ABO and H Blood Group System

adsorption

• a procedure that uses red cells (with known antigens) to remove red cell antibodies from a solution (plasma or antisera)

  • ex: group A red cells can remove anti-A antisera from a solution

• method used to remove certain antibodies to analyze the ones left behind

ABO discrepancy

when the forward grouping of red cells does not agree with reverse grouping of plasma to determine ABO phenotype

autocontrol/autologous

• testing an individual’s plasma with their own red cells

• can be used to determine if an autoantibody is present

What is the difference between cold autoantibodies and cold alloantibodies?

• cold autoantibodies

  • red cell antibodies specific to autologous (one’s own) antigens

  • reacts at room temp or below

• cold alloantibodies

  • red cell antibodies (other than ABO) specific to human red cell antigens

  • also reacts at room temp or below

elution

• a procedure that dissociates (removes) antigen-antibody complexes on red cells

• typically performed after a positive DAT

• used to identify complicated antibodies

immunodominant sugar

sugar molecule responsible for antigen specificity (fucose, galactose, and acetyl galactosamine)

incompatible crossmatch

• when agglutination or hemolysis is observed in the crossmatch of patient plasma and donor red cells

• indicates serologic incompatibility

• donor unit is not acceptable for the patient

titers

the extent to which an antibody is diluted before it loses its ability to agglutinate with antigen

clinically significant antibodies

• antibodies capable of causing decreased survival of transfused cells

  • via transfusion reactions or HDFN

• IgM and IgG are clinically significant because they are efficient at activating complement

agglutination reactions for ABO typing are usually

3+ or 4+

anti-A and anti-B antisera react in which phase of testing?

the immediate spin phase

• because the antibody is IgM

When are ABO antigens first detected in fetuses? When are antigens fully expressed?

• first detected during 5-6 weeks gestation

• fully expressed at about 2-4 years old

Describe the biochemical structure of ABO antigens

• oligosaccharide chain

  • 3 sugar molecules attached to a protein or lipid

• this chain is the building block of A, B, and H antigens

What are the two types of ABO antigen precursors?

• type 1 precursor

  • develops into ABO antigens found in body fluids and secretions

• type 2 precursor

  • develops into ABO antigens present on RBCs (and also some in body fluids)

What is the H gene responsible for?

• controlling the production of H antigen

• coding for the enzyme glucosyltransferase

  • this enzyme attaches L-fucose to the type 2 precursor antigen

  • the type 2 precursor with fucose is the foundation of A and B antigens

What is the A gene responsible for?

• coding for the enzyme N-Acetylgalactosaminyltransferase

  • this enzyme attaches N-acetylgalactosamine to the type 2 precursor antigen, to create the A antigen

What is the B gene responsible for?

• coding for the enzyme D-galactosyltransferase

  • this enzyme attaches D-galactose to the type 2 precursor antigen, to create the B antigen

What are the purposes of the ABO and Se genes?

• ABO gene

  • control the production of ABO antigens (gene found on chromosome 19)

• Se gene

  • controls the presence or absence of ABO and H antigens in body secretions (gene also found on chromosome 19) — secretor gene

  • Se and se are alleles (se is amorphic)

Which blood group has the highest amount of H antigen on their red cell surfaces?

group O

ABO phenotypes can be divided into subgroups that differ…

• quantitatively

  • differences in the number of antigen copies expressed on red cells

• qualitatively

  • differences in structure (some subgroups have highly branched structures)

How can A₁ subtype cells be distinguished from A₂ cells?

• distinguished with the lectin Dolichos biflorus

  • agglutinates with A₁ cells but not A₂ cells

Some A₂ subgroup individuals have…

• anti-A₁ antibodies

  • created due to the foreign branch structure of the A₁ antigen

  • can cause ABO discrepancy

  • optimal reactivity at room temp

When are ABO antibodies created, and when do they reach their maximum titer levels?

• created around 3-6 months of age

  • reverse typing is not performed until 4 months of age

• max levels around 5-10 years old

  • decreases as you age (due to age or disease states)

Most anti-A and anti-B antibodies in group A or B individuals are ___, while anti-A and anti-B antibodies in group O individuals is ___.

IgM, IgG

What type of RBCs and plasma are selected for transfusion?

• ABO identical RBCs (or ABO compatible if identical is unavailable)

• ABO identical plasma (or ABO compatible if identical is unavailable)

  • donor antibodies MUST be compatible with recipient RBCs

Which ABO blood group is the universal donor of RBCs?

group O (RBCs have do NOT have A or B antigen)

Which ABO blood group is the universal recipient of RBCs?

group AB (no circulating A or B antibody in plasma to interact)

Which ABO blood group is the universal plasma donor?

group AB (lack of anti-A and anti-B antibody in plasma)

Which ABO blood group is the universal plasma recipient?

group O (no A or B antigen on red cells to interact with antibody)

What are 3 indications of an ABO discrepancy (forward and reverse)?

• weaker than expected reactions (2+ instead of 3+ or 4+)

• expected reactions are missing

  • ex: group O serum does NOT react with A₁ or B cell reagent

• extra reactions occur

What are some general causes of ABO discrepancies?

• specimen mix-up/bad sample

• incorrect interpretation of test results

• reagent or equipment errors

  • ensure QC is satisfactory

  • do not under or over centrifuge tests

• procedure errors

  • failure to follow directions

  • failure to add reagents

group A with acquired B antigen

• group A₁ individuals who begin to react with anti-B reagents in red cell testing

• appears as group AB

• causes an “extra antigen discrepancy” in ABO testing

• due to certain diseases of the GI tract (cancer, obstructions, or gram negative septicemia)

B(A) phenotype

• group B individuals who begin to react with anti-A reagents during forward typing

• causes an “extra antigen discrepancy” in ABO testing

• caused by trace amounts of sugar for the A antigen being added to the H antigen in B individuals

• very rare

How are extra antigen discrepancies in ABO testing resolved?

• repeat testing

  • Same results? determine the patient diagnosis and transfusion history

• test the patient’s serum against their own red cells (autocontrol)

  • anti-B in the patient’s serum will not react with the acquired B antigen

polyagglutination

• a hidden antigen on the RBC membrane that is exposed due to bacterial infection or genetic mutation

• antigen reacts with most human antisera

• causes an “extra antigen discrepancy” in ABO testing

What can cause nonspecific aggregation of RBCs during ABO testing?

• Wharton’s jelly

  • a jelly-like tissue found in cord blood that can interfere with blood typing

• cord cells must be washed with saline before testing

What are some causes of the ABO discrepancy “missing or weak antigens?”

• missing or weak antigens: ABO subgroups may demonstrate weak or no reactivity with anti-A or anti-B reagent

1. leukemia or Hodgkin’s patients may show weakened A or B antigen

2. inheritance of an alternate allele at the ABO locus

   • can result in a reduction of antigen sites per red cell

How can the ABO discrepancy “missing or weak antigens” be resolved?

• check the patient’s diagnosis and transfusion history

• repeat testing with extended incubation time

  • 10-15 minutes at room temp to enhance antigen-antibody reaction

mixed field reactions

• a type of ABO discrepancy where the test result shows agglutinated AND unagglutinated cells (agglutinates with a cloudy red background)

• causes:

  • patient has two distinct cell populations (ex: group O cells are transfused to a group A, B, or AB individual)

  • BM transplant, stem cell transplant, or A₃ subtype

• resolution:

  • obtain patient diagnosis and transfusion history

Describe the following ABO discrepancy in plasma testing: extra antibodies

• unexpected agglutination reactions occur in reverse typing

• causes:

  • A₂ subgroup individuals with anti-A₁ antibody (anti-A₁ antibody agglutinates with A1 cells) (in theory, type A individuals should NOT agglutinate with A₁ cells)

  • cold alloantibodies and autoantibodies

  • rouleaux

How can cold alloantibodies cause extra reactions in plasma ABO testing?

• cold alloantibody: non-ABO ABs that are specific to other red cell antigens

• it is possible the reverse typing cells possess antigens from other blood groups that react with alloantibodies in the patient’s serum

How can cold autoantibodies cause extra reactions in plasma ABO testing?

• cold autoantibody: ABs specific to autologous antigens

• typically anti-A or anti-I

• these antibodies react with ALL reverse, screening, and panel cells

How can the ABO plasma discrepancy “extra antibodies” be resolved?

• obtain the patient’s diagnosis and transplant history

• test the patient plasma with screening cells (type O)

  • to detect alloantibodies

  • then perform an antibody ID test if positive

• perform an autocontrol

  • test the patient’s plasma with their red cells

  • positive autocontrol test = reverse typing results are the product of cold autoantibodies

How can rouleaux cause ABO discrepancies during plasma testing?

• abnormal amounts of serum proteins can cause false-positives in reverse typing

• ALSO: can cause problems with ABO red cell testing if unwashed cells are used

• resolutions:

  • wash RBC suspension and repeat forward type

  • perform saline replacement for reverse types

saline replacement

• used to distinguish rouleaux from true agglutination in reverse typing

• steps:

  • recentrifuge the plasma and red cells

  • replace the plasma with saline

  • observe for agglutination

    • no agglutination = positive reaction before was due to rouleaux

    • agglutination = true agglutination

Describe the following ABO discrepancy in plasma testing: missing or weak antibodies

• weak or negative agglutination in reverse typing

• can be explained by investigating the patient’s history, age, diagnosis, and Ig levels

  • newborns do not have antibodies yet, and the elderly have reduced AB levels

  • certain disease states can lower Ig levels

What are some resolutions for the ABO discrepancy “missing or weak antibodies”?

• obtain patient history and diagnosis

• incubate test for 15 min at room temp, centrifuge, and interpret agglutination

  • if agglutination is still negative: incubate at 4 degrees for 15 min with an autocontrol to verify that positive results are due to ABO antibodies (not cold autoantibodies)

What is the Bombay phenotype (O_h)?

• a very rare condition where RBCs lack H, A, AND B antigen

  • genotype is hh (amorph), therefore no expression of H antigen on red cells

• forward typing indicates patient is type O

• patient serum contains: anti-H, anti-A, AND anti-B

Why is blood transfusion difficult for those with the Bombay phenotype?

• anti-H antibody is clinically significant because it reacts at 37 C and can activate complement (leading to intravascular hemolysis)

• ONLY Bombay blood can be transfused to Bombay patients

  • group O cells are unacceptable because they have H antigen

• sources of blood for transfusion:

  • stored autologous units

  • units from siblings

  • units from the rare donor program

An individual who inherits the Se allele homozygously (SeSe) or heterozygously (Sese) is referred to as a…

secretor

• these patients have soluble H antigens in their secretions that are converted to A or B antigens