BLOOD AND RELATED PRODUCTS

 The importance of blood in health and disease has been appreciated since ancient times
but blood transfusion was not practiced on a large scale until early this century.
 Previous attempts were often frustrated by clotting and ignorance of the existence of blood
groups; therefore, for centuries bloodletting rather than transfusion remained a pillar of
medical therapy.
 The discoveries of blood groups by Landsteiner in 1900, and the non-toxic anticoagulant
sodium Citrate by Hustin in 1915 paved the way for rapid advances. The demands of the
army for blood in the First World War provided a further stimulus.
 Steady progress was made in the aseptic handling and storage of blood, and at the
beginning of the Second World War a blood transfusion service was set up under the
Ministry of Health.
 This has continued, and now Regional Blood Transfusion Centers collect, examine,
process and store blood and transport it to hospitals as required.
 Human blood and the products obtained from it are not available commercially in the
United Kingdom.
 In England and Wales immunoglobulin is obtained from the Public Health Laboratory
Service, fibrin foam and thrombin from the Blood Products Laboratory at the Lister
Institute, and other products from the Regional Blood Transfusion Centres.

 There are also

the

official preparations is shown in Table 34.1.

(A) Whole Human Blood

 This is human blood that has been mixed with a suitable anticoagulant. Any person in good
health is accepted as a donor provided that he or she-
1) Is not suffering from any disease that can be transmitted by transfusion. This includes
syphilis, malaria, and serum jaundice.
2) Is not anaemic. The hemoglobin content of the blood should not be less than 12.5 and
13.3 per cent for female and male donors respectively. This can be checked on the spot
by allowing a drop of blood to fall into a copper sulphate solution of specific gravity
1·053 (female) or 1·055 (male). If the drop sinks the sample is satisfactory.

Collection
 The blood is collected aseptically from the median cubital vein, in front of the elbow, into a
sterile container containing an anticoagulant solution. During collection the bottle is gently
shaken to ensure that blood and anticoagulant are well mixed, thus preventing the
formation of small fibrin clots.
 Not more than 420 ml is taken at one attendance. Immediately afterwards the container is
sealed and cooled to 4-6°C.
 The equipment used for taking blood is made from plastics, and is disposable. The
container is most often the Medical Research Council blood bottle but plastic bags have
been used in America for some years and are likely to be the containers of the future.

Blood Clotting
 According to classical theory blood clotting takes place in two phases:

 Although it is now known that several

other factors enter into the clotting reaction.
 In response to injury, the tissues and blood platelets free substances that activate the
clot-promoting enzyme thromboplastin.
 This, with the assistance of ionised calcium and other factors, converts prothrombin into
the active clotting enzyme thrombin which acts on fibrinogen, converting it into insoluble
fibrin, the matrix of the clot.
ANTICOAGULANTS
1. Citrates
 The solution most often used as a blood anticoagulant is known as Acid-citrate-dextrose
(ACD) and has the composition-
Sodium acid citrate ______2·0 to 2·5 G
Dextrose _______________3 G
Water for Injections __to_120 ml
 The citrate prevents clotting by binding the calcium ions as unionized calcium citrate. At
one time the normal (tri-sodium) citrate was used but it has a very alkaline pH in solution
 which causes considerable caramelisation (darkening) of the dextrose 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. In
addition, it is less likely to induce flaking of the glass of the container.
 The higher concentration (2·5 G/120 ml) is often preferred because it more effectively
reduces the formation of small clots.
 The dextrose delays hemolysis of the erythrocytes in vitro and prolongs their life after
transfusion. Its function may be connected with the synthesis of compounds, such as
adenosine tri-phosphate (ATP) that are important in making energy available to living
cells.

2. Heparin
 This is a naturally occurring anticoagulant made by the mast cells of the connective
tissue surrounding blood vessels. It inhibits clotting in the circulatory system.
 Occasionally it is used in blood for transfusion when large volumes is required for patient
and the corresponding large amounts of citrate would be harmful, e.g. in cardiac surgery.
 It quickly loses activity in blood in vitro and normal quantities are effective for about a
day.
 ADC, on the other hand, prolongs the storage life to three weeks. Heparin is expensive
and may continue its action after transfusion, necessitating the administration of
neutralizing substances such as protamine sulphate.

3. Disodium Edetate
 This is a chelating agent which has a strong affinity for divalent metals like calcium.
 It is sometimes preferred when preservation of blood platelets is essential, although the
stability of these seems to depend much more on preventing contact with glass surface:
if plastic bag" or silicon treated bottles are used, ACD is almost as effective for this
purpose.
 The survival of red cells in dextrose-edetate solutions is as good as in ACD.

TESTING
 At the time that blood is taken, two small additional amounts are collected. One, which is
often obtained by draining the collecting tube, is put into a small 5 ml bottle and is firmly
attached to the main container.
 This is for testing compatibility with the blood of the recipient before administration; using
a separate specimen avoids the dangerous procedure of attempting to remove a sample
from the main bottle without causing bacterial contamination.
 With a plastic bag it is possible to leave the blood-filled collecting tube attached to the
bag and to seal it at several points with a special tool; then a section can be separated
for testing without contaminating the bulk.
 The second, somewhat larger sample is used as soon as possible: (a) for a serological
test to confirm the absence of syphilis, and (b) to determine the ABO grouping of the
cells and plasma and the Rh grouping of the cells.

BLOOD GROUPS
 Fundamentally, the aim of blood grouping is to prevent an antigen-antibody reaction.
 Red cells carry an antigen that reacts with the corresponding antibody in the plasma of
individuals of certain other groups. If the cells are transfused into an individual with the
equivalent antibody in his plasma they are rapidly destroyed, with serious consequences.
 Although some nine blood group systems, are known, only the ABO and Rh are of major
importance as causes of haemolytic transfusion reactions.

1. ABO System
 The first sign of the haemolytic antigen-antibody reaction is agglutination and, therefore,
red-cell antigen and plasma antibodies are called agglutinogens and agglutinins
respectively.
 The agglutinated cells haemolyse, freeing hemoglobin and other constitutes and causing
jaundice and kidney damage, if the latter is extreme the patient may die. Table 43.2
shows the importance aspects of this system.
 Generally, the recipient's cells are not seriously harmed by the agglutinins in the donor’s
plasma because the latter quickly become diluted in the recipient’s blood.

2.
Rh

System
 The red cells of some individuals carry an antigen that, because it is also found in the
rhesus monkey, is known as the rhesus (Rh) factor.
 If Rh + blood is transfused into an Rh negative recipient production of antibodies to Rh +
cells may be stimulated. If this occurs, subsequent transfusion of Rh + blood to the same
patient will cause a haemolytic reaction.
 In addition, if a foetus is Rh + (from its father), and the mother is Rh negative and has
the Rh + antibody in her blood, either from a previous transfusion of Rh + blood or as a
result of stimulation by antigens of the foetus, the mother's antibodies may cross the
placenta and destroy the foetal erythrocytes. This haemolytic reaction may kill the foetus
or cause the infant to be severely anaemic.
STORAGE
 Apart from short periods for transport and examination, which must not exceed thirty
minutes, blood must be kept at 4 to 6°C until required for use.
 Even at this temperature deleterious changes take place. The leucocytes disintegrate in
a few hours and the platelets in a few days. The red cells show a fall in ATP and other
organic phosphates, a reduction in oxygen-carrying capacity and, due partly to loss of
lipid from their membranes, increased fragility.
 Storage at room temperature even for only a day, seriously reduces post transfusion
survival of the erythrocyte.
 The fitness of blood for transfusion is based on its appearance.
 On standing, the cells sediment, leaving a layer of yellow supernatant plasma.
 If the blood has been taken shortly after a heavy fatty meal the plasma may be turbid
and show a white layer of fat on its surface. On top of the red cells there may be a
complete or partial greyish layer of leucocytes.
 The most important feature, however, is the line of demarcation between cells and
plasma, which must be sharp; if it is obscured by a diffuse red coloration, indicating
haemolysis, the blood is unfit for use.
 Complete haemolysis, if it occurs rapidly, is usually a sign of bacterial infection but its
absence is not confirmation of sterility since certain psychrophilic bacteria, predominantly
pseudomonads and members of the coli-aerogenes group, can grow in blood at
refrigerator temperature without causing haemolysis.
 Many of the organisms isolated from contaminated blood have been capable of using
citrate as their sole source of carbon and as would be expected, this has led to clot
formation.

Uses
 The volume of the blood in the body can be reduced to a dangerously low level by
haemorrhage, shock, burns, and uncontrollable diarrhea and vomiting.
 Haemorrhage and certain diseases may result in deficiency or absence of vital blood
constituents such as red cells, platelets, or clotting factors.
 The transfusion of whole blood can be of great value in all these circumstances but
often, because of the risk of transfusion reactions, it is not used where the need is solely
to make up blood volume but is restricted to haemorrhage and certain diseases where
there is deficiency of the vita oxygen-carrying erythrocytes.
 Normally whole blood is not administered unless the ABO and Rh groups of donor and
recipient are known and a sample of the donor's has been tested for compatibility with
that of the recipient.
 In an emergency, group O, Rh negative blood may be given while the above precaution
are being taken.

(B) Dried Human Plasma

 Whole blood has several serious disadvantages:
1. It has poor keeping properties necessitating use within three weeks.
2. It requires refrigerated storage.
3. It must be compatible with the blood of the recipient.
 Dried plasma, on the other hand, has the following advantages-
1. Properly stored it keeps well for at least five years.
2. If protected from light it can be stored at room temperature provided this is below
20oC.
3. It can be given to patients of any blood group.
 Consequently, in suitable circumstances, dried plasma is used as a substitute for whole
blood.

PREPARATION
Two major problems have to be overcome-
(a) Transmission of Viral Jaundice.
 Viral jaundice is one of the most serious ill-effects of transfusion. There are two types:
infective hepatitis and homologous serum jaundice, distinguished by their incubation
times, approximately five and twenty weeks respectively, and their mortality rates,
approximately 0.3 and 12 per cent respectively.
 However, most infections following transfusion are mild.
 Control is partly effected by refusing to accept donors with a history of jaundice, but not all
cases are recognized and since at present there is no reliable test by which carriers can
be detected, an occasional infected bottle is inevitable.
 Attempts have been made to kill the causative viruses by ·treatment with ultra-violet light
but the method is technically difficult.
 If the preparation of a blood product involves pooling material from a large number of
donors, infection in one or two bottles will be distributed throughout the pool and appear
in each of the units made from it.
 Nowadays the pools used for making dried plasma and serum are limited to not more
than ten donations, and the incidence of jaundice is only slightly greater than when whole
blood is transfused.
 In the past, when pools of 300 or more bottles were made the incidence was 7 to 12
percent.
(b) Neutralization of Plasma Agglutinins.
 Earlier it was stated that the agglutinins in the donor's plasma usually do not damage the
recipient's red cells. Occasionally, however, the plasma agglutinins are very powerful and
can cause serious haemolysis of the cells of the recipient. This means that incompatibility
problems are not entirely eliminated by using products such as plasma and serum that
contain no cells.
 The problem has been overcome since the discovery that red cell agglutinogens also
occur as water soluble forms in plasma, saliva and other body fluids.
 Consequently, by mixing plasma from different groups in suitable proportions the powerful
agglutinins can be cross-neutralized by soluble agglutinogens, producing a preparation
that is safe to transfuse to all groups.
 The most satisfactory ratio for mixing is 9 of A: 9 of 0: 2 of B or AB.
 Dried plasma is usually prepared from time expired citrated blood. The supernatant fluid
is separated, as described under concentrated red blood corpuscles.
 Batches of not more than ten bottles are pooled, choosing the correct ratio of blood
groups to neutralize powerful agglutinins.
 The pools are kept at 4 to 6°C while samples are tested for sterility and no pool is used
unless it passes. Then 400 ml quantities are dispensed into MRC bottles and subjected to
freeze-drying. The following are special features of the plasma process-

Preliminary Freezing
 The bottles are sealed with bacteriologically efficient fabric pads covered by ring-type
closures and then centrifuged at -18°C. The liquid snap-freezes and becomes distributed
around the inside of the bottle in the manner shown in Fig. 24.12.

Primary Drying
 The bottles of frozen material are
mounted horizontally in the drying chamber and a high vacuum is applied. The ice
sublimes on to a condensing coil kept at -50°C and a small heater provides the latent
heat required for evaporation.
 This stage takes about two days, after which the residual moisture content is about 2 per
cent.

Secondary Drying
 This is done in another chamber by vacuum desiccation over phosphorus pentoxide. It
takes about a day, and the product is left with about 0·5 per cent of moisture.
 Each fabric seal is then replaced by an MRC type closure perforated by a plugged
hypodermic needle.
 The bottles are returned to the secondary drying chamber, re-evacuated, and then the
vacuum is broken with dry sterile nitrogen.
 Finally, the needles are removed and the closure is protected with a sterile viscose cap.
STORAGE
 Dried plasma, kept below 20°C and protected from light, moisture, and oxygen, remains
usable almost indefinitely, although it is customary to impose an arbitrary expiry date or
about five years.
 Its fitness for use is shown by its solubility when reconstituted in a volume of Water for
Injections, Sodium Chloride Injection or a solution containing 2·5 per cent dextrose and
0.45 per cent sodium chloride, equivalent to the original volume of plasma.
 It must dissolve completely within ten minutes at room temperature. Gel formation or
incomplete solution indicates deterioration. After reconstitution it must be used
immediately.

USES
 Reconstituted plasma is a satisfactory alternative to whole blood in conditions where
there is no loss of red cells. It is of particular value in the treatment of severe burns and
scalds where, because of extensive fluid and protein loss, there is considerable haemo-
concentration.
 It may also be given when blood is more appropriate; either because whole blood is
unavailable or, in emergency, until the results of matching tests are known.
 Because of its long storage life at a convenient temperature dried plasma is more suitable
than blood as a reserve stock in a small hospital or a remote community.

(C) Plasma Substitutes

 The limited supplies of plasma, the cost of-producing the dried form and the risk of
transmitting serum hepatitis stimulated attempts to find substitutes of non-human origin
that could be used to restore the blood volume temporarily while the recipient replaced
the lost protein.

Properties of an ideal plasma substitute

1. The same colloidal osmotic pressure as whole blood. ·
2. A viscosity similar to that of plasma.
3. A molecular weight such that the molecules do not easily diffuse through the capillary
walls.
4. A fairly low rate of excretion or destruction by the body.
5. Eventual and complete elimination from the body.
6. Freedom from toxicity, e.g. no impairment of renal function. ,
7. Freedom from antigenicity, pyrogenicity, and confusing effects on important tests such
as ·blood grouping and the erythrocyte sedimentation rate.
8. Isotonicity in solution, equal to that of blood plasma.
9. High stability in liquid form at normal and sterilizing temperatures and during transport
and storage.
10. Ease of preparation, ready available and low cost.
GUM SALINE
 This is a synonym for Injection of Sodium Chloride and Acacia, which was official in the
1932 British Pharmacopoeia.
 In the First World War, Bayliss experimented with soluble starch, dextrin, and gelatin as
plasma substitutes and finally used 6 per cent acacia in 0·9 percent sodium chloride
solution.
 It was transfused extensively until signs of liver dysfunction disclosed that the gum was
not metabolized but stored in various organs.

POLYVINY-LPYRROLIDONE
 In the Second World War the Germans introduced a synthetic colloid, polyvinylpyrrolidone
for the treatment of shock.
 It was marketed in this country in the 1950s but was later withdrawn because of
suspected carcinogenicity.

DEXTRAN
 To date this is the most satisfactory plasma substitute. It is a polysaccharide produced
when the bacterium Leuconostoc mesenteroides is grown in a sucrose-containing
medium.
 The organism secretes an enzyme that converts sucrose to dextran according to the
following equitation:

 Different strains produce dextran

of two main groups-
1. Ling practically unbranched chains of glucose units joined by 1 : 6 glucosidic linkages.
2. Highly branched polymers consisting of short chains of 1 : 6 units joined by 1 :4 and
1 : 3 linkages to branches.
 Branched chains are more likely to give rise to allergic reactions when injected, and in
dextrans used for plasma substitutes the linkages should be almost entirely of the 1:6
type.
 This is achieved by choosing a suitable specially developed strain of the organism that
produces dextran in which about 95 per cent of the linkages are 1:6.

Production
 The manufacture of dextran is similar in many respects to the production of an antibiotic
which involves laboratory culture followed, in the factory, by growth in seed tanks and
then in 4500 dm3 fermenters.
 Because synthesis of the enzyme and its action on the sucrose are rapid, the high degree
of asepsis maintained in antibiotic fermentations is unnecessary.
 Also, as the process is inhibited by aeration, there is no need for a costly supply of sterile
air.
 Another special feature is the need to prevent the hydrolysis of the sucrose to glucose
and fructose during sterilization of the culture media. If this occurs dextran will not be
produced because in nature the conversion does not involve inversion but is a straight
transglycosidation.
 Preventative measures include adjustment of the media to neutral pH before sterilization,
and the avoidance of overheating.
 When maximum conversion to dextran has been obtained it is precipitated by adding a
suitable organic solvent.
 Natural dextran consists of chains of approximately 2,00,000 glucose units with molecular
weights up to about 50 million. Very large molecules, i.e. those with molecular weights
above about 2,50,000 have serious drawbacks-
1. They yield very viscous solutions that are difficult to administer.
2. They may cause renal damage and allergic reactions
3. They interfere with blood matching and sedimentation test by causing rouleaux
formation. Rouleaux are aggregates of red cells that resemble piles of plates.
4. They produce colloidal osmotic pressures that are lower than those of small
molecules.
 Therefore, to produce a material suitable for medical use it is necessary to reduce the
size of the natural molecules. This may be done in several ways:

(a) Acid Hydrolysis: The dextran is adjusted to a pH of 2 and is heated at 90°C. As hydrolysis
proceeds, the preparation becomes less viscous and the reaction is stopped at the required
viscosity.
(b) Thermal Degradation. A solution of dextran is heated under pressure at 160°C in the
presence of sodium sulphite, to prevent oxidative deterioration, and calcium carbonate, to
neutralize acidity. The method is slower than acid hydrolysis but the yield of preferred
molecules is better and fewer reducing groups are produced.
(c) Ultrasonic Disintegration. Bombardment with ultrasonic waves splits the molecules into
fragments of approximately the same size and the product is clinically acceptable, unlike the
material from the previously mentioned methods, which requires considerable fractionation.
Unfortunately, this technique is much more expensive to use.
(d) Seeding the Fermenter. If a low molecular weight dextran is included in the culture
medium before fermentation the organism will use it as a template on which to build more
glucose molecules. The average molecular weight of the product is much lower than if no
template dextran is provided.

 Acid hydrolysis seems to be the method most often used. The hydrolyzed product
contains molecules ranging from 10,000 to 1 million in molecular weight. The very small
 molecules, i.e. those of below a molecular weight of about 60,000, also have
disadvantages:
1. They are rapidly excreted in the urine.
2. They pass into the tissue fluid’s causing an adverse osmotic pressure.
 Therefore, the product should contain the minimum of molecules of molecular weight less
than 60000.
 Molecules with molecular weights between approximately 60,000 and 1,00,000 are not
excreted in the urine but they diffuse fairly rapidly into the tissues.
 This suggests that the ideal fraction should have a majority of molecules within the range

1,00,000 to 2,50,000 with a bias towards the lower end.

 However, the American type of dextran has an average molecular weight of about
75,000. The aim was to restore the colloidal osmotic pressure quickly and to ensure fairly
rapid elimination of the foreign colloid from the body.
 The latter advantage, combined with a reduction in rouleaux formation is considered to
outweigh the disadvantage of the high dosage necessary to compensate for excretion
losses.

 The British dextran of the 1963 British Pharmacopoeia (Dextran 150 Injection) had an
average molecular weight of about 1,50,000 and produced a more prolonged effect due
to the preponderance of larger molecules that are not lost from the blood vessels.
 This preparation caused rouleaux formation, a condition known as sludging because it
slows the flow of blood in the capillaries and post-capillary veins.
 To overcome the problem, which is less evident with lower molecular weight fractions, the
1968 British Pharmacopoeia has replaced Dextran 150 with Dextran 110, which has an
average molecular weight of 1,10,000.

 To obtain the clinical ranges of molecular weight, the neutralized hydrolysate is subjected
to a long process of fractional precipitation.
 A water miscible organic solvent, in which the polysaccharide is insoluble (e.g. acetone or
alcohol) is added under very carefully controlled conditions and the required fraction is
gradually separated by repeated retreatment of -either precipitate or supernatant fluid.
 Cost decides the narrowness of the fraction finally accepted.

 In all samples, because of entrainment of one fraction with another, there are small
proportions of very large and very small molecules.
 The selected fraction still requires considerable purification to remove-
 (a) Reducing sugars-by further solvent precipitation. The main contaminant is fructose,
the by-product of the fermentation.
(b) Fractionation solvents-by evaporation under reduced pressure.
(c) Inorganic salts- by demineralization in a mixed bed ion exchanger. It is particularly
important to remove phosphates because they cause precipitation during sterilization
and storage.
(d) Colour-by adsorption on to activated charcoal.
(e) Pyrogens-by adsorption on to asbestos, or cellulose derivatives.
(f) Microorganisms-by filtration. Between each treatment the preparation is passed
through a fibrous pad and just before bottling a membrane filter is used.
 The solution is diluted to a concentration of 5 percent, in either 5% Dextrose injection or
sodium chloride injection, packed in sulphur treated soda lime bottles and closed with
lacquered rubber plugs. Finally, it is sterilized usually by heating in an autoclave.