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Ab ID & Significance

We’ve detected an unexpected antibody, but now we need to figure out exactly what it is – that’s Antibody Identification. And just as importantly, we need to understand if this antibody is actually capable of causing problems – that’s assessing its Clinical Significance. This whole process is detective work crucial for patient safety!

Think of the positive antibody screen as an alarm bell. Antibody identification is figuring out why the alarm went off, and assessing clinical significance tells us how seriously we need to take that alarm

Antibody Identification: Unmasking the Culprit

  • The Goal: To determine the specific antigen(s) targeted by the unexpected antibody(ies) detected in the patient’s plasma/serum. Is it anti-K? Anti-Fya? Anti-E? Anti-Jkb? Or maybe even multiple antibodies?
  • Why It’s Done
    • Crucial for Safe Transfusion: Knowing the antibody specificity allows us to select donor red blood cells that LACK the corresponding antigen. This is the cornerstone of preventing hemolytic transfusion reactions (HTRs)
    • HDFN Prediction & Management: Identifying antibodies known to cause Hemolytic Disease of the Fetus and Newborn (e.g., anti-D, anti-K, anti-c) allows for appropriate monitoring and management during pregnancy
    • Understanding Incompatibilities: Explains unexpected incompatible crossmatches

How It’s Done: The Antibody Panel

  • The Main Tool: Antibody Identification Panel: Similar to screening cells, but a larger set (usually 11-20 vials) of Group O reagent red blood cells. Each vial comes from a different donor with a unique and extensively typed antigen profile, detailed on an accompanying sheet called an antigram
  • The Method: Usually the Indirect Antiglobulin Test (IAT)
    1. Patient plasma/serum is mixed with cells from each vial of the panel
    2. Testing often includes different phases or enhancements (e.g., LISS or PEG incubation at 37°C, followed by the AHG phase). Reactions (agglutination or hemolysis) and their strength (0 to 4+) are recorded for each panel cell at each phase
    3. Autocontrol A vital part of the panel is testing the patient’s plasma/serum against their own red blood cells
      • Negative Autocontrol: Usually points towards an alloantibody (an antibody formed against a foreign antigen through transfusion or pregnancy)
      • Positive Autocontrol: Suggests an autoantibody (reacting with self-antigens) or perhaps antibodies reacting with recently transfused donor cells still circulating in the patient. This complicates the picture!

Interpreting the Panel: Pattern Recognition

  • The “Rule-Out” Method: This is key!
    • Look at the panel cells that did NOT react (result = 0) with the patient’s plasma
    • For each non-reactive cell, look at the antigens it possesses. If a cell is negative (0 reaction) but positive for a certain antigen (especially if homozygous, e.g., Fy(a+b-)), then the patient’s antibody is not likely directed against that antigen. You can “rule out” or cross off that antigen specificity
    • Systematically do this for all non-reactive cells across the panel
  • Matching the Pattern
    • After ruling out as many specificities as possible, look at the antigens that remain
    • Examine the pattern of positive reactions. Does the patient’s plasma react with all (or most) of the panel cells that are positive for one of the remaining antigens?
    • Does the patient’s plasma not react with the cells that are negative for that same antigen?
    • When the pattern of reactivity perfectly matches the presence/absence of a single antigen across the panel, you have likely identified the antibody
  • Confirmation: Need statistical confidence. Often requires seeing the pattern hold true across enough informative cells (e.g., “rule of three”: at least 3 cells positive for the antigen react, and at least 3 cells negative for the antigen do not react). Sometimes selected cells (additional reagent cells with specific antigen combinations) are needed to confirm or separate multiple antibodies
  • Considering Phase & Strength: IgG antibodies typically react at the 37°C/AHG phase. IgM often react stronger at room temp/IS. Reaction strength can give clues (e.g., dosage – where antibodies react stronger with cells from donors homozygous for the antigen, like Jk(a+b-), versus heterozygous, like Jk(a+b+))

Dealing with Complexities

  • Multiple Antibodies: The plasma reacts in a pattern that doesn’t fit a single specificity. Requires careful rule-outs, potentially using techniques like enzyme treatment (destroys some antigens like Fya, Fyb, M, N; enhances others like Rh, Kidd, Lewis) or adsorption/elution
  • Autoantibodies: A positive autocontrol muddies the water. Techniques like adsorption (using patient cells or other cells to pull the autoantibody out of the plasma) may be needed to see if clinically significant alloantibodies are hiding underneath. Elution (removing bound antibody from the patient’s own red cells) can help identify the autoantibody or antibodies coating transfused cells
  • Antibodies to High-Frequency Antigens (HFAs): Plasma reacts with all panel cells (and often the autocontrol is negative). Requires testing against rare cells lacking HFAs
  • Antibodies to Low-Frequency Antigens (LFAs): Antibody screen might be negative, but crossmatch with a specific unit is incompatible. Identification can be tricky

Clinical Significance: Does This Antibody Matter?

Just because we identified an antibody doesn’t automatically mean it will cause harm. We need to assess its potential to cause problems in vivo

  • What Makes an Antibody Clinically Significant?: Its ability to cause:
    • Decreased Red Cell Survival: Leading to Hemolytic Transfusion Reactions (HTRs), which can range from mild delayed reactions to life-threatening acute intravascular hemolysis
    • Hemolytic Disease of the Fetus and Newborn (HDFN): Where maternal IgG antibodies cross the placenta and destroy fetal red blood cells
  • Factors Determining Significance
    • Immunoglobulin Class
      • IgG: Most clinically significant antibodies are IgG (especially subclasses IgG1 and IgG3) because they react optimally at body temperature (37°C), can cross the placenta (causing HDFN), and can effectively activate complement or be cleared by macrophages
      • IgM: Usually react best at colder temperatures. Most are considered clinically insignificant unless they react strongly at 37°C or activate complement efficiently (e.g., ABO antibodies are IgM but highly significant!). Examples often considered insignificant include anti-M (unless reactive at 37°C), anti-N, anti-P1, anti-Lea, anti-Leb
    • Thermal Amplitude: Does the antibody react at 37°C? Antibodies only reactive below 30°C are generally not significant for transfusion unless they have a very high titer or activate complement well (causing hemolysis in vitro)
    • Proven Track Record: Has this antibody specificity been implicated in causing HTRs or HDFN before? Some have notorious reputations!
      • Generally Significant: ABO, Rh (D, C, E, c, e), Kell (K, k), Duffy (Fya, Fyb), Kidd (Jka, Jkb), Ss
      • Generally Insignificant (usually): Lewis (Lea, Leb), P1, M (most), N, Lua
      • Variable: Some antibodies can be hit or miss
  • Action Based on Significance
    • Significant Antibody Identified: MUST provide antigen-negative red blood cell units for transfusion. For pregnant patients, requires careful monitoring for HDFN
    • Insignificant Antibody Identified: Usually, antigen-negative units are not required, though it’s good practice to document the antibody. Crossmatch compatible blood (IS or electronic if criteria met) is typically sufficient. However, lab policies may vary

Key Terms

  • Antibody Identification: The laboratory process of determining the specific red blood cell antigen(s) targeted by an unexpected antibody detected in a patient’s plasma/serum
  • Antibody Panel: A set of 11-20 commercially prepared Group O reagent red blood cells, each with a unique and extensively documented antigen profile (antigram), used for antibody identification
  • Antigram: The chart accompanying an antibody panel or screening cell set, detailing the specific antigen phenotype of each reagent red blood cell vial
  • Rule-Out Method: A systematic process used during antibody panel interpretation where antigen specificities are eliminated from consideration based on the lack of reactivity between patient plasma and panel cells possessing those antigens
  • Matching the Pattern: The process of comparing the pattern of positive and negative reactions observed between patient plasma and panel cells with the antigen profiles on the antigram to pinpoint the antibody specificity
  • Autocontrol (AC): A test performed concurrently with an antibody panel where the patient’s plasma/serum is tested against their own red blood cells. Helps differentiate between alloantibodies and autoantibodies
  • Alloantibody: An antibody produced against a foreign red blood cell antigen encountered through transfusion or pregnancy. Usually associated with a negative autocontrol
  • Autoantibody: An antibody directed against an individual’s own red blood cell antigens. Usually associated with a positive autocontrol and may complicate alloantibody identification
  • Dosage Effect: A phenomenon where an antibody reacts more strongly with red blood cells expressing a double dose (homozygous) of an antigen compared to cells expressing a single dose (heterozygous). Common with Rh, Duffy, Kidd, and MNS system antibodies
  • Selected Cells: Additional reagent red blood cells with specific antigen combinations chosen to help confirm an antibody’s specificity or differentiate between multiple antibodies
  • Clinical Significance: The likelihood that a specific red blood cell antibody will cause in vivo harm, such as a hemolytic transfusion reaction or Hemolytic Disease of the Fetus and Newborn. Determined by factors like immunoglobulin class, thermal reactivity, and historical evidence
  • Antigen-Negative Blood: Donor blood units lacking the specific red blood cell antigen corresponding to a patient’s clinically significant alloantibody. Required for safe transfusion in such cases
  • Enzyme Treatment: Using proteolytic enzymes (e.g., ficin, papain) to treat reagent red blood cells, which enhances reactivity of some antibodies (Rh, Kidd, Lewis) while destroying the antigens targeted by others (MNS, Duffy), aiding in antibody identification
  • Adsorption: A technique used to remove antibodies from plasma/serum by incubating it with red blood cells possessing the corresponding antigen(s). Often used to remove autoantibodies to detect underlying alloantibodies
  • Elution: A process used to recover antibodies that have bound to red blood cells (in vivo or in vitro). The recovered antibody solution (eluate) can then be tested to determine its specificity