INTRODUCTION TO IMMUNOHEMATOLOGY

DEFINITIONS

Immunity: Protection against infection or disease caused by foreign particles

Immunology: Study of the immune system and the immune response; study of
antigen-antibody reactions in vivo

Serology: Study of antigen-antibody reactions in vitro

Hematology: Study of the cellular components of the blood

Immunohematology (Blood Banking): The detection, identification, and/or

quantitation of antibodies involved primarily with red cells, although white cells and
platelets may also be involved

TYPES OF IMMUNOHEMATOLOGY FACILITIES

Transfusion Service

blood typing

antibody detection and identification

compatibility testing (crossmatching)

blood component therapy (hemotherapy)

transfusion reaction workups

autoimmune hemolytic anemia workups

hemolytic disease of the newborn (HDN) workups

determining Rh immune globulin eligibility

Donor Center (Blood Center)

donor recruitment

donor screening

blood collection

testing (typing, infectious disease screening)

blood component preparation

component preservation

may provide reference lab services

may store rare donor blood

NATURE OF BLOOD BANKING:

Blood banking involves less automation than Hematology and Clinical Chemistry. The
results are not numerical other than grading reactions as 1+, 2+. 3+ and 4+.
There is a heavy emphasis on the thought processes of logic, interpretation, decision making
and problem solving. You need to think about what is happening in the test tube and
whether the patient's red cell antigens or antibodies are involved.
Blood banking can be very rewarding since there are visible life-saving aspects of providing
the patient with the needed blood components. Unfortunately it is also prone to human
error and the consequences of error can be fatal. There are not many places in the clinical
laboratory where a patient may die from an erroneous result, but giving A or B blood to an
O individual can do that.
BLOOD BANK ANTIGENS AND ANTIBODIES
Antigen
Antigens are defined as substances recognized by the body as foreign, causing the body to
produce an antibody to react specifically with it
Characteristics of antigens:
In order to be an antigen to you it must be foreign (not found in the host)!

Autologous antigens are your own antigens (not foreign to you)

Homologous, or allogenic, antigens are antigens from someone else (within the same
species) that are foreign to you

Antigens must be chemically complex.

Proteins and polysaccharides are antigenic due to their complexity. On the other
hand, lipids are antigenic only if coupled to protein or sugar.

Besides being chemically complex, antigens must also be large enough to stimulate
antibody production. Their molecular weight needs to be at least 10,000.

Due to the complexity of these molecules there are specific antigenic determinant
sites, or epitopes, which are those portions of the antigen that reacts specifically with
the antibody.

Factors determining whether an antigen will stimulate an antibody response:

Degree of foreignness. Only human blood is transfused to humans.

Size and complexity. Although red cells are smaller than white blood cells, they tend
to be more antigenic due to the complexity of the antigens on the cell surface. Some
are proteins and others are oligosaccharides.

Dose of antigen administered. How much antigen is the individual exposed to and
what is the frequency of that exposure.

Genetic makeup of host may also dictate whether an antibody is produced. Some
individuals have a greater ability to make antibody and others have the antigen so
they would not make the antibody.

Blood group antigens:

There are over 300 known blood group

antigens

Over 1,000,000 different antigen sites on

each red blood cell.

These antigens are attached to proteins or

lipids on the red cell membrane and are
usually complex sugar groups.

Some stick out far on the red cell

membrane and some are buried within
crypts on the membrane surface.

Antibodies
Proteins produced by lymphocytes as a result of stimulation by an antigen which can then
interact specifically with that particular antigen.
Serum components

Human serum can be separated into albumin and globulin components

Globulins can be separated into several different parts:

a. Alpha 1 and alpha 2 globulins
b. Beta globulins (serum complement)
c. Gamma globulins (immunoglobulins or antibodies)

Parts of an antibody:
1. Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains
2. Light chains - made of kappa or lambda chains
3. Disulfide bonds - hold chains together
4. Hinge region - allows antibody to flex to reach more antigen sites
5. Fab fragments - contains variable portion of antibody: antigen-binding sites
6. Fc fragment - contains constant portion of antibody; also site of complement
activation

Classes of antibodies:
1. IgG - provides long-term immunity or protection
2. IgM - first antibody produced in response to an antigenic stimulus
3. IgA - found in secretions. Protects against infections in urinary, GI, and respiratory
tracts
4. IgE - involved in allergic reactions
5. IgD - not much known about it. Surface receptor of B lymphocytes
6. Most important classes of antibodies in blood banking are IgM and IgG

Characteristics of IgG and IgM antibodies

1. Clinical significance
Clinical of red cell antibodies in blood bank depend on whether they can cause in

vivo hemolysis, which in turn will cause transfusion reactions or HDN

IgG will frequently cause in vivo hemolysis due to antibody coating red blood cells.
IgM, with a few important exceptions, usually does NOT cause in vivo hemolysis.

The most important of these exceptions are ABO antibodies.

2. Size of the antibodies
IgG is relatively small, it is comprised of only 1 immunoglobulin subunit (monomer)
IgM is relatively large, it is comprised of 5 immunoglobulin subunits. (pentamer)

3. Serum concentration
IgG is found in the largest concentration of all immunoglobulins in the plasma.
IgM is found in relatively small amounts
IgG > IgA > IgM

4. Complement activation
IgG = will do it if conditions are optimal
IgM = very good complement activator

5. Placental transfer
IgG is small enough to cross placenta and is the only immunoglobulin capable.
IgM and the other classes do not cross placenta

6. Optimum temperature of reactivity

a. IgG = 37oC
b. IgM = 4 oC (may react at any temperature below 30C)

7. Number of antigen-binding sites

IgG has 2 binding sites
IgM has 10 binding sites

Terms used to describe antibodies

Immunoglobulin:
antibody formed as a result of immune stimulus (exposure to foreign antigen)
Naturally occurring
antibody formed without prior exposure to foreign antigen
Autoantibody:
antibody formed to one's own antigens (abnormal condition)
Alloantibody (unexpected, irregular, atypical):
antibody formed to foreign antigens, but within the same species
Agglutinin:
antibody capable of causing agglutination when react with corresponding antigen
Isoagglutinin:
name commonly given to blood group antibodies anti-A and anti-B
Saline agglutinin:
antibody capable of causing direct agglutination of antigens suspended in a saline
medium without requiring any enhancement techniques
Hemolysin:
antibody capable of causing hemolysis when reacting with corresponding antigen
Cold antibody (cold agglutinin):
antibody whose optimal temperature of reactivity is less than 30oC
Warm antibody:
antibody whose optimal temperature of reactivity is greater than 35oC

Monoclonal antibodies
Monoclonal antibodies react with very specific antigenic determinants and therefore shows
no cross-reactivity. They are not produced in humans or animals, but harvested from cells in
cells grown in tissue culture. The tissue culture cells made from fusion of a plasma cell, which
is the antibody producer and the myeloma cell, which provides longevity and ability to
make large amounts of antibody

Monoclonal antibodies used in most reagent antisera today because contain high
concentrations of highly specific antibodies and lack infectious disease hazards associated
with human-source antiserum.

ANTIGEN-ANTIBODY REACTIONS IN GENERAL

Rules of Thumb For in vivo Antigen-Antibody Reactions
1. If a person's cell have the antigen, the antibody should NOT be present in that
person's serum
2. If an antibody to a blood group antigen is present in the serum of a person, his or her
cells should lack that antigen
3. The antigens are on the cells and the antibodies are in the serum
Stages of Antigen-Antibody Interaction
The first stage is sensitization. Sensitization occurs when antibodies react with antigens on the
cells and coat the cells.

The second stage of the reaction is agglutination. Agglutination occurs when antibodies on
coated cells form cross-linkages between cells resulting in visible clumping.

FACTORS AFFECTING SENSITIZATION (In vivo or in vitro)

1. Specificity depends on the spatial and chemical "fit" between antigen and antibody

2. Since the immunoglobulins and the red cell membranes both have an electrical
charge, there is an optimum pH. pH differences cause differences in chemical
structures of antigens/antibodies, affecting the "fit".

3. The optimum temperature depends on the type of antibody involved. IgG

antibodies react best at 37oC; IgM react best at 4oC.

4. Optimum incubation time: you need to incubate long enough to reach equilibrium,
but not too long

5. The antigen's accessibility is also important since the antibodies must be able to
reach antigens. Those antigens, like the ABO antigens, are on the surface of the red
cell while others may be hidden in the crypts of the cell membrane.

FACTORS AFFECTING AGGLUTINATION IN VITRO

Number of Antigen Sites
The number of antigen sites on the red cell is important since the more antigen sites result in
more antibodies being attached and forming cross-linkages. These cross-linkages result in
agglutination
Size and Structure of the Antibody
The larger antibodies (IgM) can reach between more antigen sites on different red cells and
therefore causing stronger agglutination reactions. IgM antibodies also have more binding
sites to react with antigens and potentially causing cross-linkages between 5 different cells.
Distance between Cells
Centrifugation of the cells attempts to bring the red blood cells closer together, but even
then the smaller IgG antibodies usually cannot reach between two cells. The larger
antibodies, IgM, can reach between cells that are further apart and cause agglutination.

The concept Zeta potential is important to understand why the cells will maintain a
certain distance from each other. Zeta potential refers to the repulsion between the
red blood cells. It is due to an electric charge surrounding cells suspended in saline.

It is cause by sialic acid groups on the red blood cell membrane which gives the cells
a negative charge.

The positive ions in saline attracted to the negatively charged red blood cells.

The net positive charge surrounding cells in saline keeps them far apart due to
repulsion from electric charges

Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists

To overcome zeta potential techniques need to neutralize these charges One of the
common techniques is:
1) Add albumin to test mixture
2) OH- groups of albumin neutralize positive charge

Antigen-Antibody Ratio
The optimum ratio is 80 parts antibody to 1 part antigen. There are specific terms for
variations in this ratio.
Prozone - antibody excess:
Antibodies saturating all antigen sites; no antibodies forming cross-linkages between
cells; no agglutination
Zone of equivalence:
antibodies and antigens present in optimum ratio, agglutination formed
Zone of antigen excess (Post-zone):
too many antigens - any agglutination is hidden by masses of unagglutinated
antigens

In order to get optimum antigen-antiboy concentration washed 3% saline suspension of red

cells to mix with our reagents.

LABORATORY TECHNIQUES: REAGENTS, REACTION GRADING; CELL WASHING; SAMPLE

COLLECTION
BLOOD BANKING REAGENTS
The techniques used involves mixing antigens, usually on red blood cells with antibodies. The
environment where this reaction occurs can range in temperature from 4 oC to 37oC. With
the most common being room temperature for ABO and the initial Rh(D) testing and 37oC
when screening and identifying other clinically significant antigen-antibody reactions. In a
number of situations we are looking for particular antigens on the red cell such as looking for
A or B antigens to determine a patient's ABO type. Other times we may be looking for
particular antibodies that may cause transfusion reactions or HDN. Depending on whether
we are looking for a particular antigen or antibody will determine what reagents we are
going to use. If we are looking for an A antigen on a patient's red cells, we will use known
anti-A reagent that will cause agglutination of the A antigens on the red cells. If the patient
has on B antigens or no ABO antigens, as in the case of an O individual, their cells will not
agglutinate with anti-A reagent.
Sources of Antigen Testing:
The red cells may either reagent red cells with known antigens, patient red cells, or donor
red cells. The reagent red cells are commercially prepared and have all the red cell
antigens identified.
When we use red cells where the antigens have already been determined, we can identify
the possible antibodies present. The reagent cells used for blood banking include the
following:

A1 and B cells for confirmation of the ABO type in all patients and donors other than
newborn babies

Antibody screening cells are O cells that have been studied to determine the
presence of a number of antigens for specific antibodies that are known to cause
transfusion reactions and hemolytic disease of the newborn. The antibody screening
technique is part of all compatibility tests done before blood is transfused. Some of
the more common antibodies detected are anti-D, anti-E, anti-K.

Antibody identification cell panel are again O cells with the specific antigens known.
Usually there are between 8 and 12 different cells in a cell panel. The pattern of
positive and negative reactions help identify the antibody.

Sources of Antibody for Testing

Antibody is found in serum. If it is the patient's serum that is being tested, we do not know
what antibody may be present so we are using one of the 3 types of reagent cells listed
about. If the serum is commercial reagent, the specific antibody present is already known.
The commercial serum reagent is referred to as antisera. Therefore, we use Anti-A antisera to
determine if a patient or donor is Type A. If we are trying to determine if the patient is Rh+ or
Rh-, we will use anti-Rh (D) antisera.
Known Source with known

Unknown components

components

The source is either the patient or the donor

Antigen

Reagent Red Blood Cells

Patient or Donor red blood cells

Antibody

Commercial Antisera

Patient or Donor serum or plasma

ABO/Rh(D) typing

Antigen typing from other blood group systems such as Rh antigens other than D, Kell,
Kidd, and Duffy

Antibody screening for antibodies form to blood group antigens other than A and B

Antibody identification to determine the specific antibodies detected in the

antibody screening

Crossmatch, or compatibility testing, which determines whether donor blood can

probably be safely transfused to the recipient

Procedure

Purpose

Source of Antigen

ABO/Rh typing

Detects A, B, and D

Patient's RBC's

Detects antigens of
Antigen typing

other blood group

Source of Antibody
Commercial anti-A, anti-B,
and anti-D
Commercial antisera to the

Patient's RBC's or

specific antigens

systems (examples: K, E, Donor RBC's

(examples: anti-K, anti-E,

C, Fya, Jka)

anti-C, anti-Fya, anti-Jka)

Detects antibodies with

Antibody screening specificity of RBC
antigens

Commercial
Screening Cells

Antibody

Identifies the specificity Commercial Panel

identification

of RBC antibodies

Cells

Patient's serum

Patient's serum

Determine compatible
Crossmatch

in donor and patient

before transfusion

Donor RBC's

Patient's serum

GRADING REACTIONS
Grading agglutination reactions gives an indication of the relative amount of antigen or
antibody present. All tubes tests should be graded. The technique used in the resuspension
of the cells will affect the grading of the reaction.

1. Resuspension Procedure:
read only one tube at a time
hold tube upright
very gently shake the tube and observe how

the cells come off the cell button

2. Grading Reactions
swirling off = negative
coming off in chunks = positive
continue shaking till all cells resuspended
tilt tube, read and grade reaction

3. Grading system:
4+ = solid clump
3+ = several large clumps
2+ = small to medium sized clumps; clear background
1+ = small clumps; cloudy background
+w = tiny aggregates; cloudy background
+ micro = positive upon microscopic examination only
MF = mixed field. Small clumps amidst many unagglutinated cells.
hem = hemolyzed (a positive reaction)
0 = negative, no agglutination (Never use - for negative)

WASHED CELL SUSPENSIONS

3% Red Blood Cell Concentration in Saline
Between 2-5% cell suspension provides optimum antigen concentration for the tube
method for red blood cells typing.
Washing Red Blood Cells Before Making the 3% Suspension
The purpose of washing the red blood cells is to remove plasma, which contains substance
that may interfere with antigen-antibody reaction. The following may be in the plasma and
may interfere with testing:

Soluble antigens such as A and B may be present and neutralize reagent.

Interfering proteins such as Wharton's jelly that is seen in newborn cord blood,
cold-acting autoimmune antibodies, and increased levels of immunoglobulins that
may cause either agglutination or rouleaux

Hemolyzed red blood cells due to a difficult draw will interfere in your grading
interpretation of hemolysis

Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.

Good Technique when washing and making a 3% cell suspension involves the following:

Place 1 to 3 drops of blood in the tube

Fill the tube 3/4 full of saline (there will be less splattering in the centrifuge)

Centrifuge long enough spin to pull most of cells into a button in the bottom of tube.

Decant the saline completely

Shake the tube to resuspend cell button before washing the cells again.

COLLECTING BLOOD BANK SAMPLES

Samples for Blood Bank Testing

Most samples for blood banking are drawn into a red top tube - serum is preferred.
(No clot activation tube should be used since the patient's red cells may also need to
used and no other chemicals should be present)

A few tests require an EDTA sample if complement is not to be activated.

Serum must be tested while fresh to ensure good complement activity.

Antigens on cells are stable longer (months) in a clot tube.

QUALITY ASSURANCE IN BLOOD BANKING

Quality Programs
When discussing Quality Programs it includes:

Quality Control

Quality Assurance

Quality Improvement
a. Responsibilities of the blood product requirements (anticoagulants and
preservatives, shelf life etc.)
b. Specific requirements related to independent quality control and quality
assurance for overall quality of blood products and the processes related to
dispersion of those products.

Organization: active support of quality systems must be place for the following procedures;

SOP's - Standard Operating Procedures

Training plans and development of procedures

Approval of lot release of reagents and quality control reagents

Review and approval of practices relating to personnel, equipment, selection of

suppliers, process control, final inspection and handling nonconforming components,
methods in place for handling incidents, errors, and accidents.

Equipment

Validation of new equipment

Calibration and preventative maintenance including standard equipment like

refrigerators, complex equipment and computer systems

Continual monitoring of blood bank refrigerators extremely important in both blood

centers and transfusion services

Supplier issues
Specific criteria is set for both the specificity and the potency of the reagents. For example,
anti-A will only react with A cells and will demonstrate a 3-4+ reaction with A1 reagent cells.
Other than very rare antisera, routine blood bank reagents CANNOT be used after the
expiration date. Daily quality control testing needs to be done for ABO, Rh, and Antibody
Screening. Typing antisera for other red cell antigens will be tested when performing the
antigen testing on the patient and donors since this test is not done each day.

Each manufacturer is required to provide a product insert for each reagent. The product
insert needs to include the following:

Reagent's description

Proper use procedures

What to expect in regards to performance

Limitations

When a new shipment of reagents is received, the product insert needs to be reviewed and
any changes in the standard operating procedure. Total compliance with the
manufacturer's directions must be followed.
Process control, final inspection, and handling elements

Development of SOP

Control of changes in policies, processes or procedures

Acceptance testing to new/revised software involved in blood bank procedures

Validation of new policies, processes or procedures

Monitoring and control of production processes

Participation in proficiency testing appropriate for each testing system in place

Established QC procedures for supplies and equipment

Supplier qualifications and product specification need to be in place

Control processes for nonconforming blood and blood components and products.

Documents and records (4 levels)

1. Policies (Level 1) relate to "What to do" in response to various situations
2. Processes (Level 2) relate to "How it happens"
3. Procedures (Level 3) "How to do it"
4. Forms/Records, Supporting Documents etc. (Level 4) that need to be completed
when you are performing the procedures and following the processes and policies.
Incidents, errors, and accidents

If incident occurs, the severity of the incident is determined by the facility

If it is a one-time incident: "What is the likelihood it will happen again?" and what to
do about it if it could happen again

If there are multiple similar incidents "What might be the root cause?"

Develop processes for continuous improvement to help eliminate both one-time

incidents and multiple similar incidents.

Assessments: internal and external

A Quality Assessment Program includes both internal and external assessment:

Internal assessment includes blood usage review committees within a hospital

(transfusion audits) or institutional QA teams

External assessments includes inspections, surveys, proficiency surveys performed by

agencies

Process improvement

Corrective actions that are educational not punitive

Timely corrections

Yearly reports relating to QA and CQI committees

ANTIGLOBULIN TESTING
The antiglobulin test, is to detect clinically significant unexpected antibodies that have
coated cells either in vivo or in vitro.
Principle of Antiglobulin Test
Red cells coated with complement or IgG antibodies do not agglutinate directly when
centrifuged. These cells are said to be sensitized with IgG or complement.

IgG-coated red cells

Complement-coated red cells

In order for agglutination to occur an additional antibody, which reacts with the Fc portion
of the IgG antibody, or with the C3b or C3d component of complement, must be added to
the system. This will form a "bridge" between the antibodies or complement coating the red
cells, causing agglutination.

The light-colored antibody molecule represents the

anti-globulin reagent that binds with the complement
attached to the red blood cells.
Traditionally rabbits were immunized with human gamma globulin to make this antibody to
IgG or C3d.
Types of Antiglobulin Tests

Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's

cells in vivo.

Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation step so antibodies in serum
can react with antigens on cells in vitro. After washing the cells antiglobulin reagent is
used to detect antibody coating of cells.

Reagents
Production Methods of Anti-Human globulin (AHG or Coombs) Reagent

May be made by injecting rabbits with purified human IgG or C3, then harvesting the
antibodies produced by the rabbit.

Monoclonal technology may be used to make monoclonal antiglobulin reagent

Specificity types
Polyspecific Anti-human Globulin: blend of Anti-IgG & Anti-C3b, -C3d
Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone
Note: Reagent does not contain antibodies to IgM. Information about IgM coating of cells
comes from the presence of C3 coating the cells since IgM is a strong complement
activator.

Interpretation of Antiglobulin Tests

Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is
based on the following
Positive Antiglobulin Test

Wash cells three times to

remove unbound antibody
Only antibody attached to
the cells remain

Add Anti-Human Globulin

Visible Agglutination

Summary of the reaction:

1. Antigen-antibody reaction, which can take place either in vivo or in vitro
2. Cells coated with IgG antibody and/or complement
3. Cells washed 3-4X to remove unbound or free antibody or complement
4. The only antibody or complement left is attached to red cells
5. AHG (Coombs serum) added
6. Antibodies in Coombs serum react with antibodies or complement on red cells,
causing agglutination
7. If no agglutination add Coombs control reagent cells* (CCC).

Negative Antiglobulin Test

Antibodies are not attached

to the antigens during
incubation.

Wash the cells 3 times to remove any unattached antibodies.

Add Anti-human globulin

No visible agglutination and therefore a negative test

Add Coombs Control Check

Cells
Check cells agglutinated and
original test cells remain
unagglutinated.
Coombs Control Agglutinated by Anti-Human Globulin
1. NO antigen-antibody reaction occurred.
2. No attachment of antibody or complement to red cells
3. Cells washed three to four times = all plasma or serum antibodies was washed away.
4. Anti-human globulin, Coombs, serum added, which would react with
antibody-coated cells if present.
5. But no agglutination, because no antibodies or complement on red cells for the
anti-human globulin, Coombs, serum to react with
6. Must add Coombs Control Check Cells to negative reactions
CCC are cells coated with IgG antibody
Will react with antibodies in Coombs serum still "floating around" in the tube.

 Agglutination will now result

Agglutination following addition of CCC verifies negative result

False Positives and Negatives

Anti-human globulin (Coombs) antibody prefers to react first with free antibody and
then with antibody-coated cells

If the free antibody has already reacted with the anti-human globulin, no free
Coombs serum to react with Coombs Control Check Cells (CCC)

False negatives that are detected by negative Coombs control cells includes:
Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension
that are available to neutralize the AHG (Coombs serum) so it will not react with red cells
bound with antibody.
Delay in adding Coombs serum after washing step will lead to antibody eluting off,
detaching from, cell while cells are sitting in saline. Now free antibody present in the saline
neutralizes AHG, Coombs, serum so it will not able to react with cells bound with antibody.
Small fibrin clot among the cells that were not washed away will have immunoglobulins and
complement present. The antibodies and complement in the fibrin clot neutralizes AHG,
Coombs, serum leading to a negative test.
Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be
detected by a negative reaction when adding Coombs Control Check Cells.
There are also false negatives NOT detected by negative Coombs Control Cells that
include:

Too heavy cell suspension

Delay during cell washing procedure, which can lead to antibody eluting off cells
while they are sitting in saline and then the antibody is washed away during the
remaining washes

Improper centrifugation can either lead to lost of cells during the washing or the
need to shake too hard during resuspension.

False positives

False positive reactions can also occurred when performing this test. These would not be
detected by the use of Coombs Control Check Cells. Reasons for a false positive reaction
could be the following:

Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT)

Spontaneous agglutination (cells heavily coated with IgM)

Non-specific agglutination ("sticky cells")

All of these reactions would be the result of cells appearing to agglutinate, or actually
agglutinating. Using a clotted tube for the DAT may allow complement to become
activated in the test tube since calcium ions are free to be part of the complement
cascade.

Direct Antiglobulin Testing

Principle
The Direct Antiglobulin Test detects in vivo coating of patient cells - either IgG antibodies,
complement, or both. Within the patient's blood stream antibodies attach to their specific
antigens on the red blood cells. This happens in Hemolytic Disease of the Newborn (HDN), in
transfusion reactions, and in autoimmune hemolytic anemia. Certain drugs are also known
to activate complement and it can also coat the cells in vivo.
When the blood is drawn the antibodies and/or complement have already attached to the
red cells. Those red cells from the EDTA tube will be washed 3 or more times and a 3% cell
suspension is made. A drop of cell suspension and the anti-human globulin are mixed in a
tube and then centrifuged. If agglutination occurs, it indicates the patient has a positive
Direct Antiglobulin Test due to antibody coating the cells in vivo. If IgM antibodies involved,
DAT will be identified by complement binding since the polyspecific antisera has both
anti-IgG and anti-C3. The meaning of a positive DAT is found under Clinical Causes of a
Positive DAT.

Technique

1. Add 1 drop of patient cells from EDTA tube to tube

2. Wash 3-4X to remove plasma antibodies and make 3% cell suspension.
3. Add a drop of 3% cell suspension to a clean, labeled tube.
4. Add drop of Polyspecific AHG (Coombs serum) to the tube.
5. If test is positive with polyspecific reagent, set up again using monospecific reagents
to see if it is antibody or complement or both coating the cells.
6. We want to make the test as sensitive as possible, so allow all negatives to incubate 5
minutes to enhance complement coating.
7. Read all negatives microscopically to detect weak coating.
8. False pos. possible if red top tube used to collect sample.
In-vitro complement coating frequently happens when sample clots or cools down

due to weak cold-acting auto-antibodies like anti-I

Prevent by using lavender top tube to tie up Ca+ and Mg+ ions and prevent

complement activation in vitro.

9. Whenever positive DAT is obtained, obtain the following information on the patient:
Diagnosis (particularly autoimmune hemolytic anemia, HDN, transfusion reactions)
Medications
Recent transfusion history of both red cell and plasma components
Other lab values that may indicate red cell destruction (hematocrit, bilirubin, LDH)

Clinical Causes of Positive DAT

1. Normal patient with unexplainable reasons for a positive DAT
2. Transfusion reaction work-ups require that a DAT be performed on the
post-transfusion specimen since the patient's antibodies and/or complement may
coat the transfused donor cells. These reactions are usually a weak positive or mixed
field agglutination since you are testing a mixed population of patient and donor
cells.
3. Warm-acting Autoimmune disease, can lead to patient antibodies coating their own
cells. This results in a strong positive result. A cold-acting autoimmune hemolytic
anemia would be due to IgM antibodies that in turn activate complement. The
complement-coated cells would then be detected by the antiglobulin reagent.
4. Hemolytic disease of newborn is due to the mother's IgG antibodies crossing the
placenta and coating the antigens on the fetal red cells. Cord blood collected at

time of birth would be tested, but may need to followed up by a heel stick of EDTA
blood. The reaction is usually a strong positive.
5. Complement on the red cells may be the result of antigen-antibody reactions which
may involve red cells. Complement can also be activated if immune complexes are
present in the plasma and the activated complement attaches to red cells.
Complement can also become activated by the C3 by-pass mechanism and the
lectin activation process. Again once the activation of complement occurs in the
blood stream, it can become attached to the red cells.
6. Passive transfer of antibody from donor units of plasma or platelets may attach to the
patient's red cells since recipients are given ABO compatible blood but other
unexpected red cell antibodies may not have been detected. These antibodies in
donor plasma can coat antigens on patient cells when group AB, A, or B receive
group O plasma products (and possibly platelets)
7. ABO mismatched transplants of particularly bone marrow can occur if an universal
"O" donor bone marrow is given to an A, B, or AB recipient. "Passenger lymphocytes"
from group O donor organ make antibody to group AB, A, or B recipient cells and
these in turn can activate complement. It is also more common for "O" individuals to
make an IgG anti-A,B, which would also contribute to a positive DAT.
8. Sensitization of red cells due to medications like penicillin and cephalosporins that
usually involves non-specific coating of red cells. Other drugs like tetracyclines,
antihistamines and sulphonamides cause the development of immune complexes
that are capable of activating complement. Some drugs, like ibuproten, levodopa
and methyldopa, are also known to cause autoimmunity. If a patient has a positive
DAT, drug-induced problems should be considered.
Indirect Antiglobulin Testing
The indirect antiglobulin test is one of the most important and commonly used techniques in
immunohematology. It is used to commonly for the detection of:

Weak D's in donor bloods and pregnant females of individuals who type D (-) at room
temperature when doing ABO and Rh typing.

The presence or absence of antigens on a person cells from particularly the Kell, Kidd,
and Duffy Blood Group systems.

Unexpected, clinically significant antibodies in the patient's serum during the

antibody screening procedure and the antibody identification procedure

Principle
The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells. This
is done when sensitization does not lead to direct agglutination. This occurs when there are
too few antigens on the red cell, too few antibodies in the serum and those antibodies are
in the IgG class.
Summary of the Indirect Antiglobulin Technique
1. Incubate cells with serum at 37oC for 15 to 30 minutes.
2. After incubation wash the cells three to four times.
3. Add AHG, Coombs reagent, centrifuge and read for agglutination.
4. If the test is negative, add Coombs Control Check Cells to check for false negatives.
Screening Serum for Unexpected Antibodies Procedure
1. Involves patient serum plus reagent red cells (Screening Cells)
The patient's serum potentially has unknown antibody.
Screening Cells have known antigens for common clinically significant antibodies.

2. If there is agglutination after Coombs step with either (or both) Screening Cells,
patient has an unexpected antibody.
3. If antibody screen positive, must do additional tests to specifically identify antibody
The uses for antibody screen are:

Testing donor plasma to make sure no unexpected antibodies will be transfused to

the recipient.

Testing recipient serum before transfusion to make sure patient has no unexpected
antibodies to react with donor cells.

Testing maternal serum to make sure pregnant mother has no antibodies to react
with fetal cells causing hemolytic disease of the newborn.

Red Cell Antigen Typing

Red cell antigen typing involves patient cells plus reagent antiserum. The patient's cells are
the unknown antigen and the reagent antiserum is the known antibody. The antiglobulin
technique is used for antigen typing for a weak D and a number of other clinically
significant antibodies like the Kell, Kidd, and Duffy antibodies. If there is agglutination after
the addition of anti-human globulin, patient cells had that specific antigen.The specific
procedure varies depending on what antigen is being tested for, and what brand of
antiserum is being used.
Uses for red cell antigen typing are:

Typing donors for antigen if patient has antibody.

Verifying that patient is negative for antigen if he/she has made the antibody.

Typing patient to see what antigens are lack, so can predict what antibodies is
capable of making, if they seem to be likely to make additional antibodies.
Controls

When performing red cell antigen testing always run known positive and negative controls.
This will verify that antiserum is acting properly. The positive control should be heterozygous
for the antigen to ensure antiserum is capable of detecting weaker antigens. For example,
when performing antigen typing for K, you would want a cell that is K+ and k+.

POSITIVE CONTROL

NEGATIVE CONTROL

PATIENT CONTROL

PATIENT TEST

Cells without Antigen

Patient Cells

Patient cells

2+

0 / MF

Reagent Antiserum

Reagent Antiserum

Rh control

Reagent. Antiserum

Heterozygous Positive
cells

May be Positive (2+)

Should be at least 2+

Should be Negative

Should be Negative

Negative or Mixed
Field

GENETICS IN BLOOD BANKING

Mendelian Inheritance and Significance Terms
Basic Principles:
1. Each parent contributes 1/2 of the genetic information.
2. The genetic information is contained on chromosomes composed of DNA
3. Humans have 23 pairs of chromosomes
a. 22 matched (autosomal) chromosomes and
b. 1pair of sex chromosomes (females have 2X and males XY).
4. Examples of Chromosome locations for common Blood Groups are as follows:
System

Common Genes

Located on Chromosome

ABO

A, B, O

MNSsU

M, N, S,s,U

P1

22

Rh

D, C, E, c, e

Kell

K, k, Kpa, Jsa,Kpb, Jsb

Lewis

Le, le

19

Duffy

Fya, Fyb, Fy3

Kidd

Jka, Jkb

18

Xg

Xga

5. Genes are the units of inheritance within the chromosomes.

6. At each location, or loci, on the chromosomes there are possibilities of different forms
of the genes, these different forms are called alleles.
(For example ABO Blood Group System, there are A1, A2, B, and O as common alleles.
or allelic genes)
7. When the inherited alleles are the same the person is homozygous such as OO, when
the individual inherits 2 different alleles such as AO, they are heterozygous for both A
and O genes.
8. On occasion we will see examples of dosage where some antibodies will react more
strongly with homozygous cells than with heterozygous cells. For example, an anti-E
that reacts as a 3+ with EE cells and only 1+ with Ee cells.
9. A Punnett Square is used to determine the inheritance possibilities for a particular
mating. For example if the mother's genotype (genes) are AO and the father's
genotype (genes) are BO. In this example there three heterozygous possibilities AB,
AO, and BO and one homozygous possibility OO

Dad

AB

AO

BO

OO

Mom

10. In the above Punnett Square, the AB genotype will have both A and B antigens,
therefore the phenotype is AB since both are expressed. AO and BO genotypes will
demonstrate only the A and the B antigens respectively and therefore the
phenotypes are A and B respectively. The individual that is OO will have the O
phenotype.
11. A and B genes are dominant, or co-dominant, and the O gene is recessive. The
dominant genes will be expressed if present. Recessive genes will only be expressed if
they are homozygous.
12. Most Blood Group genes are co-dominant and therefore will be expressed if present.
Mitosis and Meiosis
Two kinds of cell division:
Mitosis is cell division that leads to two identical cells that has the same number of paired
chromosomes. (In humans there are 23 pairs or 46 chromosomes)
Meiosis is the cell division that occurs when gametes (sperm and eggs) are formed and will
not have pairs of chromosomes. (In humans there will be 23 chromosomes in the sperm that
will match up with the 23 chromosomes in the egg when fertilization occurs to form the
gametocyte.). The sex of the child is determined by the X and Y chromosomes. Males
provide either X or Y chromosome and females provide only provide X chromosomes.
Genes that are found only on the X chromosome are said to be sex-linked. Genes found on
the other 22 pairs of chromosomes are autosomal.
Genotypes, Phenotypes, Amorphs, and Pedigree Charts
Here is a pedigree chart for three generations. The ABO
phenotypes are listed for the known blood types.

The mother in the first generation has the AB genes

since her phenotype is AB.

In the mating for the second generation, the

genotype for the father could either be BB or BO

since his father's phenotype is unknown. It would appear the mother is AA since both
her parents are A, but.....

There are no O individuals in above example but O is considered an amorph since it

has no detectible traits. The lack of D antigen is also considered as an amorph since
no reaction with anti-D indicates the individual is D negative. These two examples are
recessive genes that need to be homozygous for it to be demonstrated.

Other Concepts Relating to Blood Group Genetics

Contributions of Blood Genetics to the Field of Human Genetics
Certain characteristics that make Blood Genetics useful for the field of human genetics
1. Simple and unquestionable pattern of inheritance
2. Can test or determine the phenotypes readily
3. More than 1 allele occurring fairly frequently
4. Environment does not affect the expression of the genes.
Some discoveries that were found in blood genetics:

Multiple alleles seen in ABO system

Population Genetics
Linkage
Linkage between the secretor genes with the Lutheran genes on the same chromosome
was already noted.
1. We now know that the D gene is closely linked to the Cc and Ee genes. The most
frequently inherited Rh positive set of genes is CDe and the most frequent Rh negative gene
is cde or ce since d is an amorph.
2. The MNSs genes are also linked, MS, NS, Ms, Ns leading to a difference between the
expected frequency and the observed frequency.
Expected frequency

Observed frequency

MS = 0.53 (M) X 0.33 (S) = 0.17

0.24

Ms = 0.53 (M) X 0.67 (s) = 0.36

0.28

NS = 0.47 (N) X 0.33 (S) = 0.16

0.08

Ns = 0.47 (N) X 0.67 (S) = 0.31

0.39

Silent Genes
As indicated already there are some amorph blood group genes that exist and lead to
none expression of a blood antigen. The following are some examples of silent genes.
Blood Group Gene

Blood Group System

Homozygous Phenotype

ABO

Oh or Bombay

Rh

Rhnull

Ko

Kell

Knull

Lu

Lutheran

Lu(a-b-)

Jk

Kidd

Jk(a-b-)

Fy

Duffy

Fy(a-b-)

=
r

Public versus Private Genes

Public Genes are found in most of the population. In the Kell Blood Group System, the
Kpb is found in close to 100% of the population

Genes that are very rare are referred to private genes. Kpa is very rarely found (2.3%
in whites and almost never in African Americans).

Paternity Testing
Today most paternity testing is done using the following technology:

Red Cell Testing for: ABO, MNSs, Rh, Duffy, Kidd, Kell

White Cell Testing using HLA antigens

DNA testing

ABO BLOOD GROUP SYSTEM

Blood group system
A series of antigens exhibiting similar serological and physiological characteristics,
and inherited according to a specific pattern.
Importance of the ABO system
Most important (clinically significant) Blood Group System for transfusion practice Why?
This is the only blood group system in which antibodies are consistently, predictably, and
naturally present in serum of people who lack the antigen. Therefore ABO compatibility
between donor and recipient is crucial since these strong, naturally occurring A and B
antibodies are IgM and can readily activate complement and cause agglutination. If ABO
antibodies react with antigens in vivo, result is acute hemolysis and possibly death.
Indications for ABO grouping:
ABO grouping is required for all of the following individuals:

Blood Donors Since it can be life threatening to give wrong ABO group to patient.

Transfusion recipients Since we need to know the donor blood is ABO compatible.

Transplant Candidates and Donors ABO antigens are found in other tissues as well.
Therefore the transplant candidates and donors must be compatible.

Prenatal Patients To determine whether mothers may have babies who are suffering
from ABO-HDN.

Newborns (sometimes) If the baby is demonstrating symptoms of Hemolytic Disease

of Newborn, the ABO group needs to be determined along with Rh and others.

Paternity testing Since the inheritance of ABO Blood Group System is very specific, this
serves as method to determine the likelihood the accused father is the father or not.

Landsteiner's rules for the ABO Blood Group

1. A person does not have antibody to his own antigens
2. Each person has antibody to the antigen he lacks (only in the ABO system)
3. Below are the four blood groups and the antigens and the expected,
naturally-occurring antibodies present.

ABO Typing
ABO typing involves both antigen typing and antibody detection. The antigen typing is
referred to as the forward typing and the antibody detection is the reverse typing

The forward typing determines antigens on patient's or donor's cells

a. Cells are tested with antisera reagents anti-A, anti-B, (donor cells anti-A,B)
b. Reagents are made from monoclonal antibodies.
c. One advantage of the monoclonal antibodies are the antibody strength.
d. Another advantage of monoclonals: human source reagents can transmit
infectious disease (hepatitis).

Reverse typing determines antibodies in patient's or donor's serum or plasma

a. Serum tested with reagent A1 cells and B cells
b. Reverse grouping is also known as backtyping or serum confirmation
Routine ABO Typing

Reaction of Cells Tested With

Reaction of Serum Tested Against

Reverse ABO Group

Anti-A

Anti-B

A1 Cells

B Cells

AB

Discrepancies in ABO typing

1. Results of forward and reverse typing must agree before reporting
2. If forward and reverse do not agree, must identify cause of discrepancy
3. If cannot resolve discrepancy, must report blood type as UNKNOWN and give group
O blood
Characteristics of ABO antigens
ABO antigens are glycolipid in nature, which are oligosaccharides attached directly to lipids
on red cell membrane. These antigens stick out from red cell membrane and there are
many antigen sites per red blood cell. Besides the presence on red cells, soluble antigens
can be present in plasma, saliva, and other secretions. These antigens are also expressed on
tissues other than red cells. ABO antigens are only moderately well developed at birth.
Therefore ABO-HDN not as severe as other kinds.

Characteristics of ABO antibodies

1. These are expected naturally occurring antibodies occur without exposure to red
cells containing the antigen.
2. IgM antibodies, predominantly
3. They react in saline and readily agglutinate. Due to the position of the antigen and
the IgM antibodies it is not necessary to overcome the zeta potential.
4. Optimum temperature is less than 30oC, but reactions take place at body temp
5. Not only are these antibodies expected and naturally occurring, they are also
commonly present in high titer, 1/128 or 1/256.
6. They are absent at birth and start to appear around 3-6 months as result of stimulus by
bacterial polysaccharides. (For this reason, newborn blood is only forward typed.)
ABO INHERITANCE
gene: determines specific inherited trait (ex. blood type)
chromosome:
unit of inheritance. Carries genes. 23 pairs of chromosomes per person, carrying
many genes. One chromosome inherited from mother, one from father
locus: site on chromosome where specific gene is located
allele: alternate choice of genes at a locus (ex. A or B; C or c, Lewis a or Lewis b)
homozygous: alleles are the same for any given trait on both chromosome (ex. A/A)
heterozygous: alleles for a given trait are different on each chromosome (ex. A/B or A/O)
phenotype: observed inherited trait (ex. group A or Rh positive)
genotype:
actual genetic information for a trait carried on each chromosome (ex. O/O or A/O)
dominant:
the expressed characteristic on one chromosome takes precedence over the
characteristic determined on the other chromosome (ex. A/O types as A)
co-dominant:
the characteristics determined by the genes on both chromosomes are both
expressed - neither is dominant over the other (ex. A/B types as AB)
recessive:
the characteristic determined by the allele will only be expressed if the same allele is
on the other chromosome also (ex. can type as O only when genotype is O/O)

ABO Genes
The A and B genes found on chromosome 9. We inherit one gene (allele) from our father
and one from our mother. The two co-dominant alleles are A or B. Anytime an individual
inherits an A or B gene it will be expressed. The O gene is not expressed unless this gene is
inherited from both parents (OO). Therefore O gene is recessive.
Below is example of two individuals who are A. One inherited only one A gene along with an
O gene and is therefore heterozygous. The other inherited 2A genes and is homozygous.

1 = A/A

2 = A/O

Homozygous A

Heterozygous A

Phenotype A

Phenotype A

Genotype A/A

Genotype A/0

Can Contribute Only an A Gene to Offspring

Can Contribute A or O Gene to Offspring

Inheritance Patterns
1. A/A parent can only pass along A gene
2. A/O parent can pass along either A or O gene
3. B/B parent can only pass along B gene
4. B/O parent can pass along either B or O gene
5. O/O parent can only pass along O gene
6. AB parent can pass along either A or B gene
ABO phenotypes and genotypes
1. 1. Group A phenotype = A/A or A/O genotype
2. 2. Group B phenotype = B/B or B/O genotype
3. 3. Group O phenotype = O/O genotype
4. 4. Group AB phenotype = A/B genotype

BIOCHEMISTRY OF THE ABO SYSTEM

The ABO antigens are terminal sugars found at the end of long sugar chains
(oligosaccharides) that are attached to lipids on the red cell membrane. The A and B
antigens are the last sugar added to the chain. The "O" antigen is the lack of A or B antigens
but it does have the most amount of next to last terminal sugar that is called the H antigen.

Production of A, B, and H antigens

The production of A, B and H antigens are controlled by
the action of transferases. These transferases are
enzymes that catalyze addition of specific sugars to the
oligosaccharide chain. The H, A, or B genes each
produce a different transferase, which adds a different
specific sugar to the oligosaccharide chain.
1. Precursor chain of sugars is formed most frequently as
either Type 1 or 2 depending on linkage site between
N-acetylglucosamine (G1cNAc) & Galactose (Gal).
2. H gene causes L-fucose to be
added to terminal sugar of
precursor chain, producing H
antigen (Type 2 H antigen
saccharide chaine).

3.

Either A gene causes

N-acetyl-galactosamine to be
added to H substance,
producing A antigen, (shown in
this diagram) or

4. B gene causes D-galactose to be

added to H substance, producing
B antigen.

5. If both A and B genes present, some H-chains converted to A antigen, some converted
to B antigen.
6. If H gene absent (extremely rare), no H substance can be formed, and therefore no A or
B antigen. Result is Bombay blood group.

Bombay blood group

Bombay blood group lacks H gene and therefore cannot make H antigen (H substance).
Since the H substance is the precursor for the A and B antigens, these antigens also are not
made. The cells type as O and the serum has anti-A, anti-B, and anti-H since the individual
lacks all of these antigens. Anti-H agglutinates O cells. The only cells Bombay individuals do
not agglutinate are from other Bombay blood people since they lack H antigen,
Bombay phenotype

Para-Bombay phenotype

H antigen is not expressed on

H antigen is weakly expressed on RBCs.

RBCs.

H antigen may be present or absent in

H antigen is not found in saliva.

saliva.

Serum contains anti-H.

Serum contains anti-H.

Genotype: h/h se/se

Genotype: (H), Se/Se or Se/se or se/se

Subgroups of A and B
The subgroups of A and B are caused by decreased
amounts of antigen on red cells.
The most common are subgroups of A. Approximately 80%
of the A's and AB's have a normal expression of A1. Most of
the other 20% are either A2 or A2B. This subgroup has fewer
H chains converted to A antigen result is more H chains
on red cell, and fewer A antigens.
There are other, weaker subgroups of A exist: A3; Aint; Am, Ax; Ael. Each has a different pattern
of reacting with anti-A, anti-A, and various antibody-like substances called lectins.
Lectins are extracts of seeds of plants that react specifically with certain antigens.

Ulex europaeus, or lectin H, which agglutinates cells that have H substance.

Dolichos biflouros, or lectin A1, which agglutinates cells with A1.

Lectin-H reacts strongest with O cells, which has a high concentration of H antigen, and
weakest with A1 cells, which have a low concentration of H.
Bombay

Lectin

O cells

A2 cells

A2B cells

B cells

A1 cells

A1B cells

Lectin-H

4+

3+

2-3+

2+

1+ / 0

1+ / 0

Lectin-A1

4+

4+

cells

Differentiating Subgroups of A
1. Use lectin-A1 to differentiate A1 cells from all others - will agglutinate only A1 cells
2. Look for weaker or mixed field reactions
3. Look for anti-A1 in serum (serum reacts with A1 cells but not A2 cells)
4. Look at strength of reactions with anti-A,B or with lectin-H
GROUP

A1

A2

A3

Ax

Reaction with anti-A

4+

4+

mf

Reaction with anti-A,B

4+

4+

mf

2+

Reaction with Lectin-A1

4+

Reaction with Lectin-H

0-w

1-2+

2+

2-3+

Presence of anti-A1

no

may

may

often in serum

Problems with Ax:

Because Ax cells initially type as O and serum usually has anti-A1, (along with anti-B), patient
forwards and reverses as O. Unfortunately when Ax is transfused into an O individual, the
naturally occurring anti-A,B will react with the donor cells causing a transfusion reaction.
Therefore: To prevent Ax from being erroneously typed as O, confirm all group O donors with
anti-A,B.
Rh SYSTEM
Rh Antigens
D (Rh) is the most important antigen after A and B antigens. Unlike the anti-A and anti-B
antibodies, anti-D antibodies are only seen if a patient lacking D antigen is exposed to D+
cells. The exposure of D+ cells usually occurs through pregnancy or transfusion.
There are at over 50 Rh antigens that have been identified including those that are either
combinations of these antigens or weak expressions of the above antigens, but most Rh
problems are due to D, C, E, c or e.
Characteristics of Rh antigens
The Rh antigens together are proteins of 417 amino acids. These proteins cross the red cell
membrane 12 times. There are only small loops of the protein on the exterior of the cell
membrane.

Therefore Rh antigens are not as available to react with their specific antibodies and there
are fewer antigen sites than ABO. Unlike the ABO system, Rh antigens are not soluble and
are not expressed on tissues. They are well developed at birth and therefore can easily
cause hemolytic disease of the newborn if the baby has a Rh antigen that the mother lacks.
Besides the antigens being well-developed at birth, they are very good immunogens. This is
especially true to D, which if the most immunogenic after A and B antigens.

Rh Antibodies
Unlike the ABO antibodies that are mainly IgM, Rh antibodies are commonly IgG. They are
NOT naturally occurring and therefore are formed by immune stimulus due to transfusions or
baby's red blood cells during pregnancy. The most common antibody to form is anti-D in Rh
negative individuals.
Since Rh antibodies are IgG they bind best at 37oC and their reactions will be observed with
indirect antiglobulin technique. Agglutination reactions are enhanced by high protein
(albumin), low-ionic strength saline (LISS), proteolytic enzymes (papain) and polytheylene
glycol (PEG).
Rh antibodies will react more strongly with homozygous cells than with heterozygous cells.
For example, an anti-E will react with strongly with E+E+ cells and more weakly with E+e+
cells. This is called dosage effect.
Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions can occur due
the various Rh antibodies. Anti-D is the most common cause of hemolytic disease of the
newborn. Since D antigen is so immunogenic we screen all donor units for the D antigen.
Therefore if an individual is A+, it means both the A and the D antigens are present. On the
other hand, if an individual is A-, the A antigen is present and the D antigen is absent.
To prevent problems due to anti-D:

always give Rh-negative individuals Rh-negative blood

give Rh immune globulin to Rh-negative mothers to prevent formation of anti-D

during pregnancy.

The incidence of Rh antibodies

Anti-D most common antibody seen in Rh(D) negative people
Anti-E most common antibody seen in Rh pos people since only 30% of population have

the antigen
Anti-C or Anti-c less common - most people have the antigen
Anti-e often seen as autoantibody and will make it difficult to find compatible blood

since 98% of the population have the e antigen

Anti-C,e or Anti-c,E often seen in combination. If a patient lacks both a C and e and

has made an anti-C, then enhancement techniques should be done to make sure that
an anti-e is not also present.

Rh System Inheritance
Fisher-Race theory involved the presence of 3 separate genes D, C, and E and their alleles c
and e and the absence of D since an anti-d has never been found. These three genes are
closely linked on the same chromosome and are inherited as a group of 3. The most
common group of 3 genes inherited is CDe and ced is the second most common.
Weiner Theory
Weiner believed there was one gene complex with a number of alleles resulting in the
presence of various Rh antigens. According to Weiner there were 8 alleles, R, R1, R2, Rz, r, r', r",
ry , which ended up with different antigens on the red cells that he called Rh, rh', rh", hr', hr". If
a person has the Fisher-Race genotype of DCe/DCe, it is easier to refer to that type as R1R1
Tippett Theory
Tippett predicted that there are two closely-linked genes - RHD and RHCE. The RHD gene
determines whether the D antigen that spans the membrane is present. Caucasians who
are D negative have no gene at that gene loci.
The RHCE gene determines C, c, E, e antigens produced from the alleles:

RHCe

RHCE

RHcE

RHce
Rh Gene Complexes, Antigens, Possible Combinations and Percentages

Haplotypes
R1

Genes Present

Phenotype Percentage

D,C,e

R1

42%

RHce

dce

37%

R2

RHD RHcE

DcE

R2

14%

RHD RHce

Dce

4%

r'

RHCe

dCe

r'

2%

r"

RHcE

dcE

r"

1%

Rz

RHD RHCE

DCE

Rz

<1%

ry

RHCE

dCE

ry

<1%

RHD RHCe

Antigens Present

Translating From Wiener To Fisher-Race

1. R always refers to D whether it is R, R1, R2, or the very rare Rz.
2. r always refers t the lack of D
3. o refers to having no C or E
4. 1 or ' always refers to C
5. 2 or " always refers to E
6. The very rare haplotypes that have both a C and E are given letters from the end of
the alphabet z and y.
Determining Genotypes From Phenotypes
The following steps will be helpful in determining from the individual's phenotype. These rules
are based on probability so the least likely genotypes will involve Rz or ry.
1. Type patient for the five Rh antigens: D, C, c, E, e
2. Start with D: is it positive or negative?
1. If negative, the individual will be homozygous d.
2. If positive for D, you can't tell yet whether the individual is homozygous or
heterozygous for D. Therefore, put D on just one chromosome.
3. Look at C: is it positive or negative?
1. If negative, put c on each chromosome.
2. If positive, look at c result to determine if the C is homozygous or heterozygous.
If there is no c present, there would be two C and it would be homozygous.
If a c is present as well as C, they are heterozygous.
3. If homozygous, then put C on each chromosome.
4. If heterozygous, put C on same chromosome as D; put c on other.
4. Look at E: is it positive or negative?
1. If negative, put e on each chromosome.
2. If positive, look at e result to determine if homozygous or heterozygous.
3. If homozygous, put E on each chromosome.
4. If heterozygous, put E on same chromosome as the D unless the D already
has a C; put e on other chromosome. DCe is more common than DcE and
DCE is extremely rare.
5. Put C and E together on same chromosome only if no other possible combinations

Most Common Genotypes

The following genotypes are listed as the most common with 1 being the most common in
Whites and 7 the least common. Rz and ry are so rare they are not included.
Incidence of the most common genotypes
Antigens Present
D, C, c, e

D, C, e

Genotype

Incidence(%)

DCE

Weiner Haplotypes

Whites

Blacks

DCe/ce

R1r

31.1

8.8

DCe/Dce

R1Ro

3.4

15

Dce/Ce

R or

0.2

1.8

DCe/DCe

R1R1

17.6

2.9

DCe/Ce

R1r'

1.7

0.7

ce

ce/ce

rr

15

DCcEe

DCe/DcE

R1R2

11.8

3.7

DCe/cE

R1r"

0.8

<0.1

DcE/Ce

R2r'

0.6

0.4

DcE/ce

R2r

10.4

5.7

DcE/Dce

R2R

1.1

9.7

Dce/ce

Ror

3.0

22.9

Dce/Dce

R oR o

0.2

19.4

DcE/DcE

R2R2

2.0

1.3

DcE/cE

R2r"

0.3

<<0.1

DcEe

Dce

DcE

Applications of Rh genotyping
Paternity testing of blood group antigens is based on a process of exclusion. Since the RHD
and RHCE are closely linked and Ce, ce, cE are produced by a single gene, there are
limited combinations that the father can provide.
HDN predictability by testing father's Rh genotype. This helps predict likelihood of HDN due
to D when mom has anti-D. The most common Rh genotype of father will indicate whether
baby has O%, 50%, or 100% probability of being D positive.

If the father is also D negative (ce/ce), the baby will be D negative as well and there
is a 0% probability of the baby suffering from Rh HDN.
If the father's Rh genotype appears to be either, R1r, R2r or Ror, the baby has a 50%
probability of being D positive and suffering from Rho HDN.
On the other hand if a father's Rh genotype appears to be any of the following, R1R1,
R2R2, R1R2, RoRo, R1Ro, or R2Ro, the baby has a 100% probably of getting a D gene from
his father and therefore being D positive and suffering from Rh HDN.

Variants
Weak D (Du)
Weak D is a weakly expressed D antigen that will only be demonstrated after incubation at
35-37oC followed with antiglobulin testing. (ie being demonstrated only by Coombs
technique). An Rh control must always be run along with the weak D test. Always consult the
product insert to determine if Rh Control needs to be run when performing the immediate
spin D testing. The following results could be obtained when performing the D testing:
Immediate Spin

37oC Anti-D
Anti-D

Rh Co

AGT
Anti-D

Interpretation

Anti-D

Rh Co

Rh Co

Weak D

True D negative

D positive

Or any time the Rh control is

0

positive, you cannot interpret

results and need to perform
further testing

Testing for Weak D

AABB requires that all donor blood that originally fails to react with anti-D at immediate spin
must be tested for weak D. Units that test weak D positive would be labeled D positive and
would be transfused only to D positive individuals.
Hospitals may or may not test all Rh negative recipients for weak D. The cost of time and
reagents is minimized if only the immediate spin. This may create some confusion with the
recipient if their donor card indicates they are Rh positive but they type Rh negative when
they are the recipient. Recipients that type D negative at immediate spin would be given D
negative blood, which not create a problem for the patient.
When performing testing prenatal and postnatal mothers, D-negative blood at immediate
spin would be tested for weak D as well to determine if they are eligible for Rh Immune
Globulin. Since Rh Immune Globulin is actually anti-D it is safe for a true D negative, but not
for a weak D positive mother.

Why do weak D's exist?

Quantitative Weak D There are individuals that quantitatively produce fewer D

antigen sites. This is more common in Blacks and is often seen with the Dce haplotype.
On rare occasions among Whites an unusual DCe or DcE may also produce a
quantitatively decrease weak D.

Position Effect Weak D In this case the D is weakened by the position of a C on the
opposite haplotype which is called the trans position. The two Rh genotype
combinations where this type of weak D is seen are: Dce/Ce and DcE/Ce. Today this
type of weak D would type as a regular D due to the improvement of reagents.

Partial D antigen (Mosaic D) It has been found that some D-positive individuals make
an alloanti-D that reacts with other D positive cells but not their own. Many of these
will demonstrate a weak D type of reaction. In this type of weak D, the individuals are
lack some of the components of the D antigen and therefore are able to make
allantibodies to those specific components if they are transfused with D positive
blood.

Other Rh System Variants

There are presently 46 Rh antigens identified and named. The following are the most
common of those variants

Cw is a low frequency antigen found in approximately 2% of Whites and 1% of Blacks.

It is not an allele of C and c. Its allele is MAR, which is found in 99.9% of the population.

V and VS are low frequency alleles found in 1% or less of the Whites, but are more
common in Blacks. V is found in 30% of the Blacks and VS in 32%.

G is present when D or C present due to the present of serine at the 103 position of
the Rh polypeptide. Anti-G will react with both D+ and C+ cells.

f is present when c and e together on same chromosome: Dce or ce. This is the most
common of what are called cis product antigens.

Rhnull has no Rh antigens on their red cells but these individual can transmit normal Rh
antigens to their offspring. In the most common type the core Rh polypeptide is
missing. A less common type has the regulator gene that turns off the expression of Rh.
There have been at least 43 individuals in 14 families that are Rhnull. In these individuals
the red blood cell membrane is abnormal and some of these have been identified
when it was observed that they had hemolytic anemia and abnormal red cell
morphology. If these individuals develop an Rh antibody following a transfusion or
pregnancy, it is considered a anti-total Rh antibody.

Rh Typing
False Positives
1. When following through to AGT for weak D and will be identified as false positive by a
positive Rh control. This is seen when a patient/donor has strong positive DAT. The
cells are coated with antibody (not necessarily Rh antibody) in vivo. Albumin is
necessary in reagent Anti-D to overcome the zeta potential allowing cells coated
with IgG Anti-D to get close enough together to agglutinate, but cells coated in vivo
with any IgG antibody will also agglutinate in albumin.
These false positives are corrected by using form of Anti-D that does not require
albumin. There are two types of alternative types of anti-D:
Monoclonal (IgM) anti-D will cause agglutination of D positive cells without the

presence of albumin at room temperature. A number of facilities normally use this

type of anti-D and therefore do not routinely use Rh control.
Chemically modified anti-D has been modified by breaking the disulfide bonds

closest to the hinge region so antibody can reach cells that are farther apart.
2. False positive can also be caused by rouleaux formation, which will look like
agglutination macroscopically. Rouleaux would be identified microscopically due to
the "coin-stacking" appearance of the red cells. This false positive would be
corrected by washing cells 3 to 4 times and then retesting.
False Negatives
False negatives are not readily identifiable, but can occur in the following instances:

The most common is the result of too heavy cell suspension due to too many cells for
the amount of antibody in the antisera.

They may also rarely be caused by extremely strong positive DAT. In this case a the
patient's D antigen sites are coated in vivo and there are no sites left for commercial
anti-D to attach to. This can be fixed by heating cells gently to elute off antibody
without damaging cells, then re-test.

LEWIS BLOOD GROUP SYSTEM

There are distinct differences in regards to the Lewis Blood Group System

Manufactured by the tissues

Lewis antigens are secreted into body fluids

Absorbed onto red cells from the plasma

The Lewis Blood Group System is also similar to the ABO system

The antigens are part of the same oligiosaccharides that are part of the ABH antigens

The Lewis antigen (fucose) is added onto the N-acetyl-glucosamine that is just before
the galactose where the fucose is added for the H antigen in the secretions.

Lewis Antigens
The development of Lewis antigens is controlled by two alleles of Lewis blood group system.

Le is dominant and results in the presence of Lewis antigen.

The recessive le (absence of Lewis gene) is recessive and therefore 2 le/le needs to
be inherited

Genotypes
Both Le/Le or Le/le result Lewis positive antigen. Lewis antigen exists as either Lewis a (Lea) or
Lewis b (Leb). Lewis negative results from le/le.
Phenotypes
Lewis System Phenotypes and Their Incidence
Reactions with AntiLea

Leb

Phenotype

Adult Phenotype Incidence in %

Whites

Blacks

Le(a+b-)

22

23

Le(a-b+)

72

55

Le(a-b-)

22

Le(a+b+)

Rare

Rare

Formation of Lewis Antigens

What Lewis antigens are formed depends on interaction between Lewis (Le/le), Secretor (Se)
and H genes in the tissues to produce Lewis antigens in the secretions.
Lewis antigens have a similar structure to ABO antigens:
1.

Formed at terminal sugars of Type I precursor substance made by tissue cell in plasma
a. If person has the Lewis gene, it adds fucose to second sugar from the end = Lea
b. If person is also a secretor, H gene adds fucose to terminal sugar of precursor
substance = Leb antigen
c. If person NOT a secretor, no H added to precursor substance made by tissue cell, so
it remains as Lea

2.

NOT part of rbc membrane - synthesized by tissue cells, carried by plasma, adsorb onto
rbc surface

3.

Not present on newborn red cells

4.

Can disappear during pregnancy

Lewis Antibodies
I. Types of antibodies

Anti-Lewisa

Anti-Lewisb

Anti-Lewisa and Lewisb

A.

Almost always produced by Lewis a negative; Lewis b negative people

B.

Naturally occurring but not regularly occurring (unexpected antibodies)

C.

Frequently seen in pregnancy (due to loss of Lewis antigen during pregnancy)

D.

IgM, therefore usually not clinically significant

1. Does not cross placenta
2. Reacts best at RT, but some MAY react at 37C
3. If anti-Lea present at 37C, may cause hemolytic transfusion reaction
4. Anti-Leb is usually clinically insignificant

II. Secretor Status

A. Secretor status controlled by Secretor (Se) gene
1. Secretor = Se/Se or Se/se (80%)
2. Non-secretor = se/se (20%)

B. Secretors have soluble A, B and H antigens in body fluids - plasma, tears, saliva
C. Can test saliva for presence of ABH antigens
D. Practical application of saliva testing:
1. Determine ABO type in patients with ABO discrepancies
2. Determine ABO type in patients massively transfused with another blood type
3. Can also use Lewis types to determine secretor status:
E. Lewis a positive, Lewis b negative = non-secretor
F. Lewis a negative, Lewis b positive = secretor
G. Lewis a negative, Lewis b negative = can't tell secretor status from Lewis types
H. Can only use this method if patient has not been heavily transfused recently - need to
type patient red cells, not donor

III. Testing for Secretor Status

A. Utilizes principle of AGGLUTINATION INHIBITION
1. Patient's saliva boiled and cleared
2. Cooled saliva mixed with reagent anti-A, anti-B and anti-H
3. If soluble A, B, or H antigens present in saliva, these will react with antibodies in
reagent antiserum, and neutralize it, so no antibody available to agglutinate test
cells
4. if no soluble A, B or H antigens in saliva, antibodies in reagent antiserum will not be
neutralized, and will be free to react with test cells
B. Group A Secretor:
1. A antigens in saliva
2. Addition of reagent anti-A: antibodies tied up by soluble A antigens
3. Addition of known A cells results in no agglutination (no free A antibody to cause
agglutination)
NO AGGLUTINATION = POSITIVE TEST FOR SECRETOR STATUS
C. Group A Non-Secretor:
1. NO ABO antigens in saliva
2. Addition of reagent Anti-A leaves free antibody in serum
3. Addition of known A cells causes agglutination.
AGGLUTINATION = NEGATIVE TEST FOR SECRETOR STATUS

OTHER BLOOD GROUP SYSTEMS

MNS SYSTEM
Antigens and Their Inheritance
Antigens M and N are co-dominant alleles that are closely linked to the S and s antigens,
which are also co-dominant. Chromosome 4 contains these linked genes. These antigens
are inherited by a complex pattern similar to the Rh system. The Ms and Ns linkage is more
common than the MS and NS linkages. All these antigens, however are fairly frequent in the
population with the following overall frequencies:

M = 78%

N = 72%

S = 55%

s = 89%

U = Greater than 99%

U antigen is a high incident antigen NOT seen in individuals who lack both S and s antigens.
Individuals who lack this antigen (<1%) have a high likelihood of forming anti-U, -S and -s.

Phenotype

Phenotype Frequency %
Whites

Blacks

M+N-

28

26

M+N+

50

44

M-N+

22

30

S+s-U+

11

S+s+U+

44

28

S-s+U+

45

69

S-s-U-

Less than 1

Biochemistry of the MNS antigens

M and N antigens are glycoproteins containing sialic acid that cross the cell membrane.
Carboxyl terminus extends into the red cells interior, a hydrophobic segment as part cell
membrane and an amino terminal segment on the external environment of the red cell. The
external components of the antigens are destroyed by enzymes like typsin and papain.
Antigens of MNSU blood group system are well developed in newborns. Therefore a mother
who is negative for one of these antigens could be stimulated to make antibodies thatmay
cause HDN. The antibody would have to be an IgG immunoglobulin that reacts at 37oC)

MNS System Antibodies

Anti-M is frequently seen as a saline agglutinin if testing is done at room temperature.
It is predominantly IgM and can be naturally occurring. It will show dosage and

therefore M homozygous cells will react with antibody more strongly than
heterozygous cells. Therefore predominant form of antibody is not clinically
significant.
There are instances where some or all of the antibody is IgG in nature. If you have an

anti-M that strongly at 37oC and/or AHG, it should be considered to be potentially

clinically significant.

Mild to severe cases of hemolytic disease of the new born have be reported.

Since the M antigen can be removed by enzyme, the reactivity of anti-M can be
destroyed by enzyme.
When performing a crossmatch on a recipient's specimen that contains anti-M, a

pre-warmed crossmatch should be preformed.

Anti-N is very rare and has similar reactivity as anti-M.
Anti-S, anti-s, anti-U usually form red cell immunization due to transfusions and pregnancies.

They are usually IgG and react best after 37oC with AGT (Coombs) technique.

All capable of causing Hemolytic Transfusion Reactions (HTR, delayed) and HDN

S is usually destroyed by enzyme but s is variable and U is not destroyed by enzyme

Anti-U is rare but should be considered if a previously transfused or pregnant Black

patient has an antibody to a high-incident antigen.

Summary of Antibody Characteristics

Antibody

Reactivity

Bind

In Vitro

HTR**

HDN***

Comment

No

Few

Mild-Severe

Usually

(Rare)

No

Rare

Moderate

Variable

Some

No

Yes

Mild

Most

Variable

Few

No

Yes

Mild-Severe

Most

No

(Rare)

No

Yes

Mild-Severe

< RT*

37

AHG

Enzymes Complement Hemolysis

Most

Few

Few

Destroy

(Rare)

Most

Few

Few

Destroy

Some Some

Most

Few

Few

Rare Some

*Room Temperature
**Hemolytic Transfusion Reaction
***Hemolytic Disease of the Newborn

Clinically
Insignificant

KELL SYSTEM
Antigens and Their Inheritance
A number of mothers, who were believed to have babies with hemolytic disease of the
newborn, one of the reactions was not due to Anti-D. The non-Rh antigen involved in this
case was that subsequently known as Kell.
There are three pairs of alleles within the Kell system. Each pair has a high frequency and low
frequency gene that are co-dominate if present. The three pairs are:

K (Kell), or K1, and k (Cellano), K2

Kpa (K3) and Kpb (K4)(Penney)

Jsa (K6)and Jsb (K7)(Sutter)

K, Kpa and Jsa are low frequency antigens and k, Kpb and Jsb are high frequency antigens.
There is a Kell phenotype, K null (Ko or K5), is very rare and K, k, Kpa, Kpb, Jsa, Jsb antigens are
not expressed.
The Kell Systems antigens are found in only small amounts on the red cell carried on a single
protein. The function of this protein is unknown.

Phenotype

Phenotype Frequency %
Whites

Blacks

K+k-

0.2

Rare

K+k+

8.8

K-k+

91

98

Kp(a+b-)

Rare

Kp(a+b+)

2.3

Rare

Kp(a-b+)

97.7

100

Js(a+b-)

0.0

Js(a+b+)

Rare

19

Js(a-b+)

100.0

80

Ko [K-,k-,Kp(a-b-),Js(a-b-)]

Extremely rare

Kell System Antibodies

Anti-Kell is the most clinically significant antibody within this system. The Kell antigen is
considered the next most antigenic after the D antigen of the Rh system. Individuals lacking
the K antigen can make anti-Kell after only two exposures to Kell-positive blood. Because
over 90% of the population are Kell negative it is not difficult to find donor blood that is
compatible with the recipient.
Antibodies to other antigens in Kell system are very rare.
Antibody Characteristics of the antibodies to the Kell System are:

IgG

Cause HDN and HTR (delayed)

React best in Coombs after 37oC incubation

Does not show dosage (homozygous KK and heterozygous Kk cells react with the
same strength)
Enzyme has no effect

Summary of Antibody Characteristics

Reactivity

Bind

Antibody

In Vitro

Complement Hemolysis

HTR**

HDN***

< RT*

37

AHG

Enzymes

Some

Some

Most

No change

Some

No

Yes

Mild-Severe

Few

Few

Most

No change

(Some)

No

Yes

Mild

Kpa

Some

Some

Most

No change

(Some)

No

Yes

Mild

Kpb

Few

Few

Most

No change

(Some)

No

Yes

Mild

Jsa

Few

Few

Most

No change

(Some)

No

Yes

Moderate

Jsb

(No)

(No)

Most

No change

(Some)

No

Yes

Mild-Moderate

DUFFY SYSTEM
Antigens and Their Inheritance
The Duffy antigens Fya and Fyb are a pair of co-dominant alleles found on chromosome 1.
The phenotypes Fy(a-b+), Fy(a+b+), Fy(a-b+) are very common among the US white
population. Fy(a-b-) is very rare in the white population but makes up 68% of the black
population of the United States.
Biochemically the Duffy antigens are glycoproteins that has an external loop. This external
loop can be destroyed by enzymes such as ficin, papain, and trypsin. The Fya and Fyb
antigens are receptors for the malarial parasite, Plasmodium vivax. Therefore individuals
that are phenotypically Fy(a-b-) have a resistance to malaria. This particular phenotype is
found up to 100% of western Africa and of course 68% of the American Blacks.
Phenotypes and Frequencies in the Duffy System
Phenotype Frequency %

Phenotype

Whites

Blacks

Fy(a+b-)

17

Fy(a+b+)

49

Fy(a-b+)

34

22

Fy(a-b-)

Very rare

68

Duffy System Antibodies

Anti-Fya much more common than anti-Fyb and is more likely to cause HTR and HDN.

IgG

Anti-Fya can cause HDN and HTR (delayed) and anti-Fyb is milder and no HDN cases
have been reported but could possibly be a cause.

React best in Coombs after 37oC incubation

Reactions destroyed by enzyme of the red cells

Summary of Antibody Characteristics

Reactivity
Antibody

Bind

In Vitro

AHG Enzymes Complement Hemolysis

% Compatible
HTR

HDN

< RT*

37

Fya

Rare

Rare

Most Destroy

Some

No

Yes

Mild- Sever

Fyb

Rare

Rare

Most Destroy

Some

No

Yes

(Yes)

in US
Population
34 W
17 W
77 B

KIDD SYSTEM
Antigen, Antibodies and Their Inheritance
Jka and Jkb antigens are inherited on chromosome18 where urea transport mechanisms
located. Cells that are Jk(a-b-) are less likely to lyse in presence of high concentration of
urea. These antigens are inherited by co-dominant alleles Jka and Jkb (high frequency
antigen). Kidd antigens are thought to be grouped very close together in clusters on red cell
membrane. Due to close proximity of antigens when antibodies are attached complement
can be activated, which cause intravascular transfusion reactions.
Phenotypes and Frequencies in the Kidd System
Phenotype Frequency %

Phenotype

Whites

Blacks

Jk(a+b-)

28

57

Jk(a+b+)

49

34

Jk(a-b+)

23

Jk(a-b-)

Extremely rare

Both anti-JKa and anti-Jkb are hard to detect and identify since they are very weak and are
detected primarily at antiglobulin phase of testing. These antibodies are usually low titer as
well as being weak reactions. The antibodies disappear rapidly from circulation and also in
stored serum since their recognition is enhanced if complement is present. These antibodies
will often show dosage. Enzyme /PEG can enhance reaction
Characteristics of anti-Jka and anti-Jkb are as follows:

IgG , react best after 37oC with Coombs technique

Can cause HDN and Hemolytic Transfusion reactions that are acute intravascular
reactions, or they may be delayed transfusion reactions that show up after the
patient's immune system is response and the memory cells quickly produce
antibodies to the antigens. While most of the antibodies are more likely to cause
extravascular hemolytic transfusion reactions, the Kidd antibodies can activate
complement and therefore cause intravascular hemolytic transfusion reactions.

Summary of Antibody Characteristics

Antibody

Reactivity
< RT 37

AHG

Bind

In Vitro

Enzymes Complement Hemolysis

HTR

HDN Comments

Jka

Few Few Most Enhance

All

Some

Yes

Mild Associated with

Jkb

Few Few Most Enhance

All

Some

Yes

Mild Severe Delayed HTR

Bg Antibodies
Bg antibodies are not considered clinically significant, but may mask clinically significant
antibodies. They are antibodies to white cell antigen remnants on red cells

Anti-Bga reacts with Human Leukocyte Antigen (HLA)-B7

Anti-Bgb reacts with Human Leukocyte Antigen (HLA)-B17

Anti-Bgc reacts with Human Leukocyte Antigen (HLA)-A28

Reactions relating to these antibodies are weak, react possibly at room temperature or
more commonly in the antiglobulin (Coombs) phase. They react with only a few cells in a
cell panel. They do not cause hemolytic disease of the newborn or hemolytic transfusion
reactions. They are seen frequently seen in multiply transfused patients or in multiparous
women (multiple pregnancies)
A summary of these antibodies are as follows

Not clinically significant, but serological reactions make them look like they are

High Titer if the antibodies are titered. The titers are usually at least 1:64 and often will
be over 1:1000

Reactions are very weak and will break apart very readily due to the weak attraction
between the antigens and antibodies (low avidity).

HTLA ANTIBODIES = High Titer Low Avidity (Antibodies have high titer but very weak reaction)
Specific serologic characteristics of the HTLA antibodies are:

IgG

React best in Coombs after 37oC incubation

W+ to 1+ reactions

React with most cells (antibodies to high-frequency antigens)

Not clinically significant since they are not known to cause hemolytic disease of the
newborn or hemolytic transfusion reactions. Since they are antibodies to
high-frequency antigens they may mask clinically significant antibodies that are also
in the serum.

I AND P BLOOD GROUP SYSTEMS

I Blood Group System
I antigen is found on almost all adults and is part of the precursor component of the
oligosaccharide that forms the A, B, and H antigens.
There are two types of oligosaccharides: Type 1 and Type 2. These chains that are part of
the ABH antigens form the i antigen found in infants.

By the time the child reaches 2 years of age a number of b1-->6 linkages are added
between the residual galactose components. In adults these precursor components are
become branched with a b1-->6 binding between 2 galactose molecules forming a
branched oligosaccharides that make up the I antigen seen in most adults.
Therefore I (almost all adults) are formed from
branched sugar chains on precursor
substance.
All cord bloods form i antigen from straight-line
sugar chains on precursor substance of the A,
B, and H antigens.

As indicated: in most children i is converted to I

by the age of 2 years.

Anti-I, Anti-IH, and Anti-i Antibodies

Anti-I antibodies are the most common antibodies found if antibody screenings are done at
immediate spin, room temperature. Their characteristics are:

IgM immunoglobulins and therefore are saline agglutinins with their optimal
temperature at 4oC. Since they are IgM they also do not cross the placenta.

Anti-I is naturally occurring often due to a Mycoplasma pneumoniae infection or

some lymphomas and leukemias.

These antibodies are USUALLY not clinically significant

Anti-I reacts with all adult cells (including patients own, all reagent cells, all donor
cells)

Anti-I does not react with cord cells

Auto-anti-I is a common cold agglutinin

High titers of auto-anti-I can interfere with serological testing:

In ABO typing, the forward grouping may have patient cells coated with IgM autoantibody,
so all cells agglutinate. When this happens all results are positive and the group looks like
AB+. On the reverse grouping anti-I in serum reacts with all adult cells, so A1 and B cells
always agglutinate. All results are positive and the group looks like an O.
Cells +

Cells +

Cells +

Serum +

Serum + B

Cells +

Cells + Rh

anti-A

anti-B

anti-A,B

A1 cells

cells

anti-D

Cont

4+

4+

4+

4+

4+

4+

4+

Interpretation
Unknown
ABO Group

Method to resolve these Problems

Correct forward typing by washing cells in warm saline and re-testing.

Correct reverse by warming the serum before testing, or adsorbing the cold
autoantibody out of the serum.

Crossmatch results will result in all donors being incompatible. Correct by:

warming the donor cells and patients serum before crossmatching,

omitting potentiators like LISS and

using monospecific anti-IgG.

Anti-IH antibodies are directed against both I antigens and H antigens. Therefore they react
most strongly with group O adult cells, least with cord cells or A1 cells.
Comparison of Anti-I, Anti-IH, and Anti-H Reactions
Antibody

Adult A Cells

Adult O Cells

Cord A Cells

Cord O Cells

Anti-I

2+

2+

Anti-IH

2+

4+

2+

Anti-H

2+

2+

Anti-i

2+

2+

Anti-IH is seen most often in A1 adults and is not clinically significant but may cause problems
with the ABO grouping and crossmatch results just as anti-I does. Follow the same steps for
resolving the problem as indicated for anti-I.
Diseases
1. Anti-I and anti-IH are only clinically significant when they cause Cold Hemagglutinin
Disease (CHD). In this disease a strong autoanti-I is present. The blood in patient toes,
fingertips, earlobes is cooler than than 37oC and the antibody coats cooled cells,
binds complement, and causes hemolysis. This disease is treated by keeping the
patient's extremities warm so the blood is not allowed to cool.
2. High-titer anti-I also commonly associated with Mycoplasma. pneumoniae infections.
Treatment would be giving the patient appropriate antibiotics.
3. Anti-i, which is fairly uncommon and transient, is associated with infectious
mononucleosis.
P System
Antigens of the P system are Pk, P, P1 .

Basic structure is the precursor substance, but is called globoside (P) or paragloboside (p) in
the P system as the diagram below shows:

P antigens poorly developed at birth and therefore do not usually cause Hemolytic
Disease of the newborn.

They are present in variable amounts on different red cells and seems to diminish in
storage.

Like ABO and Ii antigens, they are composed of sugar chains as seen in the
accompanying figure.

Like A and B antigens, they are present on tissue cells as well as red cells.

Similar antigens found in nature (bird droppings, pigeon eggs, hydatid cyst fluid) and
these substances can be used to neutralize anti-P in a patient's serum. These similar
antigen solutions can be useful when the antibody is interfering with ABO and
crossmatch testing.

The Pk antigen occurs when the Pk antigen does not convert to P

P Phenotypes
80% of people with P antigen are P1 phenotype (have P and P1 antigens) and 20% of
people with P antigen are P2 (P antigen only - no P1). The P2 phenotype merely signifies
absence of P1 antigen in people with P antigen (globoside). Individuals who are Pk
consistantly have an alloanti-P that is an IgM antibody. The absence of the P antigen is very
rare and is designated as p.
P Antibodies
Alloanti-P1 is the most common of the antibodies in the P system. This antibody is commonly
seen in P2 people. IAlloanti-P1'ss characteristics are:

IgM class and therefore is saline reactive, enhanced by cold incubation, may bind
complement.

Not clinically significant

Naturally occurring but not regularly occurring. It is formed with no known red blood
cell stimulus - either pregnancy or transfusion. If individuals are P1 negative, they do
not automatically have anti-P1 i, but it is fairly common

Antigen-antibody reactions in P system enhanced by adding albumin to system, or

by treating red cells with enzymes

Auto anti-P is an IgG antibody seen in P1 or P2 persons and causes clinically significant
Paroxysmal Cold Hemoglobinuria (PCH). The Donath-Landsteiner Test helps diagnose
PCH. It is designed to detect biphasic anti-P. A biphasic antibody reacts with the antigen
on the red blood cell at colder temperature, binding complement. When
antigen-antibody-complement complex warms up, the red blood cells are hemolyzed.
Donath-Lansteiner test is performed by incubating patient's serum with his own cells first at
4oC, then at 37oC, and then looking for hemolysis.

A positive Donath-Landsteiner = no hemolysis at 4oC, no hemolysis at 37oC, hemolysis

only in the tube that was incubated at both temperatures

A negative Donath-Landsteiner = neg at both temps, OR hemolysis at 4oCas well as

37oC

Donath-Landsteiner Test

4oC incubation:

37oC incubation:

no hemolysis = NEGATIVE

no hemolysis = NEGATIVE

4oC + 37oC incubation:

hemolysis: = POSITIVE

PRINCIPLE AND APPLICATIONS

The patient's serum is tested for the presence of clinically significant antibodies using an
indirect antiglobulin method. The serum is tested against unpooled Group O cells selected
to possess the relevant blood group antigens.
The antibody screen is routinely performed as part of compatibility testing; it is also
performed upon request on prenatal patients, on Rh immune globulin candidates, as part
of an organ transplant workup, or on other patients for whom an "indirect Coombs" is
requested.
SAMPLE
Since the patient's serum is tested, a 10 ml clotted (red top) tube is preferred. A 4 or 5 ml red
top is acceptable; a 2 ml red top is acceptable from a newborn.
The sample should be tested when fresh; old or improperly stored samples may lose
complement activity and lead to false negatives.

PROCEDURE
1. Verify that patient information on the sample matches information on the worksheet.
2. Centrifuge the sample and separate the serum to a labeled tube.
3. Prepare a washed 3% cell suspension from the patient's cells.
4. Label 3 tubes
[ Patient last name/or #, I ] , [ Patient last name/or #, II ] , [ Patient last name/or #, III ]
5. Using a large bore pipette, add 2 drops of patient serum to all tubes. Hold the
dropper at a consistent 45-degree angle.
6. Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II
tube; add one drop Screening Cell III to the III tube.
7. Shake all tubes to mix and centrifuge at high speed the time appropriate for the
saline spin calibration in the serofuge.
8. Gently resuspend and examine macroscopically for agglutination using the lighted
agglutination viewer.
9. Record all negative reactions as ; grade positive reactions on a scale of w+ to 4+.
10. Again holding the dropper at a consistent angle, add 2 drops of PEG to all tubes.
11. Shake to mix and incubate at 37oC at least 15 but no more than 30 minutes. (NOTE: IF
A WATERBATH IS USED FOR THE 37oC INCUBATION, THE INCUBATION TIME MAY BE
REDUCED TO 10 MINUTES).
12. Wash all tubes 4 times, decanting well after each wash, mixing the cell button well
between washes, and blotting the last drop of saline after the final wash.
13. Immediately add one drop AHG to each tube, shake to mix, and centrifuge the time
appropriate for the Coombs spin calibration in the serofuge (usually the same as the
saline calibration).
14. Immediately resuspend gently and check macroscopically for agglutination using
the lighted agglutination viewer.
15. Read and record results as described above, along with your initials and the date, if
not already recorded on the worksheet.
16. Add one drop Coombs Control Cells to all negative reactions and centrifuge 15-30
seconds in the serofuge.
17. Resuspend and macroscopically examine for agglutination. There must be
agglutination in this step or the test is invalid. Record results.

INTERPRETATION

The lack of agglutination indicates the absence of antibodies to antigens on the

reagent test cells, and the test is reported negative.

Agglutination in either Screening Cell tube prior to the addition of Coombs Control
Cells indicates the presence of unexpected alloantibodies. The test is reported
positive and further testing is necessary to identify the antibody(ies).

Agglutination in the autocontrol (AUTO) indicates coating of cells circulating in the

patient. This may be due to an antibody to medications, autoantibody, passively
transfused alloantibody, or alloantibody coating transfused cells in the patient.
Perform a DAT and obtain patient's medication list, diagnosis and transfusion history.

NOTES AND PRECAUTIONS

1. Use of PEG requires a strict serum/PEG ratio. Also, add no more than 2 drops of serum
when using PEG.
2. AHG must be added to the cells immediately following washing. Antibodies may
elute from the cells if they are allowed to sit in saline without the addition of AHG.
3. The Coombs Control Cells must be positive at the end of the procedure. Reasons for
negative results here include:
Inadequate washing of cells in Coombs phase. Residual serum neutralizes AHG.
A small fibrin clot has formed in tube, neutralizing AHG. Be sure sample has

thoroughly clotted.
Coombs reagent was omitted or is inactive.

4. Weak or questionable reactions may be enhanced by:

Increasing the amount of serum used to 3-4 drops. PEG must be omitted, and the

incubation time extended to 30 minutes.

Omitting PEG and adding albumin to the test mixture. Extend the incubation time

to at least 20 minutes.
Using enzyme treated cells. See manufacturer's directions.
Repeat testing using PEG to the test mixture. See manufacturer's directions.

5. Occasionally you may get a moderately strong reaction at room temperature that
persists weakly in Coombs. This may not be clinically significant if it is due to
complement binding at room temperature from a cold-reactive antibody. To
determine if the reaction is due to complement binding, do a pre-warmed screen

THE CROSSMATCH
General Information
Crossmatch also known as compatibility testing, pretransfusion testing or typing. The
definition of a compatibility test is a series of procedures use to give an indication of blood
group compatibility between the donor and the recipient and to detect irregular antibodies
in the recipient's serum.
The main purpose for performing a crossmatch is to promote the safe transfusion of blood.
We are performing testing to demonstrate donor blood is compatible with recipient's blood.
Crossmatch procedures should be designed for speed and accuracy - get the safest blood
reasonably possible available to patient as soon as possible. The goal of compatibility test is:

Detect as many clinically significant antibodies as possible

Detect as few clinically insignificant antibodies as possible

Complete the procedure in a timely manner.

Once donor blood is crossmatched with a potential recipient, the results of crossmatch is
good only 3 days. If the physician wants the donor blood available longer, we must get a
new recipient sample and repeat tests. This protocol helps detect new antibodies that may
be forming, especially when patient has been transfused within past three months.
Parts of the Crossmatch

Identification of recipient and recipient blood sample is crucial since major of the
hemolytic transfusion reactions are due to errors in patient or sample identification.

ABO and Rh typing of recipient's blood and resolving any ABO discrepancies. If the
discrepancy cannot be resolved before patient needs transfusion type O blood
should be given. If problems arise with D testing, Rh negative blood should be given.

Performing an antibody screen on recipient's serum for clinically significant antibodies.

These antibodies are most likely to occur in 37oC and AGT phases of testing. Each
negative AGT test must be followed by "Coombs Control Check Cells." An
autocontrol may or may not be used. Some labs prefer to perform this routinely
during the antibody screen while others will only include it if an antibody needs to be
identified. The autocontrol has to be part of antibody identification procedure.

Comparing present findings with previous records for the recipient. If previous testing
has been performed on the recipient and should match current testing. These
comparisons can give assurance that no identification errors have occurred, but it is
not proof. Records would also show if clinically significant antibodies have been

detected in the past. These antibodies may be presently at undetectable levels. Any
history of clinically significant antibodies, even if undetectable now in the patient,
dictates an antiglobulin phase crossmatch needs to be done between recipient's
serum and donor's cells.

Confirmation of ABO and Rh type of red cell components being given when of blood
is received in laboratory.

Selection of appropriate ABO and Rh component units for the recipient first would be
the same ABO and Rh type. Transfused donor red cells must be ABO compatible with
the patient's plasma and whatever antibodies may be present. Transfused plasma
must be ABO compatible with the recipient's red cells.
Selection of Components When ABO-Identical Donors Are Not Available
ABO Requirements
Whole Blood
Red Blood Cells
(most plasma removed)

Must be compatible with the recipient's plasma.

Granulocytes, Pheresis

Must be compatible with the recipient's plasma.

Fresh Frozen Plasma

Must be compatible with the patient's red cells.

Platelets, Pheresis
Cryoprecipitated AHF

Must be identical to that of the recipient

All blood groups acceptable; components

compatible with the recipient's red cells preferred
All ABO groups acceptable

Rh-positive components should be given to Rh-positive individuals and Rh-negative

units should be reserved for D-negative individuals. The physician needs to be
involved in decisions relating to giving Rh-positive blood to an Rh-negative individuals
since those individuals have 80% chance of making an anti-D following transfusion.

Perform a crossmatch either serologically. If no clinically significant antibodies are

found in the recipient the institution has the option of choosing an immediate-spin
crossmatch (serologic technique) or a computer crossmatch. If clinically significant
antibodies are found, an antiglobulin crossmatch must be performed.

Type and Screen

The type and screen consists of ABO/Rh, antibody screen, and a records check. This order is
used when likelihood of needing blood is low. Therefore, no donor blood crossmatched to
patient. If need for blood suddenly arises, you can take sample that is already typed and
screened, and perform a crossmatch with donor units from the specimen. Type and screen
protocol cannot be used if patient has an antibody. Then an antiglobulin crossmatch must
be performed.

Benefits of a Crossmatch
Performing a crossmatch before transfusing blood has the following benefits:

Detects major ABO errors (ie. crossmatching an A donor with an O or B recipient )

Detects most recipient antibodies to antigens on donor red cells (if the antibody is in
high enough titer to react) One of the most common clinically significant antibodies
that are missed are the Kidd antibodies.

Limitations of a Crossmatch:

Will not detect errors in patient identification (unless a previous record exists)

Will not detect ABO mix-ups if blood types are compatible (can crossmatch group A
donor blood for an AB recipient)

Will not detect Rh errors (can crossmatch Rh+ donor blood with Rh negative recipient
with no reaction if the patient has no anti-D)

Will not detect all recipient antibodies to donor antigens (antibody may be too weak
to detect, but still cause transfusion reaction such as the Kidd antibodies)

Will not prevent alloimmunization of recipient (only ABO and Rh antigens matched patient can potentially make antibody to all the other antigens) This is why many of
the discovered antibodies are found in multi-transfused patients.

Immediate Spin versus Antiglobulin, Coombs, Crossmatch

The purpose of Immediate spin step of crossmatch is to detect major ABO incompatibility
between donor and recipient. ABO incompatibility is the most common life-threatening
type of transfusion reaction and is often due to clerical errors.
It is permissible to stop at immediate spin step of crossmatch if:
1. Immediate spin is negative and
2. Antibody screen is negative in all phases and
3. There is no record of previous antibodies
It is NOT permissible to stop at the immediate spin step and must carry crossmatch through
antiglobulin, Coombs, phase if:

Immediate Spin test agglutinated or

Patient has an antibody (screening cells are positive) or

Patient has a record of a previous antibody

ABO DISCREPANCIES
DEFINITION
Any deviation from the expected pattern of antigen on the cell and the opposite
antibody in the serum.
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

4+

4+

4+

ABO Discrepancies MUST BE RESOLVED

In RECIPIENTS the discrepancies must be resolved before any blood component is

transfused. If not resolved before blood is needed, transfuse Group O (O NEGATIVE if
there is a discrepancy in the Rh type also).

In DONORS the discrepancies must be resolved before any blood is labeled with a
blood type..

GENERAL RULES TO RESOLVE:

1. Always re-test first.
2. Check for clerical/technical errors
3. Weakest reaction is usually the one in doubt.
4. Check results of the screening cells.
5. Check the patients age.
6. Check the diagnosis
7. Check the transfusion history.

KINDS OF DISCREPANCIES

CLERICAL ERRORS (TRANSCRIPTION ERRORS)

Clerical errors are the most common.

If you do not record results as you read each tube, you run risk of recording incorrect
results. Always check which tube you are reading and record the results immediately.

Make sure you are recording results on right worksheet. One way to prevent this error
is to minimize the times working with more than one patient or donor at a time.

Recording results in the wrong spot on worksheet could occur when you put some of
the serum results in the cell typing area or vice versa. Be sure you have techniques
that will prevent you from performing this error. (This is why our labeling procedure
uses capital A and B for the forward type and a1C and bC for the reverse typing)

TECHNICAL ERRORS

Sample mix-up such as wrong serum tube with wrong clot or 3% suspension.

Failure to add serum or reagent can lead to technical errors where no reaction is
occurring where one is expected. Remember for both ABO and Rh always add your
reagent antisera and serum before adding cells.

Addition of wrong reagent such as screening cells, which are O, instead of A1 /B cells

Contaminated reagents could result in either false negative or false positive results
depending on whether reagent added neutralized or added to the reactivity of
the original reagent.

Under centrifugation could lead to a negative reaction since the cells are not
encouraged adequately to bind with the antibody. Over centrifugation can lead to
you reading the reaction as positive while there is still a button on the bottom of the
tube or your shaking to dislodge the button broke up the agglutination reaction.

Warming the test could result in a false negative reaction since ABO antibodies are
IgMs that react better in the cold.

Too many cells in your cell suspension can lead to decreased or negative reactions
since there are too many cells for the number of antibodies present in the reagents.

Failure to detect weak results can occur if you are not watching the reactions while
you are shaking them out or if you shake too hard.

Failure to detect hemolysis can be a definite problem. Remember a positive

reaction can be hemolysis as well as agglutination since the antigen-antibody
reaction can bind complement. When complement is bound it can lead to
hemolysis that is also an indication of a positive reaction.

Dirty glassware can cause the cells to artificially clump.

PROBLEMS WITH SERUM TESTING

Serum testing is more common than problems with cell typing. This is either manifest as an
extra antibody present or an expected antibody missing
Extra Antibody
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

Expected Antibody Missing

PROBLEMS WITH RED CELL TESTING

There are a number of problems that can occur with the red cell testing including:

Mixed-field agglutination

Weak or missing antigens

Unexpected antigen

Polyagglutinable cells

Mixed-field agglutination
ANTI-A

ANTI-B

A1 CELL

B CELL

MF

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

4+

4+

Weak or missing antigen

Unexpected antigen

Polyagglutinable cells

RESOLVING PROBLEMS WITH SERUM TESTING:

Weak or missing antibody(ies)
An extreme example would be no reaction for the forward and reverse typings.
ANTI-A

ANTI-B

A1 CELL

B CELL

The steps to follow to resolve this discrepancy is to:

1. Check birth date since newborns and the elderly are more likely to demonstrate this
discrepancy. Newborn antibodies are not present until at least 6 months. DON'T
ATTEMPT TO SERUM-CONFIRM NEWBORNS. As individuals ages they may also lose their
ability to maintain their antibody levels. Therefore, the very elderly have decreased
antibody levels.
2. Check diagnosis since patient conditions such as: Immune deficiencies,
Chemotherapy, Radiation Therapy, and Bone marrow transplantation may explain
the missing antibodies.
Resolution of Missing Antibodies:
1. Add two more drops of serum just in case you forgot to add them the first time and
centrifuge. If negative then incubate in cold (4-18oC) 15-30 MINUTES
2. Include autocontrol to rule out interference from natural anti-I when incubating at
(4-18oC).
3. At 4oC Anti-A and Anti-B enhanced since they are saline, cold-acting antibodies as
seen in this example for an O individual.
ANTI-A

ANTI-B

A1 CELL

B CELL

AUTOCONTROL

2+

2+

4. Compare this with a 4oC Auto-Anti-I enhanced would have a positive autocontrol as
seen in the example below
ANTI-A

ANTI-B

A1 CELL

B CELL

AUTOCONTROL

2+

2+

2+

5. Group A or Group B can serve as its own negative control.

6. 4oC Anti-B enhanced is shown below:

ANTI-A

ANTI-B

A1 CELL

B CELL

AUTO-CONTROL

4+

2+

7. 4oC Anti-I enhanced on the other hand would have a positive autocontrol.

ANTI-A

ANTI-B

A1 CELL

B CELL

AUTO-CONTROL

4+

2+

2+

2+

8. If anti-I enhanced along with anti-A or anti-B, can re-set up and incubate at 18oC. As
seen in this example of 18oC: Anti-B enhanced, anti-I nonreactive
ANTI-A

ANTI-B

A1 CELL

B CELL

AUTO-CONTROL

4+

2+

Presence Of Unexpected Anti-A

The presence of Anti-A1 should be suspected when the antibody is reactive against the A
cells but not the screening cells at immediate spin as seen in the example below.
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

Immediate Spin
Screening Cell I

Screening Cell II

Autocontrol

37oC

AHG

CCC

Naturally anti-A1 occurs in subgroups of A or are passively-transfused from Group O platelets

and other blood products.
How to Resolve the Issue of Unexpected Anti-A:
1. Check recent transfusion history for group O products, (especially platelets) that
would explain the presence of this antibody.
2. Test patient cells with lectin-A1. Subgroups will be negative with this reagent but
A1cells will be positive.
Lectin + A1 CELL = 4+
Lectin + A subgroups CELLS = 0
3. Test patient serum with three A1 cells and three A2 cells and if it is an anti-A1 the
following reactions will occur:
Anti-A1: SERUM + A1 CELLS = +
SERUM + A2 CELLS = 0
Anti-A1 will react only with the A1 cells but not with the A2 cells

4. In the case of passive Anti-A from Group O Platelets the reactions would be the
following:
SERUM + A1 CELLS = +
SERUM + A2 CELLS = +
In this case if the antibody is strong enough need to transfuse group O blood .
UNEXPECTED A OR B ANTIBODY WHEN THE IMMEDIATE SPIN ANTIBODY SCREENING IS POSITIVE
You may have a positive reaction with the reagent A1 or B cell that is due to a
room-temperature antibody reacting with an antigen other than A or B on the cells
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

Immediate
Spin
Screening Cell I

2+

Screening Cell II

Autocontrol

37oC

AHG

CCC

How to Resolve the Issue of Unexpected Anti-A that is probably another antibody due to the
results of the Antibody Screening:
1. Identify the antibody by performing an identification panel at room temperature.
2. Pre-warm away (use caution) the effect of this antibody by doing the reverse typing
with prewarmed serum and reagent cells.
3. Type reagent A1 or B cell for the corresponding antigen once the antibody is
identified.

For example, if the patient had an anti-N that was showing up at room temperature
according to the antibody identification process, you would then type for N on the reagent
cells used for the reverse typing. If anti-N is causing your problem, then the cells should have
N antigen present.
ROULEAUX FORMATION GIVING UNEXPECTED AGGLUTINATION IN ALL SERUM TESTS
Rouleaux can give unexpected agglutination in all serum tests

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

Immediate
Spin
Screening Cell I

2+

Screening Cell II

2+

Autocontrol

2+

37oC

AHG

CCC

Rouleaux may also give false positive cell typing if strong enough and cells are insufficiently
washed. This phenomenon is due to alteration in serum protein concentration such as:

Multiple myeloma

Macroglobulinemia

Liver disease (decreased albumin)

Also seen with volume expanders

Characteristics of rouleaux is that it:

Looks like agglutination macroscopically

Microscopically it appears as "stacks of coins"

How would you resolve rouleaux problems?

Do saline replacement technique:
1. Re-centrifuge the test tube.
2. Draw off serum without disturbing cell button
3. Add two drops of saline
4. Resuspend
5. Rouleax disperses in saline; TRUE AGGLUTINATION REMAINS
RESOLVING PROBLEMS WITH CELL TYPING
Mixed-field agglutination
Mixed-field agglutination is seen as large or small agglutinates with many unagglutinated
cells. Usually mixed-field agglutination means a MIXED-CELL POPULATION. The causes of
mixed-field agglutination can be:

Mixed cell populations resulting from massive transfusion of another blood group such
as an B individual receiving "O" red blood cell donor units since the transfusion center
did not have enough B donor units.

Bone marrow transplant patients may have both some of their original type of cells
and the type of the bone marrow transplant.

Weak subgroups of A3 traditionally give a mixed field reaction.

Rarely the condition called chimerism due to intrauterine exchange of erythrocyte

precursors between twins or 2 fertilized eggs fuse into one individual.

You should try to find cause of mixed field agglutination before setting up blood to transfuse
so be sure to check the patient's transfusion records and clinical history. If it appears to be a
weak subgroup performed the tests discussed under Unexpected Anti-A
Weak or missing antigen
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

Weak, or missing, antigen may be due to very weak subgroup of A or B, loss of transferase in
acute leukemia, massive transfusion of GROUP O, or bone marrow transplant
How would you resolve a weak, or missing, antigen?

Obtain recent transfusion history and any clinical history of bone marrow transplant

Read forward grouping microscopically

Use anti-A,B and incubate at 4-22oC at least 15 minutes

Use monoclonal antisera that is known to react with antigens like Ax and Bx

Perform specialized tests if the above steps do not resolve the problem:

Specialized tests would include absorption/elution techniques and saliva studies.

Acquired B antigen (with the colon or infections with Gram-negative rods)
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

2+

4+

Bacterial enzymes modify the "A" antigen to a "B" antigen and the patient forward types as
an AB but reverses as an A.
How would you resolve a possible acquired B antigen?

Set-up an autocontrol. The patient's own anti-B will not agglutinate their own AB cells.

Check clinical history to evidence of colon problems or Gram-negative rods.

Check monoclonal anti-B product inserts since some will not react with B acquired
antisera

Acidify some reagents anti-B to pH 6 and re-test. Modified (acquired) B antigens will
not react in the acidified antiserum, normal B antigens will still react

Polyagglutinable cells
Most monoclonal anti-A and anti-B will show problems with polyagglutinable cells if it is a
problem with the cell membrane that leads to the agglutination. The most likely causes of
due to Wharton's Jelly, found in cord blood, and strong positive direct antiglobulin test due
to a cold agglutinin. In the case of strong positive DAT, it would appear to be an AB in the
forward type and an O on reverse.

ANTI-A

ANTI-B

A1 CELL

B CELL

2+

2+

4+

4+

Coats newborn cord cells and the child's type may appear AB. You do not do a
reverse on newborn blood since they have not made any anti-A or anit-B yet.

If the baby types as an AB recheck by washing cells several times and re-testing since
you need to make sure you have removed the Wharton's Jelly and the baby is truly
an AB. Better yet ALWAYS WASH CORD BLOOD AT LEAST 4 TO 5 X'S BEFORE
DETERMINING THE TYPE OF THE BABY.

1. Strong positive DAT

May be seen in cold auto-immune hemolytic anemia

If due to cold agglutinin, wash several times in warm saline and re-test

Cells washed 3X at 37oC would probably look like this:

ANTI-A

ANTI-B

A1 CELL

B CELL

4+

4+

PROBLEMS WITH BOTH CELLS AND SERUM

Strong cold auto-agglutinins
ANTI-A

ANTI-B

A1 CELL

B CELL

4+

4+

4+

4+

Immediate Spin

37oC

AHG

CCC

Screening Cell I

4+

3+

3+

Screening Cell II

4+

3+

3+

Autocontrol

4+

3+

3+

A strong cold auto-agglutinin is most often due to strong auto-anti-I.

1. To resolve cell typing difficulties:

Wash cells 3-4X with warm (37oC) saline

Re-test warm-washed cells

2. To resolve serum typing difficulties:

Perform serum testing at 37oC (Use caution that weak isoagglutinins (anti-A and
anti-B) are not missed using this technique)

Autoabsorb cold agglutinins onto patient cells at 4oC.

IMMUNE HEMOLYSIS & COMPLEMENT

Complement
Complement helps fight off bacterial and viral infections, eliminates protein complexes, and
helps in the immune response complex. The three major roles of complement are:

promoting acute inflammation process that allow white cells and macrophages to
migrate to the source of the problem

altering the cell surfaces to encourage phagocytosis (opsonization)

modifying the cell surface that will eventually lead to cell lysis

Characteristics of Complement
Complement is a series of proteins found in fresh, normal serum. It is in the beta region on
protein electrophoresis and is categorized as a beta globulin.

The complement

component that is found in the highest concentration is C3. Terminology commonly used
for the various complement components:

Complement components are identified by C and their number: C1, C2, C3 etc

Complement products resulting from the splitting of these proteins during the
activation process are followed by a lower case letter: C3a, C3b, C1q

If complement complexes develop that have enzymatic activity are written with a
bar above the top: C5b678

Complement controls various biological process:

1. Cell destruction through lysis
2. Cell destruction through opsonization (enhanced phagocytosis) especially with C3b
3. Chemotaxis via certain split products acting as chemical signals to phagocytic cells
4. Anaphylaxis again through the split products (C5a and C3a), which promote
inflammation. C5a and C3a can bind with mast cells and basophils leading to the
release of histamine. This is turn:

Increases vascular permeability

Smooth muscle contraction to preserve blood for vital organs

Increases cellular membrane adhersion

Complement is normally inactive in serum or if activate the activation pathways are

inhibited by control mechanisms of other complement proteins. It becomes activated by:

Antigen-antibody reactions - most often IgM (classical pathway)

Bacterial polysaccharides, virus particles, enzymes, endotoxins (alternate and lectin

pathway)

Complement Cascade - (Classical Pathway)

Two adjacent antigen sites bound by antibody capable of binding complement:

IgM molecule since it has 5 immunoglobulin subunits

Certain IgG molecues that are capable of binding with complement. (only IgG 1, 2,
or 3) Complement needs 2 IgG molecules bound to antigen sites that are within 30-40
nm of each other.

The steps of the classical pathway

1. Antigen-Antibody reaction occurs
2. Complement senses adjacent Fc receptors
3. Complement is activated and forms C1qrs complex
4. C1qrs activates C4
5. Activated C4 breaks down to C4a and C4b
6. C4b activates C2 to form C4b2a complex
7. C4b2a complex also known as C3 convertase
8. In presence of Ca+, C4b2a activates C3
9. Activated C3 breaks down to C3a and C3b
10. C3b attaches to rbc membrane - is sensed by RE system and removed from
circulation (extravascular hemolysis)
11. Activated C3a activates many C5 molecules that leads to the development of the
Membrane Attack Complex.
12. Each activated C5 activates many more C6 and C7
molecules
13. C6 and C7 conversion proceed rapidly to C8 and C9
activation
14. Activated C8 activates many more C9 molecules
15. Activated C9 attacks red cell membrane, boring holes
in it
Contents of red cell leak out into plasma leading to intravascular hemolysis. As the
complement cascade results in biologic side-effects such as chemotaxis, opsonization, and
anaphylaxis.

What Determines Whether or Not Complement Will Be Activated

Nature of antibody coating the cell

IgM antibodies are the best because they have more antigen-binding sites. They can
achieve binding of two adjacent antigens by single IgM molecule. Only certain IgG
subclasses are capable of activating complement: IgG subclasses 1, 2, and 3. Of these IgG
subsets, IgG 3 is the best. IgG subclass 4 does not activate complement.
Number of antigen sites on red cell
The more antigen sites found on the red blood cell, the more likely two adjacent ones are
bound by antibody.
What does this mean in relationship to the ABO System?
The A and B antigens are in very high concentration on the red blood cells. The
natural-occurring, expected antibodies (anti-A, anti-B, and anti-A,B) are IgM. ABO
incompatibilities are the most likely to result in intravascular transfusion reactions from the
activation of the classical pathway.
Possible Outcomes of Complement Activation in Blood Banking

Normal survival of red cells can occur but realize the other outcomes are more likely

Intravascular hemolysis (complement cascade go to completion) and cells are lysed

Extravascular hemolysis where the complement cascade stops at C3b step. Cells
coated with C3b are removed from circulation via macrophages and neutrophils

"Damaged cells" (spherocytes and stroma fragments) lead to decreased cell survival
and possible activation of Hageman factor that leads to coagulation activation

Disseminated Intravascular Coagulation (DIC) may also occur due to small clumps of
agglutinated cells in blood stream, fibrinogen consumption, activation of fibrinogen
system and fibrin destruction.

When leukocytes are exposed to various antigen-antibody complexes they will also
respond by secreting various cytokines that will lead to: fever, a drop in blood
pressure and additional release of white blood cells from bone marrow

Renal failure, which is the most common complication of an untreated hemolytic

transfusion reaction. This occurs due to a combination of :
Hypotension + Contraction of the blood vessels in the kidney(smooth muscles that
responds in anaphylaxis) + Intravascular clots + Toxic effects of free hemoglobin

Non-Immune Hemolysis

Physical

Temperature extremes (outside body)

Hypotonic solutions (IV solutions)

Mechanical (pumps, intravascular clots)

Chemical- toxins
Immune-Mediated Cell Destruction (Hemolysis)
Causes of Immune Cell Destruction:
1. Antibodies that bind complement
2. Antibodies that do not bind complement
3. Complement activation only
Types of Immune-Mediated Hemolysis
Intravascular Hemolysis
Intravascular hemolysis is complement mediated since the hemolysis is due to
activation of the complement pathway by the classical pathway. Intravascular
indicates the cell destruction takes place within the blood vessels. This type of hemolysis
can be life-threatening due to both the possibility of anaphylaxis and renal failure.
Extravascular Hemolysis
Extravascular Hemolysis may be antibody mediated or complement mediated. The cell
destruction takes place within RE system (spleen, primarily) and is generally not
life-threatening. The survival rate of the transfused cells will be decreased.
Immune Response Following a Blood Transfusion Leading to Alloimmunization
1. Donor red cells carry foreign antigens die normally and phagocytized by RE system.
2. Foreign antigens processed and stimulate immune response.
3. IgM antibody produced after several weeks, probably no cell destruction.
4. Gradual decline of red cells results in continuous re-exposure of antigen to immune
system.
5. Primary and secondary responses can overlap each other.
6. IgG antibodies can be produced while IgM antibody productions is going on, from
the same blood transfusion (same "stimulating event").

7. Low-titer IgM antibodies may not cause cell destruction, but higher-titer IgG
antibodies will eventually lead to extravascular hemolysis.
Intravascular Hemolysis (complement mediated) Summary
Mechanism of intravascular hemolysis
Complement is activated when two adjacent antigen sites are bound by antibody. The
activated complement rapidly proceeds through several chemical changes to membrane
attack complex. The membrane attack complex bores a hole through red cell membrane,
causing hemolysis. The rate of complement activation and amount of complement
activated determines whether complement cascade goes to completion.
Signs of intravascular hemolysis

Sudden drop in blood pressure due to the anaphylactoxins: C5a and C3a

Hemoglobinuria due to the lysis of of the red blood cells. If more hemoglobulin is
released that can be carried by albumin.

Hemoglobinemia (plasma hemoglobulin levels) occurs again due to the lysis of red
blood cells in the blood vessels.

Decreased haptoglobin since haptoglobin can also carry hemoglobin. There are
limited amounts of haptoglobulin produced so once it is used, it will be removed by
the liver and the haptoglobulin levels decreased.

No rise in hematocrit following blood transfusion since the donor blood is destroyed.

Anaphylactic shock, death may result if large amounts of complement activated

Antibodies that bind complement (classical pathway)

IgM antibodies = anti-A, anti-B, anti-I, anti-Lewis a

IgG antibodies = anti-A,B, auto anti-P, anti-D, anti-Kidd, anti-Kell, etc.

Extravascular Hemolysis - Antibody Mediated

Mechanism of antibody-mediated hemolysis:
Antibodies attach to antigens on red cell membrane and are sensed by phagocytes in RE
system. The the antibody-coated red cells are ingested by the phagocytic cells and
destroyed at a rate faster than normal cell destruction. RE system plucks antibody off cells,
leaving damaged membrane. Cell becomes a spherocyte with shorter lifespan.
Antibodies that do not bind complement but promote extravascular cell destruction
Extravascular hemolysis processes are caused by IgG antibodies:

Rh antibodies (anti-D, anti-C, anti-c, anti-E, and anti-e)

Anti-Kell (anti-K, anti-k, anti-Kpa, anti-Jsa etc.)

Anti-Kidd (anit-Jka and anti-Jkb)

Anti-Duffy (anit-Fya and anti-Fyb)

Anti-S
Signs of extravascular hemolysis:

Signs of extravascular hemolysis includes a falling hematocrit, increased bilirubin

(unconjugated), increased LD, and an abnormal peripheral smear (polychromasia,
spherocytes, fragments)
Extravascular Hemolysis - Complement Mediated
In extravascular hemolysis that is complement mediated the breakdown products of
activated complement attach to red cell membrane (primarily C4b and C3b and C3d).
Presence of C3b coated cells are sensed by phagocytes in RE system. Complement-coated
cells are ingested and destroyed at a rate faster than normal cell destruction.
Causes of Complement-Mediated Extravascular Hemolysis:
Any of the IgM or IgG antibodies that are capable of activating complement can cause
this type of hemolysis. Besides the classical pathway the alternate pathway of complement
activation can also occur due to the presence of various components like cell walls of
bacteria and yeast, dialysis membranes, dextran, and some tumor cells. Certain drugs can
also activate complement and can lead to a complement-mediated extravascular
hemolysis.