Immunohematology

MLSIMHEC [LECTURE]
NATIONAL UNIVERSITY | COLLEGE OF MEDICAL TECHNOLOGY | S.Y 22-23

IMMUNOHEMATOLOGY LECTURE NOTES (MIDTERM REVIEWER)

● A normal human has 46 chromosomes (22
INTRODUCTION TO IMMUNOHEMATOGY pairs of somatic and 1 pair of sex
chromosomes).
KEY POINTS IN HISTORY
● Chromosomes: Structures that carry genetic
information encoded on double - stranded
1942 Blood was taken from three young
DNA
men and given to the stricken Pope
Innocent VII in the hope of curing ● Mitosis: Process of cell division that results in
him. Unfortunately, all four died. the same number of chromosomes in the new
(This is the first time a blood and old cells
transfusion was recorded in the ● Meiosis: Process of cell division that occurs in
history) gametes resulting in one-half the
1869 Braxton Hicks recommended Sodium chromosomes in each new cell
Phosphate.
● Blood group systems: Groups of related RBC
1901 Karl Landsteiner discovered the ABO
blood groups. Edward E. Lindeman - antigens inherited according to Mendelian
He carried out the vein-to-vein genetics
transfusion of blood. ● Phenotype: Physical, observable expression of
1914 Hustin reported the used of Sodium inherited traits; detectable products
Citrate as an anticoagulant solution ● Genotype: Inherited genes; actual genetic
for transfusion makeup
1915 Lewisohn determined the minimum
● Pedigree chart: Visual map that displays a
amount of citrate needed for
family history and can display inheritance
anticoagulant and demonstrated its
nontoxicity in small amount. patterns for individual traits
1916 Rous and Turner introduced Citrate ● Gene: Smallest unit of inheritance
Dextrose solution for the ● Genetic locus: Site on chromosome where
preservation of blood specific genes are located
1941 Dr. Charles Drew appointed as ● Alleles: Alternative forms of a gene
director of the first American Red ● Antithetical: Opposite form of a gene,
Cross blood bank at Presbyterian
different allele
Hospital
1943 Louitit and Mollison introduced the ● Polymorphic: Having two or more possible
Acid-citrate-dextrose (ACD) alleles at a locus
1957 Gibson, introduced the citrate- ● Codominant: Equal expression of both alleles
phosphate-dextrose (CPD), replaced in phenotype
ACD as the standard preservative for ● Recessive: Same allele must be inherited from
blood storage. both parents to be expressed, homozygous
● Dominant: Only one allele must be inherited
REVIEW OF BASIC GENETICS
for it to be expressed; gene product always
present
GENETICS
● Study of transmission of inherited ● Autosomal: Genes expressed with equal
characteristics. frequency in males and females, on non-sex
● Important in the study of antigen inheritance. chromosome

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DEOXYRIBONUCLEIC ACID combinations of both. Some immunogen
● "Backbone" of heredity. produce a greater response than others.
● Two-stranded duplex that runs in opposite  There are 23 RBC antigen systems containing
directions. over 200 RBC antigens. RBC antigens are
inherited and are composed of proteins,
● Composed of 4 nitrogenous bases,
glycoproteins, and glycolipids.
deoxyribose, and a phosphate group.
● Purines Adenine and Guanine  Human leukocyte antigens (HLAs)
● Pyrimidines: Cytosine and Thymine o Present on leukocytes and tissue cells
● Gene - a segment in the DNA along the o Genes that encode the HLA antigens are
chromosome at a specific position (locus). part of the major histocompatibility
● Allele - genes at a specific locus that differ in complex (MHC).
nucleotide sequence (homozygous vs.
o MHC is on chromosome 6 and is divided
heterozygous).
into Class I, II, and III.
 Class I - A, B, and C loci.
OVERVIEW OF IMMUNOHEMATOLOGY  Class II - DR, DP, and DQ.
 Class III - complement proteins.
DEFINITION
 Immunohematology is the study of blood  Immune response to transfused incompatible
group antigens and antibodies, HLA antigens HLA antigens causes fever and chills. This is
and antibodies, pretransfusion testing, known as a febrile, non-hemolytic transfusion
identification of unexpected alloantibodies, reaction.
immune hemolysis, autoantibodies, drugs,  HLA must be matched for organ, tissue, bone
blood collection, blood components, marrow, and stem cell transplant donors and
cryopreservation of blood, transfusion- recipients. If the recipient is not matched
transmitted viruses, tissue banking and organ correctly, a severe graft-versus-host disease
transplantation, blood transfusion practice, results.
safety, quality assessment, records, blood  HLA test applications include paternity testing,
inventory management, and blood usage organ and tissue transplantation, bone
review marrow and stem cell transplantation, and
platelet matching.
IMMUNE SYSTEM
 Acquired immunity is a specific response of  Platelet antigens
the immune system in which antibodies o Membranes have protein antigens.
specific to a particular antigen are produced. o Platelet antibodies occur less frequently
Plasma cells produce antibodies. in the general population because of less
 Innate immunity is a nonspecific reaction of antigen variability.
the immune system that attacks all invaders. It o Antibodies reacting with platelets may
includes physical and biochemical barriers and be ABO-, HLA-, or platelet specific.
cells such as leukocytes, including neutrophils, o Diseases: Neonatal alloimmune
monocytes/macrophages, and natural killer thrombocytopenia and posttransfusion
cells. Physical barriers include intact skin, purpura
mucous membranes, etc. Bactericidal
enzymes are biochemical barriers. ANTIBODY CHARACTERISTICS
 Molecular Structure:
ANTIGEN CHARACTERISTICS o Each molecule has two heavy chains and
 Antigens are substances that combine with an two light chains.
antibody. Antigens are found on the surface of o The heavy chain is responsible for the
platelets and WBCs as well as RBCs. immunoglobulin group specificity.
 Immunogen is an antigen that causes a o Antibody binding site is found in the
specific immune response is an. Immunogens variable region of the heavy and light
are made of protein, carbohydrates, and chains.

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 - Reaction temperature: This
IgM antibodies IgG antibodies is the temperature at which
a) Composed of five a) Single the antibody reacts best;
basic immunoglobulin unit most clinically important
immunoglobulin b) Cannot visibly antibodies react best at
units (pentameric) agglutinate RBCs 37°C.
b) Can directly bind c) Normally, cannot
- Incubation time: This is the
with RBCs and activate
produce complement unless
time allowed for the
agglutination two molecules are antibody to attach to the
c) Can activate present (i.e., IgG3) antigen. This reaction occurs
complement d) Can cross the by chance. Times will vary
d) Cannot cross the placenta according to the antibody
placenta because of e) React optimally at and media used in vitro (i.e.,
large size of 37°C albumin, LISS—low-ionic-
molecule f) Typically clinically strength saline).
e) React optimally at significant; capable - pH: The optimal pH for in
room temperature of causing
and below
vitro reactions is 7.
transfusion reactions
f) Usually clinically or hemolytic disease o Lattice formation: Random collisions of
insignificant of the newborn antibody-coated RBCs link antibodies
(HDN) together to form visual agglutination.
 Factors affecting visual
ANTIGEN-ANTIBODY REACTIONS IN VIVO agglutination
 Transfusions can lead to antigen-antibody - Reaction temperature
complex formation and complement - Incubation time
activation in vivo, if wrong type of blood is - PH
transfused. - Repelling negative charges:
 Transfusion of foreign antigens (RBC, HLA, In normal saline, RBCs have a
and platelet) into a recipient can cause an net negative charge that
immune response and antibody formation in repels other RBCs in
the recipient (alloantibodies). solution. This charge inhibits
 Antigen-antibody complexes are removed by agglutination.
the reticuloendothelial system: spleen, liver,  Antigen and antibody agglutination
and lymph nodes. o Zone of equivalence: Antigen and
antibody concentrations produce
ANTIGEN-ANTIBODY REACTION IN VITRO maximum agglutination.
 Reactions are detected by agglutination or o Prozone (antibody excess): Too much
hemolysis. antibody compared with antigen
 Some antigen-antibody complexes require concentration
two stages for detection: Sensitization and o Post Zone (Antigen excess): Too much
lattice formation. antigen compared with antibody
o Sensitization: Antibody attaches to concentration
antigen but does not produce visible
agglutination or hemolysis.  Grading agglutination reactions
 Factors affecting first stage of o 4+ RBC button is solid with a clear
agglutination supernatant.
- Serum to cell ratio: This is o 3 + RBC button breaks into several large
the amount of antibody clumps, with a clear supernatant.
compared to the number of o 2+ RBC button breaks into many
cells in solution. Increased medium-sized clumps, with a clear
amount of serum equals an supernatant.
increase in the number of o 1+ RBC button breaks into many
antibodies in the solution. medium- and small-sized clumps, with

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 background having many free RBCs  Linkage and haplotypes
(appears cloudy). o Linked genes: Genes that are close
o +w RBC button breaks into many together on a chromosome and
clumps, barely or not visible inherited as one unit. The Law of
macroscopically, with many RBCs in the Independent Assortment does not
background (use microscope to see hold with linked genes.
clumps). o Haplotype: Set of genes inherited
o 0 = no agglutinated RBCs via one of the two parental
gametes
 Hemolysis is another indication of o Amorphs: Genes that do not
antibody-antigen reactions and is caused by produce a detectable product.
complement activation. The supernatant
appears clear red, with a smaller or ANTI- HUMAN GLOBULIN (AHG) TESTING
nonexistent RBC button. “COOMB’S TEST”

MENDELIAN INHERITANCE PRINCIPLE PRINCIPLE AND PROPERTIES

 Law of Independent Segregation: Two  Principle : A Technique for detecting cell-bound
members of a single gene pair passed from immunoglobulin. It is used to detect incomplete
one generation to the next in separate antibodies (IgG)
gametes  Widely used procedure in modern blood
 Law of Independent Assortment: Traits banking because it detects antibodies which are
inherited from different chromosomes not detectable by other techniques.
expressed separately and discretely  Why cell bound immunoglobulin? It is not fully
 Inheritance patterns: The inheritance of attached to the red blood cell.
blood group antigens (A, B, O) can be  Purpose - It enhances, It is attached to the
predicted using a Punnett square. Punnett incomplete IgG which is attached to our RBC and
squares have the one person's genotype on makes it visible agglutination
the top and the other person's genotype on
the side. HISTORY
 Each square represents a possible genotype ● Before the discovery of the antiglobulin test,
for an offspring. An offspring from these only IgM antibodies had been detected. The
particular parents would have a 25% chance utilization of the AHG test permitted the
of inheriting any one of the four possible detection of non-agglutinating IgG antibodies
variants. and led to the discovery of many new blood
 Punnett squares are useful for group systems.(not only ABO or AB but also Rh
understanding inheritance of blood groups or D, other minor blood groups, minor
and ramifications of heterozygosity or antibodies, etc.)
homozygosity. ● The Coomb’s procedure involved the injection
 Homozygous: Individual inherits identical of human serum into rabbits to produce anti
alleles at the same gene locus from both human serum
parents. ● They inject the human sera (antigen) with the
 Heterozygous: Individual inherits different use of rabbit, it produces hyper immunizing the
alleles at the same gene locus from each animals, with the purified immunoglobulin or
parent. complement, it produces high titer IgG
 Dosage effect: Agglutination reactions are antibodies.
generally stronger for homozygous cells and ● Anything that is foreign to the body, it produces
slightly weaker for heterozygous cells. antibodies to that foreign antigens.
 Cis: Genes are inherited on the same ● For the rabbit, the human sera is the antigen
chromosome. (foreign); it produces antigen to human IgG.
 Trans: Genes are inherited on separate IgM, and human complement system that led to
chromosomes. Genes inherited in polyspecific coombs test sera
transposition can weaken the trait's ANTI-HUMAN GLOBULIN SERA
expression.

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POLYSPECIFIC AHG REAGENT (BROAD SPECTRUM
COOMBS TEST REAGENT)
 Contains antibody to human IgG and to C3d
Component of the complement system.
Other anti-complement antibodies such as
anti-C3b, anti-C4b or anti-C4d may also be
present. (green in color)
 It consist of a pool of rabbit anti human IgG
and mouse monoclonal anti C3b, anti C3d
 Commercially prepared polyspecific AHG
contains little or no activity against IgA and
IgM
MONOSPECIFIC AHG reagent – Contains only one
antibody specificity, either anti-IgG or antibody to
specific complement components such as C3b or
C3d.

HYBRIDOMA TECHNOLOGY
ANTIBODIES REQUIRED IN ANTIHUMAN GLOBULIN TEST  can be used to produce monospecific AHG
reagent using clones of plasma cells that will
ANTI-IGG produce either anti-IgG or anti-complement

 AHG reagent must have antibody activity to

non agglutinating blood group antibodies, the
presence of anti-light chain activity allows
detection of all IgG classes.
○ Contains no anti-complement activity

ANTI-COMPLEMENT

 involves anti-complement reagents such as

anti-C3b-C3d and are reactive against the
designated complement components only and
no activity against human immunoglobulin

PREPARATION OF AHG REAGENT

CLASSIC METHOD
 involves injecting human serum into
laboratory animals. The human globulin as
foreign antigen triggers the animal’s immune
response and an antibody is produced. This
method produces the polyspecific AHG
reagent.

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 7.
Check for presence of agglutination by
dislodging the cell button. If no
agglutination , stand for 5 minutes.
8. Centrifuge and read and record results.
9. If no agglutination was observed, place
1 drop of IgG sensitized red cells
STAGES OF ANTIGEN - ANTIBODY INTERACTION (control for checking). Centrifuge and
● The first stage is sensitization. Sensitization read the result.
occurs when antibodies react with antigens INDIRECT AHG TEST
on the cells and coat the cells.  PRINCIPLE: detect antibody in serum or
● The second stage of the reaction is plasma (red tube)
agglutination. Agglutination occurs when  Performed to determine in vitro sensitization
antibodies on coated cells form cross- of red cells (indirect antiglobulin test = indirect
linkages between cells resulting in visible coombs test)
clumping.  Useful in the following Situations:
➢ Detection of antibodies to potential donor
TWO TYPES OF AHG TEST cells, Identification of antibody specificity
➢ Determination of red cell phenotype
DIRECT ANTIGLOBULIN TEST (DAT) ➢ Titration of incomplete antibody
● PRINCIPLE: Detects in vivo sensitization
(inside the body) of RBCs with IgG and/or  SPECIMEN AND MATERIALS NEEDED:
complement components. o 5% RBC suspension
● EDTA/CITRATE- used to minimize the o AHG reagent
possibility of in vitro attachment of o NSS
complement components para di magkaroon o LISS (incubate for 10 minutes) / BSA (inc
ng false negative) for 15-30 minutes) - fasten the reaction
● CLINICAL CONDITIONS that can result in in o Centrifuge
vivo coating of red cells: o Nescofilm
1. HDN- Hemolytic disease of newborn o Centrifuge
2. HTR- Hemolytic transfusion reaction o Incubator
3. Autoimmune and drug induced o Patient’s serum
hemolytic anemia o Positive and Negative Control

● MATERIALS  PROCEDURE:
○ 5% RBC suspension 1. Labelled 4 Plain Test Tube as T, AC, PC,NC
○ AHG reagent 2. Add 2 drops of 2-5% RBC Suspension
○ NSS (Known O)
○ IgG-sensitized red cells 3. Add 100 ul of patient serum to Test Tube
○ Centrifuge labelled T
○ Nescofilm 4. Add 2 drops of Anti-D to Tube PC
5. Add 2 drops of NSS to tube NC
● PROCEDURE 6. For AC tube put 100 ul of patient serum and
1. Prepare a 5% RBC suspension using the 2 drops of Patient red cells.
patient's blood sample. 7. Then Add 2 drops of LISS to all test tubes
2. Place 3 drops of RCS in clean labelled 5 (T,PC,NC, AC). Mix and Incubate for 10 mins
ml plain tube at 37ºC. OR Add 2 drops of 22% BSA to all
3. Wash the suspension 3x using NSS. test tubes (T,PC,NC,AC). Mix and Incubate
4. Decant completely the supernatant for 30 mins at 37ºC
after the last washing. 8. Centrifuge and read and record results for
5. Add 2 drops of AHG. agglutination or hemolysis.
6. Mix and Centrifuge 9. Wash the suspension 3x using NSS.
10. Decant completely supernatant after the
last washing.

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 11. Add 1-2 drops of AHG to each tube ○ Albumin (BSA) allows antibody-coated cells
(T,PC,NC,AC) to come into closer contact with each other.
12. Mix and Centrifuge. (2 drops 22% albumin + 2 drops serum + 1
13. Check for the presence of agglutination by drop of 3-5% rbc suspension)
dislodging the cell button. ○ LISS–enhance antibody uptake (2 drops 3%
14. If no agglutination was observed, place 1 rbc suspension in LISS + 2 drops of serum)
drop of IgG-sensitized red cells. Centrifuge , ○ Polyethylene Glycol – it’s action is to
read and record results remove water, thereby concentrating the
antibody.
○ Temperature –vast majority of IgG has an
optimal reaction rate at 37’C
○ Incubation time:
■ Saline suspension – 30-120
mins
■ LISS - 10-15 mins
■ BSA - 30 minutes
○ Washing of cells – to remove unbound
serum globulins.
○ Saline for washing should be fresh and
buffered at to a pH of 7.2 to 7.4
○ Centrifugation–1000 rcf for 15-20 seconds

ERRORS

FALSE POSITIVE
● Improper Specimen (clotted)
INTERPRETATION OF RESULTS
● Auto-agglutinable cells/ polyagglutinable
cells
Negative: there is a smooth suspension of RCS ● Bacterial contamination of saline
Positive: There is agglutination Cells with positive DAT used for IAT
Dirty Glass wares
Gel technique Over centrifugation and overreading
Negative: bottom
Positive: top FALSE NEGATIVE
● Inadequate or improper washing of cells
● Deteriorated AHG reagent
● AHG reagent not added
● Serum not added in IAT
● Inadequate incubation
● Cell suspension too heavy or too weak
● Under centrifuged or over centrifuge
● Poor reading results.
● NOTE: Cells used for DAT must be collected
in EDTA or citrates

FACTORS AFFECTING AHG TEST

● Ratio of cells to serum - a minimum ratio of 40:1

should be aimed (2 drops of serum + 1 drop of
5% RBC suspension of the patient)
● Reaction medium

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 ABO AND H BLOOD GROUP SYSTEM

ABO BLOOD GROUP SYSTEM

● The most important of all human blood groups.
● The only blood group system with predictable
antibodies in the serum to antigens not present on
RBCs..
● Development of anti-A and anti-B without RBC
exposure could be due to exposure to A-and B-like
antigens from bacteria.
● Transfusion of incompatible ABO type results to
almost immediate lysis of donor RBCs (severe to
fatal)

COMPARISON OF DAT AND IAT Type O - Universal donor (RBC)

Universal Recipient (plasma)
Type AB - Universal Recipient (RBC)
DIRECT INDIRECT Universal Donor (Plasma)
○ Specimen: ○ Specimen: PLAIN TUBE
EDTA/CITRATE ○ SERUM OR PLASMA ABO PHENOTYPES, ANTIGENS, AND ANTIBODIES
○ RCS (red cell ○ After obtaining serum
suspension) (usual) or plasma, the
○ Principle: add 1 drop of patient serum will be
COOMBS REAGENT. added to the donor’s
○ (+) Result: RBC blood
agglutination sample/RCS/Known O
○ After sensitization and
washing 3 times
○ Add coombs reagent INHERITANCE
and check for visual ● Described by Bernstein (1924).
agglutination ● One (1) gene from each parent determines the
○ Application: apply to genotype.
compatibility testing. ● Follows mendelian genetics, codominantly
expressed.
● Chromosome 9.
● O gene-amorph gene
● Possible genotypes:
1. AA and AO
2. BB and BO
3. AB
4. OO
● Possible phenotypes:
1. A
2. B
3. AB
4. O

ABO GENOTYPES AND PHENOTYPES

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 IMMUNODOMINANT SUGAR
● A sugar that occupies the terminal portion of
the precursor chain and confers blood group
specificity

H, A, AND B ANTIGENS
● Formation is a result of interaction between the
.
following genes:
1. ABO and H genes: H, A, and B antigens on
AMOUNT OF H ANTIGENS ON EACH ABO TYPES
RBC membrane.
2. ABO and Se genes: H,A, and B antigens on
secretions.
● Each gene codes for a glycosyltransferase.
● Enzyme transfers an immunodominant sugar to a
basic precursor substance.
● Precursor substance:
1. Type 1- on RBC membrane H, A, AND B SOLUBLE SUBSTANCES
2. Type 2- in secretions

BASIC PRECURSOR SUBSTANCE ABO ANTIBODIES

● Predominantly IgM (produces strong
agglutination at RT and activates C' at 37°C).
● Small quantities of IgG.
● Anti-A,B-cross-reacting IgG in the sera of group
O
● Becomes detectable at 3-6 months of age

● Causes rapid intravascular hemolysis and can

cause death.
● Knowledge on the amount of IgG ABO
antibodies allows prediction of ABO HDFN.

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LABORATORY TESTING INTERPRETATION OF FORWARD GROUPING RESULTS
→ FORWARD GROUPING
● Detection of A and/or B antigens on the surface
of RBCs.
● Reagents:
1. Anti-A antisera (blue)
2. Anti-B antisera (yellow)
● Positive: Hemagglutination
→ REVERSE GROUPING
● Detection of anti-A and/or anti-B antibodies in
plasma or serum.
● Reagents: REVERSE GROUPING: TUBE METHOD
1. Known A/A, cells 1. Add one drop of serum to each labelled test
2. Known B cells tubes.
● Positive: Hemagglutination 2. Add one drop of known reagent cells to each
tube correspondingly.
LECTIN SOURCES 3. Mix and centrifuge for 20-30 seconds.
4. Resuspend Cells gently.
5. Observe for agglutination of RBCs

FORWARD GROUPING: SLIDE METHOD

1. Place one drop of whole blood to each antisera
on a glass slide separately
2. Add one drop of whole blood to each antisera
on the slide
3. Mix the antisera and blood sample
4. Observe for agglutination of RBCs
INTERPRETATION OF REVERSE GROUPING RESULTS

SUBGROUPS OF TYPE A
● Von Dungem (1911) - described two different
antigens based on reactions of group ARBCs
with anti-A, and anti-A.
FORWARD GROUPING: TUBE METHOD ● Both react equally strong in anti-A forward
1. Prepare a 2-5% red grouping.
cell suspension
2. Add one drop of
antisera on each
labelled test tubes
3. Add one drop of RCS
BOMBAY PHENOTYPE (Oh)
on each test tubes
4. Mix and centrifuge ● First reported by Bhende (1952) in India
for 20-30 seconds ● Due to the hh genotype thus H, A, and B antigens
5. Resuspend cells are not formed.
gently ● Appears as group O in the forward and reverse
6. Observe for typing. Individuals with the Bombay phenotype have
agglutination of an IgM anti-H that is potent at 37°C.
RBcs.

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● Transfusion of group O donor RBCs to a Bombay ○ Incubate test mixture at room temperature
recipient results to immediate lysis of donor cells. for 30 minutes.
● Only RBCs from another individual with the same ○ If #1 remains negative, incubate test mixture
phenotype can be used for transfusion. at 4°C for 15-30 minutes
○ In cases of acquired B phenomenon, treat
patient RBCs with acetic anhydride.
○ Wash patient RBCs with saline to remove
BGSS

ABO DISCREPANCIES GROUP III DISCREPANCIES

● Unexpected reactions in the forward and
● Unexpected reactions in the forward and reverse
reverse grouping due to increased plasma
grouping
proteins causing rouleaux formation.
● Usually resolved by repeating the test on the same
● Possible causes:
sample using a saline suspension.
1. Elevated globulin levels (eg. plasma cell
● Any technical factor should be reviewed and
dyscrasia)
corrected first
2. Elevated plasma fibrinogen
● Relevant information needed in resolving
3. Presence of plasma expanders (eg, dextran
discrepancies:
and polyvinylpyrrolidone)
1. Age
4. Wharton's jelly in card blood samples
2. Diagnosis
● Resolution:
3. Medications
1. Wash RBCs several times with saline.
4. History of transfusion
2. Wash cord RBCs 6-8x
5. History of pregnancy
● If discrepancy persists, record results but delay
GROUP IV DISCREPANCIES
release of typing interpretation until discrepancy is
● Unexpected reactions in forward and reverse
resolved.
grouping due to miscellaneous problems.
GROUP I DISCREPANCIES
● Possible causes:
● Unexpected reactions in the reverse grouping
1. Cold reactive autoantibodies
due to weak or missing antibodies.
2. Circulating RBCs of more than one ABO
● Due to depressed antibody production.
group
Common populations with group I discrepancy.
3. Unexpected ABO isoagglutinins
1. Newborn
4. Unexpected non-ABO alloantibodies.
2. Elderly patients
● RESOLUTION
3. Patients with leukemia
○ Incubate patient RBCs at 37°C for a short
4. Patients undergoing
period and wash with saline 3x and repeat
immunosuppressive therapy
forward grouping.
5. Patients in plasma transfusion or
○ Warm patient serum and reagent cells at
exchange
37ºC and repeat reverse grouping
6. Chimerism
○ Perform autoadsorption to remove
● RESOLUTION TO GROUP I DISCREPANCIES
autoantibodies from patient serum.
○ Incubate patient serum and reagent
○ Repeat reverse grouping using at least
RBCs at room temperature for 15-30
three ABO specificities ( A1, A2, B, O)
minutes.
○ Perform antibody panel
○ Autocontrol and O cell control must
also be performed (to rule out cold
agglutinin reactions that could occur in KEY POINTS ON ABO, H, SECRETOR
#1). BLOOD GROUP SYSTEM
GROUP II DISCREPANCIES
● Unexpected reactions in the forward! grouping Landsteiner's Rule: If an individual has the antigen,
due to weak or missing antigens. that individual will not have the antibody. This is a
● Possible causes: universal law and has few exceptions.
1. ABO subgroups
2. Leukemia
ABO Antigens
3. Hodgkin's lymphoma
4. Acquired B phenomenon  Found on RBCs, lymphocytes, platelets, tissue
5. Blood group-specific soluble cells, bone marrow, and organs
substances  These antigens can be secreted by tissue cells if
6. Chimerism the appropriate genes are present.
● RESOLUTION  Glycolipid or glycoprotein

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 Developed in utero at 5-6 weeks of gestation Anti-A and Anti-B. Anti-A1 lectin is active against
 Full expression of ABO antigens occurs between A1, but not the other A subgroups.
2 and 4 years of age.  Subgroup A3 characteristically produces a
 Frequencies mixed-field reaction with polyclonal Anti-A and
polyclonal Anti-A,B.
 If weak subgroups of A in recipients are not
detected, there is no harm in a person with the
subgroup receiving type O blood. However, if
the person with the weak subgroup of A donates
blood that is transfused to a group O
patient, intravascular hemolysis may result.

 A and B Are Codominant Traits:

o If the allele is present, the antigen will be
expressed.
 O is an amorph allele that produces no
INHERITANCE AND DEVELOPMENT OF A, B, AND H transferase to add sugars to the H determinant
ANTIGENS site.
 The H antigen is the building block for the A and
B antigens. There are only two alleles in the H  Anti-A and Anti-B:
gene: H and h. o These antibodies are produced by humans,
 The H allele is found in 99.99% of the world's who lack the corresponding antigen, as a
population, and h is a rare amorph allele. result of exposure to naturally occurring
 The H antigen acts as the acceptor molecule for substances that resemble A and B
the two sugars that make up the A and B antigens.
antigens.
 The A blood type is the H antigen with N-  Anti-A and Anti-B Are IgM Antibodies: This
acetylgalactosamine attached. means they activate complement and cause
 The B blood type is the H antigen with o- visible RBC agglutination. They may cause
hemolysis at room temperature.
galactose attached.
 The O blood type is the H antigen with no
 Routine ABO Grouping
additional sugar attached.
o Forward type: Person's RBCs are mixed
with reagent Anti-A and Anti-B.
ABO Subgroups
o Reverse type: Person's serum is mixed
 Subgroups differ in the amount of the antigen
with reagent A1 and B RBCs.
expressed on the RBCs. Subgroup A2 possesses
o ABO discrepancies occur when the forward
both A and A1 antigens on the RBC surface.
and reverse groupings do not agree.
Subgroup A2 only expresses A antigen.
 Problems with forward grouping
 Blood group A has two major subgroups, A1 and
(extra antigen present, weak
A2 .
antigens) could be caused by
o 80% of group A people are A1
acquired B phenotype,
o 20% of group A people are A2.
polyagglutination, rouleaux, ABO
 People with subgroups of the A antigen can
subgroups, transfusion of non-type
produce antibodies against A1 antigen.
specific blood, and bone marrow or
 Subgroups of A include A1, A2, A3, Ax, Am, Ael, and
stem cell transplants.
Abantu.
 Problems with reverse grouping
 Subgroups of A can be detected by polyclonal (unexpected antibodies or
Anti-A, B. This is produced by Group O weak/missing antibodies) could be
individuals only. Anti-A,B will agglutinate A seen in individuals with A
subgroups because it has specificity for both A subgroups with Anti-A1 cold
and B antigens but cannot be separated into alloantibodies, cold autoantibodies,

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 and rouleaux, and in a newborn or o Position effect: Occurs when the C antigen
elderly person. is inherited trans to the D antigen. This
weak D may be detected without carrying
Bombay (Oh) Phenotype the test to the antiglobulin phase.
1. Person inherits hh genotype. o Partial D: Occurs when only part of the D
2. Types as an O (forward and reverse); has antigen is inherited. There are multiple
alloanti-H capable of activating complement epitopes that make up the D antigen. A
and causing a hemolytic transfusion reaction partial-D individual lacks one or more of
3. These people can only be transfused with these epitopes and is capable of making
Bombay group blood. Blood may be collected antibody to the epitopes that s(he) lacks.
and frozen as autologous or from siblings who Partial-D individuals are usually detected
are also Bombay. because the antigen reacts strongly with
monoclonal reagents. A partial D is
Secretor Status suspected when a seemingly D-positive
1. Two alleles: Se and se person makes anti-D after transfusion with
2. People who inherit Se are secretors and are D-positive blood.
capable of expressing ABO and H antigens in o Weakly reactive D means a person is D-
their secretions. positive. AABB Standards state that all Rh-
3. A, B, and H antigens, appropriate to the negative donor units must be tested for
individual's ABO group, are found in saliva, weak D, and those units that test positive
urine, tears, bile, amniotic fluid, breast milk, must be identified as D-positive.
exudate, and digestive fluids of secretors o However, weak-D recipients are
(Se). transfused with D-negative blood.

RH BLOOD GROUP SYSTEM Other Rh system antigens

 Controlled by two genes RHD and RHCE.  f or ce: If c and e are present on the same
 RHD controls D expression haplotype, f antigen is expressed.
 RHCE controls C, c, E, e expression.  Ce or rhi: C and e are inherited as a haplotype
 Rh antigens are proteins. made by D-positive individuals who make anti-
C.
Rh Terminology  Cw: Low-frequency antigen
 The most common individual antigens are  V or ces: 30% prevalence in African-Americans
named in the Fisher-Race terminology, D, C, c,  G: In test tube appears to be anti-D and anti-C
E, e, Cw, G, etc.  Rh:29: Antibody to Rh:29 is the antibody to the
 Haplotypes are often expressed in a modified high-frequency Rh antigen made by Rhnull
Wiener terminology such as R1R1 for CDe/CDe. people.
 Phenotype: RBC antigens identified with
specific antisera; Unusual phenotypes
 Genotype: Genes present on person's  D deletion: No reaction occurs when tested with
chromosomes anti-E, anti-e, anti-C, and anti-c. Written as D —
 Rhnull phenotype: This appears to have no Rh
Rh system antigens antigens. The membranes of their RBCs are
 D antigen: Most immunogenic of Rh antigens abnormal and the RBCs have a shortened life
 Weak D span. This can result from inheriting two
o Weak D occurs when D is weakly nonfunctional RHCE alleles along with the dual
expressed. deletion of the RHD alleles. Rhnull phenotype can
o Weak D must be detected by an IAT also result from inheriting two recessive
(indirect antiglobulin test). regulator alleles at the RHAG locus. The latter
o Genetic cause: Weaker expression of the individuals pass on normal RHD and RHCE alleles
cDe haplotype may fail to react by direct to their children.
agglutination testing, but it will react
strongly by the IAT.

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Rh antibodies
 Produced in humans through pregnancy or
transfusions
 IgG antibody; Rh antibodies generally do not
activate complement
 Optimal reaction temperature: 37°C
 Reaction phase: AHG (antihuman globulin)
 Agglutination enhancement occurs with LISS,
enzymes, and PEG (polyethylene glycol).
 Stronger reactivity of antibody with cells from
homozygous individuals is shown with anti-C,
anti-c, anti-E, and anti-e (dosage).
 C and e and E and c are usually found together.
 These antibodies produce hemolytic transfusion
reactions (HTRs).
 Antibodies may not be currently detectable, but OTHER MAJOR BLOOD GROUP SYSTEMS
the person should always receive antigen
negative blood if they have a history of Rh Kell Blood Group System (006)
antibodies.
1. Abbreviation: K
 Rh antibodies can cause hemolytic disease of 2. Antibody class: IgG
the newborn (HDN), because they can cross the 3. Optimal reaction temperature: 37°C
placenta. 4. Reaction phase: AHG
 Rh immune globulin (RhIG) administered after 5. Enzyme treatment: No effect
delivery (within 72 hours) can protect a woman 6. Antigens: K (Kell), k (Cellano), Kpa, Kpb, Kpc,
from making anti-D. Jsa, Jsb Antigens, and Ku; common Kell system
antigens k, Kpb, and Jsb
TABLES TO BE FAMILIAR 7. Allelic pairs: Include K and k, Kpa and Kpb, Jsa
and Jsb
8. K is very immunogenic. Although the K
antigen is found in only about 9% of the
population, anti-K is encountered quite
frequently and can cause HTR and HDN.
9. Kellnull: This is also known as Ko. It occurs when
RBCs lack the Kell antigens but have the Kx
antigen.
10. The Kx antigen is produced by a gene located
on a different chromosome than the Kell
system genes. This antigen is inherited
independently from the Kell antigens; the Kx
antigen structure appears to be required for
the expression of the Kell system antigens.
Knull individuals have increased amounts of
Kx.
11. McLeod phenotype
a. Individuals who have an alteration of the
allele-producing Kx on the X
chromosome lack Kx on the red blood
cells and have greatly decreased
expression of Kell antigens.

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 b. These individuals have decreased RBC 4. Reaction phase: AHG
survival as well as RBC morphologic and 5. Enzyme treatment: Enhances agglutination
functional abnormalities. 6. Clinically significant: Associated with HTR and
mild HDN
Lewis Blood Group System (007) 7. Antigens: Jka, Jkb, Jk3
1. Abbreviation: Le 8. Four phenotypes: Jk(a+b -); Jk(a - b+); Jk(a+b+);
2. Antibody class: IgM Jk(a-b-)
3. Optimal reaction temperature: Most often 4°C, 9. Alleles: Jka codes for Jka and Jk3; Jkb codes for
sometimes 37°C Jkb and Jk3.
4. Reaction phase: Room temperature, 37°C, and 10. Show dosage effect: Weak antibodies
AHG agglutinate homozygous cells more strongly
5. Enzyme treatment: Enhanced agglutination than heterozygous cells.
6. Clinically significant: No 11. These antibodies bind complement.
7. Produced by tissue cells and secreted into fluids. 12. These antibodies deteriorate in storage,
The antigens are adsorbed onto the RBC declining quickly to below the detectable level
membranes. in human serum, and commonly cause delayed
8. May take 6 years to fully develop these antigens. HTR (DHTR).
9. Genetics: If Le gene inherited, Lea is adsorbed
onto RBCs—Le(a+b-). Lea is the only antigen that Lutheran Blood Group System (005)
can be secreted by a nonsecretor.
10. If Se gene is also inherited, Leb is adsorbed onto 1. Abbreviation: Lu
the RBC—Le(a - b +). 2. Antibody class: Lua IgM; Lub IgG
2. Bombay phenotypes are Lea positive if they 3. Optimal reaction temperature: Lua 4°C; Lub 37°C
inherit the Le gene. 4. Reaction phase: Lua room temperature; Lub AHG
3. Cells type as Le(a+b+) (transiently during first 5. Enzyme treatment: Variable effect.
years of life), Le(a+b-),Le(a-b+), Le(a-b-). 6. Clinically significant:
4. Lewis antibodies are sometimes formed during a. No clinical significance. Anti-Lua can be
pregnancy but weaken and disappear after present without prior transfusion or
delivery. pregnancy
b. Anti-Lub is rare and associated with HTR
Duffy Blood Group System (008) and HDN.
5. Abbreviation: Fy 7. Antigens: 18 total, including Aua and Aub
6. Antibody class: IgG 8. Alleles: Lua, Lub
7. Optimal reaction temperature: 37°C
8. Reaction phase: AHG I Blood Group System (027)
9. Enzyme treatment: Destroys Fya and Fyb 1. Abbreviation: I
10. Clinically significant: 2. Antibody class: IgM
a. Anti-Fya and anti-Fyb can cause HTR and 3. Optimal reaction temperature: 4°C
HDN. 4. Reaction phase: Immediate spin (IS) and
b. The Fy(a-b-) phenotype is more resistant to occasionally 37°C
malarial infection by Plasmodium vivax. 5. Enzyme treatment: Enhanced agglutination
11. Antigens: Fya, Fyb 6. Clinically significant: No
12. Four phenotypes: Fy(a+b-); Fy(a-b+); Fy(a+b+); 7. It can be a bothersome antibody, masking the
Fy(a-b-) reactions of a clinically significant alloantibody.
13. Alleles: Fya, Fyb, and Fy (silent allele) May need to pre-warm cell suspension and
14. Commonly show dosage effect: Weak reagent or
antibodies react more strongly with 7. do cold auto absorption to find clinically
homozygous cells. significant alloantibodies.
8. Strong anti-I is associated with Mycoplasma
Kidd Blood Group System (009) pneumoniae infection.
1. Abbreviation: Jk
2. Antibody class: IgG
3. Optimal reaction temperature: 37°C

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P Blood Group System (003) a. Clinically significant, causing HTR and HDN
1. Abbreviation: Pt b. Anti-U is rare and occurs in S-s-U- people.
2. Antibody class: IgM (anti-Pj)
3. Optimal reaction temperature: 4°C MINOR BLOOD GROUP SYSTEMS (KEYPOINTS)
4. Reaction phase: IS, 37°C and AHG 1. Diego: Dia, Dib, Wra, Wrb; Dib and Wrb are high-
5. Enzyme treatment: Enhanced agglutination incidence antigens.
6. Clinically significant: 2. Cartwright: Yta and Ytb; Yta is a high-incidence
a. Anti-Pj is not clinically significant. antigen.
b. Anti-Pi + P + Pk is an IgG clinically 3. Xg: Xga antigen has a higher incidence in females
significant antibody. than in males.
8. Phenotypes: Pp P2, p, Pf, P2k, and Luke 4. Scianna: Scl, Sc2, and Sc3; Scl and Sc3 are high-
9. Alleles: Pp P, Pk, andp incidence antigens.
10. Anti-Pj can be neutralized by soluble P1 reagent. 5. Dombrock: Doa, Dob, Gya, Hy, and Joa; Gya, Hy,
11. Autoanti-P is Donath-Landsteiner antibody. and Joa are high-incidence antigens.
Naturally occurring biphasic antibody 6. Colton: Coa, Cob, and Co3; Coa is a high-
associated with paroxysmal cold incidence antigen.
hemoglobinuria. It binds to the antigen on the 7. Chido/Rodgers: Cha and Rga are both high-
patient's RBCs in the cold and fixes complement. incidence antigens.
The RBCs are hemolyzed when the temperature 8. Gerbich: Ge2, Ge3, and Ge4 are high-incidence
reaches 37°C. antigens.
12. Patients with autoanti-P may require a blood 9. Cromer: Cra and several others are high-
warmer for transfusion. incidence antigens.
13. Anti-PPiPk is found in individuals of the p 10. Knops: Kna, McCa, Sll, andYka are high-
phenotype. It is clinically significant and incidence antigens.
associated with spontaneous abortions. Need 11. Cost: Csa and Csb; Csa is a high-incidence
compatible blood from other p phenotype antigen.
individuals. 12. Vel: Vel is a high-incidence antigen. Anti-Vel is a
hemolytic, clinically significant antibody.
MNS Blood Group System (002) 14. John Milton Hagen: JMH is a high-incidence
antigen.
1. M and N antigens
15. Sid: Sda is a high-incidence antigen.
a. Abbreviation: MN
b. Antibody class: IgM
c. Optimal reaction temperature: 4°C or 37°C
Practice Questions for Midterms
d. Reaction phase: IS, 37°C, or AHG
e. Enzyme treatment: Destroys antigens
f. Clinically significant: No
g. Antigens: M and N associated with
glycophorin A
2. S and s antigens
a. Abbreviation: Ss
b. Antibody class: IgG
c. Optimal reaction temperature: 37°C
d. Reaction phase: AHG
e. Enzyme treatment: Variable effect
f. Clinically significant: Yes
g. Antigens: S, s, and U associated with
glycophorin B
3. Anti-M
a. It is clinically significant if IgG; IgM
antibody is not clinically significant.
b. Demonstrates dosage effect
4. Anti-N is very rare.
5. Anti-S, Anti-s, and Anti-U

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