Storage and preservation of the blood.pptx

Preparation,
preservation, and
Storage of blood
and its
components
Dr. Amany M. Elshamy
Lecturer of Biochemistry and
Molecular Diagnostics
PH.D of Biochemistry and
Molecular biology
MLS, AUC, Cairo

Introduction
• The first authentic blood
preservative was
developed by two English
scientists, Loutit and
Mollison in the year 1943
and it was known as
“Acid Citrate Dextrose
(ACD)”.

Introduction
• Sterile collection systems must be used for the collection of
blood and blood components. These must be used by the
instructions of the manufacturer.
• A check must be made before use to ensure that the collection
system employed has not been damaged or contaminated and
that it is appropriate for the intended collection procedure.
• Defects in blood bags must be reported to the supplier and be
subjected to trend analysis.
• Component labeling and information

Component labeling
and information
• Before use, all blood
components must be labelled
with relevant identity
information. The type of label
to be used, as well as the
labelling methodology, must be
established in written
procedures.
• Critical information must be
provided in machine-readable
format to eliminate transcription
errors..

Labelling
• The labelling must comply with the relevant national legislation and
international agreements. The following information must be shown on the
label or contained in the component information leaflet, as appropriate:
• the producer’s identification;
• the unique identity number;
• the name of the blood component;
• the ABO and RhD groups;
• blood group phenotypes other than ABO and RhD (optional);
• the date of donation;
• the date of expiry;
• the name of the anti-coagulant solution;

Biochemic
al changes
in the
stored
blood
RBC
• The viability of the red cells depends on the 2-3-
DPG and ATP levels. The 2-3-DPG level falls
more rapidly than the ATP in the stored blood.
• The oxygen-releasing capacity of the
hemoglobin is directly proportional to the 2-3-
DPG level.
• If the 2-3-DPG level falls, less oxygen is released
to the tissues. The fall in pH of stored blood also
affects 2-3-DPG level.
The other changes which take place in RBC are:
• RBC changes its shape from discoid to
spherical
• Osmotic fragility is increased
• There is a loss of red cell membrane lipids.
Note: 2,3-DPG is the most abundant phosphate
compound in the red cell.

Biochemic
al changes
in the
stored
blood
WBC
• The WBC becomes nonfunctional after
24 hours of storage, but they retain their
antigenic properties and are quite capable
of causing non-hemolytic febrile transfusion
reactions in the recipients.
Platelets
• The platelets stored at 4-6°C lose their
hemostatic properties within 48 hours.
Coagulation factors
• The coagulation factors, mainly, V and VIII
lose their activity within 72 hours of
storage.

Biochemic
al changes
in the
stored
blood
Electrolytes
• The most important biochemical change in
the stored blood is the loss of potassium
from the red cells and the intake of
sodium from plasma.
• The choice of the preservative depends on
the percentage of “posttransfusion survival
of red cells” and its storage period. The
higher the post-transfusion survival and
longer the storage period, the better the
preservative.

Preservation solutions
• Preservation solutions contain impermeant agents, osmotic
molecules that prevent cell swelling, purine
nucleotide precursors (adenine, phosphates, ribose) to sustain
a low metabolic rate, antioxidants (glutathione, vitamin
E), enzyme inhibitors, buffers (e.g., HCO3) and electrolytes
mimicking intracellular (e.g., UW solution) or extracellular (e.g.,
IGL-1 solution) fluids.

• Acid citrate dextrose (ACD).
• Citrate phosphate dextrose (CPD).
• Citrate phosphate dextrose-adenine (CPDA-1)
Preservation solutions

Acid citrate dextrose (ACD)
• The ACD is hardly used as a blood
preservative anymore in any modern
setup, because of its shorter storage
time (21 days) and poor viability of
cells (70%).
• It was used with the glass bottles.

Citrate phosphate dextrose (CPD)
• It was discovered by Gibson, et al that the addition of phosphate
in the ACD anticoagulant increases the post-transfusion survival
of cells to 80% compared to ACDs 70% after 21 days of storage.
• The composition of CPD solution is given below:
• Trisodium citrate (dihydrate) 26.30 gm
• Sodium dihydrogen phosphate 2.28 gm
• Dextrose 25.50 gm
• Citric acid 3.27 gm
• Distilled water 1.0 L
The pH of the solution is 5.6-5.8.

Citrate phosphate dextrose (CPD)
• The citrate in the solution acts as the anticoagulant (0.2%)
citrate can prevent clotting of 2.5 liters of blood.
• Phosphate helps in maintaining the pH of the blood.
• Dextrose provides nourishment to the cells and helps in the
synthesis of ATP.
• The post-transfusion survival of cells depends on ATPs.

Citrate phosphate dextrose-adenine
(CPDA-1)
• It was observed by Simon in 1962 that the addition of adenine to
the CPD solution increases the post-transfusion survival of cells
to 80% and the storage period to 35 days.
• The ATP concentration is higher and the viability is higher in
adenine blood.
The composition of CPDA -1 is given below:
• Trisodium citrate (dihydrate) 26.30 gm
• Sodium dihydrogen phosphate 2.22 gm
• Dextrose (monohydrate) 31.8 gm • Adenine 0.275 gm
• Citric acid 3.27 gm • Distilled water 1 L

Citrate phosphate
dextrose-adenine
(CPDA-1)
• The ratio of preservative to blood
is 14 ml of CPDA-1 solution for
100 ml of blood.
• The amount of preservative
present in 350 ml and 450 ml
capacity bags is 49 ml and 63 ml
respectively.
• The other preservative CPDA-2 in
which the amount of dextrose is
increased to 44.6 gm, improves
the viability of cells further.

Benefits of
RBC
Additive
Solutions
• Extends the shelf-life of RBCs to 42 days
by adding nutrients
• Allows for the harvesting of more plasma
and platelets from the unit
• Produces an RBC concentrate of lower
viscosity that is easier to infuse

Additive
solution
• An additive solution may be defined as a
solution packaged in a blood bag set
which: (I) is contained separately in a
satellite bag, but is attached by tubing to
the primary CPD-containing bag; (2) is
an isotonic solution containing some or
all of the nutrients needed for extended
red cell storage.

Additive
solution
• The commonly used additive solutions
are:
• CPD-SAG.
• CPD-SAGM.
• CPD-Adsol.

Additive solution
• CPD-SAGM
• The SAGM stands for Saline-
Adenine-Glucose-Mannitol.
The four-bag system
introduced by Hogman, et al in
1978, contains a preservative
solution in two bags. Bag 1
contains 63 ml of CPD solution
while bag 2 contains 100 ml of
SAGM solution.

Additive solution
• CPD-SAGM
• 450 ml of blood is collected in the
primary bag containing CPD
solution, the plasma is expressed
after centrifugation to the empty
bag and then the 100 ml of the
SAGM solution is expressed in the
primary bag containing red cells.
The red cells can be stored for 42
days in SAGM.
• The post-transfusion survival of
cells in SAGM is more than 80%.

Additive
solution
CPD-Adsol
• The composition of CPD-Adsol is the
same as of CPD-SAGM, except that, it
contains higher concentrations of
glucose, adenine, and mannitol, which
improves the viability of cells
considerably.

The preparation,
use and quality
assurance of blood
components

1. Preparation
of RBCs
• For the preparation of Red Cell components,
plasma is removed from Whole Blood by
centrifugation.
• Requirements and quality control.

2. Preparation of Red Cells, Leucocyte-Depleted(Red Cells,
LD. )
• Red Cells, Leucocyte-Depleted (LD) is a red cell component derived from Whole
Blood donation, Red Cells or Red Cells, BCR by removing the leucocytes.
• Red Cells, LD contains a minimum haemoglobin content of 40 g. The hematocrit is
0.65-0.75. Red Cells, LD contains less than 1.0 × 106 leucocytes.
Preparation
• Generally a filtration technique is used to produce Red Cells, LD. Leucocyte
depletion within 48 hours after donation is the standard.
Red Cells, LD can be produced:
• by leucocyte filtration of Whole Blood, with subsequent centrifugation and removal
of the plasma;
• by leucocyte filtration of a red cell component.

3. Red Cells, Apheresis
• Definition and properties
• Red Cells, Apheresis (Aph) is a red cell component
obtained by apheresis of a single donor using
automated cell-separation equipment.
• For preparation of Red Cells, Aph, Whole Blood is
removed by an appropriate apheresis machine from
the donor and anti-coagulated with a citrate-containing
solution.
• The plasma is returned to the donor.
• Either one or two units of Red Cells, Aph can be
collected during a single procedure.

4. Platelets, Recovered, Single Unit (Rec,
SU)
• Platelets, Recovered, Single Unit (Rec, SU) is a platelet
component derived from a single Whole Blood donation. It
contains the majority of the original Whole Blood platelet
content, suspended in plasma.
• Platelets, Rec, SU contains more than 60 × 109 platelets.
• Platelets, Rec, SU contains up to 0.2 × 109 leucocytes if
prepared by the platelet-rich plasma method, and up to 0.05 ×
109 leucocytes if prepared by the buffy coat method.
• Platelets, Rec, SU can be used for neonatal and infant
transfusion.
• In order to achieve a ‘standard adult dose’, 4 to 6 units of
Platelets, Rec, SU have to be transfused.

Preparation from platelet-rich plasma
(PRP)
• A unit of Whole Blood, stored for up to 24 hours in conditions
validated to maintain the temperature between + 20 °C and +
24 °C, is centrifuged so that an optimal number of platelets
remain in the plasma and the number of leucocytes and red
cells are reduced to a defined level.
• Platelets from PRP are sedimented by hard-spin centrifugation;
the supernatant platelet-poor plasma is removed, leaving 50-70
mL of it with the platelets. The platelets are allowed to
disaggregate and are then re-suspended in the remnant
plasma.

Preparation from buffy coat
• A Whole Blood unit, stored for up to 24 hours in
conditions validated to maintain the temperature
between + 20 and + 24 °C, is centrifuged so that
platelets are primarily sedimented to the buffy coat layer
together with the leucocytes.
• The buffy coat is separated and processed further to
obtain a platelet concentrate. Single buffy coats diluted
with plasma are centrifuged so that the platelets remain
in the supernatant, but red cells and leucocytes are
sedimented to the bottom of the bag.

5. Platelets, Apheresis
• Platelets, Apheresis (Aph) is a component obtained by
platelet apheresis of a single donor using automated
cell separation equipment, which contains platelets in a
therapeutically effective dose suspended in plasma.
• Platelets, Aph contains a minimum content of 2 × 1011
platelets.
• Platelets, Aph contains a maximum leucocyte content
of 0.3 × 109 cells.

5. Platelets, Apheresis
Preparation
• For preparation of Platelets, Aph, Whole Blood is
removed from the donor by the apheresis machine, anti-
coagulated with a citrate solution and then the platelets
are harvested.
• For use in neonates and infants, Platelets, Aph can be
divided into satellite units under sterile conditions.

6. Plasma, Fresh Frozen (FFP)
• Plasma, Fresh Frozen (FFP) is a component for transfusion or
for fractionation, prepared either from Whole Blood or from
plasma collected by apheresis, frozen within a period of time
and to temperature that adequately maintains the labile
coagulation factors in a functional state.
• It must contain, on average, 70% or more of the value of the
freshly collected plasma unit and at least similar quantities of
the other labile coagulation factors and naturally occurring
inhibitors.

6. Preparation of Plasma, Fresh Frozen
(FFP)
From Whole Blood
• Plasma is separated from Whole Blood that has been collected
using a blood bag with integral transfer packs employing hard-
spin centrifugation and collected preferably within 6 hours.
Alternatively, plasma may be separated from platelet-rich
plasma.
• Plasma may also be separated from Whole Blood that,
immediately after donation, has been cooled rapidly by a
special device validated to maintain the temperature between +
20 °C and + 24 °C and is held at that temperature for up to 24
hours.
• It is prepared from a single-pack Whole Blood donation,

6. Plasma, Fresh Frozen (FFP)
By apheresis
• FFP may be collected by apheresis. The freezing
process must commence preferably within 6 hours of
collection and not more than 18 hours after completion
of the procedure if the unit is refrigerated.
• Freezing must take place in a system that allows
complete freezing within one hour to a temperature
below – 30 °C.

7. Cryoprecipitate
Definition and properties
• Cryoprecipitate is a component containing the
cryoglobulin fraction of plasma obtained by further
processing of Plasma, Fresh Frozen and then
concentrated.
• It contains a major portion of the Factor VIII, von
Willebrand factor, fibrinogen, Factor XIII and
fibronectin present in freshly drawn and separated
plasma.

7. Preparation of Cryoprecipitate
• Plasma, Fresh Frozen is thawed, either overnight between + 2
to + 6 °C or by the rapid thaw-siphon thaw technique.
• After thawing, the component is re-centrifuged using a hard spin
at the same temperature.
• The supernatant cryoprecipitate-poor plasma is then partially
removed.
• The sedimented cryoprecipitate is then rapidly frozen.

7. Preparation of Cryoprecipitate
Preparation
• When Cryoprecipitate is prepared from Whole Blood-derived
plasma, the maximal final volume of the component is 40 mL.
• Alternatively, Plasma, Fresh Frozen obtained by apheresis may
be used as the starting material and the final component can be
prepared using the same freezing/thawing/re-freezing
technique.
• Leucocyte depletion of the starting material and/or virus
inactivation, and/or quarantine is a requirement in some
countries.

8. Granulocytes,
Apheresis
Definition and properties
• Granulocytes, Apheresis is a
component that contains
granulocytes suspended in
plasma and is obtained by
apheresis of a single donor
using automated cell
separation equipment.

8. Granulocytes, Apheresis
• An adult therapeutic dose of Granulocytes, Apheresis contains
between 1.5 × 108 and 3.0 × 108 granulocytes/kg body weight of
the designated recipient.
• Granulocytes, Apheresis has a significant content of red blood
cells, lymphocytes, and platelets.
• Donors of Granulocytes, Apheresis require pre-treatment with
corticosteroids and/or growth factors.
• HLA type is required.
• Granulocytes, Apheresis must be irradiated.

RBCs storage
• Red Cells must be kept at a controlled temperature
between +2 °C and + 6 °C.
• The storage time depends on the anti
coagulant/preservative solution used. For example, the
storage time is 35 days in CPDA-1.
• Validated transport systems must ensure that at no time
during a maximum transit time of 24 hours did the
temperature exceed + 10 °C.

Long-term storage of red cells
• The red cells in a frozen state can be stored for years.
• Freezing damages red cells by intracellular ice formations and
hypertonicity. This can be prevented by the addition of glycerol.
• The glycerol limits the ice formation and provides a liquid phase
for cooling of salts, by permeating the red cells easily during
freezing.

Long-term storage of red cells
(Glycerolisation)
High glycerol solution
• 40% w/v concentration of glycerol is known as high glycerol
solution.
• The red cells are frozen at –180°C over a period of 30 minutes
by mechanical refrigeration and then stored at –40 to –50°C for
3 years.
• Low glycerol solution
• The 20% w/v concentration of glycerol is known as low glycerol
solution.
• The red cells are frozen at –190°C using liquid nitrogen for 2-3
minutes and then stored in the gas phase of liquid nitrogen for 3

Thawing and deglycerolisation
• Since the red cells are stored in frozen state, so, they must
be brought to body temperature. The process is carried out
by keeping the frozen red cells in a water bath at 37°C for 10
minutes. The procedure is known as Thawing.
• The glycerol must be removed properly (Glycerol can cause
hemolysis).
• The principle of deglycerolisation is that frozen red cells are
kept first in hypertonic solution, and then moved to less
hypertonic and finally to isotonic solution.
• Red cells from a donor with sickle cell trait form an insoluble jelly-like mass during
deglycerolization.

Procedure for deglycerolisation
• • The 40% w/v glycerolised red cells are diluted in 12% sodium
chloride buffered to pH 7.2 with 0.15% disodium phosphate and
equilibrated for 5 minutes.
• Wash the red cells in 2 litres of 1.6 % of sodium chloride
solution buffered to pH 7.2 with 0.03 gm% of disodium
phosphate.
• Finally wash the red cells in isotonic glucose solution, which is
prepared in 1 litre of 0.9 gm% of sodium chloride solution,
containing 0.2 gm% of glucose buffered with 0.0 65 gm%
disodium phosphate to a pH of 6.8.
• The shelf life of deglycerolised blood is 24 hours.

Storage of plasma
• Storage of plasma
• The fresh frozen plasma (FFP) is stored at –30°C for 1
year.
• 36 months at below – 25 °C
• 3 months at – 18 °C to – 25 °C

Storage of platelets
• The platelets are stored ( in an agitator) at
room temperature (RT) 20-25°C for 5 days
only.
• More than 5 days of storage of platelets at
RT increases the chance of bacterial
contamination of platelets and the loss of
platelet quality during storage (known as
the platelet storage lesion).

Storage of platelets
• During storage, platelets can become activated, leading to the release of
various substances such as adenosine diphosphate (ADP), thromboxane
A2, and serotonin.
• This activation can result in platelet aggregation and the formation of a platelet
plug, which is essential for clot formation and hemostasis.
• Platelet activation is a normal physiological response to injury or inflammation,
but it can also occur during storage due to various factors such as temperature
changes, exposure to certain chemicals, or prolonged storage time.

Storage of
Cryoprecipitate
• The stability of Cryoprecipitate on
storage is dependent on the storage
temperature. The optimal storage
temperature is below – 25 °C.
• Recommended storage times are:
• 36 months at or below – 25 °C;
• 3 months at – 18 °C to – 25 °C.

Storage and
transport of
Granulocytes
.
Granulocytes, Apheresis are not suitable
for storage and must be transfused as
soon as possible after collection. If
unavoidable, storage must be limited to
the shortest possible period.
The unit must be transported to the
user in a suitable container between +
20 and + 24 °C, but without agitation.

• The correct temperature for shipping RBCs is
• A. 1 to 6C
• B. 1 to 10C
• C. 20 to 24C
• D. 37C
•
• RBC storage times vary with the anticoagulant/preservative
used. Which of the following is properly paired?
• A. Citrate-phosphate-dextrose (CPD): 35 days
• B. Additive solution (AS): 47 days
• C. Citrate-phosphate-dextrose-adenine (CPDA)-1: 35 days
• D. Acid-citrate-dextrose (ACD): 35 days

• which of the following represents a change seen in a unit of
RBCs stored with CPDA-1 at the end of its shelf life?
• A.Percentage of viable cells at 24 hours after transfusion decreases to 71%
• B.Supernatant K+ (potassium ion) concentration decreases
• C.Supernatant pH increases
• D.Red cell 2, 3-DPG increases
• E. Supernatant hemoglobin decreases

• A unit of RBCs is issued to the floor and returned without being
transfused. How long can blood be out of the refrigerator and
still be used for transfusion?
• A.10 minutes
• B.30 minutes
• C.1 hour
• D.4 hours

• The maximal shelf life of irradiated RBCs is :
• A.4 hours
• B.6 hours
• C.24 hours
• D.21 days
• E.28 days

• The preferred method for generating leukocyte- reduced RBC
components is:
• A. Thawing and deglycerolizing a frozen unit
• B.Filtering using a leukocyte-reduction filter
• C.Irradiation
• D.Centrifugation
• E.Washing

• The most common high concentration of glycerol used in the
U.S. for freezing RBCs is:
• A.5%
• B.10%
• C.20%
• D.40%

• The rationale for deglycerolizing frozen RBCs with extensive
washing is:
• A. Glycerol is not approved by the FDA
• B.Glycerol is toxic to kidneys
• C.Glycerol can cause hemolysis
• D.Glycerol can cause anaphylaxis

•
Which of the following choices explains why a unit of blood may
form an insoluble jelly-like mass during deglycerolization?
• A. Inadequate deglycerolization
• B. Bacterial contamination
• C. Insufficient anticoagulant
• D. Inadvertent use of hypotonic saline for washing
• E.Red cells from a donor with sickle cell trait

• According to the AABB standards, the maximum allowable
shelf-life of platelets without gentle agitation is:
• A.1 hour
• B.4 hours
• C.8 hours
• D.24 hours

• According to AABB standards, 90% of the units of random-
donor platelets prepared from whole blood should contain a
minimum of _______ platelets per unit.
• A. 5.5 x 10^ 9
• B.5.5 x 10 ^ 10
• C.5.5 x 10 ^11
• D.3 x 10 ^ 10
• E.3 x 10 ^ 11

• The minimum acceptable pH of platelet units at the end of the
storage period is:
• A. 4.2
• B.5.2
• C.6.2
• D.7.2
• Which of the following is a change associated with platelet
storage?
• A.Decreased H+ concentration
• B.Platelet activation
• C.Change in shape from round to discoid
• D.Increased swirling effect

•
Transfusion of one platelet concentrate (ie the platelets present
in one whole blood donation) into a hematologically stable adult
of average size with no history of transfusion and/or pregnancy
is expected to increase the platelet count by:
• A. 1000 to 5000 /uL
• B.3000 to 5000 /uL
• C. 3000 to 12000 /uL
• D. 5000 to 10000/ uL

• FFP that has been thawed and is being stored at 1 to 6 C should be
transfused within
• A. 4 hours
• B. 6 hours
• C. 12 hours
• D. 18 hours
• E. 24 hours
• Which of the following is true?
• A.To prepare FFP plasma must be separated from red cells within 24 hours
• B.If an additive solution is used, the expiration date for RBCs stored at 1 to 6 C is 42 days
after phlebotomy
• C.To prepare Cryo FFP is thawed at 20 to 24 C
• D.Platelets derived from a unit of whole blood must contain 3 x 10 ^ 11 platelets in 75% of
units released

•
A group B Rh-positive patient requires FFP. Group B FFP is not
available. The substitute component of choice is:
• A. Group O FFP
• B. Group A, Rh negative cryo
• C. Group A FFP
• D. Group AB FFP
• After thawing cryo should be stored at:
• A.42C
• B.37C
• C.20 to 24 C
• D.1 to 6 C

• According to AABB standards each bag of cryo must contain a
minimum of how many Internation Units of Factor VIII?
• A.70
• B.80
• C.100
• D.120
• Cryo contains which of the following?
• A.Factor XI
• B.Protein C
• C.Protein S
• D.Factor XIII

• Which is true regarding cryo?
• A.Is prepared from FFP thawed at 20 to 24C
• B.Once thawed is stored at 1 to 6 C and given within 6 hours of thawing or 4 hours of
pooling
• C.It is prepared by filtering thawed FFP at 1 to 6 C
• D.It is prepared by centrifuging thawed FFP at 1 to 6 C

• Granulocytes are stored at
• A.1 to 6 C without continuous agitation
• B.1 to 6 C with continuous agitation
• C. 20 to 24 C without continuous agitation
• D. 20 to 24 C out continuous agitation
• The shelf life of granulocytes is:
• A.4 hours
• B.6 hours
• C.12 hours
• D.24 hours

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