of serum: Put 0.5 ml of serum in a test tube. Keep the test tube in a water bath at 56 degrees C for 30 minutes. Cool it at room temperature.

3. Preparation of Test Slide: Put 0.05 ml of inactivated serum in the well of a glass slide. Dispense the one drop of VDRL antigen suspension onto the well of the slide.

4. Performing the Test: Mix the serum and antigen suspension in the well by rotating the slide gently for 4 minutes. (Rotate 10 times in 5 seconds by hand.)

5. Incubation: Incubate the test slide at room temperature for a specific period. Observe for any visible reactions.

6. Interpretation: examine the test slide for agglutination. The presence of a fine granular precipitate or flocculation indicates agglutination. Record the results as either positive or negative based on the presence or absence of agglutination.

7. Disposal: Dispose of all biohazardous materials and contaminated supplies according to institutional or regulatory guidelines.

It's important to note that the VDRL and RPR tests are useful for screening, a positive result requires confirmation with treponemal tests due to the possibility of false positives. Additionally, these tests do not provide information about the stage of syphilis infection

Principle, techniques, and interpretation of Rheumatoid factor (RF)

Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease. Rheumatoid arthritis patient serum has Rheumatoid factor (RF). Rheumatoid factor (RF) is an antibody. Rheumatoid factor (RF) may also be present in the serum of patients suffering from other diseases.

Principle: Rheumatoid factor (RF) is an antibody that targets the Fc portion of immunoglobulin G (IgG) antibodies. Latex particles coated with IgG react with the patient’s serum containing RF. The RF acts as an antibody to IgG on the latex particles. This causes agglutination. The RF test is used as a diagnostic tool for rheumatoid arthritis (RA) and some other autoimmune disorders. The principle of the test involves detecting the presence and quantity of RF in a patient's serum.

Techniques

1. Sample Collection: Blood is collected from the patient through venipuncture.

2. Serum Separation: The collected blood is allowed to clot, and serum is separated by centrifugation.

3. RF Test Procedure:

Reagents: Latex reagents (Polystyrene latex particles coated with human IgG), Glycerine saline buffer pH 8.2.

Prepare 1:20 dilution of the patient’s serum by mixing 950 microliters of glycerine saline buffer and 50 microliters of serum. Place one drop of diluted serum on a properly labeled clean slide. Mix latex reagents thoroughly. and add one drop of serum. Mix using a wooden applicator stick. Observe agglutination (Immune complex) for 2 minutes.

The patient's serum is mixed with particles coated with IgG. If RF antibodies are present in the serum, they will bind to the Fc portion of IgG-coated particles. This forms immune complexes.

Dr Pramila Singh