BLOOD BANKING (IMMUNOHEMATOLOGY) NOTES

HISTORICAL ASPECTS

Pope Innocent VII (1492) first time a blood transfusion was duly recorded in history
Braxton Hicks (1869) recommended sodium phosphate as a nontoxic anticoagulant
Karl Landsteiner (1901) discovered the ABO blood group
Edward E. Lindemann vein-to-vein transfusion of blood using multiple syringes and a
special cannula for puncturing the vein through the skin
Unger developed a syringe-valve apparatus
Hustin sodium citrate
Lewisohn determined the minimum amount of citrate needed for anticoagulation and
determined its nontoxicity in small amounts
Rous and Turner (1916) citrate-dextrose solution
Charles Drew establishment of blood bank system. Appointed director of the American
Red Cross Blood Bank at the Presbyterian Hospital
Loutit and Mollison acid-citrate-dextrose
Gibson citrate-phosphate-dextrose
Lovric (Australia) additive solutions composed of saline, adenine, glucose, trisodium
citrate, citric acid, and sodium phosphate in a standard CP2D
Hogman (Sweden) additive solutions composed of SAG (saline, adenine and glucose) in a
standard CPD anticoagulant. Modified further as SAGM (mannitol protects against
spontaneous storage hemolysis)
Valeri and co-workers rejuvenation solutions

Blood Banking Genetics

Basic Concepts:
a) Genetics study of inheritance. Transmission of characteristics from parent to
offspring.
b) Heterochromatin dark staining bands in the nucleus under a light microscope
c) Euchromatin lighter staining bands found in the nucleus
d) Histones proteins wound around the DNA molecule
e) Chromatin complex of DNA and proteins
f) Mitosis cell division that produces two daughter cells that are identical to the
parent cell
g) Meiosis cell division unique to germinal tissues.
h) Genotype an individuals collection of genes
i) Phenotype observable traits of an individual
j) Alleles alternative forms of the same gene
k) Homozygous having identical alleles (TT, tt)
l) Heterozygous different alleles (Tt, tT)
m) Recessive trait carried but not expressed until it is inherited in the pure/
homozygous form
n) Dominant trait that can be expressed even if it is not homozygously inherited
o) Autosomal a trait not carried by the sex chromosomes
p) X-linked trait carried by sex chromosomes
Mendelian Laws:
a) Law of Dominance In a cross of parents that are pure for contrasting traits, only
one form of the trait will appear in the next generation
b) Law of Segregation during formation of gametes, the two alleles responsible for
trait separate from each other. Alleles for a trait are then recombined at
fertilization, producing the genotype for the traits of the offspring
c) Law of Independent Assortment Alleles for different traits are distributed to sex
cells and offspring independently of one another.
Inheritance Patterns:
a) Autosomal Dominant a dominant gene in autosomal chromosomes can be
expressed even if it is inherited homozygously or heterozygously
b) Autosomal Recessive two copies of the recessive gene in autosomal chromosomes
must be present in order for the trait to be expressed

c) X-linked Dominant If the father carries the trait, all his sons wont be able to inherit
the gene, however all his daughters will inherit the gene. If the mother carries the
gene, transmission is identical to an autosomal inheritance pattern
d) X-linked Recessive characteristically present in the Father but is never passed on
from father to son, instead he passes it on to all his daughters, who then become
carriers.
Population Genetics
Hardy-Weinberg Principle:
- Basic Premises: (a) the population must be large and mating
(b) mutations must not occur
(c) there must be no migration, differential fertility, or mortality
of the genotype
2
- Formula : p + 2pq + q2 = 1.0
Molecular Techniques
A. Immunological Blotting Techniques
a. Northern Blot RNA
b. Southern Blot DNA
c. Western Blot specific proteins
d. Eastern Blot analyze PTM (protein post translational modifications) such as
lipids, carbohydrates, phosphomoeties. It can also be used to analyze
enzymes
B. PCR (Polymerase Chain Reaction)
- An in vitro method for enzymatic synthesis of specific DNA sequences using
two oligonucleotide primers that hybridize to opposite DNA strands and that
flank the region of interest.
- Taq (Thermus aquaticus) thermal stable DNA polymerase obtained from
bacteria that live in hot springs
C. Cloning
- process of producing similar populations of genetically identical individuals
- Cloning Vector extrachromosomal genetic element that can carry a
recombinant DNA molecule into a host bacterial cell.
Blood Banking Proper
Notable Notes on Specimen Requirement and Specimen Processing:
Serum is the specimen of choice for blood bank procedures (because it maintains
complement activity)
The lytic potential of complement is abolished by heating the serum at 56 0C for 30
minutes (heat inactivation)
C1 and C2 are destroyed and C4 is damaged by heat inactivation
Factor B is inactivated by heating at 500
EDTA inhibit the complement activity (2 mg of EDTA : 1ml of serum)
Heparin inhibits the cleavage of C4
Potentiators aim to reduce the zeta potential of red cells
CLASSIFICATION OF
REAGENT
Protein Media
colloidal diluents
that enhance
agglutination of IgG
molecules by
increasing the
dielectric constant
(reduces the zeta
potential)

Reagent

Action

Notes

22% BSA (Bovine

Serum Albumin)

Causes
agglutination by
adjusting the zeta
potential between
red cells
Aggregrates red
cells causing a
closer proximity of
red cells to one
another assisting
in antibody crosslinking

Requires an
incubation period
usually 15-60
minutes

PEG (polyethylene
glycol)

Macromolecule
additive with LISS.
Reduces falsepositive or
nospecific
reactions. More
effective in
detecting weak

LISS decrease the

ionic strength of a
reaction medium
thus reducing the
zeta potential

Enzymes

AHG

Polybrene

Neutralizes the
charge repulsion of
cells

Low Ionic Strength

Solution

Causes a low ionic

strength
environment
causing red cells to
take up the
antibody more
rapidly
Reduces red cell
surface charge

Ficin (figs)
Papain (papaya)
Trypsin (pig
stomach)
Bromelin
(pineapple)
Antihuman globulin

Cross-links
sensitized cells

antibodies
Macromolecule
that can detect
ABO
incompatibility as
well as clinically
significant IgG
alloantibodies
Generally contain
0.2% NaCl,
incubation period
is lessened to 5-15
minutes

Enhances: Rh,
Kidd, P, Le, I
Destroys: Fya, Fyb,
M, N, S

Direct and Indirect

AHG

It is important to note that each unit of whole blood collected contains approx. 450 ml of
blood (1 pint) and 63 ml of anticoagulant
The total blood volume of most adults is 10-12 pints
Donors can replenish the fluid lost from the donation of 1 pint in 24 hours; donor red cells
are replenished within 1-2 months after donation, thus a volunteer donor can donate
whole blood every 8 weeks.
4 components can be prepared from 1 unit of blood (PRBC, Platelets, Plasma, other
clotting factors)
A unit of blood may be stored for 21-42 days (depending on the anticoagulant used)
The donation process consists of three steps:
1. Educational Reading Materials contains information on the risks of infectious
diseases transmitted by blood transfusion
2. Donor Health History Questionnaire designed to ask questions that protect the
health of both the donor and the recipient. Identifies donors who may have been
exposed to infectious diseases
3. Abbreviated Physical Examination BP, pulse, temperature readings
Red Cell Biology and Preservation
RBC 120 days life span
3 Areas of the RBC Crucial for normal survival and function:
a) RBC membrane semi-premeable lipid bilayer supported by a protein meshlike
cytoskeleton. 52% protein, 40% lipids, 8% carbohydrates
- Integral Proteins Glycophorin A,B,C and AE channel (band 3)
- Peripheral Proteins Spectrin, Actin (band 5), Ankyrin (band 2.1), Band 4.1 and
4.2, Band 6, Adducin
Review Tip: Memorize the integral proteins since it is easier to remember,
so that in any exam you wont jumble them up with the peripheral
proteins.
- Internal membrane layer amino phospholipids
- External membrane layer glycolipids and choline phospholipids
b) Hemoglobin
- 95% dry weight of red cell, has a molecular weight of 68,000 and is capable of
allosteric change as it loads and unloads oxygen
- Main/ primary function is gas transport
- Haemoglobin synthesis

*92-95% of adult Hgb is HbA (2 alpha and 2 beta chains); 2-3% HbA 2 (2 alpha and 2
delta chains); 1-2% HbF (2 alpha and 2 gamma)
-

Oxygen Dissociation Curve

Review Tip: mnemonic for shift to the right: CADET, face right (CO2, Acid,
2,3 DPG, Exercise, Temperature). Another tip is to remember that the pH
is always the opposite of the other factors (ex. Inc pH, dec CO2, dec 2,3
DPG, dec temp)
*Shift to the Left = Fetal haemoglobin; Shift to the Right = Adult Hemoglobin
*Shift to the Left = increased pH; Shift to the Right = decreased pH
* The P50 of blood is 26-30 mm Hg
State of Patient
Anemia
Acidosis
Fever
Hypoxia
Exercise
Alkalosis
Abnormal Hemoglobins
Multiple Transfusions

SHIFT TO THE LEFT/

SHIFT TO THE RIGHT
Shift to the RIGHT
Shift to the RIGHT
Shift to the RIGHT
Shift to the RIGHT
Shift to the RIGHT
Shift to the LEFT
Shift to the LEFT
Shift to the LEFT

c) RBC Metabolism
i.
Emden-Meyerhof anaerobic pathway that produces needed RBC energy. Generates
90% of the ATP needed by the cells

ii.

iii.

iv.

Pentose Phosphate generates 10% of the ATP needed by cell. The activity of this
pathway increases following increased oxidation of glutathione or decreased activity
of the glycolytic pathway.
*When the pentose phosphate pathway is functionally deficient, the amount of
glutathione becomes insufficient to neutralize intracellular oxidants thus producing
Heinz bodies (denatured haemoglobin)
Methemoglobin Reductase necessary to maintain the heme iron in the ferrous
state.
*In the absence of NAD, Methemoglobin accumulates causing a loss of oxygen
transport capabilities
Luebering-Rapaport accumulation of 2,3 DPG (affinity for oxygen)

2 Important RBC Characteristics

a. Deformability phosphorylation of spectrin
Loss of Deformability
- loss of ATP leads to a decrease in phosphorylation of spectrin
- accumulation or deposition of membrane calcium
- removed by the spleen (extravascular sequestration)
- spherocytes
b. Permeability permeable to water and anions (can transverse the membrane in less
than a second).
- Impermeable to cations such as Na+ and K+.
- Intracellular-to-extracellular ratios for sodium and potassium are 1:12 and 25:1
respectively
- Calmodulin has been speculated to control the pumps that control permeability
(prevents excessive intracellular calcium build up)

BLOOD GROUPS
A. MAJOR BLOOD GROUPS
I.
ABO most important blood group system
o naturally occurring antibodies
o IgM antibodies, cold reacting antibodies, does not cross the placenta
but very potent!!!
o Antibodies are undetectable due to low titers, but start to become
detectable at the age of 3-6 months; antibody production peaks at the
age of 5-10 years, then declines as age progresses.
o Cord blood is used to type newborns (forward typing)
o Reverse grouping is a unique test for the ABO blood group
o Summary Table for Review Purposes:
BLOOD TYPE
IMMUNODOMINANT SUGAR
ANTIBODY FOUND IN
SERUM
A
N-acetylgalactosamine
Anti-B
B
D-galactose
Anti-A
AB
N-acetylgalactosamine + DNone
galactose
O
L-fucose
Anti-A, Anti-B, Anti-A,B
hh (Bombay)
NA (unable to produce
Anti-A, Anti-B, Antifucosyltransferase)
A,B, Anti-H
RED CELL PRESERVATION
- Provide viable and functional blood components to patients who require blood
transfusion
- To consider a transfusion to be successful, 75% of cells transfused should remain
viable for 24 hours.
- Optimum temperature for storage of blood: 1-60C
- Lesion of Storage dec pH, glucose, ATP; loss of red cell function; inc lactic
acid
*the rate of restoration of 2,3 DPG is influenced by the acid-base status,
phosphorus metabolism and degree of anemia
- Red Cell Preservatives:
NAME
ABBREVIATI STORAGE
pH(initi NOTES
ON
TIME
al)
Acid-citrateACD
21
5.0
The low pH causes most
dextrose
2,3 DPG to be lost early
in the first week of

Citratephosphatedextrose
Citratephosphateadenine
Citratephosphatedouble
dextrose
-

CPD

21

5.6

CPDA-1

35

5.6

CP2D

21

5.6

Additive Solutions:
NAME
ABBREVIATION
Adsol

AS-1

STORAGE
TIME
42

storage
Red cells become low in
2,3 DPG by the second
week
Addition of adenine
seems to increase ADP
levels.
Contains 100% more
glucose than CPD, 60%
more glucose than
CPDA-1. /
pH at 370C

NOTES

6.6

Contains
buffered
adenine,
glucose and
mannitol to
retard
hemolysis. It is
coupled with
CPD as primary
bag
anticoagulant.
Nutricel
AS-3
42
6.5
Employs CP2D
as primary bag
anticoagulant
and 100ml of
buffered
adenine glucose
solution for RBC
viability.
Optisol
AS-5
42
6.5
Uses CPD as
primary
anticoagulant
and contains
adenine,
glucose and
mannitol.
*blood stored in additive solutions is NOT routinely given to newborn infants
*Additive solutions allows extraction of increased volumes of fresh plasma
*Additive solutions allow storage of whole blood at room temp for up to 8 hours
-

Rejuvenation Solutions solutions containing PIGPA (phosphate, inosine, glucose,

pyruvate and adenine) that aim to regenerate both ATP and DPG levels in red
cells stored in CPD, CPDA-1, or AS-1
Rejuvesol only rejuvenating solution that is FDA-approved.
Red cells stored in a liquid state for less than 3 days after expiration can
be rejuvenated for 1-4 hours at 370C in rejuvesol.
Units must be used within 24 hours

Red Cell Freezing used for autologous units and storage of rare blood types. It
involves the addition of a cryoprotective agent to red cells that are less than 6
days old.
*Glycerol is the most commonly used cryoprotective agent
Advantages and disadvantages of Red cell Freezing
ADVANTAGES
DISADVANTAGES
Long term storage (10 years)
Time consuming
Maintenance of red cell viability and function
Costly equipment
Low WBC and platelets
Storage temp (-65)
Removal of significant amounts of plasma
Higher cost of products
proteins
Advantages of High glycerol to Low glycerol
Advantage
High Glycerol (40% w/v)
Low Glycerol (20% w/

Initial freezing temp

Need to control freezing rate
Type of freezer
Maximum storage temp
Shipping requirements
Effect of changes in storage
temp
-

-800C
No
Mechanical
-650C
Dry ice
None. Can be thawed and
frozen again

-1960C
Yes
Liquid nitrogen
-1200C
Liquid nitrogen
Critical

Blood Substitutes
A. Stroma-Free Hemoglobin Solution (SFHS) prepared by hemolyzing
outdated red blood cells and removing all of the contaminating stroma
which may be toxic to the kidney.
B. Chemical Modified Hemoglobin cross-linking and/or polymerizing the
haemoglobin chains can increase dwell time by enlarging the molecule
and inhibiting its break up into smaller sub units which are easily filtered
by the kidney.
C. Recombinant Hemoglobin made using modified human genes expressed
in bacterial cells
D. Encapsulated haemoglobin haemoglobin is enclosed in liposomal
vesicles.
E. Perfluorochemicals (PFC) hydrocarbon structures in which all the
hydrogen atoms have been replaced with fluorine.

PLATELET PRESERVATION
- Platelet Concentrates effectively used to treat bleeding associated with
thrombovytopenia, as well as other disorders in which platelets are
quantitatively and qualitatively deficient.
- Methods for PC Prep:
A. Centrifugation
B. Apheresis
- Done within 8 hours after collection, approximately 50ml of plasma is retained.
- Which must contain a minimum of 5.5 X 1010 platelets. Has a pH of at least 6.0
- PC bags are stored at 20-240C with continuous agitation for 5 days with a pH of
7.0
- Factors: initial pH, temp of storage, total platelet count, volume of plasma,
duration of storage, agitation during storage, and lactic acid ion accumulation
*Platelets stored for several hours at 40C are associated with an irreversible discto-sphere transformation (microtubule disassembly), which is a major
contributor to decreased survival of platelets.
*Platelets progressively change shape from discs to spheres at a pH that falls
from 6.8 to 6.0 (reversible), and when the pH falls below 6.0, this change
becomes irreversible that renders the platelets nonviable after infusion in vivo.