Antibody Identification

• When an allo-antibody is detected, its specificity and clinical significance should be assessed.
• If a patient is known to have formed an allo-antibody, each new sample should be fully tested to include or exclude the presence of other, different allo-antibodies. 
• The patient’s plasma should be tested against a panel of red cells, and should always include an IAT.
• Cells in the antibody screen can be used to contribute towards assigning antibody specificity and exclusion of other antibodies.
• A check should be done to ensure that ABID results do not conflict with screening results (this may reflect the wrong sample being used, or an antibody directed against a low-frequency antigen absent on either panel).
• Inclusion of the patient’s own red cells as an auto-control may be helpful, but does not exclude the presence of an allo-antibody.
• Antibody specificity requires that the patient’s plasma react against at least 2 (ideally 3) red cells homozygous for the antigen and does not react against at least 2 (ideally 3) samples negative for the antigen. 
• Other clinically-significant antibodies must be excluded after the antibody has been identified.
• Confirmation technically requires that the laboratory demonstrate that the patient is negative for the antigen against which the allo-antibody is directed.
  • The patient’s red cells should be phenotyped using a reagent of the same specificity of the antibody assigned.
  • This must be done with suitable positive and negative controls.
  • If the test against patient’s cells is positive, consider:
    • The antibody may be an auto-antibody.
    • The patient may have been transfused recently with antigen-positive cells.
    • The patient’s cells may be coated with immunoglobulin or complement.
    • The antibody may have been wrongly identified.
    • The wrong sample may have been tested.
  • If the patient has been recently transfused, then phenotyping may be inaccurate due to antigen positivity of the transfused cells; in this case, genotyping can be considered.
• Panel cells should comprise reagent red cells from at least eight group O donors.
  • For each of the clinically-significant antibodies, at least two cells must be positive and two cells negative for those antigens.
  • The panel should support resolution of as many common antibody mixtures as possible.
• Panel cells must comprise at least:
  • One R₁R₁ cell (CDe/CDe)
  • One R₁^wR₁ cell (CDe/C^wDe)
    • These should express the antigens K, k, Fy^a, Fy^b, Jk^a, Jk^b, S and s between them.
  • One R₂R₂ cell (CdE/CdE)
  • One r’r cell (Cde/cde)
  • One r”r  cell (cdE/cde)
  • Thre rr cells (cde/cde)
  • At least one cell must be K+
  • Collectively, at least one cell must be homozygous for  k, Fy^a, Fy^b, Jk^a, Jk^b, S and s.
• Limitations:
  • Panels may not be able to definitively identify combinations of common allo-antibodies.
  • Use of two panel cells increases the probability of being able to identify multiple allo-antibodies.
• At least one panel of enzyme-treated cells should be available, as it increases the chance of identifying an antibody mixture where at least one of the antibodies is directed against antigens affected by enzymes.
• If donor red cells give a positive IAT crossmatch reaction, but the antibody screen/identification is negative:
  • Recipient plasma contains an allo-antibody against a low-frequency antigen which the donor is positive for.
  • Red cells in the donation are coated with IgG.
  • The wrong blood has been selected for cross-matching.
• If all panel cells are positive but auto-control and DAT persistently negative:
  • If blood group O: consider Bombay blood group.
  • If non-O: consider presence of anti-HI
    • anti-HI can be excluded by testing patient plasma against 

Common Red Cell Antibodies and Their Clinical Significance

Other Resources:

• BCSH Guideline: Pre-Transfusion Compatability Procedures (2012)