11 Antibody Screening & Identification

• Antibody screening identifies the specificities of antibodies causing positive serological crossmatch

  • SPECIMEN: Serum + screening cells
• Antibody screening is a key process in pre-transfusion compatibility testing.

  • It is one of the principal tools for investigating potential hemolytic transfusion reactions, HDN, and immune hemolytic anemia
  • Detects alloantibodies, which are antibodies against someone else’s red blood cells.
• Autoantibodies may complicate detection. If there is presence in their plasma or serum, it complicates detection because it does not mean that it will cause hemolytic reaction.
• Cause of Hemolytic reaction are antibodies of patient that targets the donor or someone else's red blood cells.
• Autoantibodies can cause positive screening
• Tube technique is routinely performed.

  • Recipient serum + known RBCs (screening cells)
  • IS+37°C+AHG
  • Enhancement media may be used
• Enhancement media may be used in tube technique such as LISS, BSA, PEG, in order to facilitate antigen-antibody binding
• In tube technique, there is hemolysis or agglutination in any phase.

  • In order to observe hemolysis or agglutination, we have to use serum as a plasma
• RBC reagents

  • 2-5% in preservative diluent
  • Packaged in sets of 2-3 cells with varied antigen expression.
  • Accompanied by antigen profile sheet
• Antigen profile sheet is used for basis or guide in antigens present in screening cells
• 22% Albumin is an enhancement media that reduces zeta potential
• Zeta potential is the net negative charge present in red blood cells membrane.

  • Works to repel other red blood cells from one another to avoid clumping during circulation
  • Makes your red blood cells comes together so that they will be agglutinated by your antibodies
• Low ionic strength solution is an enhancement media which is a glycine in an albumin solution.

  • It increases uptake of antibodies onto RBC surface
• Polyethylene Glycol is an enhancement media that removes water in test system thereby concentrating any antibodies present

  • Makes the RBC surrounding hydrophobic so that they can easily take up the antibodies
  • Not appropriate for recipients with increased plasma proteins
  • Generally more sensitive than LISS and albumin
• Be very careful in using PEG enhancement media because it can cause non-specific aggregation of cells (centrifugation after 37°C is omitted) and has a high chance of producing a false agglutination
• AHG reagents brings the non-agglutinating antibodies together.
• In order to visualize antigen-antibody binding, we add AHG reagents for us to see the presence of non-agglutinating antibodies in reaction vessel
• Polyspecific AHG

  • Polyvalent or broad-spectrum Coombs serum
  • Components:
  • Anti-IgG
  • Anti-C3/C4 or Anti- C3d/C3b or Anti-IgM
• Monospecific AHG

  • Components
  • Anti-IgG or Anti-C
• Coombs control cells are basically red blood cells with attached anti-D antibodies in their D antigen

  • D(+) RBCs coated with anti-D
  • Used to verify if the negative AHG is valid
• Agglutination or hemolysis in any of the phase

  • NEGATIVE (0) = the patient has no antibodies against the antigens present in the screening cells
  • POSITIVE (+1 to +4) = complete hemolysis (+4)
• IgM antibody allows reaction of cold reacting antibodies, which is detected in IS phase
• Polyspecific AHG detects IgM antibody if the AHG contains anti-complement activator
• IgG antibody is detected in 37°C/AHG phase
• In autocontrol,

  • (+)Ab screen; (-) Autocontrol = Alloantibody
  • (+)Ab screen; (+) Autocontrol = Autoantibody and/or Alloantibody
• There is single antibody if reaction is in same phase and same strength
• There is multiple antibody if reaction is in more than 1 screening cell
• This table shows that the reaction is caused by alloantibodies

  
• This table shows that the reaction is caused by alloantibodies and autoantibodies

  
• Mixed field agglutination indicates presence of potent hemolysins, specifically Anti-Sda, Anti-Lua/Anti-Lub
• Possible causes of reaction:

  • Caused by single Alloantibody
  • Two alloantibodies, antigens present only on cell II
  • Probably IgG antibody

  
• Possible causes of reaction:

  • Multiple antibodies
  • Single antibody showing dosage effect
  • Probably IgG antibody

  
• Possible causes of reaction:

  • Multiple antibodies
  • Single antibody showing dosage effect
  • Probably IgM → Anti-N, Anti-M

  
• Possible causes of reaction:

  • Multiple antibodies
  • Potent cold-reacting antibody that binds complement
  • Probable mixture of IgM and IgG

  
• Possible causes of reaction:

  • Single antibody, antigen present in both cells
  • An antibody against a high frequency antigen
  • Weak, cold-reacting antibody that binds complement

  
• Possible causes of reaction:

  • Warm-reacting allo/autoantibodies
  • Probably IgG antibodies
  • Transfusion reaction

  
• In antibody identification, 11-20 Group O cells with varied antigen expression is used as reagents
• A profile sheet specifying the antigens on each cell and providing a place to record reactions accompanies each panel.

  • (+) Sign indicates the presence of the antigen

  
• Strength does not indicate significance, only the amount of antibody available to participate in the reaction

  • Different reaction strengths = Multiple antibodies or Antibodies showing Dosage effect
• IgM antibody is clinically insignificant in IS phase