BLOOD PRODUCTS
Syllabus:
Blood products and plasma substitutes: Historical background, collection and storage of
blood, whole human blood and products
obtained from it including plasma constituents such as fibrinogen, thrombin,
globulins and albumins. Methods used for these and packages employed for them.
Quality control of blood and its constituents: Plasma substituents, their properties and quality
control.
Questions:
1. Describe official (I.P.) blood proucts [8] (2001)
2. Collection, labeling and storage of whole human blood.
3. Labeling and storage of blood products. (2000)
4. What products are obtained from normal human blood?
State briefly the standards set in collection and packaging of blood products.
HISTORICAL BACKGROUND
Blood transfusion was not practiced on a large scale
until early this century. The main problem was blood-clotting and the ignorance
of existence of blood groups.
The milestones:
·
1900: Landsteiner discovered blood groups
·
1915: Hustin discovered that sodium
citrate acts as a non-toxic anticoagulant.
The demands of the army for
blood in the World War-I provided a further stimulus. After that steady
progress was made in the aseptic handling and storage of blood.
BLOOD PRODUCTS
Blood (a)
 |
Red blood corpuscles (b) Plasma
(c) |
Clotting factors Serum (d)
 |
Fibrinogen (e) Thrombin (f)
Immunoglobulin (g)
(g - globulin)
Fibrin
(Clot)
(h)
Official products:
(a)
Whole human blood
(b)
Concentrated
human red blood corpuscles
(c)
Dried human
plasma
Human
plasma protein fraction
Dried
human plasma protein fraction
(d)
Dried human serum
(e)
Human fibrinogen
(f)
Human thrombin
(g)
Human normal
immunoglobulin injection
(h)
Human fibrin foam
WHOLE HUMAN BLOOD
COLLECTION OF BLOOD
Selection of blood donor
Any person in good health is accepted as a donor
provided he or she –
(i)
Is not suffering
from any disease that can be transmitted by transfusion. This includes
syphilis, malaria, serum jaundice and HIV.
(ii)
Is not anaemic.
The haemoglobin content of the blood should not be less than 12.5 in female
and 13.3% in male donors.
Collection
The
blood is collected aseptically from the median cubital vein, infront of the
elbow, in a sterile container containing an anti-coagulant solution. During
collection the bottle is gently shaken to ensure that blood and anti-coagulant
are well mixed, thus preventing the formation of small fibrin clot.
Not more than 420ml is taken
at one attendance. Immediately afterwards the container is sealed and cooled to
4-60C.
Anticoagulant
Blood clotting:
According to the classical theory blood cloting takes
place in two main phases:--
Thromboplastin, Ca++.
1. Prothrombin Thrombin
Thrombin
2. Fibrinogen Fibrin
After an injury, the tissues and blood platelets
releases substances that activate the clot promoting enzyme thromboplastin.
Thromboplastin with the help of calcium ion and other factors converts
prothrombin into the active enzyme thrombin. Thrombin acts on fibrinogen,
converting it into insoluble fibrin. Among the anticoagulants three
preparations are used:
(1) Citrate [Acid Citrate Dextrose (ACD)]
Composition Sodium acid citrate 2.0 to 2.5 gm
Dextrose 3.0
gm
Water for injection upto 120 ml
MOA The citrate
binds with Ca++. Thus clotting is stopped. Here citrate acts as a
chelating agent.
 |
N.B. Previously trisodium citrate was used but it
has very alkaline pH in solution which causes darkening (caramelisation) of the
dextrose during sterilization. Hence the two solutions were sterilized
separately. The acid citrate has a pH of 5.0 and causes little or no
caramelization. In addition, it is less likely to induce flaking of the glass
of the container.
The dextrose provides a
substrate for glycolysis, thus extending the storage life of RBC. ACD solution
can store the blood up to 21 days.
(3) Citrate Phosphate
Dextrose Solution (CPD)
Composition Citric acid (C6H8O7)
Sodium
citrate (C6H5Na3O7.2H2O)
Sodium
hydrogen phosphate (NaH2PO4.H2O)
Dextrose
(C6H12O6.H2O)
Water
for injection
Citrate ions chelates the
calcium ions, thus preventing coagulation of blood.
Citric acid, sodium citrate
and sodium hydrogen phosphate are in the proportions to buffer the solution at
a pH 5.0 to 6.0.
Dextrose provide a substrate
for glycolysis and increases both storage and post transfusion lives of blood
cells.
The expiration time for whole
blood with CPD solution is 21 days.
(2) Heparin
This is naturally-occurring
anticoagulant made by the mast cells of the connective tissue surrounding blood
vessels.
Advantages
·
It inhibits clotting
in the circulatory system.
·
It is used
occasionally in blood transfusion, especially when large volume of blood must
be given to the patient and where large amount of citrate is harmful e.g. in
cardiac surgery.
Disadvantages
·
It quickly loses
its activity in vitro and normal
quantity is effective for about a day.
·
Heparin is
expensive
·
It may continue
its action after transfusion. Some time it is necessary to administer some
neutralizing substance (of heparin) such as protamine sulfate.
(3) Disodium
edetate
This is a chelating agent that
binds with divalent Calcium ion firmly.
Advantages
·
It is some time
preferred when preservation of blood platelets is essential, although the
stability of these seems to depend much more on preventing contact with glass
surfaces.
·
The survival of
red blood corpuscles in dextrose-edetate
solution is as good as in ACD.
Testing
When the blood is collected two additional blood
samples are also collected. The collecting tube is drained and blood is
collected in a 5ml bottle to avoid bacterial contamination during drawing the
blood from the main container. If it is a plastic bag then two knots are given
on to the tube. The in between parts of the tube is cut and the blood sample is
taken out. These samples are kept for the following tests:
A.
First blood
sample is used for testing the compatibility with the blood of the recipient
before administration.
B. The second sample is used for (a) serological tests to
confirm the absence of syphilis, malaria, hepatitis-B and HIV and (b) to
determine the ABO grouping of the cells and plasma and the Rh grouping of the
cells.
Storage
Apart
form short period of transport and examination, which must not exceed 30
minutes, blood must be kept at 4 to 60C until required for use.
Untoward changes that may occur at room
temperature:
1.
The leukocytes
disintegrate in a few hours and platelets in a few days.
2.
The red cells
shows a fall in ATP and other organic phosphates, its oxygen carrying capacity
reduces, the membrane of the red cells become fragile due to partial loss of
lipid from their membrane.
Simple tests for fitness of blood for
transfusion:
1.
On standing, the
cells sediment, leaving a layer of yellow supernatant plasma. The line of
demarcation between the cells and plasma must be sharp. If it is obscured by a
diffuse red coloration it said that haemolysis has taken place and the blood
becomes unfit for use.
2.
If complete
haemolysis occurs it indicates a bacterial infection.
3.
Some times
haemolysis may not be found if stored in 4 to 60C. In this case pseudomonas
and aerobic coli groups may be found to grow in blood in refrigerated
condition.
Use
In
the following conditions whole human blood transfusion is carried out:
1.
In hypovolemia:
When the volume of blood is reduced to a dangerously low level by haemorrhage,
shock, burns, uncontrollable diarrhoea and vomiting.
2.
Haemorrhage
and certain other diseases may result may result in deficiency of red cells,
platelets or clotting factors.
3.
If the
requirement is only for increasing the blood volume then whole blood is not used.
CONCENTRATED HUMAN RED BLOOD CORPUSCLES
Method of preparation:
Whole
blood with anticoagulant (citrate) The blood must be less than 15
days
old.
Allowed
to stand or centrifuged The cells are sedimented and
plasma remains as supernatant 
fluid.
More
than 40% of the supernatant fluid
is
siphoned off through sterile tubes
under
strict aseptic conditions.
Storage: There is a risk of bacterial contamination, hence
the product should be used within 12 hours.
Administration: The cells are matched with the recipient’s plasma
before administration and haemoglobin content must be above 15.5%.
Uses:
1.
When
administration of whole blood may increase the volume of blood (which is not
intended) then only concentrated red cells are given e.g. in chronic anaemia.
2.
In infants if whole blood is tranfused
then large amount of citrate may enter with the blood which may be toxic. In
that case this preparation may be used.
DRIED HUMAN PLASMA
Dried plasma has the following advantages:
1.
If properly
stored it can be preserved for 5 years.
2.
If protected from
light it can be stored at room temperature provided it is below 200C.
3.
It can be given
to patients of any blood groups.
Preparation
1. Raw material: Usually time-expired whole blood is used.
2. Separation of
plasma from whole blood is done
either by allowing to stand or by centrifugation.
3. Pooling: Batches of not more than 10 bottles are pooled,
choosing the correct ratio of blood groups to neutralize powerful agglutinins
(The
most satisfactory ratio is : 9 parts of Gr. A
+
9 parts of Gr.O
+
2 parts of Gr. B or AB
4. Testing of
sterility: The pooled bloods are kept
at 4 to 60C and samples are tested for sterility.
5.
Freeze drying:
When the sterility test of the pools are passed then 400 ml is transferred to
bottles. They are sealed with bacteriologically efficient fabric pads covered
by ring-type closures and subjected to freeze drying.
(a)
Preliminary freezing: The bottles are sealed with bacteriologically efficient fabric pads
covered by ring-type closures and then centrifuged at –180C. The
liquid freezes and distributed around the inside of the bottle.
(b)
Primary drying:
The bottles of frozen are mounted horizontally in the drying chamber and high
vacuum is applied. The ice sublimes on to condensing coil kept at –500C
and a small heater provides the latent heat required for evaporation. This step
takes about 2 days, after which the residual moisture
content is about 2%.
(c)
Secondary drying: This is done in another chamber by vacuum descication over
phosphorous pentoxide (P2O5). It takes about a day and the product is left with
about 0.5% of moisture.
(d)
The fabric seal
is then replaced by a closure perforated with a hypodermic needle. The bottles
are again returned to the secondary drying chamber, 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 is kept below 200C and is protected from light, moisture and
oxygen. At this conditions the product will remain usable for indefinite time
but it is customary to put an expiry date of 5 years.
Fitness for use:
It
is reconstituted with 400ml of volume of Water
for Injection or,
Sodium
chloride injection or,
Dextrose
2.5% + NaCl 0.45% solution
The dried plasma must be dissolved with 10 minutes.
Gel formation or incomplete dissolution indicates deterioration.
After reconstitution it must be immediately used.
Use
1. Where there is no loss of RBC, in those cases
reconstituted plasma is given e.g. in burn patient where large amount of fluid
and protein loss occurs.
- If whole blood is not available or in emergency
when the results of matching test are not known.
- It is more suitable than whole blood in remote
places where the dried product can be kept as reserve stock.
DRIED HUMAN SERUM
Collection of blood:
The whole blood is collected
in dry bottles (in absence of anticoagulants) and allowed to clot. The
supernatant serum is separated after clot has retracted.
Every thing is same as the
preparation of dry plasma.
HUMAN PLASMA PROTEIN FRACTION
This is a solution of some of
the protein from liquid plasma. It contains albumin and certain globulins that
retain their solubility on heating.
It is prepared by
fractionating pooled citrated plasma and is similar to the fraction shown as
crude albumin in the following table:
TABLE Ether fractionation of
plasma
 |
Stabilization
A stabilizer, such as sodium
caprylate or acetyl-tryptophan is added. This allows heating for several hours
at low temperature.
Sodium chloride is added to
make the preparation isotonic.
Sterilization:
The solution is sterilized by filtration. The it is transferred in the bottle
and heated at
60 ± 0.50C for 10 hours to destroy the viruses.
Specification of final product:
Citrate concentration: 0.4 %
Protein content is not less
than: 4.3% w/v
Product exerts an osmotic
pressure equivalent to that of pooled liquid plasma containing 5.2% w/v of
protein.
Storage: It
must be stored between 5 an 200C and protected from light.
Dried human plasma protein fraction
It is prepared by
freeze-drying liquid human plasma protein fraction.
Use: Both the liquid and dried form are used for the same
purpose as dried plasma.
HUMAN FIBRINOGEN
Fibrinogen is the soluble
constituent of plasma that, on the addition of thrombin, is converted to
fibrin. Preparation: After separation
from plasma (see table) by fractionation, the precipitate is collected by
centrifugation, dissolved in citrated saline, and freeze-dried. The air n the
container is displaced by nitrogen.
Storage: The
solution should be used within 3 hours after preparation.
Reconstitution:
The citrate prevents
spontaneous clotting while reconstituted.
If shaken vigourously it
froths badly by forming a solid-stabilized foam, that is very slow to disperse.
Hence it should be dissolve by gentle rocking action only.
Use:
Occasionally fibrinogen is administered alone to treat fibrinogen deficiency,
but more often it is used in conjunction with thrombin.
HUMAN THROMBIN
This is the enzyme that
converts fibrinogen into fibrin.
Preparation:
The thrombin obtained after
fractionation of plasma is washed with distilled water and dissolved in
citrated saline. It is converted to thrombin by adjustment to pH 7.0 and adding
thromboplastin and calcium ions. The solution is filtered and freeze-dried. The
air in the container is replaced by nitrogen.
It is reconstituted with
saline when required.
Use:
1. The fibrin clot produced when thrombin is mixed with
fibrinogen is used in surgery to sutute severed nerves and to assist adhesion
of skin grafts.
2. The clot also acts as haemostat. Since the fibrin is
well tolerated by the body, new cells can penetrate it rapidly.
HUMAN FIBRIN FOAM
This is a sponge-like mass of
human fibrin.
Praparation:
A solution of fibrinogen is
whipped into froth by mechanical means and then thrombin is added.
The product is poured in
trays and freeze dried.
The fibrin-foam is cut into
pieces of suitable sizes and sterilized by dry heat at 1300C for 3
hours.
Storage: The
storage condition is same as dried serum but need not be kept under nitrogen.
Uses:
It is used as haemostat in
surgery, when other methods to arrest bleeding prove unsuccessful.
A piece is dipped into
thrombin solution and applied to the bleeding area. The foam can be left in situ, where it will be absorbed
because it is entirely of human origin.
HUMAN NORMAL
IMMUNOGLOBULIN INJECTION
Imuno-
or gamma- globulin is obtained from the globulin fraction separated in stage-3
of the fractionation process of plasma (see table).
The immunoglobulins are dissolved in 0.8% NaCl solution and a
preservative (0.01% thiomersal) is added.
The
solution is sterilized by filtration, packed in single dose containers and
stored at 4–60C with protection from light.
Normally
pools of not less than 1500 donations are used to ensure a satisfactory
representation of adult antibodies.
Use
It is used in the following
special cases:
1.
Measles: To
prevent the disease in children under 3 years.
2.
Rubella (German measles): To protect women exposed to infection in the first
four months of pregnancy.
3.
Infectious Hepatitis: To control outbreak of hepatitis in hospital wards.
CONTROL OF BLOOD PRODUCTS
Standards
Identification of protein:
1.
Precipitation
tests with specific antisera are used to show that only human serum proteins
are present in dried serum, dried plasma, the plasma protein fractions,
fibrinogen, thrombin, and immunoglobulin.
2.
The blood
proteins are identified by the mobilities of the proteins in electrophoretic
field. For example in plasma protein fraction there must not be less than 85%
of the protein having the mobility of albumin and not more than 1 % of gamma
globulins.
3.
Proteins can also
be identified by their sedimentation rate in an ultracentrifuge. This method is
specifically suitable for identifying different types of gamma globulins.
4.
Plasma clots when
calcium chloride is added, but serum
does not. Fibrinogen is identified by
clotting when thrombin is added in it. Thrombin
is identified by clotting when fibrinogen is added to it.
5.
The determination
of the blood groups, ABO of plasma and cells and Rh of cells, is the
identification test for whole blood.
Sterility and Pyrogen
All blood products must
comply with official tests for sterility and pyrogen.
Assays
For whole blood and
concentrated red cells the assay is a determination of the haemoglobin value.
For the remaining products, except fibrin foam (no assay) and thrombin, the
protein content is determined chemically. In thrombin there must be a minimum
number of clotting doses per mg, a clotting dose being the amount of thrombin
required to clot 1 ml of 0.1 % fibrinogen in saline buffered at 7.2 to 7.3 in
15 secs at 370C.