AHG P.2

• Antihuman globulin control


  Used to validate through negative results anytime you are using AHG reagent, and get a negative test result (by the tube method), YOU MUST ADD COOMB'S CONTROL CELLS AND SHOULD GET A POSITIVE RESULT (VALID)
• Coombs control cells


  IgG coated with RBCs, will react with AHG reagent to prove that AHG was added, AHG was working properly, and the washing step was adequate enough to remove any unbound globulins
• Sources of error resulting in negative check cell results

  • Inadequate washing of cells
  • Omission of AHG sera from test
  • Omission of check cells from test
  • Contaminated AHG
  • Contaminated saline
• DAT sensitivity


  Can detect 100-500 IgG/RBC and 400 - 1000 C3d/RBC
• IAT sensitivity


  Can detect 100 - 200 IgG or C3d / RBC
• Ratio of serum to cells

  Increased ratio of Ab:Ag = Increased sensitivity
• Ratios


  • 2 drops of serum and 1 drop of a 5% RCS (Ratio: 40 : 1)
  • 4 drops of serum and 1 drop of a 3% RCS (Ratio: 133 : 1)
• Reaction medium (potentiators)


  Enhanced detection of antibodies (therefore higher reactivity)
• Albumin


  A high MW protein that allows antibody-coated cells to come into closer contact with each other so that aggregation occurs, increases dielectric constant, and decreases zeta potential by dispersing some of the cations surrounding each negatively charged red cell
• Zeta potential


  The difference in electrical potential between the surface of the RBC and outer layer of the ionic cloud, reduction of which allows the RBCs to approach each other and aids in lattice formation
• Low ionic strength solution (LISS)


  Enhances antibody uptake and allows incubation time to be decreased from 30-60 to 10-15 minutes, may also reduce the zeta potential
• Polyethylene glycol (PEG)


  A water-soluble linear polymer used as an additive to increase antibody uptake by removing water molecules, more sensitive than LISS, albumin, and saline systems, but not recommended in patients with highly abnormal serum protein
• Temperature


  IgG and Complement activation are optimal at 37°C phase and Indirect Agglutination Test (IAT) Phase, IgM at Immediate Spin phase / Room Temperature phase
• Incubation time


  Depends on the potentiating medium: 10-120 minutes for saline, 10-15 minutes for LISS, 15-60 minutes for albumin, 16-20 minutes for PEG
• Washing of RBCs


  RBCs must be saline-washed a minimum of three times before the addition of AHG reagent to remove unwanted/free globulins, inadequate washing may result in a false-negative reaction
• pH of saline for washing

  Ideally the saline should be fresh or buffered to pH 7.2-7.4, as saline stored in plastic can decrease in pH and increase the rate of antibody elution
• Addition of AHG


  AHG should be added to the cells immediately after washing to minimize the chance of antibody eluting from the cell and subsequently neutralizing the AHG reagent
• Centrifugation for reading


  Higher relative centrifugal forces (RCFs) result in more sensitive reactions, the CBER-recommended method uses 1000 RCFs for 20 seconds
• Low ionic polybrene technique relies on low ionic conditions to rapidly sensitize cells with antibody, but has low sensitivity for the detection of Kidd antibodies
• Enzyme-linked antiglobulin test (ELAT)

  An RBC suspension is added to a microtiter well, washed, then AHG labelled with an enzyme is added, excess antibody is removed, and enzyme substrate is added, the amount of color produced is directly proportional to the amount of antibody present and can be measured spectrophotometrically
• Solid phase technology


  Used for the performance of antiglobulin tests, can be done in test tubes or microplates, allows for semiautomation with microplate readers
• Gel technology


  Invented by Dr. Yves Lapierre in France, used to minimize problems associated with conventional tube method, now used worldwide, standardizes the process of detecting RBC antigen-antibody reactions by using a chamber filled with polyacrylamide gel that traps agglutinated RBCs
• Types of gel tests

  • Neutral gel test (for antibody screening/identification, reverse ABO typing)
  • Specific gel test (for antigen determination)
  • Low ionic antiglobulin test (GLIAT) / AHG gel test (for IAT or DAT)
• Disadvantage of gel technique is expensive equipment (special centrifuge and incubator)
• Summary of routine procedures in immunohematology/blood bank

  • Reagent manufacture regulated by FDA, with requirements for specificity and potency
  • Expiration dates must be followed, with exceptions for rare antisera and red cells
  • Manufacturers provide package inserts with details on proper use, performance characteristics, and limitations