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Blood Bank

Blood Grouping

Routine blood grouping involves determining the patient’s ABO group (using forward and reverse typing) and Rh(D) type (including Weak D testing when appropriate). Accuracy is paramount to prevent potentially life-threatening transfusion reactions and alloimmunization. Always ensure your forward and reverse ABO results match!

So, what are we talking about when we say “blood grouping”? Primarily, we’re determining the patient’s ABO group and their Rh(D) type

Let’s break it down:

The ABO Blood Group System

This system is arguably the MOST important in transfusion medicine because we have naturally occurring antibodies in our plasma against the ABO antigens we lack on our red blood cells. This is known as Landsteiner’s Rule. Messing this up can cause a severe, potentially fatal, acute hemolytic transfusion reaction

  • Antigens: These are specific carbohydrate structures found on the surface of red blood cells. The main ones are A and B antigens. The H antigen is a precursor structure
  • Antibodies: These are proteins (immunoglobulins, mostly IgM for ABO) found in the plasma/serum. They are “naturally occurring” or “expected” because they develop early in life, likely from exposure to environmental substances (like bacteria) that have similar structures to A and B antigens
    • If you have A antigen, you have anti-B antibodies
    • If you have B antigen, you have anti-A antibodies
    • If you have both A and B antigens (Group AB), you have neither anti-A nor anti-B
    • If you have neither A nor B antigens (Group O), you have both anti-A and anti-B

How do we test for ABO?

We perform TWO tests concurrently, and they MUST match:

  • Forward Grouping (or Cell Typing)
    • What it tests: Patient’s Red Blood Cells (RBCs) for the presence of A and B antigens
    • How: We mix the patient’s RBCs with known commercial reagent anti-sera (anti-A and anti-B)
    • Principle: If the antigen is present on the RBCs, the corresponding antibody in the reagent will bind to it, causing agglutination (clumping) or sometimes hemolysis
    • Interpretation
      • Agglutination with Anti-A only = Group A
      • Agglutination with Anti-B only = Group B
      • Agglutination with BOTH Anti-A and Anti-B = Group AB
      • NO agglutination with EITHER Anti-A or Anti-B = Group O
  • Reverse Grouping (or Serum/Plasma Typing)
    • What it tests: Patient’s Plasma or Serum for the presence of anti-A and anti-B antibodies
    • How: We mix the patient’s plasma/serum with known commercial reagent RBCs (A1 cells and B cells)
    • Principle: If the antibody is present in the plasma/serum, it will bind to the corresponding antigen on the reagent RBCs, causing agglutination
    • Interpretation (Based on Landsteiner’s Rule - should confirm the Forward Group)
      • Agglutination with B cells only (means anti-B is present) = Confirms Group A
      • Agglutination with A1 cells only (means anti-A is present) = Confirms Group B
      • NO agglutination with EITHER A1 or B cells (neither antibody is present) = Confirms Group AB
      • Agglutination with BOTH A1 and B cells (both antibodies are present) = Confirms Group O
  • The Crucial Check: The results of the forward and reverse grouping MUST be concordant (match). If they don’t match, we have an ABO discrepancy, which needs to be investigated and resolved before a final blood type can be reported and blood components issued

The Rh Blood Group System (Focus on Rh(D))

The Rh system is much more complex than ABO (lots of antigens!), but the most clinically significant antigen by far is the D antigen. It’s highly immunogenic, meaning exposure to it can easily stimulate antibody production (anti-D) in individuals who lack it

  • Antigen: The D antigen is a protein found on the RBC surface
  • Antibodies: Unlike ABO, anti-D antibodies are generally NOT naturally occurring. They are typically “immune” antibodies, formed only after exposure to the D antigen through pregnancy (an Rh(D)-negative mother carrying an Rh(D)-positive fetus) or transfusion (an Rh(D)-negative recipient receiving Rh(D)-positive blood). Anti-D is usually IgG

How do we test for Rh(D)?

  • Rh(D) Typing (Cell Typing)
    • What it tests: Patient’s RBCs for the presence of the D antigen
    • How: We mix the patient’s RBCs with known commercial reagent anti-D
    • Control: A control test is run concurrently. This ensures the positive reaction with anti-D isn’t due to spontaneous agglutination or other factors. The type of control depends on the anti-D reagent used (e.g., Rh control reagent, or using the reagent diluent)
    • Principle: If the D antigen is present, anti-D will bind, causing agglutination
    • Interpretation
      • Agglutination with Anti-D (and a negative control) = Rh(D) Positive
      • NO agglutination with Anti-D (and a negative control) = Rh(D) Negative (at least initially - see Weak D)
  • Weak D Testing
    • What it is: Some individuals have a weaker expression of the D antigen (fewer D antigen sites or altered forms of the antigen). These used to be called Du
    • Why test for it: It’s important because these individuals can sometimes make anti-D if exposed to “normal” D-positive cells, BUT more importantly, their RBCs can immunize an Rh(D)-negative recipient
    • Who gets tested: Primarily blood donors who initially type as Rh(D) negative. This ensures their blood is labeled correctly (as Rh(D) Positive if Weak D is detected) to prevent immunizing Rh(D)-negative recipients. Testing policies for recipients vary, but often Weak D testing isn’t routinely performed on them (they are usually transfused as Rh(D) negative if they type negative initially). Weak D testing is also often done on newborns
    • How: If the initial Rh(D) test is negative, the test is incubated at 37°C and then converted to an Indirect Antiglobulin Test (IAT) by adding Anti-Human Globulin (AHG)
    • Interpretation
      • Agglutination after adding AHG = Weak D Positive (Reported as Rh(D) Positive)
      • NO agglutination after adding AHG = Rh(D) Negative

Methodologies While traditional tube testing is still common, many labs now use gel/column agglutination technology or solid-phase methods, which can be semi-automated or fully automated. Molecular genotyping can also determine ABO and Rh types, especially in complex cases, but serology remains the primary routine method

Key Terms

  • Antigen: A molecule (usually a protein or carbohydrate) located on the surface of red blood cells that can elicit an immune response. In ABO grouping, these are the A and B antigens; for Rh, the most important is the D antigen
  • Antibody: A protein (immunoglobulin) found in plasma or serum that is produced by the immune system in response to an antigen. In ABO grouping, these are Anti-A and Anti-B; Anti-D is the key antibody in the Rh system
  • Agglutination: The visible clumping of red blood cells that occurs when antibodies bind to corresponding antigens on adjacent cells, forming a lattice structure. This is the endpoint reaction we look for in many tube and slide tests
  • Forward Grouping (Cell Typing): The part of ABO testing where patient red blood cells are tested against known commercial Anti-A and Anti-B reagents to determine which ABO antigens are present
  • Reverse Grouping (Serum/Plasma Typing): The part of ABO testing where patient plasma or serum is tested against known commercial A1 and B reagent red blood cells to detect the expected ABO antibodies (Anti-A, Anti-B)
  • Landsteiner’s Rule: The fundamental principle of ABO grouping stating that healthy individuals possess ABO antibodies in their plasma directed against the ABO antigens lacking on their own red blood cells. (e.g., Group A person has Anti-B)
  • ABO Discrepancy: An inconsistency or disagreement between the results obtained from the forward grouping and the reverse grouping, requiring resolution before the blood type can be finalized
  • Rh(D) Positive: Indicates the presence of the D antigen on an individual’s red blood cells, as detected by testing with Anti-D reagent
  • Rh(D) Negative: Indicates the absence of the D antigen on an individual’s red blood cells, as detected by testing with Anti-D reagent (may require Weak D testing for confirmation, especially in donors)
  • Weak D (formerly Du): A phenotype where the D antigen is expressed weakly or in fewer numbers on the red blood cell surface, often requiring an indirect antiglobulin test (IAT) phase for detection. Individuals with a Weak D phenotype are typically considered Rh(D) positive, especially for donation purposes