MTAP - BLOOD BANKING o Some minor blood group can be inhibited

RW INTERNS EDITION by enyzme

• Monovalent
o 2 antigen binding sites.
• Usually immune in nature
Immunohematology • Can cross placenta
o Smaller size
• The study of immunologic reaction involving
• React at 37degC
components of blood.
o Clinically significant
• Deals with antigens and antibody, and antigen- • Example: Rh antibodies.
antibody reaction particularly of the blood.
o Antigen – a substance that gives rise to Illustration why IgG can coat but cannot
a formation of an antibody upon agglutinate:
introduction, considered to be a foreign
invader or substance.
o Antibody – substance produce in
response to antigenic stimulation.
Immunoglobulins produce by plasma
cells as product of immune response.
o Immune response – there is antigenic
stimulation.

Immunoglobulins
• 5 types – IgG, IgM, IgA, IgD, IgE
• Blood groups antibodies are mainly confined to
IgG and IgM
• IgG is a smaller molecule, with a pair each of - Because of the distance (24nm) why IgM after
heavy & light chains of amino acids. sensitization can form lattice formation
o monomer - It can react out based on its size.
• IgG antibody can just coat but not agglutinate the - IgG (14 nm) lacks 10 nm, it cannot reach out to
cells, because of the size. the other site. Can coat or attached to surface
• IgM has 5 such pairs joined together by the J site, but cannot reach the other site to form
chains. agglutination.
o Pentamer
• IgM antibody agglutinate the cells bearing
corresponding antigen.

IgM Antibodies (complete)

• Agglutinate in saline phase
• Pentavalent
o 10 subunits, per subunit have 2 antigenic
sites.
• Usually naturally occurring
o Because there is no known antigenic
stimulation for its formation.
• Do not cross placenta
• React at temperature varying from 4-20degC
o Active in-vitro but not in-vivo (37degC) →
cannot react.
o Need to identify the antibody in-vitro, Clinical Significance of Antibody
because it can masked significant Clinically significant Clinically Insignificant
antibody that supposed to be detected. ABO Lewis
• Example: ABO antibodies Rh M,N
Kell P1
IgG Antibodies (Incomplete) Duffy Lutheran
• Agglutinate in IAT phase Kidd A1
o Uses AHG reagent Ss
• However, may cause agglutination in saline - ABO is a combination of IgG and IgM, but mostly
phase using albumin/ enzymes IgM.
- Ss = IgG
 - Clinically significant – associated with hemolytic
transfusion reaction and HDFN (can cross
placental barrier)
- Clinically insignificant – IgM, but need to be
identified in the lab by doing antibody screening
because they can mask the clinically significant
antibody in-vitro at RT.
o Prolonged incubation results in
dissociation of antibody
Factors affecting stage of agglutination
• Size and Class of Antibody
o IgM antibody, being a pentamer can bind
antigen sites up to 35 nm apart
o IgG antibody, being a monomer can bind
antigen sites up to 14 nm apart
▪ Need to add AHG reagent to
enhance agglutination

• Antigenic sites
o Antigens located on surface of red cell
membrane (ABO) result in strong
agglutination
o Antigens embedded in membrane (Rh
antigens) result in weaker agglutination

• Zeta potential
o Electrostatic repulsive force between red
cells
▪ Repulsion force created between
negative charge cyanic acid of
the RBC and positive charge of
Stages of Ag-Ab reaction solvent (NSS)
Stage of sensitization Stage of Agglutination
Only coating of red cells Characterized by by
with IgG antibody w/o formation of bridges
causing agglutination between sensitized red
Bond holding Ag- Ab cells resulting in visible
complex may dissociate aggregate of red cells
& re-associate till the
state of equilibrium is
reached

Factors affecting stage of sensitization

• Antigen-Antibody ratio
o Should be in the zone of equivalence –
optimum Ab-Ag reaction
o Two volume of serum and one volume of
5% red cells (2:1)
▪ 5% red cells - undergone
washing, removes the interfering - Prozone and Post-zone – no precipitation since
substances and the antigen will there would be soluble immune complexes
be concentrated. formation. (False-negative reaction)
o Sensitivity of test depends upon number
of antibody molecules bound per red
cells.
• pH
o Most antibodies detected at neutral pH
o 7.35-7.45

• Temperature
o IgM antibodies react optimally at cold
temperature while IgG at 37’C

• Incubation time
o Time needed to reach ag-ab reaction at
equilibrium - Positive charge – ode 2 of solvent NSS
o Too short incubation -- weaker reaction - Negative Charge – ode 2 cyanic acid of the RBC
 - They will form clouding → 0 visibility from one
RBC to the other
- Coating will be affected by zeta potential.
Effect of adding LISS
• Reduction in zeta potential using LISS
o Enhance the Ag-Ab reaction
o Lower Ionic Strength → lesser positive
charge → less repulsion against negative
charge
• RBCs come closer together
• Strong agglutination
• Reduction in incubation time
o Because RBCs are closer
o NSS – 30 mins incubation time
o LISS – can reduced to 10-15 mins of
incubation time
- Another way of reducing the zeta potential → use
of enzyme, it reduces the negative charge of
cyanic acid. → reduce electrostatic reaction with
positive charge
- However, enzyme can destroy some minor blood
groups.
- Also, 22% w/w albumin – lesser the positive
charge of solvent
- PEG (polyethylene glycol) – removes the fluid
and concentrate the antibodies → enhances of
Ag-Ab reactions
Metabolic Pathways
• Keeps RBCs viable when stored
• Maintained its shape in-vivo (125 days) →
undergo lysis after 120-125 days
o Without Na-K pump → losses
deformability and permeability
characteristics. → spherocytes → burst
• To have deformability and permeability it needs
continuous ATP production within RBC (need
some energy)
• Mainly anaerobic, RBC must deliver not
consume O2.
• No nucleus/No mitochondria
o Mainly dependent on the glucose
entering on the cell.
• RBC metabolism may be divided into anaerobic
glycolysis and 3 ancillary pathways
o Anaerobic glycolysis: Embden-Meyerhof
o 3 ancillary pathways: Leubering
Rapoport shunt, Pentose phosphate
pathway, and Meth Hb reductase
pathway.
• RBCs contain no mitochondria, so there is no
respiratory chain, no citric acid cycle, and no
oxidation of fatty acids or ketone bodies
• The RBC is highly dependent upon glucose as its
energy source
• Energy in the form of ATP is obtained only from
the glycolytic breakdown of glucose with the
production of lactate (anaerobic glycolysis)
o Product: Lactate + ATP (within RBC)
RBC Metabolism
• Glucose transport through RBC membrane
o Generally, most of the cells in the body,
requires insulin as a transporter for
glucose to enter the cells to produce
ATP.
o However, RBCs & brain cells do not
require insulin
• Glucose is transported through RBC membrane
by facilitated diffusion through glucose
transporters (GLUT-1)
• Once it enters the RBC the anaerobic reaction
takes place. → breakdown of glucose → lactate +
2ATP (required by RBC)
•
Glycolysis
Important of glycolysis in red cells:
• Energy production: it is the only pathway that
supplies the red cells with ATP
o 90% of ATP requirement of RBC are
generated by Embden Meyerhof pathway
• Reduction of methemoglobin: Glycolysis
provides NADH for reduction of metHb by NADH-
cytob5 reductase
o if not in a reduced form (ferrous) → iron
will be in the non-functional form (ferric)
o Ferrous stage – has free arm to carry
oxygen.
o Ferric Stage – no free arm and cannot
carry oxygen.
• Leubering Rapaport Shunt: In red cells 2,3
biphosphoglycerate binds to Hb, decreasing its
affinity for O2, and helps its availability to tissues.
o 2,3 DPG – is stimulated for release once
there is a decrease oxygen tension
(Hypoxia). It will form anionic salt bridge
for the Hb to loss its oxygen → unload to
the tissue that requires oxygen.
o If hypoxia Is corrected it gives negative
feedback to Leubering Rapaport Shunt to
 stop the generation of 2,3 DPG → anionic o Ferric is the iron found in the
salt bridge will go self-destruction. methemoglobin and it should be reduced
o 2,3 DPG affects the oxygen dissociation to hemoglobin
curve. • Protect SH (sulfhydryl)/Thiol group of Hb and
membrane protein from oxidation.
Utilization of ATP
• Phosphorylation of sugars and proteins
o Associated with the deformability
characteristic of RBCs
o There’s a need to have a continuous
phosphorylation of spectrine in the
membrane of RBCs to maintain its
deformability or flexibility.
o The phosphorylation is possible if there is
enough ATP supplied to RBC. LEUBERING RAPOPORT SHUNT
o If the phosphorylation is stopped the • The hydrogen ion concentration is the most
RBC will lose its biconcave shape and important physiological modulator
fluid will go inside the cells → o Stimulated by 2,3-DPG when the
▪ form spherocyte shape. hydrogen ions are increased (Acidic)
▪ When it passes to sinusoids o Negative Feedback – decrease hydrogen
(smaller in diameter compared to ion
the size of RBC), it will not fit in
→ indentation of RBCs (Bite
cells)
▪ Spherocytes/bite cells → will be
taken by spleen together with
reticuloendothelial system as
senescence (will be removed) →
▪ shorten the life span of RBC →
anemia and BM is not ready to
release mature erythrocytes.

• ATPase driven ion pumps → Permeability of

RBC
o Na-K pump will not work without the ATP
to fuel its function.
o Must correct the proportion between
intra- and extracellular ration of Na and
K.
o Sodium: Higher outside
o Potassium: Higher Inside the cells
o If Na-K is not balanced →
▪ potassium and water leak out → - Tense from: Unload O2 from Hb going to tissues
RBC become dehydrated undergoing hypoxia.
▪ Calcium will go inside the cell → - Relax form: Loading of O2 going to the Hb.
rigid RBC.
▪ RES and Spleen will remove PENTOSE PHOSPHATE PATHWAY
senescence RBC. • Production of NADPH – “reducing power”
• Prevent RBC from oxidation
• Maintenance of membrane asymmetry • Generation of Glutathione – antioxidant
• Maintenance of red cell shape and • Glutathione is needed in reduced form for:
deformability using ATP dependent o Elimination of peroxide
cytoskeleton. ▪ Peroxide is a form of radicals.
o Cytoskeleton – meshwork that are o Protection of proteins SH group
interconnected with one another to
• This shunt also provides ribose-5-phosphate
maintain normal shape of RBC.
needed for PRPP (substrate for adenine
nucleotides required for continuing ATP
3 ANCILLARY PATHWAYS
synthesis)
METH Hb REDUCTASE PATHWAY
o 10% in ATP production.
• Maintains iron in reduce state for effective
transport of O2. **page 3 explanation glycolysis part
 o If EMP cannot supply ATP, PPP will help
to compensate to meet the required ATP
production.
HEMOGLOBIN-OXYGEN DISSOCIATION CURVE
• Dissociation and binding of O2 by Hb, not directly
proportional to pO2 that is present in the blood.
• Permits a considerable amount of oxygen to be
delivered to the tissues with small drop in oxygen
tension.
• Dependent to temperature, pH, and the
production of 2,3-DPG. (Not to the oxygen)
• Normal Curve
• Y-axis: percentage saturation of oxyhemoglobin
(O2 attached to hemoglobin)
• Shift to the right – reduced affinity of O2 to Hb
(O2 has higher affinity to tissues)
o Increase temperature, feedback to LRS
→ Increase release of 2,3-DPG
o Increase Hydrogen Ion → acidic
environment
• Shift to the left – increasing affinity of O2 to Hb
(relaxed form/loading phase)
o Decrease temperature
o Decrease hydrogen ions
• Stored blood → decrease 2,3-DPG
o LRS is in-vivo, no continuous LRS.
o However, 2,3-DPG will go back to its
normal level once the blood is transfused
(24hrs post-transfusion)
BLOOD BAGS AND ITS ANTICOAGULANT
A brief history of blood banking
• 1628 – William Harvey – circulation of blood
• 1665 – Richard Lower – first recorded
successful blood transfusion.
• 1818 – James Blundell – first successful
transfusion of human blood to a patient
o There was mortality
• 1900 – Karl Landsteiner – discover the first three
human blood groups (ABO blood group)
o Investigate what could be the possible
cause of mortality rate in blood
transfusion
o The year after, they discover type AB
blood group.
Milestones in blood preservation history
• 1914: Albert Hustin and Luis Agote kept blood
in the liquid state for 48 hours using citrate
o Anticoagulant, not a preservatives
• 1943: John F. Loutit and Patrick L. Mollison
introduced Acid-Citrate-Dextrose preservative,
the still used blood preservative.
o Citrate – anticoagulant
o Dextrose – food or source of ATP for
RBC, maintains the viability in-vitro.
• 1950: Carl Walter invented first plastic blood
bag.
o A surgeon, inventor, and a professor of
Harvard medical school.
o “Pioneer in the transfusion and storage of
blood”
o Credited with founding one of the work
first blood banks (US) and invention of
first blood bag/collection bags.
• 1963-1973: closed blood bag systems were
developed to ensure the sterility.
BLOOD BAGS
• Blood bags are designed for the collection,
processing and storage of whole blood and blood
components.
• They help in providing aseptic conditions for the
separation of blood components
o Before, whole blood component is given.
It has disadvantages.
▪ Hemoconcentration –
sometimes px only needed
plasma, not the cellular
component. It might increase
cellular component of the
recipient.
▪ Hypovolemia – increase plasma
component.
o To get rid of disadvantages, ABB
recommended blood component therapy.
Separate the components and only give
the needed blood component.
• It acts as closed system reducing the chances of
contamination.
SAFETY FEATURES OF BLOOD BAGS
Needle injury protector
• Provides immediate shielding of needle on
withdrawal from vein.
• Reduces the risk of needle stick injury from both
phlebotomy and sampling needles
o Best precautionary: treat every blood as
infectious
• Improves safety in blood needles.
Pre-donation bag (PDB)
• Diverts 10-30 ml of initial blood.
 • Enables diversion and collection of the first
amount of blood which usually contains skin
particles and bacteria.
• Risk of bacterial sepsis is minimized.
TYPES OF BLOOD BAGS
Single Blood Bag
• For whole blood collection
• The bag contains CPDA solution
o CPD – Life storage: 21 days
o Adenine – substrate for ATP production.
o With Adenine life storage will increase
with 14days (35 days)
• Available in capacity of 350ml and 450ml.
o 63ml – anticoagulant
o 500ml with 70ml of anticoagulant
Double Blood Bag
• Recommended when ABB is making guidelines
on blood component therapy → needs separation
bag and must be done in a close system to avoid
bacterial contamination.
• For whole blood collection
• Separation of 2 different blood components
(red blood cells and plasma/Platelet-rich plasma)
obtained through the process of centrifugation
and extraction.
o Light Spin – 3200 rpm for 2-3 mins
Triple Blood Bag
• Triple blood bag with SAGM. For whole blood
collection
o Has satellite bag, because while doing
research they found out that pRBC
cannot be stored on original date of
expiration.
o Based on density, preservatives solution
will go with PRP and not with the pRBC
o Additional Satellite bag has SAGM
(additive solution) → after separation
SAGM will be unlock and added to the
pRBC. (To maintain its viability and
permeability of the RBC while being
stored) → improved life storage into 42
days.
▪ Saline
▪ Adenine
▪ Glucose – sugar
▪ Mannitol – sugar
o SAGMA will also eliminate the problem of
an increase hematocrit.
▪ Product will be more viscose →
difficult to transfuse.
▪ After separation of pRBC and
PRP → increase 70-85% of hct
▪ But with addition of SAGM → it
will lower into 50-60%
• Separation of 3 different blood components
(red blood cells, plasma, and platelets)
• The primary bag contains CPD, and one satellite
bag contains SAGM
Quadruple Blood Bag
• Heavy spin (3600 rpm for 5 mins) → to expel the
plasma to the 4th bag → Platelet-poor plasma
• 2nd bag = concentrated Platelet (50-70mL)
o Should not exceed 70 mL, to maintain its
6.2 pH.
o Exceed pH 6.2 → Platelet Storage
Lesion
▪ Platelet as mitochondria and is
very dependent to aerobic
glycolysis to produce ATP (to
maintains its shape)
o The volume of 50-70mL, exact oxygen
tension within the platelet bag is enough
to supply for aerobic glycolysis good for
5 days.
o When exceeds, the O2 will decrease in
plasticizer among blood bags → lowers
the pH
▪ from aerobic to anaerobic
glycolysis for ATP production →
when anaerobic, lactic acid is
produced → lowers the pH →
irreversible discoid shape
formation of platelet →
nonfunctional platelet
o PVC plasticizer = lesser O2 tension, than
polyolefins.
• It comes with SAGM for whole blood collection
• Separation for 3 different blood components (red
blood cells, plasma, and platelet) through the
buffy coat method.
• The primary bag – CPD solution and has 3
satellite bags
• One satellite bag – 100ml capacity to prepare
platelets through the buffy coat method.
• Valid for 5 days of platelet storage
ANTICOAGULANT PRESERVATIVE SOLUTIONS
• Acid Citrate Dextrose (ACD)
o 1st to discover
o Life storage: 21 days
• Citrate Phosphate Dextrose (CPD)
o Life storage: 21 days
o But much better because ACD is acidic.
 • Citrate Phosphate Dextrose Adenine (CPDA- • Also overcome the problem of high viscosity of
1) RBC concentrates
o Increase life storage for 14 days with o Lowers the hematocrit solution
adenine. (21+14 = 35 days) o For ease of transfusion.
o 2 sources of ATP production: Dextrose • With CPD anticoagulant in the primary bag, the
and Adenine additive solution used is SAGM.
- 15mL of ACD, 14mL of CPD or CPDA is used for o Extend the storage of RBC for 42 days.
preserving 100mL of blood o Maximum amount of fresh plasma is
- 450mL of blood bag: 63mL of anticoagulant harvested.
- 500mL: 70mL of anticoagulant
• PURPOSE: GENETICS: BASICS
o To prevent coagulation • GENE
▪ Due to citrate o A biological unit of inheritance
o To preserve the life and survival of RBCs o Dominant gene – always expressed as
to have the maximum post-transfusion antigen regardless of whether it is in
survival homozygous (identical allele genes) or
▪ Continues generation of glucose heterozygous state (nonidentical)
for ATP production → to maintain o Recessive gene – produce antigen only
deformability and permeability of when in homozygous state
RBC while being stored. ▪ Cannot be expressed.
▪ QC = 75% red cell survived post-
transfusion (25% storage lesion) • CHROMOSOMES
o Linear arrangement of genes
ACTION OF INGREDIENTS OF ANTICOAGULANT
SOLUTION • LOCUS
• CITRATE: Acts by chelating calcium o Hypothetical seat of gene on a
o Factor III chromosomes
o From Pakistan to China, People Power
against Communism some Dogs have • ALLELE
Fleas o Alternative form of gene at particular
o Fibrinogen, Prothrombin, Calcium locus.
• DEXTROSE: Necessary for the metabolism of
stored RBCs. • GENOTYPE
o Contains sugar o Sum total of genes present on
- It passes from plasma into red cells and is utilized chromosome regardless of whether or
for energy production. not they produce detectable products
- The principal pathway being Anaerobic o Determined through testing of genes &
glycolysis. family study
• CITRIC ACID: Prevents caramelization of ▪ Gene mapping and DNA
glucose in citrate dextrose solution during testing
autoclaving o A,B = co-dominant
o To neutralize the effect o O = recessive
• ADENINE: Improves the viability of red cells.
o Substrate for ATP production • PHENOTYPES
o Detectable product (antigen)
CPDA-2 demonstrated through direct testing only.
• Here the amount of Adenine is increased of 0.55g
and that of Dextrose to 44.6g • HOMOZYGOUS
• This is a better anticoagulant preservative o Presence of identical pair of allelic genes
solution than CPDA-1 on both chromosomes
• Both 35 days o Double dose of antigens
o Stronger reaction with corresponding
ADDITIVE SOLUTIONS antibody
• Additive solutions are preserving solutions that o Example: KK
are added to the RBCs after removal of the
plasma with/without platelets • HETEROZYGOUS
o Via light spin, 3200 rpm for 2-3 mins o Different alleles of genes on pair of
• Reason for their development – removal of the chromosomes
plasma component during the preparation of RBC o Weaker reaction with corresponding
concentrates removed much of the nutrients antibody
needed to maintain RBCs during storge o Example: Kk
ABO BLOOD GROUP SYSTEM o Antibodies present on neonates are
• The ABO blood group system is the most antibodies of the mother.
important blood group system in the human blood 2. These “naturally occurring” Abs are mostly IgM
transfusion. class. That means that, they are antibodies
• Found in platelets, epithelium, and cells other capable of agglutinating saline/low protein
than erythrocytes. AB antigens can cause an suspended red cell without enhancement and
adverse immune response to organ may activate complement cascade.
transplantation. The associated Anti-A and anti-B
antibodies are usually IgM antibodies, produced BLOOD ANTIGEN ON ANTIBODIES
in the first year of life by sensitization to the GROUP CELLS IN PLASMA
environmental substances (food, bacteria, and A A Anti-B
viruses) B B Anti-A
• Naturally occurring (unknown antigenic AB A and B None
stimulation) O None Anti-A and B

ABO TYPING
• ABO typing involves both antigen typing and
antibody detection. The antigen typing is referred
to as the forward typing and the antibody
detection is the reverse typing.
• The forward typing determines antigen on
patient’s or donor’s cells
a. Cells (5% red cell suspension) are
tested with the antisera reagents anti-A
and anti-B (and in the case of donor cells
anti-A,B)
b. Reagents are either made from hyper
immunized human sources, or
monoclonal antibodies.
- or lectins (plant extracts seeds)
c. One advantage of the monoclonal
- Antigen present = phenotype antibodies is antibody strength.
- Antibodies, always the opposite of antigen.
FORWARD TYPING
HISTORY
Reactions of cells tested with Red cell ABO
• Karl Landsteiner discovered the ABO blood
Anti-A Anti-B group
group system in1901.
0 0 O
• He and five co-workers began mixing each other
+ 0 A
red blood cells and serum together and
0 + B
accidentally performed the first forward and
reverse ABO groupings. + + AB

LANDSTEINER’S LAW • The reverse typing determines antibodies in

a) If an agglutinogen is present on red blood cell patient’s or donor’s cells.
membrane, the corresponding agglutinin must be a. Serum tested with reagent A1 cells and
absent in the plasma B cells
b) If an agglutinogen is absent on red blood cell - A1 – increase the specificity of
membrane, then the corresponding agglutinin reaction
must be present in the plasma. b. Reverse grouping is also known as
back typing or serum confirmation.
- Antibody = agglutinin
- Antigens = agglutinogen REVERSE TYPING
Reactions of serum tested Reverse ABO
IMPORTANCE OF ABO against group
• There are two principles A1 Cells B Cells
1. Almost all normal healthy individuals above 3-6 + + O
months of age have “naturally occurring Abs” to 0 + A
the ABO Ags that they lack. These Abs termed + 0 B
naturally occurring because they were thought to 0 0 AB
arise without antigenic stimulation.
o Cannot perform reverse typing for
neonates.
 BLOOD GROUP INHERITANCE H SUBSTANCE
• H gene (FUT1* gene) leads to production of an
enzyme α-2-L-Fucosyl transferase, which
transfers fucose to the terminal galactose of the
precursor Glucose Galactose N-

ABO ANTIGEN GENETICS

• The presence or absence of ABH antigens on the *FUT1 – fucosyltransferase 1
red blood cell membrane is controlled by the H
gene FORMATION OF THE A ANTIGEN
• The presence or absence of the ABH antigens in • A gene codes for an enzyme that adds GalNAc
secretions is indirectly controlled by the Se gene (N-Acetyl-D galactosamine) to the terminal sugar
• H gene – H and h alleles (h is an amorph) of the H Antigen
• Se gene – Se and se alleles (se is an amorph) • Enzyme responsible for the attachment of the
sugar to form A antigen → 1,3-N-acetyl-D
BIOCHEMISTRY galactosaminyltransferase
• Precursor: Paragloboside/Glycan o Attached to fructose
• Type I precursor: terminal galactose linked to a
subterminal N-acetylglucosamine in a 1-3
linkage.
• Type II precursor: same sugars combine in a 1-
4 linkage
• ABH antigens in RBC are derived from Type II
chains
• Blood group substances in secretion are made
from both types I & II precursor.

PRECURSOR STRUCTURE FORMATION OF THE B ANTIGEN

• B gene codes for an enzyme that adds D-
galactose.

- If type 1 → the carbon 1 of galactose is attached

to the carbon 3 of glc NAG
- Type 2 → Carbon 1 of galactose is attached to 4
glc NAG
- Most RBC are type 2 precursor
o Type 2: 80% glycoprotein, 20%
glycolipids • For type O, no conversion of H antigen. The
o Most antigenic greatest amount of H antigen is in O individual.
o Better antigenic reaction than type 1 • No sugar is present for the conversion of H
o Yield a high immunogenic reaction antigen to either A or B.
compared to plasma
- Type 1 are mostly in Plasma BOMBAY GROUP
o 100% glycolipids • The h/h blood group, also known as Oh or the
o Not antigenic reaction Bombay blood group, is a rare blood type. This
 blood (phenotype) was first discovered in
Bombay, now known as Mumbai, in India, by Dr.
Y.M Bhende in 1952.
• The Hh blood group contains one antigen, the H
antigen, which is found in virtually all RBCs and
is the building block for the production of the
antigens within the ABO blood group
o Homozygous lowercase h.
• True type O individual cannot donate to a
Bombay individual although type.
o Blood is not compatible → cause
hemolytic transfusion.

RHESUS BLOOD GROUP SYSTEM

• The Rh blood group system is one of the most
polymorphic and immunogenic systems known in - Antibody is produced when the person who does
humans. not have that antigen.
• It is the most complex system, with over 45 - Lower case d does not mean you have anti-D, it
antigens. means that you don’t have the antigen.
• Discovered in 1940 after work on Rhesus - When a person who have a lowercase d and
monkeys exposed into the uppercase D, he or she will
• RH gene located on short arm of chromosome 1. develop an antibody anti-D → Immune response.
→ HDFN
Rh Antigens
• Rh antigens are highly immunogenic, the D FISHER-RACE
antigen is most potent. • There are 8 gene complexes at the Rh locus.
• D > c > E > C > e (Highly to Rarely immunogenic) • Fisher-Race uses DCE as the order
o 40-45 antigens, but 5 are long • Others alphabetize the genes as CDE
considered to be highly immunogenic. Possible phenotypes:
• Exposure to less than 1mL of Rh-positive red DCe dCe
cells can stimulate Ab production in a Rh- DcE dCE
negative person. Dce dcE
DCE dce
Rh Genetics - Uppercase D → Rh positive
• 2 closely linked genes control the expression of - Lowercase d → Rh negative
ALL Rh antigens (codominant alleles) - Only D are detected in the laboratory because it
o RHD gene – determines the expression is highly immunogenic.
of the D antigen
o RHCE gene – determines the expression WIENER THEORY
of the C,c,E, and e antigens.

Rh Nomenclature
• There are several different systems of
nomenclature that theorize the inheritance of the
Rh system
o Fisher – Race
o Wiener
o Rosenfield
• ISBT number – based on the first to discover.

FISHER-RACE NOMENCLATURE
• CDE terminology - Single prime = c or C
• Most commonly used (i.e. WHO) - Double prime = e or E
• Developed by Ronald Fisher and Robert Race of - Rh = uppercase
England - hr = lowercase
• They theorized that the Rh antigens are
controlled by a complex of 3 set of genes with
closely linked loci (i.e. Dce gene complex codes
for D,c,e antigens.)
ROSENFIELD NOMENCLATURE ▪ To enhance the visualization of
• Antigens are designated by number agglutination.
o Rh1:D o Spin, read, and record
o Rh2:C ▪ If “D” is positive, cells are Rh
o Rh3: E positive
o Rh4: c ▪ If “D” is negative, continue
o Rh5: e testing
o Example: • Consider the Du variant.
▪ D+, C+, E-, c+, e+ is written as • Rh negative means no D
Rh: 1,2, -3,4,5 antigen, does not mean
it has anti-D
o Add 22% albumin and incubate for 20” at
GENOTYPE vs. PHENOTYPE 37degC
• The phenotype is the result of the reaction o Spin, read, and record
between the red cells and antisera o Wash 3X in saline
• The genotype is the genetic makeup and can be o Add AHG, spin, read, and record
predicted using the phenotype and by o If “D” is positive after heat/albumin or
considering the race of an individual. AHG → cells are weak D positive; if
negative, cells are Rh negative; “C”
GENOTYPE SYMBOL Rh(D) STATUS should always be negative
cde/cde rr Negative ▪ Check cells – serves as reactant
CDe/cde R1r Positive and really check if the result is
CDe/CDe R1R1 Positive valid.
cDE/cde R2r Positive o Add check cells to neg. tubes; spin, read,
Cde/cDE R1R2 Positive and record.
cDE/cDER R2R2 Positive
- Lowercase r = absence of the D antigen ISBB PM
- Subscript 1 = uppercase C
- Subscript 2 = uppercase E BASIC TERMS TO REMEMBER:
Clinical Significance Antibodies that are
Rh Phenotype CDE associated with
decreased RBC survival
R1r CcDe
Ab are in IgG form;
R1R2 CcDEe
optimum reaction at 37oC
R1R1 CDe
(body temp)
rr ce
Transfusion reactions
R2r cDEe
HDN
R0R0 cDe
Not clinically significant Antibodies that do not
R2R2 cDE cause red cell destruction
rr" cEe in vivo
RzRz CDE optimum reaction at 4 -
rr' Cce 20oC
- Z = both uppercase C and E Needs in vitro
- 0 = both lowercase C and E identification and analysis
because it can mask
IMPORTANCE OF THE Rh SYSTEM clinically significant
• After the A and B antigens, the D antigen is the antibodies
most important red cell antigen in blood banking Cold reacting Agglutination best
o Not naturally occurring (IgG) antibodies observed at or below
o Clinically significant room temp
• The D antibody can cause transfusion reactions Mostly associated with
and hemolytic disease of Newborn the not clinically
(HDN)/Erythroblastosis fetalis. significant antibodies
Warm reacting Agglutination best
D TESTING antibodies observed at 37oC
• Protocol associated with the
o Add Anti-D to “D” tube; Rh control to “C” clinically significant
tube antibodies
▪ Always use tube test rather than
slide method.
▪ Slide method is much better if
there is Rh view box at 45degC.
SYSTEMS THAT PROODUCE COLD- REACTING o A cells: weaker rxn
ANTIBODIES ANTI-I ANTIBODIES
❖ Anti-I:
LEWIS ANTIGENS o Associated as a cause of Cold
• Soluble antigens produced by tissues and found Agglutinin Disease (similar to PCH)
in body fluids (plasma) o May be secondary to Mycoplasma
• Considered to be a tissue antigen → adsorbed pneumoniae infections
on the red cell surface ▪ Atypical pneumonia
LEWIS INHERITANCE ❖ Anti-i:
• Lewis system depends on Hh, Se, and Le genes o Rare and is sometimes associated with
• Le, h, and se do not produce products infectious mononucleosis (kissing
• If the Le gene is inherited Lea substance is disease)
produced
• Le, H, and Se genes must ALL be inherited to P ANTIGEN
convert Lea to Leb • Similar to ABO system
Examples: • The most common phenotypes are P1 and P2
Le se H Le (a+b-) o P1 – consists of P1 and P antigens
Le Se H Le (a-b+) o P2 – consists only of P antigens
le h se Le (a-b-) • Like the A2 subgroup, P2 groups can produce anti-
le hh se Le (a-b-) P1
• 75% of adults have P1
LEWIS ANTIBODIES P1 ANTIGEN
• Usually occur naturally in those who are Le(a-b-) • Strength of the antigen decreases upon storage
• Other phenotypes RARELY produce the antibody • Found in the secretions like plasma and hydatid
• IgM (may fix complement, becoming hemolytic) cyst fluid (Echinococcus granulosus)
o Cyst of a dog tapeworm
• Enzymes enhance activity
P ANTIBODIES
• May be detected soon after pregnancy because
❖ Anti-P1
pregnant women may temporarily become Le(a-
o Naturally occurring IgM
b-)
o Not clinically significant
• No clinical significance… Why? o Can be neutralized by hydatid cyst fluid
o Le antibodies in a patient can be to reveal more clinically significant
neutralized by the Lewis antigens in the antibodies
donor’s plasma (cancel each other out) ❖ Anti-P
o Do not cause HDN because they do not o Produced in individuals with paroxysmal
cross the placenta (antigens not cold hemoglobinuria (PCH)
developed well in cord blood) Le(a-b-) o PCH – IgG auto- anti- P attaches
complement when cold (fingers, toes). As
I ANTIGENS
the red cells circulate, they begin to lyse
• These antigens may be I or i (releasing Hgb)
• They form on the precursor chain of RBC o This PCH antibody is also called the
• Newborns: i antigen Donath-Landsteiner antibody
• Adults: I antigen ▪ Biphasic hemolysin
• i antigen (linear) converts to I (branched) as the • In vivo, colder temp: can
child matures (precursor chain is more linear at cause hemolysis
birth) at about 18 months • In vitro, warmer temp:
I ANTIBODIES can cause hemolysis
• Most people have autoanti-I (RT or 4oC)
• Alloanti-I is very rare MNSs BLOOD SYSTEM
• Cold-reacting (RT or below) IgM antibody • 4 important antigens (more exist):
• Clinically insignificant o M
• Can attach complement (no hemolysis unless it o N
reacts at 37oC) o S
• Prewarming the tests can eliminate reactivity o S
• Enzymes can enhance detection o U (ALWAYS present when S & s are
• Anti-I often occurs as anti- IH inherited)
• This means it will react at different strengths with • M & N (IgM) located on Glycophorin A
reagent cells (depending on the amount of H • S & s and U (IgG) located on Glycophorin B
antigen on the RBC) • Remember: Glycophorin is a protein that carries
o O cells: strong rxn → no conversion of H many RBC antigens
into A or B
MNSs ANTIGENS SYSTEMS THAT PRODUCE WARM- REACTING
• All show dosage ANTIBODIES
• M & N give a stronger reaction when KELL SYSTEM
homozygous, (M+N-) or (M-N+) • Similar to the Rh system
• Weaker reactions occur when in the • 2 major antigens (over 20 exist)
heterozygous state (M+N+) o K (Kell), <9% of population
• Antigens are destroyed by enzymes (i.e. ficin, o K (cellano), >90% of population
papain) • The K and k genes are codominant alleles on
U (Su) ANTIGEN chromosome 7 that code for the antigens
• The U antigen is ALWAYS present when S & s • Well-developed at birth
are inherited • The K antigen is very immunogenic (2nd to the D
• About 85% of S-s- individuals are U-negative antigen) in stimulating antibody production
(RARE) OTHER KELL ANTIGENS
• U-negative cells are only found in the Black • Other sets of alleles also exist in the Kell system:
population • Analogous to the Rh system: C/c and E/e
• Kp antigens
Frequency of MNSs Antigens o Kpa: low frequency antigen (only 2%)
Phenotypes Blacks (%) Whites (%) o Kpb: high frequency antigen (99.9%)
M+ 74 78 • Js antigens
N+ 75 72 o Jsa (20% in blacks, 0.1% in Whites)
S+ 30.5 55 o Jsb: high frequency (80-100%)
S+ 94 89 KELL ANTIGENS
U+ 99 99.9 • Kell antigens have disulfide-bonded regions on
High- incidence the glycoproteins
antigen • This makes them sensitive to sulfhydryl
reagents:
Can a person have NO MNSs antigens? o 2-mercaptoethanol (2-ME)
➢ Yes, the Mk allele produces no M, N, S, or s o Dithiothreitol (DTT)
antigens o 2-aminoethylisothiouronium bromide
➢ Frequency of 0.00064 or .064% (AET)
Kellnull or K0
ANTI-M AND ANTI-N ANTIBODIES • No expression of Kell antigens except a related
• demonstrate dosage antigen called Kx
• Anti-M and anti-N • As a result of transfusion, K0 individuals can
o IgM (rarely IgG) develop anti-Ku (Ku is on RBCs that have Kell
o Clinically insignificant antigens)
o If IgG, could be implicated in HDN • Rare Kell negative units should be given
(RARE) KELL ANTIBODIES
o Will not react with enzyme treated cells • IgG (react well at AHG)
ANTI-S, ANTI-s, AND ANTI-U • Produced as a result of immune stimulation
• Clinically significant (transfusion, pregnancy)
• IgG • Clinically significant
• Can cause RBC destruction and HDN • Anti-K is most common because the K antigen is
• Anti-U extremely immunogenic
o Will react with S+ or s+ red cells • K, Kpb, and Jsb antibodies are rare (many
o Usually occurs in S-s- cells individuals have these antigens and won’t
o Can only give U-negative blood units develop an antibody)
found in <1% of Black population • The other antibodies are also rare since few
o Contact rare donor registry donors have the antigen
Kx ANTIGEN
MNSs ANTIBODY CHARACTERISTICS • Not a part of the Kell system, but is related
Antibody Ig Class Clinically o Kx antigens are present in small amounts
significant in individuals with normal Kell antigens
Anti-M IgM (rare IgG) No o Kx antigens are increased in those who
Anti-N IgM No are K0
Anti-S IgG Yes • Clinically significant in RBCs and WBCs
Anti-s IgG Yes o If a red cell lacks Kx → abnormal shape
Anti-U IgG Yes (acanthocytes) and reduced in vivo
survival → senescent
 ▪ If senescent → brought to Phenotypes Blacks Whites
reticuloendothelial system for Fy(a+b-) 9 17
disposal Fy(a+b+) 1 49
o If absent in WBCs → Chronic Fy(a-b+) 22 34
Granulomatous Disease (CGD) Fy(a-b-) 68 RARE
▪ Phagocytic cell is able to engulf
the foreign invader but it cannot DUFFY ANTIBODIES
digest due to lack of Kx • IgG
▪ Recurrent bacterial infection • Do not bind complement
• Clinically significant
McLEOD SYNDROME
• Stimulated by transfusion or pregnancy (but not a
• The XK1 gene (on the X chromosome) codes for
common cause of HDN)
the Kx antigen
• Do not react with enzyme treated RBCs
• When the gene is not inherited, Kx is absent
(almost exclusive in White males) The Duffy and Malaria Connection
• Causes abnormal red cell morphologies and • Most African – Americans are Fy(a-b-)
decreased red cell survival:
• Interestingly, certain malarial parasites
o Acanthocytes – spur cells (defected cell
(Plasmodium knowlesi and P. vivax) will not
membrane)
invade Fya and Fyb negative cells
o Reticulocytes – immature red cells
• It seems either Fya or Fyb are needed for the
• Associated with chronic granulomatous
merozoite to attach to the red cell
disease
• The Fy(a-b-) phenotype is found frequently in
o WBCs engulf microorganisms, but
West ad Central Africans, supporting the theory
cannot kill (normal flora)
of selective evolution
REVIEW
KIDD BLOOD GROUP
Cold antibodies (IgM) Warm antibodies (IgG)
• 2 antigens
LIiPMABHN
o Jka and Jkb (codominant alleles)
Naturally occurring
o Show dosage
Anti-Lea Rh antibodies
Genotype Phenotype Whites (%) Blacks (%)
Anti-Leb Kell
JkaJka Jk(a+b-) 26.3 51.1
Anti-I Duffy
JkaJkb Jk(a+b+) 50.3 40.8
Anti-P1 Kidd
JkbJkb Jk(a-b+) 23.4 8.1
Anti-M S,s
JkJk Jk(a-b-) rare Rare
Anti-A, -B, -H
KIDD ANTIGENS Anti-N
• Well-developed at birth
ENZYME ACTIVITY (Papain, Bromelin, Ficin, Trypsin)
• Enhanced by enzymes
Enhanced Destroyed
• Not very accessible on the RBC membrane
Kidd Fya and Fyb
KIDD ANTIBODIES Rh M, N
• Anti-Jka and Anti-Jkb Lewis S,s
o IgG I
o Clinically significant P
o Implicated in HTR and HDN
o Common cause of delayed HTR DOSAGE
o Usually appears with other antibodies • Kidds and Duffy the Monkey (Rh) eat lots of
when detected M&Ns
• Anti-Jk3
o Found in some individuals who are Jk(a-
b-)
o Far East and Pacific Islanders (RARE)

DUFFY BLOOD GROUP

• Predominant genes (codominant alleles):
o Fya and Fyb code for antigens that are
well-developed at birth
o Antigens are destroyed by enzymes
o Show dosage
BLOOD DONOR SELECTION AND SCREENING
Blood
Why You Should Donate Blood? Donors
Transfusion
• There is no substitute for Blood
o Perfluorocarbon – synthetic; given to
individuals who have known subgroups
of ABO Collection Testing
• Every 3 seconds someone needs a blood
transfusion
• Your donation will help save the life of up to 3
Processing Screening
people
o Plasma component
o Cellular component
o Platelet PRE-DONATION INFORMATION
• Blood is the most precious gift anyone can give • Documents to give information to donors
to another person – The Gift of Life. • General advice
o Pre- and post – donation
DONOR SELECTION CRITERIA o Information about giving blood
• Donor selection determines the eligibility of a • Deferral information
donor to donate blood o Self – deferral on the basis of medical
condition
TYPES OF BLOOD DONORS o Self – deferral on the basis of risk
• Voluntary Donors – Donate blood on their own behavior
• Replacement Donors – from within the patient’s • Registration
own family or community • Consent of the donor
• Autologous Blood Donor – are patients who o Donation is not mandated/ obligated
donate their own blood for self • Demographic information
• Apheresis Donor – donate blood components o For contact tracing, if tested positive
through the process of cell separation • Medical history
• Physical examination
Our Primary concern • Laboratory tests
• Safety of Blood Donor & Blood Recipient (patient)
because “Safe Blood” gives life, “Unsafe blood” DONOR SELECTION
gives infections • Donor selection criteria are essential
• To ensure “Blood Safety” o Based on accepted regional/
o Strict “Donor Screening” international practice
o “Testing” of collected blood to WHO • Protect the donor
specified standards o Ensures that it is safe for the donor to
o Strict “cross-matching” of blood samples donate
to ensure safe transfusion • Protect the recipient
o Ensures that any risk of transfusion –
POLICIES AND GUIDELINES: RRSCC transmitted infection or other adverse
• Donor Recruitment effect is minimized
• Donor Retention
• Donor Selection DEMOGRAPHIC INFORMATION
• Blood Collection • So that the donor can be informed of any
• Donor Counselling laboratory test abnormality or can be called for
o Especially for 1st time donors future donation
o Donor’s name
DONOR RECRUITMENT PROGRAM o Father’s/ Husband’s name
An adequate supply of low risk donors require: o Date of birth/ Age
• A donor recruitment program o Gender: Male/ Female
• A dedicated recruitment section with trained o Residential & Office Address with phone
staff like Lab Technologist, Lab specialist, lab numbers
scientist or MLSO

BASIC TRANSFUSION CHAIN

• The quality of the BTS is influenced by the quality
of each of the links
DONOR SCREENING • Have had any dental treatment
• Safe Donor selection involves: • Have taken Aspirin in the past 72 hrs
o Identification of low risk populations o Antagonist for platelets → makes PLTs
o Donor education and recruitment non- functional
o Encouraging self deferral based on • Have diabetes, heart disease or high BP, cancer,
“Deferral Criteria” blood clotting problem or blood disease
o Medical Examination before donation • Have TB, bronchial asthma or allergic disorder,
• Voluntary Non-renumerated Repeat Donor liver disease, kidney disease, fits or fainting
(VNRD) is safest
o Holding a donor card DONOR SHOULD BE IN GOOD HEALTH…
• Age: 18 – 60 yrs
WHO CAN DONATE? • Hgb: not less than 12.5 g/dL
• Age: 18 – 60 years • Pulse: bet. 50 and 100/ minute with no
• Weight: >45 kgs irregularities
• Hemoglobin level: >12 gms/dL • BP:
• Body temp not more than 37oC o Systolic: 100-180 mmHg
• Pulse regular rate between 60 -100/ min o Diastolic: 50-100 mmHg
• BP between 90 – 160 systolic and 60 – 100 • Temp: Normal
diastolic • Body weight:
• Not taking medicines for HT, DM, IHD o ≥ 45 kgs (for 350 ml)
o ≥ 60 kgs (for 450 ml)
MEDICAL INTERVIEW • Both male and female can donate blood
• Give idea to possible exposure of donor • Skin at the venipuncture site should be from any
o Are you at present in good health? lesion or scar of needle. Any pricks may indicate
o When did you last eat? drug use
o Are you taking any medicine?
o Have you been vaccinated recently? TEMPORARILY DEFER THE DONOR IF THERE IS
o Have you suffered a convulsion or mental HISTORY OF…
disorder? • Major surgery – 6 mos
o Have you ever had jaundice? • Minor surgery – 3 mos
o Have you lost significant weight loss in • Donated blood – 3 mos
last six months? • Tooth extraction/ manipulation – 3 days
• Collection of information on health and behavior • Received blood or blood component transfusion
for critical assessment of donors regarding fitness – 1 yr
• What to ask and why • Aspirin – defer if taken w/in last 3 days for
o Maintain confidentiality component preparation/ plateletpheresis
o Good relationship comes from trust • History of malaria for 3 mos after treatment and
o Personal interviews should not be cessation of symptoms
overheard
• Tuberculosis for 5 yrs after complete treatment
o Maintain right of each donor to privacy
and cessation of symptoms
• Medical examination
o Haemoglobin testing PERMANENTLY DEFER DONORS WITH HISTORY
▪ Copper sulfate OF…
• Float: anemic
• Sink: can donate • Heart disease: Coronary disease, Rheumatic
o Weight heart disease
• Cardiac medication, cardiac surgery
SHOULD NOT…
• Hypertension
• Have cough, influenza or sore throat, common
• Endocrine: diabetes, hyperthyroidism
cold at the time of blood donation
• Malignancy
• Have taken any antibiotics or any other
• High risk group donors for HIV infection
medications (allopathic or Ayurveda or Sidha or
Homeo) in the past 48 hours • Chronic renal failure
• Have taken alcoholic beverages in the past 24 • Chronic liver disease
hours • Stomach ulcer
• Be pregnant or breast feeding or donate during • Bleeding tendencies
her menstrual cycles • Severe allergic disorder, Asthma
• Have donated blood in past 3 months • Medications: anticoagulants, immuno-
• Have been treated for rabies in the past one year suppressive, cardiac medications
• Be treated for malaria in the past 3 months
• Have had any immunizations in the last 1 month
ACCEPTABLE CRITERIA
• Donor should meet all the acceptable criteria for
routine whole blood donation however:
o Age of donor: 18 – 50 years
o Weight of donor: > than 55 – 60 kg
o The pre-procedure platelet count should
be more than 150,000 per cubic mm
• Still assessed by the doctor
SHORT PERIOD
• Donor should not have taken aspirin or any other
platelet inhibitor in last 72 hrs
• The donor should not be fasting prior to the
procedure
• Donor should have a prominent and easily
accessible central antecubital vein in at least one
of the arm
• The minimum time gap between two blood
donations should be 12 weeks/ 3 months
• Whole blood donation must be deferred for at
least 72 hrs after plateletpheresis
• In case of re-infusion failure, donor should not
donate whole blood for 12 weeks
PRE-DONATION CARE
• Have a sumptuous healthy meal. Avoid fatty
foods.
• Get a good night’s sleep
• Drink a lot of water
• Avoid alcohol
Donor Care: Before Donation
• Everyone involved in interviewing & counselling
should develop a friendly & tactful approach
that encourages donors to be honest &
accurate in their answers to questions about their
medical history
• The health check should always be handled
professionally so that the donors feel they are in
good hands
• Be sensitive to the donors’ feelings of fear &
embarrassment
• No chatting with other staff & ignoring the donor
Donor Care: During Donation
• Staff must be trained in interpersonal skills and
should always be smart & clean in appearance
with high standard of personal hygiene
• Staff should have pleasant manners & capable of
conversing freely with donors at the time of
donation
• An act of carelessness or lack of professionalism
by staff during or after donation can be
detrimental to the donors coming back again to
donate blood
• Observe the donor for 8 – 10 mins on the donor
chair to prevent adverse reactions
• Observe for another 10 mins in the refreshment
area whilst Donor has refreshment
• Inspect the venipuncture site before the
donor leaves the donor room
“Thank you very much”
• Ask the donor to write his comments/ suggestions
in the donor refreshment register
• Thanks donors with appreciation so that they
are motivated and encouraged to become repeat
regular voluntary blood donor
AFTER DONATION
• Drink plenty of fluids for the next 24 – 48 hrs
• Avoid strenuous physical activities
• If you feel light headed, take rest
• Enjoy the good feeling that you may have saved
4 lives by donating blood
Post Donation Instruction to the Donor Before leaving
the Blood Bank
• Drink more fluid in the next 24 hrs
• Do not smoke for 1 or 2 hr after donation
• Continue with daily routine work but avoid
strenuous exercise e.g. Weight lifting for 24 hrs
• Do not drive for at least half an hour
• Volume lost due to blood donation is replenished
by 48 hrs. Can safely donate again after 3 mos
• Report to blood bank in case of any adverse
reaction
• Document adverse reaction if any on the donor
card and donor register
HEALTH BENEFITS OF BLOOD DONATION
• The joy of saving human lives
• Reduce risk of heart diseases
• Reduce risk of cancer
• Free health check up
BLOOD COMPONENTS, INDICATIONS, &
PREPARATIONS
WHY THERE IS NEED FOR COMPONENT
SEPARATION?
1. Separation of blood into components allows
optimal survival of each constituent
2. Component preparation allows transfusing only
specific blood component that the patient
requires
3. Transfusion of only specific constituent of blood
needed avoids the use of unnecessary
component, which could be contraindicated in a
patient
4. By using blood components, several patients can
be treated with the blood from one donor, giving
the optimal use of every unit of donated blood
5. Use of blood components, supplements blood
supply – adds to the blood inventory
BLOOD COMPONENTS
 Cellular components:
o Red cell concentrate
o Leukocytes – Reduced Red cells
o Platelet concentrate
o Leukocytes – Reduced
Concentrate
o Platelet Apheresis
o Granulocytes Apheresis
 Plasma components
o Fresh Frozen plasma
o Single donor plasma
o Cryoprecipitate
o Cryo-poor plasma
o Plasma derivatives
▪ Albumin 5% & 25%
 Plasma Protein Fractions
o Factor VIII concentrate Immunoglobulins
o Fibrinogen
o Other coagulation factors
o P24: more than 8 hrs but within 24 hrs of
collection
• Factor V and Viii decrease with storage
• Fluid overload
Platelet PACKED RBCS
• Shelf-life: 35 days
• Storage temp: 2-6oC
• QC Requirements: PCV 80% (Range 65-80%)
• Volume: 250-300ml
Contents:
• Red cells: 65-80%
• Plasma: 20-35%
• Some platelets
• White blood cells and anticoagulant preservative
solutions effect: 1 unit RBC raise Hct by 3% and
Hb by 1g/dl
STEPS IN SEPARATION

PREPARATION OF BLOOD COMPONENTS IS • Store the bag at 2-6oC till processed

POSSIBLE DUE TO: 1st
• Multiple plastic packs system
• Refrigerated centrifuge • Place tha bag in buckets of refrigerated
centrifuge and balance the opposite bags
• Different specific gravity of cellular components 2nd • Centrifuge at 5000 x g for 5 mins at 2-6oC
• Red cells SG: 1.08 – 1.09
• Platelet SG: 1.03 – 1.04 • Express approx 3/4th of the plasma in the
• Plasma SG: 1.02 – 1.03 satellite bag
3rd • Keep red cell at 2-6oC and plasma at -30oC
WHOLE BLOOD
• 350 – 450 ml of blood
• 49 – 63 ml of anticoagulant solution • If also preparing platelet concentrates, the initial
• 500 ml of blood: 70 ml of anticoagulant sol’n spin will be light (2-3 mins at 3200 rpm)
• Hct: 36 -44%
• No components removed Recommended by AABB: Do light spin and heavy spin
• Stored @ 1 – 6oC in a blood bank refrigerator • Light spin: 3200 (2-3 mins)
• Shelf life: • Heavy spin: 3600 (5 mins)
o Citrate – phosphate – dextrose (CPD): 21
days Indications:
o CPDA (adenine): 35 days • Patient need urgent operation and has Hb
o AS-1, AS-3, AS-5: 42 days <10g/dl
• Anticipated surgical blood loss >1000 ml
1 unit of Whole Blood: • Other acute loss of blood
• Can increase Hb by 1 g/dl • Replacement fluid blood loss <20% of blood vol
• Rate of Blood Transfusion: 3 ml/kg/hr • Anemia associated with incipient/ established
o For nurses cardiac failure
o Depends on the weight • Hb value <6g/dl
• Patients approaching delivery and has Hb value
Whole blood indications:
<7g/dl
• Acute active blood loss with hypovolemia
• In hereditary hemolytic anemias and
• Exchange transfusion Contraindications betathalassaemia major
o Risk of Volume overload
• Chronic anemia IRRADIATED RED BLOOD CELLS
• Incipient Cardiac Failure • Gamma radiated to kill lymphocytes
• The lack of T cells prevents grafts versus host
Whole blood drawbacks: reaction
• After storage for 24 hrs, platelets and WBC are Indications:
non-functional • Severely immunocompromised patients
o FFP: 6 – 8 hrs after collection
• Lymphoma patients
 • Stem cell/ marrow transplants
• Intrauterine transfusion
• Neonates undergoing exchange transfusion
• Hodgkin Disease
Irradiated RBCs must be given a radiation of at least 25
Gy to the mid-plane of the canister, after which the
expiration date of the product changes to 28 days from
the time of irradiation or maintains the original
outdate whichever comes first.
LEUCOCYTE REDUCED RBCS
Methods of the preparation of Leucocyte-Reduced Red
cells:
• Centrifugation and removing of buffy coat
• Filtration
• Washing of red cells with saline
• Freezing and thawing of red cells
Leukocyte-Reduced RBCs are products in which the
absolute leukocyte count is less than 5 x 106.
Indications:
• Multi transfused patients like thalassemia
• Leukemia
• Aplastic anemia
• Immunosuppressed
• Immunodeficient
• Prevention of recurrent FNHTRs
• Prevention or delay of primary alloimmunization
to HLA antigen
• Prevention of CMV transmission in at risk
individual
o Thrives in lymphocytes
PREPARATION PLATELET CONCETRATE
• Random Donor Platelet: Prepared from 450ml
of Whole blood
• Single Donor Platelet: Prepared by apheresis
Random donor platelet concentrate, prepared from 1 unit
of blood collected from 1 donor
• Volume: 50 ml
• Storage temp: 22-24oC (agitation)
• Shelf life: 3-5 days
• Adult dose: 1 unit/10kg (4-6 units)
• Peads dose: 10-15 ml/kg
• ABO & Rh compatible
• Cross match not necessary
• Manual unit raises 5000-10,000 PLT in adult
Procedure:
Precautions:
• Agitation during storage helps in:
o Exchange of gases
o Maintenance of pH
o Reduce formation of platelet aggregates
• pH should never fall below 6 because:
o Change in shape of platelets from disc to
sphere
o Pseudopod formation
o Release of platelet granules
Platelet Quality Indicators:
• A platelet concentrate should contain a minimum
of 5.5x1010 platelets (in 90% of the sampled units
according to AABB standards) in a volume
routinely between 45 and 65 ml that is sufficient
to maintain a pH of 6.2 or greater at the
conclusion of the 5-day storage period
• When platelet concentrates (usually 4-6) are
pooled using an open system, the storage time
changes to 4 hrs
• A new method of pooling that uses a closed
system allows the pool to be stored for 5 days
from the date of collection
• Apheresis components contain 4-6 times as
many platelets as a PC prepared from whole
blood. They should contain a minimum of 3.0 x
1011 platelets
• Take note for the presence of swirling
o Platelets are functional (discoid shape)
• Platelet components are stored up to 5 days at
20C to 24C with continuous agitation
• During shipping, platelets can be stored without
agitation for up to 24 hrs during the 5-day storage
period
• If platelet bag is broken or opened, the platelets
must be transfused within 4 hrs when stored at
20C to 24C
• RDP - platelet of 70kg adult by 5000 to 10000.uL
• Plateletpheresis - platelet of 70kg adult by
30000 to 60000/ul
GRANULOCYTE CONCENTRATE
• Obtained by apheresis from a family member BLOOD TRANSFUSION REACTIONS
• Stored at 24C - An unfavorable and harmful transfusion related
• Must contain ≥ 1,0 x 1010 in 200 – 600 mL events occurring in the patient during or after
• Must be infused within 24 hrs transfusion of blood components is called
Indications: transfusion reaction
The role of granulocytes transfusion has decreased with:
• The availability of new and better antibiotics PRE-TRANSFUSION TESTING
• The advent of recombinant granulopoeitic growth ABO and Rh (D) Blood Grouping:
factors e.g. granulocyte stimulating factor • Patient’s and donor’s blood sample
Cross matching of blood sample:
FRESH FROZEN PLASMA • Major cross match: Pt’s serum + Donor cells
• Plasma removed from cells within 6-8 hrs of • Minor cross match: Pt’s cells + Donor serum
collectionis rapidly frozen to below
• -30oC temperature COMMON CAUSES OF TR
• Before transfusion it is necessary to thaw at 37oC • Misidentification of the patient
• Once thawed there is rapid deterioration of • Improper sample identification
clotting factors, so use immediately after thawing • Wrong blood issued
• Volume: 200ml • Administration error
• Shelf life: 1 yr • Technical error
• Storage temp: -30oC • Storage error
• Dose: 12-15 ml/kg
Indications: TYPES OF TRANSFUSION REACTIONS
• Single or multiple clotting factor deficiency Immune mediated
• Severe liver disease • Hemolytic TR
• Massive transfusion o Immediate
o Delayed
• DIC
• Non-Hemolytic TR
Preparation of FFP: o Immediate
o Delayed
Non immune mediated
• Immediate
• Delayed

HAEMOLYTIC TR (HTR)
• Hemolytic TR are most severe type of transfusion
reactions and can be categorized into 2 types:
A. Immediate HTR/ Intravascular HTR
B. Delayed HTR/ Extravascular HTR

IHTR or INTRAVASCULAR HTR

• In intravascular transfusion reaction the
hemolysis of red cells takes place within the
circulatory system
Quality Indicators • Hemolysis occur within few min after starting
Fresh Frozen Plasma transfusion (<24 hrs)
• Stored: -18C or colder (-30C, -65C) • Common cause: Incompatibility in ABO
• Volume: 200-250 ml • This type of reaction is mainly due to IgM Ab’s
• Content of 1U/ml clotting factors (anti-A, & anti-B), mediated by the rapid activation
PF24 of complement and is usually associated with the
• Same storage temp transfusion of ABO incompatible blood
• Volume: 150-250 ml
• Decrease in labile factors Signs and Symptoms:
• Fever
CRYOPRECIPITATE • Chills
Quality Indicators: • Hypotension
• Storage temp: --18C for 1 year; thawed at 20C – • Chest and back pain
24C for 6 hrs; pooled for 4 hrs • Nausea
• Volume: 10-25 ml • Dyspnea
• Content: FVIII 80- 120U; fibrinogen 150mg/ml • Vomiting
 • Haemoglobinuria Pathophysiology:
• Acute renal failure
• Pain at transfusion site
• Shock & DIC

DELAYED HTR/ EXTRAVASCULAR HTR

• Extravascular TR are rarely severe and mainly
due to IgG antibodies, e.g. Rh, Kell or Duffy
system
• These ab’s bring about the destruction of red cells
by the macrophages in the spleen or liver
• Haemolysis occurs after few hours or after about
3-7 days of transfusion
• Most common cause: Kidd
Signs and Symptoms:
• Fever
• Chills
• Malaise

URTICARIAL (ALLERGIC) TR
• A type of immediate hypersensitivity reaction
• Allergic signs and symptoms appear within few
minutes of exposure

Causes of urticarial TR
Signs & Symptoms: • The donor’s plasma contain allergens which react
with regain present in patients plasma
• Fall in Hb
• The donor’s plasma contains regain that
• Rise in bilirubin and mild jaundice within 5-7 days
combines with allergens in the patient plasma
of transfusion
• Renal failure (rare)

NON-HAEMOLYTIC TRANSFUSION REACTIONS

Non haemolytic TR can be classified as:
I. Febrile non haemolytic TR (FNHTR)
II. Urticarial (allergic) transfusion reaction
III. Anaphylactic transfusion reaction
IV. Non cardiogenic pulmonary edema (TRALI)
V. Circulatory overload
VI. Graft versus host disease (GVHD)
Signs and Symptoms:
FNHTR • Local erythema
• These reactions are the most common and • Pruritis
account for over 90% of TR • Hives (raised red wheal)
• These are benign, self limiting reaction due to the • Hypotension
presence of ab’s to WBC or PLT antigens and are • Loss of consciousness
usually seen in multi transfused patients • Shock
• These occur within minutes of starting the
transfusion ANAPHYLACTIC TR
• This is severe, life threatening reaction, which
occur in rare patients who are IgA deficient and
have developed anti-IgA ab’s
• These reaction developed quickly – within
minutes of starting the transfusion.
Signs and Symptoms:
• Respiratory Tract – cough, bronchospasm,
dyspnea
• GIT – nausea, vomiting, diarrhea
• Circulatory system – hypotension, syncope
• Skin – generalized flushing, urticaria
TRANSFUSION RELATED ACUTE LUNG INJURY
(TRALI)
• Also known as non cardiac pulmonary edema
• Altered permeability of the pulmonary capillary
bed by activation of complement, histamine
mediated events, or prostaglandins which leads
to fluid accumulation, inadequate oxygenation,
and reduced cardiac return
S&S:
• Acute onset of respiratory distress
• Dyspnea
• Cyanosis
• Fever
• Chill
POST TRANSFUSION PURPURA
• Occur with platelet concentrate transfusion
• Rapid onset of thrombocytopenia due
production of platelet alloantibodies
• Usually in multiparous female
• Duration: 7-14 days from transfusion
• Therapy: corticosteroids
GRAFT VERSUS HOST DISEASE
• Complication of blood component therapy or
bone marrow transplantation
Patients at risk:
• Lymphopenic patients
• Bone marrow suppressed cases
• Fetus receiving intrauterine transfusion
• Newborn infants receiving exchange transfusion
• Congenital immunodeficiency syndrome
S&S:
• Fever
• Rash
• Diarrhea
• Hepatitis
• Liver Dysfunction
• Bone marrow suppression
• Fatal
Therapy
• Corticosteroids
• Prevention
NON IMMUNE NON HEMOLYTIC TRANSFUSION
REACTION
• IMMEDIATE: bacterial overload, circulatory
overload
• DELAYED: iron overload
Bacterial Overload
• Bacterial contamination
• Due to toxins
• Antibiotic can be used for therapy
Circulatory Overload
• More volume of blood transfused
• Cause: fast rate
• Leads to congenital heart failure, pulmonary
edema
• Signs: chest pain, cough, hypertension
Iron Overload
• Long term complication of RBC transfusion
• Also known as transfusion haemosiderosis
• Iron accumulation: affect functions of heart, liver,
endocrine system
• Signs: muscle weakness, fatigue, weight loss,
mild jaundice, anaemia
• Therapy: iron chelating agent
LABORATORY INVESTIGATIONS
• Check all the records to ensure that the correct
unit of blood was transfused to the right patient
• This includes:
o Patient’s details
o Blood requisition form
o Compatibility report
o Labels
to • Examine the patient pre-transfusion & post-
transfusion plasma from EDTA sample for
evidence of free Hb or increased bilirubin
• Pink or red discolouration in post-transfusion
plasma indicates the presence of free Hb due to
red cell destruction
• Yellow discolouration of the sample drawn 6-8
hr after transfusion indicates increased bilirubin
• Check urine (post-transfusion) – 1st sample
INTERPRETATION OF LABORATORY FINDINGS
• If nothing abnormal, indicates that no acute
hemolytic reaction
• If any finding is positive, or clinical finding strongly
suggest a hemolytic reaction, the following
investigations to be done:
1. Repeat crossmatch, testing both pre and post
transfusion sample of the patient against the
sample from the bag by saline/ albumin,
coombs techniques
2. Repeat antibody screening and identification
of patients pre and post transfusion samples
IMMUNOHEMATOLOGICAL/ BLOOD BANKING
PROCEDURES
Methods for determination of ABO group of RBCs:
• Slide method
• Forward Method
• Tube Method
• Reverse Method
• Gel card method
• Microplate method
Red Cell Suspensions for Blood Grouping
• 50%: Slide Method
• 5%: Test Tube Method
• 1%: Gel Technology
• 1%: Microplate
Slide Method for ABO Grouping
Not recommended as a routine method
• Very rudimentary method for determining blood
groups
• CANNOT be used for transfusion purposes as
false positives and negatives do occur. Drying of
 reaction mixture can caue aggregation – false
positive
• Less sensitive, not reliable for weakly reactive
antigens and antibodies
• Can only be used for emergency ABO grouping
or for selection of plateletpheresis donors
Test Tube Method of ABO Grouping
Recommended method
• Allows longer incubation of antigen and antibody
mixture without drying
• Tubes can be centrifuged to enhance reaction
• Can detect weaker antigen/ antibody
Two steps in ABO grouping
• Cell grouping (Forward Grouping)
o Tests the patients red cells with known
Anti-A & Anti-B to determine the antigen
expressed
• Serum grouping (Reverse grouping)
o Test the patients serum with known A &
B cells to determine the presence of
antibody
Microplate Method
• It is ideal for testing large number of blood
samples
• More sensitive to detect weaker antigen- antibody
reactions
• Results can be photographed for archival storage
• Microplate can be incubated & centrifuged
• There is significant saving in time and in the cost
of disposables and reagents
• Microplates are intended to be disposable
however they can be reused after cleaning them
properly making sure that all foreign protein are
removed
• Microplates can be adapted for automation
Column Agglutination Technology
• One card is basically a set of 6 microtubes
• Microtubes contain either Sephadex Gel or glass
microbeads impregnated with antisera
• Antigen-antibody reaction takes place in the
reaction chamber of microtube
• Gel matrix or glass beads act as sieve and allow
free cells (un-agglutinated) to pass through and
settle at the bottom of microtube while
agglutinated cells are trapped in the matrix
FORWARD TYPING
TUBE TECHNIQUE FOR Rh TYPING
• Prepare 5% washed red cell suspension of test
sample
• Take 3 clean test tubes and label tubes 1 & 2 as
“test” and tube 3 as “control”
• Place 1 drop of anti-D (D1) in tube 1 and 1 drop
of anti-D (D2) in tube 2
• Place 1 drop of 22% bovine albumin/ control in
tube 3
• Add 1 drop of 5% test cell suspension to each
tube
• Mix well, centrifuge at 1000rpm for 1 min
• Resuspend cell button & look for agglutination
• Control tube should show no agglutination
• For all RhD negative test on blood donor, D u test
recommended
Monoclonal Anti-D
3 types:
1. IgM anti-D monoclonal reagent
2. Blend of IgM and IgG monoclonal antibodies
reagent
3. Blend of monoclonal IgM and Polyclonal (human)
IgG anti-D
• IgM antibodies are highly specific and saline
reacting equally at RT and 37o C but unreliable for
detection of Weak D
• Blended antibodies are now routinely used and
can be used for detecting Weak D
Weak D
- Inheritance of D genes which result in lower
densities of D Antigens on RBC membranes,
gene codes for less D
Partial D
- Absence of a portion of the total material that
comprises the D antigen (qualitative defect)
- If the partial D patient is transfused with D positive
red cells, they may develop an anti-D alloantibody
to the part of the antigen (epitope) that is missing
Method for Weak D Testing
• Add 1 drop of 10% suspensionof D negative red
cells to a test tube and add 2 drops of Anti D
(blend of IgG + IgM)
• Incubate at 37C for 30 mins
• Wash 3x with normal saline
• Make dry red cell button and add polyspecific
AHG reagent
• Look for agglutination
Results:
If there is agglutination Du Positive
If there is no agglutination Du Negative

Significance of Weak D
Donors
• Weak D testing on donors required
• Labeled as D positive
• Weak D substantially less immunogenic than
normal D
• Weak D has caused severe HTR in patient with
anti-D
Patients
• If weak D due to partial D can make antibody to
portion they lack
• If weak D due to suppression, theoretically could
give D positive
• Weak D testing on patients not required
• Standard practice to transfuse with D negative
patients

DIRECT COOMBS TEST INDIRECT COOMBS

TEST
A type of clinical blood test Another type of Coombs
used to detect the test used to detect
antibodies or complement antibodies produced
proteins attached to the against foreign rbcs
rbcs
Detects antibodies or Detects the developed
complement proteins antibodies against foreign
attached to the rbcs rbcs
Uses washed rbcs w/o Uses serum
plasma
Detects autoimmune Used prior to the blood
hemolytic anemia transfusion and in
prenatal testing of
pregnant women

- With incubation time

AHG contents
• Polyspecific AG reagent contains antibodies to
both IgG and C3
• Monospecific AHG contains antibodies to either
IgG or C3

Coomb’s Check Cells

• When IAT or DAT is negative, the anti-human
globulin (AHG) reagent is free in the tube (it did
find any coated RBCs with which to bind). RBCs
already coated with antibody are added. The free
 AHG should attach to the IgG-coated check cells
and give visible agglutination after centrifugation
• This required step ensures that the AHG reagent
was added and is working properly
Coombs Control Cells
• Rh positive cells coated with anti-D antibodies or
cells coated with the C3 portion of complement
• Coombs control cells will react with the antibody
in the AHG reagent
• The first step in reading hem-agglutination
reactions is inspection of the supernatant for
signs of hemolysis (red/ pink coloration)
Why do we need to identify?
• Antibody identification is needed for transfusion
purposes and is an important component of
compatibility testing
• It will identify any unexpected antibodies in the
patient’s serum
• If a person with an antibody is exposed to donor
cells with the corresponding antigen, serious side
effects can occur

Reagent RBCs
• Screening cells and Panel cells are the same with
minor differences:
o Screening cells
▪ Antibody detection
▪ Sets of 2 or 3 vials
o Panel cells
▪ Antibody identification
▪ At least 10 vials per set
ANTIBODY SCREENING & IDENTIFICATION Antibody Panel vs Screen
- An antibody screen consists of 2 or 3 group O - An antibody panel is just an extended version of
reagent red cells with known antigen phenotypes. an antibody screen
A positive antibody screen means that an - The screen only uses 2-3 cells
unexpected antibody is present in the patient’s
serum. If the antibody screen is positive, the
antibody must be identified by performing an
antibody panel
- Antibody screening test involve testing patient’s Antibody Panel
serum against 2 or 3 reagent red blood cell - An antibody panel usually includes at least 10
samples called screening cells panel cells:
- Screening cells are commercially prepared
group O cell suspensions obtained from
individual donors that are phenotype for the most
commonly encountered and clinically important
red blood cell
- Why group O? does not contain any antigens
- Group O cells are used so tht naturally occurring
anti-A or anti-B will not interfere with detection of
unexpected antibodies
- The cells are selected so that the following Panel
antigens are present on at least one of the cell - Group O RBCs
sample: D, C, E, c, e, M, N, S, s, P, Lea, Leb, K, k,
Fya, Fyb, and Jkb

Autologous control
• Autologous control is considered as part of the Ab
screening, it can be performed in parallel with the
Ab screen and involves testing the patient’s
serum against the patient’s red blood cells
• A positive autologous control is an abnormal
finding and usually means that the patient has a - Each of the panel cells has been antigen typed
positive direct antiglobulin test (DAT) (shown on antigram)
o + refers to the presence of the antigen
Grading Reactions o 0 refers to the absence of the antigen
• Aggregation or hemolysis of test red blood cells
is the visible end point of an Ab-Ag interaction
• Test results should be read immediately after
centrifugation as delays in reading may cause
elution of antibody and false neg results
 DO NOT FORGET…
- ADD CHECK CELLS FOR ALL NEGATIIVE
RESULTS

INTERPRETING ANTIBODY PANELS

• There are few basic steps to follow when
interpreting panels
o “Ruling out” means crossing out antigens
- An autocontrol should also be run with ALL that did not react
panels o Circle the antigens that are not crossed
out
o Consider antibody’s usual reactivity
o Look for matching pattern

- The same phases used in an antibody screen are

used in a panel

Antibody ID Testing
- A tube is labeled for each of the panel cells plus
one tube for AC:

IS Phase
- Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis
- Record the results in the appropriate space as
shown:

(LISS) 37oC Phase

- 2 drops of LISS are added, mixed and incubated
for 10-15 mins
- Centrifuge and check for agglutination
- Record results
AHG Phase
- Wash cells 3x with saline (manual/ automated)
- Add 2 drops of AHG and gently mix
o Centrifuge
o Read
o Record reactions
 Procedure
• To determine the percentage of red blood cells
containing fetal hemoglobin:

• 2000: ave number of RBC/ field

• % → x 50 = volume of hemorrhage
o

HEMOGLOBIN F STAIN
• The acid elution test is employed to assess the
distribution of hemoglobin F in the red blood cell:
• This information is useful in helping to diagnose:
o Hereditary persistence of fetal
hemoglobin
o In determining the presence of fetal red
cells in the maternal circulation during
pregnancy
▪ How many vials of rhogam
should be administered
Reagents and Equipment:
• Fetal cell fixing solution is Ethyl alcohol, 80%
• Fetal cell buffer solution is citric acid- phosphate
buffer, pH 3.2 to 3.3
• Fetal cell stain:
o Erythrosin B (eosin B) stain, 0.1&
o Ehrlich’s acid hematoxylin
• Coplin jars
• Water bath (37C)

Specimen
• Make blood smears from venous blood collected
in EDTA anticoagulant or from the fingertip (toe
or heel)
• For best result blood should be less than 6 hrs
old, although successful staining has been
achieved on specimens refrigerated for up to 2
weeks
• The smears should be fixed within 2 hrs of
preparation