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ABO Grouping
Updated: Aug 07, 2018
Author: Victoria K Gonsorcik, DO; Chief Editor: Jun Teruya, MD, DSc, FCAP more...

OVERVIEW

Overview
The ABO system is regarded as the most important blood-group system in transfusion medicine because of
severe hemolytic transfusion reactions and, to a lesser degree, hemolytic disease of the newborn.

ABO grouping is a test performed to determine an individual's blood type. It is based on the premise that
individuals have antigens on their red blood cells (RBCs) that correspond to the four main blood groups: A, B,
O, and AB

Clinical Indications/Applications
The ABO test may be part of a group of tests performed in various clinical settings.

Type and screen

A type and screen includes ABO, Rh, and antibody detection/identification.

Type and cross

ABO, Rh, antibody detection/identification, and compatible matching with a donor unit are included in a type
and cross. [5]

Transfusion recipient testing

A type and cross needs to be completed before blood is issued to prevent ABO incompatibility. [5]

Recipient organ/hematopoietic stem cell testing

A type and screen of the recipient is performed to assess ABO compatibility between the recipient and
potential donor of organ and hematopoietic stem cells. In hematopoietic stem cell transplantation, progenitor
cells engraft into the recipient's chemotherapy-induced "empty" marrow and replace the recipient's blood
ABO type and Rh type with that of the donor's if the engraftment is successful.

Blood donor testing

A type and screen of the donor is completed as one of the required tests for blood donation. This allows
blood-collection facilities to quickly assess the inventory status of ABO/Rh type by label identification. Before
a donor unit is transfused, additional compatibility testing with the intended recipient's plasma/serum is
completed. This will also act as a second validation step of the initial donor ABO/Rh typing.

Donor organ/hematopoietic stem cell testing

A type and screen of the donor is completed as one of the required tests for organ or hematopoietic stem cell
donation to assess the level of compatibility of the donor's and intended recipient's ABO/Rh types, which will
dictate the subsequent type of blood components transfused.

Evaluation of hemolytic disease of the fetus and newborn associated with ABO
incompatibility

Hemolytic disease of the fetus and newborn occurs when a fetus inherits paternal red blood cell (RBC)
antigens that the maternal immune system does not recognize as her own. A small percentage of fetal blood
may come into direct contact with maternal blood circulation through fetal maternal hemorrhage (eg,
amniocentesis, trauma, miscarriage/abortion, placental abnormalities).

The ABO system of fetal RBC antigens are not as fully developed in utero and are lesser in number. Mild
hemolysis may result if there is ABO incompatibility between the baby and mother, as the maternal immune
system does not easily recognize the incompatible ABO antigen, thus potentially averting a more serious
hemolytic reaction.

The scenario that is indicted more often for an increased severity of hemolysis is when the mother is type O
and the fetus has either type A, B, or AB. The anti-A or anti-B antibodies that are produced from type O
mothers are mainly immunoglobulin (Ig) G. IgG antibodies are smaller and are able to easily pass through the
placenta, thurs more likely to expose fetal RBCs to the mother's antibody in the right clinical setting (see also
Rh Incompatibility for information on hemolytic disease of the fetus and newborn) (Rh typing is discussed in a
separate article).

Platelet refractory evaluation

A platelet refractory evaluation attempts to evaluate why a patients platelet count did not increase as
expected after a platelet transfusion; it includes evaluating immune-mediated versus nonimmune-mediated
causes. ABO incompatibility is an example of an immune-mediated cause.

Because ABO antigens are minimally expressed on platelets and less than 2 mL of RBCs are left in a unit of
platelets, it is often unnecessary to give ABO-compatible platelets. However, if an individual requires frequent
platelet transfusions, the amount of RBC exposure and platelet ABO antigens may increase enough that an
individual's antibodies will attack and cause hemolysis.

Test Performance
ABO typing is performed by taking a sample of blood, placing it in a centrifuge, and separating red blood cells
(RBCs) from serum/plasma. A "front," or forward type, and "back," or reverse type, are then performed.

The front/forward type takes an individual's RBCs and mixes them with commercially prepared reagents of
anti-A antibody and with separate anti-B antibody. The test measures visual agglutination or lack of
agglutination. The back/reverse type mixes an individual's plasma with reagent RBCs positive for antigen A,
and separate reagent RBCs positive for B antigen. The test also measures visual agglutination or lack of
agglutination.

Most ABO antibodies are immunoglobulin (Ig) M. When IgM comes into contact with a foreign antigen, it
attaches or "coats" the antigen. Once attached, IgM can come into contact with other antigen-binding sites
and antibodies, which will bring them closer together, thus causing visual agglutination (see the following
image). The presence of this reaction is designated with a positive symbol (+) and the absence with the
number zero, as shown in Table 3, further down in this section.
Manual Tube Testing. Image created by Jaye Parsley.

Limitations
As noted earlier, the amount of sample can limit ABO typing. For standard tube testing, column agglutination
"gel card" methodology (see the image below) and solid phase test systems, at least 1 mL of blood is
required.

Column agglutination, "gel card". Image created by Jaye Parsley.

If a newborn has received a substantial amount of type O red blood cells, the baby may be typed as O even if
the baby is non-O.

Methods
For many years, manual agglutination testing using a test tube was the main methodology for ABO/Rh typing.
With the advent of automated testing, new methodology has been introduced, including column agglutination
and solid phase test systems.

Manual tube testing

Testing forward typing, an individual's red cells (in a 2%-5% saline suspension) are mixed in a test tube with
separate reagents anti-A and anti-B at room temperature. After centrifugation, the pelleted cell button is
gently resuspended and examined for agglutination. If there is agglutination, this will indicate a positive
reaction. The amount of agglutination is graded on a scale of 0 to 4+, as shown in the following image.
Testing back typing, an individual's plasma is mixed in a test tube with separate reagent A and B red blood
cells (RBCs). The remainder of the testing and interpretation is completed as stated above for forward typing.

Manual Tube Testing. Image created by Jaye Parsley.

Column agglutination ("gel," "gel card")

Testing forward typing, an individual's RBCs are initially mixed at room temperature in small, gel-filled tubes
that contain separate reagent anti-A and anti-B. The specimen is centrifuged and assessed for agglutination.
Agglutination seen at the top of the tube column indicates a strong positive reaction; agglutination seen at the
bottom signifies no reaction. The amount of agglutination is also graded on a scale of 0 to 4+, as depicted in
the image below.

Testing back typing, an individual's plasma is initially mixed at room temperature in small, gel-filled tubes that
contain separate reagent A and B RBCs. The remainder of the testing and interpretation is completed as
stated above for forward typing.

Column agglutination, "gel card". Image created by Jaye Parsley.

Solid phase test systems ("microplate test")

Separate anti-A and anti-B reagent is embedded in the bottom of microplate wells. An individual's RBCs (in a
2%-5% saline suspension) are mixed together with an enhancing reagent and centrifuged. The plates are
then assessed. If there is just one tightly packed RBC pellet at the bottom of the microplate, this is considered
to be no reaction. If there is a covering of RBCs throughout the bottom of the microplate, this would indicate
agglutination, a positive reaction. As with the previous two tests, the amount of agglutination is graded on a
scale of 0 to 4+.

Testing back typing, separate portions of reagent A and B RBCs are embedded in the bottom of the
microplate wells. An individual's plasma is mixed togeter with an enhancing reagent and centrifuged. The
plates are interpretated as stated above for forward typing.

The following table provides further description of conventional agglutination grading.

Table 4. Standard Agglutination Grading (Open Table in a new window)

Marsh Score Conventional Grading Description of Grading

12-11 4+ Complete agglutination

10-9 3+ Strong reaction: 2-3 clumps

8-6 2+ Strong reaction: several clumps

5-4 1+ Many clumps

3-2 --- Scattered agglutination

1 w Weak granularity

0 0 Even cell suspension

Adapted from Marsh WL. Scoring of hemagglutination reactions. Transfusion. 1972 Sep-Oct. 12
(5):352-3. [7]

Laboratory Resources
The following are common laboratory resources:

ARUP Laboratories

John Hopkins Medical Laboratories

 Massachusetts General Hospital Pathology Service

Mayo Medical Laboratories

LabCorp

Quest Diagnostics

Stanford University Medical Center

Questions & Answers

Overview

What is ABO grouping?

Which is the role of genetics in ABO grouping?

What are the indications for ABO grouping?

What is the role of ABO grouping in transfusion recipient testing?

What is the role of ABO grouping in recipient organ/hematopoietic stem cell testing?

What is the role of ABO grouping in blood donor testing?

What is the role of APO grouping in donor organ/hematopoietic stem cell testing?

What is the role of ABO grouping in the evaluation of hemolytic disease of the fetus and newborn?

What is the role of ABO grouping in platelet refractory evaluation?

How do ABO subtypes cause mixed field reactions in ABO grouping?

How is ABO grouping performed?

What is a mixed field reaction in ABO grouping?

How does a massive transfusion result in a mixed field reaction in ABO grouping?

How does a mixed field reaction in ABO grouping occur following a hematopoietic stem cell transplantation?

How do bacterial infections and malignancies cause mixed field reactions in ABO grouping?

What are limitations of ABO grouping?

What are the methods of ABO grouping?

What is the role of manual tube testing in ABO grouping?

What is the role of column agglutination in ABO grouping?

What is the role of solid phase test systems in ABO grouping?

What are lab resources for ABO grouping?

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