Blood Grouping

History - Karl Landsteiner

Discovered the ABO Blood Group System in 1901

He and his five co-workers began mixing each others red cells and serum together and inadvertently performed the first forward and reverse ABO groupings

Landsteiners Rule

If an antigen (Ag) is present on a patients red blood cells the corresponding antibody (Ab) will NOT be present in the patients plasma, under ‘normal conditions’.

Major ABO Blood Group

ABO Group

Antigen Present

Antigen Missing

Antibody Present

A A B Anti-B

B B A Anti-A

O None A and B Anti-A&B

AB A and B None None

ABO Basics

Blood group antigens are actually sugars attached to the red blood cell.

Antigens are “built” onto the red cell. Individuals inherit a gene which codes for specific sugar(s) to be added to the red cell.

The type of sugar added determines the blood group

Principle of blood grouping

There are two principles

1-almost all normal healthy individuals above 3-6 months of age have “ naturally occurring Abs” to the ABO Ags that they lack

These Abs termed naturally occurring because they were thought to arise without antigenic stimulation

Principle of blood grouping

2- These “naturally occurring” Abs are mostly IgM class. That means that, they are Abs capable of agglutinating saline/ low protein suspended red cell without enhancement and may activate complement cascade.

ABO and H Antigen Genetics

Genes at three separate loci control the occurrence and location of ABO antigens

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

Location

The presence or absence of the ABH antigens on the red blood cell membrane is controlled by the H gene

The presence or absence of the ABH antigens in secretions is indirectly controlled by the Se gene

H Antigen

The H gene codes for an enzyme that adds the sugar fucose to the terminal sugar of a precursor substance (PS)

The precursor substance (proteins and lipids) is formed on an oligosaccharide chain (the basic structure)

RBC Precursor Structure

Glucose

Galactose

N-acetylglucosamine

Galactose

Precursor Substance (stays the

same)

RBC

Formation of the H antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

Precursor Substance (stays the

same)

RBC

H antigen

Fucose

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 Immunodominant sugars 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 N-acetylgalactosamine to the terminal sugar of the H antigen N-acetylgalactosaminyltransferase

The “B” gene codes for an enzyme that adds D-galactose to the terminal sugar of the H antigen D-galactosyltransferase

Formation of the A antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

FucoseN-acetylgalactosamine

A antigen

Formation of the B antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

FucoseGalactose B antigen

H antigen

Certain blood types possess more H antigen than others:

O>A2>B>A2B>A1>A1B

Why do Group O individuals have more H antigen than the other groups?

Group O individuals have no A or B genes to convert the H antigen to A or B antigens….that means more H antigen sites

Group O Group A

Many H antigen sites

Fewer H antigen

sites

A

A A

AA

Most of the H antigen sites in a Group A individual have been

converted to the A antigen

Genetics

The H antigen is found on the RBC when you have the Hh or HH genotype, but NOT from the hh genotype

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

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

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

Very rare (130 worldwide)

Bombay Phenotype (Oh)

The hh causes NO H antigen to be produced Results in RBCs with no H, A, or B antigen (patient types as O)

Bombay RBCs are NOT agglutinated with anti-A, anti-B, or anti-H (no antigens present)

Bombay serum has strong anti-A, anti-B and anti-H, agglutinating ALL ABO blood groups

What blood ABO blood group would you use to transfuse this patient??

Another BombayGroup O RBCs cannot be given because

they still have the H antigenYou have to transfuse the patient with

blood that contains NO H antigen

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

ABO antibodies

IgM is the predominant antibody in Group A and Group B individuals Anti-A Anti-B

IgG (with some IgM) is the predominant antibody in Group O individuals Anti-A,B (with some anti-A and anti-B)

ABO antibodies

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

High titer: react strongly (4+) 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

ABO routine testing

Several methods for testing the ABO group of an individual exist. The most common method is:

Serology: This is a direct detection of the ABO antigens. It is the main method used in blood transfusion centres and hospital blood banks.

This form of testing involves two components: a) Antibodies that are specific at detecting a particular ABO antigen on RBCs.       

b) Cells that are of a known ABO group that are agglutinated by the naturally occurring antibodies in the person's serum.

ABO ROUTINE TESTING

DIRECT OR FORWARD GROUPING

Test for antigens• Patient’s cells containing unknown antigens tested with known antisera

• Antisera manufactured from human sera

Aantisera used:Antisera Color Source

Anti-A Blue Group B donor

Anti-B Yellow Group A donor

Anti-A,B Red Group O donor

Forward Grouping

Reaction of patient red blood cells tested with Reagent anti-A and anti-B antisera

Slide: 20-40% RBC suspension + anti-serum

Tube (12x75mm): 2-5% RBC suspension + anti-serum (centrifuge before read)

Forward Grouping

Reaction Patterns for ABO Groups

Blood group Agglutination with Anti-A

Agglutination with Anti-B

A + -

B - +

AB + +

O - -

Reverse grouping

• serum is combined with cells having known Ag content in a 2:1 ratio

• uses commercially prepared reagents containing saline-suspended A1 and B cells

Reverse grouping

Reaction Patterns for ABO Groups

Blood Group Agglutination with A cells

Agglutination with B cells

A - +

B + -

AB - -

O + +

Grading of Agglutination:

Negative (0) No clumps or aggregates

Weak (+/-) Tiny clumps or aggregates barely visible macroscopically or to the

naked eye

1+ Few small aggregates visible macroscopically

2+ Medium-sized aggregates

3+ Several large aggregates

4+ One solid aggregate

ABO blood group (forward blood grouping)

Patient Red Cells Tested With

Interpretation Anti-B Anti-A Patient

0 0 1

0 4+ 2

4+ 0 3

4+ 4+ 4

ABO blood group (forward blood grouping)

Patient Red Cells Tested With

Interpretation Anti-B Anti-A Patient

O 0 0 1

A 0 4+ 2

B 4+ 0 3

AB 4+ 4+ 4

Reverse Grouping (Confirmatory grouping

Patient SERUM Tested With

Interpretation

B Cells A1 CellsPatient

4+ 4+ 1

4+ 0 2

0 4+ 3

0 0 4

Reverse Grouping (Confirmatory grouping

Patient SERUMTested With

Interpretation

B Cells A1 CellsPatient

O 4+ 4+ 1

A 4+ 0 2

B 0 4+ 3

AB 0 0 4

Forward & reverse ABO blood grouping

Reaction of Cells Tested With

Reaction of Serum Tested Against ABO

Group

Anti-A Anti-B A1 Cells B Cells

1 0 0 + + O

2 + 0 0 + A

3 0 + + 0 B

4 + + 0 0 AB

Forward & reverse ABO blood grouping

Reaction of Cells Tested With

Reaction of Serum Tested Against ABO

Group

Anti-A Anti-B A1 Cells B Cells

1 0 0 + +

2 + 0 0 +

3 0 + + 0

4 + + 0 0

ID card system

This ID-Card contains a mixture of human polyclonal and monoclonal anti-A, human polyclonal anti-B and human polyclonal anti-D antibodies.

The microtube ctl is the negative control. Two microtubes with neutral gel serve for reverse grouping with A1 and B cells.

Fully-automatic walk-away for ID-Cards

Stand alone instrument

Continuous sample loading

Continuous reagent loading

Priority samples (STAT)

Reagent stock

High throughput

Optimized for small blood volumes

Dispense verification

Easy-to-use

Full test menu

Wi-Fi

Touchscreen 17"

Host connectivity

Internal & external validation

Capacity180 samples240 ID-Cards28 reagent vials

Thank you….