LEC 1 SIR PDF

• Inaccuracy of testing can be due to systematic errors or sporadic/isolated errors
• Errors can be false positive, false negative, or false positive/negative
• Cleaning of Dirty Glasswares
• Includes cleaning dirty tubes and microscope slides
• Preparation of diluted household bleach (Ex. 10% sodium hypochlorite solution)
• Collection & Preparation of RBC Suspension
• Principle: The concentration of erythrocytes in saline suspension is important for testing accuracy
• Small amounts for immediate use
• Cell suspension from anticoagulated specimens like EDTA: 2% RBC suspension, 5% RBC suspension
• Steps:

  • Preparation of washed red cells by washing them
  • Dilution to the right concentration for accurate results
• Issues include bacterial growth, loss of avidity and specificity, complement & anti-complement problems
• Storage methods:

  • Freeze drying
  • Frozen solid at -20°C
  • Storage at 4°C
  • Addition of bactericidal/bacteriostatic agents
• Agglutination is the antibody-mediated clumping of particles expressing antigen on their surface
• Agglutination of RBCs occurs when antibody molecules bind to antigenic determinants on multiple red cells, forming visible aggregates
• Agglutination is the endpoint for most tests involving red cells and blood group antibodies
• Stage 1: Sensitization phase - physical attachment of antibodies to antigens on RBC membrane

  • Reversible interaction when antibodies combine rapidly with antigens
• Stage 2: Lattice formation - IgM-mediated, establishment of crosslinks between sensitized particles resulting in aggregation
• Crosslinking influenced by zeta potential
• Factors Influencing the Degree of Ag-Ab Interaction: Agglutination
• Includes:

  • Physical factors, ionic strength, reaction temperature, incubation time, steric hindrance, pH
  • Zoning phenomenon - Prozone, zone of equivalence, postzone
• Physical factors like ionic strength can be influenced by Na+ and Cl- shielding effects

  • Enhancement agents like Low Ionic strength saline (LISS) and Polybrene can be used
• Set A (195) antibodies are cold reacting - IgM, Set B (75) antibodies are warm reacting - IgG
• Reaction temperature, incubation time, and binding constants affect the degree of Ag-Ab interaction
• Optimum incubation time for the detection of reactions is one hour
• Incubation time in laboratory testing ranges from 15 to 60 minutes
• Factors influencing the degree of antigen-antibody interaction in agglutination include:

  • How tightly an antibody attaches to its specific antigen
  • Physiochemical effects that occur when dissimilar antibodies compete for space in reaching their specific receptor sites
• Steric hindrance (mutual blocking) is a physical factor that influences the degree of antigen-antibody interaction in agglutination
• pH levels for reactions:

  • Usually at pH 7.0 for all reactions
  • Anti-D reacts more strongly at pH 6.5 – 7.0
  • Anti-M reacts strongly at pH 6.0 – 6.5
• Methods of enhancing agglutination include:

  • Centrifugation to force red blood cells closer together
  • Treatment with proteolytic enzymes like papain, ficin, and bromelin to eliminate antigen-binding sites for certain antibodies
  • Use of colloids such as albumin, PEG, polybrene, polyvinylpyrrolidone, and protamine to increase dielectric constant and reduce zeta potential of red blood cells
  • Anti-human globulin (AHG) reagent to cross-link sensitized red cells and facilitate agglutination
• • Use of LISS (uses 0.2% NaCl) to bring red cells closer together for strong crosslinking by cell-bound antibodies
  • Polyethylene glycol (PEG) for pseudoagglutination and rouleaux formation
• Factors enhancing rouleaux formation:

  • High protein reagent (Anti-D)
  • Abnormal increase of plasma proteins
  • Inverted albumin-globulin ratio
  • Transfusion of high molecular weight IV solution
  • Cord blood samples from newborns
  • Infection with increased erythrocyte sedimentation rate (ESR)
• Techniques in noting agglutination and/or hemolysis:

  • Observation of free hemoglobin in the supernatant fluid after centrifugation
  • Gently dispersing red blood cells to observe agglutination
  • Microscopic reading using a tube holder adaptation to the microscope
  • Holding the tube at an angle to observe cell behavior
  • Microscopic examination useful in distinguishing rouleaux from true agglutination and detecting specific patterns of agglutination
• General considerations:

  • Labeling of tubes/slides and volume of serum used
  • Cell suspension should be washed and resuspended to a 2-4% concentration of saline
  • Grading of test results or agglutination reactions should be standardized among Blood Bank staff
  • Elution procedure is used to break the antigen-antibody complex with subsequent release of the antibody into the surrounding medium