Agglutination - LM 335 - PDF

Section 2-Part 3 Agglutination LM 335 Dr. Hassan Kofahi Agglutination Agglutination is a visible aggregation of particles caused by an antigen- antibody reactions. Agglutination occurs when one of the reactants (Antigen or antibody) is particulate. The antibodies that produce...

Section 2-Part 3 Agglutination LM 335 Dr. Hassan Kofahi Agglutination Agglutination is a visible aggregation of particles caused by an antigen- antibody reactions. Agglutination occurs when one of the reactants (Antigen or antibody) is particulate. The antibodies that produce agglutination reactions are often called agglutinins. The antigen must be exposed on the surface of the particle and each particle must have multiple epitopes. Types of particles used in agglutination reactions include: Erythrocytes (agglutination of RBCs is often called hemagglutination) Bacterial cells Inert carriers such as latex particles. Agglutination is a two-step process: 1. Sensitization 2. Lattice formation Sensitization An initial reaction in which the Antigen binds to the antibody. Each antibody molecule binds to a single epitope. No cross-links yet. No visible aggregates yet. This step is affected by the affinity of the antibody and the nature of the epitopes hidden epitopes are less likely to bind to the antibody than exposed epitopes. Lattice formation At this step, Antibodies bridge the gaps between particles in such a way that one antibody molecule binds to one epitope on each of two different particles. Affected by: Ionic strength pH Temperature Ab:Ag ratio Zeta potential Erythrocytes carry a slight negative charge on their surface. In an ionic solution, red cells surround themselves with cations to form an ionic cloud. Because of these charges, cells repel one another which keeps them about 25 nm apart. IgG antibodies often cannot bridge the distance between the cells, because of their small size and restricted flexibility at the hinge region. Visible reactions with IgG often require the use of enhancement techniques. IgM molecules has a diameter of 35 nm, which makes them better agglutinins. Enhancement of lattice formation 1. Reducing the surface charges of the RBCs: Reducing the surface charges allow the RBCs to get closer to each other and enhance the agglutination. This can be achieved by: Reducing the ionic strength of the buffer by using low ionic strength saline (LISS). Addition of albumin (5%-30%) to the reaction. Albumin neutralizes the surface charges on the RBCs. Using enzymes e.g. Bromelin, papain, trypsin, and ficin Reduce the surface charges of the RBCs by cleaving chemical groups and decreasing hydration. e.g. Ficin cleaves sialoglycoproteins from the RBC surface. Enhancement of lattice formation (continued) 2. Centrifugation: Increases cell-cell contact (physically). 4. Temperature: IgM reacts best between 4°C and 27°C (cold-reacting). IgG reacts best between 30°C and 37°C (warm-reacting). 6. pH: most of Ag-Ab reactions occur when the pH is 6.5-7.5. 7. The use of antihuman globulin reagent (will be discussed later). Agglutination reactions: advantages Easy to perform Require no complicated equipment Relatively inexpensive. Quick Can be performed as needed (no need to batch specimens). Generally, qualitative. Can be semi-quantitative. Types of agglutination reactions 1. Direct agglutination 2. Passive (indirect) agglutination 3. Reverse passive agglutination 4. Agglutination inhibition Direct agglutination Occurs when the antigens are found naturally on a particle. e.g. ABO blood grouping: the antigens are found naturally on RBCs. e.g. Widal test: the antigens are found naturally on a bacteria. Can be used to test for antigens or antibodies e.g. ABO blood grouping is done by using known antisera to test the types of antigens present on the RBCs. e.g. Widal test: known bacterial suspensions (different Salmonella serotypes) are used to test the presence of antibodies in patients’ sera. Direct agglutination Antigens are present naturally Antibodies Visible agglutination on a particle Passive (indirect) agglutination Used when the antigen is soluble. The antigen is attached to an insoluble particle. i.e. the particles become coated with an antigen not normally found on their surface. Types of particles used for this purpose RBCs Latex particles Gelatin The particles are coated with a known antigen to detect antibodies. e.g. Streptolysin O-coated latex particles are used to detect the presence of antibodies in the serum against group A streptococci. e.g. Viral antigen-coated RBCs are used to detect antibodies against Hepatitis B, Hepatitis C and many other viruses. Passive (indirect) agglutination Reverse Passive Agglutination In this technique, the particles are coated with the antibody instead of the antigen. The antibody is oriented in a way that retain its activity, with the antigen-binding sites facing outward and ready to bind their antigens. This test is used to detect antigens. e.g. Grouping of streptococci by using suspensions of latex particles coated with group-specific antibodies. e.g. C-reactive protein (CRP) is detected and semi-quantified with latex particles coated with anti-CRP antibodies. e.g. The rapid slide test for detecting rotavirus in fecal sample using latex particles coated with specific antibody that reacts with the rotavirus antigen. Reverse Passive Agglutination Agglutination inhibition In this technique, soluble antigens (in the patient sample) compete with particulate antigens (indicator particles) on binding the limited antibody- combining sites. The test is performed in two steps: First, the patient sample is incubated with a limited amount of reagent antibody that is specific against the antigen to be tested. Then, indicator particles coated with the same antigen are added to the reaction. Results: Positive result: if the patient sample contains the antigen of interest, the free antigens in the sample will bind to the antibodies, leaving no free antibody to react with the indicator particle no agglutination. Negative result: If the patient sample has no free antigens, the reagent antibody is able to react with the indicator particles visible agglutination. Agglutination inhibition Reagent antibodies This technique is used for detecting drugs, such as heroin and cocaine. Hemagglutination inhibition uses the same principle except that the particles used are RBCs. Agglutination: interpretation of the results Large number of agglutination kits are commercially available for the detection of wide variety of antigens or antibodies. Semiquantitative results can be obtained by performing a serial 2-fold dilution of the sample. The titer can be determined as the reciprocal of the highest dilution giving a positive result. The agglutination reactions are performed in test tubes, microtiter plates or on glass/cardboard slides. Tubes and microtiter plates are usually used for Hemagglutination reactions. In the tube-method, the tubes are centrifuged and then shaken to see if the cell pellet can be evenly resuspended. Tube method: interpretation of the results Microtiter plate method: interpretation of the results. Interpretation of the test is done on the basis of the cell sedimentation pattern. A dark red, smooth button at the bottom of the microtiter well indicates a negative result. Positive In a positive reaction the cells spread across the well’s bottom. Negative Slide method: interpretation of results Latex agglutination results Hemagglutination results Antihuman globulin-mediated agglutination Is a technique for detecting the reactions of non-agglutinating antibodies (IgG) by adding a second antibody. Also known as coombs' test. Used when the RBCs are sensitized with IgG, but no visible agglutination occur. The second antibody is an antihuman globulin antibody Made in animals. Recognize and bind to the Fc region of human IgG bound to the sensitized RBCs. The antihuman globulin antibody is able to bridge the gaps between the sensitized RBCs and allow the lattice formation and agglutination. Direct antihuman globulin (coombs’) test Used to detect in vivo sensitization of RBCs. Used for diagnosing some autoimmune diseases. e.g. Autoimmune hemolytic anemia, hemolytic disease of the newborn. Called direct because the patient’s RBCs are tested directly as they come from the body Indirect antihuman globulin (coombs’) test Used to determine the presence of a particular antibody in a patient sample. Sensitization of RBCs occurs in vitro. You will learn more about Coombs’ test in the blood banking course. Agglutination: quality control and quality assurance General guidelines: Always run positive and negative controls parallel to patient sample. If the results of the controls are not as expected, the test is invalid. Follow the manufacturers' instructions carefully. Store the reagents properly. Do not use expired reagents.