ABO BLOOD GROUP

History: Karl Landsteiner

He and his five coworkers began mixing each others red cells and serum together and inadvertently performed the

http://www.nobelpreis.org/castellano/medizin/images/landsteiner.jpg

Why is it important?

Biochemical & Genetic Considerations

ABO ANTIGENS

ABO INHERITANCE
In 1924, Bernstein described the theory for the inheritance of ABO blood groups Codominant in expression Genotypes: Phenotypes:

ABO and H Antigen Genetics

Genes at three separate loci (ABO, Hh, and Se) control the occurrence and location of ABO antigens There are three common alleles at the ABO The H and Se (secretor) loci,

The presence or absence of the A, B, and H antigens is controlled

Location
The presence or absence of the ABH antigens on the red blood cell membrane is controlled by the The presence or absence of the ABH antigens in secretions is indirectly controlled by the

ABO Antigen Genetics

H gene
Se gene ABO genes

H Antigen
The H gene codes for an enzyme that adds the sugar fucose to the terminal sugar of a

The precursor substance (proteins and lipids) is formed on an

RBC Precursor Structure

RBC

Glucose Galactose N-acetylglucosamine Galactose

Precursor Substance (stays the same)

Formation of the H antigen

RBC

Glucose Galactose N-acetylglucosamine Galactose

H antigen
The H antigen is the foundation upon which A and B antigens are built A and B genes code for enzymes that add an immunodominant sugar to the H antigen ________________________are present at the terminal ends of the chains and confer the ABO antigen specificity

A and B Antigen
The A gene codes for an enzyme (transferase) that adds _______________________to the terminal sugar of the H antigen
N-acetylgalactosaminyltransferase

The B gene codes for an enzyme that adds ________________to the terminal sugar of the H antigen
D-galactosyltransferase

Formation of the A antigen

RBC

Glucose Galactose N-acetylglucosamine Galactose

Fucose

Formation of the B antigen

RBC

Glucose Galactose N-acetylglucosamine Galactose

Fucose

Genetics
The _____________ is found on the RBC when you have the Hh or HH genotype, but NOT from the hh genotype The _____________is found on the RBC when you have the Hh, HH, and A/A, A/O, or A/B genotypes

The _____________is found on the RBC when you have the Hh, HH, and B/B, B/O, or A/B genotypes

H antigen
Certain blood types possess more H antigen than others:

Greatest amount of H

Least amount of H

ABO Antigens in Secretions

Secretions include
Blood Group Substances are soluble antigens (A, B, and H) that can be found in the secretions. This is controlled by the

Secretor Status
The secretor gene consists of 2 alleles (Se and se) The Se gene is responsible for the

If the Se allele is inherited as SeSe or Sese, the person is called a 80% of the population are secretors

Secretors
Secretors express soluble forms of the H antigen in secretions that can then be Individuals who inherit the sese gene are called

The se allele is an amorph (nothing expressed) sese individuals do not convert antigen precursors to H antigen and has neither soluble H antigen nor soluble A or B antigens in body fluids

Secretor Status Summary

The Se gene codes for the presence of the H antigen in secretions, therefore the presence of A and/or B antigens in the secretions is contingent on the inheritance of the Se gene and the H gene

A antigen Se gene (SeSe or Sese) se gene (sese) H antigen in secretions

and/or

B antigen

No antigens secreted in saliva or other body fluids

ABO Group
Secretors (SeSe or Sese):
A B

ABH Substances
A
+++ 0

B
0 +++

H
+ +

O
AB Non-secretors (sese): A, B, O, and AB

0
+++

0
+++

+++
+

Sese + h/h (no H antigen) no antigens in secretions

COMPARISON OF ABH ANTIGENS ON RBCs AND IN SECRETIONS

ABH Antigens on Red Cells RBC antigens can be glycolipids, glycoproteins, or glycosphingolipids A, B, and H Soluble substances Secreted substances are glycoproteins

RBC antigens are only synthesized on type Secreted substances are primarily 2 precursor chains synthesized on type 1 precursor chains Type 2 chain refers to a beta1 4 linkage in which the number one carbon of the galactose is attached to the number three carbon of the N-acetylglucosamine sugar of the precursor substance Enzyme produced by the H gene (-2-Lfucosyltransferase)acts primarily on type 2 chains, which are prevalent on the RBC membrane Type 1 chain refers to a beta 1 3 linkage in which the number one carbon of the galactose is attached to the number three carbon of the N-acetylglucosamine sugar of the precursor substance Enzyme produced by the Se gene (-2-Lfucosyltransferase) preferentially acts on type 1 chains in secretory tissues

Lewis (Le)
The Lewis Blood Group System is mentioned here because it is related to secretor status Lewis antigens are plasma antigens formed by tissues and are released into plasma where they adsorb onto the RBCs (they are not an integral part of the RBC membrane) Consists of 2 antigens Lea Leb

Lewis
Lea and Leb are a single gene (Le) and its amorph (le) Lea is a precursor to Leb The Le gene codes for a transferase, which attaches L-fucose to the precursor chain to form the Lea antigen (designated Le(a+b-) If the H and Se genes are inherited, the Lea is converted to Leb and is designated Le(a-b+) In childhood, both may be on the RBC, Le(a+b+) If a person is lele, they will have no Lewis antigens in plasma or on red blood cells

ABO Subgroups
ABO subgroups differ in the amount of antigen present on the red blood cell membrane Subgroups have less antigen Subgroups are the result of less effective enzymes. They are not as efficient in converting H antigens to A or B antigens (fewer antigens are present on the RBC) Subgroups of A are more common than subgroups of B

Subgroups of A
The 2 principle subgroups of A are: A1 and A2

A2 Phenotype
Why is the A2 phenotype important? A2 and A2B individuals may produce an anti-A1 This may cause discrepancies when a crossmatch is done (incompatibility) Whats the difference between the A1 and A2 antigen?

A1 and A2 Subgroups*
Anti-A Anti-A1 Anti-H antisera antisera lectin ABO antibodies in serum # of antigen sites per RBC

A1 A2

4+ 4+

4+ 0

0 3+

Anti-B Anti-B & anti-A1

900 x103 250 x103

*Adapted from Flynn, J. (1998). Essentials of Immunohematology

Other A subgroups
There are other additional subgroups of A Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu A3 red cells cause mixed field agglutination when polyclonal anti-A or anti-A,B is used Mixed field agglutination appears as small agglutinates with a background of unagglutinated RBCs They may contain anti-A1

B Subgroups
B subgroups occur less than A subgroups B subgroups are differentiated by the type of reaction with anti-B, anti-A,B, and anti-H B3, Bx, Bm, and Bel

Other ABO conditions

Bombay Phenotype (Oh) Inheritance of hh The h gene is an amorph and results in little or no production of L-fucosyltransferase Originally found in Bombay (now Mumbai) Very rare (130 worldwide)

Bombay (Oh) Phenotype

Total Lack of H, A, and B antigens Develop strong anti-H, anti-A, and anti-B O forward, O reverse; with positive antibody screen

GENERAL CHARACTERISTICS OF BOMBAY Oh+ (Hnull) Phenotypes

Absence of H, A, and, B antigens; no agglutination with anti-A,-B, -H lectin Presence of anti-A, anti-B, and anti-A,B and a potent wide thermal range of anti-H lectin A,B,H nonsecretor (no A, B, or H substances present in saliva) Absence of -L-fucosyltransferase (H enzyme) in serum and H antigen on red cells Presence of A or B enzymes in serum (depending on ABO genotype) A recessive mode of inheritance (identical phenotypes in children but not in parents) RBCs of the Bombay phenotype will not react with the anti-H lectin RBCs of the Bombay phenotype are compatible only with the serum from another individual

H deficient phenotypes
Basic Concepts
Rare phenotypes in which the RBCs are completely devoid of H antigens or that have small amounts of H antigen present Three categories: 1) Category 1:

2) Category 2:
3) Category 3:

CLASSIFICATION

Proposed Genes inherited

Glycosyltransferase

Red Cell antigens: A, B, and H detected

A, B, and H soluble substances in secretions

Antibodies in serum

Category 1 Oh , Oh B, O h A, OhAB

hh sese

None or A and/or B in serum or RBC stroma

None detectable

None detectable

Anti-A, antiB, anti-H

Category 2 Oh, Ah, Bh, ABh

A and/or B hh sese

A and/or B in Weak A/B None serum and Residual H detectable RBC stroma when A or B immunodomi nant sugar is removed with appropriate enzyme A and/or B in Weak A/B serum/ and H RBCs, H in serum (weak) H substance (normal amounts) A/B (all normal

Anti-H, AntiA/Anti-B

Category 3 Se O h O, O h A , O h B , OhAB

Weak IH Anti-A/anti-B

ABO DISCREPANCIES
Group I Group II Group III Group IV

***Assignment: Resolution of the different group discrepancies (1/2 crosswise)

GROUP I DISCREPANCIES
Unexpected reactions in reverse grouping due to weakly reacting or missing antibodies More common than most of the other groups Suspected when reaction in the serum grouping is weak or missing Reason: Patient has depressed antibody production or cannot produce ABO antibodies Rare group I discrepancies: Chimerism: presence of two cell populations in a single individual

GROUP I DISCREPANCIES
Some of the more common populations with discrepancies in this group are:
Newborns Elderly patients Patients with leukemias demonstrating hypogammaglobulinemia or lymphomas Patients using immunosuppressive drugs that yield hypogammaglobulinemia Patients with congenital agammaglobulinemia or immunodeficiency diseases Patients with bone marrow transplantations Patients whose existing ABO antibodies may have been diluted by plasma transfusion or exchange ABO subgroups

GROUP II DISCREPANCIES
Unexpected reactions in the forward grouping due to weakly reacting or missing antigens Least frequently encountered Some of the causes are:
Subgroup of A (or B) may be present Leukemias may yield weakened A or B antigens

Hodgkins disease has been reported in some cases to mimic the depression of antigens found in leukemia
Acquired B phenomenon is most often associated with diseases of the digestive tract ( cancer of the colon)

GROUP II DISCREPANCIES
Rare Group II discrepancies
Excess amounts of Blood group-specific soluble (BGSS) substances present in the plasma in association with certain diseases such as carcinoma of the stomach and pancreas Antibodies to low-incidence antigens in reagent anti-A or anti-B Chimerism

GROUP III DISCREPANCIES

Between forward and reverse grouping caused by protein or plasma abnormalities and result in roleaux formation or pseudoagglutination attributable to:
Elevated levels of globulin from certain disease states (MM, Waldenstroms macroglobulinemia, plasma cell dyscrasias, moderately advanced cases of Hodgkins lymphomas)
Elevated levels of fibrinogen Plasma expanders (dextran and polyvinylpyrrolidone) Whartons jelly in cord blood samples

GROUP IV DISCREPANCIES
Between forward and reverse groupings due to miscellaneous problems and have the following :
Cold reactive autoantibodies in which RBCs are so heavily coated with antibody that they spontaneously agglutinate, independent of the specificity of the reagent antibody Patient has circulating RBCs of more than one ABO group due to RBC transfusion or marrow transplant Unexpected ABO isoagglutinins Unexpected non-ABO alloantibodies

GROUP IV DISCREPANCIES
Rare Group IV discrepancies
Antibodies other than anti-A or anti-B may react to form ag-ab complexes that may then adsorb onto patients RBCs Some individuals have antibodies against acriflavin in their serum
Pxs ab combines with the dye and attaches to the pxs rbcs, resulting in agglutination in the forward grouping

ABO Blood Group:

ABO Antibodies

Landsteiners Rule:
Normal, Healthy individuals possess ABO antibodies to the ABO antigen absent from their RBCs

ABO Blood Group System

The ABO Blood Group System was the first to be identified and is the most significant for transfusion practice It is the ONLY system that the reciprocal antibodies are consistently and predictably present in the sera of people who have had no exposure to human red cells

Blood Group Systems

Most blood group systems (ABO and others) are made up of: An antigen on a red cell and the absence of its corresponding antibody in the serum (if youre A, you dont have anti-A) If you do NOT have a particular antigen on your red cells then it is possible (when exposed to foreign RBCs) to illicit an immune response that results in the production of the antibody specific for the missing antigen

ABO
Remember:
The ABO Blood Group System does NOT require the presence of a foreign red blood cell for the production of ABO antibodies ABO antibodies are non-red blood cell stimulated probably from environmental exposure and are referred to as expected antibodies

ABO antibodies
group A serum contains anti-B group B serum contains anti-A group AB serum contains no antibodies group O serum contains anti-A, anti-B, and anti-A,B

Anti-A1
Group O and B individuals contain anti-A in their serum However, the anti-A can be separated into different components: anti-A and anti-A1

Anti-A,B
Found in the serum of group O individuals Reacts with A, B, and AB cells Predominately IgG, with small portions being IgM Anti-A,B is one antibody, it is not a mixture of anti-A and anti-B antibodies

ABO antibodies
Activate complement React at room temperature or colder IgM is the predominant antibody in Group A and Group B individuals

IgG (with some IgM) is the predominant antibody in Group O individuals

ABO antibody facts

Reactions phase: Complement can be activated with ABO antibodies (mostly IgM, some IgG) High titer:

ABO Antibodies
Usually present within the first 3-6 months of life Stable by ages 5-6 years Decline in older age Newborns may passively acquire maternal antibodies (IgG crosses placenta) Reverse grouping (with serum) should not be performed on newborns or cord blood

Nature of antibodies
Non-red blood cell stimulated (previously discussed) ABO antibodies Red blood cell stimulated Antibodies formed as a result of transfusion, etc Usually IgG Active at 37C Can occur in group O (may occur in group A or B) These antibodies also occur in the other Blood Group Systems

Laboratory Testing:
ABO typing

The Use of Lectins for Antigen Confirmation

Dolichos biflorus = anti-A1 Ulex europaeus = anti-H

58

ABO Blood Groups

ABO Group A Antigen Present A Antigen Missing B Antibody Present anti-B

B O
AB

B None
A and B

A A and B
None

anti-A anti-A, anti-B, anti-A,B None

Forward & Reverse Typing

Reaction of cells tested with: Reaction of serum tested with:

anti-A 1 0

anti-B 0

A cells +

B cells +

ABO group O

2
3

+
0

0
+

0
+

+
0

A
B

AB

ABO Ag LOCATION IN THE BODY

Body fluids
Saliva Tears Urine Digestive juices Bile Milk Amniotic fluid

Pathologic fluids
Pleural Peritoneal Pericardial Ovarian cyst

ABO Antigens and Antibodies

ABO antigens based on combinations of three genes: A, B, and O Antibodies are clinically significant and naturally occurring causing most fatal acute HTRs some causing HDFN ABO antibodies neutralized with secretor saliva.

Group O
Generally the most common blood group Genotype: OO Antigen: H Antibodies: anti-A, anti-B, and anti-A,B Antibodies are naturally occurring and very strong Anti-A,B (mostly IgG) may cross placenta to cause HDFN

Group A
Genotype: Antigen: Antibodies: A subgroups

Group B
Genotype: Antigen: Antibodies: B subgroups: Not important

Group AB
Genotype: Antigen: Antibodies: B subgroups: Not important A2B:

ABO Testing
Cell typing (forward grouping) to determine antigen types on RBCs Serum/plasma typing (reverse grouping or backtyping) to determine type of antibody in serum: Note the opposite reactions If the forward reactions are opposite of reverse, an ABO discrepancy is not present.

ABO Grouping Reagents

Forward Grouping Reagent Reverse or Back Tying Cells

Forward Grouping Reagent

Forward Grouping
Reagent: Monoclonal antibody Highly specific IgM Expected 3+- to 4+ reaction 1 drop Anti-A=Blue; anti-B=Yellow (Acroflavin dye) A and B antigens on patient red cells are agglutinated by known sera (anti-A, anti-B)

Reverse or Back Tying Reagent Cells

Reverse or Back Typing

Reagent Cells: Human Source Expected 2+ to 4+ reaction 4-5% cell suspension 1 drop Anti-A or anti-B antibodies in patient serum (or plasma) agglutinate with A1 and B antigens on Reagent cells

Forward Typing Procedures

To determine what antigens are present on RBCs.

Step 1. Label test tubes.

Step 2: Make a 2-5% patient red cell suspension.

Step 3: Add reagent antisera (1 drop).

Step 3A: Add reagent Anti-A antisera (1 drop).

Step 3B: Add Anti-B reagent antisera (1 drop).

Step 4: Add one drop of 2-5% suspension of patient RBC to each tube.

80

Step 5: Mix and centrifuge (approximately 20 seconds).

Group A: 4+ Agglutination with Anti-A 0 Agglutination with Anti-B

Group B: 4+ Agglutination with Anti-B 0 Agglutination with Anti-A

Group AB: 4+ Agglutination with Anti-A and Anti-B

Group O: No Agglutination with Anti-A or Anti-B

Back Typing
To determine what antibodies are present in patients plasma.

Step 1: Label Test Tubes

Step 2: Add two drops of patient serum to each tube

Step 3: Add one drop of reagent cells to each test tube

Step 3A: Add one drop of Reagent A1 cells

Step 3B: Add one drop of Reagent B cells

Step 4: Mix and centrifuge (approximately 20 seconds)

Group A: 4+ Agglutination with B Cells 0 Agglutination with A1 Cells

Group B: 4+ Agglutination with A1 Cells 0 Agglutination with B Cells

Group O: 4+ Agglutination with A1 Cells 3+ Agglutination with B Cells

Group AB: No Agglutination with A1 and B Cells

What can Cause ABO Discrepancies?

Disagreement between the interpretations of forward and reverse grouping Antigen problems Antibody problems

Antigen Problems
Lack of expected antigens

Presence of unexpected antigens

Antibody problems
Lack of expected antibodies

Presence of unexpected antibodies

A Subgroups
A1 A2 A3 Ax Aend Am etc

A1 vs A2 Phenotypes
Blood Group A1 (80%) A2 (20%) Anti-A + + Anti-A1 lectin + 0

A1 & A2 account for 99% of A group

A1vs A2 Phenotypes
Quantitative differences:

Qualitative differences between A1 and A2 antigens:

B Subgroups
Very rare and are less frequent than A subgroups. B subgroups demonstrate variations in the strength of the reaction using antiB and anti-A,B

Examples are: B3, Bx, Bm, Bel

Acquired B phenotype
Occurs in type A individuals with:

Bacteria deacetylate group A sugar (GalNAc); remaining galactosamine crossreacts with reagent anti-B.

Acquired B phenotype

Acquired B phenotype
AB forward (with weak reactions with reagent anti-B) A reverse Reaction with anti-B is negative, if:

Acquired B typing result

Forward
Anti-A Anti-B Interp

Reverse
A1 cells B cells Interp

4+

1-2+

AB

4+

AB

Blood Type: Antigens vs Antibodies

Blood Type
A B AB O

Antigens on rbcs
A B A,B None

Antibodies in Plasma
Anti-B Anti-A None Anti-A, Anti-B

Consequences of ABO incompatibility

Severe acute hemolytic transfusion reactions
One of the most frequent causes of blood bank fatalities Clerical errors

Most frequent HDFN; usually mild.

Sources of Technical Errors Resulting in ABO Discrepancies

Inadequate identification of blood samples Cell suspension too heavy or too light Clerical errors A mix-up in samples Missed observation of hemolysis Failure to add reagents Failure to follow manufacturers instructions Uncalibrated centrifuge Contaminated reagents Warming during centrifugation

Resolving ABO Discrepancies

Problems with RBCs Rouleaux MF agglutination Unusual phenotype (hh) Disease processes (Acq. B) Resolution Techniques

Resolving ABO Discrepancies (Contd)

Problems with serum Rouleaux Presence of unexpected Ab Absence of expected Ab Resolution Techniques