Blood products:Collection, Processing and Storage of whole human blood, dried human plasma, plasma substitutes

Presented by: STEFFI THOMAS
Assistant Professor
School of Pharmacy, LNCTU
Bhopal
School Of Pharmacy, LNCTU, Bhopal

BLOOD
 Main transport system in the body.
 Blood carries raw materials and finished products from where
they originate to where they are used and transports waste
products to disposal sites.
 Accounts for about 7% of the body weight of a normal adult.
 Blood is composed of plasma and cells suspended in plasma
(RBC, WBC, PLATELETS)
 Plasma is largely made up of water in which many constituents
are dissolved. These constituents include proteins:-
- ALBUMIN: The most common protein in blood.
- Blood clotting proteins made by the liver.

Cont..
 PLASMA
- 55% of blood.
- Liquid part of the blood.
- Plasma transports: Soluble food molecules, Waste products, Hormones,
Antibodies.
 RED BLOOD CELLS (RBCs)
- 5-6 million/ml.
- Transports oxygen.
 WHITE BLOOD CELLS (WBCs)
- The body’s defence.
- Part of the immune system.
- Larger than RBCs.
- Have nucleus.
- 4000-13000 per mm2.
- Two types: Phagocytes and Lymphocytes.

Cont..
 ERYTHROPOIETIN: A protein made by the kidneys
that stimulates red cell production.
 IMMUNOGLOBULINS: Antibodies made by plasma
proteins in response to infections.
 HORMONES: Such as thyroid hormone and cortisol.
 MINERALS: Such as iron and magnesium.
 VITAMINS: Such as folic acid and B12.
 ELECTROLYTES: Such as calcium, potassium and
sodium

Cont..
 BLOOD PRODUCTS: Therapeutic substance prepared from human blood.
 WHOLE BLOOD: Unseparated blood collected into an approved container
containing an anticoagulant preservative solution.
 BLOOD COMPONENT: A constituent of blood, separated from whole
blood such as
o Red blood concentrate.
o Red blood suspension.
o Plasma.
o Platelet concentrates.
o Plasma or platelets collected by apheresis.
o Cryoprecipitate, prepared from fresh frozen plasma: rich in factor VIII and
fibrinogen.
 PLASMA DERIVATIVES: Human plasma proteins prepared under
pharmaceutical manufacturing conditions such as
o Albumin.
o Coagulation factor concentrates.
o Immunoglobulins School Of Pharmacy, LNCTU, Bhopal

INDICATIONS
 Anaemia.
 Major surgical operations.
 Accidents resulting in considerable blood loss.
 Cancer patients requiring therapy.
 Women in childbirth and new born babies in certain cases.
 Patients of hereditary disorders like Haemophilia and
Thalassaemia.
 Severe burn victims.

CONDITIONS FOR BEING A DONOR
A person in good health is accepted as a donor provided that
he or she is:-
 Not suffering from any diseases that can be transmitted by
transfusion. This includes syphilis, malaria, and serum
jaundice.
 Not anaemic-The haemoglobin content of blood should
not be less than 12.5% for females and 13.3% for males.
 Not taking medications which might prove toxic or have
allergic reactions in a patient e.g. antibiotics

Anti-coagulants
Chemical substances which act on the clotting factors and
prevent the formation of blood clot, thus ensures the
fluidity of blood
 Disodium EDTA
 Citrates
 Heparin

Citrates
 The solution most often used as a blood anticoagulant is
known as Acid-citrate dextrose (ACD)
-Sodium acid citrate (2.0 – 2.5 g)
-Dextrose (3.0 g)
-Water for injection (q.s. upto 120ml)
 The citrate prevents clotting by binding the calcium ions
as unionized calcium citrate, thus preventing a vial step of
clotting (by making calcium unavailable for clotting)

Why acid citrate and not normal
citrate?
 The normal trisodium citrate has a very high alkaline pH in
solution which causes considerable caramelisation of dextrose
(darkening) during sterilization and the two solutions have to
be autoclaved separately.
 The acid citrate produces a pH of about 5 and causes little or no
caramelisation.
 The higher concentration (2.5g/120ml) is often preferred
because it more efficiently reduces the formation of small clots.
 Why add dextrose?
- The dextrose delays the hemolysis of the erythrocytes in vitro
and prolongs their life after transfusion. Its important in
making energy available to living cells.

HEPARIN
 Naturally occurring anticoagulant.
 Made by the mast cells of the connective tissue surrounded blood
vessels.
 It inhibits clotting in the circulatory system.
 Occasionally, it is used in blood for transfusion when large volumes
must be given to one patient and the corresponding amounts of
citrate would be harmful e.g. in cardiac surgery
 It quickly less activity in blood in vitro and normal quantities are
effective for about a day.
 ACD on the other hand, prolongs the storage life to three weeks.
 Heparin is expensive and may continue its action even after
transfusion, thus needing administration of neutralizing substances
such as protamine sulphate (thaat reverses the effect of heparin)

DISODIUM EDETATE
 This is also a chelating agent like ACD.
 It has a strong affinity for divalent metals, and thus will
bind to calcium firmly.
 It is sometimes preferred when preservation of blood
platelets is essential.
 ACD is almost as effective as disodium edetate.
 Mostly used as dextrose-edetate solution.
 The survival of red blood cells in dextrose edetate is as
good as in ACD

WHOLE HUMAN BLOOD
 DEFINITION: One unit whole blood contains 450ml of donor
blood plus anticoagulants.
 VOLUME: 450ml.
 STORAGE: between 2-6°C in approved blood bank refrigerator.
Transfusion should be started within 30 minutes of removal from
refrigerator.
 SHELF LIFE: 21 days.
 INDICATIONS:
o Red cell replacement in acute blood loss with hypovolemia
(decrease in volume of blood due to blood loss).
o Exchange transfusion.
 CONTRAINDICATIONS: risk of volume overload in patients with
• Chronic anaemia.
• Congestive heart failure (CHF) –in which heart doesn’t pump
blood as it should do

COLLECTION OF HUMAN BLOOD:-
Blood is collected aseptically (by cleaning the area with antiseptic)
from the median cubital vein (superficial vein) in the front elbow
A sterile needle of a blood transfusion set is carefully and skillfully
inserted into the cubital vein.
The needle delivers the blood to a sterile plastic bag or a plastic bag
or a bottle through a flexible tubing.
This blood is put into a sterile container containing an anticoagulant
solution and the bottle is gently shaken to ensure that blood (420 ml)
and anticoagulant are well mixed, thus preventing the formation of
small fibrin clots

A maximum of 450 ml of blood is taken in one attendance
Immediately afterwards the container is sealed and cooled
to 4-6°C for storage

Cont..
 On standing blood collected should have its cells sediment to
form a dark red layer occupying (about half the fluid volume)
 It should have yellowish coloured top layer (may be clear or
cloudy depending upon the amount of lipid ingested by the
donor before the blood transfusion)
 If the blood has been taken within a short period after heavy
fatty meal, the plasma may be turbid and has a white layer of
fat on its surface.
 The top surface of RBC will be a greyish layer of leucocytes.
 For healthy blood, there should be a sharp line of demarcation
between red cells and plasma.
 If the line of demarcation has an obscure (cloudy, shadowy,
hiding, not clear) appearance, this indicates haemolysis of red
cells and blood becomes unfit for the use.

Cont..
OFFICIAL REQUIREMENTS FOR WHOLE HUMAN
BLOOD:-
 Collected blood should be cooled to 4-6°C immediately after
withdrawal from the donor and should be stored at that
temperature throughout the period of 21 days
 Blood group (ABO type) and the Rhesus factor has to be
determined
 The blood should comply with sterility testing according to the
standards.
 The container bag should be labelled for its blood group and
Rh factor, the volume and anti-coagulant solution used, the
date of collection and expiry date, required storage condition,
the number/code for tracing back the original donor.

Changes in the composition during
storage
 Significant deterioration of clotting factors.
 Thus the infusion of large amount of stored whole blood
results in infusion of undesirable constituent.
 The collected blood may get damaged or hemolysed on
exposing it to heat, freezing temperature, hypotonic and
hypertonic solutions, extreme pH, excessive agitation and
beyond a particular period of time.
 If during collection the blood produces clots or there is
presence of any small clot, they can be removed by having
a filter in the transfusion set.

Contamination by Microbial organisms
 Blood cannot be made free from any microbial contamination
using dry or moist heat sterilization or by filtration.
 Even if it is contaminated by any microbes, it can be prevented
from growth of microbes by storing at a reduced temperature
like 4-6°C.
 Thus a sterile process and procedure should be handled during
collection and storage of blood.
 Sterile and usually disposable equipments and apparatus should
be used during collection of blood.
 Certain microbes (like Gram negative bacteria) can even grow
at a temperature of 4°C
 Based on the number of microbial count, it can cause any
reaction.
 Therefore, it has to be checked before transfusing the blood to
recipients body.

CLOTTING PROCESS OF BLOOD
 Clotting is a natural phenomenon exhibited by our body
when the body tissues get exposed to the environment on
an account of any mechanical injury.
 It is a defence mechanism, where blood oozes out from
the affected area for a particular period and stops,
initiating the formation of entangled mass like structure
which covers the injured area, thus preventing from
further infection.
 There is a list of clotting factors present in blood which in
one way or another favour or react to form the clot.

1.RED BLOOD CELLS
 Red blood cells contain haemoglobin and serve as the primary
agent for transport of oxygen to tissues.
 PREPARATION:- It is prepared by removing most of the
upper plasma layer from whole blood by centrifugation or
sedimentation (have been left standing). Supernatant fluid is
removed by sterile tubes.
 DESCRIPTION:- 150-200ml red cells from which most of the
plasma has been removed.
 INFECTION RISK:-Same as whole blood, so sterile
procedures have to be followed
 INDICATIONS:- Replacement of red cells in anaemic patients
(chronic anaemia).
 Available as Red Cell Concentrates (RCC)

Cont..
 It should not be older than 14 days.
 STORAGE:-At 4-6°C till transfusion takes place.
 Blood from the donor should be checked for its ABO type
group.
 LABEL:-Similar to the official requirements of the whole
human blood, but in addition, the date and time of
removal of plasma should be mentioned.
 Available in 2 types:-
(1)RCC with additive solution
(2)RCC without additive solution

Cont..
 Additive solution-acid citrate dextrose solution
 With additive solutions, RBC’s can be kept refrigerated
for up to 45 days and it reduces the viscosity, hence
making the transfusion easy.
 While in some patients, use of fresh RBC’s is important
(in case of neonatals) to ensure optimum cell function.
 RBC increases oxygen carrying capacity (without
increasing blood volume as plasma may increases allergic
or anaphylactic reactions)

2.CONCENTRATED PLATELETS
 Platelets stop bleeding by forming a plug in case of any injury.
 On centrifugation of human blood, RBC’s sediment while leaving
the plasma at the upper layer.
 The upper plasma layer has got suspended platelets.
 This upper plasma layer can be separated and used for therapy or it
can be subjected to centrifugation at high speed causing
sedimentation of highly concentrated platelets.
 USES:-For therapy of haemorrhage (bleeding) for patients of
thrombocytopenia(low platelet count e.g. in pregnancy or medication
side-effects) and leukaemia (bone marrow disorder i.e. blood
cancer).
 STORAGE:-20-24°C
 SHELF LIFE:-5 days

3.PLASMA
 Aqueous part of blood which can be derived from
separation of whole blood collection.
 Important elements of plasma:-albumin, coagulation
factors, fibrinolytic proteins, immunoglobulin and other
proteins
 Dried human plasma
 Cryoprecipitate (concentrate of cold and insoluble high-
molecular weight plasma proteins like Fibrinogen, Factor
VIII, Factor XIII)
 Fresh frozen plasma (FFP)

Cont..
 On storing blood, the RBC’s start losing their ability to survive due
to disruption of its semi-permeable membrane
 On transfusing a 21 days old blood to a recipients body, about 70%
of the cells remain in the recipients body for 24 hours, but if blood
is older than 21 days the blood will not be assured for its life in
recipients body and is unfit for use, but the plasma proteins are
stable.
 Therefore plasma can be removed and can be used
 These plasma proteins have a property of denaturing on exposure
to heat.
 Care and official requirements should be followed during
processing and storage
 Official requirements of these products include sterility, identity,
freedom from pyrogens, total protein content, electrolyte content,
presence of preservatives and stabilizers.

Cont..
(a)DRIED HUMAN PLASMA (DHP):-Prepared from expired
citrated blood. Uses:-therapy of severe burn, can be given in
case of emergency until the blood group matching is known,
extensive fluid and protein loss.
 The supernatant fluid of centrifuged blood is removed
aseptically.
 This plasma is pooled (accumulated) from donation of blood
selected so that the normal distribution of ABO group is
represented.
 Therefore a 10-donor pool contains plasma from about equal
number of A and O together with atleast on B or AB donation
is done for neutralization of agglutinins if any
 STORAGE OF POOLED BLOOD:- at 4-6°C (sample is
tested for sterility and its not used until it passes the tests)

Cont..
 Then 400ml quantities are dispensed into bottles and subjected
to freeze drying
 Virus transmission:-Virus gets easily transfused. DHP
involves pooling from various donors. Even if one donor is
being affected by virus such as hepatitis etc. it may get
transmitted into the whole pool of blood.
 Plasma agglutinins:-Presence of plasma Agglutinins can
agglutinate respective antigen in the blood and cause hemolysis
to the recipient. Even if the RBC’s have been removed, the
incompatibility of agglutinins remain in RBC free plasma.
This problem can be eliminated by mixing plasma from
different groups in suitable ratios, thus the agglutinin are being
neutralised by plasma soluble agglutinogen thus ensuring
safety of product. The ratio used is:-

Cont..
 General aspects of freeze drying are allowed with special
features of the plasma process
• Preliminary freezing
• Primary drying
• Secondary drying
GROUPS MIXING RATIO (PARTS)
A 9
B 2
AB 2
O 9

Cont..
PRELIMINARY FREEZING:- The bottles are sealed with
efficient closures and then centrifuged at -18°C. The liquid portion
freezes and becomes distributed around the inside of the bottle.
PRIMARY DRYING:- The bottles of frozen material are mounted
horizontally in the drying chamber and high vacuum is applied.
The condensed solid ice sublimes on a condensing coil kept at -
50°C and a small heater provides the latent heat required for
evaporation. This stage takes about 2 days, and resulting product
has moisture content of about 2%.
SECONDARY DRYING:- This is done in another chamber by
vacuum desiccation over phosphorous pentoxide. It takes about a
day, and the . The product is left with about 0.5% of moisture. The
product is then filled with dry sterile nitrogen.

Cont..
 STORAGE OF DHP:-Kept below 20°C and protected
from sunlight, moisture and oxygen
 EXPIRY DATE:- about 5 years
 DHP is a light to deep cream coloured powder
 Stability of the product can be seen by its solubility on
reconstituting the powder with water for injection at room
temperature. It must dissolve completely within 10
minutes.
 It should be used immediately after reconstitution.

Cont..
(b)FRESH FROZEN PLASMA:-
 On centrifugation of whole human blood, the plasma is
collected within a few hours and stored in frozen
condition (-30°C)
 When it has to be used, frozen plasma should be thawed
by immersing in a water bath at a temperature not
exceeding 37°C.
 It has got clotting factors preserved in frozen state
 USES:-for treatment of haemorrhage

4.DRIED HUMAN SERUM
 Prepared by collecting blood without addition of
anticoagulant and is allowed to clot.
Then it is centrifuged and the separated fluid is pooled,
bottled and then subjected to freeze drying
 The ratio of blood groups in the pooling process and all
the other requirements are similar to those of DHP
 They are used similar to the usage of DHP except as a
source of fibrinogen.

5.PLASMA SUBSTITUTES
 The cost of production of plasma and their fractions , their
limited supply, its risk of transmitting blood borne diseases etc.
has led to the seek of plasma and their products of non-human
origin.
 Those synthetic substances should be non-antigenic, non-
toxic, should be easily and completely excreted from body,
should be isotonic with blood.
 They can be called as blood volume expanders or substitutes
for plasma.
 Used to replace abnormal blood loss or plasma or extracellular
fluid. Used to treat patients with established hypovolemia and
surgical blood loss

Ideal properties of plasma substitute
 Should be isotonic with the blood
 Should be free from pyrogens
 Should have similar viscosity as that of blood plasma
 Should not penetrate or diffuse through the blood capillaries.
 They should be metabolized or excreted by the body
 Should get completely eliminated from our body
 Should not produce toxicity
 Should be stable in dry and in liquid form and also during
transport, storage and sterilizing conditions
 Should be easily prepared
 Manufacturing cost should be low

(i)Dextran
 It’s a polysaccharide produced by the bacterium “Leuconostoc
mesenteroides” which is subjected to fermentation in sucrose
containing medium.
 The microorganism produces an enzyme which produces dextrose
from sucrose
 Sucrose Dextrose + Fructose
Dextran sucrase enzyme (secreted by Leuconostoc
mesenteroides)
 In large scale it is manufactured using fermenter.
 When maximum coversion to dextran has been obtained, it is
precipitated by adding a suitable solvent.

Cont..
 Very large molecular weight dextran is unsuitable for use
because:-
i. They produce high viscous solution which is unsuitable
to administer through parenteral route
ii. They may produce hypersensitivity reactions
iii. They don’t produce isotonic solutions with blood.

Cont..
 Reduction in weight of dextran can be done by:-
i. Acid hydrolysis:- by treating dextran with acid and bringing the pH
to 2 by heating at 90°C. This leads to hydrolysis of dextran reducing
its molecular weight and this process is continued to achieve the
required viscosity.
ii. Thermal degradation:- slower than acid hydrolysis process, but
produces better molecules of dextran. Dextran obtained from
fermentation process is heated to 160°C under pressure. Sodium
sulphite is added to prevent oxidative deterioration. Calcium
carbonate is added to neutralize excessive acidity.
iii. Disintegration by ultrasonic waves:- expensive method, where
ultrasonic waves (of required wavelength and frequency for
particular duration of time) are used to break the long chain
polymers into smaller chain of required length.

Effect of molecular weight of dextran
 Dextran having very small molecules (molecular weight between
60,000-100,000) are rapidly excreted in urine but can diffuse into
the tissues causing adverse osmotic pressure.
 Dextran of molecular weight of 100,000-2,50,000 is ideal
 Molecular weight above 2,50,000 can cause allergy and renal
damage.
 Dextran 40 is used to treat severe burns, trauma injuries etc.
 Solution is diluted to a concentration of 5% in either 5% dextrose
injection or sodium chloride injection, packed in sulphur treated
soda-lime bottles and closed with lacquered rubber plugs. Then it
is sterilized by heating in an autoclave.

Purification of dextran
 The dextran obtained from the fermentation is made to undergo
fractional precipitation by treating it with a water miscible organic
solvent (e.g. Alcohol etc.) which does not solubilize this
polysaccharide.
 The fraction obtained has many impurities which can be purified by:-
-Solvent precipitation:- to remove reducing sugar i.e. fructose which is
the by-product of fermentation
-Passage through ion exchange resin for removal of inorganic salts
-Treating with activated charcoal for removal of any colour pigments
-Adsorbing on to asbestos fibres for removal of pyrogens
-Passing the product through sterile membrane filters for removal of
microbes.

(ii)Gum saline
 It is sodium chloride injection and acacia
 DISADVANTAGES:-Signs of liver dysfunction as gum is
not readily metabolized by various organs
INGREDIENTS QUANTITY
SODIUM CHLORIDE 0.9%
ACACIA 6%

(iii)Polyvinylpyrrolidone
 Synthetic colloid
 DISADVANTAGE:-carcinogenic

REFERENCE
 Muthuprasanna P., Dr. Manisha Mishra, “Pharmaceutical
Dosage form and Cosmetic Technology”, Birla
Publications Pvt. Ltd., 1st edition, 2009-2010, pp. 183-204

THANKS

Blood products:Collection, Processing and Storage of whole human blood, dried human plasma, plasma substitutes

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