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BLOOD BANKING REVIEW

Jan Renzo D. Besa, RMT


Table of specifications
Denise M. Harmening
Historical Overview
Ancient Egyptians bathed in it, aristocrats drank it,
authors and playwrights used it as themes, and modern
humanity transfuses it.
Historical Overview
1492
• Pope Innocent VII from 3 human blood donors
• First recorded blood transfusion in history
Historical Overview
1665
▫ The first recorded successful blood transfusion occurs in
England: Physician Richard Lower keeps dogs alive by
transfusion of blood from other dogs.

1667
▫ Jean Baptiste Denis:
▫ First recorded animal-to-human blood transfusion (calf blood)
▫ Richard Lower: Sheep’s blood

1795
▫ Philip Syng Physick:
▫ Unconfirmed first human-to-human transfusion
Historical Overview
1818
▫ James Blundel of England
▫ Successful transfusion to a woman suffering form
postpartum haemorrhage.

1869
▫ Braxton Hicks
▫ First non-toxic anticoagulant: sodium phosphate
Historical Overview
1901
▫ Karl Landsteiner, Discovery of ABO blood groups,
"Specificity of Serological Reactions”

1902
▫ Anthony Decastello and Adriano Sturli
▫ AB blood group
Historical Overview

Edward E. Lindemann
▫ Was first to successfully carry out vein to vein
transfusion of blood by using multiple syringes and a
special cannula for puncturing the vein through the
skin.
▫ Unger designed his syringe-valve apparatus.
Historical Overview
1907
▫ Richard Weil
▫ 1st to perform ABO typing and began compatibility
testing
▫ 1st to suggest ABO inheritance

1913
▫ Reuben Ottenberg stressed importance of
compatibility testing.
Historical Overview
1914
▫ Albert Hustin, sodium citrate as an anticoagulant
solution.

1915
▫ Richard Lewisohn, minimum amount of citrate
needed for anticoagulation.
Historical Overview
1916
▫ Rous and Turner, introduction of citrate dextrose
solution for RBC preservation.

1930’s
▫ Function of glucose in RBC metabolism was known.

1932
▫ First blood bank in Leningrad, Russia.
Historical Overview
1939-1940
▫ Philip Levine (together with Stetson, Landsteiner
and Alex Wiener), 1st discovery of Rh blood groups

1941
▫ Charles Drew developed techniques in blood
transfusion and blood preservation during WWII.

1943
▫ Loutit and Mollison introduced the used of ACD
Historical Overview
1945
▫ Coomb, Mourant, Race, Antihuman globulin reagent (was
first described by Carlo Moreschi in 1908)

1947
▫ Rh immune Globulin for prevention of Hemolytic Disease of the
Fetus and Newborn

1951
▫ Edwin Cohn ,development of cell separator, paved the way for
component therapy.
▫ Carl Walter, blood collection using a collapsible bag of polyvinyl
resin.
Historical Overview
1957
▫ Gibson
▫ Introduction of citrate-phosphate-dextrose (CPD)

1960
▫ Plasmapheresis (therapeutic)

1985
▫ DR. Yves Lapierre developed Gel test in Lyon,
France
Basic Genetics
Genetics
• The study of inheritance or the transmission of
characteristics from parents to offspring.

Levels of Genetics:
• Population, concerning genetic traits in large numbers
of individuals.
• Cellular, which pertains to the cellular organization of
genetic material.
• Molecular, based on the biochemistry of genes and the
structures that support them.
Deoxyribonucleic Acid

DNA carries the


primary genetic
information within
chromosomes found in
each cell.
The Central Dogma of Molecular Biology
Terminologies
Genes:
• Smallest unit of inheritance
• Section of DNA along the chromosome.
• Encode certain traits or visible characteristics

The specific location of a gene on a chromosome is called a


locus (plural = loci), and at each locus there may be only
one or several different forms of the gene, which are called
alleles.
Terminologies
Chromosomes:
• Composed of the genetic material chromatin, a complex
of the nucleic acid polymer DNA wrapped around highly
basic proteins called histones.

• The helical structure of DNA allows a lot of information


to be packaged in a very small amount of space.
Terminologies
Alleles:
• One of two or more alternative genes which may be
present at a given locus on a chromosome.

• The presence of two identical alleles results in a


homozygous genotype (i.e., AA), and the phenotype
is group A blood.

• The inheritance of different alleles from each parent


gives a heterozygous genotype.
Terminologies
Alleles:
Dominant:
• Only one allele must be inherited for it to be expressed;
gene product always present.
RR/Rr= Red colored flower
Recessive:
• Same allele must be inherited from both parents to be
expressed, homozygous.
Rr= Red colored flower
rr= White colored flower
Terminologies
Genotype:
• The sequence of DNA that is inherited.

Phenotype:
• Anything that is produced by the genotype, including an
enzyme to control a blood group antigen; the length of
long bones of the skeleton etc.
Terminologies
Amorphic gene:
• A gene with no observable effect, manifestation or
product.

Hemizygous:
• Refers to the condition when one chromosome has a
copy of the gene and the other chromosome has that
gene deleted or absent.
Terminologies
Codominant:
• Equal expression of both alleles in phenotype
• Most of the antigens in the various blood group systems
(i.e., ABO,Rh, Kell, Kidd, etc.) generally follow
straightforward inheritance patterns, usually of a
codominant nature.

AB genes= AB antigens in RBCs


Terminologies
Polymorphic:
• Having two or more possible alleles at a locus
• Example: ABO blood group

Antithetical:
• Opposite form of a gene, different allele.
Terminologies
• Cis: Genes are inherited on the same chromosome.

C in cis position to D:
DCe/dce
• Trans: Genes are inherited on separate chromosomes.
Genes inherited in transposition can weaken the trait's
expression.
C in trans position to D:

Dce/dCe
Terminologies
Linked genes
• Genes that are close together on a chromosome and
inherited as one unit.

Haplotype
• Set of genes inherited via one of the two parental
gametes.
Terminologies
Dosage:
If antibody gives a stronger reaction with RBCs double-
dosed for the target antigen, it shows the dosage effect.
Patterns of Inheritance
Autosomal dominant:
▫ Genes expressed with equal frequency in males and females, on
non-sex chromosome.
Sex-linked dominant:
▫ Carried on the X chromosome; no father-to-son transmission;
will be expressed if passed from father to daughter or from
mother to son.
Sex-linked recessive:
▫ Carried on the X chromosome.
▫ Males inherit it from carrier mothers; traits are exhibited most
commonly in males. (e.g., hemophilia A).
▫ Females can exhibit the trait but must inherit it from both carrier
mother and affected father.
Mendelian Inheritance Principles
Law of Independent Segregation
▫ Two members of a single gene pair passed from one
generation to the next in separate gametes.

Law of Independent Assortment


▫ Traits inherited from different chromosomes
expressed separately and discretely.

Law of Dominance
▫ Recessive alleles will always be masked by dominant
alleles.
Law of Independent Segregation
Law of Independent Assortment
Hardy-Weinberg Principle
Mathematical formula that allowed the study of Mendelian
inheritance in great detail.

Hardy-Weinberg formula:
p+q=1

p is the gene frequency of the dominant allele


q is the frequency of the recessive allele
Can also be stated p2 + 2pq + q2 = 1
Punnet square
The inheritance of blood group antigens (A, B, O)
can be predicted using a Punnett square.
Mother’s Genotype
A B
Father’s B AB BB
Genotype
O AO BO
Blood Banking Immunology
IMMUNOHEMATOLOGY
Serologic, genetic, biochemical, and molecular study of
antigens associated with membrane structures on the
cellular constituents of blood, as well as the immunologic
properties and reactions of blood component . (Henry,
2011)
Immune System
• Innate or natural
▫ The nonspecific primitive IS

• Acquired or adaptive
▫ The specific, evolved IS
 Humoral, mediated by B cells and antibody
production
 Cellular, mediated by T cells and lymphokines
Characteristics of Antigens
• Antigens are substances that combine with an
antibody.
• An antigen that causes a specific immune response is an
immunogen.
• 23 RBC antigen systems containing over 200 RBC
antigens.
 Proteins -Rh, M, and N
 Glycolipids - ABH, Lewis, Ii, and P
 Glycoproteins - HLA
Antibody Characteristics
Characteristics of Blood Group Antibodies
Naturally Occurring Immune Antibodies
• Found in the serum of • Found in the serum of
individuals who have never individuals who have been
been previously exposed to RBC transfused or who are pregnant.
antigens by transfusion,
injection, or pregnancy.

• IgM cold agglutinins, which • IgG antibodies that react best at


react best at room temperature 37°C and require the use of
or lower, activate complement, antihuman globulin sera for
and may be hemolytic when detection.
active at 37°C.
• Common immune antibodies
• Common naturally occurring encountered in testing include
antibodies react with antigens those that react with the Rh,
of the ABH, Hh, Ii, Lewis, MN, Kell, Duffy, Kidd, and Ss blood
and P blood group systems. group systems.
Reagent antibodies
Polyclonal Antibodies Monoclonal Antibodies
Serum antibodies and are Produced by isolating individual B
produced in response to a single cells from a polyclonal population
antigen with more than one and propagating them in cell
epitope. culture with hybridoma
technology.

Not optimal in the laboratory Preferred in testing because they


are highly specific, well
characterized, and uniformly
reactive.
Unexpected Antibodies
• Must be detected and identified before blood can be
safely transfused, no matter their reaction strength or
profile.

• The reactivity of unexpected antibodies is highly varied


and unpredictable, as they may be either isotype IgM or
IgG; rarely, both may be present in the same sample.

• These antibodies may be able to hemolyze, agglutinate,


or sensitize RBCs.
Alloantibodies and Autoantibodies
• Alloantibodies are produced after exposure to
genetically different, or nonself, antigens, such as
different RBC antigens after transfusion.

• Autoantibodies are produced in response to self-


antigens. They can cause reactions in the recipient if they
have a specificity that is common to the transfused
blood.
%
Schematic representation of the
mechanism of antibody-dependent
cell-mediated cytotoxicity (ADCC).
Blood Groups
General characteristics
• ABO and Rh blood groups are the most significant in
transfusion practice.

• There are over 300 RBC antigens that are formally


recognized internationally.

• Blood group antigens are defined by carbohydrates


(sugars) attached to glycoprotein or glycolipid structures
or by amino acids on a protein.
General characteristics
• Blood group antigen:
▫ Protein
▫ Glycoprotein
▫ Glycolipid
▫ NOTE: Antigens are not limited to RBCs

• Blood group system:


▫ Group of blood group antigens that are genetically linked
▫ 30 total systems per ISBT
General characteristics
Private Antigen
▫ Antigen that is unique to an individual or a related family of
individuals
▫ Not commonly found on all cells
▫ Less than 1% of the Population

Public Antigen
▫ Antigen found commonly among individuals
▫ More than 98% of the population
General characteristics
Low-Frequency antigen
▫ “Low-incidence”
▫ Prevalence of less than 1% of most random
populations

High-Frequency antigen
▫ “High-incidence”
▫ Prevalence of more than 90% of most random
populations
Summary of Blood Group Systems
ISBT NUMBER BLOOD GROUP CHROMOSOME NO.
001 ABO 9q
002 MNS 4q
003 P 22q
004 Rh 1p
005 Lutheran 19q
006 Kell 7q
007 Lewis 19p
008 Duffy 1q
009 Kidd 18q
010 Diego 17q
Summary of Blood Group Systems
ISBT NUMBER BLOOD GROUP CHROMOSOME NO.
011 Cartwright 71
012 Xga Xp
013 Scianna 1p
014 Dombrock 12p
015 Colton 7p
016 Landsteiner-Wiener 19p
017 Chido/Rodgers 6p
018 H 19q
019 Kx Xp
020 Gerbich 2q
Summary of Blood Group Systems
ISBT NUMBER BLOOD GROUP CHROMOSOME NO.
021 Cromer 1q
022 Knops 1q
023 Indian 11p
024 Ok 19p
025 Raph 11p
026 John-Milton Hagen 15q
027 I 6p
028 Globoside 3q
029 GIL 9p
030 RHAG 6p
ABO blood group system
ABO blood group system
• The ABO system is the most important of all blood
groups in transfusion practice.

• Karl Landsteiner in 1901 discovered the ABO blood


groups.

• Adriano Sturli and Alfred von Decastello discovered type


AB in 1902.

• Only blood group system in which individuals have


antibodies in their serum to antigens that are absent
from their RBCs.
Inheritance of the ABO Blood Groups
• ABO, like most other blood group systems, is
codominant in expression. If the allele is present, the
antigen will be expressed.

• One position, or locus, on each chromosome 9 is


occupied by an A, B, or O gene

• O is an amorph allele that produces no transferase to


add sugars to the H determinant site.
Formation of A, B, and H Red Cell Antigens

• Results from the interaction of genes at three separate


loci (ABO, Hh, and Se).

• H antigen is the precursor structure on which A and B


antigens are made.

▫ H gene must be inherited to form the ABO antigens on


the RBCs
▫ Se gene must be inherited to form the ABO antigens in
secretions.
Se gene
• “Secretor” gene (chromosome 19)
• (FUT2; FUT = “fucosyltransferase”)
• Precursor to making A or B antigens in secretions
• FUT enzyme adds fucose to type 1 chains at terminal
galactose; product is type 1 H antigen
• 80% secretors, 20% non-secretors
H gene
• Closely linked to Se on chromosome 19
• (FUT1; FUT = “fucosyltransferase”)
• FUT enzyme adds fucose to type 2 chains at terminal
galactose; product is type 2 H antigen.
• Present in more than 99.99% of the population
• H antigen required before A and/or B can be made on
RBCs (type 2 H) or in secretions (type 1 H).
Precursor chains
TYPE LINKAGE Found in:
TYPE 1 beta 1→3 linkage Body Fluids and
Secretions
TYPE 2 beta 1→4 linkage Red cell membrane
Formation of A, B, and H Red Cell Antigens
Type 2 precursor chain
Formation of H antigen

The H antigen is the


building block for the
A and B antigens.

H antigen acts as the


acceptor molecule
for the two sugars
that make up
the A and B antigens.
Formation of the A antigen

The A blood type is


the H antigen with N-
acetylgalactosamine
attached.
Formation of B antigen

The B blood type


is the H antigen with
d-galactose
attached.
Interaction of the Hh and ABO genes.
O > A2 > B > A2B > A1 > A1B

Reactivity of anti-H antisera or anti-H lectin with ABO blood groups


ABO frequencies
Formation of A, B, and H Soluble Antigens

• ABH-soluble antigens can also be found in all body


secretions.

• Dependent on the ABO genes inherited and Sese


(secretor genes) that regulate their formation.

• Se gene codes for the production of a transferase (α-2-L-


fucosyltransferase).

• People who inherit the sese genotype are termed


nonsecretors.
Fluids in Which A, B, and H Substances can be detected in Secretors

• Saliva
• Tears
• Urine
• Digestive juices
• Bile
• Milk
• Amniotic fluid
• Pathological fluids:
▫ Pleural
▫ Peritoneal
▫ Pericardial
▫ Ovarian cyst
Secretor Status
Procedure:
1. Collect 2 to 3 ml saliva in a clean 16 x 100 mm tube.
Use paraffin wax or clean rubber bands to stimulate
secretions.
2. Centrifuge at 1000xg for 8 to 10 minutes
3. Transfer supernatant in a stoppered tube.
4. Place in a boiling waterbath for 10 minutes. This
inactivates enzymes that might otherwise destroy blood
group substances.
5. Recentrifuge and collect clear supernatant.
6. Dilute saliva with NSS.
Secretor Status
8. Add one drop of the appropriate diluted antiserum to
each tube.
9. Add one drop of supernatant saliva.
10. Mix and incubate for 8 to 10 minutes.
11. Add one drop of appropriate indicator cells.
12. Mix and incubate at RT for 30-60 mins
13. Centrifuge
14. Observe for macroscopic agglutination
Secretor Status
SAMPLE: Saliva
PRINCIPLE: Agglutination Inhibition
+ Agglutination: Negative
- Agglutination: Positive

A
A
A A A
A Y Y

A SeSe Y Indicator Cells


Anti-A
Secretor Status
SAMPLE: Saliva
PRINCIPLE: Agglutination Inhibition
+ Agglutination: Negative
- Agglutination: Positive

A
A
A A A
Y Y
A
A SeSe Y Indicator Cells
Anti-A
Secretor Status
SAMPLE: Saliva
PRINCIPLE: Agglutination Inhibition
+ Agglutination: Negative
- Agglutination: Positive

No agglutination:
A A Secretor
Y A Y
A A
A SeSe Y A
Anti-A
ABO Subgroups
A Subgroups
A1 Versus A2 Phenotypes
Blood group Anti-A Anti-A1 Lectin
(anti-A plus anti-A1)
A1 + +
A2 + 0

• 80% of all group A or AB are A1 or A1B


• 20% are A2 or A2B
A1 Versus A2 Phenotypes
Lectins
Lectins Specificity
Ulex europaeus Anti-H
Dolichos biflorus Anti-A
Griffonia ( Bandeiraea) simpicifolia I Anti-B
Griffonia ( Bandeiraea) simpicifolia II Anti-Tk
Vicia graminea Anti-N
Helix promatia Anti-A, Tn, Cad
Iberis amara Anti-M
Glycine soja Anti-T, Tn
Weak A Subgroups
• A3-
▫ demonstrate a mixed-field pattern of agglutination
with anti-A and most anti-A,B reagents.
• Ax
▫ not agglutinated by anti-A reagent but do agglutinate
with most examples of anti-A,B.
• Aend
▫ demonstrate mixed-field agglutination with anti-A and
anti-A,B, but only a very small percentage of the RBCs
(10% or less) agglutinate.
Weak A Subgroups
• Am-
▫ not agglutinated, or are agglutinated only weakly, by
anti-A or anti-A,B.
• A y-
▫ not agglutinated by anti-A or anti-A,B.
• Ael
▫ unagglutinated by anti-A or anti- A,B.
Investigation of weak A subgroups
Characteristics of B subgroups
Bombay Phenotype (Oh)
• First reported by Bhende in 1952 in Bombay, India.
• hh genotype
• No H antigens formed
• Phenotypes as blood group O
• Anti-A, anti-B, anti-A,B, and anti-H present in the serum
• Can only be transfused with blood from another Bombay
• RBCs of the Bombay phenotype (Oh) will not react with
the anti-H lectin (Ulex europaeus)
Group O vs Oh
Group Anti A Anti B Anti H A cells B cells

O O O + + +
Oh
Group O vs Oh
Group Anti A Anti B Anti H A cells B cells

O O O + + +
Oh O O O + +
Para-Bombay phenotype
• RBCs are completely devoid of H antigens or have small
amounts of H antigen present.

• In patients with hh,Se genes, enzyme associated with the


FUT2 gene (α2FucT2) produces H, A, B, type 1 antigens
in secretions, including plasma.
RBC compatibility
Plasma Compatibility
Universal Donor:

Universal Recipient:

Universal donor for plasma/plasma products:

Universal recipient for plasma/plasma products:


Universal Donor: O

Universal Recipient: AB

Universal donor for plasma/plasma products: AB

Universal recipient for plasma/plasma products: O


ABO Antibodies
• Individuals normally produce antibodies directed
against the A and/or B antigen(s) absent from their
RBCs. (Naturally occurring)

• Production of ABO antibodies is initiated at birth, but


titers are generally too low for detection until the
individual is 3 to 6 months of age.
ABO Naturally occurring antibodies
• Anti-A
• Anti-B
• Anti-A,B ( IgG in nature)
• Predominantly IgM, small quantities of IgG present
• Produce strong direct agglutination reactions
• Activates complement
• React at room temperature or colder
Landsteiner’s rule
If an antigen is present on a patients red blood cells the
corresponding antibody will NOT be present in the
patients plasma, under 'normal conditions'.
What’s Your Type?
Forward grouping (front type) is defined as using
known sources of commercial antisera (anti-A, anti-B) to
detect antigens on an individual’s RBCs.

Reverse grouping (back type) is defined as detecting


ABO antibodies in the patient’s serum by using known
reagent RBCs, namely A1 and B cells.
Routine Reagents Used for ABO Testing
(FORWARD GROUPING)
ANTI-A REAGENT ANTI-B REAGENT
• Monoclonol antibody • Monoclonal antibody

• Highly specific, IgM • Highly specific, IgM

• Clear blue colored reagent • Clear yellow colored reagent

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

• Usually use 1–2 drops • Usually use 1–2 drops


Routine Reagents Used for ABO Testing
(REVERSE GROUPING)

REAGENT A1 AND B CELLS


▫ Human source
▫ 4%–5% red cell suspension
▫ Expected 2+ to 4+ reaction usually use one drop
ABO Typing
Reagent Reagent
Anti-A Anti-B A1 cells B cells Interpration

0 0 + + 0
+ 0 0 + A
0 + + 0 B
+ + 0 0 AB
ABO Discrepancies
• ABO discrepancies occur when the forward and reverse
groupings do not agree.

• The unexpected reaction can be due to an extra positive


reaction or a weak or missing reaction in the forward
and reverse grouping.
Group I Discrepancies
Associated with unexpected reactions in the reverse
grouping due to weakly reacting or missing
antibodies.
Group I Discrepancies
• Newborns
• Elderly patients
• Patients with a leukemia demonstrating
hypogammaglobulinemia
• Patients using immunosuppressive drugs
• Patients with congenital or acquired
agammaglobulinemia or immunodeficiency diseases
• Patients with bone marrow or stem cell transplantations
• Patients whose existing ABO antibodies may have been
diluted by plasma transfusion or exchange transfusion
• ABO subgroups
Example:

ABO Discrepancy Seen With Weak or Missing Antibodies

Anti-A Anti-B A cells B cells


Patient O O O O
Example:

ABO Discrepancy Seen With Weak or Missing Antibodies

Anti-A Anti-B A cells B cells


Patient O O O O

Patient’s probable group: O


(elderly patient or newborn)
Group I Discrepancies
Resolution:
• Check patient’s history
• Check for technical discrepancy
• Incubate patient’s serum with reagent A1 and B
cells at RT and at 4⁰C for 15-30 minutes
Group II Discrepancies
Associated with unexpected reactions in the
forward grouping due to weakly reacting or
missing antigens.
Group II Discrepancies
• Subgroups of A (or B) may be present
• Leukemias may yield weakened A or B antigens
• Hodgkin’s disease
• “Acquired B” phenomenon
Serologic Reactions Typical of Leukemia

Patient ANTI- A ANTI-B A CELLS B CELLS


Phenotype
A mf 0 0 3+
B O +/- 4+ 0
Acquired B phenomenon
• Group A RBCs contact with gram negative bacteria

• Colon cancer, intestinal obstruction, gram negative


sepsis

• Bacterial enzymes (deacetylases) modify the


immunodominant blood group A sugar (N-acetyl-D
galactosamine) into D-galactosamine.
Acquired B phenomenon
ANTI- A ANTI-B A B
Interpretation
CELLS CELLS
4+ 2+ o 3+ A
Acquired B phenomenon
• Testing the patient’s serum or plasma against autologous
RBCs gives a negative reaction.

• Acquired B antigen is also not agglutinated when reacted


with anti-B that has a pH greater than 8.5 or less
than 6.

• Treating RBCs with acetic anhydride reacetylates the


surface molecules.
Group II Discrepancies
Resolution:
• Incubate test mixture at room temperature for
up to 30 mins
• Check patient’s history
• Check for technical discrepancy
Group III Discrepancies
These discrepancies between forward and reverse
groupings are caused by protein or plasma
abnormalities and result in rouleaux formation or
pseudoagglutination.

Rouleaux
Group III Discrepancies
• Elevated levels of globulin from certain diseases:
▫ Multiple myeloma
▫ Waldenström’s macroglobulinemia
• Hodgkin’s lymphomas
• Elevated levels of fibrinogen
• Plasma expanders (dextran & polyvinylpyrrolidone)
• Wharton’s jelly in cord blood samples
Group III Discrepancies
Example:
Forward Typing Reverse Typing
Anti-A Anti-B A1 cells B cells
4+ 2+ 4+ 2+
Group III Discrepancies
Resolution:
• Washing the patient’s red cells with saline or adding a
drop or two of saline to the tube in case of rouleaux
formation.

• If the agglutination is true red cell clumping will remain.

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


grouping only.
Group IV Discrepancies
These discrepancies between forward and reverse
groupings are due to miscellaneous problems.
Group IV Discrepancies
• Cold reactive autoantibodies
• Unexpected ABO isoagglutinins
• Unexpected non-ABO alloantibodies
• Antibodies other than anti-A and anti-B may react to
form antigen-antibody complexes that may then adsorb
onto patient’s RBCs.
• Cis-AB
• Cis-AB refers to the inheritance of both AB genes from
one parent carried on one chromosome and an O gene
inherited from the other parent.

• RBCs with the cis-AB phenotype (a rare occurrence)


express a weakly reactive A antigen (analogous to A2
cells) and a weak B antigen.
ABO Discrepancy Caused by Cold Autoantibodies
ABO Discrepancy Caused by Cold Autoantibodies

Patient’s probable group:


B
ABO Discrepancy Caused by an Unexpected ABO Isoagglutinin
ABO Discrepancy Caused by an Unexpected ABO Isoagglutinin

Patient’s probable group:


A2B
Rh Blood Group System
Rh Blood Group System
• Rh is the second most important blood group system in
terms of transfusion, as the Rh system antigens are very
immunogenic.

• One of the most complex of all RBC blood group systems


with more than 50 different Rh antigens.
History
• In 1939 Levine and Stetson described a hemolytic
transfusion reaction in an obstetrical patient.
• Landsteiner and Wiener reported on an antibody made
by guinea pigs and rabbits when they were transfused
with Rhesus macaque monkey RBCs.
▫ Agglutinated 85% of human RBCs, was named Rh after the
Rhesus monkey.
• Rh was retained for the human-produced antibody, and
anti-Rhesus formed by the animals was renamed anti-
LW.
• Anti-LW usually reacts:
▫ Strongly with D+ RBCs
▫ Weakly with D– RBCs
▫ Not at all with Rhnull RBCs

• Anti-D is different from Ant-LW

• Distinguished by testing DTT treated D+ RBCs:


▫ D antigen is not denatured by DTT
▫ LW antigen is destroyed by DTT
Fisher-Race: DCE Terminology
• Fisher and Race postulated that the antigens of the
system were produced by three closely linked sets of
alleles.
• Each gene complex carries D or its absence (d), C or c, E
or e.
• Inherited from parents in linked fashion as haplotypes
• The gene d is assumed to be present when D is absent.
Fisher-Race: DCE Terminology
Wiener: Rh-Hr Terminology
• Wiener believed there was one gene responsible for
defining Rh that produced an agglutinogen containing a
series of blood factors.
• According to Weiner, this Rh gene produced at least
three factors within an agglutinogen.
• Each factor is an antigen recognized by an antibody.
Wiener: Rh-Hr Terminology
Converting Wiener (Rh-Hr) to Fisher-Race
R= D
r= d
1 or prime= C
2 or double prime= E
0 or blank= ce
Any superscript letter = CE

1 2
D C E
Wiener Fisher-Race Wiener Fisher-Race

R1 DCe r’ dCe
R2 DcE r’’ dcE
Ro Dce r dce
Rz DCE ry dCE
Convert:

R1r” to Fisher-Race:

R2Rz to Fisher-Race:
Convert:

R1r” to Fisher-Race:
DCe/dCe

R2Rz to Fisher-Race:
DcE/DCE
Rosenfield and Coworkers:
Alphanumeric Terminology

• Rosenfield and associates proposed a system that assigns


a number to each antigen of the Rh system in order of its
discovery or recognized relationship to the Rh system.

• Based ONLY on serologic (agglutination) reactions.

• Simply demonstrates the presence or absence of the


antigen on the RBC.

• No genetic assumptions made.


Rosenfield
• D= Rh1
• C= Rh2
• E= Rh3
• c= Rh4
• e= Rh5

D + C + E + c negative, e negative
Rosenfield
• D= Rh1
• C= Rh2
• E= Rh3
• c= Rh4
• e= Rh5

D + C + E + c negative, e negative
Rh: 1, 2, 3, –4, –5
International Society of Blood Transfusion
Committee
• Six-digit number for each authenticated antigen
belonging to a blood group system

• First three numbers represent the system and


the remaining three the antigenic specificity.

• 004 was assigned to the Rh blood group system


Antigens of the Rh Blood Group System in Four Nomenclatures
NUMERIC FISHER-RACE WEINER ISBT NUMBER

Rh1 D Rh0 004001


Rh2 C rh’ 004002
Rh3 E rh’’ 004003
Rh4 c hr’ 004004
Rh5 e hr’’ 004005
RH Genes
• Wiener hypothesized that a single gene produces a single
product that contains separately recognizable factors.
• Fisher and Race proposed that the Rh locus contains
three distinct genes that control production of their
respective antigens.
• Tippett correctly proposed two RH genes:
▫ RHD
▫ RHCE
RH Genes
Two closely linked genes located on chromosome 1
control expression of Rh proteins:
▫ RHD = RhD protein
▫ RHCE =RhCe, RhcE, Rhce, or RhCE proteins
RHAG
• Rh-associated glycoprotein
• Resides on chromosome 6, Glycosylated
• Forms complexes with the Rh proteins
• Coexpressor and must be present for successful
expression of the Rh antigens
• Mutations can result to missing or significantly altered
RhD and RhCE proteins.
Rh system antigens

D antigen: Most immunogenic of Rh antigens.

D > c > E > C >e


Immunogenicity of common Rh antigens
Number of D Antigen Sites of Cells with Various Phenotypes
Variations of D Antigen Expression
Individuals with altered D antigen are categorized into
different phenotypes defined as weakened D due to:
▫ C in trans to RHD
▫ Weak D
▫ PartialD
▫ Del
C in Trans to RHD
• “Position effect” or gene interaction effect
• Occurs when the C antigen is inherited trans to the D
antigen

Example:

Dce/dCe
Weak D
• Results from inheritance of RHD genes that code for a
weakened expression of the D antigen
• D antigens are complete but fewer in number

RBC with normal


D expression
Weak D
Partial D
• One or more D epitopes within the entire D protein is
either missing or altered.

• Partial-D individual lacks one or more of these


epitopes.

• Capable of making antibody to the epitopes that


s(he) lacks.
Partial D
Missing
portion

RBC with normal RBC with Partial


D expression D
Weak D Testing

 If negative, individual is D negative


 If positive, individual is D positive
Weak D Testing
• Weakly reactive D = D-positive.

• AABB Standards state that all Rh-negative donor


units must be tested for weak D.

• Units that test positive must be identified as D-


positive.

• Weak-D recipients are transfused with D-negative blood.


Rh Deficiency Syndrome
Rh null syndrome (- - -/ - - -)
• Fail to express any Rh antigens on the RBC surface
• Mild compensated hemolytic anemia
• Reticulocytosis
• Stomatocytosis

Amorphic
▫ RHD gene is absent, no expression of RHCE gene
Regulator
▫ Gene inherited, but not expressed
Rh Deficiency Syndrome
Rh mod phenotype
• Partial suppression of RH gene expression caused by
mutations in the RHAG gene.
• RhAG protein is altered, normal Rh antigens are also
altered, often causing weakened expression of the
normal Rh and LW antigens.
• Exhibit features similar to those with the Rh null
syndrome.
• Clinical symptoms are usually less severe.
Unusual phenotypes

Cw
▫ Results in a single amino acid change most often found
on the RhCe protein.
▫ Found in about 2% of whites and is very rare in blacks.
▫ Anti-Cw has been identified in individuals without
known exposure to foreign RBCs and after transfusion
or pregnancy.
▫ Anti-Cw may show dosage
Unusual phenotypes

f (ce)
▫ Expressed on the RBC when both c and e are present
on the same haplotype.
▫ Compound antigen
▫ Anti-f only shows postive reactivity with DCE/dce.
▫ Anti-f is generally a weakly reactive antibody often
found with other antibodies.
Unusual phenotypes
rhi (Ce)
▫ Compound antigen present when C and e are on
the RhCe protein.

▫ Anti-rhi shows positive reactivity only with


DCe/dce RBCs.
Unusual phenotypes
G antigen
▫ Antigen present on most D-positive and all C-positive
RBCs.
▫ Anti-G mimics anti-C and anti-D.
▫ Anti-G activity cannot be separated into anti-C and anti-D
▫ Anti-G versus anti-D and anti-C is important when
evaluating obstetric patients.
Unusual phenotypes
Rh17
▫ Also known as Hr0, is an antigen present on all RBCs
with the “common” Rh phenotypes.

▫ Antibody is directed to the entire protein resulting


from the RHCE genes.
Unusual phenotypes
D deletion
• Demonstrate no Cc and/or Ee reactivity.
• Unusually strong D antigen expression, called exalted D.
• Results from individuals possessing normal RHD
gene(s) and hybrid RHCE-RHD-RHCE in which the
Rhce protein is replaced with RhD. (D–/D–)
• Antibody made by D–/D– people is called anti-Rh17 or
anti-Hr0.
Rh Antibodies
• Mostly IgG, react optimally at 37°C or after antiglobulin
testing, do not bind complement.

• Produced following exposure of the individual’s immune


system to foreign RBCs, through either transfusion or
pregnancy.

• Enhanced when testing with enzyme-treated RBCs


Rh Antibodies
• Stronger reactivity of antibody with cells from
homozygous individuals is shown with anti-C, anti-c,
anti-E, and anti-e.

• C and e and E and c are usually found together.


Rh typing
• Detection of D antigen on patient’s RBC
• Specimen: Whole blood/ RBC suspension
• Four types of anti-D reagents:
▫ Saline reactive reagents
▫ High-protein anti-D
▫ Chemically modified
▫ Monoclonal source
• Presence of Agglutination:
▫ Rh Positive
• Absence of Agglutination:
▫ Rh Negative (confirmed using Weak D Testing)
Rh Typing Reagents
Four types of anti-D reagents:
▫ Saline reactive reagents
▫ High-protein anti-D
▫ Chemically modified
▫ Monoclonal source
Saline reactive reagents
• Contain IgM immunoglobulin
• Low protein-based
• Advantages:
▫ Can be used to test cells that are already coated with
IgG antibody.
• Disadvantages:
▫ Limited availability
▫ Cost of production
▫ Lengthy incubation time
▫ Cannot be used for weak-D typing.
High-protein anti-D
• Consisted primarily of IgG anti-D
• From human plasma containing high-titer D-specific
antibody
• Bovine albumin and dextran or polyvinylpyrrolidone
were added
• Advantages:
▫ Reduced incubation time
▫ Ability to perform weak-D testing and slide typing.
• Disadvantages:
▫ False positives due to autoagglutinins, abnormal serum
proteins, antibodies to additives and using unwashed RBCs.
Chemically modified
• IgG converted to saline agglutinin by weakining disulfide
bonds at hinge region, greater flexibility, increases span
distance.
• Can be used for slide, tube and weak D test.
• Negative control unnecessary unless AB positive.
• Advantages:
▫ Fewer false-positive test reactions are obtained.
Monoclonal source
• Derived from single clones of antibody-producing cells
• Blend of IgM and IgG anti-D
▫ Maximize visualization of reactions at immediate spin
testing
▫ Allow indirect antiglobulin testing for weak D

• Can be used for slide, tube, microwell, and most


automated Rh testing.
Transfusion Reactions
• D antigen is the most immunogenic antigen outside the
ABO system.

• Antibody appears within 120 days of a primary exposure


and within 2 to 7 days after a secondary exposure
Transfusion Reactions
Extravascular destruction of RBCs

Fever , Bilirubin Hemoglobin Haptoglobin

• DAT +
• Antibody screen (+) (-)
• Antibody elution
Rh HDFN
• Severe
• Rh antibodies are primarily IgG, which readily cross the
placenta

• Mother:
• Father:
• Baby:
Rh HDFN
• Severe
• Rh antibodies are primarily IgG, which readily cross the
placenta

• Mother: Rh Negative
• Father: Rh Positive
• Baby: Rh Positve
Other Blood group Systems
The Lewis (007) System
• Lewis antigens are not intrinsic to RBCs but are on type 1
glycosphingolipids that are passively adsorbed onto the
RBC membrane from the plasma.

• Le gene must be present for a precursor substance to be


converted to Lea

• Se gene must be present for conversion to Leb


Lewis antigens
• Lewis antigens produced in saliva and other secretions
are glycoproteins.

• Lewis cell-bound antigens absorbed from plasma onto


the RBC membranes are glycolipids.

• Lewis antigens are not well developed on fetal RBCs.


Lewis antigens
• Lewis antigens are not expressed on cord RBCs and are
often diminished on the mother’s RBCs during
pregnancy.

• Leb act as receptor for Helicobacter Pylori


Lewis antigens
• Lewis gene (FUT3) is linked to Se (FUT2) and H (FUT1),
all located on chromosome 19.

• The synthesis of Lewis antigens depends on the


interaction of the transferases produced by the Lewis
and secretor genes.
Type 1 Precursor Chain

GlcNAc R

GAL • Lipid
• Protein

• Secretions - glycoproteins.
• Plasma - glycolipids.
Se allele (FUT2) Secretor Enzyme
SeSe
Sese
GlcNAc R

GAL

FUC Type 1 H
Le allele (FUT3) Lewis Enzyme
LeLe
Lele FUC

GlcNAc R

GAL

Le a
Interaction between Le and Se enzymes

FUC

GlcNAc R

GAL

FUC
Le b
Lewis enzyme substrates

Substrate Antigen
Type 1 Lea
Type 1 H Leb
Type 1 A ALeb
Type 1 B ALeb
Phenotypes of the Lewis System
Le and Se genes inherited

Le(a-b-) Le(a+b-) Le(a+b+) Le(a-b+)

Development of Lewis Antigens


Le gene with no Se gene inherited

Le(a-b-) Le(a+b-)

Development of Lewis Antigens


Antigens and Phenotypes Resulting From Interaction of
Lewis, Secretor, and ABO Genes
Lewis antigens during pregnancy
• Lewis antigen strength may decline dramatically during
pregnancy.

• The transiently Le(a-b-) pregnant woman may produce


Lewis antibodies during pregnancy

• These antibodies disappear after delivery as the normal


Lewis phenotype is restored.
Lewis antibodies
• Naturally occurring and made by Le(a–b–) persons.

• Generally IgM and do not cross the placenta.

• Neutralized by the Lewis substances present in plasma


or saliva.

• Types:
▫ Anti-Lea
▫ Anti-Leb
Lewis antibodies
Anti-Lea
• Most commonly encountered of the Lewis antibodies

• Common in Le(a-b-), rare in Le(a-b+)

• Often detected in room temperature tests

• Sometimes reacts at 37°C

• Rare hemolytic transfusion reactions (HTR)


Lewis antibodies
Anti-Leb
• IgM agglutinin

• Can bind complement

• Infrequently made by Le(a+b–) individuals

• Anti-LebH and anti-LebL


Disease association
• Helicobacter pylori uses Leb to bind to gastric
epithelial cells leading to peptic ulcer disease.
Additional notes:
• Lewis antigens readily absorb to and elute from RBC
membranes and assume the Lewis phenotype of the
recipient within a few days of entering the circulation.
• Lewis antibodies in the recipient's serum are readily
neutralized by Lewis blood group substance in donor
plasma.
• It is rare for Lewis antibodies to cause in-vivo hemolysis.
• It is not necessary to type donor blood for the presence
or absence of Lewis antigens prior to transfusion or
crossmatching.
The MNS (002) System
• Discovered in 1927 by Landsteiner and Levine.

• Genes are located on chromosome 4.

• Genes behave as allelic pairs that are closely linked:


▫ MM
▫ MN
▫ NN

• Has been assigned the ISBT number 002 (symbol MNS).


MN antigens
• M and N antigens are found on glycophorin A

• Destroyed by enzymes

• M and N antigens are antithetical and differ in their


amino acid residues at positions 1 and 5
▫ M is defined by serine and glycine
▫ N is defined by leucine and glutamic acid
Ss antigens
• S and s antigens are located on glycophorin B

• Differentiated by the amino acid at position 29 on GPB.

• Less easily degraded by enzymes.

▫ Methionine defines S

▫ Threonine defines s.
U-phenotype
• U for UNIVERSAL

• High-prevalence antigen is found on RBCs of all


individuals except about 1% of African American.

• Anti-U formed by S-s- are typically IgG and has been


reported to cause severe and fatal HTRs and HDFN.
Prevalence of common MN and Ss phenotypes
MNSs Antibodies
Anti-M
• Naturally occurring saline agglutinins that react below 37°C.

• pH-dependent, reacting best at pH 6.5

• Demonstrate Dosage

• Destroyed by enzymes

• Anti-M rarely causes HTRs, decreased red cell survival, or


HDFN.
MNSs Antibodies
Anti-N
• Cold-reactive IgM or IgG saline agglutinin.

• Does not bind complement or react with enzyme-treated


RBCs.

• Demonstrate dosage

• Anti-Nf
• Found in renal patients who are dialyzed on equipment
sterilized with formaldehyde.
MNSs Antibodies
Anti-Ss
• IgG, reactive at 37°C and the antiglobulin test

• Destroyed by enzymes

• May bind complement

• May cause severe HTRs with hemoglobinuria.


Disease Associations
• GPAM may serve as the receptor by which certain
pyelonephritogenic strains of E. coli.

• Plasmodium falciparum appears to use alternative


receptors, including GPA and GPB for cell invasion.
The I (027) System and I Antigen
• I= “individuality”

• Infant RBCs are rich in i; I is almost undetectable.

• During the first 18 months of life, the quantity of i slowly


decreases as I increases .

• Adult RBCs are rich in I and have only trace amounts of i


antigen
I and i Antigen
• Expression is age-dependent.
 Simple chains found on neonates make i antigen.
 Branched chains in adults make I antigen.

• Found on the membranes of leukocytes and platelets in


addition to RBCs.

• Found in the plasma and serum of adults and newborns


and in saliva, human milk, amniotic fluid, urine, and
ovarian cyst fluid.
I and i antibodies
• Anti-I is typically a benign, weak, naturally occurring,
saline-reactive IgM auto agglutinin, usually detectable
only at 4°C.

• Like Lewis, antibodies commonly have H specificity as


well (e.g., anti-IH reacts better against O and A2)
Disease associations
• Auto-anti-I
• Cold agglutinin disease
• Mycoplasma pneumoniae infection

• Auto-anti-i
• Associated with infectious mononucleosis
• Less often a problem than auto-anti-I

• i adult phenotype
• Cataracts
• HEMPAS
The P Blood Group:
P1PK (003), Globoside (028), Related (209) Antigens

• Introduced in 1927 by Landsteiner and Levine.

• P antigen is now called P1, with the name P being


reassigned to an antigen present on almost all human
RBCs.

• RBCs lacking P1 , but shown to possess P, are of the P2


phenotype.

• P blood group comprised the P, P1,Pk and Luke (LKE)


Most common P phenotype: P1 (P+P1+k–)
P1 Antigen
• Deteriorates rapidly on storage.

• Poorly expressed at birth and may take up to 7 years to be


fully expressed.

• P1, P, or Pk may be found on RBCs, lymphocytes,


granulocytes, and monocytes.

• P can be found on platelets, epithelial cells, and fibroblasts.

• P and Pk have also been found in plasma as


glycosphingolipids and as glycoproteins in hydatid cyst fluid.
P antibodies
Anti-P1
• Common, naturally occurring IgM
• Rare examples of anti-P1 that react at 37°C can cause in
vivo RBC destruction
• Neutralized by:
▫ Hydatid Cyst Fluid (Echinococcus granulosus)
▫ Pigeon droppings
▫ Turtle dove eggwhite.
P antibodies
Anti-PP1Pk
• Originally called anti-Tja

• IgM and IgG, efficiently bind complement.

• Cause severe HTRs and HDFN

• Associated with an increased incidence of spontaneous


abortions in early pregnancy.
P antibodies
Autoanti-P
• Associated with PCH

• IgG autoantibody, described as a biphasic hemolysin.

• Only demonstrable by the Donath-Landsteiner test


Disease Associations
• P antigens serve as receptors for P-fimbriated
uropathogenic E. coli.

• The Pk antigen is a receptor for shiga toxins and E. coli.

• P is the receptor of human parvovirus B19.

• Pk provides some protection against HIV infection of


peripheral blood mononuclear cells.
The Kell (006) and Kx (019) Systems
• First blood group system discovered after the
introduction of antiglobulin testing.

• Anti-K was identified in 1946 in the serum of Mrs.


Kelleher

• Second rated to D in terms of immunogenecity.


Prevalence of Common Kell System Phenotypes
Kell antigens
• Well developed at birth, found only on RBCs.

• Destroyed by trypsin and chymotrypsin when used in


combination.

• DTT, 2-mercaptoethanol (2-ME), AET, and ZZAP


destroy Kell antigens but not Kx.

• Kell antigen expression is dependent upon the presence


of the Xk protein.
Kell antigens
The Kx Antigen
• Kx is present on all RBCs except those of the rare
McLeod phenotype.

• Ko and Kmod phenotype RBCs have increased Kx antigen.

• When Kell antigens are denatured with AET or DTT, the


expression of Kx increases.
The McLeod Phenotype and Syndrome

• RBCs lack Kx and Km, weak expression of k, Kpb, and Jsb

• X-linked inheritance, males are affected.

• RBCs in individuals with the McLeod phenotype are


acanthocytic, chronic but often well-compensated
hemolytic anemia.

• Associated with CGD.


The Duffy (008) System
• Named for Mr. Duffy, a multiply transfused hemophiliac.

• Gene is located chromosome 1q.


▫ Fya, Fyb, and Fy common alleles at the Fy locus.
▫ Fya from Fya gene; high frequency in Asians
▫ Fyb from Fyb gene; high frequency in caucasians
The Duffy (008) System
• Fy is a silent allele

• Majority of African Americans tested were Fy(a–b–)

• Fy(a–b–) RBCs resist infection by Plasmodium


knowlesi and P. vivax.
Prevalence of Common Duffy Phenotypes
Fya and Fyb Antigens
• Well developed at birth

• Can be identified on fetal RBCs as early as 6 weeks


gestational age.

• The antigen sites are destroyed by most enzymes used in


serologic test.
Anti-Fya and Anti-Fyb
• IgG and react best at the antiglobulin phase.

• Show dosage

• Associated with acute and delayed HTRs.

• Anti-Fya more common and significant than anti-Fyb.


The Kidd (009) System
• First recognized in 1951 in the serum of a woman who
had given birth to a child with HDN.

• The antibody, named anti-Jka.

• Mrs. Kidd made an antibody to a high-prevalence


antigen called Jk3.

• Jk3 is present on any RBC positive for Jka or Jkb.


Prevalence of Kidd Phenotypes
Jka and Jkb Antigens
• Commonly found on RBCs of most individuals.

• Jk(a–b–)RBCs resist lysis in 2M urea.

• Well developed on the RBCs of neonates.

• Kidd antigens are not very immunogenic.

• Not denatured by papain or ficin.

• Treatment of RBCs with enzymes generally


enhances reactivity with Kidd antibodies.
Anti-Jka and Anti-Jkb
• Have a notorious reputation in the blood bank.

• IgG, binds complement.

• Demonstrate dosage, are often weak, and are found in


combination with other antibodies.

• Enhanced by using LISS or PEG ,by using four drops of


serum instead of two and using enzymes such as ficin or
papain.
Anti-Jka and Anti-Jkb
• Titer of anti-Jka or anti-Jkb quickly declines in vivo

• Common cause of delayed HTRs.

• Rarely associated with severe cases of HDFN

• Associated with autoimmune hemolytic anemia.


Anti-Jk3
• From Jk(a–b–) individuals,

• Reacts optimally by an antiglobulin test

• Reactivity is enhanced with enzyme pretreatment of the


RBCs

• Associated with severe immediate and delayed HTRs and


with mild HDFN.
The Lutheran (005) System
In 1945, anti-Lua was found in the serum of a patient with
lupus erythematosus after transfusion of blood from a
donor named Lutheran (Lutteran).
Prevalence of Common Lutheran Phenotypes
Lua and Lub Antigens
• Produced by allelic codominant genes.

• Poorly developed at birth, HDFN is rare and only mild.

• Resistant to the enzymes ficin and papain and to glycine-


acid EDTA treatment.

• Destroyed by treatment with the enzymes trypsin and α-


chymotrypsin.

• Most individuals are Lu(b+); 8% of whites and 5% of


blacks are Lu(a+).
Lu(a–b–) Phenotypes
• Dominant Type Lu(a–b–)
▫ In(Lu) for “inhibitor of Lutheran”

• Recessive Type Lu(a–b–)


▫ Two rare silent alleles LuLu at the Lutheran locus
▫ Lack all Lutheran antigens, make anti-Lu3

• Recessive X-Linked Inhibitor Type


▫ X-borne inhibitor to Lutheran
Anti-Lua
• IgM naturally occurring saline agglutinins

• Recognized by their characteristic loose, mixed-field


reactivity in a test tube.

• Rare and mild delayed HTRs due to anti-Lua


Anti-Lub
• IgG and reactive at 37°C at the antiglobulin phase

• Implicated with shortened survival of transfused cells


and post-transfusion jaundice

• Severe or acute hemolysis has not been reported.


Anti-Lu3
• Made only by individuals with the recessive type of
Lu(a–b–).

• Rare antibody that reacts with all RBCs except Lu(a–b–)


RBCs.

• Antiglobulin reactive.
Summary
Antibody Reactivity Enzyme Treatment
MN RT Destroyed
Ss AHG Variable effect
U AHG No effect
Anti-P1 RT Enhanced
Ii RT Enhanced
Kell AHG No effect
Duffy AHG Destroyed
Lewis Most RT, some Enhanced
37˚C,AHG
LuaIgM RT Variable effect
LubIgG AHG
Kidd AHG Enhanced
MINOR BLOOD GROUP SYSTEMS
The Diego (010) System
• Used as a tool in anthropologic studies of Mongolian
ancestry.

• Composed of 22 antigens.
• Three sets of independent pairs of antithetical antigens:
Dia/Dib Wra/Wrb Wu/DISK

• Carried on band 3 (AE-1)

• Mutation in AE-1 results to:


▫ Hereditary spherocytosis
▫ Congenital acanthocytosis
▫ Southeast Asian ovalocytosis
Diego antigens
• Diego antigens are expressed on RBCs of newborns.

• Dia and Dib antithetical pair


▫ Dia very low frequency except in some South Americans
and Asians
▫ Dib very high frequency in all populations

• Wra and Wrb antithetical pair


▫ Wra very low frequency
▫ Wrb very high frequency
Diego antibodies
• IgM or IgG, reactive in the IAT

• Both anti-Dia and anti-Dib have caused HTRs and HDFN

• Anti-Wra
• Some are directly agglutinating, but most require IAT.
• Has caused severe HTRs

• Autoanti-Wrb
• Common in the serum of patients with WAIHA.
The Yt (011) System
• Cartwright, “why T” became “Yt”

• Yt antigens are antithetical, represent an amino acid


substitution on the (GPI)-linked RBC glycoprotein
acetylcholinesterase (AChE)

• Yta is the high-prevalence antigen in all populations.

• Ytb is the low-prevalence antigen.


The Xg (012) System
• Named after the X chromosome and g for “Grand
Rapids”

• Gene carried on the short arm of X chromosome (“X-


linked”)

• Prevalence of Xga is 66% in males and 89% in females

• Anti-Xga is usually IgG, has not been implicated in


HDFN or HTRs.
The Scianna (013) System
• SC gene is located on chromosome 1, gene product is
erythroid membrane associated protein (ERMAP).

• Sc1, Sc2, and Sc3

• Sc1 and Sc3 are high-incidence antigens.

• Alloantibodies to Scianna antigens are rare and little is


known about their clinical significance.
The Dombrock (014) System
• Named after Mrs. Dombrock, found in 1965.

• Antigens are carried on a mono-ADP ribosyltransferase


4 (ART4)

• Doa, Dob, Gya, Hy, and Joa

• Gya, Hy, and Joa are high-incidence antigens

• Antigens are present on cord RBCs, but are absent from


PNH III RBCs.

• Considered to be poor immunogens.


The Colton (015) System
• Colton antigens are carried on an integral membrane protein,
aquaporin 1 (AQP1).

• Coa very high frequency (near 100%)

• Cob about 10%

• Co3, present on all RBCs except on Co(a–b–) phenotype.

• Co4, a high-prevalence antigen, identified on RBCs Co(a–b–)


phenotype.
Colton antibodies
• Antibodies are usually IgG.

• Anti-Coa has been reported to cause HTRs and HDFN.

• Anti-Cob has also caused HTRs and mild HDFN.

• Anti-Co3, which reacts with all Co(a+) and Co(b+)


RBCs, has been reported to cause severe HDFN.
The Chido/Rodgers (017) System
• Ch for Chido and Rg for Rodgers

• Found on the complement C4 and are adsorbed onto


RBCs from plasma.

• Cha and Rga are both high-incidence antigens.

• Anti-Ch and anti-Rg are usually IgG and react weakly.

• Anti-Ch and anti-Rg can be neutralized with pooled


plasma.
The Gerbich (020) System
• Named in 1960 after Mrs. Gerbich

• Six high-prevalence Gerbich antigens (Ge2, Ge3, Ge4,


GEPL, GEAT, and GETI)

• Five low prevalence antigens (Wb, Lsa, Ana, Dha, and


GEIS)

• Antigens are carried on sialoglycoprotein structures GPC


and GPD, associated with RBC membrane 4.1
Gerbich-negative phenotypes
• Ge:–2,3,4 (Yus type)

• Ge:–2,–3,4 (Gerbich type)

• Ge:–2,–3,–4 (Leach type), presents with


elliptocytosis.
The Cromer (021) System
• Antigens are carried on decay accelerating factor
(DAF, CD55)

• 16 antigens: 13 high-prevalence antigens and 3 low-


prevalence antigens.

• Antibodies in the Cromer system are usually IgG, but do


not cause HDFN.

• PNH III RBCs are deficient in DAF so they also


lack Cromer antigens.
The Indian (023) System
• First In(a+) individuals were from India

• Ina is present on RBCs of 4% of Indians, 11% of Iranians,


and 12% of Arabs.

• Located on CD44

• Rare In(a–b–) phenotype has been found in only one


individual who presented with congenital
dyserythropoietic anemia.

• Antibodies are usually IgG and reactive in the


antiglobulin test and they do not bind complement.
The John Milton Hagen (026) System
• High-prevalence antigen

• Found on semaphorin CDw108

• JMH is weakly expressed on cord RBCs and is destroyed


by treating RBCs with ficin and papain.

• Anti-JMH is usually IgG, high titer but weakly reactive.


Knops blood group system
• Kna, McCa (McCoy), Sll, and Yka (York) are high-
incidence antigens.

• Found on complement receptor 1 (CD35)

• Decreased expression in cases of SLE and CAD.

• Antibodies are usually IgG, reactive in the antiglobulin


test.
Miscellaneous Antigens
• Vel Antigen
• Extremely high frequency antigen (>99% in all
populations)

• Anti-Vel is characterized by its ability to activate


complement and cause in vitro and in vivo hemolysis.

• Most often IgG but can be IgM, and it has caused


severe, immediate HTRs.
Miscellaneous Antigens
Sda
• Antigen is found on 91% of RBC samples, and Sda substance is
found in 96% of urine samples.

• Only 4% of people are Sd(a–)

• Soluble form of Sda is Tamm-Horsfall glycoprotein found in


urine.

• Anti-Sda is usually an IgM agglutinin, reactivity is described as


small, refractile (shiny) agglutinates in a sea of free
RBCs.

• Neutralization of the refractile agglutinates by urine is a


technique used to identify anti-Sda.
Miscellaneous Antigens
Bennett-Goodspeed Antigens
• Associated with HLA:
• Bga = HLA-B7
• Bgb = HLA B17
• Bgc = HLA-A28

• Antibodies: IgG, destroyed by treating RBC antigens


with Chloroquine or Glycine EDTA
HTLA (high titer, low avidity)
• Antibodies that exhibit reactivity at high dilutions, but
the strength of agglutination is weak at any dilutions.

• Examples of these antibodies include:


▫ anti-Ch
▫ anti-Rg
▫ anti-Csa
▫ anti-Yka
▫ anti-Kna
▫ anti-McCa
▫ anti-JMH

• These antibodies usually are not clinically significant but


may mask significant antibodies.
Donor Screening Selection and Processing
Types of donation
• Allogeneic donation
– blood is taken from an individual of the same species as
the recipient

• Autologous donation / Donor-patient


-one who donates blood for his or her own use

• Apheresis
– process wherein blood is withdrawn from the donor and
separated into its components
– one or more components is retained and the remaining
constituents are returned
General Requirements for Donation
• General appearance:
▫ Observe the prospective donor for presence of
excessive anxiety, drug or alcohol influence, or
nervousness.

• Age:
▫ at least 17 years old
General Requirements for Donation
• Weight:
▫ At least 110 lbs/50 kg,
▫ Maximum volume of 525 mL can be collected
▫ Maximum of 10.5 mL of blood/kg of donor weight for
WB donation

• If donor weighs <110 lbs:

blood to be collected anticoagulant


General Requirements for Donation
Calculate adjusted blood vol. and anticoagulant:

Volume to collect = (donor’s weight in kg/50) × 450 mL

63 mL – above calculated volume = Amount of solution to be removed



General Requirements for Donation
• Temperature:
▫ 37.5°C or 99.5°F

• Pulse:
▫ Between 50 and 100 bpm
▫ Athletes will have a pulse less than 50 bpm

• Blood pressure:
▫ Less than or equal 180/100 mm Hg
General Requirements for Donation
Hemoglobin:
A. Allogeneic Donation
greater than or equal to 12.5 g/dL

B. Autologous Donation
greater than or equal to 11 g/dL
General Requirements for Donation
Hematocrit:
A. Allogeneic Donation
greater than or equal to 38%

B. Autologous Donation
greater than or equal to 33%
Requirements for allogeneic donation
(Philippine standards)
• Age:
• 16 – 65 years old
▫ (16-17 yrs old requires parent’s consent)

• Temperature:
▫ ≤ 37.5˚c or ≤ 99.5 ˚F

• Pulse rate:
▫ 60-100 bpm, <50 if athlete

• BP:
▫ Systolic 90 to 160 mm Hg
▫ Diastolic 60 to 100 mm Hg
BLOOD COLLECTION PROCESS
1. Donor Registration.

2. Educational material is distributed to the donor. If the


prospective donor shows symptoms of an infectious
disease, the donor is excluded from donation.

3. Actual donor selection/Identification.


BLOOD COLLECTION PROCESS
4. Blood collection
▫ Use aseptic technique :
 PVP iodine compound or chlorhexidine gluconate and
isopropyl alchohol.
▫ Scrub site at least 4cm in all directions for 20 seconds.
▫ Apply tourniquet or blood pressure cuffs: 40 – 60 mm hg
▫ 16g needle is often used for blood donation
▫ Position the needle at 10-20 deg angle
▫ Instruct donor to open and close hand every 10-20 sec
Blood collection cont.
▫ Mix the unit periodicaly 1-2 times per minute
▫ Amount of blood collected: 450mL ± 10% (405 to 495mL)
▫ Duration of collection: 7 – 1o mins
▫ If >8 mins the unit is not suitable for platelet, FFP and
cryoprecipitate.
▫ Component must be prepared 6-8 hrs after collection
Color Coding of Blood Bags
FDA (1985) RA 1517
Blood Type Color Label Blood type Color Label

A YELLOW A BLUE

B PINK B YELLOW

AB WHITE AB PINK

O BLUE O WHITE
Donor deferral
Types of deferral

1. Temporary Deferral:
▫ Prospective donor is unable to donate blood for a
limited period of time.

▫ EXAMPLE:
Donor has received a blood transfusion; defer for 12
months from date of transfusion.
Donor deferral
2. Indefinite Deferral:
▫ Prospective donor is unable to donate blood for
someone else for an unspecified period of time due to
current regulatory requirements. These donors may be
eligible to donate autologous blood.

▫ EXAMPLE:
Donor states they have lived in England for 1 year in
1989; defer indefinitely.
Donor deferral
3. Permanent Deferral:
▫ Prospective donor will never be eligible to donate
blood for someone else. These donors may be eligible
to donate autologous blood.

▫ EXAMPLE:
Donor states that he or she has hepatitis C; defer
permanently.
Donor deferral
• PERMANENT DEFERRAL:
▫ Confirmed positive test for HBsAg
▫ Viral hepatitis after 11th birthday
▫ Repeat reactive test for anti-HBc on more than one
occasion
▫ Previous donation associated with hepatitis, HIV or
HTLV transmission
▫ Confirmed positive for HCV, HIV or HTLV
Donor deferral
• PERMANENT DEFERRAL:
▫ Behavioral risk factors for HIV infection
▫ Male-to-male sexual contact even once, since 1977
▫ Men or women who engage in sex for money or drugs
since 1977 are permanently deferred
▫ IV drug users/ Parenteral drug use
▫ Growth Hormone from Human Pituitary Glands
▫ History of Babesiosis or Chaga’s disease
▫ Intake of drug Tegison or etretinate
▫ Hematologic malignancies
Donor deferral
• 1 year deferral:
▫ Blood transfusion
▫ Transplant such as organ, tissue, or bone marrow; or a
graft such as bone or skin
▫ Mucous membrane exposure to blood, non sterile skin
or needle penetration
▫ Sexual contact with anyone who has HIV/AIDS or has
had a positive test for HIV/AIDS
▫ Sexual contact with a prostitute or anyone else who
takes money or drugs or other payment for sex
Donor deferral
• 1 year deferral:
▫ Rape victim
▫ Incarceration in a correctional institution for longer
than 72 hours
▫ History of syphilis or malaria
▫ Sexual contact or living with a person (“close contact”)
who has acute or chronic hepatitis B
▫ History of syphilis or gonorrhea
▫ Travelers to areas the CDC considers to be endemic for
malaria
▫ Hepatitis B Immune Globulin (HBIG)
Donor deferral
• 2-week deferral:
▫ Measles (rubeola)
▫ Mumps
▫ Oral polio
▫ Typhoid
▫ Yellow fever

• 4-week deferral:
▫ German measles (rubella) or chickenpox
▫ Proscar, Propecia and Accutane
Donor deferral
• 12-24 hours:
▫ After recent alcohol intake
• 3 days:
▫ Piroxicam, aspirin (platelet pheresis)
• 6 weeks:
▫ Pregnancy
• 6 months:
▫ Avodart
• 3 years:
▫ Malaria confirmed diagnosis
Autologous donation
“The safest blood that you can receive is your own”
General requirements:
1. Age: no age requirement
2. Hgb :11g/dl, Hct :33%
3. General condition:
 Patient should have no condition predisposing to
bacteremia or any form of severe
cardiovascular/pulmonary condition.
4. Single unit is removed at a time, with at least 3 day
intervals, and the final phlebotomy should be at least
72 hours before the surgery.
Types of Autologous Donation
1. Preoperative Collection (Predeposit)
▫ Occurs during the 5 to 6 weeks immediately preceding a
scheduled, elective surgical procedure.
Types of Autologous Donation
2. Acute Normovolemic Hemodilution
▫ Collection of whole blood with the concurrent infusion of
crystalloid or colloid solutions.
▫ 3:1 for crystalloids and 1:1 for colloids
Types of Autologous Donation
3. Intraoperative collection
▫ Involves collecting shed blood from the surgical site, a
device that utilizers vacuum is used to collect shed blood.
▫ Blood is the washed with saline and then concentrated to
reach hematocrit of 50 – 60%, then reinfusing those cells
immediately.
Types of Autologous Donation
4. Postoperative Collection
▫ Collected from a drainage tube placed at the surgical site.
▫ It is reinfused, with or without processing, via a
microaggregate filter to screen out any debris.
Directed Donation
A directed donation is a unit collected under the
same requirements as those for allogeneic donors,
except that the unit collected is directed toward a
specific patient.
Apheresis Donation
• From the ancient Greek aphairesis, “a taking
away”)
• An effective mechanism for collecting a specific
blood component while returning the remaining
whole blood components back to the patient.
Apheresis Donation
Methods of Centrifugation:
1. Intermittent Flow Centrifugation
▫ Blood is processed in batches or cycles
▫ Requires only one venipuncture
▫ Blood is drawn and reinfused through the same needle
▫ Once the desired component is separated, the
remaining components are reinfused to the donor.
Apheresis Donation
2. Continuous flow centrifugation (CFC)
▫ Two venipuncture sites are necessary
▫ Involves withdrawal and processing and reinfusing of
blood to the individual simultaneously
Apheresis Donation
• Amount of time for a particular procedure can range
from 45 to 120 minutes.

• Citrate (ACD) is used as the primary anticoagulant in


apheresis procedures.

• Heparin can also be used as anticoagulant


Principles of Apheresis
Apheresis Donation
Types of Apheresis:
1. Plateletpheresis (Thrombocytoapheresis)
2. RBC pheresis (Erythrocytapheresis)
3. WBC pheresis (Leukapheresis)
4. Plasmapheresis
5. Stem cell pheresis
Plateletpheresis
• Procedure typically takes 45 to 90 minutes

• Donor should have at least 150,000/μL plt count

• Donor should have not taken aspirin 3 days before donation

• Interval of at least 2 days (Not exceed twice a week or 24


times a year)

• SDP is equivalent to 6-8 RDP

• QC: ≥ 3 × 10¹¹plt, pH ≥ 6.2


RBC pheresis (Erythrocytapheresis)
• 2RBC or double RBC procedure
• Every 16 weeks
Requirements for erythrocytapheresis
Sex Height Weight Hematocrit
Male 5’1’’ 130lbs 40%
Female 5’5’’ 150lbs 40%
WBC pheresis (Leukapheresis)
• Given to patients who are severely neutropenic and
unresponsive to antibiotics.
▫ RBC sedimenting agent: Hydroxyethyl Starch(HES)
▫ Corticosteroids (Prednisone/Dexamethasone)
▫ G-CSF

• ≥1.0 x 10¹⁰ PMS/unit


• Storage: 24 hours at 20 – 24 ̊c
WBC pheresis (Leukapheresis)
• Apheresis granulocytes contain a large number of viable
lymphocytes.

• Product should be irradiated prior to administration to a


severely immunocompromised patient.

• Compatibility testing is typically performed, since the


apheresis granulocyte product contains greater than 2
mL of RBCs.
Plasmapheresis
• Equivalent of at least two whole-blood-derived plasma
units.
▫ Infrequent plasmapheresis, donation occurs no
more than once every 4 weeks.
▫ Frequent or serial, donation occurs more frequently
than once every 4 weeks.
• 2 days between procedures
• Maximum of 2 donations in a 7 day period
• FDA recommend 12 L (14.4 L for donors weighing more
than 175 pounds) as the maximum allowable plasma
volume donated per year.
Therapeutic Procedures
• The rationale of therapeutic apheresis (TA) is
based on the following:
▫ A pathologenic substance exists in the blood that
contributes to a disease process or its symptoms.

▫ The substance can be more effectively removed by


apheresis than by the body’s own homeostatic
mechanisms.
Plasmapheresis (Plasma Exchange)
• Therapeutic plasma exchange (TPE) is the removal
and retention of the plasma, with return of all cellular
components to the patient.

• The purpose is to remove the agent in the plasma, such


as an antibody, toxin, or abnormal protein, that is
causing the clinical symptoms.

• TPE is also used to replace a normal factor or substance


that may be missing or deficient in the patient’s plasma.
Factors Removed by Therapeutic Plasmapheresis
Immune complexes  Systemic lupus erythematosus
Alloantibodies  Antibody-mediated transplant rejection

Autoantibodies  Guillain-Barré syndrome


Immunoglobulins causing  Waldenström’s macroglobulinemia
hyperviscosity

Protein-bound toxins or  Amanita mushroom poisoning,


drugs barbiturate poisoning

Lipoproteins  Familial hypercholesterolemia,


hypertriglyceridemia

Phytanic acid  Refsum’s disease


Therapeutic plateletpheresis
• Can be used to treat patients who have abnormally
elevated platelet counts with related symptoms.

• Thrombocytosis (at least 500,000/μL) can occur in


myeloproliferative disorders (ET, PV, CML) or as a
reactive process in response to splenectomy, infection,
chronic inflammation, or malignancy.

• If platelet count reach levels > 1,000,000/μL, the


patient is at risk for developing thrombotic or
hemorrhagic complications.
Therapeutic leukapheresis
• Used to treat patients with hyperleukocytosis,
defined as a WBC or circulating blast count of
over 100,000/μL.

• Elevated levels place the patient at risk for


complications associated with leukostasis,
including organ dysfunction due to the
formation of microthrombi in the pulmonary
and cerebral microvasculature.
Erythrocytapheresis (Red Cell Exchange)

• Removes a large number of RBCs from the patient and


returns the patient’s plasma and platelets along with
compatible allogeneic donor RBCs.

• Most commonly performed in patients with sickle cell


disease in order to decrease the number of hemoglobin
S–containing RBCs.

• Donor RBCs should be ABO- and Rh-compatible,


relatively fresh (leukoreduced, negative for hemoglobin S
and partially phenotype-matched for the Rh (C, c, E, e)
and K1 antigens.
Adverse Effects of Apheresis
• Citrate toxicity • Depletion of clotting factors
• Vascular access • Circulatory and respiratory
complications (hematoma, distress
sepsis, phlebitis, • Transfusion-transmitted
neuropathy) diseases
• Vasovagal reactions • Lymphocyte loss
• Hypovolemia • Depletion of proteins and
• Allergic reactions immunoglobulins
• Hemolysis • Air embolus
Donor Reactions
Mild Reactions:
Syncope or fainting

Management:
1. Remove the tourniquet and withdraw needle
2. Place cold compresses on the donor’s forehead
3. Raise the donor’s legs above the level of the head
4. Loosen tight clothing and secure airway
5. Monitor vital signs
Donor Reactions
Mild Reactions:
Twitching or muscle spasms

Management:
Disengage the hyperventilation sequence by conversing
with the donor and having the donor breathe into a paper
bag.
Donor Reactions
Mild Reactions:
Nausea or vomiting

Management:
1. Instruct the donor to breathe slowly
2. Apply cold compresses to the forehead
3. Turn the donor’s head to one side and provide an
appropriate receptacle
4. The donor may be given water after vomiting has ceased
Donor Reactions
Moderate reactions:
Loss of consciousness. The donor may have a decreased
pulse rate, may hyperventilate, and may exhibit a fall in
systolic pressure to 60 mm Hg.

Management:
1. Check vital signs frequently
2. Administer 95% oxygen and 5% carbon dioxide
Donor Reactions
Severe reactions:
Convulsions can be caused by cerebral ischemia, marked
hyperventilation, or epilepsy.

Management:
1. Call for help immediately; notify blood bank physician
2. Try and restrain the donor to prevent injury to self or
others
3. Ensure an adequate airway
Donor Reactions
Severe reactions:
Cardiac or respiratory difficulties

Management:
Perform CPR until medical help arrives
Donor Reactions
Hematomas caused by the needle going through the
vein, with subsequent leakage of blood into the tissue.

Management:
1. Remove the tourniquet and needle from donor’s arm
2. Apply pressure with sterile gauze pads for 7 to 10
minutes, with the donor raising his or her arm above the
heart
3. Apply ice to the area for 5 minutes
Blood preservation and Banking
Blood preservation and Banking
RBC Biology and Preservation:
Three areas of RBC biology are crucial for normal
erythrocyte survival and function:
1. Normal chemical composition and structure of the
RBC membrane
2. Haemoglobin structure and function
3. RBC metabolism
The RBC’s metabolic pathways that produce ATP are
mainly anaerobic.

▫ Deoxyhemoglobin molecule is known as the tense


(T) form, which has a lower affinity for oxygen.

▫ Oxyhemoglobin is the relaxed (R) form of the


hemoglobin molecule, which has a higher affinity for
oxygen.
Hemoglobin Oxygen Dissociation Curve
RBC Preservation
• RBC viability is a measure of in vivo RBC survival
following transfusion.

• FDA requires an average 24-hour post-transfusion RBC


survival of more than 75%.

• To maintain optimum viability, blood is stored in the


liquid state between 1°C and 6°C.
RBC Storage Lesions
Decreased Increased

• % Viable Cells • Lactic acid


• Glucose • Plasma potassium
• ATP • Plasma hemoglobin
• pH
• 2,3-DPG, shift to the left
Approved Anticoagulant Preservative Solutions
Name of Preservative ABBREVIATION STORAGE TIME
(DAYS)

Acid-Citrate-Dextrose (formula A) ACD-A 21

Citrate-Phosphate-Dextrose CPD 21

Citrate-Phosphate-Double-Dextrose CD2D 21

Citrate-Phosphate Dextrose-Adenine CPDA-1 35


Chemicals in Anticoagulant Solutions
Chemical Function
Citrate Chelates calcium; prevents clotting
(sodium citrate/citric acid)
Monobasic sodium Maintains pH during storage; necessary
phosphate for maintenance of adequate levels of
2,3-DPG
Dextrose (Glucose) Substrate for ATP production (cellular
energy)
Adenine Increases ADP levels, driving glycolysis
toward synthesis of ATP
Additive Solutions
• Preserving solutions that are added to the RBCs after
removal of the plasma with or without platelets.

• Additives contain:
▫ Saline
▫ Adenine
▫ Glucose
▫ Mannitol (protects against storage-related hemolysis).
Additive Solutions
Name Abbreviation Storage Time (days)

Adsol AS-1 42
Nutricel AS-3 42
Optisol AS-5 42
Freezing and Rejuvenation
• RBC Freezing is primarily used for autologous units
and the storage of rare blood types.

• Involves the addition cryoprotective (Glycerol) agent to


RBCs that are less than 6 days old.

• Two concentrations of glycerol have been used to freeze


RBCs:
▫ High-concentration glycerol (40% w/v)
▫ Low-concentration glycerol (20% w/v)
RBC Rejuvenation

• ATP and 2,3-DPG levels are restored.


• RBCs stored in the liquid state can be rejuvenated at
outdate or up to 3 days after outdate by incubating
the RBC unit with 50 mL of the rejuvenating solution for
1 hour at 37°C.

• Rejuvesol contains:
 Phosphate
 Inosine
 Pyruvate
 Adenine
Blood components
Terms to know:
• Whole blood:
 Blood collected before separation into components
• Components:
 Parts of whole blood that are separated
• Closed system:
 A sterile system of blood collection
• Open system:
 When the collection is exposed to air, decreasing
expiration date
WHOLE
BLOOD
Heavy spin Light spin
5000 x g for 5 3200 x g for 2-3
mins. mins.

Packed Platelet Rich


RBC Plasma
Heavy spin
5000 x g for 5
mins.
Platelet

Plasma

Cryoprecipitate

Cryoprecipitate
Whole Blood
Indications Volume expansion, ↑ O2
for use
Storage 1- 6˚c
Temperature
Shelf life • ACD, CPD or CP2D = 21 days
• CPDA1= 35 days
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 450–500 mL
Quality Hct approx. 40%
control
Packed RBCs
Indications for Symptomatic anemia with normal blood
use volume, ↑ O2
Storage 1- 6˚c
Temperature
Shelf life • Open system= 24 hours
• ACD, CPD or CP2D = 21 days
• CPDA1= 35 days
• AS=42 days
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 250-300 ml
Quality control Hct. ≤80%
Washed RBCs
Indications Symptomatic anemia on patients with
for use severe allergic or anaphylactic conditions
(IgA-negative persons)
Storage 1- 6˚c
Temperature
Shelf life 24 hours
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 180 mL
Quality Hct 70–80%
control
RBC leukoreduced
Indications Prevents febrile transfusion reaction and
for use TRALI caused by Abs againts WBC,
Prevent CMV
Storage 1- 6˚c
Temperature
Shelf life Closed system: Same
Open system: 24 hours
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 250–300 mL
Quality • 5 × 10⁶WBCs
control • ≥ 85% RBC recovery
RBC irradiated
Indications Prevent GVHD, ↑ O2
for use
Storage 1- 6˚c
Temperature
Shelf life Original outdate or 28 days from
irradiation
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 250-300ml
Quality 25 Gy to center of canister
control
Irradiated blood components
• Irradiation inhibits the proliferation of T cells and subsequent
transfusion-associated graft-versus-host disease.

At risk for GVHD:


▫ Immunocompromised
▫ Receiving a bone marrow or stem cell transplant,
▫ Fetuses undergoing an intrauterine transfusion,
▫ Recipients of blood from relatives

• FDA and AABB recommend a minimum dose of gamma


irradiation of 25 Gy to the central portion of the blood unit,
with no less than 15 Gy delivered to any part of the blood unit.
Irradiated blood components
• RBCs, platelets, and granulocyte concentrates
contain viable T lymphocytes.

• Irradiation is generally performed using cesium-


137 or cobalt-60.

• The expiration date of irradiated RBCs is 28


days from the time of irradiation or the original
outdate, whichever is sooner.
Frozen RBCs
Indications Storage of blood with rare phenotypes
for use and autologous units
Storage -65˚c or -120˚c
Temperature
Shelf life 10 years
RBC deglycerolized
Indications Rare phenotypes
for use ↑ O2
Storage 1- 6˚c
Temperature
Shelf life 24 hr
Dosage ↑ hgb 1g/dL
↑ hct 3%
Volume 180 mL
Quality 80% RBC recovery
control < 1% glycerol
< 300 mg hgb
Random Donor Platetet
Indications • Thrombocytopenia
for use • DIC
• Cancer patients undergoing chemotherapy
Storage 20–24°C
Temperature
Shelf life 5 days
Dosage ↑5k–10k/μL
Volume 50–70 mL
Quality ≥ 5.5 × 10¹⁰ plts
control pH ≥ 6.2
Random Donor Platelet
WHOLE
BLOOD
Light spin
3200 x g for 2-3
mins.

Platelet Rich
Heavy spin
Plasma
3600 x g for 5
mins.
Allow the platelets to rest
undisturbed for 1 to 2 hours
at 20°C to24°C
Platelet
Concentrate

• 40 to 70 mL plasma
• Stored at 20°C to 24°C with
continuous agitation
Single Donor Platetet
Indications Platelet Refractoriness, Prevention of
for use HLA alloimmunization
Storage 20–24°C
Temperature
Shelf life 5 days
Dosage ↑30k–60k/μL
Volume 200–400 mL
Quality ≥ 3 × 10¹¹
control pH ≥ 6.2
Refractory Patients
• Massive splenomegaly
• High fever
• Sepsis
• DIC
• Platelet or HLA antibodies

• Corrected count increment using a 10-minute to 1-hour


post-transfusion platelet count can provide valuable
information about patient response to a platelet
component.
Corrected count increment

Corrected count increment
Fresh Frozen Plasma
Indications Multiple coagulation factor deficiency,
for use Liver disease, DIC
Storage –18°C (1 year)
Temperature –65°C (7 Years)
If thawed, store at 1-6°C for 24 hours
Shelf life 1 year or 7 years
Dosage ↑Factor 20–30%
10–20 mL/kg
Volume 200–250 mL
Quality 8 hr CPD, CPDA-1, CP2D
control 6 hr ACD
Cryoprecipitate
Indications • Hypofibrinogenemia
for use • Factor XIII def.
• Von willebrand’s disease
• Hemophilia A
Storage Frozen: –18°C (1 year)
Temperature Thawed: 20-24°C (6hours)
and Shelf life Pooled: 20-24°C (4 hours)
Dosage ↑Fibrinogen 5–10 mg/dL
Volume 10–25 mL
Quality FVIII:C 80 IU
control Fibrinogen of at least 150mg per unit
Procedure for Production of Cryoprecipitate
1. The plasma must be frozen within 8 hours of collection.
2. Thaw FFP slowly in the refrigerator at 1°C to 6°C.
▫ 14 to 16 hrs (standard blood bank refrigerator
▫ 4 hours thaw bath (4°C water bath)
▫ Endpoint is when the plasma becomes slushy
3. Centrifuge the plasma at 4°C for a “hard” spin.
4. Leave 10 -15ml plasma.
5. Separate and refreeze the cryoprecipitate immediately.
6. Store at –18°C or colder up to 12 months.

If the supernatant plasma is refrozen at –18°C, it must be


labeled as plasma cryoprecipitate reduced.
Calculation of Cryoprecipitate dose

Calculation of Cryoprecipitate dose

Granulocyte Concentrate
Indications • Neutropenia < 500 PMN/uL with
for use infection unresponsive to antibiotics
• Prevent GVHD (Irradiated)
Storage 20–24°C
Temperature
Shelf life 24 hours
Dosage 1–2 × 10¹⁰ / infusion four daily dose
Volume 200–600 mL
Quality ≥ 1.0 × 10¹⁰
control
Factor VIII Concentrate
Indications Prevent bleeding in patients with
for use Hemophilia A
Storage 1- 6 °C
Temperature
Shelf life Varies, check vial

Dosage 1U FVIII/kg body wt ↑ 2%


Volume 10–30 mL
Calculation of factor VIII dose
Formula:
(Desired level of Factor 8 – Initial level of Factor 8) x plasma volume (mL)
=units of factor VIII required
Example:
A 70-kg hemophiliac patient with a hematocrit level of 30% has an initial
factor VIII level of 4% (4 units/dL, 0.04 units/mL). How many units of factor
VIII concentrate should be given to raise his factor VIII level to 50%?

1. Blood volume = weight (kg) × 70 mL/kg


70 kg × 70 mL/kg = 4,900 mL
2. Plasma volume = blood volume (mL) × (1.0 – Hct)
4,900 mL × (1.0 – 0.30) = 3,430 mL
3. Solution: 3,430 mL × (0.50 – 0.04) = 1,578 units
Factor IX Concentrate
Indications Prevent bleeding in patients with
for use Hemophilia B
Storage 1- 6 °C
Temperature
Shelf life Varies, check vial
Dosage 1U FIX/kg body wt ↑ 1.5%
Volume 20–30 mL
Immune Serum Globulins
Indications Patients with Hypogammaglobulinemia
for use (contains gamma globulins IgG, IgM,
IgA)
Storage 3 yr IM
Temperature 1 yr IV
Shelf life 3 yr IM
1 yr IV
Normal Serum Albumin
Indications Plasma volume expansion, for patients
for use with haemorrhagic shock, severe burns
Storage 2–10°C
Temperature
Shelf life 5 yr, 25%
Content 96% albumin 4% globulin
Plasma Protein Fraction
Indications Plasma volume expansion, for patients
for use with haemorrhagic shock, severe burns
Storage 2–10°C
Temperature
Shelf life 5 yrs
Content Albumin 80–85% , Globulin 15–20%
Synthetic Volume Expanders
• Crystalloids
▫ Ringer’s lactate (sodium, chloride, potassium, calcium, and lactate ions)
▫ Normal isotonic saline (sodium and chloride)
• Colloids
▫ Dextran
▫ HES

• Useful in burn patients


Comparison of Crystalloid and Colloid solutions
Blood Administration
Blood Administration
1. Positive identification of the patient, patient’s blood specimen, and
blood unit for transfusion.
▫ Specimen: Collected within 3 days of scheduled transfusion
▫ Sealed sample of donor’s blood must be stored at 1-6°C for 7 days
after transfusion

2. Venous access should be established before the blood is issued.


(Using 18 gauge needle)

3. All blood components must be filtered (170-μm filter)

4. Blood components are infused slowly for the first 10 to 15 minutes.


(2ml/min)
Blood Administration
5. Patient’s vital signs (pulse, respiration, blood pressure, and
temperature) should be monitored periodically during the
transfusion.

6. Blood warmers should be set at 37°C, not more than


42°C.

7. Only isotonic (0.9%) saline or 5% albumin should be


used to dilute blood components.

8. The blood components should then be infused as quickly as


tolerated or, at most, within 4 hours.
Reissue of unit
1. Closure must not have been entered in any way.
2. Blood must have been kept between 1-10°C on a
continuous basis.
3. Pilot tube or sealed segment of the donor tube must
still be attached to the container.
4. The blood should be not away from the blood bank for
more than 30 minutes.
5. Records must be available that verify all inspection
criteria.
Transfusion Reactions
Transfusion Reactions
A transfusion reaction is defined as any transfusion-
related adverse event that occurs during or after the
transfusion of whole blood, blood components, or human-
derived plasma products.

 Acute transfusion reaction

 Delayed transfusion
Transfusion Reactions
• Acute/ Immediate transfusion reaction
 Transfusion reaction with signs or symptoms presenting
during or within 24 hours of transfusion.

• Delayed transfusion reaction


 Transfusion reaction with signs or symptoms
presenting after 24 hours of transfusion.
Transfusion Reactions
Immune hemolysis occurs when previously formed IgM
(ABO) or IgG (non-ABO) antibodies in the recipient
recognize the corresponding donor RBC antigen and result
in complement-mediated intravascular hemolysis.

Non-immune hemolysis occurs when the RBC suffers


mechanical or chemical damage and is manifested as an
asymptomatic hemoglobinuria.
Acute hemolytic transfusion reactions
Definition Acute hemolysis due to transfusion of incompatible
blood with accompanying presenting symptoms
within 24 hours of transfusion.
Cause Improper patient identification at the time of sample
collection or transfusion is the most common cause.
Symptoms Severe, rapid onset, fever, chills, flushing, pain at
site of infusion, tachycardia, hemoglobinemia,
hemoglobinuria, hypotension.
Prevention Proper ID of the patient and blood components
Treatment Discontinue transfusion
Maintain vascular access
Maintain blood pressure
Maintain renal blood flow
Treat DIC if present
Transfusion-Associated Sepsis
Definition Acute nonimmune transfusion reaction presenting
with body temperatures usually 2°C or more above
normal and rigors that can be accompanied by
hypotension.
Cause Occurs when bacteria (often caused by Yersinia
enterocolitica) are introduced to the patient via a
contaminated blood product.
Symptoms • Fever/chills
• Hypotension
• Shock
Prevention Aseptic techniques, Check for the presence of clots,
brown or purple discoloration or hemolysis in blood
components
Treatment Administer broad spectrum antibiotics intravenously
Febrile Nonhemolytic Transfusion Reaction
Definition An acute complication of transfusion presenting with
at least a 1°C increase in body temperature
Most commonly encountered type of
transfusion reaction
Cause • Immune mediated and is due to the presence of
preformed antibodies, release of endogenous
pyrogens.
• Due to platelet storage changes, production and
release of biologically active cytokines.
Symptoms Fever, chills, nausea or vomiting, tachycardia,
increase in blood pressure, and tachypnea.
Prevention Prestorage leukocyte reduction
Treatment Treat with antipyretics
For rigors, treat with meperidine
Allergic Mild (Urticarial Transfusion Reaction)
Definition Occurs as a response of recipient antibodies to an
allergen present in the blood component.
Cause Activation of mast cells in the recipient triggered
most frequently by an allergen present in the plasma
of the blood component.
Symptoms Weals, hives, erythema, or pruritus
Prevention For repeated reactions, consider premedication with
antihistamines, transfuse washed components.
Treatment Treat with antihistamines
Allergic Severe (anaphylactoid or anaphylactic)
Definition IgA-deficiency-related anaphylactic reaction.
Cause Caused by transfusion of IgA postive blood to an IgA-
deficient recipient who have developed anti-IgA.
Symptoms Bronchoconstriction, angioedema, diarrhea and
cardiovascular instability (hypotension, cardiac
arrhythmia, loss of consciousness, shock, cardiac
arrest).
Prevention For IgA absolute deficient patients provide IgA
deficient blood components.
Treatment Treat with subcutaneous epinephrine
Transfusion-Related Acute Lung Injury
Definition Acute transfusion reaction presenting with
respiratory distress and severe hypoxemia during or
within 6 hours of transfusion.
Leading cause of transfusion-associated
fatalities
Cause Caused by antibodies against leukocytes present in
donor plasma (antibody against human neutrophil
antigens)
Symptoms Severe hypoxemia
Prevention • Use male only plasma, exclude or screen female
platelet donors.
• Transfuse leuko-reduced components
Treatment Supplemental oxygen
Mechanical ventilation
Transfusion-Associated Circulatory Overload
Definition Iatrogenic, transfusion-induced hypervolemia
At risk:
• Patients with pre-existing CHF, elderly patients,
children, patients with renal failure.
Cause Occurs when the patient’s cardiovascular system’s
ability to handle additional workload is exceeded,
manifesting as congestive heart failure.
Symptoms Severe hypoxemia , ↑ Blood pressure
Jugular vein distension, ↑ Central venous pressure
Prevention • Slower transfusion rate
• Transfuse in smaller volumes
Treatment Give supplemental oxygen and diuretics.
In severe cases, therapeutic phlebotomy may be
indicated.
Delayed Serologic/Hemolytic Transfusion Reaction
Definition Detection of “new” red cell antibodies after 24 hours of
transfusion.
Occurs secondarily to an amnestic response but can also
occur during a primary immune response and may or may
not be associated with shortened survival of the transfused
cell.
Cause Transfusion of incompatible blood during emergency or
massive transfusion may occasionally be the cause of a
delayed hemolytic reaction.
Symptoms Flulike symptoms, Pallor, Jaundice.
↓ Hemoglobin ↑ Total bilirubin
Prevention Accurate record-keeping, obtain transfusion history and
limit transfusions.
Treatment Transfuse antigen negative, AHG crossmatched compatible
PRBC.
Transfusion-Associated Graft-Versus-Host Disease
Definition Delayed immune transfusion reaction due to an
immunologic attack by viable donor lymphocytes contained
in the transfused blood component against the transfusion
recipient.
Cause • HLA antigen difference between donor and recipient.
• Presence of donor immunocompetent cells in the
blood component.
• Recipient incapable of rejecting the donor
immunocompetent cells.
At risk: Infants ,patients with cancer or compromised immune
systems.
Symptoms Rash, fever and diarrhea
Prevention Gamma irradiation of cellular blood components as
indicated
Transfusion-associated graft-versus-host disease (TA-GVHD) in an
immunocompetent patient.
Post-Transfusion Purpura
Definition Delayed immune complication of transfusion that
presents with profound thrombocytopenia, frequently
accompanied by bleeding, 1 to 24 days after a blood
transfusion.
Cause Occurs when a patient who is previously sensitized to
human platelet antigens by pregnancy or transfusion
is reexposed via a transfusion.
Most commonly implicated is the human platelet
antigen (HPA)1a.
Symptoms Bleeding
Prevention Limit transfusions
Treatment Intravenous immunoglobulin
Iron Overload (Transfusion Hemosiderosis)
Definition Delayed, nonimmune complication of transfusion,
presenting with multiorgan damage secondary to
excessive iron accumulation.
At risk: Patients with aplastic anemia, congenital hemolytic
anemia, thalassemia.
Symptoms Multiorgan failure
Prevention • Preventing the accumulation of iron stores by
chelation
• Transfusion of neocytes
• Red cell exchange.
Treatment Use of iron-chelating agents (parenteral
deferoxamine, oral deferiprone, and oral deferasirox)
Physical or Chemical Induced
Transfusion Reaction
• Intravascular lysis caused by hypertonic or hypotonic
solutions

• Mechanical damage caused by infusion through small


bore needles

• Heat damage due to blood warmers

• Citrate toxicity
Transfusion Transmitted Diseases
Disease Transmission Prevention—Required Tests
Diseases REQUIRED TESTS
Hepatitis B HBsAg, anti-HBc
Hepatitis C Anti-HCV, HCV RNA
HIV Anti-HIV-1/2 HIV-1 RNA
HTLV Anti-HTLV-I/II
Syphilis STS
West Nile Virus WNV RNA
Other Viruses
• Cytomegalovirus
 Most frequently transmitted virus from mother to
fetus.
 ELISA, FIA, indirect hemagglutination.

• Epstein-Barr Virus
• Kissing disease
• Infectious mononucleosis
Other Viruses
• Parvovirus B19
 “Fifth disease”
 Enters the RBC via P antigen & replicates in the
erythroid progenitor cells
 PCR
• HHV-6
• Roseola infantum or sixth disease.
• blood components are not being tested for HHV-6
• HHV-8
• Kaposi’s sarcoma (KS), primary effusion lymphoma
• No evidence to support a TTD association
Transfusion-Associated Parasites
• At least three parasites have been associated
with transfusion associated infections:
▫ Babesia microti
▫ Trypanosoma cruzi
▫ Malaria (Plasmodium species)
Bacterial Contamination
• Incidence of transfusion-associated bacterial sepsis is
low, the morbidity and mortality rates are high.
• Common sources of bacterial contamination include
donor skin and blood.
• Platelets have been the most frequent source of septic
transfusion reactions.
• According to the CDC, Yersinia enterocolitica is the
most common isolate found in RBC units, followed by
the Pseudomonas species.
Bacterial Contamination
• Propionibacterium acnes, a common isolate of human
skin, was the most common bacterial contaminant in
RBCs.

• Staphylococcus epidermidis and Bacillus cereus (both


gram-positive) are the organisms most frequently
recovered from donated blood and contamination of
platelets.
Prion Disease
• Creutzfeldt-Jakob Disease
▫ One of the transmissible spongiform encephalopathies
▫ Sheep, goats, cattle, cats, minks, deer, and elk, and
humans can be affected by TSE.
▫ Iatrogenic CJD acquired through contaminated
neurosurgical equipment, cornea or dura mater
transplants, or human-derived pituitary growth
hormones.
▫ Causative agent of all TSEs is believed to be a “prion,”
which is described as a self-replicating protein.
Blood Banking Techniques
and procedures
Techniques and procedures
1. Typing
2. Compatibility testing
3. Antibody detection
4. Antibody Identification
Basic Red Cell- Antibody Interactions
Agglutination
• Basic reaction in blood banking
• Two phases/stages:
1. Sensitization/coating
2. Lattice formation
Sensitization

Lattice formation
Basic Red Cell- Antibody Interactions
Hemolysis
• Direct lysis of RBCs due to antibody coating and
complement activation.
• Uncommon, but equal to agglutination.
• Usually due to IgM antibodies.
Factors affecting antigen-antibody reaction

• Temperature
▫ Cold-reactive RT or colder (IgM)
▫ Warm-reactive 37 deg C (IgG)
▫ Must react in appropriate temperature for best
antibody detection.

• pH
▫ Optimal pH 6.5-7.5 or 7
▫ Antibodies enhanced by acidic pH (6.5):
 Anti-D, Anti-M, anti-Pr
Factors affecting antigen-antibody reaction

• Ratio of serum to cells:


▫ 40:1
▫ (2 drops of serum and 1 drop of a 5% RBC solution)

▫ 133:1
▫ (4 drops of serum with 1 drop of a 3% RBC solution)
Factors affecting antigen-antibody reaction
Factors affecting antigen-antibody reaction

• Incubation time
• Incubation times may vary between 30 and 120
minutes.

• If a LISS or PEG technique is being used incubation


times may be shortened to 10 to 15 minutes.
Factors affecting antigen-antibody reaction

• Centrifugation for Reading


• 1000 RCFs for 20 seconds

• The optimum centrifugation conditions should be


determined for each centrifuge.
Tube Method
Enhancement Reagents
Low Ionic Strength Solution
▫ Contains glycine in an albumin solution.
▫ Increases the uptake of antibody onto the RBC during
the sensitization phase.
Enhancement Reagents
22% albumin
Works by reducing the zeta potential and dispersing the
charges.
Enhancement Reagents
Polyethylene Glycol
▫ Removes water from the test system, thereby
concentrating any antibodies present.
▫ Can cause nonspecific aggregation of cells.
▫ More sensitive than LISS, albumin, or saline systems.
▫ Not recommended in patients with elevated levels of
plasma protein.
Potentiator Incubation Time
None (NSS) 30-60 minutes
LISS 10-15 minutes
Albumin 15 - 30 minutes
PEG 15 minutes
Alternatives to Tube Testing
• Column agglutination technology (Gel testing)
▫ Developed by Dr. Yves Lapierre of Lyon, France.
▫ Using gelatin, acrylamide gel, and glass beads.

• Solid-phase Red Cell Adherence Testing


▫ In 1984, Plapp and coworkers reported using solid-phase
red cell adherence (SPRCA) for detecting RBC antigens and
antibodies.
Gel Testing

Anti-IgG
Gel Testing

Anti-IgG
Gel Testing

Serum

Anti-IgG
Gel Testing

Anti-IgG

NEGATIVE
Gel Testing
Gel Testing
Solid Phase Adherence Method

Well coated with RBC antigens.

Patient’s antibody attached to the RBC antigens


Solid Phase Adherence Method

Reaction between the


patient’s antibodies
and the indicator cells.

No patient Ab present
Solid Phase Adherence Method
Solid Phase Adherence Method
Antiglobulin Test
• Antihuman globulins (AHGs) obtained from
immunized nonhuman species bind to human globulins
such as IgG or complement, either free in serum or
attached to antigens on red blood cells.

• AHG reagent is colored GREEN.


Antihuman Globulin Reagents:
• Polyspecific AHG:
▫ Antibodies to human IgG and C3d.

• Monospecific AHG:
▫ Anti-IgG or antibody to anti–C3b-C3d.

• Classic AHG sera (polyclonal) are prepared by injecting


human globulins into rabbits, and an immune stimulus
triggers production of antibody to human serum.

• Hybridoma technology is used to produce monoclonal


antiglobulin serum.
Antiglobulin Test
Indirect antiglobulin test (IAT)
Purpose:
To detect in vitro sensitization of RBCs.
▫ Compatibility testing
▫ Antibody detection
▫ Antibody ID
▫ Antibody Titration
▫ RBC phenotype
Direct Antiglobulin Test (DAT)
Purpose:
To detect in vivo sensitization of RBCs.
▫ HDN -Maternal antibody coating fetal RBCs
▫ HTR -Recipient antibody coating donor RBCs
▫ AIHA -Autoantibody coating individual’s RBCs
Factors Affecting the Antiglobulin Test
Ratio of serum to cells 2 drops serum: 1 drop of cells
Reaction medium LISS, PEG, Albumin
Temperature 37 deg or RT
Incubation time Based on Reaction medium
Washing of RBCs Minimum of three times
Saline for washing pH of 6.8 – 7.2
Addition of AHG Added after washing
Centrifugation for reading 20 seconds
Antiglobulin Test Sources of Error
• False positive tests result from RBCs being
agglutinated before the washing step (cold agglutinin),
improper RBC suspension, dirty glassware, and
overcentrifugation.

• False negative tests result from poor washing of


RBCs, testing being delayed, loss of reagent activity, no
AHG added, or use of an improper RBC suspension.
Compatibility testing
• Compatibility testing refers to the serologic aspect of
pretransfusion testing.

• It includes every serologic facet, beginning with donor


blood and ending with the recipient blood sample.
STEPS IN PRETRANSFUSION TESTING

1. Request for transfusion

2. Identification of transfusion recipient and blood


specimen collected

3. Testing of transfusion recipient’s blood specimen:


▫ Blood specimen acceptability
▫ ABO group and Rh type
▫ Antibody detection and
▫ Antibody identification
▫ Comparison of current and previous test results
4. Donor RBC unit testing:
ABO group confirmation and Rh type
confirmation for Rh-negative RBC units

5. Donor red cell unit selection

6. Compatibility testing (crossmatch)

7. Labeling of blood or blood components with the


recipient’s identifying information and issue
Positive Recipient Identification
• Major cause of transfusion-associated fatalities is
clerical error.

• Most common cause of error is misidentification of the


recipient.

• A facility-generated recipient ID wristband must always


be compared with the blood requisition form (blood
request form).
Positive Recipient Identification
If the patient does not have a wristband or if the patient’s
identity is unknown, some form of positive identification
must be attached to the patient before collecting the
samples:
▫ Temporary tie tag
▫ Wristband or ankle band

Ask the patient to state his or her full name and to spell it
out.
Collection of Patient Samples
• Blood samples should be drawn, carefully using a
technique that avoids hemolyzing the sample.

• Serum (Preferred) or plasma may be used for


pretransfusion testing.

• Disadvantages of using plasma:


• Formation of small fibrin clots
• Plasma anticoagulants may inactivate complement
Collection of Patient Samples
• About 10 mL of blood is usually sufficient for all testing
procedures.

• Venous samples are to be drawn only from below the


infusion site, not above it.
Collection of Donor Samples
• RBCs for donor pretransfusion testing can be prepared
from the segmented tubing through which the donor
blood was collected.

• Both donor and recipient samples must be stored for a


minimum of 7 days at 1°C to 6°C following transfusion.
Testing the Donor Sample
According to AABB, ABO grouping and Rh typing
(including a test for weak D) and tests intended to prevent
disease transmission must be performed on a sample of
donor blood taken at the time of collection.
Testing the Patient Sample
• ABO, Rh grouping, and antibody screening of the
patient’s serum can be performed in advance of or at the
same time as the crossmatch.

• Hemolyzed specimens are not acceptable.

• Specimens must be no older than 72 hours for


patients transfused or pregnant within the last 3 months.
ABO Grouping
• Determining the patient’s correct ABO group is the most
critical pretransfusion serologic test.

• Can be performed on slides or in tubes, using solid-


phase RBC adherence or column gel technology.
Rh Typing
• Rh typing is performed using anti-D blood typing
reagents.

• Tube or slide tests should be performed according to the


manufacturer’s directions for the reagent.
Antibody screen
• Purpose:
▫ The recipient’s serum or plasma must be tested for
clinically significant unexpected antibodies.
(Antibodies that are reactive at 37°C or in the AHG test)

Only a small percentage of the population (between 0.2%


and 2%) has detectable RBC antibodies.
Antibody screen
Indications:
1. Pretransfusion compatibility testing
2. Obstetric patients
3. Transfusion reactions
4. Blood or plasma donors
RBC Reagents (Screen cells)
• Come from group O individuals who have been typed
for the most common, and the most significant, RBC
antigens.

• Group O cells are used so that anti-A and anti-B will not
interfere in the detection of antibodies to other blood
group systems.
Antigen profile sheet
• Ideally, there will be homozygous expression of many
of the antigens within the screen cell set, allowing for
detection of antibodies that show dosage.

• Antibodies that react more strongly with cells having


homozygous antigen expression are said to show
dosage.
Homozygous inheritance vs heterozygous inheritance.
Common blood group systems with antibodies
that exhibit dosage:
▫ Rh (except D)
▫ Kidd
▫ Duffy
▫ MNSs
▫ Lutheran
Clinical Significance of 37°C-Reactive Antibodies
VERY UNUSUAL
USUALLY USUALLY SOMETIMES
(IF EVER)
• ABO • Bg Cartwright,Lutheran,
• Rh • Ch/Rg Gerbich,Dombrock,M
• Kidd • Leb NLea,Vel,LW,Ii,H,Ata,
• Duffy • JMH Inb,Mia,Csa
• S • Xga
• S
• U
• P
Antibody Screen Procedure
Antibody Screen Procedure
Check cells (Coombs control cells)
▫ Group O cells sensitized with IgG (anti-D).
▫ All AHG negative tests will have Coombs’ control
cells added to confirm the negative result.
Antibody Screen Procedure
Can be performed with the following techniques:
• Tube method
• Gel Method
• Solid Phase Adherence Method
Sample 1
Cell IS 37 Deg C AHG
Screening cells I 1+ Negative Negative
Screening cells II 2+ Negative Negative
Autocontrol Negative Negative Negative
Sample 1
Cell IS 37 Deg C AHG
Screening cells I 1+ Negative Negative
Screening cells II 2+ Negative Negative
Autocontrol Negative Negative Negative

Interpretation: IgM Alloantibody


Sample 2
Cell IS 37 Deg C AHG
Screening cells I Negative Negative Negative
Screening cells II Negative Negative Negative
Autocontrol Negative Negative 2+
Sample 2
Cell IS 37 Deg C AHG
Screening cells I Negative Negative Negative
Screening cells II Negative Negative Negative
Autocontrol Negative Negative 2+

Interpretation: IgG Autoantibody


Antibody Identification
• Once an antibody has been detected, additional testing is
necessary to identify the antibody and determine its
clinical significance.

• Antibody identification is performed by using a panel of


11 - 20 group O cells similar to the screening cells
used for antibody detection with various antigen
expression.
Antibody Identification
Patient History
Information concerning the patient’s age, sex, race,
diagnosis, transfusion and pregnancy history, medications,
and intravenous solutions may provide valuable clues in
antibody identification studies.
Antibody Identification panel
• Collection of 11 to 20 group O RBCs with various antigen
expression.

• Should include cells with homozygous expression of Rh,


Duffy, Kidd, and MNSs antigens.
Antibody identification
profile sheet
Group O panel cells
Each of the panel cells has been antigen typed
+ refers to the presence of the antigen
0 refers to the absence of the antigen

Example: Panel 6 has 11 antigens ( c, e, f, M, s, P1. Leb , k, Fya, Jka, Jkb)


Autocontrol

Autocontrol
Patient RBCs
+
Patient serum
• IS

Tested in 3 phases • 37°


• AHG
3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Add “check” cells to any negative AHG!
CC

3+ o o 
o o o 
o o o 
3+ o o 
o o o 
o o o 
3+ o o 
o o o 
3+ o o 
o o o 
o o o 
o o o 
Interpreting Antibody Panels
Ruling out:
• Negative reaction (0) indicates that the antibody(ies)
does(do) not react with any antigen on that RBC.

• Positive reaction (+) should never be used at any


phase of testing to rule out. Always use this in
identification.
Ruling out

\ 3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Ruling out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Circle antigens not crossed out

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Look for a matching pattern

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o
Look for a matching pattern

3+ o o
o o o
o o o
3+ o o
o o o
o o o
3+ o o
o o o
3+ o o
o o o
o o o
o o o

Anti-Lea
Guidelines:
Autocontrol:
• Negative: Alloantibody
• Positive: Autoantibody or DTR

Phases:
• IS – Cold (IgM)
• 37˚ - Warm reacting (IgM or IgG)
• AHG – Warm (IgG)

Reaction strength:
• Single-strength reactions usually indicate a single antibody.

• Various strength reactions usually indicate multiple antibodies


or dosage.
Guidelines:
Determining the antibody specificity:

• Single antibody:
▫ If there is only one antibody, the reactions will match the
antigen pattern on the antigram.

• Multiple antibodies:
▫ If there is more than one antibody, the reactions are
difficult to match with a single antigen pattern on the
antigram.
Rule of three
• The rule of three must be met to confirm the presence
of the antibody
• A p-value ≤ 0.05 must be observed
• This gives a 95% confidence interval

• How is it demonstrated?
Patient serum MUST be:
 Positive with 3 cells with the antigen
 Negative with 3 cells without the antigen
3+ o o
o o o
o o o
3 Positive 3+ o o
cells o o o
o o o
3+ o o
o o o
3 Negative 3+ o o
cells o o o
o o o
o o o
Additional Techniques for Antibody Identification

• Selected cell panels


• Proteolytic Enzymes
• Neutralization
• Prewarming
• Adsorption
• Elution
• Chemical Treatment
▫ Sulfhydryl reagents
▫ ZZAP
▫ Chloroquine
Selected Cell Panels
• Cells selected for testing should have minimal
overlap in the antigens they possess.

• For example:

Identified 3 different antibodies:


anti-M, anti-S, and anti-Lea
Selected Cell Panels
Selected M S Lea IS LISS AHG
cells 37°
#1 + 0 0 0 0 2+

#5 0 + 0 0 0 3+

#8 0 0 + 0 0 0

Results: Anti-M and Anti-S are pesent.


Enzymes
May destroy certain antigens and enhance expression of
others.

Enzymes:
• Ficin (Figs) – most common
• Papain (Papaya)
• Bromelin (Pineapple)
• Trypsin (calf spleen)
Enzymes

M M M
Enzymes

M M M
Enzymes

M M M
Enzymes
Y Y
M M M
Enzymes
Y Y
M M M
Enzymes
Y Y
M M M
Enzymes

Y Y
Enzymes
Enzymes modify the RBC surface by removing sialic acid
residues and by denaturing or removing glycoproteins.
Enhanced Destroyed
Rh Duffy
Kidd MNS
Lewis Xga
P1 Ch/Rg
I
ABO
Enzyme techniques
• One-step
▫ Enzyme is added directly to the serum/cell
mixture

• Two-step
▫ Panel cells are pre-treated with enzyme, incubated
and washed
▫ Patient serum is added to panel cells and tested
Neutralization
Other substances in the body and in nature have antigenic
structures similar to RBC antigens.

Antibody Neutralizing
The patient’s Serum
identification panel substance inhibits
is first incubated
is performed using reactions
with the neutralizing
the between the antibody
substance.
treated serum. and panel RBCs.

Note:
Use of a control (saline and serum) is necessary to prove that the loss
of reactivity is due to neutralization and not to dilution of antibody
strength by the added substance.
Sources of Substances for Neutralization of Certain
Antibodies
ANTIBODY SOURCE OF NEUTRALIZING
SUBSTANCE
Anti-P1 Hydatid cyst fluid, pigeon
droppings, turtledoves’ egg whites
Anti-Lewis Plasma or serum, saliva
Anti-Chido, anti-Rodgers Serum (contains complement)
Anti-Sda Urine
Anti-I Human breast milk
Prewarming
• Performing pretransfusion testing with all reagents and
samples incubated and kept at 37 C.

• Can help eliminate effects of cold auto- or alloantibodies.


Adsorption
Removal of specific antibodies from sample via incubation
with antigen positive RBCs.

• Autoadsorption:
▫ Using the patient’s own RBCs to remove autoantibodies.

• Alloadsorption:
▫ Using selected non-self RBCs to remove alloantibodies.
Autoadsorption

Autoantibody
Autoadsorption

Autoantibody

ANTI-K
Autoadsorption

PT PT
K- K-
Autoantibody

ANTI-K
Autoadsorption

Autoantibody
PT
K-
Autoantibody
Autoantibody
PT
ANTI-K K-
Autoadsorption

Autoantibody
PT
K-

Autoantibody
PT
ANTI-K
K-
Alloadsorption

ANTI-K

ANTI-C

ANTI-S
Alloadsorption

ANTI-K K+C+S-

ANTI-C K+C+S-

ANTI-S K+C+S-
Alloadsorption

ANTI-K

K+C+S-

ANTI-C

ANTI-S K+C+S- K+C+S-


Alloadsorption

ANTI-K

K+C+S-

ANTI-C

K+C+S- ANTI-S
Elution
• Removing or “dissociating” an antibody that is attached to the
surface of a red blood cell.

• Commonly used in identification of antibodies in transfusion


reactions or hemolytic disease of the newborn, as well as in
the workup of warm autoantibodies.

• Methods:
▫ Heat elution
▫ Freeze thaw
▫ Acid (Glycine-HCL-EDTA, digitonin)
▫ Organic solvents (chloroform, Xylene, ether)
Alloadsorption

ANTI-K

K+C+S-

ANTI-C

K+C+S-
Alloadsorption

K+C+S- ANTI-K

TREAT
K+C+S- ANTI-C
Sulfhydryl Reagents
Dithiothreitol (DTT) or 2-mercaptoethanol (2-ME)

▫ Cleave the disulfide bonds of IgM molecules and help


differentiate between IgM and IgG antibodies.
▫ Denatures surface RBC antigens of multiple groups:
 Kell
 Lutheran
 Dombrock
 Yt
 LW
ZZAP
• A combination of proteolytic enzymes and DTT

• Purpose:
Dissociation of IgG molecules from the surface of
sensitized RBCs and alters the surface antigens of
RBCs.
Chloroquine
• Removes IgG from coated (DAT-positive) RBCs to allow
for accurate phenotyping.

• Also removes residual HLA antigens from RBCs.


▫ Bg antigens
Selection of Appropriate Donor Units
• If possible, patients should receive blood components of
their own ABO group.

• If group specific blood is not available, units selected


must lack any antigen against which the recipient has a
clinically significant antibody.

• When a recipient must be given blood of a different ABO


group, only packed RBCs can be given.
Selection of Appropriate Donor Units

Suggested ABO Group Selection Order for Transfusion of RBCs


ABO Group 1st Choice 2nd Choice 3rd Choice 4th Choice
of Recipient
AB AB A B O
A A O
B B O
O O
Selection of Appropriate Donor Units
• Rh-positive blood is acceptable as long as no preformed
anti-D is demonstrable in their sera.

• Transfusion of Rh-negative male patients and female


patients beyond menopause with Rh-positive blood is
acceptable as long as no preformed anti-D is
demonstrable in their sera.
Crossmatch Testing
The testing of the patient’s serum with the donor RBCs,
including an antiglobulin phase or simply an immediate
spin phase to confirm ABO compatibility.

• Major crossmatch: Donors RBC + Patient Serum


• Minor crossmatch: Donors Serum + Patient RBC
• Autocontrol: Patient serum + Patient RBC

A crossmatch is only one part of


pretransfusion testing!
Crossmatch Testing
Originally the serologic crossmatch preceded antibody
screening as part of pretransfusion compatibility testing to
check for unexpected alloantibodies.

Two main functions of the serologic crossmatch:


1. It is a final check of ABO compatibility between donor and
patient.
2. It may detect the presence of an antibody in the patient’s
serum that that was not detected in antibody screening
because the corresponding antigen was lacking from the
screening cells.
Electronic crossmatch
• Recipient ABO/Rh is tested in duplicate and results are
entered into a validated blood bank computer system.
The recipient's transfusion history is researched through
the computer.

• If the recipient has not been transfused in the last 3


months and his(her) antibody screen, both current and
historical, is negative, blood for the recipient is issued
without any additional testing.
Serologic Crossmatch Tests
Immediate-spin crossmatch:
• This is used when recipient has no history of
alloantibodies and current antibody screen is negative.
• The donor cells and recipient serum or plasma are added
to a tube. This tube is spun and the reaction is graded.
▫ If negative, the recipient is transfused with this unit of
blood.
▫ If positive, the crossmatch must be carried out as an
antiglobulin crossmatch:
IS, 37°C incubation, and AHG phases.
Abbreviated crossmatch

The type and screen, coupled with an immediate


spin crossmatch, is referred to as an abbreviated
crossmatch.
Serologic Crossmatch Tests
Antiglobulin crossmatch:
▫ Performed when a history of an alloantibody or the
detection of one in the current antibody screen
warrants an antiglobulin crossmatch.

▫ Involves IS phase,addition of potentiator, 37°C


incubation phase, three washes, antiglobulin phase,
and finally IgG-coated control cells.
Number of units to crossmatch
A patient with an anti-K and an anti-Jka in her plasma
needs 2 units of RBC for surgery. How many group specific
units should be screened to find 2 units of RBC? The
frequency of Jka (+) is 77% and the frequency of K(+) is
10%.

Formula:
___________Number of units needed by the patient______________
Frequency of negativity of antigen #1 x Frequency of negativity of antigen #2
Number of units to crossmatch
Solution:

___2 units____
0.23 x 0.90

= 9.66

Number of units to crossmatch =10 units


Hemolytic Disease of the
Newborn
Etiology
HDFN is the destruction of the RBCs of the fetus and
neonate by IgG antibodies produced by the mother due to:

• Previous pregnancy
• Transfusion
• During the second and third trimester of pregnancy
Rh HDFN
• Most severe
• Rh antibodies are primarily IgG, which readily cross the
placenta

• Mother:
• Father:
• Baby:
Rh HDFN
• Most severe
• Rh antibodies are primarily IgG, which readily cross the
placenta

• Mother: Rh Negative
• Father: Rh Positive
• Baby: Rh Positve
Hemolysis, Anemia, and Erythropoiesis

Hemolysis

Anemia

erythroblastosis fetalis

Hepatosplenomegaly

hydrops fetalis
• RBC destruction releases hemoglobin, which is
metabolized to bilirubin.

• After birth, accumulation of metabolic by-products of


RBC destruction can reach levels toxic to the infant’s
brain (generally, more than 18 to 20 mg/dL).
Prevention of Rh HDFN
Rh-immune globulin
▫ RhoGAM
▫ Purified IgG anti-D
▫ Given to D-negative woman at 28th of gestation and
within 72 hours following delivery of a D-positive
fetus.
Dosage
• 1 Full dose vial
▫ 300ug/1500IU of RhIg
▫ Protects up to 30 mL D + WB (15mL D+ RBCs)

• Mini dose vial


▫ 50 ug/250 IU or RhIg
▫ Protects up to 5mL D+ WB (2.5 mL D+ RBCs)
Fetal bleed screen (Rosette Screen Test)
Kleihauer Betke acid elution stain
• HGB F Acid Resistant
• HGB A Elutes
Calculation
EXAMPLE:

6 fetal cells were counted in 2000 adult cells in a


Kleihauer-Betke acid elution test:

6 cells/2000 cells x 100 = 0.3% fetal cells

0.3%/ 100 x 5000= 15 mL FMH

15/30 = 0.5 vial Round up to 1 and add 1 = 2 vials


Short method:

% Fetal cells x 50 = RhIg vials


*assumes Maternal BV is 5000ml
ABO HDFN
• Most common form of HDN

• Maternal ABO antibodies that are IgG can cross the


placenta and attach to the ABO-incompatible antigens of
the fetal RBCs.

• Infants are treated by phototherapy to break down


excess bilirubin.

• Microspherocytes and increased RBC fragility in the


infant are characteristic of ABO HDFN
Comparison of ABO Versus Rh HDFN
Treatment of HDFN
• Intrauterine Transfusion
• Phototherapy
• Intravenous Immune Globulin
• Exchange Transfusion
Intrauterine Transfusion
Definition Performed by accessing the fetal umbilical vein
(cordocentesis) and injecting donor RBCs
directly into the vein.
Purpose To maintain fetal hemoglobin above 10 g/dL.
Indications • MCA-PSV indicates anemia.
• Fetal hydrops is noted on ultrasound
examination.
• Cordocentesis blood sample has hemoglobin
level less than 10 g/dL.
• Amniotic fluid ΔOD 450 nm results are high.
Exchange Transfusion
Definition Use of whole blood or equivalent to replace the
neonate’s circulating blood.
Purpose • Used primarily to remove high levels of
unconjugated bilirubin and thus prevent
kernicterus.
• To replace antibody-coated RBCs with
compatible donor cells
• To remove circulating maternal antibodies
• To suppress erythropoiesis
Indications • Greater than 0.5 mg/dL/hr rise in bilirubin
• Rise of 10 mg/dL in the first 24 hours
Selection of Blood for Intrauterine and
Neonatal Transfusion

• Use group O RBCs or group specific


• The RBCs must be antigen negative for the mother’s
respective antibodies.
• CMV-negative
• Hgb S negative
• Less than 7 days old
• Irradiated to prevent graft-versus-host disease
Treatment of HDFN
• Intravenous Immune Globulin
▫ IVIG competes with the mother’s antibodies for the FC
receptors on the macrophages in the infant’s spleen,
reducing the amount of hemolysis.

• Phototherapy
▫ Phototherapy at 460 to 490 nm is used to change the
unconjugated bilirubin to isomers, which are less lipophilic
and less toxic to the brain.
Immune hemolytic anemia
Immune hemolytic anemia
Shortened RBC survival mediated through the
immune response, specifically by humoral
antibody.

Three categories:
1. Alloimmune
2. Autoimmune
3. Drug-induced
Immune hemolytic anemia
Alloimmune
Patient produces antibodies to foreign or non-self
RBC antigens introduced into the circulation
through transfusion, transplant, or pregnancy.
Immune hemolytic anemia
Autoimmune Hemolytic Anemia
Production of antibodies that are directed against
the individual’s own RBCs

May be classified as:


• Warm reactive (70%)
• Cold reactive (18%)
• Drug-induced (12%)
Comparison of Warm and Cold AIHA
Comparison of PCH and CHD
Drug-induced Hemolysis
Patients produce antibody to a particular drug or drug
complex, with subsequent damage to RBCs.

Four mechanisms:
• Drug adsorption (Hapten)
• Immune complexes
• Membrane modification/Nonspecific protein adsorption
• Autoantibody formation
Drug-Adsorption (Hapten) Mechanism

• Drugs operating through the drug-adsorption


mechanism bind firmly to proteins, including the
proteins of the RBC membrane.

• Penicillins (most common)


• Streptomycin
• Cephalosporin
Drug-Dependent /Immune Complex Mechanism

• “Innocent Bystander” Mechanism


• Drugs operating through this mechanism combine with
plasma proteins to form immunogens.

• Quinidine
• Phenacetin
Membrane Modification

• “Nonimmunologic Protein Adsorption”


• Drugs modify RBCs so that plasma proteins (e.g., IgG,
IgM, IgA, and complement) can bind to the membrane.

• Cephalosporins
▫ Cephalothin (Keflin)
Autoantibody Formation
• Methyldopa alters the function of T-suppressor cells and
suggest that this upset in the immune system would
allow production of antibody against self.

• Drug alters RBC membrane components.


Mechanisms Leading to Development of
Drug-Related Antibodies
Quality Management
Quality
Defined as the degree to which a product or service meets
requirements.

Blood banks must provide quality to their customers in


many forms, including:
▫ Safe, satisfying donation experiences for blood donors.
▫ Accurately labeled and tested blood components provided
to transfusion services.
▫ Timely, accurate transfusion services provided to
physicians and other health-care personnel.
▫ Safe and efficacious blood transfusions to patients.
Quality management
• Quality management (QM) is actively and
continuously practiced by the blood bank’s leaders,
managers, and staff throughout all blood bank
operations.

• Validates its processes, monitors process performance,


knows where the problems are, continuously takes
action to determine root causes of problems and
removes them, and documents its actions.
Compliance
Compliance simply requires the correction of
identified deviations and deficiencies and usually
leaves the facility with the false sense that it has
solved its problems and has been brought into
compliance.
Quality Control
• Most blood bank technologists are familiar with routine
blood bank QC procedures.

• Daily testing of the reactivity of blood typing reagents;


calibrating serologic centrifuges; and monitoring
temperatures of refrigerators, freezers, and thawing
devices.
Monitoring of Instruments and Equipment

Requires daily monitoring when in use:


• Blood typing reagents
• Heating blocks
• Water baths
• Refrigerators and freezers(continuous)
• Platelet incubators
• Donor unit agitators
• Weighing scales
• Balances
• Hemoglobinometer
• Microhematocrit centrifuges
Monitoring of Instruments and Equipment

• Annually:
▫ Mercury thermometers
• Quarterly:
▫ Cell washers
▫ Blood warmers
▫ Centrifuge
• Monthly:
▫ Refrigerated Centrifuge
▫ Alarm activation (ref and freezers)
• Every four hours:
▫ Platelet incubators
Quality Assurance
• Set of planned actions that ensure that systems and
elements that influence the quality of the product or
service are working as expected, individually and
collectively.

• Addresses how well an entire process, which is a


sequence of activities, is functioning.
Common Blood Bank QC Activities and QA Indicators
QC Activities QA Indicators
Collection Equipment • Number of donor forms with
• Microhematocrit incomplete or incorrect
instrument information
• Hemoglobin instrument
• Apheresis equipment • Number and types of unusable
• Blood-weighing scales units and blood components
Blood Components
• Red blood cell hematocrit • Number of blood typing
• Cryoprecipitated AHF discrepancies in donors and
Reagents patients
• Copper sulfate
• Reagent antisera • Number of and reasons for
Laboratory Equipment invalid tests
Documents and Records
• Documents are approved information
contained in a written or electronic format.
▫ Written policies
▫ Process flowcharts
▫ Procedures and instructions
▫ Forms
▫ Manufacturers’ package inserts
▫ Computer software and instrument operator manuals
▫ Copies of regulations and standards
Policy
(What will be done)

Process
(How it happens)

Procedure
(How to do it)

Quality Management System Documentation


Plan-Do-Check-Act Process.
A common quality improvement process.
Step1: Plan
A mission-consistent, customer-oriented action plan
• Identify opportunities for improvement from data sources
• Prioritize improvement activities
Step 2: Do
Put the plan into action
• Implement the action plan
• Collect performance data
Step 3: Check
Has the planned and implemented change created intended improvement?
Step 4: Act
Decide what to do next
• Determine if customer needs were met
The cycle of organization-wide quality management.

Plan

Improve Implement

Assess
Laboratory Information Systems
Information processing
The proper management of information related to blood
donors, blood components, and patients receiving
transfusions is crucial to ensuring the safety and
traceability of blood products.
Common Information System Acronyms

ACRONYM DEFINITION
• CPU Central processing unit
• HIS Hospital information system
• LIS Laboratory information system
• PC Personal computer
• RAM Random access memory
• ROM Read-only memory
• SOP Standard operating procedure
System Components
Computer system includes three major
components:

• Hardware
• Software
• People
Hardware
Hardware
Three main functions performed by hardware
components:
• Processing
• Input and output
• Storage
Processing Hardware
• Central processing unit
▫ the core of the machine
• Read-only memory (ROM)
▫ contains the “start-up” instructions for the
computer.
• Random access memory (RAM)
▫ an array of chips in which data are temporarily
entered while they are being processed.
Input and Output Devices
Input devices:
▫ Keyboards
▫ Pointing devices
▫ Bar-code readers
▫ Testing instruments
Output devices:
▫ Monitor
▫ Printer
Combined input and output device:
▫ Modem
Software
Tells the computer what to do with all of the information it has
received.
• Operating system software
▫ Controls the computer’s hardware, manipulates the application
software, and coordinates the flow of data to and from disks and
memory.
• Application software
▫ Allows users to perform tasks that are specific to blood bank
operations.
• Interface Software
▫ Allow data to flow between a hospital information system
(HIS) and the blood bank system or between the LIS and the
blood bank.
People
• Human components of a blood bank information system
are the users and at least one person designated as the
system manager.

• System manager oversees the maintenance of the


system’s hardware and software.
TRUST GOD, FOCUS
and TRUST THE
PROCESS.

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