HEM 2133

Immunohaematology IImmunohaematology I

Lesson 3: ABO Typing Procedure

ABO Tube Grouping

A more sensitive and reliable method

Is used in blood banks and in clinical

laboratories

Requires dilution of the blood with saline to Requires dilution of the blood with saline to

make a 2% to 5% suspension of cells

ABO tube grouping consists of:

Direct or forward grouping, which identifies

the antigens on the cells

Reverse or confirmatory grouping, which Reverse or confirmatory grouping, which

identifies the blood group antibodies in the

serum

Performing Forward Grouping

Forward or direct grouping identifies the antigens present on red blood cells by reacting a suspensions of cells with commercial anti-A and anti-B sera and observing for agglutination after centrifugationagglutination after centrifugation

If a centrifuge is not available, the reactions can be observed after allowing the tubes to sit undisturbed at room temperature for 15 to 30 minutes

A 2% to 5% red blood cell suspension is made by

adding 18 to 19 volumes of saline to one volume

of the patients blood

Two tubes labeled A and B are set up: one drop of

anti-A serum is placed in the A tube and one drop

of anti-B serum is placed in the B tube

One drop of the patients 2% to 5% cell

suspension is added to each tube, and the

contents are mixed

The tubes are centrifuged for 30 seconds to

enhance the reaction

Interpretation of Forward Grouping

The tubes are tapped gently to loosen the

cells from the bottom of the tube and the cells

are observed for agglutination

Clumping of the cells is a positive reaction Clumping of the cells is a positive reaction

indicating the antigen present on the cells

corresponds to the antibody placed in the test

tubes

Reactions should be graded using a plus

system

neg (no visible clumps or agglutinates)

w + (a few small, very fine aggregates barely visible to the naked eye)

1 + (button breaks into numerous tiny clumps. Background becomes cloudy)

2+ (button breaks into many medium-sized 2+ (button breaks into many medium-sized clumps. Background is clear)

3+ (button breaks into a few large clumps. Background is clear)

4+ (button is one or two large clumps after being dislodged. Background is clear)

Reaction of Antisera with Red Cells

The reaction between the antiserum and red

cells can occur in various ways:

1. Agglutination

If the red cells and the antiserum contain If the red cells and the antiserum contain

corresponding antigen and antibody, they

will react to bring about agglutination of the

red cells

If either antigen or antibody is absent, there

will be no agglutination

2. Hemolysis

Sometimes, the antigen-antibody reaction

may result in hemolysis due to activation of

the complement system

Complement, if present in the antiserum, can Complement, if present in the antiserum, can

bind to the antigen-antibody complex, and

lyse the red cells

Complement can be easily inactivated by

heating at 56C for 30 minutes

3. Rouleaux formation

A high concentration of globulin in patients

serum can hold the red cells together to

appear like a stack of coins

This is rouleaux formation and can be This is rouleaux formation and can be

mistaken for agglutination

Performing Reverse Grouping

Reverse grouping identifies the antibodies

present in a patients serum or plasma by

reacting the plasma with a 2% to 5%

suspension of group A cells and a commercial suspension of group A cells and a commercial

2% to 5% group B cells and observing for

agglutination

Two drops of the patients plasma are added

to each of three tubes marked a, b and control

One drop of the group A cell suspension is

added to tube a, one drop of group B cell

suspension is added to tube b, and one drop

of a 2% to 5% suspension of patient cells is

added to the control tube

The contents of the tubes are mixed, and the

tubes are centrifuged for 30 seconds

Interpretation of Reverse Grouping The tubes are tapped gently, and the cells are

observed for agglutination and the reactions graded

A positive test, agglutination, indicates that the antibody present in the patients plasma corresponds to the antigen on cells added to the corresponds to the antigen on cells added to the tube

The control tube should always be negative for agglutination since it contains only the patients plasma and cells

Reverse grouping results should confirm the results of forward grouping

Blood Group Reactions of cells with

Anti-A Anti-B

A + 0

ABO Forward Grouping Results

A + 0

B 0 +

AB + +

O 0 0

Blood

Group

Reactions of plasma with

A cells B cells O cells

O + + 0

ABO Reverse Grouping Results

O + + 0

A 0 + 0

B + 0 0

AB 0 0 0

Forward and Reverse Grouping

Gel Typing

Automated and semi-automated systems are

available for blood grouping and

crossmatching

Solid-phase and gel or column typing methods

can be automated allowing some walk-away can be automated allowing some walk-away

testing

Gel typing

Sensitive and specific

The procedure can be standardized, verified

and validated

Testing is performed in a card prefilled with Testing is performed in a card prefilled with

gels mixed with the appropriate reagent

A dilution of patient cells is pipetted onto the

gel column and the card is incubated

The card is centrifuged and read

Agglutinated cells will not travel through the gel but remain at the top of the column, a positive reaction

Non-agglutinated cells travel through the gel Non-agglutinated cells travel through the gel to the bottom of the column, a negative reaction

Since some gel typing reactions are stable for several hours, tests can be retained and reread if necessary

Advantages of Gel Typing

Minimal handling of reagents and specimens

increases biosafety

Eliminates variables due to work technique

Clear, stable, well-defined endpoints Clear, stable, well-defined endpoints

objective and reproducible interpretation of

test results

Reduce needs to repeat test

Anomalous Results in ABO Blood

Grouping

A situation that exist when the results

between forward and reverse grouping is of

no match (odd)

May lead to false negative or false positive May lead to false negative or false positive

reactions

Technical Problems - mostly

Dirty tubes or glassware

Contamination or inactivation of reagent

Failure to add reagent or serum

Failure to follow manufacturers direction Failure to follow manufacturers direction

Warming during centrifugation

Uncallibrated centrifuge

Over/undercentrifuge

Improper cell to serum concentration

Cell suspension too light or too heavy

Failure to identify hemolysis as positive

Careless readings Careless readings

Clerical errors (incorrect recording of results

or interpretation/readings)

Failure to incubate tests at 20-25C or below

Improper technique

Missing or Weak Reaction Antibodies

Reason depressed Ab production or cannot

produce the ABO antibodies

Cause false negative

1. Age newborn infants (not producing own

Abs yet) or elderly (declined Ab)Abs yet) or elderly (declined Ab)

2. Disease patients with leukaemia,

lymphomas, using immunosuppressive drugs,

immunodeficiency disease, bone marrow

transplant

3. Chimerism possess dual population of red

cells (mixed cell population)

How to resolve?

Eliminate all technical errors

Incubate patients serum with red cells at Incubate patients serum with red cells at

room temperature for 15 minutes

Chimerism establish whether the patient has

transfusion or transplant bone marrow, fetal

maternal bleeding

Missing or Weak Reaction Antigens

Unusual genotype subgroups of A and B

Disease leukaemia, Hodgkins disease

Acquired B-like activity resulting from the

action of gram-negative organisms/ intestinal action of gram-negative organisms/ intestinal

obstruction/ colon or rectum cancer/ other

lower intestinal tract disorders

Colouring dyes

Antibodies in the reagent

Plasma Abnormalities

1. Increased gamma globulin disease e.g.

multiple myeloma, Hodgkins lymphoma

rouleaux formation

2. Abnormal proteins causing rouleaux

formationformation

3. Whartons jelly found only when cord blood

is used

Rouleaux - stacks of red blood cells (RBCs)

which form because of the unique discoid

shape of the cells

Rouleaux