BLOODBANK AND SEROLOGY PRINCIPLE OF ANTI-GLOBULIN TEST testing) or screening cells (Ab

• Ab molecules and complement screen) in serum.

Autoantibodies components are globulin. 2. Determination of RBC
- “Self” antibodies and are directed • Injecting an animal with human phenotype using Antisera (i.e.
against self-antigens. globulin stimulates the animal to Kell typing, weak D testing).
produce antibodies to a foreign 3. Titration of INC Ab.
Alloantibodies protein. - It is a two-step process compared to
- Non-self antibodies and are produced DAT’s one-step process
after exposure to non-self antigens. TYPES OF ANTIGLOBULIN TEST
IAT Steps:
Warm Antibodies 1. DIRECT ANTIGLOBULIN TEST RBC + Serum + Immediate Spin +
- Used to react and are active at body - Detects in vivo sensitization (inside) of Incubate (No visible agglutination yet at
temperature (37C) and are considered RBCs w/ IgG or complement 37C commonly) → Centrifuge again →
to be “clinically significant”. components. Wash RBC → Add AHG Reagent →
- It involves IgG (mostly). - Not a required test in routine pre- Centrifuge again → Check for
transfusion protocols. macro/micro agglutination.
Cold Antibodies - Does not include incubation as
- Used to react are active at room sensitization happens in vivo. Note:
temperature (22-24C). - Initial DATs include testing one drop of • If (-) add CCC then centrifuge. If it
- It involves IgM (mostly) a 3% to 5% suspension of washed RBC reacts positively, it means that the
with polyspecific (anti-IgG, anti-C3d) negative result is correct.
COMPLEMENT SYSTEM reagent. • Coomb’s Check Cells (CCC) –
• Classical - starts in C2 - The saline control detects spontaneous Made of Type O cells and it
• Alternative- Starts in C3 agglutination of cells or reactions validates negative results.
• Lectin binding occurring without adding AHG reagents. • IgG is not a good agglutinator
• MAC- Starts in C5 compared to IgM.
American Association of Blood Bank - This is due to the fact that IgM is
ANTIBODY PROPERTY (AABB) Technical Manual pentameric and has a larger
1. Affinity surface area for reactions
- Strength of a single antigen-antibody compared to IgG’s monomeric
bond nature.

2. Avidity Why washing is done in IAT and DAT?

- Expresses the binding strength of a - When both DAT and IAT are performed,
multivalent antigen with antisera IgM RBCs must be saline-washed a minimum
- Detected in immediate spin phase of 3x before adding AHG agent.
3. Specificity (strong agglutinator as it is Cold- - Inadequate washing may result in a
reacting. false-negative reaction because of the
FACTORS THAT INFLUENCE neutralization of AHG reagent by
AGGLUTINATION REACTION residual unbound serum globulins

1. Prozone (>Ab, <Ag) ABO BLOOD GROUP

- Antibody excess. - The most important of all blood groups
in transfusion practice.
• It is the unique blood group system.
2. Equivalence (Ag = Ab)
• It is the only blood group system in
- Optimum proportions of antigen and which individuals have antibodies in
Positive test results could indicate:
antibody. their serum to antigens that are
• Autoimmune Hemolytic Anemia
absent from their RBCs.
• Drug-Induced Hemolytic Anemia:
3. Postzone (>Ag, <Ab)
- Caused by penicillin (it causes drug • Karl Landsteiner
- Antigen excess.
adsorption to RBCs), cephalosporin, - Discovered ABO in 1901
(modifies RBC membrane), rifampin - He and his five co-workers began
ANTIGLOBULIN TEST
(immune complex formation), and mixing each other’s red cells and serum
- Used to detect RBCs sensitized with IgG
methyldopa (autoantibody formation). together and inadvertently performed
alloantibodies, IgG autoantibodies, and the first forward and reverse ABO
complement components. groupings.
2. INDIRECT ANTIGLOBULIN TEST (IAT)
- Sensitization can occur either in vivo or - “Beginning of the concept of individual
- Performed in vitro sensitization of RBCs uniqueness”.
in vitro.
and used for:
- IAT and DAT remain the most common
1. Detecting incomplete (non- “If an antigen is present on a px RBC, the
procedures performed in blood group
agglutinating) Ab to potential corresponding Ab, will not be present in
serology.
donor RBCs (compatibility the px, plasma under “normal
conditions.” – Karl Landsteiner Rule

Bloodbanking and Serology (Immunohematology)

 • Group O antibodies React best at to form the ABO antigens in
TYPES OF ABO BLOOD TYPING room temperature (22-24C) or secretions.
below in vitro and also activates • The precursor on erythrocytes is
1. FORWARD GROUPING (FRONT TYPE) complement to completion at 37C referred to as type 2.
- Using known sources of commercial – Can cause acute hemolytic • ABH antigens on the RBC are
antisera (Anti-A, Anti-B, and Anti-D) to transfusion reactions constructed on oligosaccharide
detect antigens on an individual’s RBC. chains of a type 2 precursor
- Specimen: Red blood cell. (RCS in tube If there are Anti-A or Anti-B ab in the substance.
method, Whole blood or PRBC for slide newborn serum, where did they most
method). likely originate? What source? Se gene
Note: Agglutination cannot be graded • Most antibodies found in cord - Required in typing secretions.
in the slide method as it does not utilize blood serum are of maternal origin. - If absent, an individual is None
centrifugation. Slide typing is only used in • Results of serum ABO testing before secretors.
rapid or emergency cases. 3 to 6 months of age cannot be
considered valid because some or Interaction of Hh and ABO genes
2. REVERSE GROUPING (BACK TYPE) all of the antibodies present may be - Individuals who are in blood group O
- Detecting ABO Ab in the px serum by IgG maternal antibodies that have inherit at least one H gene (genotype HH
using known reagent RBCs, namely A1 crossed the placenta. As a result, it or Hh) and two O genes.
and B cells. is logical to perform only forward
- Specimen is serum/plasma and the grouping on cord blood from Note: The O gene at the ABO locus
reagents are Known A or Known B newborn infants. which is sometimes referred to as an
(human source). “amorph” does not elicit the production
INHERITANCE OF ABO BLOOD GROUPS of a catalytically active polypeptide
ABO ANTIBODIES transferase; Therefore, the H substance
- “Naturally occurring”. Bernstein (1924) remains unmodified.
- ABO Ab are mostly IgM. - Demonstrated that an individual
- The production of ABO Ab is initiated at inherits one ABO gene from each parent 1. Hh GENES – H and h ALLELES
birth, but titers are generally too low for and that these two genes determine • “H” allele
detection until the individual is 3-6mos of which ABO-Ag are present in the RBC - Codes for fucosyltransferase enzyme
age. membrane. that adds fucose on type 2 chains
- Ab production peaks when an • One position, or locus, on each (primarily) to form the H antigen onto
individual is between 5-10yrs of age and chromosome 9 is considered by an which A and B antigens are built on
declines later in life. A, B, or O gene. RBCs.
- Although and contains predominantly • O gene is considered amorph, as no
IgM Ab, there may be small quantities of detectable antigen is produced in • “h” allele
IgG present. response to the inheritance of this - Silent allele (amorph)
gene.
Elderlies (>65y.o) and newborns – Have • The designations group A and B H Antigen
low Ab titer. refer to phenotypes, whereas AA, - It is the foundation upon which A and B
BO, OO denote genotypes. antigens are built.
• Anti-A, B Antibody • In the case of an O individual, both - A and B genes code for enzymes that
- Was thought to be just a mixture of Anti- phenotype and genotype are the add an immunodominant sugar to the H
A, and Anti-B, and cannot be separated same, because that individual antigen.
into a pure specificity when adsorbed would have to be homozygous for
with either A or B cells but it was proven the O gene. A Antigen
that it is not a combination of Anti-A and A gene
Anti-B but is a separate “cross-reacting” FORMATION OF A, B, AND H RED CELL - It codes for an enzyme that adds
antibody that is usually IgG in nature. ANTIGEN GalNAc (N-Acetyl-D galactosamine) to
• Results from the interaction of genes the terminal sugar of the H Antigen. (This
• RBC Immune Anti-A, B at 3 separate loci (ABO, Hh, and Se). sugar is responsible for A specificity)
- When exposed to Group A or B Ag (or • Produce specific
both) Group O persons will have an glycosyltransferases that add A gene → a-3-N-
immune response that results in the sugars to a basic precursor acetylgalactosaminyltransferase → N-
production of separate immune anti-A substance. acetye-D-galactosamine
and/or anti-B ab. • A, B, and H antigens are formed
- This could be seen in Fetomaternal from the same basic precursor B Antigen
Bleed of a Group O mother with a Group material (called a paragloboside or B Gene
A baby. (ABO HDN). glycan) to which sugars are - Codes for an enzyme that adds D-
attached in response to specific galactose to the terminal sugar of the H
Note: enzyme transferases elicited by an antigen.
• Knowing the amount of IgG anti-A, inherited gene. - When both A and B genes are
anti-B, or anti-A,B in a woman’s • The H antigen is actually the inherited, the B enzyme (a-3-D-
serum sometimes allows prediction precursor structure on which A and galactosyltransferase) seems to
or diagnosis of hemolytic disease of B antigens are made. Inheritance of compete more efficiently for the H
the fetus and newborn (HDFN) the H gene results in the formation substance than the A enzyme (α-3-
caused by ABO incompatibility – of the H antigen. Nacetylgalactosaminyltransferase).
Cord blood from O+ mother • H and Se Gene are closely linked B gene → α-3-Dgalactosyltransf erase
• For Group A and Group B persons, and located on chromosome 19. → D-galactose
the predominant Ab is IgM. • The H gene must be inherited to
• Group O is predominantly IgG (with form the ABO antigens on the RBCs
some IgM). and the Se gene must be inherited
Bloodbanking and Serology (Immunohematology)
 - Controls the presence of H, A, and B THE BOMBAY PHENOTYPES (Oh)/Hnull
antigens on both RBCs and in Secretions • In RBC testing using anti-A and anti-
B, the Bombay would phenotype as
2. Se Gene an O blood group.
- Se and se alleles (se is an amorph) • However, the RBCs of the Bombay
- Controls the presence of H antigen in phenotype (Oh) do not react with
the secretions the anti-H lectin (Ulex europaeus)
• Bombay serum contains anti-A,
3. ABO Genes anti-B, anti-A,B, and anti-H.
- A, B, and O alleles
- Inherit 1 gene from each parent that CATEGORIES OF ABO DISCREPANCIES
codes for an enzyme that adds sugar to
the H antigen. 1. GROUP I DISCREPANCIES (MOST
COMMON)
ABO Subgroups - Associated with unexpected reactions
- Represents phenotypes that show in the reverse grouping due to weakly
weaker variable serologic reactivity with reacting or missing antibodies.
commonly used human polyclonal anti- - When a reaction in the serum grouping
A, anti-B, and anti-A, B reagents. is weak or missing, a group I discrepancy
should be suspected, because,
A SUBGROUPS normally, RBC and serum grouping
reactions are very strong (4+).
Von Dungern (1911) - Obtaining the patient’s history may
- Described two different A antigens resolve this type of discrepancy.
based on reactions between group A - Includes: Chimerism, Bruton Disease
RBCs and anti-a and anti-A1
2. GROUP II DISCREPANCIES (LEAST
• A1: Group A RBCs that react with COMMONLY ENCOUNTERED)
both anti-A and anti-A1 - Are associated with unexpected
• A2: Only react with Anti-A reactions in the forward grouping due to
weakly reacting or missing antigens
Note: RBC from A1 and A2 individuals - Usually due to weak antigen
react equally strong with current
reagent monoclonal anti-A in ABO
3. GROUP III DISCREPANCIES
forward typing tests
- These discrepancies between forward
and reverse groupings are caused by
• Anti-A strongly reacts on both A1
2. Se Genes – Se and se alleles protein or plasma abnormalities and
and A2
result in the rouleaux formation or
• Differentiation of A1 and A2
“Se” Allele pseudoagglutination
phenotypes can be determined by
- Codes for a fucosyltransferase enzyme Rouleaux - is a stacking of erythrocytes
using a reagent made from the
that adds fucose onto type 1 chains that adhere in a coinlike fashion, giving
seeds of the plant Dolichos biflorus,
(primarily) in secretory glands. the appearance of agglutination.
which serves as a source of anti-A1.
- Controls expression of H antigens in – Caused by unexpected Ab reaction
This reagent is known as Anti-A1
secretions (saliva, body fluids, etc.) – Se Lectin.
allele is an amorph Cord blood samples received in the
- People who inherit these “sese” laboratory can also pose a problem in
Lectins
genotypes are termed nonsecretors. ABO testing, since cord cells may be
- Seed extracts that agglutinate human
contaminated with a substance called
cells with some degree of specificity. This
Wharton’s jelly, which may cause the red
reagent agglutinates A1 (or A1B) cells
cells to aggregate. Washing cord cells six
but does not agglutinate A2 or A2B cells.
to eight times with saline should alleviate
spontaneous rouleaux due to Wharton’s
jelly.

4. GROUP IV DISCREPANCIES
- These discrepancies between forward
BOMBAY BLOOD GROUP and reverse groupings are due to
- Discovered by Bhende in 1952 in miscellaneous problems:
Bombay, India. • Cold reactive autoantibodies
- Homozygous inheritance of the h gene • Patient has circulating RBCs of more
3. ABO genes – A, B, and O alleles than one ABO group due to RBC
• A and B alleles (hh) results in the inability to form the H
antigen and subsequently the A or B transfusion or marrow/stem cell
- Code for glycosyltransferase enzyme transplant
antigens.
that adds sugar onto H antigens to • Unexpected ABO isoagglutinins
produce A and B antigens. - No H, A, or B antigens on the RBC
membrane, only an abundant amount • Unexpected non-ABO
of precursor substance. alloantibodies
ABO ANTIGEN GENETICS
1. Hh Gene Potent cold autoantibodies
- H and h alleles (h is amorph) - Can cause spontaneous agglutination
of the patient’s cells. These cells often
Bloodbanking and Serology (Immunohematology)
yield a positive direct Coombs’ or • Two terminologies are based on
antiglobulin test. postulated genetic theories of Rh
– Cis AB phenotype inheritance.
• The third common terminology used
Crypt Antigen – “Hidden antigen”.
describes only the presence or
Hubener-Thomsen Syndrome – Presence absence of a given antigen.
of polyagglutination (Crypt antigen). The • The fourth is the result of the
cell membrane covering the crypt International Society of Blood
antigen will express the crypt antigen. Transfusion (ISBT) combined efforts.

RH BLOOD GROUP SYSTEM 1. Fisher-Race: DCE Terminology

- They postulated that the antigens of
Rh the system were produced by three
- It refers to a specific red blood cell closely linked sets of alleles.
(RBC) antigen (D) and to a complex - Fisher and Race named the antigens of
blood group system currently composed the system D, d, C, c, and E, e.
of over 50 different antigenic
specificities. “d” = Absence of D antigen
• The second most important blood • The phenotype of a given RBC is
group system in terms of transfusion defined by the presence of D, C, c,
• Antigens are very IMMUNOGENIC E, and e expression.
• Rh specific Ags reside on PROTEINS • According to the Fisher-Race
• Produced only after exposure to theory, each person inherits a set of
foreign red blood cells. Rh genes from each parent (one D
or d, one C or c, and one E or e)
HISTORY • DCE not CDE
• Before 1939, the only significant • Fisher-Race terminology represents
blood group antigens recognized the easiest way to think about the
were those of the ABO system. five major Rh system antigens, but it
• It began when Levine and Stetson has shortcomings.
described a hemolytic transfusion • The person expressing no Rh
reaction in an obstetrical patient. antigens on the RBC is said to be
• One year later, Landsteiner and Rhnull, and the phenotype may be
Wiener reported on an antibody 3. Rosenfield and Coworkers:
written as “—/—".
made by guinea pigs and rabbits Alphanumeric Terminology
• Weakened expression of all Rh
when they were transfused with • As the Rh blood group system
antigens of an individual has also
Rhesus macaque monkey RBCs. expanded, it became more difficult
been reported. These individuals
• This Ab w/c agglutinated 85% of to assign names to new antigens
are said to have the Rhmod
human RBCs, was named Rh after using existing terminologies.
phenotype. Placing parenthesis
the Rhesus monkey. • In the early 1960s, Rosenfield and
around (D), (C), and (e) indicates
• Levine and coworkers associates proposed a system that
weakened antigen expression.
demonstrated that the agglutinin assigns a number to each antigen
causing the HTR and the Ab of the Rh system in order of its
2. Wiener: Rh-Hr Terminology
described by Landsteiner and discovery or recognized relationship
- Wiener believed there was one gene
Weiner appeared to define the to the Rh system.
responsible for defining Rh that
same blood group. • For the five major antigens:
produced an agglutinogen containing
• Anti-Rhesus and Anti-LW ➢ D is assigned Rh1,
a series of blood factors, this Rh gene
• Further research resulted in defining ➢ C is Rh2,
produced at least three factors within an
Rh as a primary cause of hemolytic ➢ E is Rh3,
agglutinogen.
disease of the fetus and newborn ➢ c is Rh4, and
- It is important to remember that an
(HDFN, also called erythroblastosis ➢ e is Rh5
agglutinogen in Wiener nomenclature
fetalis) and a significant cause of • A minus sign preceding a number
actually represents the presence of a
hemolytic transfusion reactions. designates the absence of the antigen.
single haplotype expressing three
• By the mid-1940s, five antigens If an antigen has not been typed, its
different antigens.
made up the Rh system. number will not appear in the sequence.
• R=D
• r = absence of D (d)
TERMINOLOGY • 1/’ = C
Terminologies used to describe the Rh • 2/” = E
system are derived from four sets of • z/y = CE
investigators. • rh = C/E
• hr = c/e

Bloodbanking and Serology (Immunohematology)

 Rh FUNCTIONS • Rh antigens are highly
RhD and RhCE proteins and RhAG are immunogenic; the D antigen is the
exclusively on red blood cells. As they most potent then (c, E, C, e)
are transmembranes, it is not surprising D>c>E>C>e
they play a role in maintaining the Exposure to less than 0.1 mL of Rh-
structural integrity of red cells, they may positive RBCs can stimulate antibody
also be transporters. production in an Rh-negative person.
• 4 subclasses → IgG1, IgG2, IgG3
Weak D: Variations of D Antigen and IgG4
Expression • IgG1 and IgG3 are of the greatest
- When Rh-positive RBC samples are clinical significance because the
typed for the D antigen, they are reticuloendothelial system rapidly
expected to show strong positive clears RBCs coated with IgG1 and
4. International Society of Blood reactivity (3+ to 4+) with anti-D reagents. IgG3 from the circulation.
Transfusion Committee: Updated However, some individuals have RBCs • DO NOT BIND COMPLEMENT
Numeric Terminology that possess D antigen that requires an
- Its mandate was to establish a uniform indirect antiglobulin test to detect the Rh Typing Reagents
nomenclature that is both eye- and presence of D antigen. • The reagents may be high-protein-
machine-readable and is in keeping - Du based or low-protein-based, saline-
with the genetic basis of blood groups. based, chemically modified,
1. Weak D monoclonal, or blends of
Found in the Reference Book: table 7-6 • Inheritance of RHD genes that code monoclonals.
for a weakened expression of the D • The goal is to use a reagent
RH GENES antigen. anti-D that will allow for typing
- It is now known that only two closely • The D antigens expressed appear to individuals’ RBCs as quickly
linked genes located on chromosome 1 be complete but fewer in number. and accurately as typing for
control the expression of Rh proteins— • Quantitative problem ABO.
namely, RHD and RHCE.
2. C in Trans to RHD CLINICAL CONSIDERATIONS
RHD and RHCE → controls the • The allele carrying RHD is trans (or in
expression of Rh proteins the opposite haplotype) to the TRANSFUSION REACTIONS
allele carrying C; for example, • The D antigen is the most
RHD codes for the presence or absence Dce/dCe. immunogenic antigen outside the
of the RhD protein, • The Rh antigen on the RBC is normal, ABO system. When anti-D is
RHCE codes for either RhCe, RhcE, Rhce, but the steric arrangement of the C detected, a careful medical history
or RhCE proteins antigen in relationship to the D will reveal RBC exposure through
antigen appears to interfere with pregnancy or transfusion of
• RHAG – Rh-associated Glycoprotein the expression of D antigen. products containing RBCs.
(RhAG) DCe/dce • Circulating antibody appears within
 RhAG is termed a coexpressor and 120 days of a primary exposure and
must be present for successful 3. Partial D within 2 to 7 days after secondary
expression of the Rh antigens. • D antigen expression can be exposure.
weakened when one or more D • Rh-mediated hemolytic transfusion
Rh-POSITIVE PHENOTYPES epitopes within the entire D protein reactions, whether caused by
- As predicted, RH genes are inherited as are either missing or altered. primary sensitization or secondary
codominant alleles. Rh-positive • Anti – D typing immunization, usually result in
individuals inherit one or two RHD genes, extravascular destruction of
which result in the expression of RhD 4. Deletion (Del) immunoglobulin-coated RBCs.
antigen and are typed Rh-positive. - Del is a phenotype occurring in • The direct antiglobulin test is usually
• In addition to the RHD gene(s), two individuals whose red blood cells possess positive, and the antibody screen
RHCE genes are inherited. an extremely low number of D antigen may or may not demonstrate a
sites that most reagent anti-D are unable circulating antibody.
Rh-NEGATIVE PHENOTYPES to detect.
- Rh-negative individuals can arise from - Adsorbing and eluting anti-D from the Hemolytic Disease of the Fetus and
at least three different mutations. These individual’s red cells is often the only way Newborn (HDFN)
mutations are most often found in to detect the D antigen. • Levine and Stetson postulated that
individuals falling into three different Rh Antibodies the antibody causing the
ethnic backgrounds: European, African, • Most Rh antibodies are IgG transfusion reaction also crossed the
and Asian. immunoglobulins and react placenta and destroyed the RBCs
optimally at 37°C after AHG testing. of the fetus, causing its death. The

Bloodbanking and Serology (Immunohematology)

 offending antibody was
subsequently identified as anti-D.
• HDFN caused by Rh antibodies is
often severe because the Rh
antigens are well developed on
fetal cells, and Rh antibodies are
primarily IgG, which readily crosses
the placenta.

Rhogam (RhIg)
- Method was developed to prevent
susceptible (D-negative) mothers from
forming anti-D, thus preventing RhD
HDFN.
Rh-immune globulin – A purified
preparation of IgG anti-D, is given to D-
negative women during pregnancy and
following delivery of a D-positive fetus.
• Rh-immune globulin is effective
only in preventing RhD HDFN.

RH TERMINOLOGY ACTIVITY

NOTE:
• R = Presence of D-antigen
• r = Absence of D-antigen aka (d)
• 1 or ’ = C
• 2 or ” = E
• z=C
• y=E
• rh’ = C
• rh” = E
• hr’ = c
• hr” = e

Correction: 1. DCe/dCe

Rosenfield
A.) : 1,2,-3,4,5
B.): +1,+2,+3,+4,+5
C.) : -1,2,3,-4,5
D.) : 1,-2,3,4,5
E.) : 1,2,-3,-4,5

Bloodbanking and Serology (Immunohematology)