SBGV Blood Banking – KELEDUKIDI

Chapter 1
History
Blood Transfusion History
• Together with 3 young men died with blood transfusion in 1492
• First recorded blood transfusion in history
• Clotting was the principal obstacle to overcome
• In 1892, he recommended a nontoxic anticoagulant, sodium phosphate
• First example of blood preservation research.
• In 1901, he discovered the ABO blood groups
• Explained the serious reactions that occur in humans as a result of incompatible transfusion
• He carried out vein-to-vein transfusion by using multiple syringes and a special cannula for puncturing
the vein through skin = time consuming, required many skilled assistants
• Designed syringe-valve apparatus
• Transfusions from donor to patient by an unassisted physician became practical
• In 1914, reported the use of sodium citrate as anticoagulant for transfusion
• In 1915, determined the minimum amount of citrate needed for anticoagulation and demonstrated its
toxicity in small amounts
• In 1916, introduced citrate-dextrose solution for blood preservation.
• Pioneered on developing techniques in blood transfusion and blood preservation during World War 2.
• In February 1941, he was appointed director of the first American Red Cross blood bank at Presbyterian
Hospital.
• Introduced the formula for preservative acid-citrate-dextrose (ACD)
• In 1957, introduced an improved preservative solution called Citrate-Phosphate-Dextrose (CPD)

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Groups of antigen catalogue
according to ISBT
Chapter 2
Common Blood Groups
Blood Group Terminology
1. Blood group system
2. Collection
3. High-prevalence series (901)
4. Low-prevalence series (700)
Controlled by a single gene locus or by two or more closely linked genes
Share a biochemical, serologic, or genetic relationship
Found in more than 90% of the population
Found in less than 1% of the population

Common Blood Groups

ABO (001)
Rh (004)
MNS (002)
P1PK (003), Globoside (028), and Globoside Collection
Lutheran (005)
Kell (006) and Kx (019)
Lewis (007)
Duffy (008)
Kidd (009)
I (027) and i Antigen

ABO Blood Group System (001)

Description • Most important of all blood groups in both transfusion and transplant medicine.
• Even today, transfusion of the wrong ABO group remains a cause of death in hemolytic transfusion reaction
fatalities reported to FDA.
• First blood group system discovered by Karl Landsteiner.
• ABO grouping is the most frequently performed test in the blood bank.
• Both ABO forward and reverse grouping test must be performed on all donors and patients.
• It has been postulated that bacteria, pollen particles, and other substances present in nature are chemically
similar to A and B antigens
Antigens • Expression of A and B antigens on the RBCs is fully developed by 2 to 4 years of age and remains constant
throughout life.
• Phenotypic expression of ABH antigens may vary with race, genetic interaction, and disease states.
• Are integral parts of the membranes of RBCs, endothelial cells, platelets, lymphocytes, and epithelial cells.
• First individuals to perform forward and reverse grouping

• The only blood group system in which individuals already have antibodies in their serum to antigens that are
absent from their RBCs without any prior exposure to RBCs through transfusion or pregnancy.

ABO Inheritance
Description Theory of inheritance was first described in 1924
Follows simple Mendelian genetics
Expression Codominant
Chromosome 9 ABO genes
O gene Amorph
Genotype

Phenotype 1. A
2. B
3. AB
4. O

Formation of A, B, and H Red Blood Cell Antigens

ABH Antigens • Results from interaction of genes at three separate loci (ABO, Hh, and Se)
Precursor substance on RBC • Terminal Galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1-> 4 linkage
Type 2 = Type 2
• Terminal Galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1-> 3 linkage
Type 1 = Type 1
H antigen • Precursor structure on which A and B antigens are made.
• Inheritance of the H gene = formation of H antigen
H and Se genes • FUT 1 gene(H) = Chromosome 19
• FUT 2 gene (Se) = Chromosome 19
• Not part of the ABO system
• Their inheritance influences A and B expression

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• Must be inherited to form ABO antigens on the RBCs
• Must be inherited to form ABO antigens in secretions

H (018) Blood Group System

H gene • Located on chromosome 19
• Must be inherited to form the ABO antigens on the RBCs
• Inheritance influence A and B antigen expression
• Not part of the ABO system
• HH/Hh gene = ______________________
• Precursor structure on which A and B antigens are made
• Recessive gene
• hh gene = ____________________

Secretor (Se/se)
Se gene • Located on chromosome 19
• Dominant gene
• Must be inherited to form the ABO antigens in secretions
• Sese and SeSe gene = presence of ABO antigens in secretions
se gene • Recessive gene
• sese gene = absence of ABO antigens in secretions
Secretions

ABO Antibodies
Description • Naturally occurring antibodies = because they are normally produced without any exposure to RBCs.
• Predominantly IgM, activate complement, and react at room temperature or colder.
• ABO antbodies production is initiated at birth, but titers are generally too low for detection until infants are 3
to 6 months. = Therefore most antibodies found in cord blood serum are of maternal origin.
• Results of serum ABO testing before 3 to 6 months of age cannot be considered valid because some or all of
the antibodies present may be IgG maternal antibodies that crossed the placenta. As a result, it is logical to
perform only forward grouping on cord blood from newborn infants.
• Antibody production peaks between 5 and 10 years of age and declines later in life.
• Elderly people usually have lower levels of anti-A and anti-B; therefore, antibodies may be undetectable in
the reverse grouping
• ABO antibodies can cause rapid intravascular hemolysis if the wrong ABO group is transfused, potentially
resulting in patient death.
• Although anti-A (from a group B individual) and anti-B (from a group A individual) contain predominantly IgM
antibody, small quantities of IgG may also be present.
• Serum from group O individuals contains anti-A, anti-B, and anti-A,B.
• Anti-A,B reacts with both A and B cells. Anti-A,B antibody activity, originally thought to be just a mixture of
anti-A and anti-B, cannot be separated into a pure specificity when adsorbed with either A or B cells.
• For example, if group O serum is adsorbed with A or B cells, the antibody eluted will react with both A and B
cells.
• Anti-A,B antibody is not a combination of anti-A and anti-B but is a separate “cross-reacting” antibody that is
usually IgG in nature.
• Anti-A,B reagent is routinely used for performing ABO confirmation of group O donor units simply because it
is more economical to use one reagent instead of both anti-A and anti-B.

ABO Genotypes and Phenotypes

Genotype Phenotype
A1A1
A1A2
A1O
A2A2
A2O
A1B
A2B
OO
BB
BO

Glycosyltransferases and Immunodominant Sugars Responsible for H, A, and B Antigens Specificities

Gene Glycosyltransferase Immunodominant Sugar Antigen
H (FUT1) α-2-L-Fucosyltransferase L-Fucose H
A α-3-N-Acetylgalactosaminyltransferase N-Acetyl-D-Galactosamine (GalNac) A
B α-3-D-Galactosyltransferase D-Galactose B

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A Subgroups
• Group A RBCs that react with both anti-A and anti-A1
• Group A RBCs that react with anti-A and not anti-A1
• Weak A subgroup
• Characteristically demonstrate a mixed-field pattern of agglutination with anti-A and most anti-A,B reagents
• ___________________ = small agglutinates within predominantly unagglutinated red cells.

A1 vs A2 Phenotype
Blood group Anti-A reagent (Anti-A and Anti-A1) Anti-A1 lectin reagent
A1
A2

Associated Conditions
A individuals

B individuals

O individuals

Depress expression

Bombay Phenotype (Oh)

Description • The term Bombay has been used to refer to the phenotype that lacks normal expression of the ABH antigens
because of the inheritance of the hh genotype.
• Can only be transfuse with blood from _________________
Phenotype
Antibodies Anti-A
Anti-B
Anti-A,B
Anti-H

The Para-Bombay Phenotypes

Description • Due to absence of H antigens or decreased expression of H antigens
• RBCs of these individuals express weak forms of A and B antigens, which are primarily detected by adsorption and
elution studies.
• If a person is genetically A or B, the respective enzymes can be detected, but no H enzyme is detectable, even though it
has been shown that there is limited production of H antigen on the RBC.
• Shows few or no ABH antigens on RBCs, sometimes accompanied by normal expression of ABH antigens in secretions
and body fluids.

Acquired B Phenomenon
Description • It is an acquired enzymatic modification of group A1 red cells in vivo.
• The acquired B phenomenon is found most often in individuals with the A1 phenotype.
Cause Harmening
• Individuals with intestinal obstruction, carcinoma of the colon or rectum, or other disorders of the lower intestinal
tract may have increased permeability of the intestinal wall, which allows passage of the bacterial polysaccharides
from Escherichia coli serotype O86 into the patient’s circulation.
• This results in the acquired B phenomenon in group A1 individuals.
• The patient’s group A red cells absorb the B-like polysaccharide, which reacts with human-source anti-B.

Henry’s
• Usually occurs in the setting of bacterial infection or cancer and reflects enzymatic deacetylation of group A antigen to
form a B-like antigen on RBCs.
• The acquired B phenotype arises when microbial deacetylating enzymes modify the A antigen by altering the A-
determining sugar (N-acetylgalactosamine) so that it resembles the B-determining galactose.
Bacteria
Enzyme

Methods
Forward grouping • Also known as _________________
• Using known sources of commercial antisera (Anti-A, Anti-B) to detect antigens on an individual’s RBC
Reverse grouping • Also known as _________________
• Detecting ABO antibodies in the patient’s serum by using known reagent RBC’s, namely A1 and B cells.

Routine Reagents Used for ABO Testing

Anti-A Reagent Anti-B Reagent
Monoclonal antibody Monoclonal antibody

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Highly specific Highly specific
Forward Grouping IgM IgM

Expected 3+ to 4+ reaction Expected 3+ to 4+ reaction

Usually 1 to 2 drops Usually 1 to 2 drops
Reagent A, and B cells
Reverse Grouping Human source
4% to 5% RBC suspension
Expected 2+ to 4+ reaction usually use 1 drop

Forward and Reverse Grouping

Forward Grouping Reverse Grouping
Blood Group Anti-A Anti-B Antigen(s) in RBCs A1 Cells B Cells Antibody(ies) in
Serum
O No A or B antigen A and B
A A B
B B A
AB A and B No A or B
Antibodies

ABO Discrepancies
Group 1 Group 2 Group 3 Group 4
Reason Unexpected reactions in the Unexpected reactions in the Discrepancies between forward Discrepancies between
reverse grouping due to weakly forward grouping due to and reverse groupings are due forward and reverse
reacting or missing antibodies weakly reacting or missing to protein or plasma groupings are due to
antigens abnormalities and result in miscellaneous problems
rouleaux formation or
pseudoagglutination
Conditions • Newborns = (ABO antibody • Subgroups of A or B may • Elevated levels of • Cold reactive
production is not be present globulin from certain autoantibodies in which
detectable until 3 to 6 • Leukemias may yield disease states, such RBCs are so heavily
months of age) weakened A or B as multiple myeloma, coated with antibody
• Elderly Patients antigens and Hodgkin’s Waldenström’s that they spontaneously
(production of ABO disease has been macroglobulinemia, agglutinate,
antibodies is reported in some cases other plasma cell independent of the
depressed) to mimic the depression dyscrasias, and certain specificity of the
• Patients with a leukemia of antigens found in moderately reagent antibody
(e.g., chronic lymphocytic leukemia. advanced cases of • Circulating RBCs of
leukemia) or lymphoma • The “acquired B” Hodgkin’s lymphomas more than one ABO
(e.g., malignant phenomenon will show • Elevated levels of group due to RBC
lymphoma) weak reactions fibrinogen transfusion or
demonstrating with anti-B antisera and • Plasma expanders, such marrow/stem cell
hypogammaglobulinemia is most often associated as dextran and transplant
• Patients with a leukemia with diseases of the polyvinylpyrrolidone • Unexpected ABO
(e.g., chronic lymphocytic digestive tract (e.g., • Wharton’s jelly in cord isoagglutinins
leukemia) or lymphoma cancer of the colon). blood samples • Unexpected non-ABO
(e.g., malignant alloantibodies
lymphoma) demonstrating
hypogammaglobulinemia
• Patients with congenital or
acquired
agammaglobulinemia or
immunodeficiency diseases
• Patients with bone marrow
or hematopoietic
progenitor stem cell (HPC)
transplants (patients
develop
hypogammaglobulinemia
from therapy and start
producing a different RBC
population from that of the
transplanted bone
marrow)
• Patients whose existing
ABO antibodies may have
been diluted by plasma
transfusion or exchange
transfusion
• ABO subgroups

Lectins used in Blood Banking

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Lectins • Seed extracts that agglutinate human cells with some degree of specificity
• Agglutinates A1 or A1B

• Agglutinates B cells

• Agglutinates O cells (H specificity) and other ABO blood groups depending on the amount of H
antigen available

Rh Blood Group System (004)

Description • The most complex RBC antigen in human, encompassing some 50 antigens, many phenotypic variants, and
complex serologic relationships.
• Second most important blood group system in terms of transfusion, as the Rh system antigens are very
immunogenic.
• Can produce HDN and HTR
• RHD, RHCE, RHAG are exclusively found on RBC membranes
History • Landsteiner and Wiener reported on an antibody made by guinea pigs and rabbits when they were transfused
with Rhesus macaque monkey RBCs.
• The antibody agglutinated 85% of human RBCs was named Rh after the Rhesus monkey.
• The name Rh was retained for the human-produced antibody.
• Anti-Rhesus formed by the animals was renamed anti-LW in honor of those first reporting it (Landsteiner and
Wiener).
Rh-positive Individual’s RBC antigen possess the __________
Rh-negative Individual’s RBC antigen lacks the D-antigen
Rh antibody • Most are IgG, some are IgM
• Produced only after exposure to foreign red blood cells.
• Primary cause of hemolytic disease of the fetus and newborn (HDFN) or erythroblastosis fetalis
Rh antigen property • Protein
Rh function in RBC • Maintains structural integrity of RBCs
• Have a role in transporting ammonia
Rh gene • Inherited as codominant alleles
• Most common phenotype in black
• Most probable genotype in any population

Less Encountered Rh Antigens

• Antithetical to MAR antigen
• Express in combination with both C and c and in the absence of either allele

Anti-Cw
• Identified in individuals without known exposure to foreign RBCs and after transfusion or pregnancy.
• May show dosage
• f antigen is expressed on the RBC when both c and e are present on the same haplocyte

Anti-f
• Causes HTR and HDN
• No reagent to test anti-f
• c negative and e negative are f negative
• Present on most D-positive and all C-positive RBCs
• Also known as Crawford antigen
• Low prevalence and found in African Descent

Terminology
1. Fisher-Race Terminology
2. Wiener Terminology
3. Rosenfield Terminology
4. International Society of Blood Transfusion (ISBT) Terminology

Fisher-Race Terminology
Description • DCE Terminology
• Represents the easiest way to think about the five major Rh system antigens
• Antigens of the system were produced by three closely linked sets of alleles
• Each gene was responsible for producing a product(or antigen) on the RBC surface
• Each antigen and corresponding gene were given the same letter designation (when referring to the gene, the
letter is italicized).
• Fisher and Race named the antigens of the system D, d, C, c, and E, e.
• “d” represents the absence of "D" antigen
• Each person inherits a set of Rh genes from each parent (One D or d, one C or c, and one E or e)
• Rh genes were thought to be codominant, each inherited gene expresses its corresponding antigen on the RBC.
• The combination of maternal and paternal haplotypes determines one’s genotype (the Rh genes inherited from
each parent) and dictates one’s phenotype.
• An individual’s Rh phenotype is reported as DCE rather than CDE because Fisher postulated that the C/c locus lies
between the D/d and E/e loci.

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• An individual’s Rh phenotype is reported as DCE rather than CDE because Fisher postulated that the C/c locus lies
between the D/d and E/e loci.
Disadvantage • As the number of Rh antigens continues to grow, the original Fisher-Race terminology is becoming too limiting.

Wiener Terminology
Description • Rh-hr Terminology
• One gene responsible for defining Rh that produced an agglutinogen containing a series of blood factors.
• Rh gene produced at least three factors within an agglutinogen.
• The agglutinogen may be considered the phenotypic expression of the haplotype.
• Each factor is an antigen recognized by an antibody.
• Antibodies can recognize single or multiple factors (antigens).
• Fisher-Race may be converted to Wiener and vice versa
Agglutinogen • When describing an agglutinogen, the uppercase R denotes the presence of the original factor, the D antigen.
• The lowercase r indicates the absence of D antigen.
• The presence of uppercase C is indicated by a 1 or a single prime (').
• Lowercase c is implied when there is no 1 or ' indicated.
• That is, R1 is the same as DCe; r' denotes Ce; and R0 is equivalent to Dce.
• The presence of E is indicated by the Arabic number 2 or double prime ('').
• Lowercase e is implied when there is no 2 or '' indicated—that is, R2 is the same as DcE; r'' denotes cE, and r is
equivalent to ce.
• When both C and E are uppercase, the letter z or y is used.
• Rz denotes DCE, whereas ry represents CE.

Rosenfield Terminology
Description • Alphanumeric terminology
• This system has no genetic basis, nor was it proposed based on a theory of Rh inheritance, but it simply
demonstrates the presence or absence of the antigen on the RBC.
• A minus sign preceding a number designates the absence of the antigen. If an antigen has not been typed, its
number will not appear in the sequence.
• D is assigned Rh1, C is Rh2, E is Rh3, c is Rh4, and e is Rh5.
• For RBCs that type D + C + E + c negative, e negative, the Rosenfield designation is Rh: 1, 2, 3, –4, –5.
• If the sample was not tested for e, the designation would be Rh: 1, 2, 3, –4.
• All Rh system antigens have been assigned a number.

International Society of Blood Transfusion Committee

Description • Updated numeric terminology
• Committee on Terminology for Red Cell Surface Antigens.
• Its mandate was to establish a uniform nomenclature that is both eye- and machine-readable and is in keeping
with the genetic basis of blood groups.

Immunogenecity of Common Rh Antigen

RHAG (030) Blood System

Description • Newest blood group system
• Does not have Rh blood group antigens; however, its presence in a complex with the Rh proteins is essential
for Rh antigen expression.
RhAG gene • Another gene that is important for the expression of Rh antigen
• RHAG gene product: Rh-associated glycoprotein (RhAG)
• Coexpressor of Rh antigen
• Must be present for successful expression of Rh antigens
• Chromosome 6
Antigens 1. Duclos (RHAG1)
2. Ola (RHAG2)
3. DSLK (Duclos-like)
Antigen property Glycoprotein

Rh Anomaly
• Fail to express any Rh antigens on the RBC surface
• Negative for LW and FY5
• S, s, U antigen on glycophorin B may be depressed
• Mutation in RHAG gene
• Can be transfuse with _____________

Clinical manifestation:
• Mild compensated hemolytic anemia
• Reticulocytosis
• Stomatocytosis
• Slight-to-moderate decrease in hemoglobin and hematocrit levels
• Increase in hemoglobin F
• Decrease serum haptoglobin

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• Increase bilirubin
• Exhibit a severely reduced expression of all Rh antigens
• Partial suppression of RH gene expression caused by mutations in the RHAG gene
• S, s, U antigen on glycophorin B may be depressed
• Less severe than Rhnull

Weak D
• Historically known as Du
• Characterized by weak or absent RBC agglutination by anti-D during routine serologic testing.
• In weak D individuals, the D antigen usually requires enhancement with AHG owing to a quantitative
decrease in RhD protein.
• Weakly reactive D means a person is D-positive.
• Rh-negative donor units must be tested for weak D, and those units that test positive must be identified as
D-positive.
• Weak-D recipients are transfused with D-negative blood.
• Also known as D mosaic
• Are RHD proteins with missing D epitopes.
• Although they type as D-positive, persons with partial D antigens can make alloanti-D.
• The alloanti-D produced by these individuals recognizes D-specific epitopes missing on their own RBCs
(antibodies reactive with allogeneic, but not autologous, RBCs.

RH Typing
Description • Routine Rh typing for donors and patients involves only the D antigen.
• Tests for the other Rh antigens are performed when identifying unexpected Rh antibodies, obtaining
compatible blood for a patient with an Rh antibody, investigating parentage or other family studies, selecting
a panel of phenotyped cells for antibody identification, or evaluating whether a person is likely to be
homozygous or heterozygous for RHD.

Rh Typing Reagents
Reagents • High protein based or low protein based
• Saline-based
• Chemically modified
• Monoclonal
• Blends of monoclonal
Saline-based reagent • Contains ______________
• First typing reagent available to test for the D antigen

High-protein anti-D reagent
Disadvantage:
1. Limited availability
2. Cost of production
3. Lengthy incubation time
• Consist of IgG anti-D
• Human plasma containing high-titer D-specific antibody was used as the raw material.
• Potentiators of bovine albumin and macromolecular additives such as dextran or polyvinylpyrrolidone were added
to the source material to optimize reactivity in the standard slide and rapid tube tests to allow for direct
agglutination of red cells using an IgG anti-D.
• This media causes RBCs to be in closer proximity to each other and allows IgG anti-D to crosslink and cause direct
agglutination.

Disadvantage:
• False positive due to presence of potentiators and higher protein concentration

Major advantage:
• Reduced incubation time
• The ability to perform weak-D testing and slide typing with the same reagent

MNS Blood Group System (002))

Description • Second blood group to be discovered after ABO
• Discovered in 1927
• Antibodies discovered by Landsteiner and Levine in rabbit sera
• May show dosage effect
• Glycophorin A and glycophorin B may play a role in Plasmodium falciparum infection
• Plasmodium falciparum can adhere to RBCs via sialic acid, which is highly expressed in glycophorins
• Glycophorin-deficient phenotypes, such as En (a−), are relatively resistant to P. falciparum in vitro.
Commonly encountered
antigens
MNSs antigen Expressed only in the RBCs
Chromosome 4
Glycophorin A (GPA) • M, N
• The major RBC sialic acid–rich glycoprotein (sialoglycoprotein, SGP)
• Destroyed by enzymes (ficin, papain, bromelin, trypsin, pronase)
• Contribute to zeta potential of RBCs (negative charge)

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Glycophorin B (GPB) • S, s, U
• U means _________________
• U is a high incidence antigen found in every individuals except Blacks
• Contribute to zeta potential of RBCs (negative charge)

Antibodies
Anti-M • pH dependent
• Reacts best at pH 6.5
• Most are IgM
• Commonly encountered antibody in the bloodbank
• Formed by N/N individual
Anti-N • Most are IgM
• Formed by M/M individual

Anti-S • Most are IgG (significant)

• Can cause HTR and HDFN
Anti-s • Most are IgG (significant)
• Can cause HTR and HDFN
Anti-U • Most are IgG (significant)
• Can cause HTR and HDFN
• Common in ____________

GPA- and GPB- Null phenotypes

1. U- phenotype • U antigen is found on GPB
• RBCs are S-s-U-
• Antibody: Anti-U = Most are IgG
• Can cause HTR and HDFN
• Found in Blacks
2. En(a-) phenotype • Ena = envelope
• Ena is a high incidence antigen
• M-N- individual (due to deletion of GPA)
• Antibody = anti-Ena
3. Mk phenotype • Mk is a silent gene
• Named after Metaxas and Metaxas-Buhler when they found an allele
• M-N-S-s-U-En(a-) = due to deletion of GPA and GPB

P1PK (003), Globoside (028), and Globoside Collection

Description • Traditionally, the P blood group comprised the P, P1, and Pk antigens and, later, Luke (LKE).
• P1 antigen is uniquely expressed on RBCs and can be lost with in vitro storage.
• The P system antigens also serve as receptors for P-fimbriated uropathogenic E. coli
History • The P blood group was introduced in 1927 by Landsteiner and Levine.
• In their search for new antigens, they injected rabbits with human RBCs and produced an antibody, initially
called anti-P, that divided human RBCs into two groups: P+ and P–.
Antigen type
High-frequency antigens
P1, P, Pk antigens Can be found on RBCs, lymphocytes, granulocytes, and monocytes
P, Pk antigens • Have been found in plasma as glycosphingolipids and as glycoproteins in hydatid cyst fluid
• Pk and P antigens are also expressed on nonerythroid cells, including lymphocytes, platelets, plasma, kidney,
lung, heart, endothelium, placenta, uroepithelium, fibroblasts, and synovium.
• Receptors for shiga toxins, produced by Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC)
strains.
• Associated with α-interferon, and major histocompatibility class II receptors
• The Pk antigen is a marker of apoptosis in germinal center B cells, Burkitt lymphoma, and lymphoblastic
leukemia
• Can bind human immunodeficiency virus (HIV) and may confer resistance to HIV infection
• Serves as a receptor for Streptococcus suis (it causes meningitis) and Pseudomonas aeruginosa.
• Marker of embryonic and mesenchymal stem cells and is implicated in adhesion, cell signaling, and metastasis
in renal cell and breast carcinoma
• Receptor for Parvovirus B19
• Parvovirus B19 is the causative agent of fifth’s disease
• People lack P antigen are resistant to parvovirus B19
• Can be found on platelets, epithelial cells, and fibroblasts.

P Blood Group: Phenotypes, Antigens, and Antibodies

Phenotype Antigens Present Possible Antibodies
P1 P1, P, Pk
P2 P, Pk
Null Phenotypes (Absence of P antigen)
p None
P1k P1, Pk

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P2k Pk

Formation of P antigens
Description • Like ABH antigens, the antigens result from the sugars added sequentially to precursor structures.

Biosynthetic Pathways of P Blood Group Antigens

Lactosylceramide (Gb2) →Lactotriaosylceramide →Paragloboside (type 2 precursor) →P1 antigen
→Pk antigen = Globotriaosylceramide (Gb3) →P antigen = Globoside (Gb4) →Luke (LKE)

Antibodies
Anti-P1 • Associated with Echinococcus granulosus, Fasciola hepatica = parasitic infections
• Common, naturally occurring antibody in the sera of P1– individuals.
• Most are IgM, rarely IgG
• Interfering anti-P1 antibody can be removed by neutralization with hydatid cyst fluid
Anti-PP1Pk • Formerly known as anti-Tja
• T = ________________
• J = ________________
• A = __________________
• Produced by p individuals
• IgG, IgM
• Associated with early abortions
Alloanti-P • Anti-P is found as a naturally occurring alloantibody in the sera of Pk individuals.
• IgG class anti-P is associated with habitual early abortion
Autoanti-P • Due to cold reactive IgG
• The IgG autoantibody in PCH is described as a biphasic hemolysin: In vitro, the antibody binds to RBCs in the
cold, and, via complement activation, the coated RBCs lyse as they are warmed to 37°C.
• Anti-P = D_____________________
• Associated with __________________________
• Autoantibody does not react in routine test systems
• Can be confirmed by ______________________

Lutheran Blood Group System (005)

Description • Lutheran is a minor constituent of RBC membranes
• Lutheran antigens are ubiquitously expressed on human tissues.
• Expressed by colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle,
liver, lung, placenta, brain, heart, and bone marrow
History • Anti-Lua was found in the serum of a patient with _____________________
• The new antibody was named Lutheran for the donor
• The donor’s last name was_____________ but the donor blood sample was incorrectly labeled.
Antigen property
Biologic role • Lutheran is a high-affinity receptor for laminin, a basement membrane protein involved in cell differentiation,
adhesion, migration, and proliferation.
• Overexpression of LU glycoprotein in ovarian carcinoma and other cancers is hypothesized to facilitate tumor cell
adhesion and metastasis
• In sickle cell patients, increased Lutheran expression on reticulocytes and sickle cells may contribute to the
pathophysiology of vaso-occlusive crises.
• IgM
• Characteristic loose, mixed-field reactivity in a test tube.
• Can cause mixed-field agglutination
Anti-Lub • Most are IgG, some are IgM and IgA

⌂⌂⌂
Kell (006) and Kx (019)

Kell (006) Blood Group System

Description • First blood group system discovered after the introduction of antiglobulin testing
• Anti-K was identified in 1946 in the serum of Mrs. Kelleher.
• In 1949, anti-k, the high-prevalence antithetical partner to K, was described.
• The discovery of the null phenotype in 1957, designated Ko, helped associate many other antigens with the Kell
system.
• Kell blood group antigens are found only on RBCs. (Harmening)
• The Kell antigen is found on RBCs, erythroid and megakaryocyte progenitors, skeletal muscle, and testis. (henry’s)
• The associated Xk protein is found in erythroid tissues and in other tissues, such as brain, lymphoid organs, heart,
and skeletal muscle.
• Despite its lower quantity, K is very immunogenic.
• Excluding ABO, K is rated second only to D in immunogenicity.
• K0K0 is an autosomal recessive, null phenotype that completely lacks all Kell antigens
• Ko and Kmod phenotype RBCs have increased Kx antigen.
• As a consequence, these individuals can make an alloantibody to the Kell glycoprotein (anti-Ku).
• K0K0 RBCs have enhanced expression of Kx antigen, present on the XK protein.
• Kell antigens are significantly depressed/absent on McLeod RBCs, an X-linked recessive phenotype characterized

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by the absence of XK protein on RBCs (Kx antigen, XK1; ISBT 019), acanthocytes, and neuromuscular disorders.
• Because McLeod individuals lack XK and Kell proteins, these individuals can make alloantibodies directed against
both proteins.
• As a consequence, McLeod individuals are incompatible with both Kell-positive and K0K0 RBCs.
• RBCs may appear to acquire Kell antigens.
• K- patient who acquired a K-like antigen during a Streptococcus faecium infection.
Antigen property
Antigen function • Unknown, but it has structural similarities to a family of zinc-binding neutral endopeptidases.
• It has most similarity with the common acute lymphoblastic leukemia antigen (CALLA or CD10), a neutral
endopeptidase on leukocytes.
Antigens 1. K antigen = __________
2. k antigen = __________
3. Others Kpa, Kpb, Kpc, Jsa, Jsb, and Ku
Common Kell antigens k, Kpb, and Jsb
Antithetical to k antigen
Antithetical to K antigen
Kell antibodies • Most are IgG, rarely IgM
• Clinically significant
• Anti-Kell antibodies are also associated with HDFN.
• The most commonly encountered antibody against the Kell blood group system is anti-K1, which is second only to
Rh D in immunogenicity.
• They can be associated with both immediate and delayed hemolytic transfusion reactions.
• Most anti-K appear to be induced by pregnancy and transfusion.
• Anti-K is usually IgG and reactive in the antiglobulin phase, but some examples agglutinate saline-suspended
RBCs.

Anti-K IgM
• Naturally occurring IgM examples of anti-K are rare and have been associated with bacterial infections.
• Marsh and colleagues studied an IgM anti-K in an untransfused 20-day-old infant with an E. coli O125:B15
infection whose mother did not make anti-K.
• The organism was shown to have a somatic K-like antigen that reacted with the infant’s antibody, so the bacterial
antigen was thought to have been the stimulus.
• The antibody disappeared after recovery.
• The most reliable method of detection is the indirect antiglobulin test.

KX (019) Blood Group System

KX antigen • Kx is present on all RBCs except those of the rare McLeod phenotype
• The associated antigen Kx is the only antigen in the Kx system, ISBT number 019 and symbol XK.
• In the absence of XK protein (McLeod phenotype), Kell expression is decreased on RBCs, suggesting that XK
protein may help transport Kell.
• The XK protein is expressed on several nonerythroid tissues, including liver, skeletal muscle, heart, brain, and
pancreas.
• Characterized by both hematologic and neuromuscular abnormalities, typically presents with areflexia, dystonia,
and choreiform movements late in life.
• Late-onset muscular dystrophy and cardiomyopathy can also be seen.
• Both hematologic and neuromuscular defects in McLeod patients are believed to result from the absence of XK
protein on red cells, brain, heart, and skeletal muscle.
• It is interesting that the Huntington’s disease gene, another neurodegenerative disorder, is located near the XK
gene on the X chromosome.
• The McLeod phenotype can also be associated with chronic granulomatous disease (CGD), a functional neutrophil
defect resulting in severe, recurrent, life-threatening bacterial infections.
• Because of the proximity of the CYBB and XK genes on the X chromosome, approximately 7% of patients with X-
linked CGD also express a McLeod phenotype.
• Exclusive to male.
• Inheritance is X-linked recessive.
• Not all males with the McLeod phenotype have CGD, nor do all patients with CGD have the McLeod phenotype.

Lewis Blood (006) Group System

Description • Unique because the Lewis antigens are not intrinsic to RBCs but are on type 1 glycosphingolipids that are passively
adsorbed onto the RBC membrane from the plasma.
• Associated with secretor gene
Antigens • Lea and Leb are not antithetical antigens produced by alleles; rather, they result from the interaction of
independently inherited alleles.
Other lewis antigen Leab
• Present on all Le(a+b–) and Le(a–b+) RBCs
• The antigen was previously known as ____
• Common and is frequently found with anti-Lea or anti-Leb.
• The antibody is heterogenous and occurs mainly in Le(a–b–)
• Amorphic/silent allele
• Regardless of the secretor allele inherited, no Lea or Leb is produced.
Antigen biologic role • Helicobacter pylori, a causative agent of gastritis and ulcers, binds H, Leb, and Ley antigens
• Increased incidence of ulcers and stomach cancer among blood group O secretors

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• A Lewis null and/or nonsecretor phenotype has also been linked with a higher incidence of recurrent Candida
vaginitis and urinary tract infection.
• A Le (a−b−) phenotype is associated with an increased incidence of heart disease
• Sialyl-Lea is also the epitope for the tumor marker CA 19-9, a useful serologic marker for monitoring patients with
gastrointestinal and other malignancies
• A nonsecretor phenotype protects against Norovirus infection
Antibodies • Most are IgM
• Naturally occurring
• Seldom clinically significant

Lewis and Secretor Gene Interaction

Lewis Gene Secretor Gene Phenotype Lewis Substance in Saliva and
Plasma
Le/Le or Le/le se/se Le (a+b-)
Le/Le or Le/le Se/Se or Se/se Le (a-b+)
le/le Se/se Le (a-b-)
le/le se/se Le (a-b-)

Antigens and Phenotypes resulting from Interaction of Lewis, Secretor, and ABO Genes
Genes Antigens in Secretions RBC Phenotype
Le, Se, A/B/H Lea, Leb, A, B, H
lele, Se, A/B/H A, B, H
Le, sese, A/B/H Lea
lele, sese, A/B/H None
Le, sese, hh, A/B Lea
Le, Se, hh, A/B Lea, Leb

Duffy (008) Blood Group System

Description •In addition to RBCs, the Duffy antigens are expressed on cerebellar Purkinje cells and postcapillary venule
endothelial cells.
• Duffy antigens have also been reported on endothelial cells of renal glomeruli, vasa recta, thyroid, and
pulmonary capillaries, as well as on alveolar type 1 squamous cells and epithelial cells of renal collecting tubules.
• Member of the superfamily of chemokine receptors
• Duffy antigen is also known as Duffy antigen receptor for chemokines (DARC)
• Duffy glycoprotein binds a variety of proinflammatory cytokines.
Antigen property Glycoprotein
Antigens • Codominant: Fya, Fyb
• High incidence: Fy3, Fy5, Fy6
• Fy4 = Distinct, unrelated antigen and is no longer included in the Duffy system.
Fy (a-b-) • Also known as ____________
• Predominant phenotype in blacks
• Resistant to __________________________________________
Antibodies • Clinically significant
• Associated with HTR and HDFN
• Most are IgG
• Dosage

Anti-Fya
• Most commonly encountered antibody
• Associated with acute and delayed HDN
Anti-Fyb
• Associated with acute and delayed HDN

Anti-Fy5
• Discovered in the serum of an Fy(a-b-) black child who was later died of leukemia

Kidd (009) Blood Group System

Description • Antibody was first found in the serum of Mrs. Kidd, whose infant had HDFN.
• Jka and Jkb are antithetical antigens
Antigens 1. Jka = Jk (a+b-)
2. Jkb = Jk (a-b+)
3. Jk (a-b-) = null phenotype
• Null phenotype
• Found in Filipinos, Indonesians, Chinese, and Japanese
Antibodies • Dosage
• Most are IgG
• Associated with immediate and delayed HDFN

Anti-Jka
• Most commonly encountered antibody
• Formed by Jk(a-b+)

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Anti-Jkb
• Formed by Jk(a+b-)

I Blood Group System (027) and i antigen

Description • Contains 2 biosynthetically related antigens: I, i
• I = ______________________
• I and i antigens are not antithetical antigens, they have a reciprocal relationship.
• At birth, infant RBCs are rich in i; I is almost undetectable; over the next 18 months of development, the infant’s
RBCs will convert from i to I antigen.
• I and i antigens are found on the membranes of leukocytes and platelets in addition to RBCs.
• I and i have also been found in the plasma and serum of adults and newborns and in saliva, human milk,
amniotic fluid, urine, and ovarian cyst fluid.
iadult phenotype Associated with ______________________
i antigen • L___________________
• Elevated i antigen is also observed on cord RBCs and reticulocytes and in megaloblastic anemia, leukemia, and
chronic hemolytic states as a sign of stressed erythropoiesis
• Elevated i antigen is also observed in HEMPAS (hereditary erythroblastic multinuclearity with positive acidified-
serum test), a congenital dyserythropoietic anemia associated with chronic hemolysis, binucleated erythroblasts,
and altered red cell glycosylation
I antigen • Derived from i antigen
• B___________carbohydrate
• Adult i
Antibodies Anti-i
• IgM
• Reacts best at room temperature
• Uncommon but has been reported in CAIHA, infectious mononucleosis, choriocarcinomatosis, and alcoholic
cirrhosis.

Testing for antibodies
Anti-I
• Auto anti-I is common
• IgM
• Reacts best at room temperature
• Associated with Cold autoimmune hemolytic anemia (CAIHA), Mycoplasma pneumoniae
Testing for anti-I or anti-I antibodies is done at 4oC using group O RBCs or cord RBCs

Antibody Characteristics
Naturally occurring
Clinically significant
Warm antibodies
Cold Antibodies
Usually react in AHG
Can react in any phase of testing
Detection enhanced by enzyme treatment of test cells
Not detected with enzyme treatment of test cells
Enhanced by acidification
Common cause of delayed HTR
Associated with Mycoplasma pneumoniae
Associated with infectious mononucleosis
Associated with PCH

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Groups of antigen catalogue
according to ISBT
Blood group system
Collection
High-prevalence series (901)
Low-prevalence series (700)
Chapter 3
Uncommon Blood Groups
Blood Group Terminology
1. Blood group system
2. Collection
3. High-prevalence series (901)
4. Low-prevalence series (700)
Controlled by a single gene locus or by two or more closely linked genes
Share a biochemical, serologic, or genetic relationship
Found in more than 90% of the population
Found in less than 1% of the population

ISBT System
010 Diego
011 Yt
012 Xg
013 Sciana
014 Dombrock
015 Colton
016 Landsteiner-Wiener
017 Chido-Rodgers
020 Gerbich
021 Cromer
022 Knops
023 Indian
024 Ok
025 Raph
026 John Milton Hagen
029 Gill
030 Rh-Associated Glycoprotein
031 FORS
032 JR
033 LAN
034 VEL
035 CD59
036 Augustine
Collections • Cost Collection 205
• Ii Collection 206
• Er Collection 207
• Globoside Collection 209
• Collection 210 (Unnamed)
• MN CHO Collection 213
Others • ISBT 700 series
• ISBT 901 series

Uncommon Blood Groups

Description Routinely used only by an advanced BB reference laboratory

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Diego System (010)
Description • Was named after the first antibody maker in a Venezuelan family during an investigation of HDFN
• Carried on ______
• Also known as RBC anion exchanger 1 (AE1) or solute carrier family-4 anion exchanger 1 (SLCA1)
• Had caused HDFN in a Venezuelan baby
• Found in Mongolian ancestry and South American Indians
Antibodies • Usually IgG
• Sometimes IgM

Yt System (011)
Description • Named after the first antibody maker, Cartwright
• Used the last letter “t” in the patient’s last name
• Apparently “why T” became “Yt.”
Antigens • Represent an amino acid substitution on the glycosylphosphatidylinositol (GPI)-linked RBC
glycoprotein acetylcholinesterase (AChE)
• AChE is an important enzyme participating in neurotransmission, but the function of RBC-bound AChE
is not known
• Sensitive to ficin and papain
• Sensitive to DTT
• Resistant to glycine-acid EDTA treatment
• Not present on the RBCs of people with PNH type III
Antibodies • Does not cause HDFN

Xg System (012)
Description • X = ______________
• G = ___________________
• Anti-Xga was discovered in the serum of a multiply transfused man
Antigens • Xga and CD99
• Sensitive to ficin and papain
• Resistant to DTT treatment
Antibodies • Anti-Xga = naturally occurring = do not cause HTR or HDFN

Scianna System (013)

Description • Named after the first antibody maker
Location
Antigens • Resistant to ficin and papain
• Resistant to DTT treatment
Antibodies • Not clinically significant

Dombrock System (014)

Description • Named after the first antibody maker, Mrs. Dombrock (anti-Doa)
Location
Antigens • Resistant to ficin, papain, and glycine-acid EDTA
• Sensitive to 0.2 M DTT treatment
• Absent from PNH III RBCs
Antibodies • Usually IgG
• Anti-Doa and Anti-Dob have caused delayed HTR

Colton System (015)

Description • Was named after the first antibody maker
• It should have been named Calton, but the handwriting on the tube was misread!
Location
Antibodies • Usually IgG
• Anti-Coa = can cause HTR and HDFN

Landsteiner-Wiener System (016)

Description • Human anti-Rh = Anti-D
• Rabbit and guinea pigs’ anti-Rh = Anti-LW
Location • Intracellular adhesion molecule 4 (ICAM-4)
Rhnull RBCs • Lacks the LW glycoprotein
Antigens • Depressed during pregnancy, Lymphoma, and Leukemia
Autoanti-LW • Common in WAIHA

Chido-Rodgers System (017)

Description • Was named after the two antibody producers
• Ch = _________
• Rg = _________
Antigens • Not intrinsic to the RBC membrane
• Adsorbed into RBC from plasma

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• ____ =Chido
• ____ = Rodgers
Antibodies • Usually IgG
• Clinically not significant

Gerbich System (020)

Description • Was named after the first antibody producer, Mrs. Gerbich
Location • ____________________
• The glycoproteins help to maintain the RBC membrane integrity through interaction with protein
band
• 4.1 and p55, and because they are rich in sialic acid, they contribute to the net negative charge of
the RBC membrane (as do GPA and GPB of the MNS system)
Antibodies • Sometimes clinically significant and sometimes not
• Most are IgG
• Some are IgM
• Anti-Ge2 = most common

Cromer System (021)

Description Was named after the first antibody maker, Mrs. Cromer (Anti-Cra), a black pregnant patient
Location
Condition
Antibodies Usually IgG

Knops System (022)

Description Was named after the first antibody maker, Mrs. Knops
Location
Serologic null phenotype
Antibodies Usually IgG
Clinically insignificant

Indian System (023)

Description Was named after the first In(a+) were from India
Location
In(a-b-) phenotype Found in only one individual with __________________________
Antibodies Usually IgG

Ok System (024)
Description • Was named after the first antibody maker, Mrs. Kobutso
• Because Ko was already in use, the first two letters were switched to Ok
• Her parents were cousins from a small Japanese island
Location • ______ or basigin
• Basigin is a receptor essential for invasion by _________________
Antibody IgM

Raph System (025)

Description Was named after the first antibody maker, Raph (Alloanti-MER2)
Location CD151 = a tetraspanin, which appears to be essential for the assembly of basement membranes in the
kidney and skin
Antigen MER2
M = _______________
ER= _______________
2 = Originally defined by _________________
Antibody • Alloanti-MER2 = found in Jews originating from India and living in Israel (had end-stage renal
disease)
• Not clinically significant

John Milton Hagen System (026)

Description • Was named after the first antibody maker, John Milton Hagen
Location
Antigen • Decline during the later years of life, sometimes to the point of not being detected serologically
• Once JMH expression is reduced, anti-JMH can be made
Antibody • Usually IgG (predominantly IgG4 in acquired JMH-negative people)
• Clinically insignificant

Gill System (029)

Description GIL is genetically discrete from all other blood group systems.
Location Glycerol transporter aquaporin 3 (AQP3) = member of the major intrinsic protein family of water and
glycerol channels
Antigen
Antibodies Not clinically significant

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Rh-Associated Glycoprotein (030)
Description Does not have Rh blood group antigens; however, its presence in a complex with the Rh proteins is
essential for Rh antigen expression.
Antibodies Not clinically significant
Rhnull phenotype Absence of RhAG due to inactivating mutations in RHAG gene

FORS System (031)

Description • Named in honor of John Forssman, who discovered the antigen
• Was first thought to be a subgroup of A called Apae
• Unrelated to ABO System
Antigen • FORS1 = only one antigen
• The Forssman glycolipid can serve as a receptor for pathogens such as Escherichia coli, making
one believe that human cells that express the FORS1 may have an increased susceptibility to E.
coli infection.
Antibodies • Mostly IgM
• Unknown clinical significance

JR System (032)
Description • Was named after the first antibody maker, Rose Jacobs
Location • ABCG2 = member of the adenosine triphosphate (ATP) binding cassette transporters broadly
distributed throughout the body
• Involved in multidrug resistance in tumor cells, presenting a problem in chemotherapy
Antigen • ________________________
• Jr(a-) phenotype = common in _______________
Antibodies • Usually IgG
• Unknown clinical significance

LAN System (033)

Description • Was named after the first antibody maker, Langereis (Anti-Lan)
Antigen • Lan = only one antigen
Antibodies • IgG
• __________ = Considered as clinically significant
• Caused an immediate HTR

Vel System (034)

Description • Was named after the first antibody maker
Antigen • Vel
Antibodies • Anti-Vel = characterized by its ability to activate complement and cause in vitro and in vivo
hemolysis
• Most are IgG

CD59 System (035)

Location • Membrane inhibitor of lysis (MIRL)
• CD59 plays a key role in protecting against complement regulated hemolysis by binding to C8 and
C9 thus interfering with the formation of the membrane-attach complex (MAC)
Antigen
Antibodies • Anti-CD59 = IgG
Congenital CD59 deficient individuals (No • Have been identified among Japanese, North Africans, Jews, and Turks
PNH)

Augustine System (036)

Description Was named after the first antibody maker, a black woman named Mrs. Augustine
Antibodies Anti-Ata = usually an IgG

ISBT Blood Group Collections

Description • Collections are antigens that have a biochemical, serologic, or genetic relationship but do not
meet the criteria for a system
• Antigens classified as a collection are assigned a 200 number
• Most antigens are either high or low prevalence.
• Csa antigen is a high-prevalence antigen in black
• Antibodies are IgG and not clinically significant
• Historically consisted of two antigens I and i
• I was promoted to ISBT 027
• i antigen = unbranched/linear carbohydrate chain, and precursor for I antigen
• I and i are found on most human cells
• Anti-i = Usually IgM
• Autoanti-i is a cold agglutinin associated with infectious mononucleosis
• Antibodies: IgG
• One Antigen: LKE
• Related to P1PK (003) and Globoside (028)
Antigens:

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• Lec and Led = Precursors to Lewis antigens and show increased expression in Le(a-b-)
• Glycosphingolipids and are adsorbed onto RBCs

Antibodies:
• Anti-Lec = Humans and goats
• Anti-Led = Discovered from injecting goats with saliva from Le(a-b+) individual
MN CHO Collection 213 • Antigens: Associated M and N antigen in the MNS (002)
Antibodies: React optimally at room temperature

ISBT 700 Series

Description • Antigens in the 700 series of low-prevalence antigens represent those with a prevalence of less than 1% of most
random populations.
• The responsible gene for these antigens is unknown; therefore, the antigens cannot be placed in a system.
• When the genetic basis of the antigens in the 700 Series is identified and the antigen placed into a blood group
system, the series number becomes obsolete.
• Finding a compatible blood for transfusion is not difficult because of the low prevalence of the antigen, making
compatible blood readily available.

ISBT 901 Series

Description • Unlike low-prevalence antigens, finding compatible units negative for antigens in the 901 series can be
challenging.
• Antigens in the 901 series of high-prevalence antigens represent those with a prevalence of more than 90% of
most random populations.
• The responsible gene for the antigen is unknown, and the antigen cannot be placed in a system.
• The difficulty in identifying and finding compatible units makes antibodies to the high-prevalence antigens a
potential transfusion risk.
Antigens • Emm, AnWj, Sda, PEL, ABTI, and MAM
• Antigen is not present in PNH III RBCs
AnWj • An = Anton
• H_________________ uses the AnWj antigen as a receptor to enter RBCs
• Anti-AnWj = IgG
Sd a • Named after Sid, head of maintenance department at the Lister Institute in London
• High-prevalence carbohydrate antigen
• Soluble form: found in ________________

Anti-Sda
• _____________ agglutinates under the microscope
• Inhibited with urine from Sd(a+) individuals and guinea pigs.

HLA Antigens on RBCs

Description • HLA class I antigens (A, B, C) are present on all nucleated cells
• Mature RBCs do not have nucleus and generally do not have detectable levels of HLA antigens, thus HLA antigens
are not considered a blood group antigen
Bg • Types: Bga, Bgb, Bgc
• Was given to HLA class I antigens that are detectable on mature RBCs
HLA Bga
HLA Bgb
HLA Bgc
HLA Antibodies

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Chapter 4
Antihuman Globulin Test (AHG)
History of AHG
Moreschi • First to described the principle of the test in 1908
• Used rabbit anti-goat serum to agglutinate rabbit RBCs that were sensitized with low nonagglutinating doses
of goat anti-rabbit RBC serum.
Coombs, Mourant, and Race • Described the use of AHG for the detection of weak and nonagglutinating Rh antibodies in the serum in 1945
• Describes the use of AHG to detect in vivo sensitization of the RBCs if neonates suffering from HDFN in 1946
• The test was called Coomb’s test but later was called the DAT
Dacie • Presented the first evidence that influence the final reaction in 1951= Warm (37oC) and Cold (<22oC)

Antihuman Globulin Test (AHG)

Description • Also known as Coomb’s test
• Before AHG was developed, only IgM antibodies were detected by direct testing methods.
• AHG test permitted the detection of nonagglutinating IgG antibodies and led to the discovery and
characterization of many new blood groups systems.
• Coomb’s test involved the injection of human serum into rabbits to produce antihuman serum.
• Most commonly performed procedures in Blood Group Serology (IAT and DAT)
Principle • Obtained from immunized nonhuman species bind to human globulins (IgG or complement, either free in
serum or attached to antigens on RBCs)
• Adding AHG reagent containing anti-IgG to RBCs sensitized with IgG antibodies allows for hemagglutination
of theses sensitized cells.
Purpose • To detect RBCs sensitized with IgG alloantibodies, IgG autoantibodies, and complement components. (In
vivo or in vitro)
Methods

IgM • Pentamer
• Binds to corresponding antigen and directly agglutinate RBCs suspended in saline
IgG • Nonagglutinating or incomplete antibodies = because their single monomer structure is too small to directly
agglutinate sensitized RBCs

AHG Reagents
Polyspecific AHG Reagents • Contain antibody to human IgG and to the C3d component of human complement
• Other anticomplement antibodies (anti-C3b) may also be present

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• Contains little, if any, activity against IgA and IgM heavy chains
Monospecific AHG Reagents • Contain only one antibody specificity
• Either anti-IgG or antibody to specific components of complement, such as C3b or C3d
• Commonly used: Anti-IgG, and Anti-C3b, -C3d

DAT and IAT

• One-stage procedure
• Detects in vivo sensitization
• Two-stage technique
• Detects in vitro sensitization

Classic method
Polyclonal antiglobulin serum
production
Polyclonal antibodies
Monoclonal antibodies
Preparation of AHG
• Injecting of human serum or purified globulin into laboratory animals (rabbits)
• Human globulin behaves as foreign antigen
• The rabbit’s immune response is triggered, and an antibody to human globulin is produced.
• Human IgG is injected into a rabbit results in anti-IgG production
• Human complement components injected into a rabbit result in anticomplement
• Mixture of antibodies from different plasma cell clones
• Derived from one clone of plasma cells and recognize a single epitope

Polyspecific AHG
Description Can be made using polyclonal or monoclonal antibodies
Production Usually prepared from rabbit

Antibodies Required in AHG

1. Anti-IgG • Mixture of IgG1 and IgG3
• IgM (Rarely found) = Fix complement and can be detected by anti-complement
2. Anti-Complement -

DAT
Principle Detects in vivo sensitization of RBCs with IgG or complement components
Tube for collection EDTA = to avoid in vitro complement attachment associated with refrigerated, clotted specimen
Detection IgG: 100 to 500 per RBC
C3d: 400 to 1,100 per RBC
Conditions 1. HDFN
2. HTR
3. AIHA
HDFN Maternal antibody coating fetal RBCs
HTR Recipient antibody coating donor RBCs
AIHA Autoantibody coating individual’s RBCs

IAT
Principle • Detects in vitro sensitization of RBCs
Detection • IgG or C3: 100 to 200 per RBC
Conditions • Detection of incomplete (nonagglutinating) antibodies to potential donor RBCs (compatibility testing) or to
screening cells (antibody screen) in serum
• Determination of RBC phenotype using known antisera (weak D, any other antigen testing that requires IAT)
• Titration of incomplete antibodies

Application
Antibody detection
Antibody identification
Antibody titration
RBC phenotype
IAT Application
Tests In Vitro Sensitization
Compatibility testing Recipient antibody reacting with donor cells
Antibody screening Antibody reacting with screening cells
Antibody panel Antibody reacting with panel cells
Rh antibody titer Antibody and selected Rh cells
RBC antigen detection (weak D, K, Fy) Specific antisera + RBCs to detect antigen

Factors Affecting the Antiglobulin Test

1. Ratio of serum to cells
2. Reaction medium
(enhancement media)
• Increased ratio of serum to cells = increases the sensitivity
• Minimum ratio: 2 drops of serum to 1 drop of 5% (v/v) of RBC suspension
• In saline: 4 drops serum to 1 drop of 3% (v/v) of RBC suspension (ratio: 133:1) = to detect weak antibodies
• Mechanism: The macromolecules of albumin allow antibody-coated cells to come into
closer contact with each other so that agglutination occurs.
• Rarely used
• Incubation: 30 minutes

Disadvantages:
1. Costly than LISS
2. May miss several clinically significant antibodies
• Mechanism: Reduce the zeta potential
• Introduced by Low and Messeter

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3. Temperature
4. Incubation time
5. Washing of RBCs
6. Saline for washing
7. Addition of AHG
8. Centrifugation for reading
• Optimum: 2 drops serum to 2 drops 3% (v/v) RBCs in LISS
Purpose:
1. Enhance antibody uptake
2. Allow incubation times to be decreased from 30 to 60 minutes to 10 to 15 minutes
• PEG is a water-soluble linear polymer and is used as an additive to increase antibody
uptake
• Incubation: 10 to 15 minutes
• Purpose: To remove water molecules surrounding the RBC = to concentrate the
antibody
• AHG reagent: Anti-IgG = to avoid false-positive
• In IAT: Reading for agglutination following 37°C incubation in the IAT is omitted =
Because PEG may cause aggregation of RBCs
• Incubation: 60 minutes
• IgG reacts best at 37oC
• Optimum temperature for complement activation
• Majority of clinically significant antibodies can be detected after 30 minutes of incubation
• Extended incubation times are not necessary
• NSS: __________
• LISS and PEG: __________
• Albumin: ___________
• Minimum: 3 times (IAT and DAT)
• Washing removes free unbound serum globulins
• Inadequate washing = False-negative result = Due to neutralization of AHG reagent by residual unbound
serum globulins
• One of the most important steps in testing
• The wash phase can be controlled using check cells, or group O cells sensitized with IgG
• Should be performed immediately after incubation = to minimize the elution of low-affinity antibodies
• All saline should be discarded after the final wash = because residual saline dilutes the AHG reagent
• Ideally, NSS for washing should be fresh (suggested open expiration of 30 days)
• pH: ______
• Effect of saline stored for long periods in plastic containers = Decreased pH = will result to increase rate of
antibody elution during washing phase = false negative
• AHG should be added immediately after washing = to minimize the chance of antibody eluting from RBC
• CBER recommended method: ___________________
• Suggested: 500 RCFs for 15 to 20 seconds
• Higher RCF = ______________

Sources of Errors in AHG Testing

False-Positive Results
• Improper specimen (refrigerated, clotted) may cause in vitro
complement attachment
• Overcentrifugation and overreading
• Centrifugation after the incubation phase when PEG or other
positively charged polymers are used as an enhancement medium
• Bacterial contamination of cells or saline used in washing
• Dirty glassware
• Presence of fibrin in the test tube may mimic agglutination.
• Cells with a positive DAT will yield a positive IAT
• Polyagglutinable cells
• Saline contaminated by heavy metals or colloidal silica
• Using a serum sample for a DAT (use EDTA, ACD, or CPD
anticoagulated blood)
• Samples collected in gel separator tubes may have unauthentic
complement attachment.
• Complement attachment when specimens are collected from
infusion lines infusing dextrose solutions
• Preservative-dependent antibody directed against reagents
False-Negative Results
• Inadequate or improper washing of cells
• Failure to wash additional times when increased serum volumes are
• used
• Contamination of AHG by extraneous protein (i.e., glove, wrong
dropper)
• High concentration of IgG paraproteins in test serum
• Early dissociation of bound IgG from RBCs due to interruption in
testing
• Early dissociation of bound IgG from RBCs due to improper testing
• temperature (i.e., saline or AHG too cold or hot)
• AHG reagent nonreactive because of deterioration or neutralization
• (improper reagent storage)
• Excessive heat or repeated freezing and thawing of test serum
• Serum nonreactive because of deterioration of complement
• AHG reagent, test serum, or enhancement medium not added
• Undercentrifuged or overcentrifuged
• Cell suspension either too weak or too heavy
• Serum-to-cell ratios are not ideal.
• Rare antibodies are present that are only detectable with
polyspecific AHG and when active complement is present.
• Low pH of saline
• Inadequate incubation conditions in the IAT
• Poor reading technique

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Chapter 5
Donor Selection
Types of Deferral
Temporary Deferral • Prospective donor is unable to donate blood for a limited period of time.
Indefinite Deferral • Prospective donor is unable to donate blood for someone else for an unspecified period of time due
to current regulatory requirements.
• This donor would not be able to donate blood until the current requirement changes.
• These donors may be eligible to donate autologous blood.
Permanent Deferral • Prospective donor will never be eligible to donate blood for someone else.
• These donors may be eligible to donate autologous blood.
• Some permanent deferrals may result from the testing performed on a previous donation.

Deferrals
No deferral • Toxoids or killed or synthetic viral, bacterial, or rickettsial vaccines such as diphtheria, hepatitis A, hepatitis B,
influenza, Lyme disease, pneumococcal polysaccharide, polio injection (Salk), anthrax, cholera, pertussis, plague,
paratyphoid, rabies, Rocky Mountain spotted fever, tetanus, or typhoid injection if the donor is symptom-free and
afebrile.
2 days • Feldene = 2 days for platelet
3 days • Aspirin = inhibits platelet function = deferred only in platelet apheresis
• Piroxicam = inhibits platelet function = deferred only in platelet apheresis
14 days • Plavix and Ticlid
14 to 21 days or until scab • Smallpox vaccination
has fallen off
2 weeks • Live attenuated or bacterial vaccine such as measles (rubeola), mumps, oral polio, typhoid, yellow fever.
4 weeks • Live attenuated vaccine for German measles (rubella), and chickenpox
6 weeks • Termination of pregnancy
• A first-trimester or second-trimester abortion or miscarriage is not cause for deferral.
8 weeks or 56 days • Allogenic whole blood transfusion
12 weeks • Woman received a transfusion during her pregnancy
1 month • Proscar, Propecia, and Accutane
6 months • Avodart
12 months or 1 year • Hepatitis B Immune Globulin (HBIG)
• Experimental medication or unlicensed (experimental) vaccine

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• Organ, tissue, and bone marrow transplant
• Bone or skin graft
• Accidental needle-stick injury
• Tattoo
• Ear and body piercing
• Had sexual contact with person with HIV/AIDS and prostitutes in the past 12 months
• Had sex with anyone who has ever used a needle to take drugs or steroids
• Incarcerated / imprisoned
• Close contact with person with HBV and HCV
• Traveled to an area where malaria is endemic
• Clotting factor concentrates
• Males who have had sex with another male (MSM)
3 years • Soriatane
• Malaria
• Individuals who have lived longer than 5 years in countries considered malaria-endemic by CDC
Indefinite • Insulin from cows (bovine, or beef, insulin) = can cause Mad Cow disease
• Anti-HBc positive
• Chagas disease
• Babesiosis
• Leukemia, cancer, lymphoma
• Diseases of blood such as Hemophilia, von Willebrand disease, sickle cell anemia, thalassemia, Kaposi’s sarcoma,
polycythemia, or a history of receiving clotting factor concentrates
Permanent • Tegison
• Growth hormone from human pituitary gland
• HBsAg Positive
• Has a clinical history or diagnosis of viral hepatitis at age 11 or later
• Dura mater graft
• Xenotransplantation
Teratogenic drugs

Physical Examination
General appearance • The donor center representative should observe the prospective donor for presence of excessive anxiety, drug or
alcohol influence, or nervousness
Weight • At least ___________________
• 10.5 mL of blood/Kg donor weight for whole blood collection
• Volume to collect = (donor’s weight in kg/50) × 450 mL
• Volume to collect/450 × 63 mL = reduced volume of anticoagulant.
• 63 mL (14%) – above calculated volume = amount of solution to be removed.
Temperature • _________________
• Coffee or hot beverages
Hemoglobin Women
• HGB: _______
• HCT: _______
Men
• HGB: _______
• HCT: _______
Autologous
HGB: _______
HCT: _______
Skin Lesions • Multiple puncture marks = ________________
• Not cause for deferral = poison ivy and other rashes (but should not present on the venipuncture site)

Autologous Donation
Description • One who donates blood for his or her own use; thus, such a donor is referred to as the donor-patient.
Advantage • Decreased risk of disease transmission, transfusion reactions, and alloimmunization.
• Patients with rare blood types and with multiple antibodies
Disadvantage • Bacterial contamination, circulatory overload, cytokine-mediated reactions, and misidentification of the product
or patient.
• Higher cost = due to added administrative charges
• Special labeling requirements
Conditions • Preoperative collection
• Acute normovolemic hemodilution
• Intraoperative collection
• Postoperative collection

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