Hemagglutination Methods Course Materials

Hemagglutination Methods 1 In immunology, Antigens (Ag); The substances (structural molecule) specifically bound (recognize) by Antibodies (circulating Abs) or a cell surface version of Ab (B cell receptor [BCR]). Additionally, It was expanded today to refer to any molecule or a linear molecular fragment after processing the native antigen that can be recognized by T-cell receptor (TCR). Type of Antigen based on solubility: Particulate (insoluble) or Soluble Particulate Ags Soluble Ags 2 Classification of antigen-antibody interactions [Serology LAB] 1. Primary serological tests: (Labeled Immunoassays) e.g.     2. 3 Enzyme linked immuono sorbent assay (ELISA) Immuno flurescent antibody technique (IFAT) Chemiluminescence immunoassay (CLIA) Radio immuno assay (RIA) Secondary serological tests: e.g.      Agglutination/Hemagglutination tests Complement fixation tests (CFT) Precipitation tests Fluccolation tests Serum neutralization tests (SNT) Agglutination: Particulate Antigens/Antibody Reactions Agglutination reactions involve particulate antigens (such as bacteria, viruses, Latex particles, …) capable of binding antibody molecules. Since antibody molecules are multivalent, suspended particulate antigens form large clumps (network) or aggregates, easily visible without magnification, when exposed to specific antibodies. Antibodies that cause this reaction are referred to as agglutinins. Agglutination assays can be used to determine concentrations of specific antibodies in a patient's immune sera (quantitative or qualitative). 4 Type of Haemagglutination  Active (Direct) Hemagglutination assay  (blood grouping or blood typing)  Indirect haemagglutination assay (Coombs test)  (Direct & Indirect Coomb's Test)  Haemagglutination (or inhibition) assay & Passive haemagglutination assay (Less used)  Determine the infectious diseases specially viral diseases or antibodies to viral antigens 5 Active/direct Hemagglutination test: Blood Group or Blood type One more shot of AB positive blood please 6 Direct Hemagglutination(haemagglutinin) Assay Hemagglutination, (or haemagglutination), • A specific form of agglutination that involves red blood cells (RBCs) instead of Latex particulates. • It has two common uses in the laboratory: blood typing and the quantification of virus dilutions in a haemagglutination assay (less). 7 The process of the RBC's involved in agglutinating is called Hemagglutination . • The ABO blood group system is used to denote the presence of one, both, or neither of the A nor B antigens on the erythrocytes. • The associated anti-A and anti-B antibodies (mostly IgM) depend on blood type are usually produced in the first years of life by sensitization to environmental substances such as food, bacteria, and viruses. 8 Different techniques for Hemagglutination assay • Blood typing can be performed using test tubes, microplate, or blood typing slides. • The procedure of Hemagglutination is simple, inexpensive and the interpretation of the results is also convenient. Slide Tube Microplate Point-of-care testing (POCT) Immunochromatography 9 ABO Typing 10 Cell Type (Forward Typing) (sample is whole Blood) the Ags are presented on RBCs surface detected by commercial Abs(antisera) Back Type (Reverse Typing) (sample is isolated plasma) the Antibodies are presented in plasma detected by determined RBCs The back type method is not used to determine Rh antigens. ABO Typing     Matrix: Tubes or slides Commercial Antiserums Applicator Samples • The tube method involves mixing a suspension of RBCs with commercial antisera (or plasma, for reverse grouping) in a test tube. The mixture is centrifuged to separate the cells from the reagent, and then re-suspended by gently agitating the tube. • If the antigen of interest is present, the RBCs agglutinate, forming a solid clump in the tube. If it is absent, the RBCs go back into suspension when mixed. • The slide method is similar to the tube method, except rather than using individual test tubes, blood typing is carried out in a slides RBCs and commercial antisera (or plasma, for reverse grouping) are mixed together by applicator. After 3 to 5 minutes, clump formation and Hemagglutination are checked. 11 Rhesus (Rh) Blood Group  Rh or D antigens is clinically and medically important.  According to presence or absence of Rh antigen on the RBCs surface, the individuals classify to Rh+ (if present) or Rh- (if absent). Anti-Rh (anti-D) • If agglutination occurs Drop of blood • If No agglutination Rh+ Rh- (or Weak D) The weak D phenotype (Du) is a weakened form of the D antigen that in routine D typing will react with some anti-D but not with others. Weak D red cells have the D antigen, but have fewer D antigens per cell than normal Rh positive cells. For determine weak D (or Rh negative), After negative Rh test, the Coombs test will be performed (when an immediate spin or 37° incubation with coombs reagent is done). 12 Coombs reagent (also known as Coombs antiglobulin or antihuman globulin [AHG]) AHG; the IgG Abs against Fc(constant fragment) of incomplete antibodies IgG and IgA that fail agglutinate RBCs or other particulates suspended in test.  AHG is used in both the direct and indirect Coombs test.  Coombs reagent is made by injecting human globulin into animals (boat or rabbit), which produce polyclonal (Poly-specific) antibodies specific for human immunoglobulins (usually IgG) and human complement components [C3b, C3d, C4b, C4d]).  More specific Coombs reagents or monoclonal antibodies can be used. 13 AHG Weak D (Du) Incubation in 37°C (AHG) 14 Anti-human globulin (AHG) 15 Cell Type (Forward Typing) anti-D+ coombs reagent anti-A anti-B anti-A + anti-B A(-) Sample 1 B(+) Sample 2 AB(-) Sample 3 O(+) Sample 4 16 Cell Type (Forward Typing) anti-D+ coombs reagent anti-A anti-B anti-A + anti-B A(-) Sample 1 B(+) Sample 2 AB(-) Sample 3 O(+) Sample 4 17 Indirect hemagglutination assay (Coombs test) also known as Antiglobulin test (AGT) • Coombs test is either of two blood tests used in immunohematology. They classified into the direct and indirect Coombs tests. • The direct Coombs test detects antibodies that are stuck (adhered) to the surface of the RBCs. Since these antibodies sometimes destroy RBCs, a person can be anemic and this test can help clarify this condition. • The indirect Coombs tests detects antibodies that are floating freely in the blood. These antibodies could act against certain RBCs and the test can be done to diagnose reactions to a blood transfusion. 18 Applications of coombs test (Indirect Hemagglutination test)  These include detection of anti-rhesus factor (Rh) antibodies.  RBCs from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed.  To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed. AHG 19 20 Direct Coombs (Antiglobulin) Test (DAT) Incomplete Abs (antigen/antibody ratio being in antigen excess or antibody excess or electrical charges) on the RBCs preventing the effective cross linking of the cells consequently there is no visible hemagglutination. Detects antibodies on erythrocytes (detection in vivo attachment) ↔ + AHG Patient’s RBCs 21 Coombs Reagent (Antiglobulin) DAT; Direct Antiglobulin Test (Polyspecific) or Direct Coombs Test 22 Indirect Coombs (Antiglobulin) Test  Detects anti-erythrocyte antibodies in serum  This test is done by incubating the RBCs with the serum washing out any unbound antibodies and then adding a second antiimmunoglobulin reagent (AHG) to cross link the cells.  sample Step 1 ↔ + Patient’s Serum Target RBCs Step 2 + 23 ↔ Coombs Reagent (AHG) IAT; Indirect Antiglobulin Test (Polyspecific) or Indirect Coombs Test 24 DAT and IAT Useful For DAT; Demonstrating in vivo coating of RBCs with IgG or other complement components such as C3d ; -Autoimmune hemolytic anemia -Hemolytic transfusion reactions -Drug-induced hemolytic anemia IAT; Demonstrating in vitro antibodies that can coating the RBCs - Cross match - Detection of Alloantibodies - Antenatal antibody screening - Antibody screening 25 Principle of Hemagglutination assay • Influenza virus particles have an envelope protein called the hemagglutinin, or HA, which binds to sialic acid receptors on cells. • The virus will also bind to erythrocytes (red blood cells), causing the formation of a lattice. • This property is called Haemagglutination, & is the basis of a rapid assay to determine levels of influenza virus present in a sample. • To conduct the assay, two-fold serial dilutions of a virus are prepared, mixed with a specific amount of red blood cells, and added to the wells of a plastic tray. 26 Haemagglutination (HA) Test + HA Virus RBC Suspension 27 Settling Pattern HA and HIA Procedure  Label plates (8 or 12 wells/row) Add 50 µL PBS to all wells  Add 50 µL prepared virus to 1st well   Mix and dilute (50 µL) – 1st through last well  Add 50 µL 0.5% washed RBCs to all wells Mix by shaking plates  Read at 20 – 30 minutes (when RBCs buttoned)  28 Interpretation  The red blood cells that are not bound by influenza virus sink to the bottom of a well and form a button.  The red blood cells that are attached to virus particles form a lattice that coats the well.  The assay can be performed within 30 minutes, and is therefore a quick indicator of the relative quantities of virus particles. 29 A Brief History of Blood Transfusion 30 Serological Testing before blood transfusion  ABO/Rh  Cross-match  In the ABO typing, the forward and reverse MUST match  In the Rh typing, the control must be negative  Both of these will indicate what type of blood should be given The crossmatch “shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and shall include an antiglobulin phase” Two types of crossmatches  Major – routinely performed in labs  Minor – not required by AABB since 1976 31 32 Major VS. Minor Crossmatch Major: Patient serum crossmatched with donor red cells. Minor: Donor serum crossmatched with patient red cells. Antibody screen testing on donor samples has replaced the minor crossmatch.  Why is the minor crossmatch unnecessary?  Donated units are tested for antibodies  Most blood is transfused as packed cells, having little antibodies  The plasma volume is small, and Abs will be diluted in recipient circulation 35 Crossmatch Purposes:  Prevent transfusion reactions  Increase in vivo survival of red cells  Double checks for ABO errors  Another method of detecting antibodies No agglutination ~ compatible Agglutination ~ incompatible 34 Donor RBCs (washed) Patient serum  Donor cells are taken from The procedure segments that are attached to the unit itself  Segments are a sampling of the blood and eliminate having to open the actual unit Will Verify donor cell ABO compatibility Detect most antibodies against donor cells Will Not Guarantee normal survival of RBCs Prevent patient from developing an antibody Detect all antibodies Prevent delayed transfusion reactions Detect ABO/Rh errors 37 Procedure of Major Cross-match before blood transfusion 36 37 Hemovigilance is defined as a set of surveillance procedures covering whole transfusion chain from the collection of blood and its components to the follow up of its recipients, intended to collect and access information on unexpected or undesirable effects resulting from the therapeutic use of labile blood products. 38