IMMUNOHEMATOLOGY

COLMC INTERNS
IMMUNOHEMATOLOGY
Demonstration of red cell antigen red cell antibody reactions is the
key to immunohematology
More commonly known as blood banking is a branch of
Hematology which studies antigen-antibody reactions and
analogous phenomena as they relate to the pathogenesis and clinical
manifestations of blood disorders
Combination of antibody and antigen can result in observable
reactions, most commonly
Agglutination
Hemolysis
Precipitation
 Pre transfusion testing
ABO/Rh typing
Other blood group antigen typing
Detection of red cell alloimmunization
(unexpected antibodies)
Compatibility testing (crossmatching)
Transfusion reaction work up
(direct antigolubin test, eluate)
Immune mediated red cell destruction
(DAT, eluate)
 ALLELIC pairs of genes located at the same site on chromosome pairs
CENTROMERE a constricted region of a chromosome that connects the
chromatids during cell division
CHROMATID one of the two potential chromosomes formed by DNA
replication of each chromosome before mitosis and meiosis. They are joined
together at the centromere
CHROMATIN the deeply staining genetic material present in the nucleus of
a cell that is not dividing
CHROMOSOME a linear thread made of DNA in the nucleus of the cell
CO-DOMINANT a gene that expresses a trait regardless of whether or not an
alternative allele at the same locus is also expressed on the other paternal
chromosome
CROSSING OVER the process of breaking single maternal and paternal
DNA double helics in each of two chromatids and rejoining them to each other
in a reciprocal fashion, which results in the exchange of parts of homologous
chromosomes
DOMINANT a gene that expresses a trait that does not allow the expression
of a trait encoded by an alternative allele at the same locus of the other
parental chromosome
 GENE the basic unit of heredity, made of DNA. Each gene occupies a
specific location on a chromosome
LOCUS - the site of a gene on a chromosome
LYONIZATION the inactivation of one of the female X chromosomes
during embryogenesis. This inactivated chromosome forms the Barr body in
the cell nucleus
MEIOSIS a process of two successive cell divisions producing cells, egg, or
sperm that contain half the number of chromosomes found in somatic cells
MITOSIS division of somatic cells resulting in daughter cells containing
the same number of chromosomes as the parent cell
RECESSIVE a gene that in the presence of its dominant allele does not
express itself. A recessive trait is apparent only if both alleles are recessive
SEX-LINKED a gene contained within the X or Y chromosome
SOMATIC CELL nonreproductive cells or tissues
X-LINKED a gene on the X chromosome for which there is no corresponding
gene on the Y chromosome
BASIC PRINCIPLE

Genetic material that determines each trait is found in the

nucleus of a cell
Human 46 chromosome (23 pairs)
22 of the pairs are alike in both males and females
Sex chromosomes (XX) females, (XY) males
Each chromosome consists of two arms
p short or petite arm
q long arm
MITOSIS AND MEIOSIS

MITOSIS
Process whereby the body grows or replaces dead or injured somatic cells
Five stages
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
MEIOSIS
a process of two successive cell divisions producing cells, egg, or sperm that
contain half the number of chromosomes found in somatic cells
It is during meiosis that genetic diversity occurs
GENETICS AND HERDITARY
GENETICS
Study of heredity and explains inheritance of all bodily characteristics
ALLELES
Alternative forms of genes, any one of which may occupy a single
locus on homologous chromosomes
HOMOZYGOUS
Individuals who have identical alleles at a given locus on both
chromosomes
AA, KK, kk
HETEROZYGOUS
The alleles present at the particular locus on each chromosome are
nonidentical
AO, AB, Kk
 DOSAGE EFFECT
A significant difference in antibody reaction strength
depending on the quantity of the target antigen present
on a target red blood cell
FREQUENCY OF ALLELLES
Is the proportion that it contributes to the total pool of
alleles at that locus within a given population at a given
time
HARDY-WEINBERG LAW

Based on the assumption that genotypes are

distributed in proportion to the frequencies of
individual alleles in a population and will remain
constant from generation to generation if the
processes of mutation, migration, etc do not occur
p2+2pq+q2 = 1
Used to calculate allele and genotype frequencies
in a population when the frequency of one gene
trait is known
 Law of Segregation
Two members of a single gene pair (alleles) are
never found in the same gamete but always
segregate and pass different gametes
Law of Independent Assortment
States that genes determining various traits are
inherited independently from each other
PATTERNS OF INHERITANCE
TRAIT
Observed expression of genes
DOMINANT
a gene that expresses a trait that does not allow the expression of a trait encoded
by an alternative allele at the same locus of the other parental chromosome
A trait that is observable when the determining allele is present
CO-DOMINANT
a gene that expresses a trait regardless of whether or not an alternative allele at
the same locus is also expressed on the other paternal chromosome
Different alleles on homologous chromosomes each produce an observable trait
RECESSIVE
a gene that in the presence of its dominant allele does not express itself. A
recessive trait is apparent only if both alleles are recessive
Observable only when the allele is not paired with a dominant allele
IMMUNOLOGY

IMMUNE RESPONSE
It is a highly evolved innate and adaptive system that is
fundamental for survival
IMMUNE SYSTEM
Bodys defense mechanism against foreign invaders
IMMUNITY
Process by which a host organism protects itself from
attacks by both external and internal agents
Necessary to protect the host from obvious invaders such as
parasites and also against external noxious elements and
sun exposure
 BRANCHES OF IMMUNE SYSTEM

BRANCH DEFINITION DEFENSE CELLS EXAMPLES

AGAINST INVOLVED
Cellular Cell mediated Viruses, fungi, T lymphs, Graft
mycobacteria, other macrophages rejection,
intracellular hypersensitivi
pathogens, tumor ty reaction,
cells elimination of
tumor cells
Humoral Antibody Bacteria B lymphs, Antibody
mediated (extracellular) plasma cells production
 TYPES OF IMMUNITY
TYPE EXPLANATION COMPONENTS
Natural or Defense mechanisms External defense system: intact skin,
innate present at birth. Not mucous membranes, cilia & mucus in
antigen specific respiratory tract, stomach acid, flushing
of urine, lactic acid in vagina, lysozyme
in tears & saliva, normal flora
Internal defense system: neutrophils,
macrophages, acute phase reactants,
complement

Acquired or Defense mechanisms T cells, B cells, plasma cells, antibodies,

adaptive that are antigen specific cytokines
 ADAPTIVE IMMUNITY

TYPE EXPLANATION EXAMPLE

Naturally acquired Individual infected with microorganism Clinical or subclinical
active immunity produces antibody infection

Artificially acquired Individual exposed to antigen through
active immunity vaccine develops immunity without
having infection
Naturally acquired Individual protected by antibodies
passive immunity produced by another person
DTaP, MMR, polio, tetanus
Maternal antibodies that
cross placenta and are
present in breast milk

Artificially acquired Individual receives immune globulin Rh immune globulin,

passive immunity containing antibodies produced by HBIG, antitoxins
another person
PRIMARY VERSUS SECONDARY
RESPONSE

PRIMARY SECONDARY
(ANAMNESTIC)
Stimulus 1st exposure to antigen Subsequent exposure to
antigen
Lag phase Days to months Hours
Type of Antibody IgM at first. May IgG
switch to IgG after 2-3
weeks
Titer Rises slowly, peaks Rises faster & higher,
then decline stays elevated longer
T LYMPHOCYTES

Have the TCR, which is usually associated with the CD3

complex.
Require APCs to respond to antigens
T HELPER
Have CD4 marker on their cell membrane
Provide B cell help to stimulate the immune response
T CYTOTOXIC
Have CD8 marker on their membranes
Can eliminate specific target cells without the help of
antibody
B LYMPHOCYTES

Stimulated B cells differentiate into plasma

cells to secrete humoral immunoglobulin
B cells receive T cell help for antibody
production and for immunologic memory
NK CELLS

Also called large granular lymphocytes

Play a role in host resistance to tumor and
viral infections
ANTIGENS

A substance recognized by the body as being foreign,

which can cause an immune response.
Blood group antigens exist on the surface of red blood
cells (RBCs)
TYPES OF ANTIGENS

AUTOANTIGENS
An antigen that despite being a normal tissue constituent is the
target of a humoral or cell-mediated immune response, as in
autoimmune disease.
ALLOANTIGENS
A genetically determined antigen present in some but not all
individuals of a species (as those of a particular blood group) and
capable of inducing the production of an alloantibody by
individuals which lack itcalled also isoantigen
HETEROPHILE ANTIGENS
An antigen or antigenic determinant that is found in different
tissues in more than one species.
PROPERTIES OF ANTIGEN

CHEMICAL COMPOSITION AND

COMPLEXITY OF THE ANTIGEN
DEGREE OF FOREIGNESS
SIZE
DOSAGE AND ANTIGEN DENSITY
ANTIBODIES

A protein substance secreted by plasma cells that is

developed in response to, and interacting specifically with,
an antigen
Produced and secreted by activated B lymphocytes
Basic structure two heavy chains and two light chains
A.K.A Immunoglobulins
In blood banking, it is found in serum, from either
commercial manufacturer or a patient
 IgG IgM IgA IgD IgE
Form Monomer Pentamer Monomer & Monomer Monomer
dimer

Molecular 150,000 900,000 160,000 or 180,000 190,000

Weight 400,000

Heavy chain Gamma Mu Alpha Delta Epsilon

Light chain Kappa or Kappa or Kappa or Kappa or Kappa or
Lambda Lambda Lambda Lambda Lambda

Antigen 2 10 2 or 4 2 2
binding sites

Complement YES YES NO NO NO

fixation

Cross placenta YES NO NO NO NO

 IgG IgM IgA IgD IgE

Role Defense against Neutralizes toxins First line of May play Role in allergic
bacteria & Opsonin defense. Patrols role in B cell reaction. Binds to
viruses, mucosal surfaces. maturation basophils & mast
Neutralizes Prevents cells. When 2
toxins. adherence of adjacent molecules
Opsonin, bacteria and on mast cell bind
Passive neutralizes toxins ag, degranulation
immunity in of cell with release of
newborns histamine & heparin
Others More efficient First Ig produced in In tears, sweat, On surface Type I immediate
at precipitation immune response. saliva, respiratory of B lymphs hypersensitivity
than Only Ig produced by & GI mucosa, reaction
agglutination newborn. Most breastmilk
efficient Ig at
initiating
complement cascade.
More efficient at
agglutination the IgG
IgG versus IgM

IgG IgM
Structure Monomer Pentamer
Number of Antigen binding sites 2 10
Type of Antibody Immune Naturally occurring
Optimum temperature of reactivity 37C 25C or lower
Reacts in saline? No Yes
Reacts best by IAT? Yes No
Complement fixation Moderate Strong
Causes transfusion reactions? Yes Not usually, except
ABO
Crosses placenta? Yes No
Causes haemolytic disease of the newborn? yes No
 MAJOR HISTOCOMPATIBILITY COMPLEX

Large cluster of genes

System of genes that control expression of MHC
molecules found on all nucleated cells; originally
referred to as human leukocyte antigens (HLA)
COMPLEMENT
Role in immunohematology is their ability to lyse the cell
membrane of antibody-coated RBCs
Unstable & heat labile
Classic pathway
activated by both IgG and IgM when the C1 component binds to
the fc portion of the antibody molecule
Alternate pathway
does not require specific antibody for activation, triggered by
polysaccharides and lipopolysaccharides on the surface of certain
target cells
RED CELL ANTIGEN-ANTIBODY REACTION

Agglutination
Antibody mediated clumping of particles that express
antigen on their surface
Hemolysis
Rupture of red cells with release of intracellular
hemoglobin
Precipitation
Formation of an insoluble, usually visible, complex
when soluble antibody reacts with soluble antigen
 FACTORS AFFECTING RED CELL
AGGLUTINATION

SENSITIZATION STAGE ATTACHMENT OF ANTIBODY TO ANTIGEN

Temperature Clinically significant antibodies react best at 37C
ph Most antibodies react at pH 5.5-8.5
Ionic strength Reducing ionic strength of medium facilitates interaction of
antibodies with antigen
Ag/Ab ratio Too much antibodies can cause prozone
Too much antigen can cause postzone
Incubation time Depends on medium. Usually 10-30 min.
AGGLUTINATION STAGE FORMATION OF Ag-Ab BRIDGES BETWEEN RBCs
Type of Ab molecule IgM is larger, can span distance between RBCs more easily
Density of Ag & location on RBC surface Affects ease of attachment of antibodies
Zeta potential Difference in charge netween neg-charged RBC surface & cloud of
pos ions that surround RBCs. Reducing zeta potential allows RBCs
to move closer together
ENHANCEMENT OF ANTIBODY
DETECTION
Albumin Additives
22 % bovine serum albumin. Reduces net negative charge of RBCs allowing
them to come closer together

Enzymes
Ficin & Papain most commonly used. Reduce RBC surface charge by cleaving
sialic acid molecules.

Polyethylene Glycol
Increases antibody uptake

LISS Low Ionic Strength Solution

Lower ionic strength of suspending medium, allowing antigen and antibody
to move closer together more rapidly. Reduces incubation time for IAT
ABO SYSTEM

Discovered by Karl Landsteiner

A and B were the first red cell antigens to be discovered
Von Decastello and Sturli discovered the fourth group
AB
Chromosome #9 ABO locus
Three major alleles A, B, O
ABO antigens and antibodies
Most significant for transfusion practice
ANTIGEN INHERITANCE

ABO gene are inherited in a codominant manner

The genes for all of the carbohydrates antigens
encode specific glycosyltransferases

H gene = L fucose
A gene = N-acetylgalactosamine
B gene = D galactosamine
ANTIGEN DEVELOPMENT

H antigen
Precursor for the A and B antigens
The presence of H substances in body secretions is
controlled by the Se gene (secretors)
Group O secretors have H antigen in their secretions
Group A secretors have A and H antigen in their
secretions
Group B secretors have B and H antigen in their
secretions
Group AB secretors have A, B and H antigen in their
secretions
ABO ANTIBODIES

Naturally occuring antibodies

Predominantly IgM
Activate complement
React at room temperature or colder
Initiated at birth
Titers are generally too low for detection until the
individual is 3 to 6 months of age
ABO BASICS

Blood group antigens are actually sugars

attached to the red blood cell.
Antigens are built onto the red cell.
Individuals inherit a gene which codes for
specific sugar(s) to be added to the red cell.
The type of sugar added determines the blood
group.
 O INDIVIDUALS
HIGHEST CONCENTRATION OF H ANTIGEN
AB INDIVIDUALS
LOWEST CONCENTRATION

O > A2 > B > A2B > A1 > A1B

A PHENOTYPE

Homozygous (AA) or Heterozygous (AO)

A antigens on their RBCs and anti-B antibodies in their serum or
plasma
Two main subgroups: A1 and A2
Can be differentiated by using the lectin anti-A1 reagent made of Dolichos
biflorus seeds

1. The reagent agglutinates RBCs with the A1 antigen, but not cells with the
A2 antigens
2. Approximately 80% of group A individuals are A1; approximately 20% are
A2
3. Anti-A1 can be found in 1% to 8% of group A2 individuals
4. Several other subgroups of A exist, but are extremely rare. The most common,
A3, shows mixed field agglutination with anti-A or anti-AB reagents.
B PHENOTYPE

Homozygous (BB) or Heterozygous (BO)

B antigens on their RBCs and anti-A
antibodies in their serum or plasma
A few weak subgroups of B exist, but are less
common than the rare A subgroups.
AB PHENOTYPE

A and B antigens on their RBCs and

lack both anti-A and anti-B antibodies
in their serum or plasma
O PHENOTYPE

They dont have neither A nor B antigens on

their RBCs and have both anti-A and anti-B
antibodies in their serum or plasma
Anti-AB antibodies are also found in the
serum or plasma of Group O individuals.
BOMBAY PHENOTYPE Oh

Discovered by Bhende in Maratitie, India

Observed in patients who are married by consanguinity (relative)

Lack the H gene and are homozygous for the h gene

Classification of bombay
Classical bombay
H deficient bombay non-secretors (parabombay)
H deficient bombay secretors
H deficient bombay secretors (dominant)
LECTINS

Anti A1 Dolichos biflorus

Anti B Bandiera simplicifolia

Anti H Ulex europeus

BASIC LAWS GOVERNING ABO
BLOOD GROUP INHERITANCE
No offspring can have a gene that is
absent from both parents (an individual
belonging to blood group AB cannot be
the offspring of a group O parent)

Every individual must transmit to every

offspring one of the two genes that make
up his genotype
 WITH HETEROZYGOUS PARENTS, THEIR CHILDREN
WILL HAVE NO MORE THAN 4 POSSIBLE
GENOTYPES

WHEN ONE OF THE PARENT IS HOMOZYGOUS,

ONLY TWO GENOTYPES COULD OCCUR

WHEN BOTH PARENTS ARE HOMOZYGOUS, ONLY

ONE GENOTYPE CAN OCCUR
FORWARD TYPING

Using known sources of commercial antisera

(anti-A, anti-B)
To detect antigens on an individuals RBCs
Anti A dye = Blue Tryphan blue
Anti B dye = Yellow Acriflavin yellow
BACKWARD TYPING

Detection of ABO antibodies in the

patients serum
Using known reagent RBCs, namely
A1 and B cells
ABO DISCREPANCIES
Discrepancies encompass both results that are unexpectedly positive or
negative, and results that are weaker than usual
All discrepancies between cell and serum grouping must be resolved
before a definitive type can be assigned to the patient
Should blood be required before discrepancies can be resolved, group O
red cells and group AB plasma must be transfused
Discrepancies can be grouped according to their probable causes to
facilitate resolution of the discrepancy
Occur when unexpected reactions occur in the forward and reverse
grouping
Patients age, diagnosis, transfusion history, medications and history
of pregnancy
COMMON CAUSES
Clerical errors
Improper identification of patient sample / testing
Improper recording of reactions
Technical errors
Failure to follow manufacturers directions
Contaminated reagents
Improper concentration of subject red blood cells
Failure to add reagents or improper amounts
Improper centrifugation
Warming of test

* ABO discrepancies can usually be resolved by repeating the test on

the same sample by using a saline suspension of RBCs
GROUP I DISCREPANCIES

Associated with unexpected reactions in the reverse

grouping due o weakly reacting or missing
antibodies
Serum grouping is weak or missing
These kind of discrepancies are the most common.
The reason for the missing or weak isoagglutinins
is that the patient has depressed antibody
production or cannot produce the ABO antibodies
 NEWBORN
ELDERLY PATIENTS
PATIENTS WITH LEUKEMIAS
PATIENTS USING IMMUNOSUPPRESSIVE DRUGS
THAT YEILD HYPOGAMMAGLOBULINEMIA
PATIENTS WITH CONGENITAL
AGAMMAGLOBULINEMIA OR IMMUNODEFICIENCY
DISEASES
PATIENTS WITH BONE MARROW TRANSPLANTATION
PATIENTS WHOSE EXISTING ABO ANTIBODIES MAY
HAVE BEEN DILUTED BY PLASMA TRANSFUSION OR
EXCHANGE
ABO SUBGROUPS
RESOLVING DISCREPANCIES

Eliminate all technical errors

Enhancing the reaction in reverse grouping
Incubate the patients serum with reagent
cells at room temp. for 15 mins.
GROUP II DISCREPANCIES

Associated with unexpected reactions in the forward grouping due to weakly

reacting or missing antigens. This group is the least one. Can be caused by some
subgroups of A or subgroups of B or both.

SUBGROUPS OF A (OR B) MAY BE PRESENT

LEUKEMIAS MAY YEILD WEAKENED A OR B ANTIGENS
HODGKINS DISEASE HAS BEEN REPORTED IN SOME
CASES TO MIMIC THE DEPRESSION OF ANTIGENS FOUND IN
LEUKEMIA
ACQUIRED B PHENOMENON IS MOST OFTEN ASSOCIATED
WITH DISEASES OF THE DIGESTIVE TRACT
To resolve the problem wash the patients cells with saline.
GROUP III DISCREPANCIES
These discrepancies are between forward and reverse grouping caused by
protein or plasma abnormalities and result in rouleaux formation or
pseudoagglutination

ELEVATED LEVELS OF GLOBULIN FROM CERTAIN DISEASE

STATES, SUCH AS MULTIPLE MYELOMA, WALDENSTROMS
MACROGLOBULINEMIA, OTHER PLASMA CELL DYSCRASIAS AND
CERTAIN MODERATELY ADVANCED CASES OF HODKINS
LYMPHOMA
ELEVATED LEVELS OF FIBRINOGEN
PLASMA EXPANDERS, SUCH AS DEXTRAN AND
POLYVINYLPYRROLIDONE
WHARTONS JELLY IN CORD BLOOD SAMPLES
 Rouleaux or red cells result from a stacking of erythrocytes
that adhere in a coin-link fashion giving the appearance of
agglutination.

To resolve this kind of problem, washing the patients red cells

with saline or adding a drop or two of saline to the tube in case
of rouleaux formation.

If the agglutination is true red cell clumping will remain.

Cord blood must be washed 6-8 times in forward grouping

ONLY.
GROUP IV DISCREPANCIES
These discrepancies between forward and reverse groupings are due to
miscellaneous problems

COLD REACTIVE AUTOANTIBODIES IN WHICH RBCs ARE SO

HEAVILY COATED WITH ANTIBODY THAT THEY SPONTANEOUSLY
AGGLUTINATE, INDEPENDENT OF THE SPECIFICITY OF THE
REAGENT ANTIBODY
PATIENT HAS CIRCULATING RBCs OF MORE THAN ONE ABO
GROUP DUE TO RBC TRANSFUSION OR MARROW TRANSPLANT
UNEXPECTED ABO ISOAGGLUTININS
UNEXPECTED NON-ABO ALLOANTIBODIES
 Polyagglutination can occur due to exposure of
hidden erythrocyte Ag. (T antigen) in patients
with bacterial or viral infection.

Bacterial contamination in vitro or vivo produces

an enzyme that alters and exposes the hidden Ag.
on red cell leading to T activation.
 HISTORY OF Rh BLOOD GROUP
1939 LEVINE AND STETSON
FIRST REPORTED AN ANTIBODY FOUND IN THE SERUM
OF A WOMAN WHOSE FETUS HAD HEMOLYTIC DISEASE
OF THE NEWBORN (HDN) AND EXPERIENCED
TRANSFUSION REACTION AFTER RECEIVING BLOOD
FROM HUSBAND
Syndrome in fetus is now referred to as hemolytic disease of the
fetus and newborn (HDFN).
Syndrome had complicated pregnancies for decades causing
severe jaundice and fetal death, erythroblastosis fetalis.
Erythroblastosis fetalis (HDN) linked with Anti-Rh by Levine
in 1941.
 1940 LANDSTEINER AND WEINER
DESCRIBED AN ANTIBODY OBTAINED BY IMMUNIZING
GUINEA PIGS AND RABBITS WITH RED CELLS OF A
RHESUS MONKEY
Immunized animals to Rhesus macaque monkey RBCs.
Antibody agglutinated 100% of Rhesus and 85% of human
RBCs.
Reactivity paralleled reactivity of sera in women who delivered
infant suffering from hemolytic disease.
Later antigen detected by rhesus antibody and human
antibody established to be dissimilar but system already
named.
RHESUS MONKEY
DESCRIPTION OF Rh-Hr SYSTEM

Polymorphic
Protein in nature
Located at chromosome 1
Rh D gene behaves as autosomal dominant while CE genes
behaves as autosomal co-dominant alleles
Rh antigens are genetically determined
Present in 85% of the population
The second most important red cell antigen in transfusion
practice
Most immunogenic of all RBC antigens
Development of antibody always results from immune
response
ANTIGENS OF RH SYSTEM

Terms D positive and D negative refer only to presence or absence of the Rh

antigen D on the red blood cell.
Terms Rh pos and Rh neg are old terms, although blood products still labeled
as such.
Early name Rho less frequently used.
Four additional antigens: C, c, E, e.
Named by Fisher for next letters of alphabet according to precedent set by
naming A and B blood groups.
Major alleles are C/c and E/e.
MANY variations and combinations of the 5 principle genes and their products,
antigens, have been recognized.
The Rh antigens and corresponding antibodies account for majority of unexpected
antibodies encountered.
Rh antibodies stimulated as a result of transfusion or pregnancy, they are
immune.
CLINICAL SIGNIFICANT

Primary cause of HDN

Helps in the maintenance of the integrity of
RBC membrane
Rh proteins has an active role in
transporting ammonium cations across the
red cell membrane
Involved in transfusion reaction
NOMENCLATURES OF THE Rh SYSTEM
FISHER RACE
D-C-E NOMENCLATURE
PRODUCED BY A SET OF CLOSELY LINKED SETS OF ALLELES
WIENER
Rh Hr NOMENCLATURE
A SINGLE GENE IS RESPONSIBLE IN PRODUCING AN
AGGLUTINOGEN THAT CONTAINED A SERIES OF BLOOD
FACTORS
ROSENFIELD AND COWORKERS
ALPHA NUMERIC NOMENCLATURE
PHENOTYPE IS DETERMINED BASED SOLELY ON THE RESULT
OBSERVED WITH THE ANTI-SERA EMPLOYED
INTERNATIONAL SOCIETY OF BLOOD TRANSFUSION
NUMERIC TERMINOLOGY
ASSIGNS 6 DIGIT NUMBER FOR EACH AUTHENTICATED
SPECIFICITY
Fisher-Race: CDE Terminology

Fisher Race
Suggested that antigens are determined by 3 pairs of
genes which occupy closely linked loci.
Each gene complex carries D or its absence (d), C or c, E
or e.
Each gene (except d, which is an amorph) causes
production of an antigen.
The order of loci on the gene appears to be DCE but
many authors prefer to use CDE to follow alphabet.
Inherited from parents in linked fashion as haplotypes
The gene d is assumed to be present when D is absent.
Fisher-Race

Below an offspring of the Dce/dce individual will

inherit EITHER Dce or dce from the parent, never dCe
as this would indicate crossing over which does not
occur in Rh system in man.
Fisher-Race

With the exception of d each allelic gene controls presence

of respective antigen on RBC.
The gene complex DCe would cause production of the D, C
and e antigens on the red cells.
If the same gene complex were on both paired chromsomes
(DCe/DCe) then only D, C and e would be present on the
cells.
If one chromsome carried DCe and the other was DcE this
would cause D, C, c, E and e antigens to be present on red
blood cells.
Each antigen except d is recognizable by testing red cells
with specific antiserum.
Wiener

Postulated that TWO genes, one on each chromosome pair,

controls the entire express of Rh system.
Each gene produces a structure on the red cell called an
agglutinogen (antigen).
Eight (8) major alleles (agglutinogens): R0, R1, R2, Rz, r, r,
r and ry.
Each agglutinogen has 3 factors (antigens or epitopes)
The three factors are the antigens expressed on the cell.
For example the agglutinogen R0= Rh0 (D), hr (c), hr (e)
Each agglutinogen can be identified by its parts or factors
that react with specific antibodies (antiserums).
WEINER AND FISHER-RACE

The two theories are the basis for the two notations currently
used for the Rh system.
Immunohematologists use combinations of both systems when
recording most probable genotypes.
You MUST be able to convert a Fisher-Race notation into
Wiener shorthand, i.e., Dce (Fisher-Race) is written R0.
Given an individuals phenotype you MUST determine all
probable genotypes and write them in both Fisher-Race and
Wiener notations.
R1r is the most common D positive genotype.
rr is the most common D negative genotype.
ROSENFIELD

In 1962 proposed a nomenclature based ONLY on serologic

(agglutination) reactions.
Antigens are numbered in the order of their discovery and
recognition as belonging to the Rh system.
No genetic assumptions made
The phenotype of a given cell is expressed by the base symbol of Rh
followed by a colon and a list of the numbers of the specific antisera
used.
If listed alone, the Antigen is present (Rh:1 = D Ag)
If listed with a -, antigen is not present (Rh:1, -2, 3 = DcE)
If not listed, the antigen status was not determined
Adapts well to computer entry
INTERNATIONAL SOCIETY OF BLOOD
TRANSFUSION

Abbreviated ISBT
International organization created to standardize
blood group system nomenclature.
Assigned 6 digit number for each antigen.
First 3 numbers indicate the blood group system,
eg., 004 = Rh
Last 3 numbers indicates the specific antigen,
eg., 004001 = D antigen.
For recording of phenotypes, the system adopts the
Rosenfield approach
 EXAMPLES
r before h big rh = C
h before r little hr = c
R Presence of D Rh1 = DCe
r Absence of D rh = dCe
1 or C Rh1 = DCe
(if no 1 or , then c) rh = dCe
Rh0 = Dce
2 or E Rh2 = DcE
(if no 2 or , then e) rh = dcE
Rh0 = Dce
0 c+e Rho = Dce
Z or y C+E rhy = dCE
EQUIVALENT NOMENCLATURE
PHENOTYPING AND GENOTYPING

Five reagent antisera available.

Only anti-D required for routine testing.
Other typing sera used for typing rbcs to resolve
antibody problems or conduct family studies.
Agglutination reactions (positive and negative)
will represent the phenotype.
No anti-d since d is an amorph.
Use statistical probability to determine most
probable genotype.
RH PHENOTYPING

Uses
Parentage testing
Predicting hemolytic disease of the fetus and newborn (HDFN)
Confirmation of Rh antibody specificity
Locating compatible blood for recipients with Rh antibodies.
Protocol
Mix unknown RBCs with Rh antisera
Agglutination indicates presence of antigen on cell and
determines phenotype.
Use published frequencies and subject information to determine
genotype.
WEAK EXPRESSION OF D

Not all D positive cells react equally well with anti-D.

RBCs not immediately agglutinated by anti-D must
be tested for weak D.
Incubate cells with anti-D at 37C, coating of D
antigens will occur if present.
Wash X3 add AHG
AHG will bind to anti-D coating cells if present.
If negative, individual is D negative
If positive, individual is D positive w
SIGNIFICANCE OF WEAK D

Donors
Labeled as D positive
Weak D substantially less immunogenic than normal D
Weak D has caused severe HTR in patient with anti-D
Patients
If weak D due to partial D can make antibody to portion they
lack.
If weak D due to suppression or genetic expression theoretically
could give D positive
Standard practice to transfuse with D negative
Weak D testing on donors by transfusion service not required.
Weak D testing on patients not required except in certain situations
RH ANTIBODIES
Except for rare examples of anti-E and anti-Cw which may be
naturally occurring, most occur from immunization due to
transfusion or pregnancy.
Associated with HTR and HDFN.
Characteristics
IgG but may have MINOR IgM component so will NOT react in
saline suspended cells (IS).
May be detected at 37C but most frequently detected by IAT.
Enhanced by testing with enzyme treated cells.
Order of immunogenicity: D > c > E > C > e
Do not bind complement, extravascular destruction.
 Anti-E most frequently encountered antibody followed by
anti-c.
Anti-C rare as single antibody.
Anti-e rarely encountered as only 2% of the population is
antigen negative.
Detectable antibody persists for many years and
sometimes for life.
Anti-D may react more strongly with R2R2 cells than
R1R1 due to higher density of D antigen on cells.
CONCOMITANT RH ANTIBODIES

Antibodies which often occur TOGETHER.

Sera containing anti-D may contain anti-G (anti-C + -D)
Anti-C rarely occurs only, most often with anti-D.
Anti-ce (-f) often seen in combinatiion with anti-c.
MOST IMPORTANT is R1R1 who make anti-E frequently
make anti-c.
Patients with anti-E should be phenotyped for c antigen.
If patient appears to be R1R1 should be transfused with R1R1
blood.
Anti-c frequently falls below detectable levels.
DETECTION OF D ANTIGENS

Four types of anti-D reagents

High Protein - Faster, increased frequency of false positives;
requires use of Rh control tube, converts to weak D testing
IgM (Low protein/Saline reacting) - Low protein (fewer false
positives); long incubation times; cannot convert to weak D
testing
Chemically modified - Relaxed form of IgG Anti-D in low
protein medium; few false positives; saline control performed;
converts to weak D testing
Monoclonal source, low protein, blends of mAbs
Must know the preparation, use, advantages and limitations
of each.
HIGH PROTEIN ANTI-D

IgG anti-D potentiated with high protein and other

macromolecules to ensure agglutination at IS.
May cause false positives with rbcs coated with
antibody.
Diluent control REQUIRED.
False positives due to autoagglutinins, abnormal
serum proteins, antibodies to additives and using
unwashed rbcs.
Can be used for weak D test.
IGM ANTI-D (LOW PROTEIN/SALINE)

Prepared from predominantly IgM antibodies,

scarce due to difficulty obtaining raw material.
Reserved for individuals giving false positive with
high protein anti-seras.
Newer saline anti-sera require incubation at 37.
No negative control required unless AB positive.
CANNOT be used by slide test OR weak D test.
CHEMICALLY MODIFIED

IgG converted to saline agglutinin by

weakining disulfide bonds at hinge region,
greater flexibility, increases span distance.
Stronger reactivity than IgM antibodies.
Can be used for slide, tube and weak D test.
Negative control unnecessary unless AB
positive.
MONOCLONAL ANTI-D

Prepared from blend of moncolonal IgM and

polyclonal IgG.
IgM reacts at IS
IgG reacts at AHG (weak D test)
Most frequently utilized reagent.
Used for tube, slide and weak D test.
Negative control unnecessary unless AB positive.
CONTROL FOR LOW PROTEIN
REAGENTS

Diluent used has protein concentration equaling human

serum.
False positives due to immunoglobulin coating of test rbcs
occurs no more frequently than with other saline reactive anti-
sera.
False positives do occur, patient will appear to be AB positive on
forward type.
Must run saline or manufacturers control to verify.
PRECAUTIONS FOR RH TYPING

MUST follow manufacturers instructions as testing

protocols vary.
Cannot use IAT unless explicitly instructed by
manufacturer.
Positive and negative controls must be tested in parallel
with test rbcs.
QC performed daily for anti-D
QC for other anti-seras performed in parallel with test
since these are usually not tested each day, only when
necessary.
SOURCES OF ERROR FALSE POSITIVE

Spontaneous agglutination
Contaminated reagents
Use of wrong typing sera
Autoagglutinins or abnormal serum proteins
coating rbcs.
Using anti-sera in a test method other than that
required by the manufacturer.
SOURCES OF ERROR FALSE
NEGATIVES

Use of wrong anti-serum

Failure to add anti-serum to test
Incorrect cell suspension
Incorrect anti-serum to cell ratio
Shaking tube too hard
Reagent deterioration
Failure of anti-serum to react with variant antigen
Anti-serum in which the antibody is directed against
compound antigen, often problem with anti-C.
LEWIS SYSTEM

Are not synthesized by the RBCs

These antigens are made by tissue cells, secreted into
body fluids and plasma, and absorbed onto the RBC
membrane
Genes are Le and le

The Lewis gene does not actually code for the

production of Lewis antigens but rather produces a
specific glycosyltransferase namely -4-l
fucosyltransferase
Locus assigned on chromosome #19
EXPRESSION

The amount of antigen present is variable and is

partially dependent on the individuals ABO
phenotype and age, because Lewis antigens develop
gradually.
Lewis antigen expression is affected by H, Se, and
Le genes. Lewis antigen expression may decrease
dramatically during pregnancy.
 MOST COMMON PHENOTYPES
Le (a+b)
Le (ab+)
Le (a+b+)
Le (ab)
Most neonates type as Le (ab) regardless of
which Lewis genes they have inherited.
LEWIS ANTIBODY

Anti-Lea antibodies are usually IgM, but may also

be IgG in total or in part.
Most of these antibodies react best at room temperature, but may
react at 37C.
Although most Lea antibodies are considered clinically
insignificant, they can bind complement and are, therefore,
capable of triggering in vitro hemolysis. Although Lea antibodies
are not associated with HDN, the antibodies that react at 37C or
that cause in vitro hemolysis may be associated with haemolytic
transfusion reactions (HTR).
Anti-Lea activity is enhanced by enzyme treatment.
 Anti-Leb antibodies are usually IgM and react best at room
temperature. They bind complement poorly. Anti-Leb activity
is enhanced by enzyme treatment. Leb antibodies are not
associated with HDN and rarely cause HTR.
Lewis antibodies may appear transiently during pregnancy
in Le (ab) women, but disappear after delivery.
Lewis antigens neutralize Lewis antibodies. Because both anti-
Lea and anti-Leb react with most cells on routine RBC panels,
Lewis substance can be used to neutralize the antibodies and
allow the detection of any other antibodies present.
MNS BLOOD GROUP SYSTEM

2ND BLOOD GROUP SYSTEM TO BE DISCOVERED

DISCOVERED IN 1927 BY LANDSTEINER AND
LEVINE
BY INJECTING HUMAN RED CELLS INTO RABBITS
ANTIGENS ARE KNOWN TO BE GLYCOPROTEIN IN
NATURE
GENETICS AND BIOSYNTHESIS

THE ANTIGENS OF THIS SYSTEM ARE CONTROLLED BY

TWO SETS OF ALLELIC GENES AND ARE BELIEVED TO BE
CLOSELY LINKED
1ST LOCUS M,N
2ND LOCUS S,s
THE MNS GENE COMPLEX IS LOCATED ON CHROMOSOME #4
THE ANTIGENS OF THE MNSs SYSTEM ARE LOCATED ON
TWO MAJOR SIALIC ACID-RICH GLYCOPROTEINS OF THE RBC
MN-SIALOGLYCOPROTEIN (MN-SGP) CARRIES MN
ANTIGENS GLYCOPHORIN A
Ss-SIALOGLYCOPROTEIN (Ss-SGP) CARRIES SS
ANTIGENS GLYCOPHORIN B
ANTIBODIES

ANTI M
USUALLY NATURALLY OCCURING, COLD
REACTIVE SALINE AGGLUTININS AT 5-25C
DO NOT BIND COMPLEMENT
SHOWS DOSAGE EFFECT
ANTI N
SEROLOGIC CHARACTERISTICS ARE SIMILAR TO
ANTI-M
COLD REACTING IgM OR IgG SALINE
AGGLUTININ
NOT COMPLEMENT BINDING, EXHIBITING
DOSAGE
FOUND IN RENAL PATIENTS UNDERGOING
DIALYSIS TREATMENT
 ANTI S AND ANTI s
IMMUNE MEDIATED, IgG
DETECTED AT 37C AND THE AHG PHASE
SOME FORMS REACTIVE AT COLDER TEMP
VERY RARE BUT CAN CAUSE HDN AND HTR WITH
SEVERE HEMOGLOBINURIA
ANTI U
RARE ANTIBODY FOUND IN INDIVIDUAL WHO ARE
S-s-
CAN CAUSE HDN AND HTR
REACT BEST BY INDIRECT ANTIGLOBULIN TEST
BLOOD GROUPING REAGENTS

THE ANTIGENS ARE DETECTED BY THE USE OF

SPECIFIC ANTISERA NAMELY ANTI-M, ANTI-N, ANTI-S
AND ANTI-s
SOURCES:
IMMUNIZED ANIMALS AND HUMANS
LECTINS
ANTI M: Iberis amara, Iberis Umbellate, Iberis Semperiveas Japanese turnips
ANTI N: Vices gramenea, Bauhinia variegato, B. candicars, B. bonatia, B. purpurea

IMPORTANCE: ANTIBODIES ARE IMPLICATED IN HDN

AND HTR, IMPORTANT IN FORENSIC MEDICINE,
MEDICO-LEGAL CASES ESPECIALLY IN DISPUTED
PARENTAGE
P BLOOD GROUP SYSTEM

DISCOVERED IN 1927 BY LANDSTEINER

Antigens P1 P p pk Luke
Antibodies Anti-P1 Anti-P Anti-pk
Anti- P + P1 + pk
P ANTIGENS

IMMUNODOMINANT SUGARS ADDED SRQUENTIALLY TO A

PRECURSOR SUBSTANCE
P1
POORLY DEVELOPED AT BIRTH AND MAY TAKE UP TO 7 YEARS TO BE
FULLY EXPRESSED UNLIKE P ANTIGEN

P1 subs
FOUND ON RC, PLASMA, DROPPINGS OF PIGEONS AND
TURTLEDOVES, AS WELL AS EGG WHITE OF TURTLEDOVES

P1 AND Pk
SOLUBLE FORMS ARE FOUND IN HYDATID CYST FLUID
 P ANTIBODIES
ANTI P1
COMMON NATURALLY OCCURRING IgM ANTIBODY OF P2 INDIVIDUALS
WEAK COLD REACTIVE SALINE AGGLUTININ
BIND COMPLEMENT DETECTABLE AT AHG PHASE

ANTI P
FOUND IN THE SERA OF ALL PK INDIVIDUALS
RARELY FOUND IN BLOOD BANK, IT IS SIGNIFICANT IN TRANSFUSION BECAUSE
IT IS A POTENT IgM HEMOLYSIN WITH A WIDE THERMAL RANGE OF REACTIVITY

ANTI Pk
ISOLATED FROM SOME EXAMPLES OF ANTI PP1Pk BY SELECTIVE ABSORPTION
WITH P1 CELLS

ANTI PP1Pk
PRODUCED BY ALL P INDIVIDUALS EARLY IN LIFE WITHOUT RED CELL
SENSITIZATON AND REACTS WILL ALL RED CELLS EXCEPT OF OTHER P NULLS
I BLOOD GROUP

TWO ANTIGENS I AND i

I ANTIGEN PRESENT ON ALMOST ALL HEALTHY ADULTS
I ANTIGEN VARIES IN STRENGTH ON ADULT CELL
IN THE FETAL LIFE, i IS THE MAIN RED CELL ANTIGEN AND I
ANTIGEN IS NOT READILY DETECTABLE
 Newborns do not have much I antigen
Newborns have i antigen
At about 18 months the i is replaced with I
Some transitional antigens
I substance can be found in saliva and human milk
and on lymphocytes and platelets
During disease, the I antigens may alter
ANTI - I

COMMON BUT WEAK AUTO ANTIBODY FOUND IN

SERUM OF MOST HEALTHY
IgM, NATURALLY OCCURRING
SALINE REACTIVE REACTS BEST AT 4C
ASSUMES PATHOLOGIC SIGNIFICANCE IN MANY
CASES OF COLD HEMAGGLUTININ, DISEASE IN
MALIGNANCY, ACQUIRED HEMOLYTIC ANEMIA AND
MYCOPLASMA INFECTIONS
ANTI - i

RARE IgM AGGLUTININ THAT REACTS OPTIMALLY AT 4C, POTENT

EXAMPLES MAY BE ASSOCIATED WITH INFECTIOUS
MONONUCLEOSIS
KELL BLOOD GROUP SYSTEM

Many antigens in this system and has been given a

numerical nomenclature
Six most important
Numeric Alpha Name Incidence
KEL 1 K Kell 10%
KEL 2 k Cellano 99.8%
KEL 3 Kpa Penny 2%
KEL 4 Kpb Rautenberg 99.9
KEL 6 Jsa Sutter Rare (19% Blacks)
KEL 7 Jsb Matthews 99.9%(99.8% Blacks
 Mc Leod syndrome

Reduced expression of Kell antigens

association with hemolytic anemia and chronic granulomatous disease
DUFFY BLOOD GROUP SYSTEM

Discovered in early 1950s

Fy antigen locus on chromosome 1 with Rh locus
Antigens
codominant inheritance
Fya Fyb Fyx
Others Fy3 Fy 4 Fy5 Fy6 Fs -
 Fy (a- b-) RBCs were shown to resist infection by the malaria organisms
P. knowlesi and P. vivax
Anti-Fya and anti-Fyb are usually IgG antibodies and react optimally at
the antiglobulin phase of testing; both antibodies have been implicated
in delayed hemolytic transfusion and HDN
KIDD BLOOD GROUP SYSTEM

Anti-Jka and anti-Jkb may demonstrate dosage, are

often weak, and are found in combination with other
antibodies; both are typically IgG and antiglobulin
reactive
Kidd system antibodies may bind complement and are
made in response to foreign RBC exposure during
pregnancy or transfusion
Kidd system antibodies are a common cause of delayed
hemolytic transfusion reactions
Kidd system antibody reactivity is enhanced with
enzymes, LISS and PEG
LUTHERAN BLOOD GROUP SYSTEM

Lua and Lub are antigens produced by allelic codominant

genes; they are poorly developed at birth
Anti-Lua may be a naturally occuring saline agglutinin
that reacts optimally at room temperature
Anti-Lub is an IgG antibody reactive at the AHG phase;
usually produced in response to foreign RBC exposure
during pregnancy or transfusion
The Lu(a-b-) phenotype is rare and may result from
three different genetic backgrounds
MISCELLANEOUS
BLOOD GROUPS
DIEGO

Discovered in 1955
Dia / Dib
Dia antigen has served as a useful tool in anthropologic studies
of Mongolian ancestry
Dia & Dib described causing moderate to severe transfusion
reactions and HDN
CARTWRIGHT

Yt
Yta high frequency
Ytb low frequency
Anti-Yta has been associated with transfusion reaction
XG

Discovered in 1962
Sex Linked Blood Group System
Xg allele is located on the petite (short) arm of the X chromosome
Anti-Xga antibodies predominantly IgG
SCIANNA

Sc1 high incidence Antigen

Anti Sc1 and Anti Sc2
Rare antibodies, reactive at AHG phase
Not implicated in HDN and HTR
DOMBROCK

Doa and Dob

Antibodies to the DO antigens have been reported to occasionally
cause moderate HTR
COLTON

Coa and Cob

The rare antibodies in the CO system have been associated with
causing mild HTR and HDN
CHIDO/RODGERS

Nine antigens in the Chido/Rodgers system are located on the

complement fragments C4B and C4A respectively
The clinically insignificant CH and RG antibodies present with weak and
nebulous reactivity at IAT and may be identified by plasma inhibition
methods and adsorption with C4- coated cells
CROMER

11 Cromer antigens are carried on the decay accelerating factor and

are distributed in body fluids and on RBCs, WBCs, platelets and
placental tissue
The rare anti-CROM antibodies have been noted only in black
individuals and associated with mild HTR
KNOPS

The KN antigens are located on complement receptor 1 (CR1) with

expression being depressed by the In(Lu) gene and autoimmune
disease
The clinically insignificant KN antibodies present with weak and
nebulous reactivity at IAT
INDIAN

The Ina antigen is more prevalent in Arab and Iranian populations, with
Ina and Inb antigen expression being depressed by the In(Lu) gene
Bg

Bga, Bgb and Bgc WBC antigens correspond with the class I HLA
antigens B-7, B-17 and A-28 respectively
The clinically insignificant anti-Bg antibodies present with weak and
variable reactivity at IAT, which may be destroyed by chloroquine or
EDTA glycine-HCL treatment